Pectins and Pectinases

PECTINS AND PECTINASES Progress in Biotechnology ~~ Volume 1 New Approaches t o Research on Cereal Carbohydrates (Hi...

Author: A. G. J. Voragen | A. G. J. Voragen | J. Visser

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PECTINS AND PECTINASES

Progress in Biotechnology ~~

Volume 1 New Approaches t o Research on Cereal Carbohydrates (Hill and Munck, Editors) Volume 2 Biology of Anaerobic Bacteria (Dubourguier et al., Editors) Volume 3 Modifications and Applications of Industrial Polysaccharides (Yalpani, Editor) Volume 4 lnterbiotech '87. Enzyme Technologies (Blaiej and Zemek, Editors) Volume 5 In Vitro Immunization in Hybridoma Technology (Borrebaeck, Editor) Volume 6 lnterbiotech '89. Mathematical Modelling in Biotechnology (Blafej and Ottova, Editors) Volume 7 Xylans and Xylanases (Visser et al., Editors) Volume 8 Biocatalysis i n Non-Conventional Media (Tramper et al., Editors) Volume 9 ECB6: Proceedings of the 6th European Congress on Biotechnology (Alberghina et al., Editors) Volume 10 Carbohydrate Bioengineering (Petersen et al., Editors) Volume 11 Immobilized Cells: Basics and Applications (Wijffels et al., Editors) Volume 12 Enzymes for Carbohydrate Engineering (Kwan-Hwa Park et al., Editors) Volume 13 High Pressure Bioscience and Biotechnology (Hayashi and Balny, Editors) Volume 14 Pectins and Pectinases (Visser and Voragen, Editors)

Progress in Biotechnology 14

PECTINS AND PECTINASES Proceedings of an International Symposium, Wageningen, The Netherlands, December 3-7, 1995

Edited by J. Visser Section of Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, Wageningen, The Netherlands

A.G.J. Voragen Department of Food Science, Section of Food Chemistry and Food Microbiology, Wageningen Agricultural University, Wageningen, The Netherlands

ELSEVIER Amsterdam - Lausanne - New York - Oxford - Shannon Tokyo 1996

-

Published by: Elsevier Science B.V. P.O. Box 21 1 1000 AE Amsterdam The Netherlands

Library o f Congress Cataloging-in-Pub

ication Data

P e c t i n e s and pec inases : p r o c e e d i n g s o f an i n e r n a t i o n a l svmDosium. . . Wageningen, t h e N e t h e r l a n d s , December 3-7, 1995 / e d i t e d by J. V i s s e r . A . G . J . Voragen. rn -- (~.P .r nI=.n r o a a i, ,n, h i n t o r h n n l sn vnyQy 8 ,. l a ) rn . r-.... Includes b i b l i o g r a p h i c a l references. ISBN 0-444-82330-1 ( a l k . p a p e r ) 1 . Pectin--Congresses. 2. Pectinase--Congresses. I.V i s s e r , J. 11. Voragen. A . G. J. 111. S e r i e s . TP248.P4P43 1996 664'.25--d~20 96-36458 CIP

--.,

" . " . I " . I I I I I

, - I

ISBN 0-444-82330-1 01996 Elsevier Science B.V. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher, Elsevier Science B.V., Permissions Department, P.O. Box 521,1000 A M Amsterdam, The Netherlands. No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances i n the medical sciences, the Publisher recommends th'at independent verification of diagnoses and drug dosages should be made. Special regulations for readers in the USA - This publication has been registered with the Copyright Clearance Center Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01293, USA. Information can be obtained from the CCC about conditions under which photocopies of parts of this publication may be made in the USA. All other copyright questions, including photocopying outside the USA should be referred to the copyright owner, Elsevier Science B.V., unless otherwise specified. This book is printed on acid-free paper. Printed in the Netherlands

PREFACE The Intemational Symposium on Pectins and Pectinases held in Wageningen, The Netherlands from December 3-7, 1995 was a very stimulating and succesful meeting. Attended by 190 participants representing 25 different countries, the Symposium provided the platform for many multidisciplinary interactions. Scientists working in applied fields such as pectin manufacturing, enzyme production and food processing often met for the first time with molecular biologists and X-ray crystallographers who have in recent years started to discover how interesting pectin degrading and modifying enzymes are and how complex the substrate is on which these enzymes act. Progress in pectin and pectinase research has been most prominent in two areas over the past 5 years viz in analyzing and elucidating the complex chemical structure of pectin and in unraveling the mode of action and the structure of various pectin degrading enzymes as well as in cloning the corresponding genes. It also became evident that we still know far too little about the biosynthesis of pectin and the role of pectin in plant cell wall architecture. The Symposium was further highlighted by an initiative of IPPA to honour Prof. Walter Pilnik for all his activities in the past in the interest of pectin research and of the association of pectin producing industries. This was done by awarding a talented, junior scientist in the field to present the so-called Walter Pilnik Lecture. This award, which will be given every second year, was given to Dr Maureen McCann. The editors of this volume hope that the Proceedings of this Symposium will not only reflect the present status of research in this area but that it will also remain a useful reference book.

Wageningen, July 11, 1996. J. Visser A.G.J. Voragen

This Page Intentionally Left Blank

O R G A N I Z I N G COMMITTEE A. G.J. Voragen

(chairman)

M.A. Kusters-van Someren

(secretary) J. Visser

(treasurer)

Dept of Food Science Section of Food Chemistry and -Microbiology Wageningen Agricultural University Section of Molecular Genetics of Industrial Microorganisms Wageningen Agricultural University Section of Molecular Genetics of Industrial Microorganisms Wageningen Agricultural University

G. Beldman

Dept of Food Science Section of Food Chemistry and -Microbiology Wageningen Agricultural University

J. Benen

Section of Molecular Genetics of Industrial Microorganisms Wageningen Agricultural University

H.A. Schols

Dept of Food Science Section of Food Chemistry and -Microbiology Wageningen Agricultural University

SCIENTIFIC ADVISORY COMMITTEE W. Pilnik

Wageningen, The Netherlands

(honorary chairman) P. Albersheim A.B. Bennett H.P. Heldt-Hansen J. Jenkins M. Rinaudo J. Robert-Baudouy K. Roberts J. SOderberg J.F. Thibault A.J. Vroemen

Athens, USA Davis, USA Copenhagen, Denmark Reading, UK Grenoble, France Villeurbanne, France Norwich, UK Copenhagen, Denmark Nantes, France Seclin, France


ACKN OW LED G EMENT S The Organizing Committee of the International Symposium on Pectins and Pectinases acknowledges the following organizations for their support. Without their contribution this Symposium would not have been possible. Citrus Colloids Ltd. Copenhagen Pectin A/S Cooperatieve Suiker Unie U.A. Dalgety S.F Ltd - Food Technology Center Danisco Ingredients Dionex B. V. Gist-brocades France S.A. Groupe Danone Herbstreith & Fox K.G. Hercules European Research Center B. V. IPPA Koninklijke Nederlandse Chemische Vereniging Landbouwuniversiteit Wageningen Cluster Biomoleculaire Wetenschappen Nederlandse Unilever Bedrijven B. V. Novo Nordisk A/S Obipektin A. G. Quest International Systems Bio-Industries VLAG The Graduate School for Advanced Studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences -


xi CONTENTS.

STRUCTURE, PHYSICAL AND CHEMICAL PROPERTIES OF PECTINS

Complex pectins: Structure elucidation using enzymes. H.A. Schols and A.G.J. Voragen.............................................................................................

3

Physicochemical properties of pectins in solution and gel states. M. Rinaudo...........................................................................................................................

21

Interactions of pectins with multivalent cations: Phase diagrams and structural aspects. M.A. K Axelos, C. Garniel; C.M.G.C. Retiard and J. -F: Thibault ..........................................

35

An hypothesis: The same six polysaccharides are components of the primary cell walls of

all higher plants. f? Albersheim, A. G. Darvill, M.A. 0 'Neill, H.A. Schols and A. G.J. Voragen ........................

47

Acetylation of rhamnogalacturonan I and homogalacturonan: Theoretical calculations. M. Kouwijzel; H. Schols and S. Pkrez ...................................................................................

57

The pectic polysaccharide rhamnogalacturonan I1 is a major component of the polysaccharides present in fruit-derived products. I? Pellerin, 7: Doco, S. Kdal, I? Williamsand J-M. Brilloiret ...............................................

67

Partial characterization of xylogalacturonans from cell walls of ripe watermelon fruit: inhibition of endopolygalacturonase activity by xylosylation. L. Yu and A.J. Mort ..............................................................................................................

79

PECTIN BIOSYNTHESIS AND BIOLOGICAL EFFECTS OF (DEGRADED) PECTIN

Plant cell wall architecture: the role of pectins. M. C. McCann and K. Roberts ..............................................................................................

91

Cell free synthesis of the pectic polysaccharide homogalacturonan. D. Mohnen, R.L. Doong, K. Liljebjelke, G. Frnlish and J. Chan ........................................

109

Biosynthesis in vitro of pectic ( 1 + 4)-R-D-galactan. L.S. Brickell at1dJ.S.G. Reid ..............................................................................................

127

Cell wall pectins: From immunochemical characterization to biological activity. P Van Cutsem and J. Messiaen ..........................................................................................

13 5

xii

Methyl-esterification, de-esterification and gelation of pectins in the primary cell wall. R. Goldberg, C. Morvan, A. Jairneaii and M. C. Jarvis .......................................................

15 1

Contribution of pectins on health care.

H. Yamada .........................................................................................................................

173

The role of polygalacturonase, PGIP and pectin oligomers in hngal infection. F: Cervone, G. de Lorenzo, B. Aracri, D. Bellincampi, C. Caprari, A.J. Clark, A. Desiderio, A. Devoto, F: Leckie, B. Mattei, L. Nirss and G. Salvi ...................................

191

Biologically active pectin oligomers in ripening tomato fruits.

E. Melotto, L. C. Greve and J.M. Labavitch ........................................................................

207

IDENTIFICATION, MODE OF ACTION AND 3-D STRUCTURE OF PECTINASES Kinetics and mode of action of Aspergilhrs tiiger polygalacturonases. J.A.E. Benen, H. C.M. Kestel; L. Parenicovci and J. Msser ..................................................

22 1

New enzymes active towards pectic structures. G. Beldman, M. Muttel; M.J.F:Searle-van Leeintien, L.A.M. van den Broek, H.A. Schols and A. G.J. Voragen .........................................................................................

23 1

The Bsubunit of tomato fruit polygalacturonase isoenzyme 1 defines a new class of plant cell proteins involved in pectin metabolism: AroGPs (Aromatic amino acid rich Glyco Proteins). D. DellaPenna, C. Watson,Jl? Liir and D. Schirchman......................................................

247

Characterisation of RG degradation products of new RGases using RG-rhamnohydrolaseand RG-galacturonohydrolase. M. Muttel; C.M. G. C. Renard, G. Beldnian, H.A. Schols and A. G.J. Voragen ......................

263

The effect of glycosylation of endopolygalacturonasesand polygalacturonase inhibiting proteins on the production of oligogalacturonides. C. W! Bergmann, B. Cook, A.G. Darvill, I? Alhersheim, D. Bellincampi and C. Caprari .....275 Envinia pectate lyase differences revealed by action pattern analyses. S.Bartling, P Derkx, C. Wegener and 0. Olsen .................................................................

283

Functional implications of the three-dimensional structures of pectate lyases. F: Jurnak, N. Kita, M. Garrett, S.E. Heffron, R. Scavetta, C. Boyd and N. Keen .................295

xiii

MOLECULAR GENETICS AND REGULATION OF PECTINASE BIOSYNTHESIS IN SAPROPHYTIC AND PHYTOPATHOGENIC MICROBIAL SYSTEMS

Regulation of pectinase biosynthesis in Eminia chrysanthemi. N. Hugouvieux-Cotte-Pattat, S. Reverchon, W Nassel; G. Condemine and J. Robert-Baudouy .............................................................................................................

311

Molecular genetic and biochemical aspects of pectin degradation in Aspergillus. J. Benen, L. Parenicova,M. Kusters-van Someren, H. Kester and J. Ksser ........................

33 1

Expression of polygalacturonase and pectinesterase in normal and transgenic tomatoes. G. nicker andJ. Zhang ......................................................................................................

347

Role of pectin methylesterase in tomato fruit ripening and quality attributes of processed tomato juice. A.K. Handa, D.M. Tiemann, K.K. Mishra, B. R. Thakur and R.K. Singh .............................

355

Molecular characterization and expression of CoIletoh.ichum lindemiithianum genes encoding endopolygalacturonase. S. Centis, K Hugouvieux, J. Foiirniec C. Lnfiite, M. 7: Esquerre-Eigaye and B. Dirmas ...........................................................................................................................

369

Cloning of genes encoding pectin-degrading enzymes in Azospirillum irakense. M.A. Bekri, J. Desail; L. van Lonimel andJ. Vnriderleyden ................................................

377

Transgenic potatoes that express an Erwinia pectate lyase isoenzyme.

C. Wegenel; S. Bcrrtling, J. Webel; S. Hoffnintiri-Beiiiiirig and 0. Olsen ..............................

385

Pectins and pectolytic enzymes in relation to development and processing of green beans (Phaseolus vulgaris L.). K. Recourt, T Stolle-Sniits, J.M. Lnats, J. G. Beekhriizen, C.E.M. Ehbelaal; A.G.J. Voragen, H.J. Wichers and C. van Dijk ....................................................................

399

APPLICATIONS. A) DEVELOPMENTS IN PECTIN MANUFACTURING AND APPLICATIONS

Rheological methods to characterize pectins in solutions and gels.

H.-U. Endress, C. Doschl-Volle andK. Dengler .................................................................

407

Effects of extrusion-cooking on pectin-rich materials. J.-I? Thibault,M.-C. Ralet, M.A. I.:Axelos and G. Della VXle ............................................

425

xiv

Pectin degradation in UF-membrane reactors with commercial pectinases. C. Dinnella, A. Stagni, G. Lanzarini, E Alfaiii, M. Cantarella and A. Gallijiuoco...............439

B) APPLICATION OF PECTINASES IN BEVERAGE, FOOD, FEED AND NOVEL TECHNOLOGIES

Application of pectinases in beverages. C. Grassin andP Fauquembergzie .....................................................................................

453

Application of tailormade pectinases. H.P Heldt-Hansen, L. !F Kofod, G. Biidolfen, PM. Nielsen, S. Hiittel and i? Bladt ............ 463 Cations increase activity and enhance permeation of pectinesterase in ultrafiltration.

L. Wicker............................................................................................................................

475

Production, characterization and application of rhamnogalacturonase. H. Hennink, H. Stam andM.G. van Oort............................................................................

485

Effects of a new canning process on cell wall pectic substances, calcium retention and texture of canned carrots. H. Siliha, K Jahn and K. Gierschner.................................................................................

495

SHORT COMMUNICATIONSBASED ON POSTER PRESENTATIONS. STRUCTURE, PHYSICAL AND CHEMICAL PROPERTIES OF PECTINS

Isolation and sequential extraction of cell wall polysaccharides from soy meal. M.M.H. Huisman, H.A. Schols and A. G.J. Voragen............................................................

5 11

Modelling a pentasaccharide fragment of rhamnogalacturonan I. M. Broadhurst, S. Cros, R. Hofsniann, W Mackir and S. Perez ..........................................

5 17

Influence of some cations on the reaction of apple pectin with ammonia in homogeneous media. P Denev and Chl: Kratchatiov ...........................................................................................

527

Heavy metals binding by pectins: selectivity, quantification and characterisation. KM. Dronnet, C.M. G.C. Renard, M.A. !L Axelos and J. -E Thibault ....................................

535

Quantitative Raman spectroscopy. Prediction of the degree of esterification in pectins. S.B. Engelsen and L. Norgaard ..........................................................................................

54 1

xv

Polysaccharides from Chorisia speciosu St. Hill. E.B. Beleski-Carneiro, M.R. Sierakowski,J.L.M.S. Ganter;S,F: Zawadzki-Baggio and F: Reicher ....................................................................................................................

549

Rigid and flexible pectic polymers in onion cell walls. M.-A. Ha, B. W Evans, D.C. Apperley at1dM.C. Jarvis ......................................................

561

Changes in pectic polysaccharides during elaboration of table olives. A. Heredia, R. GuillCn, C. Sanchez, A. JimCnez andJ. Fernandez-Bola2os ........................

569

Pectins from different tissue zones of apple: characterisation and enzymatic hydrolysis.

P Massiot. A. Baron at1dJ.E Drilleau ...............................................................................

577

Investigations of the influence of various cations on the rheological properties of high-esterified pectin gels. S. Neidhart, C. Hannak and K. Gierschner.........................................................................

5 83

Changes in molecular weight and carbohydrate composition of cell wall polyuronide and hemicellulose during ripening in strawberry fruit. I.:Nogata, K. Yoza, K. Kusirnioto and H. Ohf a ...................................................................

59 1

Autoclave extraction of sugar beet pulp yields gel-forming pectic hairy regions. A. Oosterveld, G. Beldman, J.M. de Bruijii at1dA.G.J. Voragen .........................................

597

Pectins in mild alkaline conditions: l3-elimination and kinetics of demethylation. C.M.G.C. Renard and J. -I? Thibairlt ..................................................................................

603

Potentiometric titration of poly(a-D)galacturonic acid. D. Rudan-Tasi? and C. Klojirtar ........................................................................................

609

Structural studies of a pectic polysaccharide from Plantago major L. A.B. Samuelsen, E.H. Cohen, B.S. Pairlsen and J.K. Wold..................................................

6 19

Structural characterization of a novel rhamnogalacturonan I1 with macrophage Fc receptor expression enhancing activity from the leaves of Patiax ginseng C.A. Meyer. K.-S. Shin, H. Kiyohara*T Matsunloto atid H. Yaniada......................................................

623

Structural features of pectic polysaccharides of red beet (Beta vzrlgaris conditiva). G.R. Strasser;D.E. Wechsler and R. Aniado .......................................................................

63 1

Isolation and physicochemical characterisation of xylose-rich pectic polysaccharides from wheat straw. R. Sun, J.M. Lawther and WB. Batiks ................................................................................

637

Chemical synthesis of oligosaccharides related to arabinogalactan-proteins(AGPs). J.E Valdor and W Mackie ..................................................................................................

645

xvi Structural features of pectic substances during growth and ripening of apples. D.E. Wechslel: G.R. Slrasser and R. Aniado .......................................................................

65 1

PECTIN BIOSYNTHESIS AND BIOLOGICAL EFFECTS OF (DEGRADED) PECTIN Metabolism of pectin in the gastrointestinal tract. G. Dongowski and H. Anger ..............................................................................................

659

Cell wall properties of transgenic tobacco plants that express a yeast derived acid invertase in their vacuole. S. Hoffmann-Benning, R. Ehrwald, L. Willniitzeratid J. Fisahn .........................................

667

Pectic polysaccharide from roots of Glycyrrhiza iiralensis:Possible contribution of neutral oligosaccharide in the galacturonase-resistant region to anti-complementary and mitogenic activities. H. Kiyohara, N. Takenioto, J. -E Zhao, H. Kmuaniiira and H. Yantada................................

673

Immunologically active polysaccharides from cell suspension of Heliarithiis 1805. M. Kratchnnova, M. Zlieva, E. Pnvlova, A . Pavlov and N. Markova ...................................

679

Pectins and pectinases in stem rust-infected wheat. M. Mierair, B. GraeJtier;A . J. Mort atid B.M. Moerschhacher...........................................

687

Bioactive fragments from pea pectin. 0. Zabotiti, N. Ibragimova, D. Ayiipova, 0. Gzirjatm), K Lozovaya, G. Beldmati and A. Voragen .................................................................................................................

693

atitniiis

IDENTIFICATION, MODE OF ACTION AND 3-D STRUCTURE OF PECTINASES Stereochemistry of hydrolysis of glycosidic linkage by three Aspergillus polygalacturonases. 19 Bieb, J.A.E. Benen, H.C.M. Kester; K. Heinrichova andJ b s e r ..................................

705

Pectin methyltransferases from suspension-cultured cells and seedlings of flax (Linum usitatissimiim L,), l? Bruyant, M.I? Briiymt-Vannier; 7: Boirrlard, A . Gnirdi~iet-Schaiininti, B. Thoiron atid C. Morvan .................................................................................................

711

Pectinases from Rhizopzis sp. efficient in enhancing the hydrolyzation of raw cassava starch: Purification and characterization. L. Chitradon, t! Pooiipairoj, t! Mahakhan, b! Kitpreechavatiichatid N. Lotong.. ...............71 5

xvii

Isolation, characterization and immuno localization of orange fruit acetyl esterase. T.M.I. E. Christensen, J.E. Nielseri and J.D. Mikkelseri .......................................................

723

Enzyme-mediated substrate immunolocalization of polygalacturonic acid within barley epidermal cell walls utilizing endopolygalacturonase of Cochliobobrs sativus and a monoclonal antibody specific for the enzyme. R.l? Clay, C. W Bergmarin and M.S. Firller ........................................................................

13 1

Influence of glucose and polygalacturonic acid on the synthesis and activity of the polygalacturonase from the yeast strain SCPP. A. Gainvors and A. Belarbi ................................................................................................

739

Pectin lyase from Fusariiim oxysporiini f. sp. radicis lycopersici: purification and characterization. M.A. Guevara, M. T Gonzdez-fat;ir andP Estivez ............................................................

747

Enzymic release of ferulic acid from sugar beet pulp using a specific esterase from Aspergillus niger. PA. Kroon, C.B. FaiiJds9C. BredJon orid G. WilJianison...................................................

761

Characterization of some endo-polygalacturonases from Sclei'otinia sclerotiorum. M.B. Martel, R. Letoltblori andM. Fivre ...........................................................................

769

Analysis of the interaction between PGIP from Phaseohis viilgaris L. and fungal endopolygalacturonases using biosensor technology B. Mattei, G. Salvi, C. Caprari, G. De Lorenzo, L? Crescenzi and E Cervone .....................

775

Rhamnogalacturonan a-L-rhamnopyranosyl-( 1 -+4)-a-D-galactopyranosyluronide lyase, a new enzyme able to cleave RG regions of pectin. M. Mutter; I.J. Colqiihoiiii, G. Reldninir, H.A. Schols and A.G.J. Vorngen..........................

783

Purification and characterisation of galactose-induced pectinases from the exo-l mutant strain of Neiirospora crassci. L.B. Crotti, J.A. Jorge, H.E Ereriri aridM.L. TM. Polizeli .................................................

787

Acetyl esterases ofdspergilliisn i p : purification and mode of action on pectins. M.J.E Searle-van Leeimen, J . 2 Mncken, D. Schipper; A.G.J. Vorngenand G. Beldman ........................................................................................................................

793

A polygalacturonase inhibitor of Dieflenhachia niacirlata. A . Chitre arid N. I.:Shastri ..................................................................................................

799

Multiple forms of carrot exopolygalacturonase.

E. Stratilova, M. Dziirova and D. Mi.slovic.?ova...................................................................

807

xviii

MOLECULAR GENETICS AND REGULATION OF PECTINASE BIOSYNTHESIS IN SAPROPHYTIC AND PHYTOPATHOGENIC MICROBIAL SYSTEMS Primary structure and characterization of an exo-polygalacturonase from Aspergillus tubingensis. H.C.M. Kestec M.A. Kirsters-van Someren, K Miiller and J. Ksser ....................................

8 17

pgaE from Aspergillus niger encodes a fourth polygalacturonase. Molecular cloning and biochemical characterisation of the gene product. L. Parenlicova, J.A.E. Benen, H.C.M. Kester andJ. Ksser ................................................

825

Identification of a seventh endo-pectate lyase of the phytopathogenic bacterium Erwinia chrysanthemi. C. Pissavin, J. Robert-Baiidoiiyand N. Hiigorivieiix-Cotte-Pattat.......................................

83 1

Synthesis of new methyl esters of 3-deoxy-D-erythro-2-hexulosonicacid (KDG) analogs, inducers of the expression of pectinase genes in bacteria Erwinia chrysanthemi. F: Alessi, G. Condemine,A. Doirtheaii, J. Robert-Baiidoiiy and D. Anker ...........................

845

Pectin methylesterase B of Erwinia chrysanthemi, the first pectinase characterised as a membrane lipoprotein. FE. Shevchik, G. Condeniine,N. Hirgoii~iieiix-Cotte-Pattatand .J. Robert-Baiidoiry ........... 837 Production of pectinases from Rhizopris sp. in solid substrates. L. Chitradon,I? Mahakhan, I? Poonpairoj, I? Kitpreechavanich and N. Lotong .................853 Endo-polygalacturonase of the yeast Klriyveromyce.~rnarxianirs is constitutive, highly active on native pectin and is the main extracellular protein. R.F: Schwan, R.M. Cooper andA.E. Wheals ......................................................................

861

Polygalacturonase and pectinmethylesterase activities during growth of Helianthirs annirirs 1805 cell suspension. M. Ilieva, M. Kratchanova, E, Pavlovc~,T Diniovcr and A. I’avlov .....................................

869

Differential expression of Erwinia chryscrnthenii strain 3937 pectate lyases in pathogenesis of African violets: importance of low iron environmental conditions. C. Mascbirx, N. Hiigoiivieirx-Cotte-Pattat nnd D. Expert...................................................

875

Regulation of polygalacturonases in two isolates of Fiisariiini oxysporiim fsp. radicis Iycopersici (FORL). B. Patiiio, M.L. Posada, M. 7: Gonzdez-JaPn, M.J. Martinez and C. Vazqirez ....................

88 1

Endo-pectinase production by intraspecific hybrids of Aspergillus sp. CH-Y- 1043 obtained by protoplast fusion. S.Solis, E. Flores-Sanchez and C. Hiiitrdn ........................................................................

893

xix

Candida boidinii - a new found producer of pectic enzymes complex. E. Stratilova, E. Breierova and R. Vadkertiovci...................................................................

899

Cloning, sequence and expression of the gene coding for rhamnogalacturonase (RHG) of Aspergillus aculeatus: a novel pectinolytic enzyme. M.E.G. Suykerbuyk, PJ. Schaap, H. Stant, K Musters and J. Ksser ..................................

907

Pectinase secretion by Aspergillus FP- 180 and Aspergillus niger N-402 growing under stress induced by the pH of culture medium. B.A. Trejo-Aguilal;J. h s e r and G. Agiilar 0 ...................................................................

9 15

Selection of a constitutive hyper-pectinolytic mutant from a Penicillium strain. N. HadjTaieb, M. Ayadi and A. Gargouri ..........................................................................

921

APPLICATIONS. A) DEVELOPMENTS IN PECTIN MANUFACTURING AND APPLICATIONS

Production of hypocaloric jellies of grape juice with sunflower pectin. M.L. AlarcLTo-Silva, H. Gil Azitiheira, M.I.N. Jatnrcirio, M. C.A. Leitco and TC. Curado .......................................................................................................................

931

Influence of microwave pretreatment of fresh orange peels on pectin extraction. M. Kratchanova, E. Pavlova, I. Patrchev and Chi: Kratchatiov ..........................................

94 1

Properties of pectinesterase from Penicilliimifellutatiuni Biourge and new developments in pectin applications. KL. Aizenberg, S.A. Syrchin, S.A. Sedina K N . Vasil 'ev, L.N. Shitikareiiko, RI. Demchenko and 19 N. Htte ............................................................................................

947


Enzymatic maceration of apple parenchyma: modelling of the degradation. A. Baron, R Massiot, C. Ella Missatig and J.E Drilleaii ....................................................

957

Enzymatic treatment in the extraction of cold-pressed lemon peel oils. L. Coll, D. Saiira, J.M. Ros, M. Moliner and J. Laencitia...................................................

963

Immobilized pectinase efficiency in the depolymerisation of pectin in a model solution and apple juice. C. Dinnella, A. Stagni, G. Lanzarini aiidM. Lniis ..............................................................

971

xx

Pectinases in wood debarking. M. Ratto and L. Piikari ......................................................................................................

979

Oligouronides of pectins in membrane reactor by enzymatic degradation of pectins from Citrus peel. A preliminary study. J.M. Ros, D. Saura, L. Coll, M. Molirier mid J. Laenciim. ......................... .........................

983

STRUCTURE, PHYSICAL AND CHEMICAL PROPERTIES OF PECTINS


J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All fights reserved.

Complex Pectins: Structure elucidation using enzymes H.A. Schols and A.G.J. Voragen Wageningen Agricultural University, Department of Food Science, Bomenweg 2, 6703 HD Wageningen, The Netherlands

Abstract

A pectic fraction, retained by ultrafiltration of the juice from enzyme treated apple tissue and resistant to further enzymic degradation, was isolated and characterized using chemical and enzymic methods. The fraction was termed MHR (modified hairy regions) and this fraction was characterized by a high arabinose content, next to a high rhamnose to galacturonic acid ratio and a high acetyl content and smaller proportions of xylose and galactose. Rhamnogalacturonase (RGase), an enzyme able to hydrolyze galacturonic acid-(1-,2)rhamnosyl linkages within the rhamnogalacturonan backbone of MHR was used to obtain both oligomeric and polymeric degradation products. These RGase-oligomers consist of a tetrameric or hexameric backbone of alternating rhamnose and galacturonic acid residues with a galactose residue substituted at C-4 of part of the rhamnose moieties. Next to the subunit from which these oligomers were released, two other subunits were recognized: a highly methyl esterified xylogalacturonan segment and residual stubs of the backbone rich in branched arabinose side chains. Comparison of the MHR with non-modified pectic hairy regions of apple cell wall, isolated in a mild and defined way, revealed great resemblance indicating that the modifications of the MHR during enzymic liquefaction were only minor. Analogous MHR fractions could be isolated from potato fibre, pear, carrot, leek, and onion tissue. Finally, an adapted model is presented for the prevailing population of apple MHR having the highest molecular weight. The universal validity of this model for pectic hairy regions from other plant sources is discussed.

1. INTRODUCTION Plant cell walls form a single continuous extracellular matrix through the body of the plant and the walls of many cells together form the skeleton of plant tissues. The cell matrix consists of various types of polysaccharides, proteins and lignin in varying amounts, organized in such a way that the cell wall is chemically rather stable and physically robust. Primary walls can best be described as reinforced, multi-component gels [1]. Cell walls control cell growth by influencing cell size and shape, but also act as a pre-existing structural barrier to invasion of micro-organisms [2]. When infection or wounding of the wall occurs,

the cell may respond by thickening, lignification or suberization of the cell, production of phenolic acids and extensins, pigments, etc. [ 1]. When edible parts of plants are considered; the quality attributes of fresh fruits and vegetables (e.g. ripeness, texture) and their processing characteristics in the manufacture of foods (juices, nectars, purees, preserves) are determined to a large extent by the cell walls of plant raw materials. These cell walls also influence the extractability of important constituents of plant raw materials like sugar, oil, proteins, etc. Detailed knowledge of the major constituents making up the cell walls (e.g. pectic substances, hemicelluloses, cellulose, and structural proteins) and the ultra-structure of the cell wall is important to control and improve processing and utilization of plant products. One of the important constituents of plant cell walls is formed by the group of pectic substances [3], which has in common that all polymers contain a high proportion of galacturonic acid residues. Pectins are extracted on industrial scale from suitable plant (waste) materials e.g. apple pomace and citrus peel and used by the food industry due to their ability to form gels under certain circumstances and to increase the viscosity of drinks. They are also widely applied as stabilizers in acid milk products. Food nutritionists are interested in pectins in foods because they are considered as dietary fibre and have been shown to lower blood cholesterol levels. Interest is also directed to their pharmaceutical activities [4]. From the above, it may be clear that scientists from various disciplines are interested in the precise structure of both extracted pectins as well as pectic substances in plant cell walls. In the following, a short summary will be given of the existing literature on the structure of pectic substances, and recent studies on the structure elucidation of highly ramified regions of pectins using enzymes will be reported.

2. PECTIC SUBSTANCES The pectic polysaccharides are probably the most complex class of wall polysaccharides [1] and comprise a family of acidic polymers like homogalacturonans, rhamnogalacturonans with several neutral polymers like arabinans, galactans and arabinogalactans attached to it [1,3,5]. The pectin consists of a backbone, in which "smooth" t~-D-(1--,4)-galacturonan regions are interrupted by ramified rhamnogalacturonan regions, highly substituted by neutral sugar rich side chains. It is suggested by De Vries et al [6] that only up to 10% of the galacturonosyl residues are included in the ramified (hairy) regions while these regions carry almost all of the neutral sugar residues. Individual classes of the pectic substances will be discussed below.

2.1 Homogalacturonans Homogalacturonan segments are defined as polymers consisting predominantly of ot-(1--,4)linked galacturonosyl residues [2]. Pure homogalacturonans have been rarely reported [1], although one of the reasons might be the poor solubility of these polymers [2]. On the other hand, more and more indications are found that homogalacturonans are covalently linked to RG-I and other cell wall polymers [1,6,7]. Homogalacturonans are usually extracted from plant material by mild acid treatment [7,8]. Depending on the extraction method used, some modification of the polymer may occur. Uninterrupted homogalacturonan regions with a degree of polymerisation (DP) of approximately 70-100 have been isolated from various plant

tissues like carrot [9], apple, beet and citrus [7,10]. An important feature of galacturonans is the esterification of the galacturonic acid residues with methanol and/or acetic acid. The DM is defined as the number of moles of methanol per 100 moles of galacturonic acid. Pectins are called high methoxyl pectins when the value for DM is 50 or higher. In the other cases, the pectin is called low methoxyl pectin. For native apple pectins a random distribution of the methoxyl groups over the galacturonan chain was found [11-13]. For commercially extracted pectins, the distribution was found to be slightly different since the relative amounts of mono-, di-, and trigalacturonides in endo PGdigests suggested small blocks of unesterified galacturonosyl residues [14]. Kiyohara et al. [14] found galacturonic acid oligomers up to octamer after PG-digestion of homogalacturonans from Angelica acutiloba Kitagawa, suggesting a more blockwise distribution. Acetyl groups are usually only present in low amounts in pectins from e.g. apple and citrus, but are present in much higher amounts in pectins from sugar beet [15] and potato [16].

2.2 Rhamnogalacturonan I and II The group of Albersheim devoted a high number of publications to RG-I type of pectic substances which is conveniently reviewed by O'Neill et al. [5]. RG-I is the major polysaccharide solubilized from suspension-cultured sycamore cell walls after treatment with PG and was found to represent 7-14 % of the cell wall [5]. The RG-I polymer is composed of alternating rhamnose and galacturonic acid residues. The length is unknown, but it could contain as many as 300 rhamnose and 300 galacturonic acid residues [2]. Polymers containing this backbone are present in most if not all higher plant cell walls [5]. Next to the rhamnose and galacturonic acid residues in the backbone, RG-I is composed of side chains containing arabinofuranosyl-, galactopyranosyl- and minor quantities of fucopyranosyl residues [5]. Next to RG-I, the group of Albersheim also described rhamnogalacturonan II (RG-II). Characteristic for RG-II is the presence of rare sugars like 2-O-methyl-fucose, 2-0methyl-xylose, apiose, aceric acid, 2-keto-3-deoxy-D-manno-octulosonic acid (KDO) and 3deoxy-D-lyxo-2-heptulosaric acid (DHA), next to the more common sugar residues rhamnose, fucose, arabinose, galactose, galacturonic acid and glucuronic acid [2]. 2.3 Other galacturonic acid-containing plant cell wall polysaccharides The presence of xylogalacturonans in which terminal xylose is linked directly to the galacturonosyl residues has been reported for mountain pine pollen [ 17], soy beans [18], and kidney beans [19,20]. The presence of terminal xylose residues linked directly to galacturonic acid moieties in pectic substances from apple has also been suggested [21,22]. Apiogalacturonan regions are proposed to be present in pectins extracted from eel grass [23] and duckweed [24]. Side chains containing glucuronic acid, galacturonic acid linked to galactose or rhamnose residues, and galactose side chains linked via a galacturonosyl residue to the C-4 of rhamnose in the backbone have been described to be present in pectic fragments from leaves and roots of several plants having an anti-complementary activity [25].

3. PECTIC ENZYMES Pectic enzymes are classified according to their mode of attack on the galacturonan part of

the pectin molecule. The main classes of pectic enzymes are pectin methylesterases (PE), polygalacturonase (PG), pectin- and pectate lyases (PL, PAL)[26-29]. Recently, in analogy to the enzymic degradation of the homogalacturonan regions of pectin, a whole array of enzymes specific for the degradation of rhamnogalacturonan regions within the pectin molecule has been recognised and these enzymes will be discussed in more detail in this book by Beldman et al [30]. The availability of these novel enzymes, next to the known pectic enzymes, offer new opportunities to use them as analytical tools in revealing the structure of oligo- and polysaccharides [31,32]. In contrast with frequently used chemical degradation methods, which usually have a poor selectivity, these enzymes act in a defined way. To be able to recognize different structural units within the polymer, endo-acting types of enzyme are preferred, although accessory enzymes might be essential as well [30]. In this study, we used purified polygalacturonase, pectin methylesterase, rhamnogalacturonan hydrolase and rhamnogalacturonan acetylesterase from fungal origin to study the structure of polymeric pectin fragments, remaining after degradation of apple tissue by a technical enzyme preparation.

4. PECTIN FRAGMENTS, RESISTANT TO ENZYMIC DEGRADATION DURING FRUIT PROCESSING During the preparation of apple juices either by direct pressing of the pulp or by the use of enzymes, it was found that significant amounts of polymeric pectic fragments were present in the enzyme-treated juices [33]. The amount and sugar composition of the fragments depend strongly on the enzyme mixture used. Obviously, the technical enzyme preparations solubilized high amounts of cell wall polysaccharides in the juice, but were lacking in enzyme activities to degrade the solubilize fragments further. Due to their relatively high concentration in the juice (4 g/L), the polysaccharides may lead to problems during processing and storage of the juice (concentrate). This was an incentive to study the enzyme resistant pectic fragments and to compare the structural features with those found for unmodified pectic molecules in the apple cell wall. This knowledge was considered to be essential in understanding the role of plant cell wall polysaccharides during processing and facilitating the introduction of new tailor-made enzymes and to develop new applicationdirected strategies in fruit and vegetable processing.

4.1 Modified Hairy Regions The enzyme-resistant fraction was in first instance characterized by its sugar composition" the fraction was rich in arabinose, while next to galacturonic acid (21 mol %) also rhamnose, xylose and galactose residues were present in significant amounts. A degree of methylation (DM; calculated on uronic acid content) of 40% and a degree of acetylation (DA) of 60% was calculated [34]. Such a high content in acetyl groups was not reported before in apple tissue. Another remarkable characteristic of the fraction was the high rhamnose:galacturonic acid ratio. The sugar composition revealed that the fragments resembled the so-called "hairy regions" as described by De Vries et al. [6], although De Vries did not mention the presence of acetyl groups. Since the fraction was isolated after treatment of the entire cell wall with various polysaccharide-degrading enzymes present in the technical enzyme preparation used,

which might have caused modifications, our fraction was designed MHR (modified hairy regions) further on. MHR also has nearly the same sugar composition and sugar linkage composition as the above mentioned RG-I. However, due to the fact that we used a quite unconventional way of extraction and since RG-I is considered to consist of strictly alternating rhamnose and galacturonic acid residues [5] we chose to use the term MHR rather than RG-I. The MHR fraction was characterized by its sugar linkage composition and it was found that the arabinose residues were less branched as mentioned in literature, probably due to debranching during the liquefaction process. Other bindings were quite representative for apple pectins [35] and RG-I type of polymers from several sources [5]. Attempts to degrade MHR by specific chemical methods like fl-elimination, treatment with metallic lithium, etc, did not result in more structural information. Also, purified enzymes like polygalacturonases, lyases, xylanases and galactanases available at that time, were not able to degrade MHR further. However, assaying more than 40 different crude enzyme preparations resulted in the recognition of one preparation which was able to hydrolyze linkages within the backbone of MHR. The enzyme responsible for this activity, rhamnogalacturonan hydrolase (RGase), was isolated from this preparation (Pectinex Ultra SP, obtained from Aspergillus aculeatus) and was further characterized [30,36]. HPSEC analysis of the RGase digest of saponified MHR revealed that only part of the glycosidic bonds (ca 4%) could be hydrolysed by RGase resulting in a mixture of polymeric material and oligomers of ca 1-2 kDa. These oligomers could be separated conveniently by high-performance anion-exchange chromatography (HPAEC) using pulsed amperometric detection (Figure 1)[37]. The structure of the oligomeric reaction products was elucidated by NMR spectroscopy following preparative HPAEC [37,38]. The structures found are summarized in Table 1. It can also be seen that the backbone of all oligomers consists of alternating rhamnose and galacturonic acid residues, with a galactose residue linked to C-4 of (part of) the rhamnose moieties. It was shown that Table 1 Structures of identified oligomers, obtained after degradation of apple MHR-S by RGase. a - R h a p - ( 1 --* 4 ) - a - G a l p A - ( 1 -~ 2 ) - a - R h a p - ( 1 --, 4 ) - G a l p A a - R h a p - ( 1 ~ 4)-ot-GalpA-(1 --* 2 ) ~ a _ R h a p _ ( 1

4)-GalpA /3-Galp-(1 ~ 4) / /3-Galp-(1 ~ 4 ) - a - R h a p - ( 1 --~ 4 ) - a - G a l p A - ( 1 -* 2 ) - a - R h a p - ( 1 -* 4 ) - G a l p A

fl-Galp-(1 ~ 4 ) - a - R h a p - ( 1 -* 4)-ot-GalpA-(1 --~ 2 ) ~ a _ R h a p _ ( 1

4)-GalpA /3-Galp-(1 ~ 4) / a - R h a p - ( 1 ~ 4)-ot-GalpA-(1 ~ 2)-ot-Rhap-(1 ~ 4 ) - a - G a l p A - ( 1 --~ 2 ) - a - R h a p - ( 1 ~ 4 ) - G a l p A a - R h a p - ( 1 ~ 4 ) - a - G a l p A - ( 1 ~ 2 ) - a - R h a p - ( 1 ~ 4)-ot-GalpA-(1 --~ 2 ) ~ a _ R h a p _ ( 1 /3-Galp-(1 ~ 4 ) " /3-Galp-(1

4)-a-Rhap-(1

4)-GaipA

4)-ot-GalpA-(1

2 ) x ' a - R h a p - ( 1 --* 4 ) - a - G a l p A - ( 1 ---, 2)-o~-Rhap-(1 ~ 4 ) - G a l p A /3-Galp-(1 ~ 4) t

/3-Galp-(1 ---, 4 ) - a - R h a p - ( 1 ---, 4 ) - a - G a l p A - ( 1 ---, 2 ) - a - R h a p - ( 1 ---, 4 ) - a - G a l p A - ( 1 ---, 2 ) ~ a _ R h a p _ ( 1 /3-Galp-(1 ~ 4) /

4)-GalpA

/3-Galp-(1 --, 4 ) - a - R h a p - ( 1 ~ 4)-tr-GalpA-(1 ---, 2)~ot_Rhap_( 1 ~ 4 ) - a - G a l p A - ( 1 ---, 2 ) ~ a _ R h a p _ ( 1 /3-Galp-(1 ---, 4) I

/3-Galp-(1 ---, 4) /

_..r

4)-GalpA

1000 900 8OO ..-..

700 50% forming gels at low pH in presence of saccharose to reduce the water activity and the low methoxyl pectins (LM with DMC*, the overlap concentration C* is in the range of [11]-1) in the presence of calcium by the egg-box mechanism first suggested for alginates [4850]. The gelation depends on the distribution of the carboxylic groups in addition of the average DM [48] and on the pH [51-53]. General behaviours describing physical gels were developped previously [54,55]. The gel strengh depends on the method used to prepare the gels, and specially the way the pectins were deesterified ; an exemple on sugarbeet pectins gives very clear data on the role of DM and compares enzyme treated and chemically modified pectins [56]. The two methods enhanced gelling ability but chemically modified pectins had a grainy texture, and became more brittle due to skrinkage ;the enzymatically modified pectins gave no skrinkage. The role of pectins treatment on gel stiffness was also demonstrated on sugar beet and citrus pectins [57].

20 The rheological behaviour in the range of LM pectin was analyzed and the solgel diagram established [59] for different stoichiometric ratios. In their paper, these authors determined the gel times for sodium pectate during calcium-induced gelation and the variation of the gel time with polymer concentration, stoichrometric ratio and temperature. Table 3 Comparison of pectin gelation Conditions Mechanism

HM Low water activity (65% sacharose) pH (kcal/mol) for the RG-I oligomers given in Scheme 1 with the different substituents at the monomers marked in bold face .........................

02-H,

O2-H, O3-H O2-Ac, O3-H O2-H, O3-Ac O2-Ac, O3-Ac

03-H

........

O2-Ac, O3-H O2-H,O3-Ac O2-Ac,O3-Ac -1.1 -2.3 -1.8 -3.0

-1.1 -0.6 -1.8

-0.7 -0.7 -1.3 -1.5

-1.9 -2.3 -2.5 -3.1

Table 2 < E c a l c - Epred> (kcal/mol) for the HG oligomers given in Scheme 2 with the different substituents at the monomers marked in bold face O2-H, O3-H O2-Ac, O3-H O2-H, O3-Ac O2-Ac, O3-Ac O2-H, O3-H -1.1 -0.9 -2.0 O2-Ac, O3-H -1.1 -2.1 -2.0 -3.1 O2-H, O3-Ac -0.9 -2.0 -1.8 -2.9 O2-Ac, O3-Ac -2.0 -3.0 -2.9 -4.0

..........

........

-..-~

-.

........................

-~-~- .......

: ....................

: .........

-

.....................

-~--.---::::::-.-~

.................

-~-~-

....................

~ = ~ .

...........

==_.~

...........

-~- ........................

: ............................

-.

.

.

.

.

.

.

.

.

.

.

: ..............

---

~

..

-

.

..............

.

.

----:

. . . . . . . . . . . . .

.

.

.

.

: .

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

From the low-energy conformations calculated for the acetylated disaccharides, helical backbone models could be build and optimised to the nearest integer helix with the POLYS program [ 15]. The A conformation of the GalA(1---~2)Rha disaccharide combined with either the A or the B conformation of the Rha(1---~4)GalA disaccharide resulted in the RG-I helices shown in Figure 5. The AA type is a left-handed three-fold helix with an advance per repeat, h, of 6.75 ,~; the AB type is two fold, h is here 7.48 ,~. These are in close agreement with calculations on helical conformation of the RG-I backbone with the galacturonic acids not acetylated, but methylated at 06 [16]. Since these authors found that methylation does not influence the conformational behaviour significantly, the agreement between their and our results is not surprising. When our conformation B' is used, a helix closely resembling the AB helix is calculated: two folded with h equal to 7.53/k. Two stable helices were generated by optimising acetylated HG from the two low-energy conformations to integer helices. The results are shown in Figure 6. The A type is a two-folded helix with h equal to 4.14 ,~. This may be compared with a proposed 2-folded helix with h equal to 4.35/k for calcium gels (on the basis of circular dichroism, calcium stoichiometry and competitive inhibition) [17,18]. The B type is a right-handed three-fold helix with h equal to 4.45/k; in close agreement with results from fibre diffraction performed on sodium and calcium pectate gels [19,20], which yielded right-handed three-folded models with h equal to 4.3 ,~. Also, our results are in good agreement again with calculations on methylated homogalacturonan [21 ].

63

Figure 5. Acetylated RG-I helices generated from the low-energy conformations of the disaccharides (see Figures 2 and 3). Left the AA type with n = -3 and h = 6.75 .~; right the AB type with n = 2 and h = 7.48 .~.

64

Figure 6. Acetylated HG helices generated from the low-energy conformations of the disaccharide (see Figure 4). Left the A type with n = 2 and h = 4.14/k; right the B type with n = 3 and h = 4.45 ~.

65

4. C O N C L U S I O N S On the basis of our energy calculations we conclude that acetyl groups at both 0 2 and 0 3 of galacturonic acid in the backbone of rhamnogalacturonan I and homogalacturonan are energetically favourable. The favourable influence of an acetyl group at 0 2 is usually somewhat bigger than at 0 3 . This is in good agreement with Keenan et al. [2], Schols et al. [4] and Ishii [6], but less with Komalavilas and Mort [3] and Lerouge et al. [5]. Next to this, we found that the stabilising energy arises from interaction of the acetyl groups with both neighbouring rhamnose and galacturonic acid. The substitution of galactose at rhamnose in the rhamnogalacturonan backbone did not have a significant effect on the calculated results. Finally, the acetylation of galacturonic acid in RG-I and H G did not lead to significantly different helical models of these backbones. Integer helices with low-energy conformations could be build in nice agreement with other experimental or theoretical results.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

A.G.J. Voragen, W. Pilnik, J.-F. Thibault, M.A.V. Axelos and C.M.G.C. Renard; in Food Polysaccharides and Their Applications, Ed. A. M. Stephen; Marcel Dekker Inc., New York 1995; 287 M.H.J. Keenan, P.S. Belton, J.A. Matthew and S.J. Howson; Carbohydr. Res. 138 (1985) 168 P. Komalavilas and A. Mort; Carbohydr. Res. 189 (1989) 261 H.A. Schols, M.A. Posthumus and A.G.J. Voragen; Carbohydr. Res. 206 (1990) 117 P. Lerouge, M.A. O'Neill, A.G. Darvill and P. Albersheim; Carbohydr. Res. 243 (1993) 359 T. Ishii; Mokuzai Gakkaishi 41 (1995) 561 MM3(1992), QCPE, Creative Arts Building 181, Indiana University, Bloomington, IN 47405, USA N.L. Allinger, Y.H. Yuh and J.H. Lii; J. Am. Chem. Soc. 111 (1989) 8551 S. P6rez, A. Imberty and J.P. Carver; Adv. Comput. Biol. 1 (1994) 146 A.D. French and M.K. Dowd; J. Mol. Struct. (Theochem) 286 (1993) 183 M.L.C.E. Kouwijzer and P.D.J. Grootenhuis; J. Phys. Chem. 99 (1995) 13426 IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN), Eur. J. Biochem. 131 (1983) 5 A.L. Spek; Acta Crystallogr. A46 (1990) C34 S. P6rez and M.M. Delage; Carbohydr. Res. 212 (1991) 253 S.B. Engelsen, S. Cros, W. Mackie and S. P6rez; Biopolymers (1996) in press S. Cros, C. Garnier, M.A.V. Axelos, A. Imberty and S. P6rez; Biopolymers (1996) in press E.R. Morris, D.A. Powell, M.J. Gidley and D.A. Rees; J. Mol. Biol. 155 (1982) 507 D.A. Powell, E.R. Morris, M.J. Gidley and D.A. Rees; J. Mol. Biol. 155 (1982) 517 M.D. Walkinshaw and S. Arnott; J. Mol. Biol. 153 (1981) 1075 M.D. Walkinshaw and S. Arnott; J. Mol. Biol. 153 (1981) 1055 S. Cros, A. Imberty and S. P6rez; Int. J. Biol. Macromol. 14 (1992) 313


J. Visser and A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V.All rights reserved.

67

The Pectic Polysaccharide Rhamnogalacturonan II is a major Component of the Polysaccharides present in Fruit-derived Products. Patrice Pellerin, Thierry Doco, St6phane Vidal, Pascale Williams and Jean-Marc Brillouet

Institut National de la Recherche Agronomique, Institut des Produits de la Vigne, Laboratoire des Polym~res et des Techniques Physico-Chimiques 2 Place Viala, F-34060 Montpellier Cedex, France

Abstract

Rhamnogalacturonan II (RG-II) was present in three fruit-derived products, a red wine, an apple juice and a tomato juice, at proximate respective concentrations of 100, 200 and 30 mg/L. Total polysaccharides were recovered by ultrafiltration and RG-II was isolated by sizeexclusion and anion-exchange chromatography. The composition of the three isolated fractions included the rare monosaccharides, apiose, 2-O-methyl-L-fucose, 2-O-methyl-D-xylose, Kdo, Dha and aceric acid that are characteristic of RG-II. Glycosyl-linkage analyses of neutral and acidic sugars were in accordance with the known structure of this complex pectic polysaccharide. Our results indicate that RG-II is released in an undegraded form from pectins during fruit processing and that its concentration increases with the use of liquefying enzymes.

1. INTRODUCTION

Pectins, a family of polysaccharides present in all plant cell walls, are composed of three galacturonic acid-rich polysaccharides, homogalacturonan, rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II). Rhamnogalacturonan II, the smallest and most complex pectic polysaccharide [1], can be isolated after treatment of plant cell walls by fungal endopolygalacturonase [2]. Its presence has been reported in the cell walls from sycamore (Acer pseudoplatanus), douglas fir (Pseudotsuga menziesii) [3], rice (Oryza sativa) [ 4], onion

68 a-D-GalA p -(I--->4)- lOt-D-GalA p -(I---~4)] -~-D-GalA p -(I--->4)- ~-D-GalA p n v

9

v

d~

3

3

2

2

1"

I"

1"

1"

2 fl-D-Dha p 5

2 Kdo p 5

1 fl-L-Araf

1

1" a-L-Rhap

1 fl-D-Api f 3'

1" ~L-Rhap 3

I Ac-O---> 3-~L-AceAf 2

I" 1 Ac-O--->Ot-L-Fucp -(I--)2)- ot-D-Gal p 2 4

I

1"

1

I"

Me

1 fl-D-Api f 3' 1

Ot-D-GalAp-(l-->2)-i~L-Rhap-(3 7. The percentage of 14C-recovered in the final pellets of 20-30 minute reactions using this procedure ranged from 2.5-20% of the UDp-14C-GalA added.

116

3.3. Synthesis of homogalacturonan in cell free membranes The incubation of UDP-[14C]GalA with tobacco microsomal membranes results in a time-dependent incorporation of radioactivity into precipitable product (18). When membranes are heated to 60~ for 15 minutes prior to incubation with UDP-[14C]GalA, the PGA-GalAT activity is destroyed, providing evidence that the time dependent production of product is enzyme-catalyzed. The reaction proceeds at an optimal rate at pH 7.8 and at temperatures ranging from 25~176 The enzyme in cell free membranes has an apparent K m of 8.9 + 3.5 (mean + SEM) for the enzyme-catalyzed incorporation of UDP-[14C]GalA into [14C]GalA-labeled product and a Vmax of 150 + 5 pmol min-1 mg-1 protein (mean 5: SEM) (18). Since it had been reported that 1.7 mM MnC12 stimulates PGA-GalAT activity in Phaseolus aureus membranes (81), we tested the effect of MnC12 concentration on PGAGalAT activity in tobacco microsomal membranes. Maximal PGA-GalAT activity was obtained at 0.25 mM MnC12 yields. Monovalent salts were also found to stimulate PGAGalAT activity with both NaC1 and KC1 giving approximately 76% stimulation of activity at 25 mM.

4. CHARACTERIZATION OF THE PRODUCTS SYNTHESIZED BY PGA-GALAT

IN MICROSOMAL MEMBRANES

4.1. Scheme for characterizing synthesized homogalacturonan An enzyme that specifically hydrolyzes r homogalacturonan, a homogeneous EPGase from Aspergillus niger, was used to establish that the synthesized [14C]-labeled product contained 1,4-1inked o~-D-galactosyluronic acid residues. Hydrolysis of polygalacturonic acid using this EPGase generates mono- di- and tri-galacturonic acid (11,64). Thus, treatment of synthesized HGA product with EPGase was expected to generate [14C]-labeled mono-, di- and tri-galacturonic acid. HGA in cell walls, however, is partially methyl-esterified (26,62) and is, therefore, partially resistant to EPGase treatment since EPGase requires several adjacent non-methylesterif'led galacturonic residues in order to fragment HGA (12,26). The possibility therefore existed that the tobacco microsomal membranes contained sufficient endogenous substrate (e.g. S-adenosylmethionine) to methylesterify the synthesized HGA (39,41) and, that such a product might not fully susceptible to fragmentation by EPGase. To test this possibility, the synthesized product was treated with base at 4~ to hydrolyze esters (26) and the amount of mono-, di-, and trigalacturonic acid released by EPGase digestion of intact and base-treated product was analyzed. This strategy yielded information on both the amount of [14C]GalA incorporated into HGA, and the proportion of HGA that was potentially esterified.

4.2. Sensitivity of product to cleavage by endopolygalacturonase The PGA-GalAT assay precipitates HGA larger than a trimer (18), thus, any HGA product completely hydrolyzed by EPGase treatment would be lost when re-precipitated in

117 the assay. The digestion of the synthesized product with EPGase followed by reprecipitation of the product in the PGA-GalAT assay resulted in only 82% of the radioactivity being recovered in the pellet compared to the re-precipitated control (18). These results revealed that at least 18% of the synthesized product contained t~l,4-galacturonic acid linkages. Treatment of the product with base to de-esterify galacturonic acid followed by cleavage of the base-treated product by EPGase resulted in release of 44% of the radioactivity in the base-treated pellet as apparent mono-, di- and tri-galacturonic acid. These results suggest that at least 44% of the synthesized product recovered is HGA and that a significant amount of product (~47%) is esterified.

4.3. Thin layer chromatography homogalacturonan

evidence that

the synthesized

product is

The synthesized product was further analyzed by TLC and autoradiography (18). UDPGalA and oligogalacturonides of DP of 2 to 10 were separated by TLC in the following order: UDP-GalA ran just behind the solvent front, followed by galacturonic acid and, sequentially, oligogalacturonides of DP 2 through 10. Commercially available polygalacturonic acid and oligogalacturonides of DP >11 remained at the origin. Intact radio-labeled product synthesized by tobacco microsomal membranes remained at the origin, as expected for polygalacturonic acid. Synthesized product that was base-treated ran in a similar fashion. Product that was treated with EPGase, however, ran at two locations on the TLC. Approximately 20% of the EPGase-treated product ran with a similar retention time as mono-pentagalaturonic acid. The remainder of the product remained at the origin. Product that was base-treated and subsequently treated with EPGase yielded the greatest amount of radioactivity that co-migrated with mono-pentagalacturonic. Quantitation of the radioactivity in three separate TLC plate experiments demonstrated that 34-50% of the base+EPGAse treated product runs as galacturonic acid and/or small oligogalacturonides, suggesting that at least 34-50% of the product is HGA. The fact that treatment of the synthesized product with base increased the amounts of oligogalacturonides released following EPGase treatment suggests that some of the synthesized product (--45%) is esterified.

4.4. High performance anion-exchange chromatography evidence that the synthesized product is homogalacturonan Another approach to characterize the [14C]GalA-labeled products synthesized by tobacco microsomal membranes was to filter the products through a 0.2 pm nylon filter to remove particulates, fractionate the products in the filtrate by high performance anion-exchange chromatography (HPAEC) over a Dionex CarboPac PAl column, collect the fractions, and determine the amount of radioactivity in each fraction. Fractionation of the intact products in this fashion revealed that only ,-10% of the radioactive product passed through the nylon filter, while the remaining 90% was retained on the filter. This result suggested that the synthesized HGA formed aggregates that did not pass through a 0.2 pm filter. Such an aggregation of HGA could be caused by cations (Mn ++) in the synthesis buffer and/or the dehydrating action of the organic solvents. A cation-dependent aggregation of the synthesized HGA was shown to be responsible since resuspension of intact product in 10 mM EDTA enabled ~60% of the product to pass through a 0.2 pm filter. The aggregation of the

118 synthesized product by cations adds further support that the product contained HGA since the aggregation of oligo- and poly-galacturonic acid in the presence of divalent cations is well documented (57). A larger percentage of the base-treated synthesized product was able to pass through the 0.2 )am filter (15-55% depending on the experiment). Treatment of oligogalacturonides with alkali is known to dissociate aggregates (57). Separation of the base-treated synthesized product by HPAEC revealed that most of the radioactivity eluted with retention times similar to oligo- and polygalacturonic acid. Approximately 36% of the EPGase-treated synthesized product passed through the 0.2 )am and was separated by HPAEC. The bulk of the radioactivity (58%) on the column eluted with retention times identical to authentic mono-, di-, and tri-galacturonic, suggesting that at least 21% of the initial product was HGA. Finally, 77% of the base+EPGase-treated synthesized product passed through the f'dter with 83% of the radioactivity eluting as mono-, di-, and tri-galacturonic acid upon HPAEC. These results showed that at least 64% of the product was homogalacturonan and that a large part of the homogalactur0nan (-67%) was esterified. Qualitatively similar results were obtained in a total of four experiments with an average of 24% of the HPAEC recovered-EPGasetreated product, and 52% of the Base+EPGase treated product, co-fractionating with authentic mono-, di-, and tri-galacturonic acid. Taken together the results confirm that a major part of the synthesized product consists of ~-l,4-1inked galacturonic acid, and thus, demonstrates that PGA-GalAT has been identified in tobacco cell free membranes. In all four HPAEC experiments there was a significant increase (49% on average) in the amount of total product that could be enzymatically hydrolyzed into mono- to tri- galacturonic acid following de-esterification with base. This suggests that on average-49% of the HGA synthesized was esterified. The amount of the base-labile linkage in the synthesized product that could be attributed to a methyl ester was examined by comparing the abilities of base-treatment and treatment with pectin methylesterase (PME) to render the product sensitive to fragmentation by EPGase (18). Base-treatment is not specific for the type of ester linkage while treatment with PME is relatively specific for the type of ester linkage cleaved. PME most readily hydrolyzes methylesters at the C6 carboxyl but can also hydrolyze ethyl esters at a reduced (--6-16%) rate (64). The radiolabeled product was synthesized, treated with base, EPGase, Base+EPGase, PME, and PME+EPGase, and intact and treated product was filtered and separated by HPAEC. The results of intact, base-, EPGase-, and Base+EPGase-treated product were similar to those described above. Treatment of product with purified EPGase allowed 54% of the product to pass through the falter with 42% of the product eluting as mono-, di- and tri-galacturonic acid. Treatment of product with base+EPGase resulted allowed 92% of the radioactive product to pass through the falter with 89% of total product being converted to mono-, di- and tri-galacturonic acid. Taken together these results suggest that --89% of the product synthesized was HGA and that ~53% of the HGA was esterified. Treatment of product with PME+EPGase allowed 54% of the product to pass through the filter with 53% of the radioactive product co-fractionating with authentic mono-, di- and trigalacturonic acid. Thus, a significant portion of the esterified HGA (-40%) appeared to be methylesterified. The possibility that synthesis of the HGA in tobacco membranes might be limited by the lack of methyl donor was ruled out since the addition of exogenous S-adenosylmethionine, a methyl donor known to function in the methylesterification of pectin (41,70), did not

119 stimulate the rate of synthesis, nor did it increase the amount of product produced. Thus, it appears that the synthesis of pectin in tobacco results in a partially methylesterified HGA.

4.5 The product synthesized in microsomai membranes is large The relative mass of the intact and treated synthesized product was determined by gel f'lltration chromatography over a Superose 12HR 10/30 column (18). The intact product eluted as a single peak with an apparent molecular mass of ~105,000 daltons compared to dextran. Product that was treated with base or PME eluted with an apparent molecular mass of ~201,000 daltons. We hypothesize that the apparent increased mass of the base- and PMEtreated product compared to intact product is due to different interactions of intact or treated product with the column matrix, including hydrophobic interactions (esterified product) or charge effects (base- and PME- treated product). In either case, however, the apparent mass of the synthesized product is large (-105,000 daltons). If the entire 105,000 daltons corresponds to homogalacturonan (i.e. there is no non-HGA primer and no RG-II- or RG-Ilike sugars are covalently attached to HGA), the HGA would have a degree of polymerization of approximately 600 galactosyluronic acid residues. Product treated with base+EPGase or PME+EPGase had a much smaller apparent mass, a mass equivalent to mono-, di- and tri-galacturonic acid. EPGase-treated product co-eluted with authentic mono-, di- and tri-galacturonic acid, as well as eluting at a slightly later retention time. We conclude that the EPGase-treated product is a mixture of mono-, di- and tri-galacturonic acid and small oligomers of esterified galacturonic acid. Such esterified galacturonic oligomers may interact with the column matrix resulting in a later elution time. The drastic reduction in the size of the intact, base-treated, or PME-treated product upon EPGase-treatment c o n f m s that the product synthesized in tobacco microsomal membranes is largely polymeric HGA.

5. SOLUBILIZATION OF PGA-GALAT

PGA-GalAT, like most cell wall synthases, appears to be a membrane protein. The purification of a membrane protein requires solubilizing the membrane with a concentration of a detergent that allows the formation of protein-detergent or protein detergent-lipid micelles that retain enzymatic activity. The establishment of optimal solubilization conditions is an empirical process. Our initial strategy involved screening eleven detergents including both zwitterionic (CHAPS, CHAPSO, Sulfobetaine SB12) and nonionic (n-Octylglucoside, n-dodecylmaltoside, deoxyBigCHAPS, BIGCHAPs, Triton X- 100, Triton X- 114, Thesit and Genapol X-080) detergents. In all cases, the addition of a 10xCMC concentration of the detergents to microsomal membranes (i.e. dispersed membranes) resulted in a drastic (2499.5%) inhibition of PGA-GalAT compared to the activity in microsomal membranes. In addition, almost no PGA-GalAT activity (4)-13-D-galactan Laura S Brickell arid J S Grant Reid Department of Biological and Molecular Sciences, University of Stirling, Stirling FK9 4LA, Scotland, United Kingdom

Abstract Membrane preparations from mung bean (Vigna radiata) hypocotyls catalysed the incorporation of label from UDP-14C-galactose (UDP-Gal) into a polymeric product which released only 14C-galactose on acid hydrolysis. The product was characterised as (1---~4)-13-Dgalactan by hydrolysis using two pure enzymes, a novel exo-(1--->4)-13-D-galactanasefrom the cotyledons of germinated Lupinus angustifolius seeds [ 1] and an endo-(1---~4)-13-D-galactanase from A spergillus niger, followed by TLC separation and quantitative digital autoradiography of the labelled digestion products. Digital autoradiographic methods were used also to show that other UDP-Gal utilising enzyme activities, including UDP-Gal-4-epimerase and phosphodiesterases, were present in the catalytic membrane preparations, and competed significantly with the galactan synthase for the UDP-Gal substrate. The galactan synthase had an apparent pH optimum at pH 6.5 in the presence of Mg +2. Galactan synthase activity peaked two days after the seeds were set out to germinate, and this was followed by a marked increase in the (1---~4)-13-D-galactan content of the tissue cell walls.

I. INTRODUCTION Linear (1---~4)-13-D-galactan is a common structural feature of the side-chains of pectins from many sources. The galactan may be unsubstituted or may form the core of the so-called type I arabinogalactans, which carry short side-chains of (1---~5)-linked L-arabinofuranosyl residues attached mainly to C-3 [2,3]. In the cotyledons of certain lupin seeds, galactan has a storage function. The cell walls of the storage mesophyll cells are massively thickened [4], and consist mainly of (arabino)galactan attached, apparently, to a rhamnogalacturonan core [5,6,7]. After germination most of the galactose- and arabinose-containing polysaccharides are mobilised from the cotyledonary mesophyll walls, leaving a galactose-depleted, rhamnose- and uronic acid-rich residual wall [4]. The mobilisation of galactan occurs in the absence of any endo-cleaving galactanase and is catalysed mainly, if not exclusively, by a novel exo-(1 --->4)-13D-galactanase [1]. This enzyme catalyses the cleavage of single galactose residues consecutively from the non-reducing ends of (1 ---)4)-13-D-galactan and (1 ---)4)-13-1inkedgalactooligosaccharides, and does not hydrolyse other plant cell wall polysaccharides [1 ]. An enzyme with similar properties has since been isolated from ripening tomato fruits, and is believed to be ripening-associated [8]. It is now our intention to make use of the natural amplification of galactan in lupinseed cotyledons to investigate the enzymatic mechanism of (1---~4)-13-D-

128 galactan biosynthesis in the developing seeds. The work described here was carried out to develop suitable analytical procedures using a convenient non-seasonal source of plant tissue. The choice of mung bean (Vigna radiata) hypocotyls was based on earlier observations that membrane preparations from that tissue catalysed the efficient incorporation of label from UDP-~4C-galactose into a polymeric product which gave ~4C-galactose on acid hydrolysis but was not characterised further [9]. Membrane preparations from cultured flax cells also catalyse the synthesis of polymeric material hydrolysing to galactose [10]. 2. MATERIAL AND METHODS

2.1. Enzyme preparation. Mung beans, purchased from a local supermarket, were soaked overnight and planted in moist vermiculite. The plantlets were normally harvested and weighed 3 days after planting, and ground (without cotyledons) with sand in the presence of 50 mM Tris-HCl pH 7.5 containing 1 mM EDTA. After a brief centrifugation at 1000g to remove sand and grossparticulate material, homogenates were routinely centrifuged at 5000g and 40,000g. The 40,000g pellet was resuspended, normally in 50 mM Tris-HCl, pH 7.5 (0.3 ml per 50 seedlings). 2.2. Routine assay conditions. Membranes (50 lal in a total assay volume of 100 pl) were incubated with UDP-Gal (0.1 raM) and MgSO4 (10 raM) in 25 mM Tris-HCl buffer pH 7.5, for 10 or 60 rain. Reactions were stopped by heating at 100~ for 3 min. Lupin galactan (0.1 mg) was added as a 0.1% solution, methanol was added to give a final concentration of 70% by volume, and the tubes were capped, heated at 70~ for 5 min and centrifuged (13000g; 5 min). Supematants were discarded or retained for analysis. Pellets were washed twice more with 70% methanol at 70~ and the supernatants were discarded. The final pellets were either dissolved in preparation for scintillation counting, or were suspended in water and freeze dried in preparation for analysis. 2.3. Analysis of pellets and su~matanls. Pellets were prepared for scintillation counting by dissolution in concentrated hydrochloric acid (15 lal, 70~ 10 min). The solution was then dissolved in scintillation fluid (Emulsifier Safe, Packard) and counted in a Packard Tri-Carb 2000CA scintillation spectrometer. Samples of supematants were dissolved directly in the scintillation fluid. All counts were corrected to dpm. Freeze-dried pellets were resuspended by heating at 100~ in 50 mM ammonium acetate buffer, pH 4.5, and digested exhaustively either with the pure exo(1---~4)-13-D-galactanase from germinated lupin seeds, prepared according to Buckeridge and Reid [1] or with a pure commercial endo-(1---~4)-fJ-D-galactanase from Aspergillus niger (Megazyme). Aliquots of the centrifuged galactanase digests or, when appropriate, the first 70% methanol supematants were subjected to liqid scintillation counting and were examined by thin layer chromatography (TLC) and digital autoradiography [11].

2.4. Analysis of seedlings for cell wall (l-->4)-13-D-galactan. Each batch of seedlings (without cotyledons) was ground in 24% (weight/volume) KOH to which a few crystals of NaBH 4 had been added, and the mixture was centrifuged

129 (15000g, 30 min). Ethanol (3 volumes) containing sufficient glacial acetic acid to neutralise the KOH was added to the supernatant. The precipitate formed on chilling was collected by centrifugation as above, washed three times with 70% (by volume) ethanol and freeze dried. The freeze dried material was dissolved in water, the volume noted, and an aliquot subjected to exhaustive digestion with the lupin exo-(1---~4)-13-D-galactanase[1 ]. Any galactose released was determined quantitatively [12].

3. RESULTS

3.1 Preparation of the membranes. Mung bean tissues were ground in a mortar with sand in the presence of extraction buffer and l mM EDTA. Incorporation of label from UDP-Gal into polymeric products was routinely assayed by incubating membranes prepared from mung bean hypocotyls with UDP~4C-Gal (0.1 mM) and Mg § (10 mM). After heat-deactivation of the enzyme, lupin galactan was added as carrier and any label in macromolecular products (70% methanol-insoluble) was determined. Optimisation of protocols for membrane isolation led to the routine adoption of a very simple procedure, involving a "clean-up" spin at 5000g, and recovery of a 40,000g pellet which contained over 80% of the total galactan synthase activity. Very little activity was recovered either in a subsequent 100,000g pellet or in the final supematant (Table 1). Incorporation of up to 15% of the UDP-Gal substrate was obtained routinely. Table 1 Galactan synthase activity distribution using two centrifugation protocols. Post-1000g supernatants were centrifuged as indicated. Centrifugations were at 4~

Sedimentation

Incorporation (% of activity* recovered)

4-step protocol 10,000g 40,000g

100,000g supernatant

3O 59 3.0 7.7

3-step protocol 5,000g 40,000g supernatant

14 81 5.7

*Calculated using total counts in macromolecular material - products not characterised.

130

3.2. Analysis of macromolecular products. Our standard incorporation assays contained resuspended particulate enzyme, labelled UDP-Gal (0.1 mM) and Mg § (10 mM) in resuspension buffer (Tris, pH 7.5). After incubation, reaction mixtures were heated briefly to 100~ and soluble lupin galactan was added, to ensure the precipitation of small amounts of galactan formed in the enzyme reaction and dissolved during the heating step. Precipitation of macromolecular products was achieved by adding methanol to a final concentration of 70%. The pellet was freed of soluble labelled products, including residual UDP-Gal, by repeated extraction with hot 70% methanol and was then analysed for labelled (1---~4)-13-D-galactan. The supernatant was analysed for soluble labelled products. Pellets were analysed for total incorporation of label by scintillation counting. They were also hydrolysed with hot trifluoroacetic acid (TFA). When the TFA hydrolysates were separated by TLC and the chromatograms were examined by digital autoradiography [ 11 ], the only labelled compound detected comigrated exactly with galactose. To determine their (1 ---~4)13-D-galactan content, pellets were digested with the pure exo-(1---~4)-13-D-galactanase from germinated lupin seeds, and with the endo-(1 ---~4)-D-galactanase from A spergillus niger, under conditions optimised to give complete digestion of the lupin galactan carrier. With each enzyme, digestion brought over 90% of the radioactivity originally in the pellet into solution. The structure of the remaining insoluble material was not investigated. When the soluble digests were separated by TLC and analysed quantitatively by digital autoradiography, the labelled saccharides comigrated exactly with, and were in the same proportions as, those formed from the carrier galactan. Moreover when time-course digestions were carried out with the endo-galactanase, the labelled oligosaccharide intermediates present at each time-point corresponded, qualitatively and quantitatively, with those from the lupin galactan carrier (Fig.

1). O

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Figure 1. Time course digestion, using A niger endo(1-M)-13-D-galactanase, of the pellet containing labelled biosyntheticproductand carrier lupin galaetan. Samples taken at different times were separated by TLC. The plates were subjected to digital autoradiography to reveal labelled compounds, and were then charred with H2SO4 to reveal lupin galaetan digestion products. A: Galaetan digestion products. B: Digital autoradiogram. Ga = galaetose; Ga2-Ga4 = (1---M)-I~-galactobiose-galaetotetraose.

Also the pattern of release of labelled galactose brought about by the exo-galactanase corresponded exactly to that observed for the carrier galactan. At the end of the enzyme incubations, less than 1% of the dissolved radioactivity remained at the origin of the TLC,

131 apparently inaccessible to enzyme digestion. Thus over 90% of the label incorporated from UDP-Gal into macromolecular products was (1--~4)-13-D-galactan and, at least under the conditions of our routine incorporation assay, the radioactivity solubilised by endo- or exogalactanase digestion gave an accurate estimate of the amounts of (1 --}4)-13-D-galactan formed. 3.3. Stability of the enzyme and substrate under assay conditions Fig. 2 shows a progress curve for the incorporation of label from UDP-Gal under the conditions of our routine assay. Clearly the rate of incorporation was high initially, but decreased very quickly, essentially to zero after 60 minutes. Constant rates of incorporation were observed only over short times, but plots of incorporation versus enzyme amount were acceptably linear for 10 minute incubations. The observed (Fig. 2) decrease in incorporation rates at longer incubation times was not due to depletion of the substrate by the galactan synthase reaction, since the maximum incorporation observed (Fig. 2) corresponded to less than 15% of the added substrate. Furthermore the decrease in reaction rate was not due to equilibrium being reached, since glycosyl transfers from high-energy sugar nucleotide substrates to polysaccharide acceptors are essentially irreversible. Thus the enzyme was possibly unstable under assay conditions, or else the substrate was being removed in competing reactions which gave 70% methanol-soluble products. The data in Fig 3 demonstrate clearly that the enzyme was highly unstable at the assay temperature (30~ in the absence of substrate, exhibiting a half life time of about 10 minutes (log plot not shown). ~-,

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Figure 3. Stability of the galactan synthase activity. Samples were pre-incubated at 30~ prior to assay.

However it is possible that the stability of the enzyme was enhanced under the conditions of the routine assay, because of the presence of its substrate. To investigate the possibility of substrate depletion due to competing reactions, the supematants corresponding to each of the time-course points were separated by TLC, and the chromatograms subjected to digital autoradiography. The results (Fig 4) showed that the labelled UDP-Gal substrate was depleted rapidly, with the increase of a major labelled compound of higher migration, and a minor slower-moving component (Fig 4). The minor component comigrated with unlabelled UDPGlc, and resulted presumably from the action of a UDP-Gal 4-epimerase present in the

132 membrane preparation. There was, however, no transfer of labelled glucose residues to polysaccharides, as witnessed by the absence of labelled glucose in TFA hydrolysates of our 70% methanol-insoluble pellets. The fast-moving labelled compound was not resolved from galactose and galactose-l-phosphate. The latter would be a product of phosphodiesterase action on UDP-Gal, the former of phosphatase action on Gal-l-P. The time-course of depletion of the UDP-Gal substrate corresponded closely with the observed time-dependent decrease in the rate of galactose incorporation. Thus it is probable that enzyme instability and substrate depletion both contributed to the observed (Fig. 2) tailing off of incorporation with time.

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3.4. Apparent pH optimum of the galactan synlhase When the activity of the enzyme was assayed at a range of pH values between pH 5.0 and pH 9.5 in Tris (pH 6.3 to 9.5) and MES (pH 5.0 - 6.5) buffers (Fig 5) there was a fairly sharp apparent optimum of label incorporation at about pH 6.5. The same result was obtained using PIPES buffers, pH 5.5 - 7.5. Analysis ofpellets confirmed that (1-->4)-13-D-galactan was the main product over the range covered by the Tris buffer. Examination of the Tris-buffer supernatants by TLC and digital autoradiography showed appreciable substrate depletion. However it was relatively constant at pH values at or below pH 7.5, indicating that the apparent optimum at pH 6.5 probably did reflect the variation of galactan synthase activity with pH. Above pH 7.5 substrate depletion increased, and was almost total at pH 9.5. The decrease in labelled UDP-Gal over this range was accompanied by a corresponding increase in the labelled compound which comigrated with UDP-GIc. 3.5. Galactan synthase in relation to (1--~4)-~D-galactan levels in tissue cell walls Mung bean seedlings at different stages of development were treated with 24% (by weight) KOH to extract non-cellulosic polysaccharides as fully as possible. After neutralisation of the extracts, the polysaccharides were isolated by ethanol precipitation, and

133 their (1---~4)-13-D-galactan contents were determined by digestion with the lupin exo-(1---~4)-13-Dgalactanase with quantitative determination of the D-galactose released. Further samples of the same seedlings were used to prepare membranes, which were tested for galactan synthase activity. At day 1, when the seeds had not completed germination but were fully imbibed, radicles were removed for extraction. By day 2 radicles were approximately 0.3 cm long: thereafter seedlings elongated linearly, reaching 5.8 cm at day 6. The galactan content of seedlings (Fig 6) was very low at days 1 and 2, but then increased almost linearly up to 130 n moles galactose equivalent at day 6. Galactan synthase activity was very low at day 1, then peaked at day 2, just before the beginning of the increase in galactan content. From day 3 galactan synthase paraUed galactan content. 4 DISCUSSION We have confirmed earlier observations (9) that membrane preparations from mung bean seedlings catalyse the formation of a galactan from UDP-Gal. Using novel enzymatic approaches we have shown further that the predominant (c. 90%) polysaccharide formed is a (1---~4)-13-D-galactan. Under the conditions of the incorporation assay adopted as standard in this work, there is significant conversion of UDP-Gal into soluble products, notably compounds comigrating on TLC with GaI-1-P and/or Gal, and with UDP-GIc. Furthermore the synthase activity is unstable at the assay temperature. The enzyme activity shows an apparent pH optimum at pH 6.5. Galactan synthase activity in membrane preparations from seedlings at different stages of development correlates positively with the content of (1---}4)-13D-galactan in the seedling cell walls. In future work we shall optimise the assay conditions to enhance enzyme stability, and to minimise other enzymatic reactions competing for UDPGal, and shall attempt the solubilisation and purification of the enzyme from mung bean and developing lupin seeds. The data in Fig. 6 allow a comparison of the rate of galactan accumulation in mung bean tissues in vivo, with rates of galactan synthesis in vitro. There are numerous reasons why such a comparison may not be valid. Galactan may be turned over rapidly, so that true synthesis rates may exceed accumulation rates significantly. Also our assay was not optimised and is unlikely to reflect maximum rates achievable in vitro. Nonetheless it is encouraging to note that accumulation rates in vivo (130 nmol galactose equivalent per seedling in 4 days 1.4 n mol per hour) are similar to synthesis rates in vitro (300 dpm per seedling per 10 min 0.78 n mol per hour). The galactan synthesised in vitro from UDP-Gal by flax membranes (10) was partially characterised. Part of it was water-soluble and part required NaOH to bring it into solution. The size-fractionated water and NaOH extracts with the highest associated radioactivity were subjected to methylation analysis to determine the predominant linkages in the total (as opposed to labelled) polysaccharide included in these fractions. Results indicated that watersoluble fractions contained a preponderance of (1---~4)-galactosyl linkages, whereas NaOH soluble fractions contained branched galactans. If the in vitro synthesised labelled flax polymers had the same solubility and structural characteristics, then the water-soluble labelled galactan was probably (l~4)-linked. Our results provide some support for this conclusion, since the flax enzyme exhibited two pH optima for (total) galactan synthesis, one of them (at pH 6.5) identical with the apparent pH optimum for (1---}4)-13-galactan synthesis in our experiments.

134 8000

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Figure 5. Incorporation at different pH values. Tris-HCl buffers were used over the range pH 6.3 - 9.5 and MES buffers from pH 5.0 - 6.5. (10 min assays)

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Figure 6. Galactan - - o - - synthase activity in relation to (1---}4)-13-D1so ~ ~, galactan content in the o~ cell walls of mung bean "~ seedlings

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seedling age (days) 5 REFERENCES

4 5 6 7 8 9 10 11 12

M.S. Buckeridge and J.S.G. Reid, Planta, 192 (1994) 502 M. McNeil, A.G. Darvill, S.C. Fry and P. Albersheim, Ann. Rev. Biochem., 53 (1984) 625 A.G.J. Voragen, W. Pilnik, J.-F. Thibault, M.A.V. Axelos and C.M.G.C. Renard. In A.M. Stephen (ed.), Food Polysaccharides and their Applications, Dekker, New York, 1995 L.A. Crawshaw and J.S.G. Reid, Planta, 160 (1984) 449 B. Carr6, J.-M. Brillouet and J.-F. Thibault, J. Agr. Food Chem., 33 (1985) 285 M.T. AI-Kaisey and Wilkie, K.C.B, Carbohydr. Res., 227 (1992) 147 N.W.H. Cheetham, P.C.-K. Cheung and A.J Evans, Carbohydr. Polymers, 22 (1993) 37 A.T. Carey, K. Holt, S. Pieard, R. Wilde, et al., Plant Physiol., 108 (1995) 1099 J.M. McNab, C.L. Villemez and P. Albersheim, Biochem. J., 106 (1968) 355 F. Goubet and C. Morvan, Plant Cell Physiol., 34 (1993) 1297 M. Becker, C. Vincent and J.S.G. Reid, Planta, 195 [1995] 331 M. Edwards, Y.J.L. Bowman, I.C.M. Dea, J.S.G. Reid, J. Biol. Chem., 263 (1988) 4333

J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.

135

Cell wall pectins 9 from immunochemical characterization to biological activity P. Van Cutsem and J. Messiaen D6partement de B!ologie, Facult6s Universitaires de Namur, rue de Bruxelles 61, B-5000 Namur, Belgium

Abstract

The plant cell wall contains high and low affinity sites for the adsorption of bivalent cations. High affinity sites constitute a significant amount of the constitutive homopolygalacturonic acids (PGA). Monoclonal antibodies have been raised to PGA and they recognize oligomers with degrees of polymerization (DP)>_9 under a calcium-induced conformation. Immunocytochemistry indicates the presence of this acidic pectin at cell corners mainly, unless deesterification of the primary wall. The same pectic fragments with DP_>9 trigger the activation of defense mechanisms in plant cells by a signal transduction pathway involving calcium and phosphoinositides. The binding of fragments by the antibodies suppresses the response, which means that the epitope recognized by the antibody is part of the pectic signal perceived by plant cells.

1. ION EXCHANGE PROPERTIES OF THE CELL WALLS The galacturonic acids of a plant cell wall mainly belong to smooth chains of homopolygalacturonic acid (PGA) and to hairy regions of rhamnogalacturonan I (RGI). In green plants, other uronic acids can be found in hemicelluloses. Provided they are not methylesterified, all these carboxylic acids deprotonate at the more or less acidic pH of wall water. The electrostatic charges of these polyanions are then compensated by cations ultimately derived from the environment. The adsorption of cations obeys several rules. The first of them is the electroselectivity : higher charged cations interact electrostatically with anions much more than monovalent cations. The hydration of the ions also plays a prominent role : the large hydration sphere of magnesium keeps the ion at a higher distance from the negative charges of the pectin than an other bivalent cation such as calcium. Other factors are involved, such as the heterogeneity and/or the local geometry of the adsorption sites, the contribution of nearby

136

ligands, the charge distribution over the molecule in the case of organic cations, etc... It is therefore not surprising to observe that plant cell walls discriminate precisely between cations adsorbed from the surrounding solution. It is possible to accurately quantify the selectivity of this cell wall adsorption by performing ion exchange isotherms. The procedure consists in equilibrating isolated cell walls with renewed solutions of two competing cations. After several changes, the cell walls are removed, dried, weighed, and the cation content of their acidic extracts tested by atomic absorption spectrophotometry. In absence of any selectivity, the amount of each cation adsorbed in the cell wall must be exactly proportional to the amount of each cation present in the treatment solution. For example, the proportions of two bivalent cations adsorbed in the cell wall must be the same as in the equilibrium solution. In other words, the ion exchange isotherm must be diagonal. Such a study has been performed on a model plant system, the Nitella flexilis cell wall [ 1, 2, 3 ]. This freshwater alga has giant internodal cells whose easily isolated cell walls constitute a simplified model of higher plant cell walls : it has no lignin and its pectin is not methylesterified. Isolated cell walls are cut in pieces and distributed in different lots over the whole exchange isotherm to reduce variability between experimental points.

1.0 1 mN

o e~

10 m N O

0.5

0

0.5 1.0 Copper in solution

Fig. 1. The proportion of uronates that bind copper in the isolated Nitella cell wall is plotted as a function of the fraction of copper in a mixed solution of copper and calcium.

137

The results of a typical isotherm between chlorides of copper and calcium ions is shown on Fig. 1. The strong departure of the curves from the ideal diagonal exchange reflects the selectivity of the wall uronates for Cu 2+. For example, when the solution contains 5% Cu 2+ and 95% Ca 2+ chlorides, about 40% of the ion exchange sites of the wall bind copper. It is possible to calculate the selectivity coefficient of this exchange for each point of the isotherm and to express its logarithm as a function of the amount of cation in the cell wall. Unexpectedly, the plot of this coefficient displayed on Fig. 2.a shows that the selectivity is all but constant : it decreases linearly when more uronic acids release calcium for copper, with a break when about the first 30% of the negative charges are compensated by the preferred cation. This is a first indication of an heterogeneity in the pool of uronic acids of the walls, and suggests that there may exist two types of exchange sites. A similar selectivity curve is observed for a K + - Ca 2+ exchange (Fig. 2.b).

2.5

2.0 .

z

2.0 1.5 lmN

I

10mN

1.5 @

1.0

I

i

i

I

i

0

t

10m i

i

i

10t

i

0.5

1.0 cellwall N Cu2+ .,

0

i

i

i

i

i

0.5

i

I

i

I

m cell wall

N Ca2+

1.0 --"

Fig. 2. The logarithm of the selectivity coefficients of a calcium-copper (a) or a calcium-potassium (b) exchange isotherm is plotted as a function of the proportion of the preferred ions adsorbed in the cell walls.

2. EPR SPECTRA

Since cupric ions are paramagnetic, it is possible by electron paramagnetic resonance (EPR) to obtain information on the status and the environment of the Cu 2+ ions adsorbed on uronic acids [4, 5]. Nitella cell walls with uronate charges compensated to 9 or 100% with copper in equilibrium with mixed copper and zinc chloride solutions had their EPR spectra recorded at two different temperatures, 93 and 293 ~ (Fig. 3.a, b).

138

a

/T

b

=93K

/T

l~cu2+ : 0"09

Ul

NZn2+ = 0.91

[!

=93K

NCu2+ = 1.00

w Fig. 3. EPR spectra of Nitella flexilis cell walls with cupric ions on 9% (a) or 100% (b) of the uronates. The spectra have been recorded at two temperatures (arbitrary intensities). The sharp small peaks indicate g = 2.0028.

When little copper was present in the walls, the lineshape of the spectra was well resolved and did not change much on thawing. This is characteristic of ions immobilized on their adsorption sites. The complete substitution of zinc for cupric ions (Fig. 3b) modifies the lineshape that is now strongly affected by increasing the temperature. This indicates a higher mobility of the last ions adsorbed on sites of lower affinity, as deduced from the exchange isotherms. Strongly bound cations would be predominant at low coverage; a second population of more weakly adsorbed cations, preserving some mobility at room temperature, would develop as the Cu 2+ content increases.

a

b

100G H

Fig. 4. The EPR spectra at 93 ~ of cell walls saturated with copper have been best fitted to theoretical lineshapes assuming only one type (a) or two types (b) of exchange sites for the adsorption of the ions. Vertical arrows at g = 2.0028.

139

The EPR spectra of cell walls saturated with copper has been fired to the numerical solutions of the spin hamiltonian describing the EPR lineshape of cupric ions. Two simulations have been performed. The first one (Fig. 4.a) considers that all uronic acids of the cell walls are similar : the best fit is rather poor. The second one assumes existence of two populations of exchange sites with different parameters. In this case, the optimization is much better and confirms the existence of two different types of uronic acids in the cell wall (Fig. 4.b). As the copper content of cell walls increases, the Cu 2+ ions distribute between the two types of uronates : most of them on high affinity sites first, at low copper contents; more on low affinity sites afterwards. The percentage of the uronic acids that bind Cu 2+ with a high affinity is plotted on Fig. 5 as a function of the relative amount of copper in the walls. We can see that about 30% of the uronic acids consist of high affinity sites.

l l00 IZ

I

I

I

I

I

I

i

I

i

I

9

2.0

T -

-

I

i

i

i

0

Z

Zn2+

0

1.5

50

1.0

i "

i

I

I

I

i

50

I

I

I

-- Total N CuZ+

I

100

Fig. 5. Distribution of cupric ions in the cell wall between high affinity CuI and low affinity CUli exchange sites.

2.38

2.39

2.40

I

2.41

g#

Fig. 6. The logarithm of the selectivity coefficient of the exchange in the wall is plotted as a function of a magnetic parameter of copper adsorbed on high affinity sites.

These high affinity sites determine most of the selectivity of the ion exchange. This is deduced from Fig. 6 in which the selectivity of the whole ion exchange (ln KN) is plotted as a function of gill, a magnetic parameter of cupric ions adsorbed on high affinity sites. This parameter is particularly sensitive to the degree of covalence of the bound between copper and its

140

ligands. We can thus conclude that the shape of the exchange isotherms is largely determined by the selectivity of the high affinity sites for the cations. The biochemical nature of these high and low affinity sites can be deduced from the literature. The high affinity sites (about 30% of all exchange sites, Fig. 5) have EPR spectra characteristic of homopolygalacturonic acid polymers [6]. The walls also comprise about 40% of RGI and hemicellulosic uronates [7, 8]. The remaining 30% thus represent low affinity homopolygalacturonic acids. In other words, the homopolygalacturonic acids of the cell walls distribute in two equal amounts of high and low affinity exchange sites. This is compatible with the dimer formation predicted by the egg box model between pectin chains in the 2~ helical conformation [9, 10]. According to this model, we would associate the high affinity sites with the inner faces of the dimers.

2. MONOCLONAL ANTIBODIES TO PGA Knowing the importance of PGA for the ion exchange in the cell wall, we considered the production of monoclonal antibodies (Ab) to homopolygalacturonic acid. We produced one IgG1 hybridoma, named 2F4, that appeared to be specific of PGA [ 11 ]. Its specificity strongly depends on the nature of the cations present during the tests. A calcium/monovalent cation ratio of about 1/150 must be kept constant during all the steps of the ELISA. Higher and lower ratios strongly decrease the binding of the antibodies. The substitution of calcium by magnesium annuls the recognition of pectin by the antibody (Fig. 7) as well as the addition of EDTA to the buffers (not shown). This is strong evidence that the Ab binds a calcium-induced supramolecular conformation of PGA. This supramolecular conformation corresponds to the formation, under low calcium concentrations, of dimerized chains of polygalacturonates in 2~ helical symmetry, according to the egg box model. The internal faces of these dimers that bind calcium cooperatively would correspond to the high affinity sites deduced from ion exchange and EPR data. In this model, the external faces of the dimers would bind monovalent cations. The fact that higher calcium contents lower the binding of the Ab, suggests that the additional calcium, adsorbed on low affinity sites (Fig. 2b), modifies or masks the conformation of the high affinity sites that contain the relevant epitope. The nature of this modification is unclear : multimerization of preformed dimers [ 10], change of helicity of the pectate chains ... ?

141 0.8

1.2 0.6 t-

t'3

~

0.8

O (D (:1.

,_

t--"

"Q

"O t-

0.4

150 000 bearing 27 _+5 % of the total activity; MMa 100-120 000 with 25+_5% of the activity; MMa 50-80 000 with 12 +3% of the activity; and MMa < 10 000 accounting for 35+5% of the total activity). Thus the isoforms of pectin methyltransferase are numerous and varied in their physical properties. The basic isoforms which were the largest single group would be expected to interact electrostatically with their pectic substrates, and the strength of the interaction will increase with the density of negative charges on the pectic galacturonan chain. It may be significant that at the optimum pHs for trans-methylation onto high-ester and low-ester pectins, pH 7 and pH 5.5 respectively, about half of the carboxyl groups were ionised in each case. 2.3 Pectinesterases from active and resting cells. When cell-wall fragments are incubated in molar NaC1, ionically bound proteins are released into the incubation medium. All investigated crude cell extracts deesterified Citrus pectin (Table 2) but the deesterification rates were clearly higher when the enzymes were still bound to the cell walls, indicating a major loss of activity during the solubilization process.

Table 2. PME activity solubilized from cell walls of active and resting cells. Activities as neq. H +. mg protein-1 Active cells Cell suspensions Hypocotyls

12.6 12.5

resting cells 16.2 34

All cell-wall extracts contained several PME isoforms differing in their isoelectric points. The isoenzyme patterns changed significantly during cell ageing. As illustrated in Fig. 3, in both poplar stems and mung bean hypocotyls, basic isoforms became prevalem in mature, resting cells whereas in young, growing cells, neutral isoforms were predominant.

157 70

~

v'

,

!

60 !" a~

50

-

4O

-

30

-

,

>~

_-

Ill'

Iz

Ds

.9~ . ~

...

0

r~

~.7(~%; :

:~0 ~10.

~:

i ,,

A

B

C

D

may

hypocotyl levels

december

march

Figure 3. PME isoform patterns in cell wall extracts from active and resting cells. a: cell wall extracts from successive segments (A, B, C and D) sectioned along mung bean hypocotyls and exhibiting decreasing elongation rates; u, 13, and 7 are the main PME isoforms present in the extracts, their pI are respectively around 7.5, 8.5 and above 9.1. b: cell wall extracts obtained from poplar cambium and inner bark tissues during cambial active (may) and rest (december and march) periods; 1, 2 and 3 represent the activity of 3 groups of PME isoforms with pI around 5-6, 7.5 and above 9.1. Activities expressed as percent of total PME activity present in each extract. In flax cells, young cells as well as mature cells contained both neutral and basic isoforms, the proportions of which varied slightly. Acidic isoforms were also detected. Some properties of the different isoforms extracted from mung bean hypocotyl cell walls (eaUed respectively PEa, PEI3 and PET, ot for the neutral isoforrn, 13 for the PME with a pI around 8.5 and 7 for the most basic one) are reported in Table 3. The three esterases differed not only in their pI but also by their Mr, their pH optimum and the ionic strength necessary for their solubilizatiorL Table 3. Properties of mung bean puritied PME isoforms Enzyme

pI

Mr (Da)

PEa PEI3 PET

7.5 8.5 above 9.5

47,000 34,000 35,000

optimal pH 5.6 to 8. 7.6 5.6

solubilized with 0.2 M NaC1 1.0 M NaC1 0.4 M NaC1

When comparing the two basic isoforms PE[5 and PET, it can be noted that the more basic one, PET, surprisingly requires a lower ionic strength for its solubilization than PEI3. The

158 activities of the three isoforms exhibited different kinetic parameters and different ionic sensitivity (Table 4, figure 4). The neutral PME which predominates in young active cells was the least affected by the pH and the ionic composition of the microenvironment and was readily solubilised. Table 4. Kinetic parameters of mung bean PME isoforms. Assays performed at pH 7.6 in 150 mM NaCI; values between brackets have been obtained at pH 5.6. Enzymatic fractions

Km Vmax Vmax / Km (10.3 M methyl groups) (tteqH+ min" mg protein")

PEot PEI3 PF_q

1.32 0.23 11.8

'~-

~e

0.20 (0.11) 1.74 (0.17) 0.12 (0.65)

MgCl 2

800

_ ~--" _

700

t~

~oo c-

300

~

"E

200

r

0.27 0.40 1.43

--o--

y

600

400

c

0

0

50

1O0

150

200

0

50

1 O0

150

200

salt c o n c e n t r a t i o n (mM)

Figure 4. Rates of pectin hydrolysis as a function of salt concentration at pH 7.6; rates as meq H §min-1 lag protein". (e) PEa, (11) PEI3 and (o) PEY, a for the neutral isoform, 13for the PME with a pI around 8.5 and Y for the most basic one. Differences were also observed in the internal amino acid sequences of the three isoforms. Figure 5 shows the sequences of the oligopeptides obtained after tryptic hydrolysis of the three purified isoforms. Only one N-terminal sequence (from PEy) could be obtained, the two others being blocked. Using oligonucleotide primers deduced from the N-terminal and one of the internal oligopeptides of PET, a eDNA fragment was obtained by PCR, cloned and sequenced [24]; all the PET peptides could be aligned with the aminoacid sequence of the mature protein deduced from the eDNA. The comparison of the amino acid sequence of PEY with other known pectin methylesterases from tomato [25], Petunia [26], Arabidopsis [27,28] and Brassica napus [29] shows that a glycosylation site is present in all cases and that all primary structures were

159 most homologous at the C-terminal region. This corresponds to a domain, the 'esterase-like domain' of Albani [29] that specifies the pectinmethylesterase function for the proteins. In contrast, the N-terminal regions are less conserved between species. In several cDNAs this region is relatively long and seems to be more susceptible to post-translational N-glycosylation. It has been suggested that this N-leader sequence may have a role in targeting the protein towards the apoplasm and also in inactivating or stabilising the protein during export. All the cloned and sequenced cDNAs corresponded to basic proteins. Figure 5. Partial peptidic sequences of peptides resulting from tryptic cleavage PME isoforms. The peptides were separated by reverse-phase HPLC. N-terminus (N-ter) was obtained with the entire PE T

PEa XGAYFENVEVI

PEI3 TVSEAUDA

PET

XNLMXVGDGI

ENVEVP

TVTEAVASAPDNGK YVIYVK

HQAVALR

DITFQNTAGPSK

VGADQSVINR

GVTFENSAGPSK

HQAVALR

IDAFQDTLYAHSNR

XYSR

PWK

EDPNQNTGTSIQ

NYLGR

TYLGR

TVIMQSSID

XLFTAQGR

EYQNTGPGAGTSNR

VNWPGYHIITSAAEASK

VTWPGYR

VITDAREAR TTTIITASR

PE T N-terminus DVKANVVAQDGSGSGI~KTVTEAVA

It would be very interesting to obtain sequence data on neutral PMEs. In spite of this lack of information it can be suggested, taking into account the important differences in the ionic and substrate-binding characteristics of the three mung bean isoforms, that they may also progress differently along the polygalacturonic backbone. Basic isoforms have been shown to act in a bloekwise manner, de-estedfying their way along the galacturonan chain while remaining attached to it by virtue of their positive charge, so that they give rise to acidic blocks that can aggregate in the presence of calcium ions [18]. Marcovic [18] demonstrated that in contrast, fungal PMEs deesterify randomly. If we hypothesise that neutral plant PMEs might also act randomly, lacking the tendency to remain ionically bound to acidic galacturonans, we can propose the following scheme to explain the observations on mung bean hypocotyls (Fig. 6). In young cells, at the top of the hypocotyl, neutral PME isoforms predominate. Their action might be restricted either by their localisation away from the substrate, or by the initial structure of their substrate e.g. a particularly high DE. In older cell walls, the activity of basic isoforms, in particular PE~/ whose catalytic efficiency is high at acidic pH, increases and generates free carboxyl groups.

160

-r

Pectin o o . . . . . o r 9 9 *. 9

neutral PMF /\

~H~0434MH~I40~

\

/~

alkaline PME /x

/\

8 ~. ~ 8 ~ 8

/\

I

-~ ~176

J Ii o0..ooooooooooooo

. . . . . . . . . . . . . .

Figure 6. Hypothetical action pattern of neutral and alkaline PME isoforms. 9 methylated galacturonic units; O unesteritied galacturonic acids; EPG, endopolygalacturonase.

These results show that both the pectin methylesterases and the pectin methyltransferases are heterogeneous groups of enzymes, so that there is ample opportunity for the developmental control of methylesterification by the balance between the different isoforms of the two enzymes and by their response to environmental conditions and to the charge density of the existing substrate. In particular, it appears that the high level of methylesterification of pectins in young tissues is brought about by a relatively high total methyltransferase activity in the Golgi, producing pectins that are already too heavily esterified to be attacked by the neutral PME isoforms that predominate in these tissues. Galacturonan chains with a random distribution of carboxyl groups show less tendency to aggregate, which is compatible with the high extensibility of the young cell walls. This example illustrates the complexity of the enzymic mechanisms needed to control the methyl ester content of pectins, and their potential for spatial and developmental regulation. 3. GEL FORMATION BY PECTINS WITHIN THE CELL WALL The quantity and distribution ofnon-esterified carboxylate residues in pectins control the binding of counterions, and thus affect the propensity of the pectins for forming aggregates, gels and precipitates. Indeed the percentage of non-esterified residues is more informative about the properties of pectins than the percentage of esterified residues (DE)" increasing the DE from 60% to 80% halves the mean charge-density and the cation-binding capacity. Cations mediate what is almost certainly the principal form of crosslinking between pectic molecules, although covalent bonds of various suspected kinds [30-35], and probably others as yet unknown, can also link pectic molecules together. We will concentrate here on galacturonans. The capacity of rhamnogalacturonans to gel in the presence of cations is uncertain. Lolium RGI at high

161 concentration formed a gel with calcium ions [A. Chesson and M.C. Jarvis, unpublished] but this may have been due to a small proportion of galacturonan present. When contaminating galacturonan segments were excluded the aflSfity of flax RGI for calcium ions was relatively low [C. Morvan and M.C. Jarvis, unpublished]. At present, therefore, there is no clear evidence that the branched segments of pectins participate in the calcium-mediated junction zones, although they may well be capable of other forms of cross-linking.

3.1 Chain conformation in galacturonan gels Shared cations will align galaeturonan chains alongside one another if the chain conformation allows holes at regular intervals for the cations, matched to their size and preferred coordination geometry, and adjacent to negative charges. The range of conformations possible for the pectic (1,4)-ot-D-galacturonosyl linkage has been explored by modelling and proton NMR in disaccharides [36-40] and the polysaccharide itself [41]. It is centred on the 21 conformation which has a 180~ rotation between galacturonosyl residues. With little change in energy or chain length, polymers starting in this conformation can twist in either direction to give a fight-handed (31) or a lett-handed (32) helix with three residues per turn, or slightly further to give a 41 helix. Insertion of a single a(1,2)-L-rhamnosyl residue introduces a sharp kink in the chain [41, 42]. Thus a rather wide range of linkage conformations is sterically possible and direct experimental evidence is needed to distinguish those that give stable aggregates with any cation.

Figure 7. The 'egg-box' structure for the junction zones of dilute calcium pectate gels: two galacturonan chains in the twofold (21) helical conformation with calcium ions (shaded circles) locked between them. For gels of pectate with calcium ions the 21 helical 'egg-box' structure (Figure 7) [43,44] has become widely accepted, but the evidence supporting it is derived mainly from gels of 5)-KDO

60 - 30 Not reported Ara-(1 9 -->5)-DHA ]

5"1 1 "1 [ AceA containing nonasaccharide ] "1 : $

: :3 Ara f( 1~ 2)-Rha-(1 -~2),,'Ara-(1~ 4)iGal-(1 ~ 2)-AceA-(1 ~3)-Rha-(1 -,3')-Api

:

9

o. = , ,

,, , , . . , ,

...,p

: 2 ' T

1 2-O-Methyl-Fuc

........... ' .: : : Rha-(1 -~2)iAra-(1-~4)!Gal-(1 -,2)-AceA-(1-,3)-Rha-(1 -~3')-Api RG-II

i

i

.......... ~ 1

2-O-MethyI-Fuc

Fig. 6 Structural differences between GL-4IIb2' and RG-II

180

3.2 Complement activating (anti-complementary) activity The complement system consists of over 20 serum proteins including 9 complement components (C1-C9) and their regulators. The complement proteins are activated by a cascade machanism of classical or alternative pathways. The classical pathway is activated by the binding of C1 to the Fc region of immune complexes containing IgM and IgG antibodies and is followed by further activation. On the other hand alternative pathway is directly activated from C3 by some activators such as lipopolysaccharide, and is follwed by further activation. Complement activation appears to be intrinsically associated with several immune reactions such as the activation of macrophages and lymphocytes, immunopotentiation and so on [25]. Several complement activating polysaccharides have been discovered in the medicinal herbs such as the roots of Angelica acutiloba Kitagawa, the leaves of Artemisia princeps PAMP, the roots of Bupleurum falcatum L, the roots and leaves of Panax ginseng, the roots of Glycyrrhiza uralensis Fisch et DC, berries of Viscum album var. album (L.) and so on [3, 26, 27]. Six kinds of complement activating pectins (AR-2IIa, 2IIb, 2IIc and 2IId) and pectic arabinogalactans (AGIIa, AGIIb-1) have been purified from the hot water extract of the roots ofAngelilca acutiloba [28-30]. AGIIa and AR-2IId showed the most potent complement activating activity. AGIIb-1 showed moderate activity and others were weak. These four pectins commonly consisit of over 90% a-(1-~4)linked galacturonan and a small amount of ramified region which contains rhamnogalacturonan core with neutral carbohydrate side chains such as arabinan, galactan and arabinogalactan which are assumed to be linked to position 4 of rhamnose [30]. Digestion with endo-a-(1-->4)-polygalacturonase after deesterification gave ramified region as an enzyme resisitant fraction and several oligogalacturonides [30]. The ramified region from each pectin had a more potent complement-activating activity than the corresponding original pectin, but the oligogalacturonides had weak or negligible activities (Table 2) [30]. These results suggest that for AR-2IIa, 2IIb, 2IIc and 2IId, ramified region might be essential for expression of the activity since each ramified region from four pectins showed similar potent activity. Because (l~3,6)-linked long ~galactosyl chains and oligosaccharides containing ~-(1-~6)-galactose, which were obtained from ramified regions, showed potent or significant activity, for AR-2IIa, 2IIb, 2IIc and 2IId, the neutral carbohydrate side chains attached to the rhamnogalacturonan core might be essential for expression of the activity (Fig. 7) [31]. Although AR-2IIa~-IIc activated complement through the classical pathway but not the alternative pathway, all ramified regions (PG-1) activated complement through both pathways. AR-2IId had a different methyl-ester distribution in the galacturonan region compared to the other three pectins [32]. These results suggest that this galacturonan moiety may modulate the activation of an alternative pathway by the ramified region, and this modulation may be controlled by the distribution of the methyl-ester on the polygalacturonan moiety. Both arabinogalactans, AGIIa and AGIIb-1, were characterized to contain arabino~-3,6-galactans by structural analysis [28, 29]. These carbohydrate structures seem to be common for complement activation by pectins and pectic polysaccharides. Acidic arabinogalactan from berries of Viscum album also strongly activated

181

complement [27]. Table 2 Anti-complementary activity of enzymic digestion products from the pectins of

Angelica acutiloba Concentration (pg/mL) 1000 500 100

Product

Product

Inhibition of TCHs0 (%)

Inhibition of TCHso (%) AR-2IIa Original PG-1 PG-2 PG-3 AR-2IIb Original PG-1 PG-2 PG-3

59.5 82.5 38.0 19.2

30.8 74.5 23.8

0.6 25.0 2.0

62.6 82.2 22.0 4.8

31.8 74.5 2.0 1.5

2.0 25.5

Concentration (~g/mL) 1000 500 100

AR-2IIc Original PG-1 PG-2 PG-3 AR-2IId Original PG-1 PG-2 PG-3

54.3 84.5 28.5 1.8

29.2 82.8

3.0 41.8

84.5 48.8 33.0 n.d. a

85.0 81.8 10.0 n.d.

52.5 40.0 n.d.

"Not determined. PG-1, ramified region; PG-3, oligogalacturonides

/

lO0

I RG core I

Gal-~3Gal--,3Gal O

h~ o3 .i--i

f

50-

\

r 03

g

Gal~6Gal-~6Gal Gal~6Gal~6Gal~6Gal Gal~6Gal~Rha Gal-~6Gal-~6Gal~Rha Gal--,6Gal~Rha-~Rha

.~

O

0 0

!

!

!

100

200

300

Concentration (mg/ml)

6

$

6

Gal

Gal

Gal

Gal

Gal

Gal

$

Gal

(reproduced with permission from reference 6. copyright 1994

Carbohydrate Polymer).

Figure 7. Complement activating potency of the ramified region and its neutral oligosaccharides from the pectins of Angelica acutiloba

182 3.3 Other immunostimulating activity Wagner et al. obtained immunostimulating pectic polysaccharides from plant cell culture of Echinacea purpurea [5]. From the extracellular polysaccharide mixture, acidic arabinogalactan (Echinacea-polysaccharide II) was purified [33]. Echinacea-polysaccharide II, which has a molecular weight at 75,000, was effective in activating macrophages to cytotoxicity against tumor cells [34] and in vitro as well as in vivo against microorganisms such as Leishmania enriettii and Candida albicans. This polysaccharide induced macrophages to produce tumor necrosis factor (TNF-a), interleukin-1 (IL-1), interferon-~2 and oxygen radicals. Echinacea-polysaccharide II consists of arabino-3,6-~-galactan part, rhamnogalacturonan part and arabinan part [5], therefore it is suggested that the polysaccharide may be certain ramified region of pectic polysaccharide. Compounds of plant origin which modify immunological responses have also been shown to influence natural cytotoxicity against tumor cells. Viscum album extracts (Iscador-M| contain a component which strongly increases the cytolytic activity of peripheral blood mononuclear cells (PBMCs) from human [35]. The rapid formation of conjugates between effector cells and tumor cells in the presence of V. album extracts appears to involve bridging by V. album rhanmogalacturonan which enhances the cytotoxicity of human NK cells [36]. Pre-incubation of NK cells with the rhamnogalacturonan did not activate the ~lling potential. Therefore, a synergistic effect of NK cell-tumor cell bridging by the rhanmogalacturonan, together with the activation of NK cytotoxicity by physiological response modifiers such as interleukin-2 might offer a new basis for the effective treatment of cancer [36]. Mitogenic polysaccharide was obtained form the roots of Glycyrrhiza uralensis [26]. Endo-a-(1-~4)-polygalacturonase digestion indicates, this mitogenic polysaccharide has a pectic nature and the enzyme resisitant ramified region showed more potent mitogenic activity than the original polysaccharide. This mitogenic polysaccharide also contained RG-II like region. Several arabino-3,6-~-galactans, which were obtained from Panax notoginseng and Saposhinikovia divaricata, were observed to activate the reticuloendothelial system (RES) in vivo by carbon clearance test [37, 38]. Pectins from Glycyrrhiza uralensis, Euchommia ulmoides and Angelica acutiloba also had RES activating activity, and arabino-3,6-~-galactan rich pectin showed more potent activity [39-41]. 4. ANTI-ULCER ACTIVITY During a study of the polysaccharides from Chinese herbs, potent anti-ulcer activity was observed in the acidic polysaccharide fraction (BR-2) from Bupleurum falcatum, and the active polysaccharides, bupleuran 2IIb and 2IIc were purified [42]. Hundred mg/kg ofbupleuran 2IIb and 2IIc showed the significant anti-ulcer activity against HCl-ethanol induced ulcerogenesis in mice (Fig. 8). This activity was almost same with positive control, sucralfate. The activity of bupleuran 2IIc was higher than that of sucralfate. Bupleuran 2IIc, which has a molecular

183 weight of 63,000, consists of 85.8% of galacturonan region comprising of 70% of a-(l~4)-linked galacturonic acid, 30% of carboxymethylated galacturonic acid and branched galacturonic acid [43]. Bupleuran 2IIc also contained ramified region which consisted of rhamnogalacturonan core and several arabino- and galactooligosaccharide side chains attached to either 2-1inked rhamnosyl residue through 4-1inked galacturonic acid or 2-1inked rhamnose directly in the rhamnogalacturonan core. RG-II like region was also contained in bupleuran 2IIc as a minor region [43, 44]. The oral administration of BR-2 at doses 50 to 200 mg/kg prevented the formation of gastric lesions induced by HCl-ethanol by dose dependent manner [45]. The intraperitoneal and the subcutaneous administrations of BR-2 also dose dependently reduced this gastric lesion. These results suggested that BR-2 exerts through not only a local action but also a systemic action in the stomach. BR-2 also inhibited a variety of acute and chronic experimental ulcer models such as ethanol induced ulcer, indomethacinHC1 induced ulcer, pyrolus ligated ulcer, water-immersion stress ulcer and acetic acid induced ulcer by oral administration [45]. The collective results suggested that the major mechanism of muc0sal protection by BR-2 may be due to its anti-secretory activity on acid and pepsin, its increased protective coating and its radical scavenging effects but not involved in the action of endogenous prostagrandins and mucus synthesis [45, 46]. The ramified region seemed to be one of active site in bupleuran 2IIc. Therefore anti-polysaccharide antibody was made by immunization of its ramified region (PG-1) to the rabbits. Then highly sensitive ELISA method using the purified antibody was developed in order to detect the active polysaccharide. This method is very useful for quality control of the active polysaccharide and for study of absorbtion to the body and pharmacodynamics of the polysaccharide [47]. In this ELISA system, anti-bupleuran 2IIc-PG-1 antibody, which was purified by Protein G-Sepharose, was coated as first antibody on the microtiter plate, and the ramified region was detected by the biotynylated anti-bupleuran 2IIc-PG-1, which was purified by both Protein-G Sepharose and bupleuran 2IIcPG-1 immobilized Sepharose, and the enzyme labelled streptavidin. Bupleuran 2IIc-PG-1 at concentrations greater than lng/well could be measured by this two site sandwich ELISA method. Immunohistograph showed that lymph-follicle in payer's patch and liver both were stained with anti-bupleuran 2IIc-PG-1 antibody specific IgG after oral administration of bupleuran 2IIc. Bupleuran 2IIc-PG-1 was also detected in the liver homogenate one week after oral administration by sandwich ELISA method (Fig. 9). These results indicate that at least a part of bupleuran 2IIc was absorbed to the body after the oral administration. Ginseng leaves also contained an unique anti-ulcer polysaccharide [48, 49]. The most active anti-ulcer polysaccharide, GL-BIII, had a molecular weight at 16,000, and 56.6% of neutral sugar and 33.1% of uronic acid were contained. As major component sugars, rhamnose, arabinose, galactose, galacturonic acid and glucuronic acid were detected. Although most of phamacologically active

184

" " I "

30 [

]

100 mg/kg, p.o. mean + s.e.m. (n=8) ** pLys elicited glyceollin accumulation in the soybean cotyledon assay, suggesting that this biological activity of endopolygalacturonase is not due solely to its ability to release oligogalacturonides from the plant cell wall. Replacement of either Ser-237 or Ser-240 with Gly reduced the enzymatic activity to 48% and 6%, respectively, of the wtY-endcpolygalacturonase. The interaction between the variant enzymes and the PGIP purified from P. vulgaris was investigated using a biosensor based on surface plasmon resonance (BIAlite*)

201

(Mattei et al., this book). The three variant enzymes were still able to interact and bind to PGIP with association constants comparable to that of the wild type enzyme. 5.2. A family of PGIPs is expressed in P. vulgaris L. In the attempt to elucidate the role of PGIP in plant resistance to fungi, different pgip-related genes are being characterized. It has been shown that a small family of pgip genes, likely clustered on chromosome 10, is present in the genome of P. vulgafis [78] and several pgip-related clones have already been isolated in our laboratory. Polymorphisms were found in the lengths of the fragments that hybridised to the pgip probe [78]. Characterization of all pgip members could give a significant contribution to the role of PGIP and correlated proteins in the communication between plant and pathogen.The structural and functional analyses of these clones are in progress in our laboratory. In collaboration with MOGEN (The Netherlands), we have obtained transgenic tomato plants expressing high levels of P. vulgafis PGIP. A chimeric gene has been constructed possessing the structural beanpgip-1 gene under the control of the CaMV 35S promoter, that allows constitutive and high-level expression in most plant tissues. The chimeric gene was introduced into tomato plants by A. tumefaciens-mediated transformation. The transgenic plants widely varied in terms of PGIP levels in the tissues; very high expression (levels from 60 to 100 folds higher than those of untransformed tomato plants) were observed in some of them. In the plants analysed, levels of PGIP well correlated with levels of transcripts of the inserted pgip gene. The PGIP purified from tomato transgenic plants exhibited a specificity different from that of PGIP purified from P. vulgafis (Figure 4).The possibility that a family of PGIPs with different specificities is expressed in P. vulgaris is being investigated.

100

~

100

o

o

o~..l

F

x~ 50

~

PGA. niger

50 PG A. niger

PG F. monili~rme

-" PG F. moniliforme 0 A

200 PGIP (ng)

400

0

20

40

60

80

PGIP (ng)

Figure 4. Inhibition of A. niger and F. moniliforme endopolygalacturonase by PGIP purified fom tomato transgenic plants (A) and from bean (B).

202

6. CONCLUSIONS

Specificity in plant-fungus interactions is likely to be determined by recognition steps involving pathogen-derived signals and complementary sensor (receptor) molecules of plant origin. Both signals and receptors are thought to play their roles at the contact surfaces between the two organisms. A while ago, we suggested that a clever strategy of the plant would be to recognize, as fungal signals, those factors that are required for basic compatibility and therefore have to be maintained by the microorganism during evolution for successful parasitism [6]. As possible signals in plant-fungal interactions, we suggested the endopolygalacturonases. The interaction between fungal endopolygalacturonase and PGIP has the requisites for functioning in a perception mechanism that leads to incompatibility. Both molecules are synthesized very early during an attempted infection and physically interact to give rise to the formation of oligogalacturonides that act as elicitors of several defense responses [54]. The recent cloning of several resistance genes has shed some light how the plant recognize molecules from pathogenic microorganisms. The products of all the isolated resistance genes share the common characteristic of being LRR proteins. It seems therefore that plants have selected this special structure for their immunological performances. PGIP is the first LRR protein characterized in plants and clearly shares an evolutionary relation with some of the resistance gene products. For example, the Cf-9 product is homologous to PGIPs not only in the leucine-rich repeat region but also outside. The PGIP gene could therefore belong to a super-family of genes, which includes the resistance genes and has the role of recognising non-self molecules. The PG-PGIP relationship offers the opportunity to dissect a known signalling interaction using powerful molecular techniques. One pgip gene of P. vulgaris has been characterized but the evidence points to there being a family of expressed genes and hence a family of PGIP proteins which could well differ in their specificities and in their expression patterns. It is essential to the elucidation of the endopolygalacturonases-PGIP interaction to discover how many PGIP proteins are involved, under what circumstances they are expressed and their specifities upon expression. 7. A C K N O W L E D G E M E N T S

This work was supported by the Ministero delle Risorse Agroindustriali e Forestali (MIRAAF) and by The European Community Grants CHRX-CT93-0244 and AIR 3-CT94-2215. 8. REFERENCES

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j. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.

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B i o l o g i c a l l y A c t i v e P e c t i n O l i g o m e r s in R i p e n i n g T o m a t o Fruits Eunice Melotto a, L. Carl Greve b, and J.M. Labavitchb aDepartamento de Botanica, Escola Superior de Agricultura Luiz de Queiroz, Universidade de Sao Paulo Piracicaba bpomo1ogy Department, University of California, Davis, CA 95616

Abstract

Recent work has shown that pectic oligosaccharides extracted from tomatoes are capable of promoting ripening in mature green tomato fruit explants (11). A water soluble, ethanolinsoluble extract of autolytically-inactivated tomato pericarp tissue will elicit a transient increase in ethylene biosynthesis when applied to pericarp discs from mature green fruit. Treatment of Na2CO3.soluble but not chelator-soluble pectin from tomato with pure tomato polygalacturonase 1 generates oligomers that are similar to those extracted from ripening fruit. The endogenous concentration of these oligomers is low, but in excess of that necessary to promote ethylene biosynthesis in this system (29). A major difficulty in assigning a regulatory role to such pectic oligomers is that at the onset of ripening in tomato fruit little evidence of polygalacturonase activity exists. However our work demonstrating the presence of neutral sugar-rich acidic oligomers suggests that other enzymatic activities capable of degrading pectic substances exist in developing tomatoes. The recent report (22) of a rhamnogalacturonase-type activity in developing fruit supports this supposition. The possibility of a role for pectic oligomers in fruit ripening regulation will be discussed.

INTRODUCTION Oligosaccharides as potential regulatory signals. In the 1970s Ayers, Albersheim and colleagues showed that oligosaccharides derived from the cell wall of the fungus Phytophthora megasperma sojae could elicit the production of phytoalexins from tissues of its soybean host and suggested that these carbohydrates represented endogenous signals for turning on host defenses (2,3). Since that time a considerable amount of research has shown that the addition of various carbohydrates (mono-,oligo, and polysaccharides) to various parts (cells, tissues, organs) of assorted plant species can activate various aspects of development. Initially, the interactions of pathogens and their hosts were studied perhaps because cell wall breakdown is so often evident in these situations (14). That these interactions lead to elicitation of the production of pathogenesis-related proteins, hydroxyproline-rich glycoproteins, phytoalexins, lignin, ethylene and other potential

208 aspects of host defenses has also been clearly demonstrated (reviewed in 15). The possibility that carbohydrate elicitors could regulate "non-pathological" aspects of plant development has also been examined. Studies have suggested a regulatory role in morphogenesis (at least in tissue culture), cell elongation, ethylene synthesis, and fruit ripening. More recent work has shown that oligosaccharides can promote ion pumping, production of active oxygen species, and phosphorylation of proteins; responses proposed to be components of pathways for transduction of plant hormone signals (reviewed in 31). Specific binding of the heptaglucoside elicitor derived from P. megasperma cell walls to soybean membranes has been demonstrated with the use of techniques analogous to those employed to identify receptors for plant hormones (13). Although much work remains, it is reasonable to propose that cell wall-derived oligosaccharides can be endogenous regulators of plant development. Cell wall breakdown is certainly a feature of fruit ripening (12, 16, 21, 24, 25) and thus it is easy to propose a source for active oligosaccharides. Ripening is a complex array of processes that together characterize the terminal stages of fruit development. Fruits like the tomato are classified as climacteric because ripening is preceded by dramatic increases in ethylene synthesis and respiration, with all of the ripening-related events (alterations of sugar and organic acid concentrations, changes in color, aroma and flavor, and flesh softening) following in a relatively short time (19). While ethylene promotes ripening and ripening involves the differential expression of many genes, not all of them are ethylene-responsive. Thus other regulatory factors must interact with ethylene to coordinate the ripening process. The cellular event(s) that promotes the initial increase in ethylene production is unknown. Each of the individual ripening events has been studied extensively (7). Of all fruits, the ripening of the tomato has probably received the most attention. This is particularly true of the softening phenomenon where examination of cell wall breakdown - including analysis of polysaccharide changes and production of putative cell wall-degrading enzymes - has been extensive (reviewed in 7, 16). It is clear that the breakdown of pectic polysaccharides, apparently catalyzed, at least in part, by the combined action of PG and PE is a feature of this cell wall metabolism. However, studies of the ripening of transgenic tomatoes with altered PG or PE production (17, 27, 35) make it equally clear that softening is not solely dependent on pectin breakdown. Other aspects of cell wall component breakdown, including digestion of xyloglucan (25) and pectic galactan (21), may also be involved. The question of how (mechanistically) tissue softening is linked to cell wall metabolism remains unanswered. While ripening-related changes are dramatic in tomato and clearly mark the onset of cellular senescence they do not occur simultaneously throughout the fruit. It is easy to follow the development of red pigment as an indicator of ripening, and each tomato variety displays its own pattern of pigmentation change which can be perturbed by environment and growing season (19). The 'Castlemart' variety (the subject of this report) begins its reddening in the center (columella and locules) and then a wave of color sweeps up through the outer pericarp, proceeding from the blossom to stem ends. Over the past several years we have used discs cut from the outer pericarp of tomatoes to study several metabolic aspects of tomato fruit ripening (10). While disc excision causes a wound response that must be allowed to dissipate before testing can begin, the use of

209 discs reduces the tissue variability that comes because ripening is not uniform throughout the fruit and provides several uniform experimental "units" from each fruit used. Discs cut from the outer pericarp of MG fruits will individually ripen and show the typical ripening characteristics (color change, ethylene synthesis, and softening - including changes in wall components and hydrolytic enzymes) of intact fruit. Nevertheless, discs cut from the blossom end of the MG fruit "ripen" a bit sooner than those cut from the stem end of the same fruit (unpublished observations). Clearly there are aspects of the regulation of ripening that depend more on position and developmental time than on the specific biochemical pathways that are routinely studied at the whole fruit or whole fruit extract levels. Our current concept of the way tomato fruit ripening is promoted and coordinated relies on the observations that (a) treatment of mature fruits with ethylene stimulates them to ripen and (b) an increased ethylene production is the first of the characteristic ripening changes one can measure (7, 8). Furthermore, tomato fruit whose ethylene production is largely blocked by transgenic modification of its synthesis show retarded ripening that can be reversed by ethylene application (26). These studies further demonstrate the importance of ethylene in regulation of tomato ripening but also raise several questions. Some facets of ripening (e.g., the production of the mRNA for PG). occur in spite of the reduced ethylene synthesis and fruits whose ripening off the plant is delayed, ripen normally when left attached (26). Ethylene production of intact fruit does not begin simultaneously throughout the organ. It apparently begins in the fruit's central tissues and proceeds to the outer pericarp (9). The duration of an intact fruit's increased ethylene production is longer than that of individual ripening pericarp discs (10), further suggesting that the ethylene production pattern of the intact fruit is a composite integration of the patterns of individual fruit parts/sectors. Finally, it is not clear what signals the fruit cells to begin producing ethylene. In earlier work we showed that oligosaccharides produced by acid hydrolysis of citrus pectin could stimulate the ethylene production and ripening of tomato pericarp discs (11). In this report we describe the isolation of neutral sugar-containing pectin oligomers from ripening tomato fruits and show that these can promote aspects of ripening in isolated discs. Our observations thus far are consistent with the idea that one component of the regulation of ripening in tomato fruits is pectic oligosaccharides.

MATERIALS AND METHODS Tomato fruits (Lycopersicon esculentum Mill. var. 'Castlemart') were collected from vines grown in the field at the University of California, Davis. Pericarp discs were cut from surface sterilized MG stage fruit (10). Droplets (10 ul) of test solutions (see below) were applied to the cut surface of discs and disc ethylene production was measured as described previously (11). The amounts of test materials used were based on colorimetric assay (6) of uronic acid content. Small sections (0.5 cm 3) of pericarp tissue from B stage fruit were cut and placed immediately into boiling 95% (v/v) ethanol and refluxed for 15 min. The ethanol was then decanted and the boiled tissue was homogenized in cold water. The sample was

210 centrifuged (680g, 20 mm) and the supernatant (water-soluble fraction) was saved for analysis and subsequent use. The pellet was then sequentially extracted with CDTA and Na2CO3, as previously described (12, 29) Tomato oligomers. The water-soluble fraction was made 80% (v/v) with ethanol, stored in the cold (4~ overnight, and precipitated materials were collected by centrifugation. The precipitates were air dried to remove ethanol and stored until needed. Preparations were redissolved and analyzed by analytical HPLC by a modification (29) of the technique of Hotchkiss et al. (23). Our assessment of the degree of polymerization of individual oligomeric peaks was based on the relative retention time of a galacturonic acid octomer (a gift from the Complex Carbohydrate Research Center, Athens, Georgia; Spiro et al. [33]). The G7 and G12 citrus pectin oligomers used in earlier work (11) were also analyzed in this fashion. In some cases individual oligomer peaks were collected for testing. For the tomato extracts, this further purification was accomplished by collection of individual peaks following preparative scale HPLC, followed by concentration and desalting using YCO5 ultrafiltration. For the more plentiful citrus preparations, the initial separation was based on anion exchange chromatography in an imidazole-HC1 gradient (29). Uronic acid peaks were identified and the middle portions of individual peaks were pooled and ultrafiltered. The CDTA- and Na2CO3-soluble pectins were also used as test materials. Extracts were dialyzed against water and freeze-dried. They were then tested directly or after digestion with purified tomato PG1 ( treatment at 37 ~ in sodium acetate, pH 4.5). Qualitative analysis of selected fractions was based on GLC separation of alditol acetates (5). Alditol acetates from uronosyl residues were prepared by methanolysis followed by reduction (4).

RESULTS AND DISCUSSION Previous work in our laboratory (11) showed that acidic oligosaccharides from citrus pectin could promote ethylene production and ripening (accelerated red color development) when applied to excised tomato pericarp discs. Furthermore, the acceleration of ripening was not due solely to the promotion of ethylene synthesis. Red color development at the inner (cut) tissue surface to which the treatment was applied was greater in response to oligomer treatment than was the response to a treatment with the ethylene precursor, ACC. Thus pectin oligomers have the potential of serving as ripening regulators. We (29) asked if analogous pectin oligomers were present in ripening tomato tissues in order to extend our examination of the potential endogenous role of oligomers as ripening regulators. The effect of a water-soluble, uronic acid-containing fraction extracted from B tomato fruit tissue on ethylene production of pericarp discs cut from MG tomatoes is shown in Figure 1. As little as l~zg of uronic acid in the extract can cause a doubling of ethylene production. Figure 2c shows the HPLC separation of the components in this active extract and compares it to the G7 and G12 preparations (Figs. 2a,b) used in earlier work. While this side-by-side comparison does not provide proof of the similarity of the active tomato and citrus preparations, it certainly suggests that each represents a series of

211

"--'0"-~, --=

0

Control (water) Extract, 1 ug Extract. tO ug

i

T

3 Hours

alter

treatment

Fig. 1. Promotion of ethylene synthesis by MG pericarp discs following treatment with water or varying concentrations (uronic acid equivalents) of B fruit water soluble fraction. Bars indicate SEs for the means of measurements of sets of 8 discs/teatment fr wt, Fresh weight. acidic oligosaccharides having similar size distributions. Based on HPLC elution of an octomer of galacturonic acid, the range of DPs represented by the tomato extract is 4 to 12. When peaks were integrated and quantified in relation to the octomer standard (Table I), it becomes clear that the cleanly resolved peaks represent only a small portion of the uronic acid in the preparations. This is particularly true for the tomato extract. Only 2 % of its uronic acid is represented by the roughly-identified, oligomer peaks. Can we be confident, then, that the oligosaccharides shown by HPLC are the active components in our extracts? Two lines of evidence suggest that the answer is yes. When the oligomercontaining tissue extract is subjected to gradient anion exchange chromatography, a heterogeneous population of uronic acid-containing materials is indicated (Fig. 3). HPLC analysis of pooled fractions from this analysis revealed oligomers only in fractions 26-41, and only those fractions promoted disc ethylene synthesis (data not shown). When individual peaks were collected following preparative scale HPLC, those chromatographing in the DP 7-10 range have ethylene-inducing activity (in relationship to their uronic acid content) greater than that of the unfractionated fruit extract (Fig. 4). We conclude, therefore, that the activity of the extract (Figure 1) is primarily due to its oligomer content. Huber and O'Donoghue (25) have reported that pectins extracted from tomato cell walls that have been prepared in Tris-buffered phenol (to remove the potential for autolytic digestion by residual, active PG) do not contain oligomers such as those we described above. While we have taken care to inactivate PG (boiling tissue pieces for 15 minutes in ethanol) and have found no active PG or cell wall autolytic activity following this treatment, we recognize that our treatment is probably less complete than theirs. Because the oligomer population we have described is quite small, we have to accept that active PG might have been present below our level of detection. If so, the oligomers we have described could have been generated in vitro. We do not think that this is the case, however. The preparation that Huber and O'Donoghue used in their analyses was a

212

2400

@ 5

1800 c t200

6

7

8

S 600

I

~0 '1

9

s

.

,

2400 I

L

l

1

6

8OO

8

5

I 9,

4

200

,o,,

600 i

5

300

l

i

L

z'8

3'2

Fig. 2. HPLC ion exchange gradient separation of samples (25 ug of uronic acid) of smaller (G7) citrus pectin oligomers (a), larger (G12) citrus pectin oligomers (b), and the B. fruit extract (c). Detection was by PAD. For each sample the peak co-eluting with the standard galacturonate octamer was designated peak 8, and the remaining peaks were numbered consecutively.

6

225 9 =

150

;

75

'0 II

4

8

~2 Relentton

6

20

~,,

Ttme ( m,n I

CDTA extract of cell walls. That extract (based on the work of Carrington et al. [12]) would have included the uronic acid we have identified as water soluble (and containing active oligomers) and wall-bound, chelator-soluble polymers containing uronide not included in our analysis. Thus, our oligomer-containing fraction would have been diluted to 40% of its initial concentration (to 0.8% of the uronic acid, based on the data in Table I and Fig. 4) in the material they analyzed. The extract was then subjected to gel filtration (Sepharose CL-2B-300) and fractions containing lower molecular weight uronic acids (in the range of 100,000 daltons and lower, according to manufacturer's specifications) were pooled for subsequent oligomer separation on Bio-Gel P-4. Fractions from this separation were then assayed for uronic acid, and none was found in the column's included volume. Given the low concentration of pectin oligomers we report in our extracts, and the fact that they, apparently, began their serial analysis with 500 ~zg of galacturonic acid equivalents, we would be surprised if that analysis had revealed tomato fruit oligomers. It was sufficient to identify pectin oligomers in extracts of ripening avocados, however. One might ask why we are stressing this point. The answer is simple. The report of Huber and O'Donoghue has been interpreted by many to be an

213

Table 1. Distribution of uronic acids in acid oligomers resolved by HPLC gradient ion-exchange chromatography (Fig. 2) Quantitation is based on comparison of integrated areas with the peak area for a known amount of galacturonic acid octomer (quantitation is approximate because of uncertainties with PAD detection of different sized oligomers). Samples(25 ug, galacturonic acid equivalents) of G7, G12, and B extract were chromotographed. Yield is the sum of the uronic acid measured in all oligomer peaks for a given sample expressed as a percentage of the 25 ~g injected. Oligomer peak numbers are shown in Figure 2 and the values shown are in ng of galacturonic acid equivalents. Peak No. Sample

4

5

6

7

8

9

10

11

12

13

G7

902

1190

694

485

347

274

232

201

104

90

14

G12

462

563

619

614

463

341

295

286

189

129

B extract

50

80

93

106

67

42

26

23

12

Yield 18.00

83

16.70 2.00

Table II. Carbohydrate compositions (weight percentage) of individual oligomer peaks purified (QAE-Sephadex or HPLC ion-

exchange separation, respectively)from mixtures of citrus pectin oligomers or B fruit extracts Compositions shown are for peaks whose biological activity is described in Figure 4. Uronic acid values are based on colorimetric assay. Proportions of neutral sugars were determined by GC and adjusted so that totals equal 100%. In fact, some oligomers (G7: peaks 8, 9 and 10. B extract: peak 10) produced small (less than 1% of the total integrated area), unknown peaks in the GC chromatograms. Component Source

Galacturonic Acid

Rha

Ara

Xyl

Man

Gal

Glc

Citrus oligomers Peak 8

93.9

I. 1

2.0

0.2

0.3

1.0

1.4

Peak 9

95.5

0.9

1.9

0.1

0.2

0.5

0.8

Peak 10

86.7

2.5

3.3

0.8

1.7

1.9

3.2

Peak 7

71.1

6.2

3.3

1.2

4.5

4.6

9.1

Peak 8

58.2

5.8

3.1

1.8

2.4

15.0

13.8

B extract

Peak 9

55.1

13.2

4.4

2.6

5.0

11.8

7.9

Peak 10

56.4

10.1

5.9

1.5

2.9

18.5

4.7

Peak 11

60.8

3.3

3.3

1.9

3.5

14.5

12.8

indication that pectin oligomers are not present in ripening tomato pericarp and, thus, that any consideration of a role for them in regulation of ripening is folly. We do not accept that oligomer presence can be ruled out by the described analysis. We accept that if they are endogenous (i.e., were not produced by PG that remained active after boiling pericarp in ethanol), the presence and biological activity we have reported here do not prove, p e r s e , that pectin oligomers are endogenous ripening regulators, and this point will be examined further. How might these oligomers be produced in the fruit? The easiest answer is that limited PG action on cell wall pectins generates them, and we have tested this point in vitro. CDTA- and Na2CO3-soluble pectins and PGA (included as a positive control) were incubated with purified tomato PG1. Surprisingly, only the carbonate-soluble material and PGA were digested (based on HPLC analysis of reaction mixtures and generation of

214

,0!!I

50 A .--I

do

1 t

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20 tO 1.,,. 10

0 r 0

9

|

"

20

|

"

|

40

-

60

' i

-

80

Fraction

l

100

120

Number

Fig. 4. Ethylene production by MG pericarp discs treated with 50 /~g (uronic acid equivalents) of partially purified G7 citrus oligomers (a) or 30/~g of individual B fruit oligomers purified by HPLC. Water was used for the control. Peak numbers correspond to those shown in Fig. 2. Bars indicate SEs for the means of measurements of 8 discs/treatment.

30

Control (water)

--.---e,-----

20

-----O m

G7

.... g--"

Peak 8

.... O---

Peak 9

- - "dr" -

P e a k 10

16

e"

e-

=T= ..-" T'o.

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14

----e---

Control (water)

---0-----

Breaker extract

.... m---

Peak 7

----.O'--

Peak 8

- - -z~--

P e a k 10

- - .t- -

P e a k 11

"

12

,c."

i 10 i

r162

sI

.~'/" ~r"

4

T

0

1

2

Mourn after

3

Fig. 3. QAE-Sephadex gradient separation of the B fruit extract. An 18 mg (uronic acid equivalents) sample of extract was dissolved in 20 ml of 125 mM imidazole-HC1 buffer (pH 7.0) and applied to the column. The column was then eluted with 50 ml 125 mM buffer followed by a 125 mM to 1.5 M buffer gradient (500ml), and, finally, 50 ml of 1.5 M buffer. Fractions of 5 ml were collected and assayed for uronic acids. Groups of fractions (26-41, 45-50, 53-75 and 84-100) were pooled, concentrated by ultrafiltration and analyzed by HPLC.

4

treatment

215 Fig. 5. Effect of PG1 digestion on the ethylene synthesis-inducing activity of CDTA-soluble tomato pectin (a), Na2CO3-soluble tomato pectin (b) and polygalacturonic acid (c). Controls were treated with solutions of heatinactivated PG1. Treatment doses were 10/tg of uronic acid equivalents. The line legends shown in panel a apply to all panels. Bars indicate SEs for the means of measurements of sets of 8 discs/teatment, fr wt, Fresh weight.

'i PG-treated --tl--

.'~ o.o

L_

Not digested

"'"" t~~176

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.~ oi

10

t-

In

o C

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3 after

4 treatment

5

6

reducing sugars; data not shown). The digested substrates, but not the undigested material, showed ethylene inducing activity in our pericarp disc assay system (Fig. 5). We have not yet compared the structures of the extracted and PGl-generated active oligomers. If the endogenous material is a participant in the initiation of ripening, however, it is unlikely the fruit PG is responsible for its origin. Several studies (e.g.; 8, 20) have shown that tomato fruit synthesis of PG follows (by as much as a day or more) the increase in fruit ethylene production that is accepted as a marker of ripening's onset. Of course, ripening is not a simultaneous, whole fruit phenomenon. It is a developmental event the passes through the fruit (its different tissue areas, perhaps even from cell to

216 cell) in a wave and so it is possible that an undetectable bit of ripening-initiating PG preceeds the rise in ethylene production as the first cells begin to ripen. However, even though studies have shown wave-like patterns of PG gene activity (30) and protein (34) in ripening tomatoes, we are not aware of data indicating that this PG "appearance" preceeds increased synthesis of ethylene. However, Schols et al. (32) have identified a pectin hydrolase produced by cultures of the fungus Aspergillus aculeatus and identified it as an RGase because it hydrolyzes the linkage between rhamnosyl and galacturonosyl residues in rhanmogalacturonans, and Gross et al. (22) have recently reported the presence of RGase activity in MG tomato fruits. Studies of in vitro RGase action on complex pectin substrates show that the enzyme often needs assistance from an assortment of "pectin processing" glycosidases to cleave the pectin backbone and that the breakdown products that result are relatively neutral sugar-rich (1, 32). MG and ripening tomato fruits contain a substantial variety of glycosidases (36). When we analyzed the oligomers in our B stage fruit extract we found them to contain an assortment of the neutral sugar residues that are normally associated with complex pectins (Table II). It is conceivable, therefore, that tomato RGase action (perhaps supported by glycosidases) could explain the presence of the oligomers we have described, and that limited pectin hydrolysis could lead to the initiation of ripening. On the other hand, recent work has shown that pectic oligomers can be transported over limited distances in tomato plants (28) and work in our lab has shown that limited pectin synthesis in tomato fruits even after ripening has begun (18). This means that the hydrolytic origin for endogenous, active pectin oligomers of tomato fruits is only one of several possible explanations for the presence we have reported here. Our laboratory is continuing its examination of the possibility that oligosaccharides contribute to the regulation of tomato fruit ripening. Figures and tables from Melotto et al. (1994) are reprinted with permission from Plant Physiology. Abbreviations: ACC, 1-aminocyclopropane-l-carboxylic acid; B, breaker stage (beginning to ripen); CDTA, trans-l,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid; DP, degree of polymerization; G7, set of smaller pectin oligomers generated by acid hydrolysis of G12, set of larger citrus pectin oligomers; MG, mature green stage (ready to ripen); PE, pectin methylesterase; PG, polygalacturonase; PGA, polygalacturonic acid; RGase, rhamnogalacturonan hydrolase.

REFERENCES An J., L. Zhang, M.A. O'Neill, P. Albersheim, A.G. Darvill. 1994. Isolation and structural characterization of endo-rhamnogalacturonase-generated fragments of the backbone of rhamnogalacteronan I. Carbohydr. Res. 264:83-96.

217 2. Ayers, A.R., J. Ebel, F. Finelli, N. Berger, P. Albersheim. 1976. Plant Physiol. 57:751-759. 3. Ayers, A.R., B.S. Valent, J. Ebel, P. Albersheim. 1976. Plant Physiol. 57:766-774. 4. Bhat, U.R., H. Mayer, A. Yokota, R.I. Hollingworth, R.W. Carlson. 1991. J. Bact. 173:21552159. 5. Blakeney, A.B., P.J. Harris, R.J. Henri, B.A. Stone. 1983. Carbohydr. Res. 113:291-299. 6. Blumenkrantz, N., G. Asboe-Hansen. 1973. Anal. Biochem. 54:484-489. 7. Brady, C.J. 1987. Ann. Rev. Plant Physiol. 38:155-178. 8. Bardy, C.J., G. MacAlpine, W.B. McGlasson, Y. Ueda. 1982. Aust. J. Plant Physiol. 9:171-178. 9. Brecht, J.K. 19897. HortScience 2:476-479. 10. Campbell, A., M. Huysamer, H.U. Stotz, L.C. Greve, J.M. Labavitch. 1990. Plant Physiol. 94:1582-1589. 11. Campbell, A., J.M. Labavitch. 1991. Plant Physiol. 97:706-713. 12. Carrington, C.M., L.C. Greve, J.M. Labavitch. 1993. Plant Physiol. 103:429-434. 13. Cheong, J.J., M.G. Hahn. 1991. Plant Cell. 3:137-147. 14. Cooper, R.M. 1984. In: (Woods and Jillis, eds.) "Plant Diseases: Infection, Damage and Loss." Blackwell, Oxford, pgs. 13-27. 15. Darvill. A.G., P. Albersheim. 1984. Ann. Rev. Plant Physiol. 35:234-275. 16. Fischer, R.L., A.B. Bennet. 1991. Ann. Rev. Plant Physiol. and Mol. Biol. 42:675-703. 17. Giovannoni, J.J., D. DellaPenna, A.B. Bennett, R.L. Fischer. 1989. Plant Cell 1:53-63. 18. Greve, L.C., J.M. Labavitch. 1991. Plant Physiol. 97:1456-1461. 19. Grierson, D., M. Kader. 1986. In: (Atherton and Rudich, eds.). "The Tomato Crop - A Scientific Basis for Improvement" Chapman and Hall, london, New York. pgs. 241-280. 20. Grierson, D., G.A. Tucker. 1983. Planta 157:174-179. 21 Grierson, D., G.A. Tucker. 1983. Planta 157:174-179. 22. Gross, K.C., D.A. Starrett, H-J. Chen. 1995. Acta Hort. 398:121-130. 23 Hotchkiss, A.T., K.B. Hicks. 1990. Anal. Biochem. 184:200-206. J 24. Huber, D.J. 1983. J. Amer. Soc. Hort. Sci. 108:405-409. 25. Huber, D.J., E.M. O'Donoghue. 1993. Plant Physiol. 102:473-480. 26. Klee. H.J. 1993. Plant Physiol. 102:911-916. 27. Kramer, M., R. Sanders, H. Bockan, C. Waters, R.E. Sheehy, W.R. Hyatt. Postharv. Biol. technol. 1:241-255. 28. MacDougall, A.J., N.M.Rigby, P.W. Needs, R.R. Selvendran. 1992. Planta 188:566-574. 29. Melotto, E., L.C. Greve, J.M. Labavitch. 1994. Plant Physiol. 106:575-581. 30. Montgomery, J., V. Pollard, J. Deikmen, R.L. Fischer. 1993. Plant Cell 5:1049-1062. 31. Ryan, C.L., E.E. Ramer. 1991. Ann. Rev. Plant Physiol. and Mol. Biol. 42:651-674. 32. Schols, H.A., C.C.J.M. Gereads, M.F. Searle van Leeuwen, M.F. Kormelink, A.G.J. Voragen. 1990. Carbohydr. Res. 206:105-115. 33. Spiro, M.D., K.A. Kates, A.L. Koller, M.A. O'Neill, P. Albersheim, A. Darvill. 1993. Carbohydr. Res. 247:9-20. 34. Tieman, D.M., A.K. Handa. 1989. Plant Physiol. 90:17-20. 35. Tieman, D.M., R.W. Harriman, G. Ramomohow, A.K. Handa. 1989. Plant Cell 4:667-679. 36. Wallner, S.J., J.E. Walker. 1975. Plant Physiol. 55:94-98.


IDENTIFICATION, MODE OF ACTION AND 3-D STRUCTURE OF PECTINASES


J. Visser and A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All fights reserved.

Kinetics and mode of action of

221

AspergiUus niger polygalacturonases

Jacques A.E. Benen, Harry C.M. Kester, Lucie Parenicovfi and Jaap Visser. Section Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, Dreyenlaan 2, 6703 HA Wageningen, The Netherlands.

Abstract Endo-polygalacturonases I and II (PGI and PGII) isolated from recombinant A. niger were characterized with respect to pH optimum, activity on polygalacturonic acid (pga), mode of action and kinetics on oligogalacturonates. Vmax and Km values using pga as a substrate at the optimum pH 4.1 were calculated as 500 U/mg and 0.15 mg/ml and 2000 U/mg and 0.15 mg/ml for PGI and PGII, respectively. Mode of action analysis revealed a random cleavage pattern for PGII while for PGI multiple attack on a single chain was observed. For PGII a partial subsite map was obtained. Site directed mutagenesis of His223 of PGII with subsequent analysis of the mutated PGII revealed that His223 is essential for catalysis.

Introduction Polygalacturonases from primarily fungal origin have been studied since several decades. Nowadays many genes encoding polygalacturonases, both exo- and endo-acting, from numerous different species have been cloned. Despite the large number of genes available and the long record of polygalacturonase studies most studies were directed at the purification of the enzymes and a limited characterization comprising mostly whether the enzyme is exo- or endo-acting, the activity on polygalacturonic acid and the pH, temperature and ionic strenght optima while very few studies were carried out toward the understanding of the mode of action, determined by the characteristics of the individual subsites of the enzymes. First studies addressing this problem via the determination of the number of subsites of an Aspergillus niger polygalacturonase and identification of catalytically important residues of this enzyme were described by Rexov~i-Benkovfi [1, 2]. Unfortunately, these studies have found only little follow up by other research groups [3]. Therefore detailed knowledge about 'subsite architecture' of these industrially important enzymes is scarce. Bussink et al. [4] and Kusters-van Someren et al. [5] have shown that in A. niger for both polygalacturonases (PGs) and pectin lyases (PLs) families of genes are present: seven

222 PG and six PL encoding genes were identified [4, 5]. The occurrence of families of genes encoding different enzymes raises the possibility of a concerted action of the enzymes of one or both families in the degradation of pectin. In order to clarify the role of the individual enzymes in the pectin degradation a comparative study in this respect was initiated. For this, individual genes were fused with the pkiA promoter of the glycolytic pyruvate kinase gene that allows expression of the individual genes under conditions where all other pectinases are repressed. Careful analysis of mode of action, kinetic parameters and subsite affinities of the enzymes on model and natural substrates will reveal the role of the individual enzymes in pectin degradation. Here we report on the characterization of recombinant PGI and PGII, the two most abundant PGs in the commercial Rapidase preparation [3] and of a site specific mutated PGII in which His223 was changed into Ala.

Materials and Methods Molecular biology. All DNA manipulations were performed using standard techniques. Promoter-gene fusions were constructed as described by Kusters-van Someren et al. [6]. Site directed mutagenesis of His223 of PGII was carried out in essentially the same way. PCR generated DNA fragments were checked for undesired mutations by sequence analysis. Transformation of A. niger NW228 (pyr, prtF) with appropriate plasmids was done as described before [7]. Growth and purification. A. niger strains transformed with either the pki-pgaI or pla'-pgalI fusion were grown in batch in 1L flasks containing 350 ml minimal medium according to Pontecorvo et al. [8] supplemented with Vishniacs spore element solution, 0 . 1 % yeast extract, 70 mM NH4C1 and 4 % fructose as a carbon source. Cultures were inoculated with 1 • 106 spores/ml and grown at 30 ~ in a rotary shaker for 18 hrs. Mycelium was separated from the medium by filtration over a nylon membrane. The filtrate was adjusted to pH 6.0 and loaded onto a DEAE-Sepharose Fast Flow column pre-equilibrated at pH 6.0 (10 mM Bis-Tris/HC1). Elution was performed with a linear gradient, 0-600 mM NaC1, in 10 mM Bis-Tris pH 6.0. SDS-PAGE of individual fractions demonstrated that the enzymes were pure after this separation. Fractions containing the enzyme were pooled and dialysed against 50 mM Na-acetate pH 4.5 and stored at either -20 ~ or 4 ~ until use. Mode of action and kinetics. Routine polygalacturonase assays were performed in a reaction mixture containing 50 mM Na-acetate pH 4.2 and 0.25 % w/v pga at 30 ~ The release of reducing sugars was determined according to Stephens et al. [9]. For determination of pH optima the 50 mM Na-acetate buffer was replaced by Mcllvain buffers. For the determination of kinetic parameters and for the mode of action of the enzymes reaction products were analysed on a Dionex BioLC/high-performance chromatography system using a Carbo Pac PA-100 anion-exchange column (25 cm x 4 mm) with pulsed amperometric detection. The samples loaded were eluted with a linear gradient of 0.15-0.60 M Na-acetate in 0.1 M NaOH at 1 ml/min in 22 min. Products were quantitated via calibration mixes containing oligogalacturonates with DP 1-8 (G1-G8) at 0.1 mM each and via an internal standard of 0.1 mM glucuronic acid (eluting between G1 and G2) with 50 pl injections.

223

Results and Discussion The PGI and PGII produced from strains transformed with the promoter gene fusion are in all respects tested identical to those enzymes obtained from the wild type strain when grown on pectic substances. For both PGI and PGII the pH optimum is 4.1-4.2 in 50 mM Na-acetate buffer, 30 ~ All further kinetic analyses were performed under these conditions. PGII. Using pga as a substrate Km and Vmax of PGII were calculated as 0.15 mg/ml and 2050 U/mg, respectively. The high Vmax and low Km demonstrate that pga is a good substrate for PGII. By following the product formation as a function of time (Fig. 1) it was demonstrated that PGII is a randomly cleaving endo polygalacturonase. The progression of substrates is characterised by an initial transient increase of higher oligogalacturonates which are gradually converted to oligomers with lower DP, eventually resulting in a mixture of G1, G2 and G3. The rather strong transient increase of G4 and G5 is not a result of transglygosylation, since PGII is an inverting enzyme (see Biely et al. elsewhere in this volume), but is merely due to the slow hydrolysis of these compounds as will be discussed below.

0.4

G1 IS

G2

G3

G4

0.3 90 min

~- 0.2 0.1 0.0

_

0

J A 5

-

-

~ _ . _ . _ . ~ _ A

10

15

-

35 min 10 min I 20

minutes

Figure 1. HPLC analysis of product progression during hydrolysis of 0.25 % polygalacturonate by PGII. Aliquots were withdrawn from the reaction mixture at timed intervals and reactions were stopped by raising the pH of the sample to pH 8.0 by mixing with 1 volume 25 mM Na-phosphate pH 9.5. G1 to G5 indicate the oligogalacturonates with corresponding degree of polymerization. The vertical axis shows the responce of the pulsed amperometric detector and the horizontal axis the elution time. Times of sampling are indicated above the trace.

224

Table I. Mode of action of PGII. Bond cleavage frequencies (BCF) in percentage for oligogalacturonates. The reducing or end of the products is indicated with a solid circle. The position of cleavage is indicated with a solid triangle. DP

BCF (%)

4 5

6

o

o

o o

Products

o

o

o

9

100

o o

o o

o o

9 o

9

67 33

dimer

o o o

o o o

o o o

9 o o

9 o

35 57 8

dimer trimer

9

A

In order to estimate the number of subsites, the binding affinities, the location of the active site and the cleavage patterns reduced and non reduced oligogalacturonates of DP 4 to 6 were used as substrates and the resulting products analysed by HPLC and TLC. Table I lists the bond cleavage frequencies for PGII. G4 is exclusively split in 1-3 mode while reduced G4 was not hydrolysed. G5 is cleaved in the 1-4 and 2-3 mode at 67 % and 33 % respectively, while reduced G5 is only split into reduced G2 and G3. The reduced G6 is not cleaved in the 1-5 mode, while reduced G2 and reduced G3 are readily formed. The non-reduced G6 is cleaved in 1-5, 2-4 and 3-3 modes yielding equimolar product pairs as was also seen for the cleavage of G4 and G5. These data demonstrate that cleavage of the glycosidic bond occurs from the reducing end. In time course experiments using different oligogalacturonates at several concentrations the turnover numbers and Km values were estimated for each oligomer in the individual binding modes and used for the calculation of the thermodynamic parameters of PGII according to Thoma et al. [10] and Hiromi et al. [11]. In Table II the data are presented. An approximation of the intrinsic rate constant, kint, was calculated from Vmax on pga (kint = kcat). From kint and Km and the turnover number for each binding mode the binding energies for each mode were calculated which were in turn used for the calculation of the individual subsite affinities listed in Table III. Since G4 is the smallest substrate used in this study, it is not possible to obtain information of the subsites at positions -3 to 1.

225 Table II, Kinetic and thermodynamic parameters of PGII using oligogalacturonates as substrates. Kp was calculated from Kp=[ko/Km]/kint while kint was obtained as described in the text. AG was calculated from -AGp=RTlnKp + 10 kJ/mole. Mode indicates the cleavage mode DP

Mode

Km • 10-6 (M)

ko

ko/Km x 10-6 Kp

(s -1)

(M -1 s-1)

(M -1)

AGp (kJ/mole)

4

G3 + G1 22

103

4.7

8048

-22.6

5

G4 + G1 13 G3 + G2 25

315 159

24.2 6.4

41438 10960

-26.7 -23.4

G5 + G1 40 G4 + G2 16 G3 + G3 31

148 386 56.5

3.7 24.1 1.8

6336 41267 3082

-22.0 -26.7 -20.2

Table III. Subsite affinities Ai for PGII (i denotes the subsite number). Subsites with a '-' prefix are located towards the non-reducing end of the substrate while subsites with a ' + ' prefix are located towards the reducing end. The active site is located between subsites -1 and + 1. Subsites -3 to + 1 were determined as one value. Subsite (Ai) Affinity (kJ/mole)

-4 +4.2

-3 / + 1 +22.6

+2 +0.8

The subsite map and the data on reduced oligomers, which showed formation of reduced G2 on reduced G5, indicate that the number of subsites is 5, stretching from position-4 to 1. The sum of the individual turnover numbers for each oligomer also shows that the rate of hydrolysis of the oligomers with DP4 and DP5 is much slower than of those with higher DP which is reflected in the product progression curves in Fig. 1. Rexov~i-Benkov~i [1] studied an A. niger endopolygalacturonase which might have been the same as the PGII described here; the cleavage pattern described is very much the same as found here and the pH optimum is exactly the same. The number of subsites for that enzyme was found to be four which is not in agreement with the number found for PGII. However, bearing in mind that in the study of Rexovgt-Benkovgt the individual binding modes of each oligogalacturonate were not addressed and therefore no subsite map was obtained the additional fifth subsite might have been overlooked.

226 PGI.

In a similar way as for PGII the kinetics for PGI were addressed. Using pga as a substrate Km and Vmax were 0.15 mg/ml and 500 U/mg, respectively. Fig. 2 shows the product progression upon pga hydrolysis. There is a strong increase of G1, G2 and G3 which is accompanied by a transient increase of G4 and G5 while there is only a small increase of oligomers with DP higher than 5. The transient increase of G4 and G5 is not due to transglycosylation since like PGII, PGI is also an inverting enzyme (see Biely et al. elsewhere in this volume). The profiles of PGI suggest that the enzyme after first random cleavage of the polymer substrate degrades the higher oligomers formed preferentially via hydrolysis of terminal residues at the reducing end. The latter was demonstrated by hydrolysis of reduced oligogalacturonates while the former was investigated in more detail by analysing the mode of action and cleavage rates on defined oligomers ranging from DP 4 to 8 . PGI hydrolyses G4 mainly to G1 and G3 at equimolar amounts as expected and a small amount is digested into G2 (Results not shown). G5 is cleaved to G1 and G4 in again equimolar amounts and to almost the same extent to G2 and G3 also in equimolar amounts (Results not shown). Thus, for G4 and G5, PGI is not much different from PGII, only the bond cleavage frequencies being slightly different. Upon hydrolysis of G6 to G8 the formation of equimolar product pairs is not observed anymore. With G6 as substrate G1 is formed at least twice as fast as G5 while G4 is formed IS G2

0.14 0.12 0.10 0

0.08

G3 G4 G5

0.06 0.04

J

0.02

IS

0.00 0

G2 G3 G4 G5 I

10

I

15

10 min I

20

minutes

Figure 2. HPLC analysis of product progression during hydrolysis of 0.25 % polygalacturonate by PGI. Aliquots were withdrawn from the reaction mixture at timed intervals and reactions were stopped by raising the pH of the sample to pH 8.0 by mixing with 1 volume 25 mM Na-phosphate pH 9.5. G1 to G5 indicate the oligogalacturonates with corresponding degree of polymerization. The vertical axis shows the responce of the pulsed amperometric detector and the horizontal axis the elution time. Times of sampling are indicated above the trace.

227 faster as G2 (Fig 3). A similar behavior was observed by Robyt and French [12, 13] for cxamylase. They demonstrated that this type of product ratios, that differ from the beginning of the reaction, are due to multiple attack on a single chain. A ratio-plot according to Robyt and French [13] for G1 and G5 formation from G6 supported that PGI exibits multiple attack on G6 (not shown). The fact that still G5 is formed upon hydrolysis of G6 demonstrates that not all G6 bound in the G5-G1 mode is cleaved in a repetitive way. Thus, only a fraction of the substrate bound in this mode is subject to repetitive attack. The ratio between cleavage in this way and normal cleavage into G1 and G5 is determined by the dissociation constant of the G5 generated upon cleavage and the first order rate constant that is responsible for the shift of the bound G5 into the G4-G1 mode. Since still a considerable amount of G5 is formed, the dissociation constant of G5 and the first order 'shift' rate constant are of the same magnitude. However, from G7 and G8 as substrates it is clear that the ratio between the dissociation constant and 'shift' rate constant is completely in favor of shift when DP equals 6 or 7. With G7 as a substrate quite large amounts of G1 are formed while there is no detectable formation of the corresponding G6, only a rapid accumulation of G5 is observed. Similarly with G8 as a substrate quite large amounts of G1 and G2 are formed while there is no detectable formation of the corresponding G7 and G6, only again a rapid accumulation of G5 occurred which indicates that the multiple attack ceases when DP is down to 5 or 4, hence when dissociation is favored over shift. The previous data allow a clear interpretation of Fig. 2. PGI initially hydrolyses pga in a random endolytic way generating higher oligomers which is accompanied by rapid multiple attack of the higher oligomers in an exolytic way to yield mainly monomers and dimers from the reducing end. Therefore no transient accumulation of higher oligomers takes place. The appearance of transient G5 and G4 is due to the fact that the multiple attack of the higher oligomers stops when DP is 4 or 5 and these product are released. The transient accumulation of the tetramer partly originates from the particular binding mode of the higher oligomers. At present we are working on a kinetic model that describes the action pattern of PGI. It has been suggested that the multiple or single attack can be distinguished based on the relative rate of accumulation of mono- di- and trimers upon hydrolysis of polymeric substrate. At first glance this appears to be valid when comparing for example the mode of action of PGI and PGII (see Fig. 1 and Fig. 2). In a comparative study of three polygalacturonases by Pasculli et al [14] the enzymes were classified according these criteria while the rate of accumulation was by far not as clearcut differing as found for PGI and PGII. The observed profiles in that study can also be explained by assuming different rates of hydrolysis of the smaller (DP 4-6) oligogalacturonates. If for instance the rate of hydrolysis of G4 and G5 of PGII would have been higher then the progression curves would have more resembled those of PGI. The only way to discern between multiple or single attack is by analysis of the stochiometry of product pairs upon hydrolysis of oligomeric substrates at initial stages of reaction.

228 40 o

,.i-,

(D c

gl

30

.,.-~

(/) 0

20

g5

E 0c -

~c -

g4 g2 g3

10 ~n-'~ 0

T 5

I I0

I 15

I 20

I 25

I 30

minutes

Figure 3. HPLC analysis of product progression during hydrolysis of 0.5 mM hexagalacturonate by PGI. Aliquots were withdrawn from the reaction mixture at timed intervals and reactions were stopped by raising the pH of the sample to pH 8.0 by mixing with 1 volume 25 mM Na-phosphate pH 9.5. G1 to G5 indicate the oligogalacturonates with corresponding degree of polymerization.

PGII His223Ala. Apart from mode of action and kinetics of wild type enzymes structure function relationships of these industrially important enzymes is of high interest to provide the necessary knowledge for genetic engineering of desired properties. As a first approach the identification of catalytically important residues was addressed in conjunction with the elucidation of the three dimensional structure [15]. Rexovfi-Benkov~i and Marckov~t [2] obtained evidence for the possible involvement of a histidine in catalysis of a fungal endopolygalacturonase. The same results were obtained for PGII using diethyl pyro-carbonate as a modifying agent in the presence or absence of substrate (Kester, unpublished). However chemical modification studies can never give conclusive evidence for the participation of the modified residue in catalysis or binding. The method to dissect the role of individual residues is site directed mutagenesis. Upon alignment of 25 endo- and exo-polygalacturonases taken from the swiss.prot. database only one histidine appeared to be conserved throughout. The conserved histidine at position 223 in PGII was changed into alanine, a small apolar residue. The mutation has a dramatic effect on Vmax using pga as substrate which decreases from 2050 U/mg to 10 U/mg; the Km did not change significantly, however. The mutation also affected the pH optimum of the enzyme as is shown in Fig. 4. The pH optimum narrows down to one pH unit. Since the polygalacturonate concentration used is well above the Km the apparent velocities can roughly be regarded as Vmax values at each pH. So, the fall and rise of the activity within one pH unit strongly suggests that catalysis is governed by only one ionisable group in the mutated enzyme, a glutamate or an aspartate. Thus, the

229

100 >,

80

9 40

~

"~9 rr

20 0

I

I

I

I

2.5

3

3.5

4

I

I

I

I

4.5

5

5.5

6

pH

Figure 4. Relative activities of wild type PGII and His223Ala mutated enzyme as a function of pH. Wild type enzyme, solid circles; His223Ala mutated enzyme, open circles. 0.30 0.25

' -

IIs

gl.

g3

0.20 0:=L

G

0.15

IS

0.10 0.05 0.00

g2 j~

0

I

5

i

I

10

,

!

15

!

20

minutes

Figure 5. Selected HPLC elution profile of products obtained after incubation of 0.25% polygalacturonate with PGII, upper trace, and PGII H223A, lower trace, respectively, demonstrating the effect of the mutation on catalysis. G1 to G3 indicate the peaks of the corresponding oligogalacturonates. IS indicates the internal standard, glucuronate. The vertical axis shows the pulsed amperometric detector response while the horizontal axis shows the retention time.

230 strong effect on Vmax, the absence of an effect on Km and the striking effect on the pH optimum, the shift in the direction expected, provide evidence for the involvement of His223 in catalysis rather than binding. This is again affirmed by the analysis of the products formed after hydrolysis of pga. In case of an effect on binding it is expected that the product distribution is different from the wild type enzyme however in case of involvement in catalysis only a slight effect, if any, on the product distribution is expected. In Fig. 5 two selected HPLC profiles are presented showing the product distribution when pga is hydrolyzed to the same extent by wild type PGII and mutated PGII. Apart from a small change in G1 contribution there is hardly any change in product distribution. A small effect on G1 is to be expected since the active site histidine is located at the junction of subsites -1 and + 1. These data demonstrate that His223 is involved in catalysis and not in binding.

Acknowledgement This work was financially supported by the European Community grant no. AIR2-CT941345.

References 1) Rexov/t-Benkov~i, L. (1973) Eur. J. Biochem. 39, 109-115. 2) Rexovfi-Benkov~t, L. and Marckov~i, M. (1978) Biochem. Biophys. Acta 523, 162-169. 3) Kester, H.C.M. and Visser, J. (1990) Biotechn. Appl. Biochem. 12, 150-160. 4) Bussink, H.J.S., Buxton, F.P., Fraaye, B.A., de Graaff, L.H. and Visser, J. (1992) Eur. J. Biochem. 208, 83-90. 5) Harmsen, J.A.M., Kusters-van Someren, M.A. and Visser, J. (1990) Curr. Genet. 18, 161-166. 6) Kusters-van Someren, M.A., Flipphy, M.J.A., De Graaff, L.H., van den Broek, H.C., Kester, H.C.M., Hinnen, A. and Visser, J. (1992). Mol. Gen. Genet. 234, 113-120. 7) Goosen, T., Bloemheuvel, G., Gysler, C., de Bie, D.A., van den Broek, H.W.J and Swart, K. (1987) Curr. Genet. 11,499-503. 8) Pontecorvo, G. Roper, J.A., Hemmons, L.J., MacDonald, K.D. and Bufton, A.W.J. (1953) Adv. Genet 5, 141-238. 9) Stephens, B.G., Felkel, H.J.Jr. and Spinelli, W.M. (1974) Anal. Chem. 692-696. 10) Thoma, J.A., Rao, G.V.K., Brothers, C. and Spradlin, J. (1971) J. Biol. Chem. 246, 5621-5635. 11) Hiromi, K., Nitta, Y., Numata, C. and Ono, S. (1973) Bioch. Biophys Acta 302, 362375. 12) Robyt, J.F. and French, D. (1967) Arch. Biochem. Biophys. 122, 8-16. 13) Robyt, J.F. and French, D. (1970) Arch. Biochem. Biophys. 138, 662-670. 14) Pasculli, R., Geraeds, C., Voragen, F and Pilnik, W. (1991) Lebensm. Wiss. u. Technol. 24, 63-70. 15) Schrtiter, K.-H., Arkema, A., Kester, H.C.M., Visser, J. and Dijkstra, B.W. (1994) J. Mol. Biol. 243,351-352.


231

New enzymes active towards pectic structures G. Beldman, M. Mutter, M.J.F. Searle-van Leeuwen, L.A.M. van den Broek, H.A. Schols and A.G.J. Voragen.

Wageningen Agricultural University, Department of Food Science, Bomenweg 2, 6703 HD Wageningen, The Netherlands.

Abstract

Microbial pectinases have been used for fruit and vegetable processing for already more than half a decade. With respect to application as well as to fundamental research, most attention has been paid to those enzymes acting towards the 'smooth' homogalacturonan part of the pectin molecule (i.e. polygalacturonase, pectin lyase, pectate lyase, pectin methyl esterase). More recently, enzymes active towards the 'hairy' rhamnogalacturonan part of pectin gained attention, since it was found that in juice processing those structures foul the ultrafiltration membranes used in a final clarification step. Two different rhamnogalacturonases (RGases A and B) were identified and purified from an A~pergillus aculeatus preparation, using apple pectic hairy regions (MHR) as substrate. Based on the structure of the products, RGase A was identified as a hydrolase, splitting the ot-GalAp-(l-2)-o~-Rhap linkage in rhamnogalacturonan, while RGase B appeared to be a lyase splitting the ot.-Rhapct-(1-4)-GalAp linkage by 13elimination. Rhamnogalacturonan oligosaccharides were used to identify and purify two other novel enzymes with high specificity towards rhamnogalacturonan fragments: a rhamnogalacturonan rhamnohydrolase and a rhamnogalacturonan galacturonohydrolase. As an accessory enzyme for the RGases, rhamnogalacturonan acetyl esterase (RGAE) was discovered in the same A. aculeatus preparation. This enzyme appeared to be specific for the de-acetylation of MHR and essential for the degradation of MHR by RGases A and B. The same enzyme (RGAE) could be purified from A. niger, together with two other esterases: a feruloyl esterase (FAE) and an acetyl esterase (PALE) specific for the removal of one type of acetyl group present in the 'smooth' regions of sugar-beet pectin. Finally, the A. aculeatus preparation was found to contain an enzyme releasing the dimer 13Xylp-(1-3)-GalAp from a soluble soy cell wall polysaccharide. The enzyme was partially purified and appeared to be active towards saponified MHR and gum tragacanth as well. It was concluded that this enzyme degraded the xylogalacturonan part in MHR by an exo-fashion.

232 1. INTRODUCTION Enzymatic liquefaction is a relatively new process for the production of juices from fruits and vegetables [ 1]. Essentially the process is as follows: the material is crushed to obtain a pulp which is treated with a combination of pectinases and cellulases. After a certain incubation time, the material becomes a liquid and the juice can be recovered by decantation. Subsequently, a clear juice is obtained by ultrafiltration. A serious problem in this process is the fouling of the ultrafiltration membrane, causing a reduced flux rate. For apple processing, the material responsible for this effect has been isolated and extensively characterized [2-4]. It appeared to consist mainly of ramified pectic 'hairy' regions (MHR), which were not degraded by the pectolytic enzymes present in the technical pectinase preparation.

2. RHAMNOGALACTURONAN DEGRADING ENZYMES FOR FRUIT AND VEGETABLE PROCESSING MHR was used as a substrate to identify and purify novel enzymes able to degrade this structure. A first endo-acting enzyme was found in an A3pergillus aculeatus preparation (Pectinex Ultra SP-L, Novo Nordisk Ferment, Dittingen, Switzerland), purified and identified as a rhamnogalacturonase acting on the rhamnogalacturonan part of MHR [5]. Subsequently this enzyme has been recognised by several other investigators [6, 7]. A second enzyme from the same A. aculeatus strain, which was able to degrade MHR was identified more recently [referred to in ref. 8]. Both enzymes have been cloned in A. oryzae and named RGaseA and RGaseB, respectively [8]. The crude A. aculeatus preparation was able to degrade MHR as such, however the purified RGases were only active towards MHR, after a chemical saponification of the substrate (MHR-S), specifically after removal of the acetyl esters. From this observation it was concluded that also a rhamnogalacturonan acetylesterase (RGAE) should exist in the original preparation. Indeed such enzyme could be purified from it [9].

2.1 MODE OF ACTION OF RGASES A AND B FROM A S P E R G I L L U S A C U L E A T U S High-performance anion-exchange chromatography (HPAEC) of the reaction products from MHR-S showed that RGaseA and B acted differently towards this substrate (Fig. 1). RGaseB produced a series of oligosaccharides with longer retention times than those obtained with RGaseA. The latter products were already isolated and identified by Schols et al. [ 10]. Using the same isolation procedure, we also purified the degradation products from MHR-S made with RGaseB, and identified their structures by NMR spectroscopy. Fig. 2 shows the primary structures of both types of products. The oligosaccharides made by RGaseA are known to consist of a backbone of alternating rhamnose and galacturonic acid residues, with

233 rhamnose at the non-reducing side and a galacturonic acid at the reducing side. The rhamnose residues are either branched or not branched with a galactose residue.

oo

rt

RGase A .

0

.

.

.

.

.

.

.

.

l , l

10

20

30

40

Retention time (min)

Fig. 1. High-performance anion-exchange chromatography of the reaction products from MHR-S, produced by RGasesA and B.

The products obtained with RGaseB contained essentially the same building blocks, but in this case the rhamnose residue was located at the reducing side and a unsaturated galacturonic acid residue was found at the non-reducing side [ 11 ]. Backbone lengths of 4 to 10 units were identified. The conclusion from this work is that RGaseA is a hydrolase, cleaving the backbone of the rhamnogalacturonan part in MHR between a galacturonic acid and rhamnose unit, while RGaseB is a lyase cleaving the rhamnogalacturonan between a rhamnose and a galacturonic acid residue. Lyase activity of RGaseB could be confirmed by measuring the increase of A235of the reaction mixture (Table 1). Using this method, it could be shown that RGaseB was very

234

RGase

A

RGase

( t i t (- ") )

B

| (

n=l -4

)

ct-Rhae-(l-4)-~

H

ct-GalAe-(l-2)-(x-Rha e ot-us-GalAe-(1-2)-ct-Rhae

B-Gale-( 1-4)-ot-Rhae

Fig. 2. Oligomeric reaction products from MHR-S, produced by RGases A and B. specific for the degradation of rhamnogalacturonan structures, either present in MHR-S or as linear oligosaccharides with a DP>18, which were isolated from a beet pulp hydrolysate [ 12]. No activity was found towards highly methylated pectin or polygalacturonic acid, neither at pH 6 or pH 8, both in the presence or absence of Ca z+. So it could be concluded that RGaseB was different from the already known pectin and pectate lyases.

Table 1. Lyase activity ofRGase B toward various substrates (U mg ~ ), determined from the increase in A235 using an e of 4800 M 1 cm ] Substrate MHR-S Linear RG oligomers pectin DM 92.3% PGA n.d., not determined" a 50 mM NaOAc pH 6 b, 20 mM Tris.HCl pH 8; c, 1 mM CaCI2;

pH 6 a

pH 8b

- Ca

+ Ca c

- Ca

+ Ca c

8.8 3.9 0 0

9.8 n.d. 0 0

10.6 n.d. 0 0

11.8 n.d. 0 0

235

Blank RGAE RGaseA Comb. rr #,',

'

........

....

/~

....

-.

j:

~,.~,.~"

-..

/

"'~, .

....... I

~.~':

2O

..~"

. . . . -.-- . . . .

.-~"" ~.

"\

1

25

"~..--1..

30

Time

....j.I

35

(m~n)

Fig. 3. High performance size exclusion chromatography of MHR after degradation with RGAE from A. aculeatus, RGaseA and a combination of these enzymes. The importance of the aforementioned rhamnogalacturonan acetylesterase for the degradation of MHR by RGaseA is presented in Fig. 3. From the high performance size exclusion chromatogram of MHR it can be seen that essentially no degradation occurs when RGaseA or RGAE are used alone. Only the combination of RGaseA and RGAE is able to degrade MHR extensively. With respect to substrate specificity, a high preference of RGAE for acetyl esters in a rhamnogalacturonan was observed (Table 2). No activity was found towards other acetylated polysaccharides such as beet pectin or acetylated xylan, nor towards smaller substrates such as triacetin and acetylsalicylic acid. From MHR about 70% of the acetyl groups could be removed after prolonged incubation. Table 2. Acetyl release by RGAE from different substrates" i

Substrate

Activity (mU/mg)

MHR

Acetyl content (w/w) 4.4

921

Release (% of total) 42 (70) b

Beet pectin Xylan Triacetin Acet~,l salicylic acid

3.0 13.3 81.0 30.0

0 0 0 0

0 0 0 0

' 20 h, 30 ~

pH 5.0.

b after prolonged incubation

236

-

B-Galactosidase

(B-Gal-ase)

-

Rhamnogalacturonan rhamnohydrolase (RG-Rha-ase)

-

Rhamnogalacturonan galacturonohydrolase (RG-GalA-ase)

(Van de Via et al., 1 9 9 4 )

(Mutter et al., 1994)

(Mutter et al., in preparation)

, B-Gal-ase

"

-

..

,.T_.]. " "

-

RG-GalA-ase

/

/ 0

< RG-Rha-ase

Fig. 4. Degradation of rhamnogalacturonooligosaccharides by several exo-acting enzymes. For symbols see Fig. 2. 2.2 RHAMNOGALACTURONOOLIGOSACCHARIDES AS SUBSTRATES TO IDENTIFY NOVEL EXO-ACTING ENZYMES IN ASPERGILLUS ACULEATUS Rhamnogalacturonooligosaccharides were also used to identify and purify new enzymes which are able to degrade these structures by an exo-fashion. All essential enzymes for their degradation have been found (Fig. 4). A rather non-specific 13-galactosidase was purified from an A. niger preparation by Van de Vis et al. [ 13]. This enzyme could split off the galactose side chain from the branched oligosaccharides. The resulting linear oligosaccharide was used to identify and purify a new rhamnogalacturoan rhamnohydrolase (RG-Rha-ase) from A. aculeatus, which cleaves off the rhamnose unit located at the non-reducing end of an oligosaccharide [ 14]. The resulting new oligosaccharide was again used to purify another new enzyme from the same source: the rhamnogalacturonan galacturonohydrolase (RG-GalA-ase), which removes the galacturonic acid unit from the non-reducing end of this oligosaccharide [Mutter et al., in preparation]. Although we do not know their role in enzymatic fruit processing, in which complex mixtures of technical pectinases are being used, these enzymes have been found to be very useful for the identification of hydrolysis reaction products from rhamnogalacturonan, as presented elsewhere in this proceedings [Mutter et al.]. Next to the RG-rhamnohydrolase, an other rhamnose releasing enzyme, active towards p-nitrophenyl-c~-rhamnoside has been purified from

237 for the presence of RGAE. This time we focused on the identification of acetyl esterases in general, using, next to MHR, also other acetylated substrates. In the rhamnogalacturonan part of MHR the acetyl groups are probably linked to the 0-2 and/or 0-3 of the galacturonosyl residue [4] (acetylated pectic 'hairy' regions). As a substrate of which the acetyl groups are located at the 'smooth' homogalacturonan regions, a beet pectin (a gift fromGrindsted, Denmark) was taken. Table 4 shows the sugar composition as well as the degrees of acetylation and methylation of these polysaccharides. From the high galacturonic acid content and the relatively low amount of rhamnose and arabinose, it can indeed be concluded that this beet pectin consists mainly of'smooth' regions with a high degree of esterification. Table 4. Sugar composition (mol%) and degree of acetylation (DA) and methylation (DM) of the pectic substrates. Rha Ara Xyl Man Gal Gluc GalA DA DM

Beet pectin 2 8 tr. tr. 5 tr. 83.8 34 6O

MHR 6 55 8 9 1 21 60 42

A crude A. niger preparation (Rapidase C80, Gist brocades, Seclin, France), was fractionated by column chromatography, using Bio Gel P10, DEAE-Bio Gel A, Cross-linked alginate, Bio Gel HTP, Bio Gel P 100 and Mono-Q (Searle-van Leeuwen et al, in preparation; see also elsewhere in these proceedings). On the DEAE-Bio Gel column three acetyl esterase peaks were detected, based on the activity towards MHR (Fig. 5). The activity of these fractions, named AEI, II and III, was determined toward several acetylated substrates (Table 5). AEI showed a relatively high activity towards beet pectin, acetylated xylan and triacetin, and a relatively low activity towards MHR. Compared with AEI, AEII was very active towards MHR, and had a much lower activity towards the other substrates. For AEIII no clear substrate preference could be observed. Since it was only a minor fraction, no further purification of AEIII was attempted. Making use of the chromatographic methods mentioned above, two different acetyl esterases could be purified from AEI. These esterases were named PAE and FAE. A third esterase was purified from AEII and this esterase was named RGAE, since it appeared to be similar to the

238 the same source. This enzyme was named pnp-rhamnohydrolase [ 14]. Again high specificity of either enzyme for one type of substrate was observed (Table 3). Next to activity towards pnpct-rhamnoside, the pnp-rhamnohydrolase was also active towards naringin and hesperidin, but was not able to act on rhamnogalacturonan-like substrates in which the rhamnose unit is ct-1,41inked to an ct-GalA, such as in RG oligomers, RG polymers, MHR or MHR-S, in galactosylated or degalactosylated form. RG-rhamnohydrolase on the other hand was only active towards RG-like structures and showed no activity when other rhamnosides, as mentioned in Table 3, were used as substrates.

Table 3. Activity of pnp-rhamnohydrolase and RG-rhamnohydrolase towards various Rha-containing substrates. Substrate

Type of linkage involving Rha

pnp-Rha Naringin Hesperidin ot-Solanin a-Chaconin

ct- 1 c~-1,2 to B-Gluc a-l,6 to B-Gluc c~-1,2 to B-Gal ct- 1,2 and c~- 1,4 to B-Gluc

RG-oligo degal. RG oligo RG-hexamer degal. RG hexamer MHR HR-Saponified degal. MHR-S RG-poly degal. RG-poly

c~-1,4 to ct- alA

pnprhamnohydrolase (U/m~) 2.3 2.2 2.2 0 0

RGrhamnohydrolase (U/m~;) 0 0 0 0 0

0 0 0 0 0 0 0 0 0

3.2 32.9 6.1 52.6 2.5 12.9 57.6 3.0 24.3

3. THE ACETYLESTERASES FROM ASPERGILLUS NIGER From the previous paragraphs it is clear that the crude A. aculeatus enzyme preparation contains a whole set of enzymes able to degrade rhamnogalacturonans, including the acetyl esters present in this substrate as isolated from apple (MHR). Rhamnogalacturonan acetyl esterase (RGAE) is an important accessory enzyme for the RGases A and B ofA. aculeatus. A. niger, an other important production organism for pectolytic enzymes, was also investigated

239

Activity " Protein (E280) 1.50

NaCI

MHR

(M)

--e--

Acetylesterase 1.50

AEII

II

AEI

~j

towards

.....

m I

!

1.00

|

1

"~

I: i. I

I

~1 I,

P. il I! i \

0.50

_..=

:1 I; :1 I"

E i\

:1

I~

,

AEIII

:

--

n /I

..-

l_l

.-"

200

250

:

J

l -1 o.so

1

o.oo

"?

0.00 0

50

100

150

Fraction

300

number

Fig. 5. DEAE-chromatography of a desalted crude enzyme preparation from A. niger.

Table 5. Acetyl release (nmol/ml) from acetylated substrates by acetyl esterases at several stages of purification. Conditions protein concentration 2 lag/ml in 20 mM piperazine buffer pH 6.0, 20 h incubation. Solid and dotted lines indicate from which fraction the esterase has been purified. Substrate

Beet pectin

MHR

Acetyl xylan

Triacetin

Substrate concentration (%)

0.4

1.0

0.2

0.4

AEI AEII ................ AEIII PAE FAE RGAE .................. Total acetyl

398 146 195 443 0 6 1452

97 1800 100 267 0 1820 2598

489 169 238 177 590 0 1894

447 136 136 33 1963 0 55000

240 RGAE from A. aculeatus, based on molecular weight and high specificity for MHR (see elsewhere in these proceedings). The FAE was active towards acetyl xylan (with a low molecular weight) and triacetin. In an additional experiment it could be shown that FAE also released ferulic acid from an endoxylanase hydrolysate of sorghum cell walls. It was therefore named feruloyl acetyl esterase (FAE). The PAE was the only one of the three purified esterases from A. niger with activity towards beet pectin. In addition, some activity towards MHR and acetylated xylan was observed. From beet pectin only about 30% of the acetyl groups could be removed by PAE, while just like for the RGAE from A. aculeatus, about 70% of the acetyl esters in MHR were hydrolysed by A. niger RGAE. Fig. 6 shows a schematic structure of the rhamnogalacturonan part of MHR, in which the acetyl groups are located at the 0-3 as well as at the 0-2 and 0-3 positions of the galacturonic acid residue. This could be concluded from some preliminary NMR studies in which several structurally different acetyl esters could be assigned. After treatment with RGAE the overall pattern of the NMR spectrum remained unchanged, indicating that RGAE does not preferentially attack an acetyl ester at a specific position. This would include that all ester groups are equally degradable by RGAE. In beet pectin we are dealing with a homogalacturonan in which the acetyl groups are probably located at the 0-2 and/or the 0-3 positions (Fig. 6). From NMR spectra, essentially two different resonances from acetyl groups could be distinguished. Upon incubation with PAE one of these signals disappeared. It was concluded that only one type of acetyl groups could be removed by this esterase, probably either at the 0-2 or the 0-3 position and/or either present as a single branch point of a galacturonic acid residue or at a double branched galacturonic acid residue. Until now it is not clear how the acetyl esters are distributed over the different OH-groups of the galacturonic acid residues and which of the acetyl groups is being released by PAE. This is still under investigation. However, these results explain at least partially why PAE can only release 30% of the total acetyl groups in beet pectin, probably due to its high selectivity. This in contrary to the unselective action of RGAE towards MHR, resulting in a much higher degree of deacetylation (70%) upon extensive treatment with this enzyme.

241

RGAE: random removal of A c - g r o u p s

in MHR

( PAE: specific removal

of either 0 - 2 or 0 - 3 in beet pectin

B_Gale_(l_4)_ct_Rhae

linked A c - g r o u p s

~--.l

o~-GalAe-(1-2)-ot-Rhar

H

r

e

Fig. 6. Points of attack of RGAE on acetyl esters in H R (upper part) and possible points of attack of PAE on acetyl esters in beet pectin (see text for explanation).

4. ENZYMATIC DEGRADATION OF A SOLUBLE 'HAIRY' PECTIC POLYSACCHARIDE FROM SOY An area of application of pectinases, other than in fruits and vegetable processing, is the use in the production of pure protein from dehulled and defatted soy meal [ 15]. A soluble polysaccharide from soy (SPS) tends to bind to the soy proteins, complicating the purification of this protein. SPS was prepared from defatted soy meal by a protease treatment, after which the insoluble part was treated with a pectinase. The obtained product was again separated in a soluble and insoluble fraction, from which the soluble fraction was ultrafiltered. The obtained high molecular weight material was designated as SPS. In Table 6 the sugar composition of SPS is presented. The material contained a considerable amount of galacturonic acid, xylose, fucose, galactose and a small amount of rhamnose. From a methylation analysis after reduction, it was concluded that at least a considerable part of the galacturonic acid residues was branched, and that about 30% of the xylose residues were terminal. Also a considerable portion of the rhamnose and fucose residues appeared to be terminal. SPS could be degraded by the crude A. aculeatus preparation Pectinex Ultra-SP, giving a product consisting mostly of the monomeric sugars xylose, galactose, fucose, arabinose and

242 galacturonic acid. Next to these products also an unknown peak was observed upon HPAEC analysis (Fig. 7). This unknown product was isolated by preparative HPAEC and identified by NMR spectroscopy. It turned out to be a dimer of xylose 13-(1-3)-linked to galacturonic acid (Fig. 8). Tabel 6. Sugar composition and some structural aspects of SPS Mol %

Structural information (methylation analysis)

Ara Rha Xyl Gal

3 4 24 8

100% terminal 40% terminal 30% terminal Gal/GalA 40% 1,3,41inked

Man Fuc GalA

1

DM" DA b

11 49

72% terminal Gal/GalA 40% 1,3,41inked

21 7

a degree of methylation b degree of acetylation

The enzyme responsible for the production of this dimer from SPS was partially purified by a series of chromatographic steps, using the following columns: DEAE-Sepharose, cross-linked alginate, Q-Sepharose, Phenyl-Sepharose, Bio Gel HTP and Mono-Q. Two fractions were characterized further: the fraction after the HTP column (HTP2) and a purer fraction, obtained after the Mono-Q column (Q2). The HTP2 fraction produced no other products than galacturonic acid and the dimer 13-Xylp-(1,3)-GalAp (Fig. 7). Several electophoretic methods (SDS-PAGE, native PAGE and IEF) showed that fraction Q2 was almost pure, some faint contaminating protein bands were found (Fig. 9). The major band upon SDS-PAGE was at 42 kDa. IEF indicated an isoelectric point at pH 4.3 for the most prominent band. It was observed that the dimer 13-Xylp-(l,3)-GalA t, together with monomeric galacturonic acid, was not only released from SPS by the action of this enzyme, but also from MHR and gum tragacanth. The activity increased after alkaline saponification of the substrates. It is known that one of the subunits of apple H R is a xylogalacturonan, consisting of a galacturonan backbone, of which a part of the galacturonic acid residues is substituted at the 0-3 position with a 13-xylose [16]. The formation of the dimer 13-Xylp-(1,3)-GalAp confirms the presence, as well as the structure of this subunit in MHR.

243

IA

Dimer

(D

0

5

10

15 20 Time (min.)

25

30

Fig. 7. High-performance anion-exchange chromatography of the reaction products from alkali saponified SPS, produced by the crude A. aculeatus preparation (Ultra-SP) and the partially purified fraction HTP-2.

Ho~O~o

J---o o

H

Is'

I~-Xylp-(1-3)-GalAp(cdl~) Fig. 8. The structure of the unknown dimer, released from SPS by the crude A. aculeatus preparation or fractions thereof.

244 Based on HPSEC, it was observed that we are dealing with an enzyme that degrades xylogalacturonans in an exo-manner, releasing the dimer and galacturonic acid. Also beet pectin and polygalacturonic acid were degraded by this enzyme, giving galacturonic acid as the only product from the start of the reaction. It can be concluded that the enzyme is an exogalacturonase, which is not hindered by side-chains of xylose.

SDS-PAGE

kDa

Native

31 20.1 14

IEF

pH

kDa 699

-

440

-

8.157.35

-

6,85

-

140 -

5.85

-

5,20

-

67-

4.55

94

67 43

PAGE

232 -

4.55 --

..~:~:......

3.50 -

Fig. 9. Electrophoresis of the dimer releasing enzyme fraction Q2.

5. CONCLUSIONS As a very simplified model, we can consider the pectin molecule to be build up of a 'smooth' homogalacturonan part, next to a 'hairy' region containing subunits of a rhamnogalacturonan and, in the case of for instance apple, a xylogalacturonan [4, 16]. Much is already known about the wide range of enzymes able to degrade the homogalacturonan part of the pectin molecule. Here we show that in analogy to the degradation of the smooth homogalacturonan regions, a whole array of enzymes is present in A.spergillus preparations, specific for the degradation of hairy regions of pectin.

6. ACKNOWLEDGEMENTS The authors wish to thank Dr. Ian Colquhoun (AFRC, Norwich, England) and Dr. Joke Venekamp (TNO, Zeist, The Netherlands) for their NMR spectroscopic analysis, leading to the

245 primary structures of the oligosaccharides derived from MHR-S by the RGases and of the dimer 13-Xylp-(1,3)-GalAp, respectively. We thank Dr. Dick Schipper (Gist brocades, Delft, The Netherlands) for NMR spectroscopic analysis of the RGAE treated acetylated pectic substrates. Ir Jean-Paul Vincken (WAU, The Netherlands) is acknowledged for useful discussions.

7. R E F E R E N C E S

1. W. Pilnik and A.G.J. Voragen In: J.J.Jen (Ed.): Quality factors of fruits and vegetables. Chemistry and Technology. ACS American Chemical Society Symposium series 405 (1989) ' 250. 2. H.A. Schols, M.A. Posthumus and A.G.J. Voragen, Carbohydr Res 206 (1990) 117. 3. H.A. Schols, E. Vierhuis, E.J. Bakx and A.G.J. Voragen, Carbohydr Res 275 (1995) 343. 4. H.A.Schols, Structural characterization of pectic hairy regions isolated from apple cell walls, Thesis Wageningen Agricultural University (1995). 5. H.A. Schols, C.C.J.M. Geraeds, M.F. Searle -van Leeuwen, F.J.M. Kormelink and A.G.J. Voragen, Carbohydr Res 206 (1990) 105. 6. J. An, L. Zhang, M.A. O'Neill and P. Albersheim, Carbohydr Res 264 (1994) 83. 7. M. Sakamoto, Y. Shirane, I. Naribayashi, K. Kimura, N. Morishita, T. Sakamoto and T. sakai, Eur. J. Biochem 226 (1994) 285. 8. L.V. Kofod, S. Kauppinen, S. Christgau, L.N. Andersen, H.P Heldt-Hansen, K D6rreich and H. Dalboge, J Biol Chem 268 (1994) 29182. 9. M.J.F. Searle-van Leeuwen, L.A.M. Van den Broek, H.A. Schols, G. Beldman and A.G.J Voragen, Appl Microbiol Biotechnol 38 (1992) 347. 10 H.A. Schols, A.G.J. Voragen and I.J. Colquhoun, Carbohydr Res 256 (1994) 97. 11. M. Mutter, I.J. Colquhoun, H.A. Schols, G. Beldman, and A.G.J. Voragen, Plant Physiol 110 (1996) 73. 12. C.M.G.C. Renard, M.Mutter, H.A. Schols, A.G.J.Voragen and J-F. Thibault, Int. J Biol Macromol (in press). 13. J.W. Van de Vis, Characterization and mode of action of enzymes degrading galactan structures of arabinogalactans, Thesis Wageningen Agricultural University (1994). 14. M. Mutter, G. Beldman, H.A. Schols and A.G.J. Voragen, Plant Physiol 106 (1994) 241. 15. H. Gurtler, H.A.S. Olsen, M Schulein, J.L. Adler-Nissen, G.W. Jensen and S. Rijsgaard, UK Patent Application GB 2115820. 16. H.A. Schols, E.J. Bakx, D. Schipper and A.G.J. Voragen, Carbohydr Res 279 (1996) 265.



247

The flSubunit of Tomato Fruit Polygalacturonase Isoenzyme 1 Defines a New Class of Plant Cell Proteins Involved in Pectin Metabolism: AroGPs (Aromatic Amino Acid Rich Glyco Proteins) Dean DellaPenna a, Colin Watson, JiPing Liu and David Schuchman Department of Plant Sciences, University of Arizona, Tucson, Arizona, USA, 85721 aAs of March 1, 1996: Department of Biochemistry/200, University of Nevada, Reno, Nevada, USA, 89557-0014

Abstract Understanding the biochemical and molecular mechanisms governing the synthesis, regulation, structure and function of plant-encoded cell wall modifying enzymes, particularly those involved in pectin metabolism, has been a long term goal of research in my laboratory. The ripening tomato fruit has proven to be a highly tractable system for this work and several laboratories have focused on studying a single, ripening-induced pectin degrading enzyme, tomato fruit polygalacturonase or PG. This chapter first reviews the field of tomato fruit PG and then focuses on our recent work with the 13subunit of tomato PG isoenzyme 1. Two PG isozymes can be isolated from ripe fruit, PG2 and PG1, the latter of which is formed by the association of a catalytic PG2 polypeptide with an ancillary glycoprotein, the l~subunit. Multiple lines of evidence suggesting the 13subunit plays an important role in regulating polyuronide degradation have prompted us to clone the 13subunit and study its structure, expression and function. 13subunitmRNA accumulates to high levels in developing fruit and is to a large degree temporally separated from PG2 expression. The 13subunit is encoded as a large precursor protein whose mature domain is composed almost entirely of the novel repeating motif FTNYGxxGNGGxxx in which the phenylalanine residues are post-translationally modified. Results from Bsubunit antisense experiments have conclusively demonstrated that the protein plays a major role in restricting PG2 catalytic activity in vivo during fruit ripening. We have isolated the complete tomato 13subunit gene family, related genes from Arabidopsis and identified homologous sequences in a number of dicots and monocots, suggesting that Bsubunit structure and function may be evolutionarily conserved in plants. We propose the tomato 13 subunit as the archetypal member of a new class of plant cell wall proteins, AroGPs (for Aromatic Amino Acid Rich GlycoProteins). AroGPs are defined by: 1) their overall protein sequence homology and common precursor structure, 2) a conserved 14 amino acid repeating motif in the mature AroGP protein and 3) a high percentage of aromatic amino acids in the mature AroGP protein. Based on the available data, AroGPs appear to be specifically expressed in PG-producing tissues, where they presumably interact with cell wall components and/or catalytic enzymes to regulate cell wall enzymatic activities in vivo.

1. B A C K G R O U N D 1.1. Pectin and Cell Wall Structure Plant cell walls provide the obvious functions of structural support and integrity and can vary tremendously in size, shape, composition and structure depending on cell type, age and function within the plant body. Despite this diversity, plant cell walls are composed of only three major classes of polysaccharides: cellulose, hemicellulose and pectins. Pectins, or polyuronides, are imbedded throughout the cell wall matrix and are particularly abundant in the middle lamella region. Pectins generally account for 10-30% of the cell wall dry weight and

248 collectively constitute a complex group of polysaccharides whose primary component is dgalacturonic acid with a number of other sugars being present as minor constituents. Polyuronides, especially those of the primary plant cell wall, have been the focus of extensive structural analyses in recent years which has provided a detailed characterization of the linkages and composition of "smooth and hairy" pectin blocks, both of which can be present in a single pectin polymer. Such studies have greatly increased our understanding of structural aspects of pectins and their role in cell wall architecture (reviewed in 4).

1.2. Pectin modifications during growth and development Although polyuronides are clearly important structural components of the cell wall they are also a dynamic class of molecules that are constantly being modified during the plant life cycle and are intimately involved in many aspects of plant growth and development. The principal plant enzymes mediating polyuronide modifications are pectin methylesterases, which remove methyl ester groups from the polymer, and polygalacturonases (PGases), which cleave the polymer chain in either an endo or exo fashion. During the past several years efforts by many laboratories in studying plant mediated polyuronide degradation have focused on the structure/regulation/function of a single catalytic PGase activity produced in large amounts during tomato fruit ripening. This system has been targeted due to the large tissue mass, high levels of developmentally regulated PGase activity and extensive physiological and biochemical background. These studies have greatly increased our understanding of the molecular regulation of pectin modification during fruit ripening and led to molecular genetic experiments to test the function of PGase in fruit ripening (reviewed in 8 and 10). However, developmentally-regulated, tissue specific polyuronide modifications by endogenous plant enzymes occur throughout the plant body and the analysis of PGase activity and function in non-fruit tissues is now being critically addressed. In recent years a substantial number of reports have described developmentally regulated, tissue specific PGase activities or cDNAs (many of which show significant homology to tomato fruit PG) in a variety of non-fruit tissues including leaf abscission zones, root cap border cells, newly initiated lateral roots and developing pollen and growing pollen tubes (1, 3, 11, 13, 21, 23). Particularly notable is the Sambucus nigra abscission zone in which two isozymes were identified and characterized that are analogous in their size, structure and sequential appearance to PG isozymes in tomato fruit (see below). These combined reports clearly demonstrate that developmentally regulated, tissue specific, pectin modifying activities/genes are expressed in a variety of cell and tissue types where extensive modification of cell wall structure or loss of cell wall adhesion occurs and suggest a broad role for pectin degrading enzymes in plant growth and development. Furthermore, the fact many non-fruit PG cDNAs show significant homology to tomato fruit PGase suggests that non-fruit PGase activities may be regulated in a fashion similar to tomato fruit PGase. Ongoing work with tomato fruit PGase may therefore provide insight into the regulation/function of PGase activities in other plant tissues.

1.3. PG and pectin degradation during tomato fruit ripening During the ripening of climacteric fruits such as tomato, an increase in the level of chelator-soluble polyuronides, and a corresponding decrease in their molecular size is well correlated with a dramatic increase in extractable endo-PGase activity. The catalytic PG protein responsible for these changes is probably the best characterized pectin degrading enzyme in plants and a variety of molecular and biochemical approaches have conclusively demonstrated that it is indeed the primary enzymic activity responsible for cell wall polyuronide degradation in ripening tomato fruit (10, 22). The catalytic PG polypeptide is encoded by a single gene (the PG gene) whose expression and function have also been extensively studied in recent years (reviewed in 11). The PG gene is transcriptionally activated at the onset of ripening and both PG mRNA and protein accumulate to high levels during the ripening process (7, 8, 11). Although there is only a single gene for the catalytic PG polypeptide, the total PG activity

249

A

B

1

2

1

3 STD ..

~..

PG2~

kD

~. ,,.~

~

-66

~-45

13-Subr ~

9. . . . . . o :

Figure 1. SDS-PAGE and Immunoblot Analysis of Purified PG1 and Separated PG2 and ~Subunit Proteins. (A) Proteins were resolved by SDSPAGE and visualized by Coomassie blue staining. Lane 1, 4 ~tg purified PG1; Lane 2, 2 ~tg purified PG2; Lane 3, 2 ~tg purified 13subunit.

'~,-31 (B) Proteins were resolved by SDSPAGE, blotted and PG2 polypeptides detected by reaction with anti-PG2 antibodies. Lane 1, 2 lxg purified PG1; Lane 2, 1 lxg purified PG2; Lane 3, 1 lxg purified fisubunit. isolated from ripe tomato fruit is attributable to a mixture of several closely related, posttranslationally derived isoenzymes, PG1, PG2A and PG2B (2, 6). The PG2A and PG2B isoenzymes (herein referred to as the PG2 isoenzyme) accumulate late in the ripening process and are each composed of a single catalytic PG polypeptide differing only in degree of glycosylation (2, 6). The PG 1 isoenzyme accumulates first during ripening and is thought to be composed of one or two catalytic PG2 polypeptides tightly associated with an ancillary glycoprotein, the 13subunit protein (13). The level of PG1 produced during ripening is apparently determined by the level of 13subunit protein present in the fruit tissue (8, 10, 17).

1.4. The [~subunit modifies the activity of the catalytic PG protein The [~subunit has been purified from PG 1 by ourselves and others and is a heat stable, acidic, heavily glycosylated protein with an apparent molecular mass of 37-39 kD (19, 26). No enzymatic activity has been identified for the protein. The 13subunit can be extracted from the cell walls of both green and ripe tomato fruit by high salt buffers (13, 14, 18, 19, 20), and in the latter case is associated with PG2 polypeptide(s) in the form of PG 1. Purified 15subunit can also associate with and convert PG2 in vitro into an isoenzyme that closely resembles PG 1 ( 13, 14, 24). Biochemical studies have shown that in vivo and in vitro formation of PG1 by the association of PG2 with the 13-subunit alters the biochemical and enzymic properties of the associated catalytic PG2 polypeptide including its pH optima, response to cations and thermal stability (summarized in Table 1). This later property provides a convenient assay for the levels of PG 1 and PG2 in total cell wall protein extracts.

Table 1: Selected biochemical properties of PG2 and PGI Isozyme

Mol. wt. *50% * 100% pH NaC1 kD inactive inactive optima pI Optima PG 1 --100 79~ 90~ 3.6 7.0 300 mM PG2 45/46 57oC 63oC 4.4 8.0 200 mM 9Heat inactivations are 5 min at the given temperature. Data are from Knegt et al. 1988.

........

250

Figure 2: EDTA soluble polyuronides, PG1/PG2 levels and size fractionation profiles of polyuronides isolated from ripening wild-type fruit. 0, 7 and 20 days after harvest are mature green, mid-ripe and over-ripe stages, respectively.

80 ~

60

~ ~40 2O

Upper panel: EDTA soluble polyuronides were extracted from identically treated tissues and measured. Note the increase in polyuronide solubility during ripening.

0

PG1-

Middle panel: Cell wall proteins were isolated, 10 ggm of each resolved by non-denaturing polyacrylamide gel electrophoresis and PG1 and PG2 isoforms detected by activity staining.

PG20

~

8

~4

_~ ,

3 7 11 20 Days After Harvest

Wild-typePolyuronideSizeProfile ~/,,0Days ~ Dyi7s 2~Days

g2

20

40 60 FractionNumber

80

Lower panel: One mg of chelatorsoluble polyuronides from the indicated stages was size-fractionated on a Sepharose CL4B column, and the uronic acid content of column fractions determined. 0, 7 and 20 day old fruit contain no PG, PG1 only and PG1 and PG2, respectively. Note the similarity of 7 and 20 day profiles despite the large amount of PG2 activity in the latter.

1.5. Several lines of data suggests that the Bsubunit regulates PG activity Several independent lines of physiological, biochemical, and molecular evidence have been put forth to support the hypothesis that PG1 is the active isoenzyme in vivo and have implicated the gsubunit as playing an important role in regulating pectin metabolism. These include the observation that only PG 1 can be extracted when maximal pectin solubilization and depolymerization are observed in both wild-type fruit and tin fruit expressing an inducible PG2 transgene (7). Subsequent accumulation of high levels of extractable PG2 activity in both tissues is not accompanied by further pectin solubilization or depolymerization (9). Furthermore, the in vivo biphasic loss of PG activity during heat treatment of intact fruit tissue mimics the in vitro heat inactivation profile of mixtures of PG1 and PG2 isoenzymes, suggesting that the PG1 complex exists in vivo (17). Finally, analysis of transgenic tomato plants constitutively expressing an antisense PG2 transgene has shown that the residual 1% PG enzyme activity extracted from ripe fruit is exclusively in the form of PG 1 and is sufficient for wild-type levels of pectin solubilization to occur during ripening. Based on this result, it was

251 proposed that only very low levels of PG 1 were required for normal pectin solubilization (22). These combined studies suggest that the presence of extractable PG 1 activity is correlated with pectin solubilization and in some cases with pectin depolymerization during tomato fruit ripening and therefore implicate the Bsubunit protein as an important factor in regulating or restricting the catalytic PG2 protein in vivo (7, 9, 13, 14). However, the data supporting this thesis is largely correlative in nature and definitive proof is lacking regarding the in vivo existence of PG 1, the contribution of the various isoenzymes to ripening-associated polyuronide degradation, and the physiological consequences of PG-dependent polyuronide degradation. 1.6. Conclusions and Directions from Prior Work on Tomato Fruit PG Despite the brevity of the above discussion, it is clear from previous studies that the large increase in PG activity during ripening is the primary enzymatic activity responsible for polyuronide degradation during tomato fruit ripening. Results in transgenic systems have suggested that a single isozyme, PG1, may be the physiologically active PG isoform in vivo. As PG1 is a complex of PG2 and the [~subunit, these data implicate the [~subunit protein as playing an important function in regulating pectin degrading enzyme(s) and hence in the process of pectin solubilization and depolymerization. A clearer molecular and biochemical understanding of the fruit ~subunit, its role in determining PG isoform levels, and the role of related proteins in other non-fruit tissues that contain PGase activities was an obvious and necessary next step in furthering our knowledge of PG function and polyuronide modifications during fruit ripening. To extend our understanding of the assembly, activity, and physiological function of the individual PG isoforms during tomato fruit ripening, and directly address the role of the Bsubunit in pectin metabolism we purified the [~subunit from PG 1 and isolated eDNA clones that encode the protein (26). The remainder of this chapter reviews our progress to date in studying the structure, expression and function of the 13subunit (25, 26, 27). Finally, we describe additional members of the tomato 13subunit gene family, related genes in other plant species and propose that the 13subunit defines a new class of cell wall proteins which we call AroGPs for Aromatic Amino Acid Rich G__lycoProteins_. 2. ISOLATION AND C H A R A C T E R I Z A T I O N OF A BSUBUNIT cDNA CLONE 2.1. Purification of the [3subunit Protein of Tomato Fruit PG1 When analyzed by SDS-PAGE, purified PG 1 was found to contain several polypeptides ranging in size from 37 to 45 kD, as shown in Figure 1A, lane 1. The 44 and 45 kD polypeptides reacted strongly with antiserum raised against purified PG2 (Figure 1B, lanes 1 and 2), and their size and immunoreactivity are consistent with their being the catalytic PG2 polypeptides. Using cation exchange chromatography in urea containing buffers, proteins in the purified PG1 sample could be further separated into two species: the PG2 polypeptides and the ~subunit polypeptide(s) (Figure 1A, lanes 2 and 3, respectively). The [~subunit protein does not react with anti-PG2 antibodies (Figure 1B, lane 3), indicating that it is immunologically distinct from the catalytic PG2 polypeptides. Protein sequencing of purified [3subunit and of several proteolytically derived ~subunit peptide fragments yielded the sequences shown in Table 2. Blank cycles were consistently obtained at specific cycles for each peptide which, in retrospect, invariably correspond to phenylalanine residues in the eDNA sequence for the protein (refer to Table 2 and bold Phe residues in Figure 3). Moreover, amino acid composition analysis of purified ~subunit protein agrees with that deduced from the ~subunit eDNA with the exception of a severe underestimate of the phenylalanine content of the mature protein (3 residues versus 23 from eDNA sequence). These data indicate that most of the phenylalanine residues in the mature ~subunit protein are posttranslationally modified in an as yet undetermined fashion.

252

Table 2. Summary of Protein Sequence Data from the Amino Terminus and Internal Proteolytic Fragments of Purified Tomato Fruit ~-Subunit. Peptide Name

Peptide Sequence

Amino terminus

NH 2- Glu-Lys-His-Ser-Gly-Asp-Ile-His-[ .9 ]-Ala-Thr-Tyr

Lys-C

NH 2- Asn-Gly-Asn-Gly-Ala-Asn-Gly-Gln-[ ? ]-Val

Glu-C- 1

NH 2- Ala-Asn-Ala-Gly-Asp-Gln-Tyr-[ ? ]

Glu-C-2

NH 2- Asn-His-[ ? ]

Arg-C

NH 2- Gly-Ser-Pro-Arg-Asp-Asn-Lys-[ ? ]-Asp-Asn-Tyr-Ala

Underlined sequences indicate amino acid sequences used for the generation of degenerate primers. Bracketed question marks represent blank cycles from the Edman degradation reaction. Additional sequence was obtained after blank cycles in all cases except the Glu-C-1 and Glu-C-2 peptides.

2.2 Cloning of a Fruit BSubunit cDNA and Analysis its Primary Sequence Purification and characterization of the [~subunit permitted the subsequent isolation of eDNA clones encoding the protein. Using oligonucleotides derived from the data in Table 2, numerous [~subunit eDNA clones were isolated and characterized from a green fruit eDNA library, pBsub2.2, the longest eDNA clone encodes a 69-kD protein containing 630 amino acids, more than twice that required to encode the mature gsubunit protein. Included in this open reading frame are all of the protein sequences shown in Table 2, confirming that the eDNA encodes the ~subunit protein. A hydropathy profile of the encoded 69-kD precursor protein is shown in Figure 3. The ~subunit precursor contains at least four distinct protein domains: a 30 a.a. amino-terminal signal sequence, a 78 a.a. amino-terminal propeptide, the mature protein domain, and a large (25 kD) carboxyl-terminal propeptide domain. The presence of a signal sequence is consistent with the protein being targeted to the endomembrane system, as would be expected for a cell wall protein. The predicted 31.5 kD unglycosylated mature domain has a calculated pI of 4.9 and contains all six identified N-linked glycosylation consensus sequences (Figure 3, branched structures), consistent with the reported heavy glycosylation of the mature ~subunit protein. The most striking structural feature of the primary sequence of the mature ~subunit protein domain is the presence of a novel repeating 14-amino acid motif with the general consensus: FTxYGxxxN(x)4_6, where the Phe and Tyr residues are invariable and Thr, Gly and Asn occur with a greater than 50% frequency at positions 2, 5, and 9, respectively. Within the core consensus region there is a strong bias for specific amino acids or amino acid group at certain "x" positions in the motif, such as Ser, Thr, or Asn at position 3, Gly at positions 8, 10, and 11, and charged or uncharged polar amino acids at positions 6, 7, 12, 13, and 14. Inclusion of these residues yields a core consensus of FTNYGxxGNGGxxx where "x" is most often a charged or uncharged polar amino acid. This motif accounts for almost the entire mature protein domain (Figure 3, boxed region) and is not found in other domains of the precursor protein. The five posttranslationally-modified phenylalanine residues discussed earlier are all located in position 1 of this motif (bold phenylalanine residues in Figure 3).

253

5-

~o' I~ "- "31 r

u., l / ,,,"~'~

"5 - i f ' / " ' / * -'~ 100

~,~t,r YY

....

' "l

~,a.latai

I

' ' 1'

Y Y YY Matu.re~-subunitprotein ~ 200 300 400 A m i n o Acid N u m b e r

i ,L~~,1 ,,,,

q,r * ~r","~T,'I ..........

,I

{ ~ } ] 500

600

Mature [$-Subunit Protein Domain Sequence .M2-EKH S ~ D

IH GVNT VNS NK DQS SGK NLH VQK DQY NGE GST DQK ENH SET DDT EAN TDV HIN

L M-cooH Core Consensus

FTNYGxxGNGGxxx

LAS

Figure 3. Hydropathy Profile and mature protein primary sequence of the deduced 13Subunit Precursor Protein. 9Upper panel: The hydropathy profile of the entire 69 kD precursor protein is shown. The abscissa is amino acid residues and the ordinate, positive values indicate hydrophilic. The black and hatched rectangles at the bottom of the figure denote the calculated signal sequence and amino-terminal propeptide domains, respectively. The mature and carboxyl-terminal domains are labeled. N-linked core glycosylation consensus sites are depicted by branched structures. Lower panel: Optimal alignment and consensus sequence of the mature [~-subunit protein repeating motif. The entire contiguous sequence of the mature domain (amino acids 109-397) is shown from top to bottom and left to right. Periods represent gaps in the alignment. Shadowed residues indicate amino acids that occur with a high frequency at a given position. Bold phenylalanine (F) residues are those which yielded blank cycles in protein sequence analysis. The boxed area defines the region of the protein from which the core consensus, FTNYGxxGNGGxxx was derived. The bold amino acids of the core consensus occur with a 50% or greater frequency.

2.3. [3Subunit and PG Expression Are Separated during Fruit Development In addition to structural studies, we have analyzed the expression patterns of ~subunit and PG mRNA during wild-type tomato fruit development (Figure 4). ~subunit and PG differ dramatically in their temporal regulation during wild-type fruit development. [~subunit mRNA is detectable as early as 10 DAP and increases gradually during development to its highest level at 30 DAP, just prior to ripening. During the following 5-day period, ripening is initiated and [~subunit mRNA decreases below detectable limits, while PG mRNA increases to its highest level. A more detailed analysis of the ripening period, using ethylene production as a marker, indicated that ~subunit and PG expression do overlap slightly early in ripening, however, PG protein is not detected until [~subunit mRNA levels decrease (27, results not shown). One likely explanation for the near complete temporal separation of 13subunit and PG expression is that ~subunit expression occurs early in fruit development to allow transport, attachment and localization of the 13subunit protein to specific regions of the cell wall in the absence of PG2.

254

~o~| 9

13-Sub-

...

PG-

.

~.

o

.

!i iI

(2.3 kb)

(1.9 kb)

,,o~~ Fruit(DAP) 0 15 20 25 30 35 40 ..

.

mW "

~

"

,

m

~.~.: :o:.7

~~:~

9

:~i~i ~(~

:9 9149 ~99149 i9~9149149

I:)21 -

(1.0 kb)

Figure 4. RNA Blot Analysis of [~Subunit, PG, and D21 Expression during Fruit Development and Ripening. Total RNA (25 ~tg) isolated from the indicated tomato tissues was probed with either a 13subunit eDNA clone, a cDNA for the catalytic PG polypeptide, or a eDNA for the constitutively expressed mRNA D21. Identical specific activities were used in each hybridization and all blots were exposed for 8 hr.

2.4. Analysis of BSubunit Function Using Transgenic Systems Despite extensive prior studies of the structure, regulation, and function of the catalytic PG protein during tomato fruit ripening, it is still unclear if PG 1 isoenzyme formation occurs in vivo or whether PG1 and/or PG2 alone is sufficient for pectin solubilization and dcpolymerization in vivo. To test the hypothesis that the B subunit is involved in pectin depolymcrization or solubilization in vivo and that the formation of PG1 is required for PG activity in vivo, several independently transformed tomato 13subunit antisense lines with substantially reduced levels of B subunit protein in mature green fruit were generated. Total cell wall proteins isolated from several independently transformed lines were assayed to determine their immunologically detectable levels of Bsubunit and PG proteins as well as the extractable PG 1 isoenzyme activity. Those lines with less than 1% of their total PG activity as PG 1 in ripe fruit were analyzed in greater detail with respect to pectin solubilization and depolymerization during fruit ripening. The antisense line TA8, in which 20kb that is not contiguous with group 1 clones and contains two closely related gsubunit genes in tandem (tomato genes 2 and 3 in Figure 11). The coding regions of all three genes have been sequenced and this data is summarized in Figure 11. All three tomato genes encode precursor proteins of approximately the same size and contain a single intron of variable size at the same location. Tomato Genes 2 and 3 show 91% and 84% amino acid identity, respectively, at the protein level to the fruit gsubunit protein (tomato gene 1) with the carboxyl domains of each gene showing higher identity than the mature protein domains. The mature domains of both tomato gene 2 and 3 exhibit the 14 amino acid repeating motif shown in figure 4 for the fruit gsubunit with minor variations being restricted largely to "x" residues of the repeating motif consensus FTNYGxxGNGGxxx. In contrast, the consensus glycosylation sites in genes 2 and 3 are more numerous and not conserved in their placement relative to those in gene 1. Preliminary RNAse protection analysis indicates that Gene 2 is expressed at high levels in floral tissue and in fruit tissue at levels near that of Gene 1. Gene 1 is expressed at high levels in fruit tissue, as expected, and at much low levels in floral tissues. Expression of Gene 3 has not been observed in either tissue and it may represent a pseudo gene or an inducible gsubunit gene that responds to stress, pathogen ingress or other stimuli. When the Arabidopsis Expressed Sequence Tag (EST) Database was searched with the tomato fruit gsubunit protein sequence two related cDNAs were identified (Figure 11). eDNA 2 is near full length and has been completely sequenced, eDNA 1 has also been sequenced but currently lacks approximately 100 amino acids of coding region. The two Arabidopsis cDNAs are 81% identical at the protein level and have lower identity to the protein encoded by tomato gene 1, 64 and 63% for eDNA 1 and eDNA 2, respectively. However, both cDNAs encode

260 proteins that contain the repeating 14 amino acid core mature protein motif, FTxYGxxxN(x)4_6, with F, Y and N being 100% conserved. The other residues in the repeating motif show more variation than the tomato proteins, as reflected in the overall identity of the Arabidopsis cDNA 1 and cDNA 2 proteins tomato gene 1 protein. As with tomato genes 2 and 3, the carboxyl domain of both Arabidopsis proteins shows greater identity to tomato gene 1 than does their mature protein domains. However, it is important to stress that the size, precursor protein structure, amino acid identity and conserved, repeating motif structure contained in the two Arabidopsis cDNAs make it clear that they are homologs of the tomato 13subunit protein. Finally, genomic southern analysis using the fruit 13subunit cDNA as a probe has identified homologous sequences in a wide variety of monocots and dicots suggesting that the 13subunit is an evolutionarily conserved cell wall protein found in most, if not all higher plants. % homology with AroGP1

Tomato Gene I (AroGP1)

100%

Tomato Gene 2 (AroGP2)

91%

Tomato Gene 3 (AroGP3)

84%

Arabidopsis cDNA1

64%

Arabidopsis cDNA2

63%

Structural Domains/Homology

YYY

BBrfff/I

YYY

Mature protein oomain 100J~ 200 300

Y YYYY

~ 400

Y Y

"~ "A

Y 9

"

I

Y

o

Y

600

o

Y Y

li~!!~~~,

78%/94%

Y

,

YY

~ ! 500

80% / 92%

Y YYYY Y Y

~///A

~

YY

Y ,

52% / 76%

Y

YY

"

9

9

Y

53% 177%

Figure II: Diagrammatic representation of the tomato Bsubunit gene family members and related cDNAs in Arabidopsis thaliana. The four domains of the respective precursor proteins are coded as in Figure 4. The large triangles represent introns. "Y"s represent the position of glycosylation consensus sequences. Tomato Gene 1 is the fruit 13subunit cDNA. Percentages underneath each mature and carboxyl domain indicate the respective identity to the mature and carboxyl domains of Tomato Gene 1. 3.3. Conclusions from Structural Studies We propose that the tomato fruit 13 subunit is the archetypal member of a new class of plant cell wall proteins, which we have named AroGPs, for Aromatic Amino Acid Rich GlycoProteins. AroGPs have the following characteristics: 1) AroGPs are encoded as a large four domain precursor protein that is more than twice the size of the mature AroGP protein. The precursor contains a large carboxyl terminal extension. 2) The mature AroGP protein domain is composed almost entirely of a repeating 14 amino acid protein motif of the sequence FTxYGxxxN(x)4_6 where F and Y are 100% conserved. 3) The mature domain contains a very high percentage of the aromatic amino acids phenylalanine and tyrosine (> 10% on a molar basis).

261 With regard to structural considerations, AroGPs somewhat resemble plant structural cell wall proteins such as hydroxyproline rich glycoproteins, glycine rich proteins, and proline rich proteins which also contain repetitive amino acid motifs and in some cases posttranslationally modified amino acids such as hydroxyproline and isodityrosine. However, AroGPs differ from these classes of proteins in several respects. First, although AroGPs do contain a repeating amino acid motif, the minimum repeating unit is much longer and shows much greater variability than those found in HRGPs, PRPs and GRPs. Second AroGPs are expressed at high levels in specific tissues (fruit or flowers) at precise developmental times. Other structural proteins have tissue and cell type specific expression of individual gene family members but as a group are generally expressed throughout the plant. Finally, and most importantly, unlike structural proteins such as PRPs and GRPs, AroGPs can bind tightly to and either directly or indirectly affect the activity of another protein with enzymatic activity (for the fruit AroGP this protein is PG). Based on the available data we propose AroGPs represent a new class of plant cell wall proteins specifically expressed in various PG-containing tissues where they interact with cell wall components and catalytic enzymes to regulate cell wall enzymatic activities in vivo. Some questions we are addressing in current and future research include: where and when are the individual tomato gene family members expressed, how similar are the proteins encoded in tomato and other species, what is the function of AroGPs in nonfruit tissues, what is the nature and structural consequence of the modified phenylalanine residues in the protein, and finally, do AroGPs interact with and regulate other catalytic proteins besides PGases? Such studies will further address our basic questions of cell wall enzyme structure, function and regulation in higher plants. 5. REFERENCES

1. 2. 3. 4. 5. 6. 7.

Alan R.L. and Lonsdale D.M. (1992) Plant Mol Biol 20:343-345. Ali, Z.M., and Brady, C.J. (1982). Aust. J. Plant Physiol. 9, 155-169. Brown S.M. and Crouch M.L. (1990) Plant Cell 2:263-274 Carpita N. and Gibeaut D.M. (1993) Plant Journal 3:1-30 DellaPenna, D., and Bennett, A.B. (1988) Plant Physiol. 86, 1057-1063. DellaPenna, D., Kates, D.S., and Bennett, A.B. (1987) 85:502-507. DellaPenna, D., Lashbrook, C.C., Toenjes, K., Giovannoni, J.J., Fischer, R.L., and Bennett, A.B. (1990). Plant Physiol. 94, 1882-1886. 8. Fischer RL and Bennett ABB (1991) Annu. Rev. Plant Physiol. Plant Mol. Biol. 42:675603. 9. Giovannoni, J.J., DellaPenna, D., Bennett, A.B., and Fischer, R.L. (1989) Plant Cell 1, 53-63 10. Giovannoni, J.J., DellaPenna, D., Bennett, A.B., and Fischer, R.L. (1990) Annu. Rev. HortSci. 108, 405-409. 11. Hawes, M.C. and Lin H.J. (1990) Plant Physiology 94:1855-1859. 12. Kalaitzis, P., Koehler, S.M. and Tucker, M.L. (1995) Plant Molecular Biology 28:647656. 13. Knegt E., Vermeer E. and Bruinsma J. (1988) Physiol Plant 72:108-114. 14. Knegt, E., Vermeer, E., Pak, C., and Bruinsma, J. (1991). Physiol. Plant. 82, 237-242. 15. Osteryoung, K.W., Toenjes, K., Hall, B., Winkler, V., and Bennett, A.B. (1990) Plant Cell 2, 1239-1248. 16. Peretto R., Favaron F., Bettini V., De Lorenzo G., Marini S., Alghisi P., Cervone F. and Bonfante P. (1992) Planta 188:164-172. 17. Pogson, B.J., and Brady, C.J. (1993) Postharvest Biol. Technol. 3, 17-26. 18. Pogson, B.J., Brady, C.J., and Orr, G.R. (1991) Aust. J. Plant Physiol. 18, 65-79. 19. Pressey, R. (1984a) Eur. J. Biochem. 144:217-221. 20. Pressey, R. (1984b) Hortscience 19:572.

262 21. Pressey, R. (1988). Reevaluation of the changes in polygalacturonases in tomatoes during ripening. Planta 174, 39-43. 22. Smith, C.J.S., Watson, C.F., Morris, P.C., Bird, C.R., Seymour, G.B., Gray, J.E., Arnold, C., Tucker, G.A., Schuch, W., Harding, S., and Grierson, D. (1990). Plant Mol. Biol. 14, 369-379. 23. Taylor, J.E., Webb, S.T.J., Coupe, S.A., Tucker, G.A. and Roberts, J.A. (1993) J of Expt. Botany 44:92-98. 24. Tucker, G.A., Robertson, N.G. and Grierson, D. (1980) Eur. J. Biochem. 112:119. 25. Watson, C.J., Zheng L.S. and DellaPenna D. (1994) The Plant Cell. 6:1623-1634. 26. Zheng L.S., Heupel R. and DellaPenna D. (1992) Plant Cell 4:1147-1156. 27. Zheng, L.S., Watson C. F. and DellaPenna D. (1994) Plant Physiology. 105:1189-1195.


263

Characterisation of RG degradation products of new RGases using RG-rhamnohydrolase and RG-galacturonohydrolase' M. Mutter', C.M.G.C. Renard b, G. Beldman', H.A. Schols', A.G.J. Voragen" "Wageningen Agricultural University, Department of Food Chemistry, Bomenweg 2, 6703 HD Wageningen, The Netherlands, fax +31 317 484893 t'Laboratoire de Biochimie et Technologic des Glucides, Centre de Recherche Agroalimentaire, Institut National de la Recherche Agronomique, B.P. 1627, 44316 Nantes c6dex 03, France lFinancial support was from Novo Nordisk A/S (Copenhagen, Denmark)

Abstract Linear rhamnogalacturonan (RG) fragments (RGO's) isolated from an acid hydrolysate of saponified sugar beet pulp were treated with the enzymes RG-hydrolase and RG-lyase. Major tools in the characterisation of the formed degradation products were the exo-acting enzymes RG-rhamnohydrolase and RG-galacturonohydrolase. These exo-enzymes were used to prepare a series of standards of RG oligomers and furthermore to confirm structure assignments, made using high-performance anion-exchange chromatography (HPAEC). The RG-hydrolase was active toward RGO's when the degree of polymerisation (DP) was 12 or higher, while the RG-lyase was only active when the DP was 14 or higher. The alternating RG sequences have to be at least 16 to 18 units long to produce similar oligomers as RG-hydrolase and RG-lyase liberate from apple modified hairy regions (MHR). 1. I N T R O D U C T I O N Schols et al. (1990a) were the first to describe the enzyme rhamnogalacturonase (RGase), able to degrade the hairy (ramified) regions of pectin. Several papers from other workers have been published dealing with RGase activity (Matsuhashi et al., 1992; D0sterh6tt et al., 1993; An et al., 1994 and Sakamoto et al., 1994). Since that time, a set of various enzymes, all with high specificity toward hairy regions of pectin and 'no activity toward homogalacturonan regions, has been found in the authors' laboratory. Searle-van Leeuwen et al. (1992) described an RG-acetylesterase; an RG-rhamnohydrolase was found by Mutter et al. (1994); and recently a newly found RGase, previously called RGase B, turned out to be an RG

264 tx-L-rhamnopyranosyl-(1---~4)-ot-D-galactopyranosyluronide lyase, abbreviated RG-lyase (Mutter et al., 1996). RGase as described by Schols et al. (1990a) is now termed an RG ot-Dgalactopyranosyluronide-(1---~2)-ct-L-rhamnopyranosyl hydrolase, abbreviated RG-hydrolase. A fitth enzyme in this series is an RG-galacturonohydrolase (Mutter et al., in preparation) specific for the removal of the terminal nonreducing GalA unit of RG chains. This enzyme contains no activity toward homogalacturonan structures at all. The discovery of these enzymes enables a better structural characterisation of the hairy (ramified) regions of pectin, as already demonstrated by Schols et al. (1990b) and also of native plant cell wall pectin (Schols et al., 1995). In this study we show how the two exo-enzymes of the above described series, the RG-rhamnohydrolase and the RG-galacturonohydrolase, can be used as tools in the characterisation of unknown RG fragments. These unknown fragments were the products of RG-hydrolase or RG-lyase action toward linear RG oligomers (RGO's), which were produced by acid hydrolysis of sugar beet pulp. 2. M A T E R I A L S

AND METHODS

RG oligomers Sugar beet pulp was saponified and then hydrolysed with 0.1 N HCI at 80 ~ for 72 h. Linear RG oligomers of DP 6 to 18, abbreviated RGO 6 to 18, were isolated by ion-exchange chromatography and size-exclusion chromatography by Renard et al. (1995). Saponified apple MHR was treated with RG-hydrolase, and the branched RG-hydrolase MHR oligomers were isolated using size-exclusion chromatography as described in Mutter et al. (1994). A similar procedure was carried out with RG-lyase, and the branched RG-lyase MHR oligomers were isolated as described in Mutter et al. (1996).

Enzymes RG-hydrolase and RG-lyase from Aspergillus aculeatus were purified using the method of Schols et al. (1990a) and of Kofod et al. (1994) respectively. A 13-galactosidase from Aspergillus niger was purified essentially according to Van de Vis (1994). Purification of an RG-rhamnohydrolase from Aspergillus aculeatus of the type described by Mutter et al. (1994) involved dialysis, DEAE Sepharose fast flow at pH 4.25, SP Sepharose fast flow at pH 4.25, Q Sepharose high performance at pH 6.0, and finally Chelating Sepharose high performance loaded with Cu 2+ ions. In the last purification step, the RG-rhamnohydrolase was separated from an RG-galacturonohydrolase. These purification steps and the characterisation of the RGgalacturonohydrolase will be discussed in detail elsewhere.

265

Incubations with enzymes All substrates (varying between 0.018 and 0.05% w/v) were incubated in 50 mM sodium acetate buffer pH 5.0, containing 0.01% w/v sodium azide, at 40 ~ for 24 h. RGO's were treated with 2.6 lag RG-galacturonohydrolase per mg substrate. When RGO's were sequentially treated with the exo-enzymes to form smaller oligomers, the RGgalacturonohydrolase and the RG-rhamnohydrolase were used in amounts between 2.4 and 2.8 lag and between 9 and 18 lag per mg substrate respectively. RGO's were incubated with 0.18 lag RG-hydrolase and with 0.42 lag RG-lyase per mg substrate. Subsequent incubation of the RG-hydrolase/RG-lyase digest with the exo-enzymes was carried out with 6 lag of RGgalacturonohydrolase and with 16 lag RG-rhamnohydrolase per mg substrate. Removal of Gal from the RG-hydrolase MHR oligomers was performed as described in Mutter et al. (1994). From the RG-lyase MHR oligomers the Gal was removed using 9.3 lag 13galactosidase per mg substrate. Derhamnosylation of the degalactosylated RG-hydrolase MHR oligomers was carried out as described in Mutter et al. (1994).

Analysis using HPAEC HPAEC was carried out using a Dionex Bio-LC system equipped with a Dionex CarboPac PA-100 (4 x 250 mm) column and a Dionex pulsed electrochemical detector in the pulsed amperometric detection (PAD) mode. A gradient of sodium acetate in 100 mM sodium hydroxide (1 ml/min) was used as follows: 0 to 50 min, 0 to 450 mM; 50 to 55 min, 450 to 1000 mM; 55.1 to 70 min, 0 mM. 3. R E S U L T S A N D D I S C U S S I O N

Preparation of a series of standards of RG oligomers Several types of RG oligomers, obtained by acid hydrolysis or enzymically, were prepared in order to have a series of standards available for the characterisation of the reaction products from linear RG oligomers by the action of RG-hydrolase and RG-lyase. These linear RG oligomers are of the type: ot-D-GalpA-(1---)2)-(ot-L-Rhap-(1-->4)-ot-D-GalpA-(1-->2))n-L-Rhap with n = 2 to 8, being oligomers with an even number of sugar residues (DP 6 to 18), abbreviated RGO's 6 to 18. They were purified from sugar beet pulp and characterised (Renard et al., 1995). The RGO's were treated with an RG-galacturonosidase from Aspergillus aculeatus, a new enzyme specific for RG fragments. This RG-galacturonohydrolase has a molecular mass of 66 kD and an isoelectric point of 5.1. The enzyme is able to remove the terminal nonreducing GalA from RG fragments and not from homogalacturonan fragments

266 (Mutter et al., in preparation). This enzyme was used to remove the nonreducing GalA from these RGO's to obtain oligomers of the type: (a-L-Rhap-(1-~4)-a-D-GalpA-(1--~2)).-L-Rhap with n = 2 to 8, defined as degalacturonosylated RGO's with an uneven number of sugar residues (DP 5 to 17). RGO 6 was sequentially treated with the RG-galacturonosidase and the RG-rhamnohydrolase (Mutter et al., 1994) to form oligomers ofDP 5 and 4. Branched RG oligomers of the type: a-D-us GalpA-(1 -->2)-(a-L-Rhap-(1 ---M)- a-D-GalpA-( 1--~2)).-L-Rhap

1"

1"

13-D-C~p-(1--M)

B-D-C~p-(1--)4)

with n = 1 to 4 (us = 4,5-unsaturated), were purified from saponified apple MHR by treatment with RG-lyase (Mutter et al., 1996 and unpublished results). These RG-lyase MHR oligomers were linearized using a 13-galactosidase from Aspergillus niger (Van de Vis, 1994). These linear RG-lyase MHR oligomers were of the type: a-D-us C~pA-(1---~2)-(a-L-Rhap-(1-+4)-a-D-GalpA-(1-~2))n -L-Rhap with n = 1 to 4, giving oligomers with a DP of 4 to 10. Oligomers with a DP of 4 to 8 were used as standards. A mixture of branched RG oligomers of the type: a-L-Rhap-(1--->4)-(a-D-GalpA-(1--~,2)-a-L-Rhap-(1---~4))n-D-GalpA

1' (I3-D- Galp-( 1--94))q

1' (I3-D- Galp-( 1--~4))~

with n = 1 to 3 and q/r = 0 or 1, was obtained from saponified apple MHR by treatment with RG-hydrolase (Schols et al., 1990a). These RG-hydrolase MHR oligomers were linearized by treatment with a 13-galactosidase from Aspergillus niger. These linear RG-hydrolase M]-IR oligomers were of the type: (a-L-Rhap- ( 1-~4)-a-D- GalpA-( 1---~2))n-a-L-Rhap- ( 1--~4)-D- GalpA with n = 1 to 3 (Schols et al., 1994; Mutter et al., 1994). These oligomers were derhamnosylated with the RG-rhamnohydrolase (Mutter et al., 1994) to form oligomers of the type:

267

((x-D-GalpA-(1-~2)-tx-L-Rhap-(1-->4)), -D-GalpA with n = 1 to 3, giving final DP's of 3, 5 and 7. The order of elution of the 24 different standard RG oligomers is shown in table I. The Rha residues are indicated by E! and the GalA residues by O, whereas the us-GalA units, which are introduced by RG-lyase action, are indicated by | The symbols are explained below the table, and will be used throughout the paper, although the shades of the symbols are different in the figures.

Table I Eluaon behaviour of different types of RG oligomers on HPAEC Structures in ascending order of elution on HPAEC DP of RGO or degalacturonosylated RGO n O-O-O-O-El DP 5 n-o O-I"l-O-El DP 4 n-o-n-o-n-o-ci DP 7 I"1-O-i"1-O

Ret. time (min) 3.0 12.2 12.5 13.3 19.8 20.8

O-I-I-O-I-I-O-l"!

21.2

DP 6

O-13-O 22.5 I-I-O-I-I-O-I"1-O-I"1-O-I"! DP 9 25.2 1"1-O-1"1-O-!"1-O 26.3 O-I-I-O-I"l-O-I"l-O-I"! DP 8 27.1 o-r'l-O-I"l-O 28.4 l-'i-O-I-I-O.r'l-O-I-'l-O-I--i-O-l-'l DP l 1 29.3 O-I"l-O-I"l-O-I-l- O-I"l-O-r'! DP 10 31.0 O-El-O-El 31.0 O-O-O-I"1-O-O-O 32.6 O-o-n-o-n-o-El-O-El-O-El-O-El DP 13 32.8 O-El-O-El-O-El-o-n-o-El-O-El DP 12 34.4 O-I"l-O-n-o-o 35.4 El-o-n-o-n-o-n-o-o-o-o-o-o-O-l-I DP 15 35 6 o-n-o-n-o-I"l-O-O-o-n-o-El-O-El DP 14 36 8 El-o-n-o-n-o-o-o-n-o-r'l-O-CI-O-CI-O-Cl DP 17 37 6 | O-l"l- O-I"i 38.4 O-El-o-n-o-n-o-El-O-O-O-O-O-Cl-O-El I DP 16 39.1 O-El-o-n-o-El-o-r'l-O-El-O-El-O-I"l-O-o-o-rl i DP 18 40.8 O, (z-D-CralpA (1-->2) linked to Rha, or D-GalpA at the reducing end; El, (z-L-Rhap (1-->4) linked to GalpA, or L-Rhap at the reducing end; | c~-D-us GalpA (1-->2) linked to Rhap. L

268 Since all oligomers in Table I have an alternating RG sequence, the differences in structure are determined by the sugar units at the reducing and nonreducing end. From Table I it can be seen that the retention time of the oligomers increases with DP within a series of similar structure, e.g. a Rha at the reducing end and a GalA at the nonreducing end. Differences in the end sugars influence the retention times drastically. When the RGO's are compared with the degalacturonosylated RGO's it can be seen that an additional Rha at the nonreducing end reduces retention time by a minute (e.g. DP 5 compared with DP 4, up to DP 17 compared with DP 16), even though the DP is higher. Structure I"1-O-I"!-O compared with !"1-O-I"1-O17 (DP 5), and structure I"1-O-I"1-O-i"1-O compared with I"!-O-!"1-O-I"1-O-!"! (DP 7), shows that an additional Rha at the reducing end has an even larger effect: the retention times are 6 to 8 minutes shorter. On the other hand, the removal of a GalA from the reducing end of O-17O-!"1-O (28.4 min) reduces the retention time much more (to O-I"!-O-I"! at 13.3 min) compared with removal from the nonreducing end (to I-1-O-i"l-O at 20.8 min). In conclusion, the reducing sugar seems to be of more influence on the retention time than the nonreducing sugar. For different types of oligosaccharides both the nonreducing and the reducing end have been reported to have the largest influence on HPAEC retention (Lee, 1990). The us-GalA at the nonreducing end causes strong retention on the CarboPac column: the largest RGO that elutes earlier than an oligomer with a nonreducing us-GalA is six units larger.

Degradation of RGO's by RG-hydrolase RGO's 6 to 18 were incubated with RG-hydrolase, under conditions estimated to be sufficient to reach an end-point situation. RG-hydrolase was not active toward RGO 6 to 10, although minor amounts of reaction products were released from RGO 10. RG-hydrolase was very active toward RGO 12 and higher. From RGO 12 two major products were formed (Fig. 1). When compared with the available standards, the earliest eluting peak corresponded with I-l_O_l-'l-O_r-I-O-I"l. The later eluting product had the same retention time as O-I"l-O-r-l-O. Together they match the DP 12 from which they originate. The designation of the products was confirmed by subsequent incubation of the reaction mixture with either the RGrhamnohydrolase or the RG-galacturonosidase. Figure 1 shows that upon incubation of the RG-hydrolase digest with the RG-rhamnohydrolase, the peak of O-I-I-O-El-O remains, a new peak is formed while l--I_O.r-l-O_l-l-O-l"i has disappeared and Rha is released. The newly formed peak has the same retention time as RGO 6, which can be explained by removal of the nonreducing Rha from l-I_O-EI-O-r-I-O-r-! by the RG-rhamnohydrolase. In case of incubation with the RG-galacturonosidase, shown in Figure 1 as well, the peak of I-1-O-!-1-O-1"1-O-I-1 remains,, while the peak of o-r-l-o-r-l,O has disappeared and a new peak and GalA are formed. The newly formed peak has the same retention time as I-I-O-r-I-O. This can be explained b y removal of the nonreducing GalA unit from O-17-O-1"!-O by the RGgalacturonohydrolase. In this manner, using the two exo-enzymes, the original assignments were confirmed.

:.

269

Since the original oligomer contains GalA at the nonreducing and Rha at the reducing end, O - D - O - D - O should result from the nonreducing end and D - O - D - O - D - O - D should originate from the reducing end and can therefore be positioned as follows: O-D-O-D-O

D-O-D-O-D-O-D.

Rha

/

3

5

5

g P

~

3

~ v

m u

.h v

n m

. V

m

~

m

~ v

~ ~

~,

0

.m ~ . .m .y . u. .w .L v

m m

~,

m

~. v

m m

v

~

m

m

m

4

II.

..r

I

0

10

20

30

m

~

m

v

v

,.I

---r

m

m

m

y

,,

m

m

v

~,

~

m

m

v

~,

m

m

m

40

Retention time (min) Figure 1: HPAEC elution patterns of RGO 12 (bottom chromatogram); RGO 12 incubated with RGhydrolase (second chrom, from bottom); RGO 12 incubated with RG-hydrolase and subsequently with RG-rhamnohydrolase (third chrom, from bottom); and RGO 12 incubated with RG-hydrolase and subsequently with RG-galacturonohydrolase (top chrom.). Numbers in chromatograms on the left correspond with structures on the right: Explanation of symbols see table I.

The results confirm that RG-hydrolase is a true rhamnogalacturonase, as it splits an alternating RG chain, and furthermore that it cleaves between GalA and Rha in the main chain by a mechanism of hydrolysis (Schols et all, 1990a). Similarly, the products from the RGO's with higher DP's were characterised. Figure 2 shows the cleavage of the various RGO's by RG-hydrolase. Differently dotted arrows indicate different cleavage options. No bond-cleavage frequencies could be given since the response factors of the various products were not known. The enzyme cleaves the chains five units from the nonreducing end. When the oligomer is larger, also seven units from the nonreducing end are cleaved off. From RGO 16 and 18 products were released whose formation could only be explained by assuming that a secondary cleavage of the reaction products occurred. Since RG-hydrolase cleaves between GalA and Rha, all primary cleavage

270 products from the RGO's have to be of uneven DP. In case of RGO 16 and 18 also products were formed that had similar retention times as the oligomers D - O - D - O (released from RGO 16 and 18) and D - O - D - O - D - O (released from RGO 18 only). In Figure 2 the secondary cleavage is indicated by a short arrow. The cleavage sites shown as primary in case of RGO 16 and 18 were considered as such for the following reasons. The smaller RGO's were cleaved five or seven units from the nonreducing end, and therefore it seemed logical that this also was the case for RGO 16 and 18. Furthermore, the resulting products after cleaving five or seven units from the nonreducing end were large enough to be cleaved by the RG-hydrolase (in case of cleavage of RGO 16, the resulting DP 11 was shown in separate experiments to be indeed cleaved by the RG-hydrolase). The (unbranched) RG-hydrolase MHR tetramer and hexamer as known to be liberated by RG-hydrolase from apple MHR-S (Schols et al., 1994) were both formed from RGO 18.

100--

12~ E 1 4 ,W= -i- , l- , = = l

r

r162162

9 ~ ~Jt ~, ~~, ~ C, ~ l

16r162162162162

/

/

i

I

i

D

i

I

i

i

|

I

/

i

|

I

l

I

r162162

I

18

Figure 2: Locations of cleavage of RGO's by RG-hydrolase. Differently dotted arrows indicate different cleavage options. A short arrow indicates a secondary cleavage, see text. Explanation of symbols, see table 1. Numbers refer to degree of polymerisation.

Degradation o f RGO's by RG-lyase When RG-lyase was incubated with RGO's 6 to 18, activity was observed toward RGO 14 and higher. From RGO 14 two peaks were formed, eluting at approximately the same retention time (Fig. 3). This retention time corresponded with | and RGO 10,

271 together matching with RGO 14. The designation of RGO 10 was confirmed by subsequent incubation of the reaction mixture with the RG-galacturonosidase, which resulted in release of GalA and the formation of a new peak with the retention time of D-O-l"l-O-D-O-D-O-D (DP 9) (Fig. 3). No confirmation of the unsaturated product could be given, since no enzyme was available (nor known) able to remove the us-GalA from the nonreducing end of RG fragments. For all oligomers, the structures of the products were assigned in this manner.

I

Gala

4,,,,4/ ~ j 3 4

P

.

21.

I1.

-

r

r

r162

1~. r162 . . . . . . . . .

0

i

. . . . . . . . .

10

,

. . . . . . . . .

20

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30


-

r

-

r

-- 3 Q - B - ~ I I

r

r

-- 3 Cg--B-O~-IB

r

r162162

,

40

Figure 3: HPAEC elutionpatterns of RGO 14 (bottom chromatogram); RGO 14 incubated with RG-lyase (second chrom, from bottom);); and RGO 14 incubated with RG-lyase and subsequently with RG-galacturonohydrolase (top chrom.). Numbers in chromatograms on the left correspond with numbers of structures on the right. Explanation of symbols see table I.

It is clear that the unsaturated nonreducing GalA unit must result from the cleavage by RG-lyase, since the original RGO contains a saturated GalA at the nonreducing end. The structures therefore can be positioned as follows: O-D-O-D-O-D-O-D-O-D | The results confirm that RG-lyase is an RGase, able to cleave alternating RG chains, by a mechanism of 13-elimination, as already described by Mutter et al. (1996). Figure 4 shows the cleavage pattern of RG-lyase toward the various RGO's. Again no bond-cleavage frequencies could be given since the response factors of the various products were not known. The RG-lyase cleaved the chain four units from the reducing end. When the

272 oligomer is larger, also six units from the reducing end are cleaved off. This is in contrast with RG-lyase, which cleaved closer to the nonreducing terminus. Secondary cleavage products were observed only in case of RGO 18. The cleavage indicated as primary was considered as such for the following reasons. Since the smaller RGO's were cleaved four or six units from the reducing end, this seemed likely also to be the case for RGO 18. Furthermore, the resulting oligomer after a first cleavage at four units from the reducing end was RGO 14, which could be cleaved by the RG-lyase.

10

A

w

m

mm

A

~

m

A

m

~

m

mm~

A

m

mm

A

~

m

m

120--m-~ ~ ~, ~ ~r ~ ~, ~ ~' ~ 14

16

t

18

Figure 4: Locations of cleavage of RGO's by RG-lyase. Differently dotted arrows indicate different cleavage options. A short arrow indicates a secondary cleavage, see text. The thin arrow indicates a less abundant cleavage option. Explanation of symbols, see table I. Numbers refer to degree of polymerisation.

The data collected in this study can provide more insight in the structure of the RG part of native pectic hairy regions. Apparently the stretches of RG need to be at least 16 units long for RG-hydrolase in order to produce the tetramer and at least 18 units long to produce the hexamer known to be liberated from apple MHR. The stretches of RG need to be even longer for RG-lyase, since from RGO 18 only the tetramer is released, while from apple MHR oligomers with a backbone of four to ten units are released. It is also interesting to see that RG-lyase is able to degrade linear fragments, while from apple MHR only oligomers branched with single unit Gal side chains are released (Mutter et al., 1996). Together with the fact that the linear RG-hydrolase MHR hexamer is usually only released in small amounts (Schols et al.,

273 1994 and Schols et al., 1995), the results suggest that in apple MHR no strictly linear RG regions are present larger than about 16 units. 4. ACKNOWLEDGMENT The authors wish to thank Marjo Searle-van Leeuwen for purification of the 13-galactosidase from Aspergillus niger and Novo Nordisk Ferment Ltd Dittingen (Switzerland) for assistance in the purification of the RG-hydrolase and Novo Nordisk A/S Bagsvaerd (Denmark) for the girl of the crude recombinant RG-lyase. 5. REFERENCES An J, Zhang L, O'Neill MA, Albersheim P (1994) Isolation and structural characterisation of endo-rhanmogalacturonase-generated fragments of the backbone of rhanmogalacturonan I. Carbohydr Res 264:83-96 Diisterh6tt E-M, Bonte AW, Venekamp JC, Voragen AGJ (1993) The role of fungal polysaccharidases in the hydrolysis of cell wall materials from sunflower and palm-kernel meals. World J Microbiol Biotechnol 9:544-554 Kofod LV, Kauppinen S, Christgau S, Andersen LN, Heldt-Hansen HP, D6rreich K, Dalboge (1994) Cloning and characterization of two structurally and functionally divergent rhanmogalacturonases from Aspergillus aculeatus. J Biol Chem 269:29182-29189 Lee, YC (1990) High-performance anion-exchange chromatography for carbohydrate analysis. Anal Biochem 189:151-162 Matsuhashi S, Inoue S-I, Hatanaka C (1992) Simultaneous measurement of the galacturonate and neutral sugar contents of pectic substances by an enzymic-HPLC method. Biosci Biotech Biochem 56:1053-1057 Mutter M, Beldman G, Schols HA, Voragen AGJ (1994) Rhamnogalacturonan a-Lrhamnopyranohydrolase. A novel enzyme specific for the terminal nonreducing rhamnosyl unit in rhamnogalacturonan regions of pectin. Plant Physiol 106:241-250 Mutter M, Colquhoun IJ, Schols HA, Beldman G, Voragen AGJ (1996) Rhamnogalacturonase B from Aspergillus aculeatus is a rhamnogalacturonan ct-L-rhamnopyranosyl-(1---~4)-ct-Dgalactopyranosyluronide lyase. Plant Physiol 110:73-77 Renard CMGC, Thibault J-F, Mutter M, Schols HA, Voragen AGJ (1996) Some preliminary results on the action of rhamnogalacturonase on rhanmogalacturonan oligosaccharides from beet pulp. Int J Biol Macromol in press Sakamoto M, Shirane Y, Naribayashi I, Kimura K, Morishita N, Sakamoto T, Sakai T (1994) Purification and characterization of a rhamnogalacturonase with protopectinase activity from Trametes sanguinea. Eur J Biochem 226:285-291 Schols HA, Geraeds CCJM, Searle-van Leeuwen MF, Kormelink FJM, Voragen AGJ (1990a) Rhanmogalacturonase: a novel enzyme that degrades the hairy regions of pectins. Carbohydr Res 206: 105-115 Schois HA, Posthumus MA, Voragen AGJ (1990b) Structural features of hairy regions of pectins isolated from apple juice produced by the liquefaction process. Carbohydr Res 206:117-129

274 Schols HA, Vierhuis E, Bakx EJ, Voragen AGJ (1995) Different populations of pectic hairy regions occur in apple cell walls. Carbohydr Res 275:343-360 Schols HA, Voragen AGJ, Colquhoun IJ (1994) Isolation and characterization of rhanmogalacturonan-oligomers, liberated during degradation of pectic hairy regions by rhamnogalacturonase. Carbohydr Res 256:97-111 Searle-van Leeuwen MJF, Van den Brock LAM, Schols HA, Beldman G, Voragen AGJ (1992) Rhamnogalacturonan acetylesterase: a novel enzyme from Aspergillus aculeatus, specific for the deacetylation of hairy (ramified) regions of pectins. Appl Microbiol Biotechnol 38:347-349 Van de Vis JW (1994) Characterization and mode of action of enzymes degrading galactan structures of arabinogalactans. Koninklijke Bibliotheek, The Hague, The Netherlands, pp 89-108

J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V.All rights reserved.

275

The Effect of Glycosylation of Endopolygalacturonases and Polygalacturonase Inhibiting Proteins on the Production of Oligogalacturonides

C.W. Bergmannl, B. Cook1, A.G. Darvill 1, p. Albersheim 1, D. Bellincampi:, and C. Caprari 2

1 Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, The University of Georgia, Athens, Georgia 30602-4712, USA

2Dipartimento di Biologia Vegetale, Universita di Roma "La Sapienza", Piazzale Aldo Moro, 00185 Rome, Italy

Abstract Fungal endopolygalacturonases (EPC~) are considered to be major pathogenicity factors, facilitating the breakdown of the plant cell wall and releasing biologically active oligogalacturonide elicitors which are also substrates for EPG. The inhibition of EPGs by plant polygalacturonase inhibiting proteins (PGIPs) has led to the hypothesis of PGIPs as general defense factors as a consequence of their proposed role in increasing the lifetime of elicitor active oligogalacturonides. Plant PGIPs exist in a variety ofglycoforms in which the peptide backbone is conserved with differing degrees of glycosylation. The population of the glycoforms change depending on the species, cultivar, and tissue investigated. Fungal EPGs also exist in a variety of glycoforms, which resulted in us studying the possible role that the glycosylation of EPG and PGIP may play in affecting the production of biologically active oligogalacturonides via their action on pectic substrates. Using HPAEC-PAD, we studied the oligogalacturonide products formed by the interaction of a variety of glycoforms of EPGs and PGIPs on polygalacturonic acid, allowing us to determine the effect of glycosylation on the sizes or lifetimes of the oligogalacturonides produced.

Results and Discussion Endopolygalacturonases (EPGs) are important pathogenicity factors of fungi and are among the first enzymes secreted when fungi are grown on isolated cell walls as their carbon source [1]. EPGs fragment homogalacmronan and solubilizc the other cell wall pectic polysaccharides, rhamnogalacturonan I (RG-I) and rhamnogalacturonan II (RG-II), and appear to make cell walls more susceptible to the action of other endoglycanases [2,3]. Homogalacturonan fragments (i.e., a-l,4-1inked oligogalacturonides) with DPs 12-14 are oligosaccharins that, depending on the plant being studied, exhibit a wide range of signal functions [4]. EPGs m vitro rapidly degrade de-esterified homogalacturonan to mono-, di-, and

276 trigalacturonides, resulting in little opportunity for bioactive oligogalacturonides to accumulate [5,6]. All dicotyledonous plants have proteins in their cell walls that are able to inhibit fungal EPGs by forming one-on-one (stoichiometric) complexes [7-11 ]. We have demonstrated that in beans the concentration ofpolygalacturonase inhibitor proteins (PGIPs) rapidly increases in the cell walls of those cells immediately surrounding the site at which a fungus penetrates the plant, and that the epidermal cells accumulate the most PGIP [ 12]. The bean PGIPs, which we have studied extensively [ 12-15], are able in vitro to inhibit greater than 99% of the activity of a variety of fungal EPGs. The low amount of EPG activity remaining in the EPG-PGIP complex is sufficient to slowly depolymerize homogalacturonan and allow for the accumulation of bioactive oligogalacturonides [ 13,16]. Purified P. vulgaris PGIPs exist as a series of isoforms, and although the protein contains four potential N-linked glyeosylation sites, it has been speculated that these isoforms may be the result of multiple gene products [ 17]. The four PGIP isoforms of P. vulgaris have now been shown to be glycoforms, in agreement with work on tomato PGIP isoforms by Stotz et al. who estimate the presence of up to seven glycoforms [ 10]. P. vulgaris PGIPs from a variety of cultivars and tissues, including PGIP isolated from cv. cannelino cells grown in liquid culture, were compared and shown to have identical N-terminal sequences. These PGIPs also all yielded the same MW band, following chemical deglycosylation with TFMS, on SDS-PAGE silverstained gels as well as on Western blots probed with antibodies raised against a peptide from the N-terminus of P. vulgaris PGIP (Fig. 1).

1

2

3

4

5

Figure 1. Lane 1) cv "pinto" pod PGIP (native), Lane 2) deglycosylated cv "cannelino" cultured cell PGIP, Lane 3) deglycosylated cv "cannelino" pod PGIP, Lane 4) deglycosylated cv "blue lake" pod PGIP, Lane 5) deglycosylated cv "pinto" pod PGIP.

Comparative Western analysis of samples directly extracted from tissue without intermediate purification demonstrate that the relative amounts of the four P. vulgaris glycoforms vary from cultivar to cultivar, but their MWs and presence are invariant (Fig.2).

277

=~..,,,, ....

-' .~ ~( ":":'%~.;,~),~,(:~")~:~';'~...:10 for each isoenzyme similar to that of the basic Ech PLs (15).

287

3.4. Substrate specificities

Plant cell walls, particularly the middle lamellae and primary cell walls, contain numerous chemically diverse pectic substances (1). Detailed analyses of potato tuber cell walls revealed that these account for approximately 50% of the total cell wall material, with around 20% of the pectins being 43% esterified (16). The unesterified form of pectin, pectate, is considered the natural substrate for PL. Highest enzymatic activity towards this compound was measured at 50~ pH 8.0 for PL2, while PL1 and PL3 exhibited optima at 40~ pH 8.5. The KM and Vmaxvalues for the PLs were analysed using various pectic substrates. Towards PGA, the kinetic d a t a - measured at 4 5 ~ hint at a significantly higher activity of PL1 compared to PL2 and PL3 (Table 1). Differences were also observed by analysing time courses for the depolymerization of pectate following addition of PL (results not shown). PL1 displayed the highest initial velocity which, however, leveled off rapidly. While PL3 degraded less substrate in the first 80 s, the total degradation after 160 s of incubation was higher. The time course for degradation of pectate by PL2 was between those of PL1 and PL3. Additional examinations of depolymerization using various esterified pectins supported this assumption. Unexpectedly, application of identical enzyme units to either PGA or esterified pectins revealed a significantly higher efficiency in degradation of 31% esterified pectin. Despite its low Vmaxvalue towards PGA, PL3 was more active in depolymerizing pectin with 31% esterification than PL1 and PL2 (Table 1). Pectin with 68% esterification was also attacked by the three enzymes, however, less efficiently, PL3 again being more active than the other two enzymes (data not shown). No degradation of 93% esterified pectin could be detected. Table 1

Kinetics of Eca PL isoenzymes using pectate and pectin 31% esterified pectin

PGA

PL1 PL2 PL3

KM (mg ml 1)

Vmax (#mol min 1 mg -~)

KM (mg ml 1)

Vmax (#mol min 1 mg ~)

0.27 0.30 0.27

846 641 634

0.20 0.20 0.10

1228 1041 2115

Intrigued by the finding that Eca PLs exhibit notable differences in their kinetics, HPAEC analyses were carried out to examine the products from the depolymerization of PGA and 31% esterified pectin. After 18 h of incubation with PGA, PL1 and PL2 had produced mainly di- and trimers. Similarly, main products of PL3 action were trimers, followed by dimers. Moreover, it was the only enzyme found to produce monomers from unesterified substrates with a degree of polymerization ___3.Using 31% esterified pectin as a substrate, similar end products were released by the PLs as from PGA. In addition to the products described, traces of tetra- up to octamers were detectable. While PL1 and PL2 released di- and trimers at almost

288

equal amounts from both PGA and 31% esterified pectin, quantities of trimers were again most substantial after PL3 action. Further evidence for the novel finding that pectate lyases depolymerize pectin more actively than pectate was provided by examination of scanning electron micrographs (6). Separate application of the three Eca PLs to potato tuber tissue not only degraded the middle lamellae, but also effected disintegration of pectic components localized in the primary cell wall. Moreover, the isoenzymes revealed activity differences in the degradation of potato tuber pectic material depending on the plant variety used. While the action of PL3 resulted in a breakdown of the middle lamellae, incubation of tuber tissue with PL1 or PL2 gave rise to disintegration of single cells and a subsequent liberation of the cell contents. These results support the notion that Eca PLs exhibit major roles for the depolymerization of pectins of intermediate esterification in vivo, thus being specially adapted to the chemical composition of potato tuber cell walls. Possibly, various individual isoenzymes degrade pectin subdomains of diverse cell wall types. Since the sequence motif (S/A/T)XXhWVDHXXh (h representing I, L or V, and X any amino acid), suggested to be part of the catalytic site or the substrate binding in several PL or PNL enzymes (17), is highly conserved in the pectin and pectate degrading Eca PL1, PL2 and PL3 at the positions 159-169, other subtle amino acid substitutions in substrate binding or catalytic residues may explain the differences in their substrate specificities. This is supported by the hypothesis that the substrate binding site of Ech PL C is located in the structure formed by loops 1 to 6 (18). Provided that similar loops are also involved in substrate binding of the Eca PLs, differential degradation of pectins by individual isoenzymes might be delineated to amino acid differences in these domains. The degradation of pectin is initiated by the action of PME, an enzyme removing methylester groups from highly methoxylated pectins. Production of incompletely deesterified pectin, probably due to end-product inhibition of PME (19, 20), may explain that an affinity of PL for both pectate and pectin is required for full pathogenicity of the bacteria. An intriguing environmental feature of an Eca invasion in potatoes is the change from pH 5.0 in fresh tissue to pH 8.5 in the infected tissue after --72 h. We propose that the involvement of PLs in the degradation of pectin is an evolutionary consequence of the alkalinization which inactivates PG [optimal activity for Eca PG is at pH 6.0 (unpublished results)]. Moreover, secretion of PL isoenzymes may ensure successful biological activity of Eca in diverse types of host cell walls. 3.5. Synergistic action between Eca PLs

Synergism is evident not only between enzymes utilizing identical substrates [e.g. cellulases (21, 22)], but also among enzymes with different specificities. For degradation of cell walls, strong synergism was found between pectolytic and cellulolytic enzymes (23, 24). Since naturally occuring pectins are structurally heterogeneous regarding side chains and degree of esterification, synergism among pectolytic enzymes displaying either identical or different modes of action might facilitate pectin depolymerization (19). To test this possibility the effect of exogenous PLs on the depolymerization of pectin was examined. All possible PL enzyme

289

combinations were added to solutions containing either PGA or pectin, followed by determination of product formation. Table 2 shows activity values of 70-90% of the sum of the single enzyme activities when using PGA or 31% esterified pectin as a substrate. Although the monoenzymic degradation of 68% esterified pectin was low compared to the less esterified compounds, combination of some PLs revealed significantly enhanced depolymerization of the substrate. Notably, synergism only occured in enzyme combinations comprising PL3, resulting in up to 64% increase in activity. Supplementation with PL1 and PL2 together showed no higher degradation of pectin compared to depolymerization by the individual enzymes (Table 2). Table 2

Synergistic depolymerization of pectic components by combined Eca PLs Synergism

PL1/PL2 PL1/PL3 PL2/PL3 PL1/PL2/PL3

PGA

31% esterified pectin

68% esterified pectin

0.89 0.99 0.83 0.92

0.78 0.72 0.73 0.67

0.96 1.37 1.32 1.64

Synergism is calculated by dividing the measured activity (enzyme combinations) by the expected activity (individual activities, data not shown). Values >1 indicate positive synergism. More detailed information was obtained from inspection of the time-course for product formation following degradation of pectin with enzyme combinations. Supplementation with PL1 and PL2 together caused high initial activities followed by a significant reduction after around 150 s. Further addition of PL1 or PL2 after 160 s effected no increase in product formation, probably due to exhaustion of available substrate. Alternatively, supplementation with PL3, either initially or after 160 s, stimulated a pronounced enhancement of pectinolysis. To obtain insight into the action pattern of pectin degradation by PL combinations, time-course experiments were carried out to analyse by HPAEC the products released from 68% esterified pectin. Supporting the findings described above, enzyme mixtures initially containing PL3 caused a notable degradation of 68% esterified pectin (Fig. 2a, b). Supplementation of the enzyme mixtures with PL1 or PL2 after 120 s of incubation showed no or only little enhancement in the depolymerization (Fig. 2a, b). Compared with mixtures comprising PL3, product formation by the combination of PL1 and 2 leveled off rapidly. Supplementation of PL3 after 120 s of incubation resulted in an increased formation of tri- and dimers (Fig. 2c). It is notable that the amount of oligomers, produced by the action of PL1 and 2 decreased upon addition of PL3, suggesting PL1 and PL2 to cleave pentamers that function as substrate for PL3 (Fig. 2c), off their polymeric substrate.

290

60 a

40-

i

i

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i

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I

l

i

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I

1

L3

20

P

0

10" 0 "P r p + L1

' 2O0

'

300

60

g 402

PL1 + PL3

20

13_

+PL2

200

300

200

300

60, 40-

PL1 + PL2 I

_

_.--

20,

o

1;o

PL3

Time (s) Rgure 2 Action pattern analyses of pectin degradation. HPAEC data of oligomers released from 68% esterified pectin by combinations of Eca PLs are graphically represented. Arrows indicate addition of the third enzyme. Products with degrees of polymerization ranging from 2 to 9 were detected. The graphs illustrate the generation of dimers (A), trimers ( I ) and pentamers (~).

291

These data show that PL1 and PL2 generate products which can be further degraded by PL3, implying differences in substrate binding and action patterns among the isoenzymes, possibly based on structural differences in the loop regions of the enzymes. Thus, PL3 is likely to be less affected by structural features of the substrate, such as esterification and sidechain length. Because of the relatively low activity towards 68% esterified pectin, it remains to be established if these observations are of importance for the degradation of pectin in vivo. 3.6. Conclusion

To date, over 50 pel sequences have been determined. Functional analysis of the corresponding enzymes should help to further clarify the functional role of conserved residues within the pectate lyase/pectin lyase family. The results of the present study indicate not only that Eca PLs are good model enzymes in the elucidation of the structure-function relationship of pectolytic enzymes with special emphasis on protein-pectin interactions, but also that they have the potential for use in biotechnology. Aspects of possible application are provided in the accompanying contribution by C. Wegener et al. describing cell maceration and enhancement of resistance against pathogen attacks by Eca PL3 in transformed potato plants. The involvement in vivo of Eca PLs in degradation of esterified pectins in the presence of PME and PNL remains to be elucidated.

4. ACKNOWLEDGEMENTS

Diter von Wettstein is thanked for support and encouragement, Ib Svendsen for protein sequencing and Dr. Jacques Benen, Wageningen Agricultural University, for providing unsaturated oligogalacturonides. Eva Gertman is acknowledged for excellent technical assistance. This work was financially supported by grant 9315048 of the Danish Research Council to professor D. von Wettstein. 5. REFERENCES

1 Varner, J. E. and Lin, L.-S. (1989). Plant cell wall architecture. Cell 56, 231-239. 2 P6rombelon, M. C. M. and Kelman, A. (1980). Ecology of the soft rot Erwinias. Ann Rev Phytopathol 18, 361-387. 3 Collmer, A. and Keen, N. T. (1986). The role of pectic enzymes in plant pathogenesis. Ann Rev Phytopatho124, 383-409. 4 Rombouts, F. M. and Pilnik, W. (1980). Pectic enzymes. In Economic Microbiology. Vol. 5: Microbial enzymes and bioconversions, pp. 228-282. Edited by A. H. Rose. New York: Academic Press. 5 Yoder, M. D., Keen, N. T. and Jurnak, F. (1993). New domain motif: The structure of pectate lyase C, a secreted plant virulence factor. Science 260, 1503-1506.

292

6 Bartling, S., Wegener, C. and Olsen, O. (1995). Synergism between Erwinia pectate lyase isoenzymes that depolymerize both pectate and pectin. Microbiology 141,873-881. 7 Nasuno, S. and Starr, M. P. (1966). Polygalacturonase of Erwinia carotovora. J Biol Chem 241,5298-5306. 8 Cornish-Bowden, A. and Eisenthal, R. (1978). Estimation of Michaelis constant and maximum velocity from the direct linear plot. Biochim Biophys Acta 523, 268-272. 9 Studnicka, G. M. (1987). Nucleotide sequence homologies in control regions of prokaryotic genomes. Gene 58, 45-57. 10 Nasser, W., Reverchon, S., Condemine, G. and Robert-Baudouy, J. (1994). Specific interactions of Erwinia chrysanthemi KdgR repressor with different operators of genes involved in pectinolysis. J Mol Bio1236, 427-440. 11 Hugouvieux-Cotte-Pattat, N. and Robert-Baudouy, J. (1992). Analysis of the regulation of the pel BC genes in Erwinia chrysanthemi 3937. Mol Microbiol 6, 2363-2376. 12 Wegener, C., Bartling, S., Olsen, O., Thomsen, K.K., Bahlow, R. and von Wettstein, D. Differences in cell wall degradation patterns by Erwinia carotovora pectate lyase isoenzymes. Submitted to Protoplasma. 13 Studier, F. W., Rosenberg, A. H., Dunn, J. J. and Dubendorff, J. W. (1990). Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185, 60-89. 14 von Heijne, G. (1985). Signal sequences. The limits of variation. J Mol Biol 184, 99-105. 15 Ried, J. L. and Collmer, A. (1986). Comparison of pectic enzymes produced by Erwinia chrysanthemi, Erwinia carotovora subsp, carotovora, and Erwinia carotovora subsp, atroseptica. Appl Environ Microbio152, 305-310. 16 Weber, J. (1976). Untersuchungen 0ber Zellwandgehalt und -zusammensetzung der Kartoffelknollen. Biochem Physiol Pflanzen 169, 589-594. 17 Barras, F., van Gijsegem, F. and Chatterjee, A. K. (1994). Extracellular enzymes and pathogenesis of soft-rot Erwinia. Annu Rev Phytopatho132, 201-234. 18 Lietzke, S.E., Yoder, M.D., Keen, N.T. and Jurnak, F. (1994) The threedimensional structure of pectate lyase E, a plant virulence factor from Erwinia chrysanthemL Plant Physiol 106, 849-862. 19 RexovA-BenkovA, L. and Markovic, O. (1976). Pectic enzymes. In Advances in carbohydrate chemistry and biochemistry 33, pp. 323-385. Edited by R. S. Tipson and D. Horton. New York: Academic Press. 20 Pitk&nen, K., Heikinheimo, R. and Pakkanen, R. (1992) Purification and characterization of Erwinia chrysanthemi B374 pectin methylesterase produced by Bacillus subtilis. Enzyme Microb Technol 14, 832-836.

293

21 Irwin, D. C., Spezio, M., Walker, L. P. and Wilson, D. B. (1993). Activity studies of eight purified cellulases: specificity, synergism, and binding domain effects. Biotechnol Bioeng 42, 1002-1013. 22 Vincken, J.-P., Beldman, G. and Voragen, A. G. J. (1994). The effect of xyloglucans on the degradation of cell-wall-embedded cellulose by the combined action of cellobiohydrolase and endoglucanases from Trichoderma viride. Plant Physiol 104, 99-107. 23 Renard, C. M. G. C., Searle-van Leeuwen, M. J. F., Voragen, A. G. J., Thibault, J.-F. and Pilnik, W. (1991a). Studies on apple protopectin. I1. Apple cell wall degradation by pure polysaccharidases and their combinations. Carbohydr Polymers 14, 295-314. 24 Renard, C. M. G. C., Schols, H. A., Voragen, A. G. J., Thibault, J.-F. and Pilnik, W. (1991b). Studies on apple protopectin. II1. Characterization of the material extracted by pure polysaccharidases from apple cell walls. Carbohydr Polymers 15, 13-32.


J. Visser and A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996Elsevier Science B.V.All fights reserved.

295

Functional Implications of the Three-Dimensional Structures of Pectate Lyases F. Jurnak a, N. Kita b, M. Garrett c, S.E. Heffron a, R. Scavetta a, C. Boyd d and N. Keen d

aDepartment of Biochemistry University of California Riverside, California 92521 bKanagawa Institute of Agricultural Science 1617 Kamikisawa Hiratsuka, Kanagawa 259-12, Japan c244 Manville Ave. Bowling Green, Ohio 43402 dDepartment of Plant Pathology University of California Riverside, California 92521

Abstract

The three-dimensional structures of two Erwinia chrysanthemi pectate lyases, PelC and PelE, have been refined to a resolution of 2.2 A. A superposition of the two structures has been used to correct the multiple sequence alignment of the extracellular pectate lyase superfamily. The corrected alignment has revealed two clusters of 'potentially catalytic' invariant amino acids, compatible with two active sites. Site-directed mutagenesis studies have confirmed that the pectinolytic active site includes the region around the Ca 2+ binding site. Furthermore, mutagenesis studies suggest catalytic roles for individual amino acids. Although PelC and PelE are structurally similar in the overall fold of the polypeptide backbone, there are significant differences in the size and conformation of the loops that comprise the pectinolytic active site. The differences in the surface charges between PelC and PelE in the groove extending from the Ca ~+ site suggest that the optimal oligosaccharide substrates are different for each enzyme. 1.

INTRODUCTION

Extracellular pectate lyases are virulence factors produced by plant pathogens that cause tissue maceration and cell death [1-4]. The enzymes are presumed to function by cleaving the a-l,4-polygalacturonic acid (PGA) component of plant cell walls. Among the most studied are the Erwinia chrysanthemi enzymes which are coded by multiple gene families that are independently regulated by complex mechanisms [5-7]. The extracellular isozymes can be grouped into two major subfamilies, pelBC and pelADE, that are characterized by their pI values and number of disulfide bonds. Within each subfamily, the sequence homology is 60% or greater but, between the subfamilies, the sequence homology is as low as 20%. Although the enzymatic mechanisms for all pectate lyases are believed to be similar, subtle differences between the subfamilies have been reported [8]. The peIADE subfamily cleaves PGA to a dimer and the pelBC subfamily produces a trimer as the major product. In addition to the sequence homology among the extracellular pectate lyases, the enzymes share sequence similarity with fungal pectin lyases as well as with plant pollen and style proteins [9]. The pectin lyases are related in function, cleaving pectin, a neutral methylated form of PGA. The function of the plant pollen and style proteins has not been elucidated but it is presumed to be important in development. Although it is generally

296 assumed that the plant proteins have pectinolytic activity, given the sequence similarity with the pectate lyases, the assumption has been proven for only one plant pollen homologue to date [10]. In contrast to the extensive genetic information, there is relatively little known about the details of the enzymatic mechanism of the pectate lyases. The presumed substrate is PGA, a plant cell wall component that is also commercially available. Since the late 1950's, it has been postulated that pectate lyases utilize a #-elimination mechanism to cleave PGA by exo- and endolytic mechanisms, resulting in an unsaturated bond between Ca and Cs at the nonreducing end of the polysaccharide [11]. Ca 2 § is required for in vitro pec'tinolytl'6 activity, but ils role is unknown. Prior to the structural studies, it had been commonly assumed that Ca z § bound only to PGA, inducing a substrate conformation that could be recognized by the enzyme [12]. The pH optimum of in vitro pectinolytic activity for all pectate lyases is rather high, ranging from pH 8 to pH 10. No amino acids had been identified that participated in catalysis or in saccharide recognition. Furthermore, there have been no reports of inhibitors which could be used to probe details of the enzymatic mechanism. Given the paucity of biochemical information, it is not surprising that the pectinolytic active site could not be delineated unambiguously from the three-dimensional structures of pectate lyases.

The E. chrysanthemi pel ~enes have been cloned and inserted into plasmid constructs with convenient properties for protein overproduction and purification [13-14]. The purity and quantity of the recombinant forms have facilitated structural studies. Although the recombinant enzymes are isolated from the periplasm of Escherichia coli, they have in vitro properties that are the same as the natural forms secreted from E. chrysanthemi. Two representative pectate lyases from different subfamilies, PelC and PelE, have been the focus of structural studies. PelC is composed of 353 amino acids with a molecular mass of 37,676 daltons and two disulfide bonds, Cys72-Cys155 and Cys329Cys352 [14]. PelE is composed of 355 amino acids with a molecular mass of 38,118 daltons and one disulfide bond, Cys291-Cys320 [7]. Although the enzymes crystallize readily, both require the presence of sulfate anions for large, ordered crystals suitable for X-ray diffraction studies [15-16]. 2.

METHODS

PelC was purified from the periplasm of E. coli cells containing the high-expression construct pPEL410 of the E. chrysanthemi EC16 pelC gene [7] and crystallized from ammonium sulfate [16]. PelE was purified in a similar manner using the high expression construct pPEL748 of the EC16 pelE gene [14] and crystallized from polyethylene glycol in the presence of lithium sulfate [15]. The PelC mutants were prepared by site-directed olig.onucleotide mutagenesis using PCR by overlapping extension methods [17-18]. The various PelC mutant proteins were prepared by overexpression of the respective genes cloned in pINK1 plasmid constructs expressed in Escherichia coli HB101 or in pRSET5A plasmid constructs expressed in E. coli HMS174(DE3). The pectinolytic activity of PelC wildtype and mutants was determined by monitoring the absorbance increase at 232 nm of a 1 ml reaction mixture containing sodium polypectate at 22 oC as previously described [13]. For each mutant, the Ca 2+ concentration was optimized to gwe a maximum specific activity. Protein concentration was determined by the method of Bradford [19]. Plant tissue maceration assays were done using mesocarp_ tissue of cucumber fruit according to the method of Mussell and Morre [20]. The Ca~z+ affinity for the PelC wildtype and mutants was determined by monitoring the changes in the intrinsic tryptophan fluorescence of PelC in response to the addition of Ca 2§ using a SPEX 112 Fluorolog spectrofluorimeter with a 150 Watt Xenon Arc lamp as source.

297 The three-dimensional structures of PelC and PelE were solved using multiple isomorphous replacements methods as previously described [21-22]. The PelC and PelE structures were superimposed using the command, 'lsq_exphcit ' in the program O [23]. The PILEUP multiple sequence alignment program o-f the GCG package was used to create the alignment of the extracellular pectate lyase superfamily as described previously [9]. All structural figures were generated by MOLSCRIPT [24]. 3.

RESULTS

The first three-dimensional structure of a pectate lyase, that of E. chrysanthemi PelC, revealed a novel topology, termed a parallel ~ helix [21]. The polypeptide backbone folds into parallel # strands that are wound into a large right-handed coil. Each level of the coil is composed of three distinct # strands, each of which forms backbone hydrogen bonds with neighboring # strands of adjacent levels. The overall appearance of the secondary structure resembles that of three parallel # sheets as shown in Figure 1A. Loops of various size and conformation protrude from the core structure. Another striking feature of the PelC structure is the high degree of internal organization. Every side chain that is oriented towards the center of the core participates in a stacking interaction with its neighbor [25]. There are extended hydrophobic stacks composed of valine, alanine, isoleucine or leucine; polar stacks of asparagme or serine; and aromatic stacks resembling DNA base-pair Interactions. Prior to the PelC structure, there were no biochemical data which identified any amino acids that participate in pectinolytic activity. Therefore, the active site could not be deduced unambiguously from the structural results alone. However, the structure provides two clues as to which region of PelC might participate in catalysis. First, the environment surrounding each of two heavy atom derivatives is compatible with that of a Ca 2 § binding site. The uranyl and Lu 3+ derivatives are coordinated directly to the enzyme through two carboxyl oxygens from Aspl31 and one carboxyl oxygen from each of Glu166 and Aspl70. Secondly, the putative Ca 2§ site is located in a narrow, highly charged groove, approximating the shape and length of 9-12 saccharide units of galacturonic acid as shown in Figure lB. The groove represents the only localization of charged amino acids on the surface of PelC and as a consequence, has been proposed as the substrate binding site. The spatial distribution of the positively-charged amino acids on PelC is particularly suggestive of an oligogalacturonic acid binding site. The periodicity of negative-charges on oligogalacturonic acid is anticipated to necessitate a pectate lyase-substrate interaction that is primarily stabilized by regularly-spaced electrostatic interactions. In 1994, two additional pectate lyase structures were reported, Bacillus subtilis Pel [26] and E. chrysanthemi PelE as shown in Figure 2A [22]. Both fold into the novel # helix topology with extensive stacking interactions among the internal side chains. The structure of B.s. Pel is complexed directly to Ca 2+ which is located at the .putative Ca 2+ site proposed for PelC. There is a groove in all three structures in the region of the proposed pectinolytic active site. However, a comparison of the PelC and PelE structures has revealed that, unlike PelC, the charged amino acids are randomly distributed over the entire surface of PelE as shown in Figure 2B. The difference in the distribution of the surface charges suggests that either the proposed pectinolytic active site region is incorrect or the ootimal substrate is not oligogalacturonic acid for both PelC and PelE. In particular, the surf'ace charges surrounding the Ca 2§ site in PelE are compatible with a substrate length of 3-4 adjacent galacturonic acid units, not 9-12 negatively charged units compatible with the PelC structure.

298

,.:%"-,-.

.... _

-:k--

A

II~

".\

il

B

Figure 1. The three-dimensional structure of PelC. A. A schematic diagram illustrating the major secondary structural features of the PelC polypeptide backbone. The three parallel/~ sheets are represented by arrows in light, medium and dark gray. B. Space-filling models of PelC. Neutral amino acids on the surface are shown in gray, negatively-chargec~ residues in white and the positively-charged residues in black. The location of the Ca: + is illustrated by cross-hatching.

A

B

Figure 2. The three-dimensional structure of PelE using the same view as Figure 1. A. A schematic ribbons diagram illustrating the major secondary structural features of the PelE polypeptide backbone. The three parallel ~ sheets are represented by the arrows in light, medium and dark gray. B. Space-filling models of PelE. Neutral amino acids on the surface are shown in gray, negatively-char~ed residues in white and the positively-charged residues in black. The location of the Ca 2 u is illustrated by cross-hatching.

299 In order to uncover additional clues as to the location of the pectinolytic active site, the structures of PelC and PelE have been superimposed and compared in atomic detail [9]. As shown in Figure 3, those aCs which superimpose within a deviation of 1.5 A or less comprise the backbone of the parallel ~ helix. Those aCs which deviate by 1.5 A to 3.0 A are located on one side of the core and comprise three to four loops, including the N- and C-terminal branches. The structural regions which differ by more than 3.0 2k include the loops protruding from the parallel/3 helix in the vicinity of the Ca 2§ binding site and the proposed pectinolytic active site. The results were ominous because the active site regions of functionally-related proteins are generally the most conserved structurally. Yet for the three known pectate lyase structures, the proposed active site region is the most diverse! In addition to an atormc comparison, the superposition of the PelC and PelE structures highlighted errors in the multiple sequence analysis of the extracellular pectate lyase superfamily based primarily upon evolutionary relationships [4,6]. The superfamily includes the extracellular pelBC and pelADE subfamilies, the fungal pectin lyases and the plant pollen proteins [9]. Approximately 252 amino acids, or the regions ranging from Gly6 to Phe257 in PelC nomenclature, could be aligned with confidence based on highly probable structural similarities. In the corrected sequence alignment, 10 amino acids are mvariant among all superfamily members. Such results are significant because amino acids which remain unchanged during evolution usually have a critical functional role. In PelC nomenclature, the 10 invariant amino acids include Gly6, Glyl2, Glyl3, Aspl31, Trp142, Asp144, His145, Thr206, Arg218 and Pro220 [9]. In addition to the latter 10 invariant amino acids, another 14 are invariant within the extracellular p.ectate lyase family [27], but not within the pectin lyose or plant pollen/style protein families. These 14 amino acids include three in the Ca ~+ binding site, Aspl70, Lysl90 and Arg223, as well as 11 in the vWiDH region: Thrl0, Thr92, Gly95, Asnll7, Thr179, Serl81, His228, Asn232, Ala338, Gly339 andLys342.

.:-

:..

..

...

Figure 3. A comparison of the a C backbone of PelC with PelE. The a Cs which superimpose within a root-mean-square deviation of 1.5 A are shown in black and those, within 3.0 A are shown in dark gray. The remaining backbone regions are shown in light ra),. The largest structural differences occur in the loops capping one end of the parallel ehx as well as in those comprising the putative substrate binding groove.

300 The locations of the set of 10 invariant amino acids cluster into two distinct regions as shown in Figure 4A and 4B. The first cluster includes Asol31, Arg218 and Pro220, all located in the vicinity of the Ca 2§ binding site. The remaining 7 amino acids are located on the opposite side of the parallel ~ helix, centered about the region containing the vWiDH pattern. Both clusters contain subsets of invariant amino acids which have chemically reactive properties and thus are potential candidates for catalytic residues. The finding suggested that either the pectate lyase superfamily members have two catalytic functions or the pectinolytic active site comprises two distinct regions, one for properly aligning the substrate via an interaction with t2az § and the other for catalyzing the cleavage reaction. The latter possibility has been eliminated by considering the distances separating the two regions. The shortest distance between the two clusters is approximately 25 2k, a distance which could accommodate binding of one end of an oligomer of 5-6 saccharide units at the Ca 2+ binding site and the other end at the vWiDH region. However, to span the shortest distance, the substrate would need to pass through the center of the parallel helix, a very unlikely possibility as a consequence of the extensive internal stacking interactions and the extensive hydrogen bonding network stabilizing the parallel ~ helix core. If the substrate were to span from one region to the other, then it is more probable that the substrate would lie on the outside of the parallel # helix core, but then the distances would be 95 2k. Such a distance would require that pectate lyases cleave substrates of 18-22 saccharide units in length. This analysis has led to the conclusion that the pectate lyase superfamily members have two active sites, one for pectinolytic function and the other as yet unidentified.

...............

...

........

:.....:::-: .........

:

...........

..

~,...~ "'....:::.:....~..~::...-~,~:....~,!..~..iiii.....~.-~i~>.. :....

':,,,,a ..

:i

..

.

4

~.. '":: ::i""i...~.i'~~'i..i:II........

.:.:ii....iiii:..,.

:.

::.... '....

.:.

......

r'

..~.::.~.~.i~.:..:.i.i--i ~---...... .... ?o .......

A

.

B

Figure 4. Superposition of the invariant amino acids in the superfamily upon the c,C tracing of PelC. A. Side view, with Aspl31, Arg218 and Pro220 o~the Ca z+ binding site on the left and Gly6, Glyl2, Glyl3, Trp142, Asp144, His145 and Thr206 of the vWiDH r e . o n on the right. B. Top view, looking down the axis of the parallel ~ helix, with the Cffz+ binding site on the left and the vWiDH region on the right. The loops covering the opening to the core of the parallel ~ helix have been removed for clarity.

301

To determine which site is the pectinolytic active site and what the function of the second site might be, many site-specific mutants of PelC have been prepared and characterized. Table 1 lists the most interesting mutations made in the region of the Ca 2 § binding site. All mutants in Table 1 crystallized isomorphously with wildtype PelC, indicating that the mutation did not result m a loss of structural integrity. In all mutants, the maceration activity on plant tissue parallels the observed pectinolytic activity, supporting the widely-held hypothesis that pectate cleavage is responsible for the observed "soft2+rot" damage. Because C~tz+ is required for m" vitro" pectinolytic activity, the affinity of Ca for the mutant has been quantitated by an intrinsic tryptophan fluorescence assay. As expected, pectinolytic activity is significantly reduced or abohshed in those mutations

ai

i~

(Lys172, Lys190, Arg218).

Table 1 PelC mutants near Ca 2 + binding site Tissue Maceration

%

' Max. Specific Activity %

Wildtype 1oo :too Mutations in Amino Acids Directly Coordinated to Ca 2 +:

Ca 2. Affinity

%

:too

DI31E DI31N EI66D EI66Q DI70N

0.5 A 1.6mg/mLC>l I'-'] 0.2 mg/mL C>I O No fraction added

0.05

0.00 0

I

I

I

100

200

300

t, S Figure 4. Batch hydrolysis of pectin in the presence of oligomers. concentrations. Finally, these oligomers apparently are not substrates for the enzyme since the slopes of the corresponding straight lines (product formation rate) are not different from the reference one. Preliminary experiments of or-l,4 oligogalacturonide separation by anion exchange chromatography have been done aiming to prepare pure fractions of oligomers at a defined polymerisation degree. Polygalacturonic acid was enzymatically degraded for 12 hours at 25~ The reaction mixture was then separated on a DEAE Sephadex column. The pertinent chromatogram is reported in Figure 5. The peak contents were pooled individually, freeze dried, desalted and then analysed by TLC. This analysis showed that peak 1 corresponds to the digalacturonic acid while peak 2 to the trigalacturonic acid, which also resulted the main components in the sample loaded to the column.

4. CONCLUSIONS

Enzymatic degradation of pectin can be satisfactory performed in UF-membrane reactors which have been proved to be helpful tool for laboratory scale investigations. Reaction products can be continuously recovered in a sequence of filtration stages. The obtained product distribution depends on the enzyme to substrate ratio, which affects particularly the

447 1,2 _

peak

1

_

1_

_

0,8

_

,r, 0 , 6 t ' " ,

peak

2

-

0,4-

_

0,2_

-

0 -' ,"W'm,'-',~" , " ~ 0 50

-

100

-

~

150 200 fraction number

~

"

,

.....

250

.,=~=Mea,

,

300

Figure 5. Elution profile of PA degradation mixture. yield of oligomers between 1 and 3 KDa. Evidences were found that products ranging from 3 to 5 monomer subunits do not depress the initial rate of pectin degradation. Research is in progress aiming to clarify the influence of other operational parameters such as residence time and molecular weight cut-off the membrane reactor on conversion yield and product distribution.

5. APPENDIX: MASS-BALANCE EQUATIONS The vessels were indexed by the subscript "j" (j = 0 refers to the reactor and j from 1 to 4 to the UF cells) and oligomers were lumped in two categories: "P" (Permeated) and "R" (Rejected). Let label "in" species entering a cell and "out" those leaving it. Instantaneous massbalance in the stream leaving a cell and feeding the following one is: Pj-l,out = Pj,in + Rj,in

(1)

The transient mass balances in the four filtration stages for the rejected and the permeated chemicals can be respectively written: dR./dt = R.. /x j j,m

(2)

dPj,out/dt = Pj,in/X "5,out/1:

(3)

448 where ~ is the mean retention time, i.e. the ratio of cell volume to volumetric flow rate. The corresponding equations for the first stage (enzymatic reactor equipped with the membrane at the highest molecular weight cut-off) are: dR o/dt = rR

(4)

rp = P0/x

(5)

+

dP0/dt

where r.. and r_ represent the net rates of production of oligomers respectively at molecular weight l~gher and e lower than 20 KDa. The system of differential mass balance equations (2)- (5) should be solved, provided that the "in" time functions were known, with the initial conditions: t = 0, P. =R. = 0 [j = 1, ..4] J J

(6)

Even if more than five time constants are elapsed when the system is shut down, the transient should not be vanished in each vessel, because at any time, the "0" cell could still furnish inlet species to the following ones due to the terminal, low rate reaction. Therefore, the constitutive rate equations are need for the solution of the above model system. Nevertheless, the complete knowledge of permeated and rejected product concentration is not necessary. In order to quantify product distribution in the whole system, it is sufficient to make an integral analysis and measure all "P" values and the overall concentration inside the cells at the end of a fixed process time (24h). In fact, if one considers the working conditions for the last stage, on the basis the perfect mixing hypothesis, P, .(24h) represents an instantaneous concentration permeate species at molecular weight le~'~t~en 0.5 KDa equal to the one still present in the cell. Subtraction of this value from the concentration detected inside the cell gives the amount of rejected species. This procedure can be extended backwards to the other stages.

Acknowledgement Research supported by National Research Council of Italy, Special Project RAISA, subproject 4, Paper N~

6. REFERENCES

1. 2. 3. 4.

W.F. Raymond and P. Larvor (eds.), Alternative uses for agricultural surpluses, Elsevier applied Science, London, 1986. M.K. Dowd, S.L. Johansen, L. Cantarella and P.Y. Reilly, J. Agric. Food Chem., 42 (1994) 283. J.H. Janssens, A. Bernard and R.B. Bailey, Biotech. Bioeng., 26 (1984) 1. M. Moresi, F. Clementi, J. Rossi, R. Medici and G.L. Vinti, Appl. Microbiol. Biotech., 27 (1987) 37.

449 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19 20. 21 22. 23 24. 25. 26. 27. 28. 29. 30. 31. 32.

M. Moo-Young, H.W. Blanch, S. Drew, D.I.C. Wang (eds.), Comprehensive Biotechnology, vol.3, The practice of biotechnology: current commodity products, Pergamon Press, Oxford (1985). A. Caridi, Abstract Book Convegno congiunto ABCD, AGI, SIBBM, SIMGBM, 1995 Montesilvano Lido (PE), Italy, 319. N. Nishio and S. Nagai, European Appl. Microbioi. Biotechnol., 11 (1981) 156. Y. Hadar, Z. Keren and B. Gorodecki, J. Biotech., 30 (1993) 133. A. Ginterov~ and A. MaxianovL Folia Microbiol., 20 (1975) 1974. E. Sanjust, R. Pompei, A. Rescigno, A. Rinaldi and M. Ballero, Appl. Biochem. Biotech., 31 (1991) 223. N. Nishio and S. Nagai, European Appl. Microbiol. Biotechnol., 6 (1979) 371. E. Chalutz, E. Kapulnik and I. Che, European Appl. Microbiol. Biotechnol., 18 (1983) 293. M. Moriguchi, Agric. Biol. Chem. 46-4 (1982) 955. G.G. Birch, N. Blakebrough, K.J. Parker (eds.), Enzyme and Food Processing, Applied Science Publishers, London (1980). A. Manganelli, Emmegi Agro-Industriale srl, private communication (1995). J.N. Bertho and R. De Baynast, Abstract Book of 7th European Congress of Biotechnology, Nice, France, 2 (1995) 42. A. Collmer, Ann. Rev. Phytopatol., 24 (1986) 283. S. Tipson and D. Horton (eds.), Advances in Carbohydrate Chemistry and Biochemistry, Academic Press, New York, 33 (1976). F. Alfani, M. Cantarella, V. Scardi, J. Membr. Sci., 16 (1983) 407 F. Alfani, L. Cantarella, A. GaUifuoco and M. Cantarella, J. Membr. Sci., 52 (1990) 339. F. Alfani, A. Gallifuoco and M. Cantarella, Chem. Eng. J., 43 (1990) B43 S. Todisco, V. Calabr6 and G. Iorio, J. Molecular Catalysis, 92 (1994) 333 P. Albersheim, P.U. Killias, Arch. Biochem. Biophys., 97 (1962) 191. M.F. Chaplin and J.F. Kennedy (eds.) Carbohydrate analysis: a practical approach, I.R.L. Press, Oxford (1986). Z.I. Kertesz, J. Biological Chem. 121 (1937), 589 E. Sthal (ed.), Thin layer chromatography, Springer Verlag Berlin, Heidelberg New York (1969). D.F. Jin and C.A. West, Plant Physiol., 74 (1984) 989. C. Dinnella, G. Lanzarini, A. Stagni and C. Palleschi, J. Chem. Tech. Biotech., 59 (1994) 237. C. Dinnella, G. Lanzarini and P. Ercolessi, Process Biochem., 30-2 (1995) 151. C. Dinnella and G. Lanzarini, Int. J. Food Science and Technol., 30 (1995) 391. E.A. Baldwin and R. Pressey, Plant Physiol., 90 (1989) 191. E. Le.derer (ed.), Chromatographic en Chimie Organique and Biologique, vol.2, Masson and C~eEditeurs, Paris (1960).


APPLICATIONS.




453

A p p l i c a t i o n of P e c t i n a s e s in B e v e r a g e s Catherine Grassin and Pierre Fauquembergue Gist-Brocades, Beverages Ingredients Group, 15 rue des Comtesses, P.O.Box 239, 59472 Seclin cedex, France

Abstract Pectinases are necessary processing aids in the production of clear and con(:entrated fruit juices. Improving pressing and clarification, they are biochemical tools of first importance. In combination with hemicellulases and cellulases, pectinases can lead to fruits liquefaction. The improvement of our knowledge on fruits cell wall has allowed us to describe new enzyme activities to specifically degrade these substrates to optimize the performance of industrial equipments and to allow to produce juices and concentrates with a higher quality in terms of oxidization, aromas level and stability.

1. ENZYMES IN FRUIT JUICE INDUSTRY Among the food industries, the fiuit juice industry has to deal with the most delicate problems. Its role consists in processing raw material whose composition varies widely, to process fruits as much as possible at the lowest cost while maintaining or even improving the organoleptic quality and stability of the finished product. This ambitious objective has been made possible for the past years through the use of enzymes at different stages of the production of juice. Because the pectin structure and composition have been extensively described by specialists during this symposium, we will directly show you the role of pectinases in fruits processing by taken examples of different technologies. Each fruit has specific quantities and ratio of pectin, hemicelluloses and cellulose. These polysaccharides are important concerning enzymes activities required to produce juices and concentrates. Moreover, even if molecular weight and methylation degree of the pectin are specific for each fruit, during the fruit maturation, endogenous pectinases depolymerases and esterase are changing the pectin characteristics. This broad variability of raw material makes difficult the standardisation of fruits processing. Commercial enzymes are tools now used all over the world and widespread to every industry transforming fruits since 1930s. Whatever would be the finished product, the producer can take advantage of a wide range enzymatic activities contained in the commercial preparations. Since a few years, new activities have been discovered in relation with fruit cell wall architecture, like new pectinases [l] (i.e. rhamnogalacturonase for the apple) new glycosidases [2](i.e. apiosidase for the grape). The improvement of the knowledge of fruit composition, cell wall chemistry, enzymology of endogenous and exogenous enzymes allow to supply the juice producers with preparations more and more purified and specific, blended in optimal combinations.

454 Enzymes most frequently proposed to fruit juices producers are pectinases coming from Aspergillus. Pectinases are exocellular enzymes and are the main activities produced among numerous side activities type hemicellulases, glycosidases. The Table 1 gives the spectrum of enzymatic activities contained in three commercial preparations A, B and C.

Table 1 Comparison between commercial enzymatic preparations Enzyme activity U/g PG PL PE exo B ARA endoGlucanase exoGlucanase beta-Glucosidase

A

B

C

400 115,000 1,000 10 6,000 3 40

800 30,000 300 10 4,500 4 2

500 115,000 95 28 11,000 25 400

It appears from these results than tile enzyme spectrum is very different depending of the starting strain of Aspergillus and ratio are very important since enzymes act in synergy. It is necessary to choose the right enzymes preparation in relation with the fruit composition and with the final product people want to obtain. In this presentation, we will take examples of different fi-uit juice processes and will try to relate enzyme activities, the role they play and tile transformation they occur in terms of finished products. These examples will concern apple juice, french cider, pineapple and wine.

2. APPLE JUICE PRODUCTION About 40 millions tons of apples are gathered per year and about 10% are processed mostly to produce clear concentrate but a little part as cloudy juice. The aim of pectinases application is to increase the plants productivity by processing apples quantity as high as possible along the season with high yields (see Table 2).

455 Table 2 Comparison of juice yields without or with enzymes maceration with Rapidase Press 50g/ton - ih - 20~ after one pressing

Fresh apples Stored apples

Juice yield %

Horizontal presse

Belt presse

no enzyme with pectinases no enzyme with pectinases

83 86 72 84

72 75 60 70

2.1. Classical process with maceration and depectinisation In the classical process, enzymes are used at two stages of" processing: maceration of the pulp and depectinisation of the juice. Because endogenous enzymes have too low activities and no quick technological effect, the application of exogenous enzymes allows to regularize the production and allows a better productivity of the plant. Along the maturation, the unsoluble protopectin is slowly transformed into soluble pectin through the endogenous pectinases of the apple, the molecular weight and the degree of methylation of the pectin are decreasing. Apples become very difficult to press since the soluble pectin content increases. The producer solves this problem by an increase of enzymes dosage. Pectinases are essential tools to produce clear concentrates (see Table 3).

456 Table 3 Technological role of enzymes Enzyme Pectinlyase endo exo B Arabanases Rhamnogalacturonase P ect in acetylest erase Pectinmethylesterase Polygalacturonases Pectinlyase endo exoB Arabanases Arabinogalactanases Rhamnogalacturonase P ecti nacetyl est erase id pressing + endoGlucanases exoGlucanases cellobiohydrolase 13-Glucosidase Xylanase .p ecti nm ethyl est erase endoPolygalacturonase Galactomannanase Arabinogalactanases Polygalacturonases Polygalacturonases P ecti nmet hyl est erase Pectinlyase Arabinogalactanases exo B Arabinosidase Apiosidase Glucosidase Rhamnosidase

Importance

Role

Application

Fruits pressing

Apple juice extraction

+++ + + + +++ +++ ++ + +

Depectinisation of juices

+

Clear concentratable juices Apple, Pear, Grape

+

++ +

Cell wall destruction Fruits liquefaction

Apple Pear

Ca pectate formation Pulp maceration

French Cider Purees Clear Pineapple juice

+ +

, ,

+++ +++ +++ ++ + +++ + + + +++ +++ +++ +++

Gum hydrolysis

Must depectinisation

Grape must clarification

Aromas release

Wine quality

For apple maceration, the main activity is the endopectinlyase (PL) which decreases the viscosity due to the colloidal pectin by decreasing the molecular weight of this substrate methylated at about 90%. In association to this PL, exo B and endoarabanase (ARAs), pectinacetylesterase (PAE), rhamnogalacturonase (RG) and other hemicellulases allow to hydrolyse side chains of the pectin, decrease the steric hindrance and make the access of pectinases to the rhamnogalacturonic backbone more easy.

457 Added to the mash, these enzyme activities decrease its viscosity and improve its pressability. The free run juice volume is larger. The yield and then the capacity of the press are increased. Pectinmethylesterase (PME) and polygalacturonases (PGs) are key activities for the juice clarification. PME produces pectic acid the substrate of the PG which starts to work below a methylation degree of 60%. The ratio PE/PG is important also because a too high PE activity would make a too fast demethylation inducing the formation of unsoluble calcium pectate before hydrolysis of the pectic acid by PGs. The following curve shows the effect of the different pectinases on the decrease of viscosity of apple juice along the time. In addition, ARAs and other pectinases make partially hydrolysed hairy regions and side chains of the pectin to settle down. In the juice, pectinases hydrolyze the residual pectin decreasing the viscosity, leading to the clarification of the juice through the neutralisation of electrostatic charges of pectin, proteins and phenolic compounds. The sedimentation necessary for the clarification of an apple juice only occurs after enzymatic degradation of the colloidal pectin and the starch. The cloud is composed of 30-40% proteins with positive charges at the pH of the juice (3.5-4.0). These proteins are associated to polyphenolics surrounded by the partially hydrolysed pectin which has negative charges. The formation of these macromolecules induce the floculation of the cloud, leading to the clarification of the juice at an optimal pH of 3.5. The clarification is perfected by fining agents and filtrations or by ultra-filtration. In conclusion, the use of exogenous pectinases allows to produce a clear and stable apple juice easily concentrated. 2.2 Total liquefaction process The enzymatic liquefaction of apple consists in the hydrolysis of cell wall polymers by exogenous enzymes into soluble components, mainly acid and neutral sugars, thus increasing the extraction level and the yield. The aim of enzymes application is to decrease within three hours the viscosity of the pulp to be able to use a decanter to separate tlle juice from the solids. Total liquefaction occurs during two to three hours at 50~ under stirring. Enzymes work close to their optimal temperature. The liquefied pulp which contains 20% solids (in volume) is centrifuged with a decanter giving a juice 1. The pomace is added with about its weight of water and pumped to a second decanter giving a juice 2. Both juices are mixed and contain less than 2 g per liter solids. After pasteurization and aromas recovery, active carbon and gelatin are sometimes added to decolourize the juice and are then removed by filtration. Tile juice is then ultra-filtered and concentrated. If we compare liquefaction to maceration, more activities are needed to liquefy the cell wall. Since 1991, new pectinases activities such as rhamnogalacturonase, pectin acetylesterase and xyloglucanases complex have been found to be important in the apple liquefaction by Henck Schols, Jean-Paul Vincken and Voragen [3]. The cellulose-xyloglucan complex accounts approximatively 57% of the apple cell-wall matrix. In a liquefaction process, an efficient enzymic degradation of this complex is crucial to increase the sugars extraction, to decrease the viscosity of the pulp then to be able to ultra-filtrate the juice without second depectinisation, at last to have negative alcohol tests required by some concentrate customers. However, if theoritically, the combination of pectinases to cellobiohydrolases plus endoglucanases should release more than 80% of all polysaccharides from the cell walls (according to Voragen and al. [4]), in industrial conditions, we arrive ahnost at this level of degradation but only for the pectin. Commercial enzymes preparations contain pectinases, hemicellulases and cellulases.

458 Enzymes like Rapidase LIQ + contain enough pectolytic activities to depectinize the juice during the liquefaction time, giving an average yield of 93-95% according to this type of process. The juice has a good quality and its composition fits with RSK values. In conclusion, the technology of total liquefaction of apple allows to work with a continuous process with less labour and faster than with a classical one, to get a high and constant yield during the whole processing season at a very high level (93- 95%), to get a pulp with a low content of solids (about 20% in volume) which can be centrifuged instead of pressed (lower investment in equipment), to decrease the quantity of waste pomace, to decrease the production costs. Liquefaction technology allows to process different fruits with the same process, at last to liquefy fruits for which no equipment had been developed to extract the juice or for which the use of pectinases did not allow to get juice such as tropical fruits.

2.3. Pomace liquefaction process This new technology is interesting because it allows to produce different juices and concentrate with classical equipment with very high yields. Pomace liquefaction is the application of liquefying enzymes on the pomace a~er the extraction of premiurn juice. Hot water is added to the pomace, and the second juice is extracted with a decanter after enzymatic treatment. The first juice is extracted without enzyme by pressing with an average yield of 50-75%. It can be processed as cloudy or clear juice and concentrate. The first extraction yield can be adjusted to get a pomace with a certain quantity of residual juice to avoid to add too much water. The pomace with water and enzymes is stirred during about three hours at 50~ The liquefied pomace is centrifuged with decanters. The juice obtained is acidic and the brix degree increases from about 6.0 to 8.5 mainly due to the degradation of pectin and cellulose. This juice is added to the first pressing juice to be depectinized in the case of clear juice production (this second acidic juice can be added to increase the acidity of juices with low acidity). At last, the quantity of waste pomace is reduced in this process. The final yield can reach 100 to 102%. Added to the pomace after a first juice extraction, enzymes preparations such as Pomaliq contain pectinases, hemicellulases and higher cellulases activities than for total liquefaction. After a first pressing and elimination of the first juice, enzymes allow to decrease the viscosity of the pomace and to extract a maximal quantity of sugars from a substrate which is still pressable. In conclusion, this process allows the producer to diversify the types of products and to give a better value to his raw material. The producer can perform pomace liquefaction with his existing presses. With a low investment in equipment (decanter) the efficiency of the plant can be greatly improved. This process is very flexible regarding the diversity of products that can be made: single strength juices with high organoleptic quality from the first extraction (clear or cloudy juices) and clear concentrates from the second extraction. The benefits brought by this process to the juice producer is the highest in comparison with other processes, if we calculate theoritically the additional weight of concentrate per ton of apples he can produce and sell, out of the cost of enzymes (see Table 4).

459 Table 4 Comparison of technologies to produce apple juice

Technologie

Enzymes

Dose

Juice quantity

(kg) Pressing Depectinization Maceration Depectinization Liquefaction

no enzyme Rapidase C80 Rapidase Press Rapidase C80 Rapidase Liq +

0 3 g/hi 50 g/T 3 g/hi 125 g/T

Liquefaction Depectinization

Pomaliq Rapidase C80

450 g/T 2 g/hl

Juice brix

(~

70~ concentrate

Increase

(kg)

750

12.5

134.0 kg

reference

850

12.5

151.8 kg

+17.8 kg

860 750 455

13.5 12.5 7.0

165.9 kg

+31.9 kg

179.5 kg

+45.5 kg

Even if these liquefaction processes are still not accepted worlwide (for instance in Europe), they should grow within the next few years. We do really believe that they are the processes of the future, and especially the process of pressing / pomace liquefaction because it is an easy process, it allows the production of quality juice combined with high yields within a great flexibility. Such high yields, low production cost and flexibility to process different fruits make that fruit juice producers are more and more choosing tlle pomace liquefaction. 2. 4. Production of traditional fl'ench cider The french cider is produced with apples from cider varieties type bitter-sweet, sour. Traditionally, the clarification of the cider was carried out by defecation: the natural demethylation of the pectin by the apple pectinmethylesterase was giving pectic acid which became insoluble after linkage with the calcium in the juice. However, the clarification was uncertain due to variable endogenous pectinmethylesterase (PME) activity of apples and variable calcium content. The process was very slow. After fermentations, tile cider was aromatic but with a low acidity and sometimes contaminations. Nowadays, this process is completely under control. It has been developped by INRA Le Rheu (Mr Drilleau) and Gist-Brocades. Rapidase CPE a purified pectinmethylesterase is added with calcium chloride allows to clarify in a shorter time and to produce french ciders of high constant quality [5]. After a few hours, there is a formation of calcium pectate after the pectin demethylation. The bubbles at the beginning of the fermentation induce a retraction of the gel which goes slowly up at the surface of the tank. This coagulum entraps micro-organisms, phenolics and main part of the natrium content. After discarding, fermentations take place at low temperature to get maximum aromas (a few weeks until 3 months). Then the cider is centrifuged, clarified by fining and filtrated, carbonated or with a prise de mousse in bottles. About 30% of french producers are using this process.

460 With the use of exogenous pectinmethylesterase, the methanol content is lower because the demethylation of the pectin is not complete, part of the methylated pectin remaining in the coagulum and because the contact time between the PME and the pectin is shorter than in the past with the natural enzyme.

3. CLEAR PINEAPPLE JUICE PROCESS The pineapple fruit is mainly processed for canning as slices or cubes. After cutting, the residual pulp is removed from the peel for cloudy juice production. Then, by-products are used to produce clear juice for slices cover or as clear concentrate. In the process of clear juice, by-products are crushed and pressed. The juice is pasteurized, cooled down and depectinized with enzymes at 50~ before ultra-filtration and concentration. This pineapple juice contains a small amount of pectin but a high hemicelluloses content type galactomannans, arabinogalactans and galactoglucomannans when insoluble parts are rich in arabinoxylans. The presence of a natural gum in pineapple juice was found. This is a neutral polysaccharide containing 70% sugars which are predominantly galactomannans (2.25 mannose: 1 galactose). Because of this gum, the ultra-filtration flux rate quickly drops down and becomes the bottle neck of the process. Gist-Brocades has developped Rapidase Pineapple which contains high galactomannanases and xylanases activities with low pectinases activities. Its application decreases the juice's viscosity, the ultra-filtration flux rate and allows to process a clear concentrate.

4. WINEMAKING The winemaker is always facing problems due to the weakness of grapes which composition is variable and different for each vintage. He tries to prevent oxidation and to work with soft conditions to preserve grapes components important for the wine's equilibrium. The sanitary state of the harvest is of first importance. Grapes composition depends on the variety, terroir, viticulture and climatic conditions. The main objective for the winemaker is to keep and valorize grape components like aromas which will determine the quality of the wine.

4.1. Enzymes in must clarification Grape's cell wall and pectin structure have been extensively described by Saulnier, Brillouet and Pellerin [7,8]. The pectin content depends on the grape variety, maturity and the technology used after the harvest. Pectin as protector colloid makes the settling of particles and the clarification very slow, related to endogenous pectinases activities of tile grape which are weak and variable. Exogenous enzymes are now widely used (i.e. 20 millions hectoliters out of 60 are treated with enzymes in France). Enzymes are used to obtain a better initial extraction of the must. They allow to increase the free run juice volume by about +20%. Pectinases are the enzymes with the most important technological effects. They enforce endogenous PME and PG of the grape, allow to extract more juice by decrease of its viscosity, to improve the clarification of the must and the wine filtration. Because the methylation percentage of the grape's pectin is lower than for the apple, polygalacturonases activities (exo and endo types) play the main role (when it is the PL for apple) and are responsible of the decrease of the must's viscosity

461 The ratio PG/PL in enzymatic preparations for enology must be higher for the grape juice than the classical preparations sold to depectinize the apple juice. In association with hemicellulases, pectinases speed up the natural process of winemaking, make the fullest use of facilities and equipment and improve the quality of the wine. The turbidity can be adjusted to 100-150 NTU to allow a good alcoholic fermentation and elimination of particles which could induce off-flavors. This shorter processing time and better clarification reduce also the oxidation of the must since the contact of the juice with grape tyrosinase bound to particles is limited. The wine well depectinised is easily filterated and has an intensive clean and fruity flavor. 4.2. Enhancement of Wine Aromas with Enzymes Aromas coming from tile grape, tile yeast or aging are essential for the final equilibrium of the wine. The wine typicity is partially due to grape aromas. This is why the winemaker tries to enhance their level, to improve the fruity and fresh character of the wine. Grape aromas a r e present in the skin and bound to sugars. Aromas bound to glycosides called "precursors of aromas" is the major part. Glycosidic precursors in tile grape berry have been described by Cordonnier, Bayonove and Gunata [8,9]. These precursors are odourless. Their content goes from 6.5 to 28 mg per liter must. However, tile ratio between different types of precursors varies greatly depending of grape varieties. Moreover, this potential of aromas under glycosidic form is very stable during the alcoholic fermentation and remains soluble and stable in wines. Other precursors of volatile compounds exist such as linalol oxides, terpenic diols and triols, linear or cyclic alcohols, norisoprenoids in C13 and acid or volatile phenols. It is possible to enhance the wine in aromatic cornpounds. Aromas release is sequential. The sequential enzymatic hydrolysis of these precursors releases free terpenols very odorant. There are mainly linalol, nerol and geraniol. In the grape, glycosides are numerous and abundant. The osidic part is formed by rutinose 6-0-ot-L-rhamnopyranosyl-13-D-glucopyranose for rutinosides, by 6-0- cz-L-arabinosyl-13-D-glucopyranose for arabinosylglucosides and by 6-0-[3-Dapiofuranosyl-13-D-glucopyranose tbr apiosylglucosides. In a first step, one ot-L-rhamnosidase, one c~-L-arabinosidase or one 13-D-apiosidase must cut the terminal sugar. In a second step, one [3D-glucosidase must release the aromatic aglycon. This can be achieved by exogenous commercial enzymes. The preparation AR2000 is tile only commercial preparation containing pectinases and these four glycosidases types. Present in the right proportions to release the major part of terpenols, these enzymes come fi-om Aspergillus niger. Wines which have been added with AR 2000 are always different from the control, contain more terpenols, terpenic diols, norisoprenoids in C13. The addition of exogenous glycosidases enhance greatly aromas in wines in relation with the aromatic potential of grape varieties. Tastings confirm that the improvement is obvious for red and for white wines. Wines are always judged more fruity and more intense. The future will be to manage tile extraction of precusors of aromas from the grape during the skin contact steps of white and red winemakings.

5. CONCLUSION Exogenous enzymes are used to produce fruit juices more easily during different stages of the process i.e. maceration, liquefaction or juice depectinisation. These biochemical tools induce specific degradations that the processor can integrate into his process line to manage and valorize the fruits transformation into juice.

462 Enzymes allow to improve the quality of the final product and the productivity in the same time. Associated to new technologies, industrial enzymes allow to give value to raw material in Food industry and to reduce the wastes quantity. They are specific tools as important as the equipment. Thanks to specialists to help us to better understand fruits composition and to associate biochemical structures to acurate enzyme activities able to answer to technical needs. Gist-Brocades as enzymes producer is associated to european fruit juice producers, INRA and Wageningen University Professor Voragen, in a Eureka project dealing with "Fruits liquefaction with specific enzymes". With the description of new enzyme activities, we will be able to create very soon new fruits derivates and new types &beverages.


1 H. Schols, C. Geraeds, M. Searle Van Leeuwen, F. Kormelink and A. Voragen, Carbohydr. Res., 206 (1990) 105. 2 Z. Gunata, C. Bayonove, C. Tapiero and R. Cordonnier, J. Food Chem., 38 (1990) 1232. 3 J.P. Vincken, G. Beldman and A. Voragen, Plant Physiol., 104 (1994) 99. 4 A. Voragen, R. Heutink and W. Pilnik, J. Applied Biochem., (1980) 452. 5 J.F. Drilleau, IAA, (1985) 885. 6 L. Saulnier, J.M. Brillouet and M. Moutounet, Conn. Vigne Vin, 22 (1988) 135. 7 P. Pellerin, E. Waters and J.M. Brillouet, Carbohydr. Polym., 22 (1993) 187. 8 R. Cordonnier, C. Bayonove and R. Baumes, Rev.Fr.Oenol., 26 (1986) 29. 9 Z. Gunata, C. Bayonove, R. Baumes and R. Cordonnier, J. Chromatogr., 331 (1985) 83.


463

Application of Tailormade Pectinases. H.P.Heldt-Hansen a, L.V. Kofod a, G. Budolfsen a, P.M.Nielsen a, S. H0ttel a'b, and T. Bladt ~'c. aNovo Nordisk A/S, Novo All6, 2880 Bagsva~rd, Denmark

Abstract Heterologous expression of pectinolytic enzymes has made it possible to tailormake pectinolytic enzyme preparations. Composition of pectinase products for existing applications can be improved, and products for new applications can be made. Apple mash treatment with galactanase, rhamnogalacturonase and rhamnogalacturonan esterase improves the cloud stability of the juice. Pectin methyl esterase is useful to gelate fruits, and the gelling effect can be enhanced by a pectin hairy region degrading enzyme combination. Rhamnogalacturonase releases soluble high molecular rhamnogalacturonans from the soy cell wall, and the use of this enzyme to produce a fibre enriched soy protein product is demonstrated. Carrot mash treatment with combinations of polygalacturonase, pectin lyase, and rhamnogalacturonase gives a puree.

1. INTRODUCTION Pectinolytic enzyme products have been commercially produced for more than two decades. These enzyme preparations are typically produced by fermentation of black Aspergilli on complex media which ensures that the pectinolytic enzymes are well expressed. The compositions of the pectinolytic enzyme preparations are optimized for their applications by strain selection, breeding, and fermentation optimization. Such enzyme preparations contain several pectinolytic enzyme activities, often with the homogalacturonan depolymerizing enzyme activities, pectin lyase, polygalacturonase and pectin esterase, present in high amounts, but also other pectinolytic enzymes like arabinanase and galactanase are often present. Furthermore rhamnogalacturonases and rhamnogalacturonan acetyl esterase have been reported from a pectinolytic enzyme preparation produced by e.g.A, aculeatus [1,2,3]. Not only pectinolytic enzymes, but also hemicellulolytic and cellulolytic enzymes are often present in such commercial pectinolytic enzyme products.

bCurrent address: Royal Veterinary and Agricultural University, B01owsvej 13, 1870 Frederiksberg C, Denmark. r address: Danish Crown, Marsvej 43, 8900 Randers, Denmark

464 The pectinolytic enzymes degrade the pectic substances of the plant cell wall. The pectic substances are characterized by a high content of galacturonic acid, which is present in homogalacturonan as well as rhamnogalacturonan [4]. In homogalacturonan long stretches of galacturonic acid are only occasionally interrupted by rhamnose, whereas rhamnogalacturonan is a polymer of alternating rhamnose and galacturonic acid often with arabinan, galactan and arabinogalactan sidechains [4]. The rhamnogalacturonan rich regions and the homogalacturonan rich regions of the pectin substances are often designated "hairy regions" and "smooth regions" respectively [5]. Pectinolytic enzyme products have been developed for numerous applications [6], most well known are mash treatment of fruits like apples and pears for higher yield of juice, mash treatment of grapes for higher colour release, clarification and depectinization of juice and wine. Depolymerization of the homogalacturonan by pectin lyase and/or by polygalacturonase and pectin methyl esterase plays an important role in such applications. Furthermore the arabinanases are known to be important to prevent the formation of arabinan haze in apples and pear juice [7]. The rhamnogalacturonan degrading enzymes have not been described to be of applicational importance. During the recent years modern biotechnology has made it possible to clone the individual components of the pectinolytic enzymes and to express them in new hosts, like Aspergillus oryzae, by the use of promoters, which do not require pectic substances for their expression. It is thereby possible in commercial scale to produce single pectinolytic enzymes, which are essentially free from most other pectinolytic enzymes. By combining such monocomponent enzymes, one can design tailormade enzyme preparations with a composition optimized for the established applications of the present pectinolotytic enzyme products. Furthermore one can design combinations very different from the present commercial enzyme preparations, tailormade for new enzyme applications. Examplewise pectinolytic enzyme preparations without homogalacturonan depolymerization activity can be made for applications where the homogalacturonan needs to be intact. The present paper demonstrates applications of combinations of cloned monocomponent enzymes, including combinations with rhamnogalacturonases, for production of cloud stable apple juice, gelation of fruits, degradation of soy cell walls, production of dietetic soy and production of carrot puree are demonstrated.

2. MATERIAL AND METHODS

2.1 Enzyme preparations The pectinolytic enzymes have been cloned from A.aculeatus. P,hamnogalacturonase B (RGase B), arabinanase, galactanase, arabinofuranosidase, polygalacturonase (PG) I and II, pectin methyl esterase (PME), pectin lyase (PL), endo-glucanase III, and endo-protease II have been cloned by expression cloning as described [2,8], whereas rhamnogalacturonan acetyl esterase (RGAE), and rhamnogalacturonase A (RGase A) were cloned by probes as described [2,9]. The genes were isolated and transformed into A. niger (for the polygalacturonases) or A. oryzae (for all the other enzymes) as described [10,11]. The transformants were fermented as described [2]. Enzyme preparations were recovered from the culture broth, and were shown to be essentially free from most interfering activities (rhamnogalacturonase, rhamnogalacturonan acetyl esterase, polygalacturonase, pectin lyase, pectin methyl esterase,

465 and arabinanase) other than the heterologously expressed enzyme. The enzyme preparations were used in all experiments, except for the degradation of soy CWM, for which purified enzymes were used (purified by ion exchange chromatography). 2.2. HPLC analysis The molecular weight distribution of enzyme digests was determined by high pressure size exclusion chromatography (HPSEC) which implied separation on three TSK gel filtration columns (POW G3500, PW G3000 and PW G2000 obtained from TosoHaas) connected in series followed by refractive index detection (RID) on a RID6A (Shimadzu). The saccharides were eluted with 0.4M Sodium acetate buffer pH 3.0 at a flow rate of 0.8ml/min using a Dionex gradient pump (Dionex Corporation). The chromatograms were processed by Dionex software AI450, and Dextran standards (Serva) were used for estimation of the molecular weight (Mw) and degree of polymerization (DP). The amount of soluble saccharide in the sample could be estimated from the area of the chromatogram. Oligomers obtained from the different substrates after enzyme digestion were separated by High Pressure Anion Exchange Chromatography (HPAEC). Oligomers were eluted from a CarboPac PAl column (Dionex Corporation) with a gradient of sodium acetate in 0.1M NaOH. Gradient mixing was controlled by the Dionex gradient pump. 25~tl were injected and eluting saccharides were detected by Pulsed Aperometric Detection (PAD) [ 12]. Rhamnogalacturonan oligomers were eluted with an acetate gradient according to Schols et al., 1994 [13]. Stachyose and raffinose were analysed according the the recommedation of Dionex [141 For the determination of monosaccharide composition enzyme digests were hydrolysed in 2M tri-flouroacetic acid (TFA) for 1 hour at 121~ followed by evaporation. The hydrolysate was redissolved in water and 25 !~1 was injected into the CarboPac PAl column. The monosaccharides were eluted with a step gradient from 0-12 min 5mM NaOH, from 12-28 min water, from 28-35 min 0.1M NaOH, and a linear gradient from 35-54 min from 0-300mM sodium acetate in 0.1M NaOH. The column was rinsed from 54-64 min with 0.5M NaOH and equilibrated from 64-70 min in 5mM NaOH. The eluting saccharides were detected by Pulsed Amperometric Detection (PAD). For calibration of the detector response standard solutions of 0.25mM, 0.5mM and lmM rhamnose, fucose, arabinose, galactose, glucose, mannose, xylose, galacturonic acid and glucuronic acid (all obtained from Sigma) were hydrolysed in TFA and analysed as described. The content of the individual monosaccharides in the enzyme digests was calculated from linear regression.

2.3. Production of cloud stable apple juice Apples (Red Belle de Boskoop, Jonagold or Mutzu) were cut and milled (1.5 mm) and 5% (w/w) of a 2% ascorbic acid solution was added immediately. Enzyme preparations (25 mg enzyme protein / kg mash) were added and the mash was incubated for 2 hours at 20~ whereafter it was pressed. The resulting apple juice was pasteurised at 85~ to discontinue further enzyme degradation. The cloud was measured as turbidity in EF/F units [15]. The cloud stability was determined by a centrifugation test as the amount of turbidity remaining after centrifugation at 4,200 x g for 15 minutes [15].

466 2.4. Gelation of oranges The oranges were washed, chopped in a meat mincer and homogenised by a Fryma mill. Water (0.6 volumes) were added before the slurry was heat treated by steam injection at 100~ for 2 minutes. The enzyme treatment was carried out for 1 hour at 40~ with 10 IU/g slurry of PME and 25 gg enzyme protein/g slurry of the other enzymes for each of the enzymes. The gelated orange slurry were treated at 85~ for 3 minutes to inactivate the enzymes before the strength of the gel was measured by a SMS Texture Analyser TA-XT2 (Stable Micro Systems, XT. RA Dimensions, Operations Manual versions) by compression analysis using a flat cylinder (20 mm dia.) with a speed of 2 mm/s. The force to provide a 20% compression was recorded. 2.5. Enzyme degradation of Soy CWM Soy cell wall material (Soy CWM) was isolated by Alcalase| treatment and jet cooking (115~ 4 minutes) of soy meal followed by centrifugation and recovery of insolubles. Aliqots of 1% suspensions of soy CWM in 0.1M acetate buffer pH 5.0 were incubated with enzymes (40gg of each to 1.5 ml of substrate) at 30~ for 24 hours and the solubilized material was analyzed by HPSEC and HPAEC. 2.6 Production of Dietetic soy. Defatted soy flour was suspended in water to 8% (v/w) protein, pH was adjusted to 6 by HC1 before 640 m g ~ Novozyme 415 (o~-galactosidase), 10 mg/L Phytase L (Novo Nordisk) and 53 mg enzyme protein/L rhamnogalacturonase B were added. After 4 hours at 50~ the pH was adjusted to 8 by NaOH, and the slurry was centrifuged. The supernatant was pasteurized (85~ 5 minutes) and freezedried. Protein was measured as 6.25 x Kjeldahl N. Phytate was measured as described in [16], and dietary fibres were analysed as described in [ 17]. 2.7. Production of Carrot puree Carrots (Boleo) were peeled by 2% NaOH at 88-96~ for 4 minutes, minced by a meat mincer (2 mm) and homogenised for 2 minutes by an ULTRA-TURRAX T25 homogenizer (from Jahne & Kunkel). The carrot mash was preheated to 45~ (20 minutes) before the enzyme preparations, 25 mg enzyme protein/kg mash, were added. The enzymes were dissolved in water to give a dilution of 5% (v/v) of the carrot mash. The mash was incubated at 45~ under stirring (60 rpm) for 2 hours, before the enzymes were inactivated at 86~ for 5 minutes in a microwave oven. Finally the purees were homogenised for 1 minute by ULTRA TURRAX. The viscosity of the puree was measured by a BROOKFIELD viscosimeter Model DV-H + with spindle A from HELIPATH SPINDLE SET at 2.5 rpm thermostated at 50~ The stability of the puree was measured as the sediment (in %) after centrifugation in 10 ml tube a 1660 x g for 10 minutes.

3. RESULTS AND DISCUSSION 3.1 Cloud stable apple juice Several combinations of pectinolytic and cellulytic enzymes were tested for their ability to give a cloudy apple juice of Belle de Boskoop apples. Selected results are shown in table I.

467 Table I Production of cloud stable apple juice from Belle de Boskoop Enzyme

Turbidity before centrifugation

Increase in turbidity relative to untreated control %

Cloud stability %

Untreated

1061 + 112

100

56 + 3

Galactanase

1212 + 28

114

77 + 24

1333 + 102

125

86+ 10

RGase RGAE

A

+

Gal

+

RGase A + Gal + RGAE + PG-II + PME

783•

74

5+

5

Average of 3 measurements. Gal: galactanase. The galactanase gives an improvement of the cloudiness and cloud stability. The cloud stability is further improved when combined with rhamnogalacturonase A and rhamnogalacturonan acetyl esterase. RGase A alone does not give any significant effect. When the cloud stabilising enzyme combination is mixed with the homogalacturonan depolymerisation enzyme combination of polygalacturonase and pectin methyl esterase, a very poor cloud stability is observed. The size distribution of the high molecule substances in the serum of the juices was studied by HPSEC, as shown on Fig 1. The HPSEC chromatograms of the sera from untreated and galactanase treated mash show a peak (A) at an apparent molecular weight of 200.000 Mw. The retention time and size of this peak is unaffected of treatment of the serum with arabinanase, galactanase and a combination of RGase A and RGAE, but disappears after treatment with a combination of polygalacturonase and pectin methyl esterase (data not shown), peak A is consequently assumed to consist of pectin. A peak (B) with an apparent molecular weight of 15.000 Mw appears in the serum after mash treatment with the combination of galactanase, RGase A, and RGAE. The retention time of this peak is unaffected of treatment of the serum by all the tested enzymes, but the size of the peak is about 35% reduced by treatment with arabinanase (data not shown). This peak is assumed to consist of rhamnogalacturonase resistant pectic structures with arab• side branches. The viscosity of both the juice and the serum increases after the mash treatment with the combination of galactanase, RGase A and RGAE (data not shown). It is assumed that the enzyme combination of galactanase, RGase A, and RGAE releases high molecular substances to the serum including the substances seen on the chromatograms as the B-peak. The high molecular substances are then assumed to increase the viscosity of the juice resulting in a stabilisation of the cloud particles.

468

+RGase A + RGAE + Gal + PG + PME

AA ~vl

AB

Ill

i ~ V ~ /"'il ++RGaseA+RGAEGa, + Gal

Untreated Uw DP

>500,000 125,000 >3,200 800

8,000 50

500 3

Fig. 1: HPSEC of serum of juice of Belle de Boskoop mash treated with enzymes. Chromatograms of the serum after mash treatment with galactananse (Gal), RGaseA + RGAE + galactanase, RGaseA + RGAE + galactanase + PG II + PME and the untreated reference are shown. The chromatogram of the serum from juice with poor cloud stability obtained after mash treatment with the enzyme mixture including the pectin depolymerisation enzyme combination of polygalacturonase and pectin methyl esterase is seen to be lacking the A-peak. High molecular pectin is therefore considered to be a prerequisite for a cloud stable juice. The cloud stabilisation effect of the enzymes depends on the apple sort. The cloud stabilisation obtained with three apple sorts with galactanase and with the combination of galactanase, RGase A and RGAE respectively is shown in Table II.

469 Table II Cloud stabilisation of three apple sorts Apple sort

Enzyme combination

Cloud stabilisation relative to untreated control %

Belle de Boskoop

Gal Gal + RGaseA + RGAE

138 153

Jonagold


105 150

Mutzu


78 115

Average of 3 measurements. Gal: galactanase None of the tested enzyme preparations gave a significant stabilisation effect on Mutzu, and galactanase did not give significant cloud stabilisation effects on Jonagold and Mutzu. In a study on apple protopectin, RGase A alone was shown to solubilize some pectic material (homogalacturonan as well as rhamnogalacturonan) and no synergism was seen with galactanase [18]. The synergistic results with galactanase and rhamnogalacturonan degrading enzymes obtained with Belle de Boskoop does therefore not seem to be general, but rather dependent on the source of substrate. 3.2. Gelation of oranges.

For production of jams and similar types of products, pectin is usually added as a gelling agent. An alternative is to let pectin methyl esterase react on the pectin containing material. This principle has been used in traditional oriental cooking, where the fruits of Ficus awkeotsang are used to form a gel. This has been shown to be accomplished by the reaction of the pectin methyl esterase originating from the red tepals with the pectin from the fruit. The hereby obtained low methoxy pectin forms a gel by natural content of calcium [ 19,20,21]. The same gelling effect can be obtained when PME is added to fruits like strawberry and oranges. The gelling effect must depend on the availability of pectin from the fruits. A mixture of possible pectin releasing enzymes was therefore tested to see whether it could improve gelling properties of oranges. The pectin releasing enzymes were a mix of pectin hairy region degrading enzymes, an endo-glucanase and a protease intended to degrade other parts of the plant cell wall than the homogalacturonan, in order to improve the availability of this part of the pectin. Oranges were chopped, minced and pasteurised, before the PME and/or the pectin releasing enzyme mixture were added to react. The texture of the orange treated with PME had a jam like appearance, with gel strength of 2 N (Table III), the gel strength was significantly improved by the addition of the pectin releasing enzyme mix. The gelling properties of pectin methyl esterase is thereby demonstrated as well as the ability of other enzymes to improve the gel strengh. The ability of the RGase and galactanase containing enzyme mixture

470 to increase the availability of the pectin might be comparable to the cloud stabilising effect of these enzymes in apple mash treatment, where pectin is believed to improve the cloud stability. Table III Gel strength of enzyme treated orange mash Gel strength (N)

Enzyme treatment None (control)

0.1

PME

2.1

Pectin releasing enzyme mix

0.1

PME + Pectin releasing enzyme mix

5.6

Pectin releasing enzyme mix: RGase A + RGAE + galactanase + arabinanase + a-arabinofuronasidase + endo-glucanase III + protease II. 3.3. D e g r a d a t i o n of Soy C W M The solubilisation of soy cell wall material (CWM) by the two rhamnogalacturonases (RGase A and RGase B) in combination with other pectinolytic enzymes were compared in order to identify enzymes for new soy processes or products. The experiments were carried out at pH 5.0, where both rhamnogalacturonases have about 25% of their maximum activity, and with high enzyme dosages to ensure that the maximum effects are obtained.

Table IV. Amount and composition of the released material from soy CWM by pectic hairy region degrading enzymes. Enzyme

Monosaccharide composition. Ratio

Released amount %

Gal A

:

rha

:

gal

:

ara

0.2

9

2

9

1

Soy CWM untreated

0

1

9

RGase A + RGAE + Gal + Ara + Araf

7 17 44 46

1 1 1 1

: : : :

1 1 1 1

: : : :

7 10 15 14

: : : :

6 7 8 8

RGase B + RGAE + Gal + Ara + Araf

37 52 48 47

1 1 1 1

: : : :

1.3 1.3 0.8 1

: : : :

14 19 15 14

: : : :

9 11 8 8

Gal: galactanase, Ara: arabinanase, Araf: t~-arabinofuranosidase

471 As seen on Table IV RGase A is only able to solubilize 7% of the CWM, whereas addition of RGAE and galactanase increases the solubilisation to 17% and 44% respectively. The solubilisation of the CWM by RGase B is less dependent on the presence of the other pectinolytic enzymes, as 37% are solubilized with RGase B alone. The ratio of galacturonic acid to rhamnose, galactose and arabinose is 1:0.2:2:1 for the untreated soy CWM, whereas the ratio is about 1:1:14:8 for the enzymatically solubilised material for most of the used enzyme combinations. The high content of galactan in Soy CWM is well known [22,23,24]. The RGase (A or B) and RGAE released material has a relatively high molecular weight, corresponding to a DP of 300 (data not shown). This polymer is depolymerized by addition of galactanase, arabinanase and arabinofuranosidase. Thus, it must be anticipated that the RGase and RGAE solubilized material is almost entirely composed of fragments of rhamnogalacturonan backbone with an almost 1:1 ratio of rhamnose and galacturonic acid and with long side chains of arabinogalactans and arabinan. The lower solubilization of Soy CMW obtained by RGase A alone compared to RGase B alone is most likely explained by a steric hindrance by the acetylation of the rhamnogalacturonan backbone and of the arabinogalactan and arabinan side branches. The explanation is verified by the monosaccharide composition which shows a lower galactose and arabinose to galacturonic acid ratio after hydrolysis with RGase A alone, compared to the ratio obtained with RGase B. Thus, RGase A preferentially cleaves in rhamnogalacturonan which is not extensively substituted with side chains. 3.4. Dietetic soy protein The ability of RGase B to solubilise soy fibres is useful for the production of a fibre enriched dietetic soy product. Soy milk has the disadvantages that it has a low fibre content and a high content of phytate and of stachyose and raffinose. To overcome these disadvantages tx-galactosidase was used to reduce the amount of stachyose and raffinose, phytase to degrade the phytate, and RGase B to release fibre material.

Table V Composition of dietetic soy product Produced with enzymes

Reference produced without enzymes

Dry matter, %

95.2

98.2

Protein, %

59.2

60.2

Dietary fibre, %

5.7

2.5

Stachyose, %

0.4

5.3

Raffinose, %

0.2

1.4

Phytate, %

0.2

1.5

472 The composition of the resulting soy product is shown on Table V, where it is seen that the protein content was unchanged, whereas raffinose, stachyose and phytate were almost removed, and the amount of dietary fibres was improved. This demonstrates that the availability of relatively pure pectinolytic enzymes, like the used RGase B, opens up for the new types of soy processes and products. 3.5. Carrot puree Enzymatic maceration, which is a softening of plant tissue by the use of enzymes, has some potential quality advantages over mechanical-thermal disintegration as maceration is obtained with less damage to the cell walls. The major part of the plant cells remains intact by enzymatic maceration [25], as the enzymes attack only the space between the cells, and with only rare injury to the cell membrane [26]. The intact cells protect nutritional components within the cells which minimise flavour changes and deterioration on storage [27,28]. Several pectinolytic and cellulytic enzymes were tested in the treatment of carrot mash for their ability to give a puree with a low degree of syneresis. The enzymes were divided into two groups: a) Pectin backbone degrading enzymes: PG I, PG II, PL, PME, and RGase B + RGAE, and b) RGase B + RGAE, arabinanase, galactanase and endo-glucanase, and all the possible enzyme combinations of each of the two groups were tested in carrot mash treatment. The ability to form a puree was evaluated subjectively as well a by the viscosity. Enzyme treated carrot mash with a viscosity lower than 2xl 0 5 cP, was found to correlate to what was subjectively classified as a nice puree-like consistency (fine structured homogeneous mass). Such a puree was obtained with 17 of the 31 enzyme combinations from group a), whereas none of the 14 enzyme combinations from group b) were able to give a puree-like consistency. The purees were obtained with combinations including either PG + PL, PG-I + RGase B + RGAE, or PL + RGase B + RGAE, which implies that either two enzymes depolymerizing the smooth regions of pectin or one enzyme degrading the smooth region and an enzyme system degrading the backbone of the hairy regions of pectin are needed in order to form a puree. A puree should preferably have a low degree of syneresis. The stability against syneresis were tested in a forced syneresis assay, where the sediment was measured after centrifugation. The results obtained with the purees with a viscosity lower than 2xl 0 5 cP is given in table VI. The most stable puree was found with combinations of either PG-I + PL or PG-I + RGase B + RGAE, whereas combinations including PL + RGase B + RGAE were found to give less stable purees.

4. CONCLUSIONS Heterologous expression of pectinolytic enzymes has made it possible to tailormake pectinolytic enzyme preparations. Such tailormade combinations without homogalacturonan depolymerizing pectin lyase and polygalacturonase activity were shown to be useful for the apple mash treatment for cloud stable apple juice and for gelation of fruits. Rhamnogalacturonase activities were found useful for these applications and for the solubilization of soy cell wall, which can be used for production of a dietetic soy product. Carrot mash treatment with combination of 2-3 pectin backbone degrading enzymes was shown to give a puree, and also here rhamnogalacturonase was shown to play a role. It is concluded that cloned pectinases can

473

be used to combine enzyme products very different from the present commercial enzymes, and that such new products can be used in applications where the present commercial enzymes have a suboptimal or no performance, like for production of cloud stable apple juice and for gelation of fruits.

Table VI. Stability against syneresis Enzyme combination

Sediment after centrifugation (1660 x g) %

PG-I + PL + RGase B + RGAE

63

PL + RGase B + RGAE

68

PL + PME + RGase B + RGAE

68

PG-I + PME + RGase B + RGAE

70

PG-I + PG-II + PME + RGase B + RGAE

70

PG-I + PL + PME + RGase B + RGAE

71

PG-II + PL + PME + RGase B + RGAE

71

PG-I + PG-II + PL + PME + RGase B + RGAE

71

PG-I + PG-II + PL + RGase B + RGAE

75

PG-II + PL + PME

76

PG-I + PG-II + RGase B + RGAE

80

PG-I + PL

81

PG-I + RGase B + RGAE

85

PG-II + PL + PME

85

8PG-I + PG-II + PL

86

PG-I + PL + PME

87

PG-I + PG-II + PL + PME

87

Standard deviation s = 2.8 (determined by multiple test with one of the enzyme mixtures).

5. A C K N O W L E D G E M E N T Thanks are due to Hanne L. Larsen, Helle F. Petterson, Susanne G. Jacobsen for skillful technical assistance, and to Gerda Jensen for secretarial assistance with this manuscript.

474 6. REFERENCES

1. H.A. Schols, C.C.J.M. Geraeds, M.F. Searle-Van Leeuwen, F.J.M. Kormelink, and A.G.J. Voragen, Carbohydr. Res., 206 (1990) 105. 2. L.V. Kofod, S. Kauppinen, S. Christgau, L.N. Andersen, H.P. Heldt-Hansen, K. D6rreich, and H. Dalboge, J. Biol. Chem., 269 (1994) 29182. 3. M.J.F. Searle-van Leeuwen, L.A.M. van den Brock, H.A. Sehols, G. Beldman, and A.G.J. Voragen, Appl. Microbiol. Biotechnol., 38 (1992) 347. 4. N.C. Carpita and D.M. Gibeaut, Plant J., 3 (1993) 1. 5. J.A. de Vries, F.M. Rombouts, A.G.J. Voragen, and W. Pilnik, Carbohydr. Pol., 2 (1982) 25. 6. A.G.J. Voragen and W. Pilnik, ACS Symp., 379 (1989) 93. 7. A.G.J.Voragen, F.M. Rombouts, M.F. Searle-van Leeuwen, H.A. Schols, and W. Pilnik, Food Hydroeolloids, 1 (1987) 423. 8. S. Christgau, T. Sandal, L.V. Kofod, and H. Dalboge, Curr. Genet., 27 (1995) 135. 9. S. Kauppinen, S. Christgau, L.V.Kofod, T. Halkier, K. D6rreich, and H. Dalboge, J. Biol. Chem., 270 (1995) 27172. 10. H. Dalboge and H.P. Heldt-Hansen, Mol. Gen. Genet., 243 (1994) 253. 11. T. Christensen, H. Woldike, E. Boel, S.B. Mortensen, K. Hjortshoj, L. Thim, and M.T. Hansen, Bio/Teehnology, 6 (1988) 1419. 12. K. Koizumi, Y. Kubota, T. Tanimoto, and Y. Okada, J. Chrom., 464 (1989) 365. 13. H.A. Schols and A.G.J. Voragen, Carbohydr. Res., 256 (1994) 97. 14. Dionex Technical Note TN 20. Dionex Cooperation. P.O. Box 3609. Sunnyvale, C.A. 94088-3603 15. S. Hamatschek, Dissertation Hohenheim (1989). 16. W. Haug and J. Lantzseh., J. Sci. Food Agric., 34 (1983) 1423. 17. AOAC 15 (1990) 985.29. 18. C.M.G.C. Renard, J.-F. Thibault, A.G.J. Voragen, L.A.M. van den Brock, and W. Pilnik, Carbohydr. Polym., 22 (1993) 203. 19. K. Komae and A. Misaki, Agric. Biol. Chem., 53 (1989) 1237. 20. K. Komae, Y. Sone, M. Kakuta, and A. Misaki, Agric. Biol. Chem., 53 (1989) 1247. 21. T.-P. Lin, C.-C. Liu, S.-W. Chen, and W.-Y. Wang, Plant Physiol., 91 (1989) 1453. 22. A.M. Stephen, The Polysaecharides (Aspinall, G.O., Ed.) Academic Press (1983) 97. 23. J.-M. Brillouet and B. Carr6, Phytoehemistry, 22 (1983) 841. 24. H.A. Sehols, G. Lucas-Lokhorst, and A.G.J. Voragen, Carbohydrates in the Netherlands, 9 (1993) 7. 25. E. von Grampp, Decliema Monographien, 70 (1972) 175. 26. K. Zetelaki-Horv/tth and K. G/ttai, Acta Alimentaria, 6 (1977) 227. 27. H. Uhlig, Swiss Food, 6 (1984) 25. 28. H.K. Streenath, F. Kogel, and B.J. Radola, J. Ferment. Technol., 64 (1986) 37.


Cations

Increase

Activity

and

Enhance

Permeation

475

of

Pectinesterase

in

Ultrafiltration Louise Wicker Department of Food Science and Technology, Food Process Research and Development Laboratory, University of Georgia, Athens, GA 30602, USA 1. ABSTRACT The effect of pH and cation concentration on pectinesterase (PE) activation and permeation on 30 kD MWCO ultrafiltration (UF) membrane was evaluated. In order of increasing effectiveness, PE activity was stimulated by monovalent and divalent cations, polyamines and trivalent cations. A similar trend was observed for permeation on UF membranes. Cation addition and higher pH releases PE from an inactive complex, increases activity, and increases permeation. Higher cation concentration decreases activity and permeation. These results suggest a common mechanism is involved in PE activation and permeation. 2. INTRODUCTION The citrus industry is a multi-billion dollar industry in the U.S. Citrus juice is highly valued as a refreshing beverage with good nutritional value, high in Vitamin C, and excellent aroma and flavor. Citrus juice (100%) has a high consumer appeal because it is one of few, processed food products that has no food preservatives or other additives. Another quality attribute of citrus juice is "cloud", which contributes mouthfeel and is due in part to the colloidal suspension of pectin, protein, lipid and hesperidin (1). Cloud loss or clarif'lcation in single strength juices appears as precipitates of insoluble material and a clear serum. Clarification is typically attributed to de-esterification of pectin by pectinesterase (PE). Formation of insoluble calcium pectate destabilizes the colloidal suspension and clarification results. In citrus juice processing, disruption of the cell wall matrix releases PE, enhances deesterification of pectin, and leads to cloud loss in single strength citrus juice and gelation of concentrates (1). In juice, PE is firmly associated with the particulates in juice (11, 15, 19) and PE activity is proportional to pulp content (18, 20, 21). Thermostable pectinesterases (TSPE), operationally defined as activity that survives 5 min at 70~ contribute most to cloud loss in juices at low temperatures and juice pH (26). The percentage of total activity that is thermostable is highly variable and differs in kinetic properties, (22, 26), ease of solubilization (28, 29), stability to low pH (25) and stability to freeze-thaw cycles (23). Some of the variability in reported total PE and TSPE may be related to limitations of current methods to quantify activity. Any processing treatment or assay condition that increases cell wall breakdown or release PE from a pectin complex would enhance detection of total and TS-PE activity. Commercially, PE is inactivated by pasteurization in a plate heat exchanger or during concentration in the TASTE evaporator.

476 Other methods to inactivate or inhibit PE have been identified, but violate standards of identity for 100% citrus juice (1,6), are highly variable (1), and change flavor (2). As a size separation process in juice processing, ultrafiltration (UF) is versatile, has lower labor costs, energy costs, and has higher ease of clean-up and maintenance (12). UF membranes come in a wide variety of materials, pore sizes and configurations. Polysulfone membranes are widely used due to chemical and physical stability, including a pH tolerance from 2-12 (8). Ultrafiltration (UF) has been used to separate juice particulates and concentrate serum (12) and to make a more flavorful juice serum (10). In citrus juice, PE did not permeate 100 kd (12) or 500 kd (10) UF membranes. The molecular weights reported for PE range from 27,000 to 54,000 (9, 14, 22, 27). Retention of PE in high MWCO membranes was attributed to fouling of the membrane by pectin (10). In this study, we show that PE permeates an UF membrane and discuss some of the biochemical parameters for PEpectate interactions, activity and permeation. 3. MATERIALS AND METHODS Marsh grapefruit (MGF) pulp was homogenized in 5 volumes of extraction buffer at 4~ and maintained at pH 8.0 (28). The homogenate was stirred for one hour, centrifuged and the supernatant used as the PE extract. Activity was measured by titration with a Brinkman (Westbury, NY) pH stat titrator at pH 7.5 and 30~ in 25 mL of 1% high methoxyl pectin (Citrus Colloids Limited, Hereford, UK) with 0.1M NaC1. PE units are expressed as the microequivalents of ester hydrolysed per minute. Uronic acid analyses were conducted based on the m-phenyl phenol (4) as modified for microplate reading (30). Activation studies were conducted at pH 7.5 at 30~ in 20 mL of 0.5 % high methoxyl citrus pectin (Citrus Colloids, Hereford, U.K.). Final cation concentration in PE extracts used for activation studies was less than 2 mM as measured by potentiometry. Controls were conducted to correct for non-enzymic alkali consumption, with no polyamine/no PE and polyamine added/no PE. PE activity was normalized as a percentage of activity with no cation addition. A bench top polysulfone hollow fiber membrane (0.0325m 2) with molecular weight cutoff (MWCO) of 30K (A/G Technology Corp., Needham MA) was used (24). UF was run in a total recycle mode at a rate of 1.2 L/min (flow speed of 0.73 m/sec), cross membrane pressure of 25 PSIG and 10 + I~ PE permeability is expressed as the fraction of PEU/mL in the permeate to PEU/mL in the retentate. Data presented are representative of at least duplicate replications. 4. RESULTS AND DISCUSSION There is strong evidence that PE-pectate complexes exist within the cell wall as well as in plant extracts. The formation of these complexes and release by cations and/or cell wall potential is thought to influence cell wall growth and extension (16, 17). The data in Fig. 1 and Fig. 2 depicts the effect on PE activity of calcium or ferric chloride and the effect of the

477 triamine, spermidine or lead acetate, respectively. In all cases, there is an initial activation followed by inhibition of activity at higher cation concentration. Not only do the cations differ qualitatively in the level of activation, they also vary in the concentration required for maximal activation as well as the range of concentrations prior to inhibition. PE activity was increased approximately 125% over a wide range of concentration by calcium (10-50 mM). Ferric chloride stimulated the activity by over 300% at approximately 10 mM, but was inhibited with a slight increase in concentration. Polyamines are naturally occurring, ubiquitous cations which are involved in cell wall regulation and growth (7). The spermidine increased PE activity about the same percentage as calcium but over a narrow range (0.5-2 mM). Likewise, lead acetate stimulated PE activity by about 175%, although to a lesser extent than ferric chloride and PE activity was inhibited within a narrow range of lead acetate concentration.

350 300 -~..4

250 200

*"~ 150 100 50 J f

0

I t

50 100 Cation conc (mM)

150

Figure 1. Effect of calcium chloride (0) and ferric chloride ( II ) on PE activity in 0.5 % pectin at pH 7.5. Qualitatively, the results observed with ferric chloride and lead acetate were highly variable. It was first noted with ferric chloride that gels were forming within the reaction vessel We observed the formation of translucent gels in the reaction vessel with calcium chloride, spermidine and ferric chloride. In the case of lead acetate, a feathery type precipitate formed in the reaction vessel. Macdonald et al. (14) observed formation of a clear gel and flocculated precipitate in high ester pectin treated with lemon endocarp and peel PE isozymes, respectively. They hypothesized that the different gel structures were due to unique mechanism of deesterification by the PE isozymes. Our results with different cations and formation of different gel structure or precipitate seems to be similar to that reported by Macdonald et al. (14). If there is a different mechanism of de-esterification for plant PEs,

478 then de-esterification and subsequent interaction with pectate may also be influenced by the type of cation present.

200 175 150 ~

125 100 75

N

50 25 0 0

2

4

6

8

10

Cation conc (mM)

Figure 2. Effect of spermidine ([3) and lead acetate ( 0 ) on PE activity in 0.5 % pectin at pH 7.5. MacDonnell et al. (15) first suggested competitive displacement for the stimulation of PE activity. An increase in activity was ascribed to competitive displacement of PE from an inactive complex followed by a decline in activity at higher concentrations due to competition for carboxylic acid binding sites between PE and cations. Competitive displacement of PE from pectic acid by cations was conclusively shown with Lineweaver-Burke plots (7). The effect was moderated by pH (7, 15). We have shown that other polyamines, including putrescine, a diamine, and spermine, a tetramine, "activate" PE activity similarly to inorganic cations (13). Ultrafiltration of heterogenous colloidal suspensions such as citrus juice is complex and many factors other than molecular weight contribute to fouling and permeation. For example, low MW aroma compounds were unevenly distributed in the permeate and retentate in UF in 500 kd MWCO system (10). The authors observed that the 500 kd MWCO UF removed all suspended solids, including pectin and PE. If PE is complexed to pectate in an inactive complex, then it is conceivable that release of PE from pectin with cations will enhance permeation in UF. At optimum salt concentration, less PE activation was observed at lower pH values than at higher pH (15). In juice systems, it is difficult to separate the effect of juice particulates on PE activity. Model studies with PE extracts allows UF in the absence of large or insoluble particulates and control of composition of the ultrafilter. In

479 these extracts, pectin was not completely removed and approximately 0.18 + 0.03 mg uronic acid/ml was present before ultrafiltration. Even this amount of pectin prevented complete permeation of PE at typical juice pH. The results presented in Table 1 summarize the effect of cation and pH on permeation of extracts of PE. Permeability is expressed as the ratio of PE in the permeate to that in the retentate. The permeability after three hours (P18o) is reported as a point of comparison. Flux was measured by timing collection of a fixed volume. The flux, LMH, after three hours 018o) is reported. At pH 8.0, P18o increased and J18o decreased with an increase in NaCL concentration. At pH 3.8, no permeability was observed at NaC1 concentrations less than 0.3M, although J18o values were higher than J18o at pH 8.0. P18o observed at pH 3.8, 0.4M NaCL was about 80% of the P~8o at pH 8.0, 0.15M NaC1. Table 1. Cations and pH Affects PE Flux and Permeability ab Permeability and Flux with NaC1 at pH 8.0 Conc., M

J18o

P18o

0.0

41 ___ 4.1

0.25 ___ 0.03

0.15

29 + 0.9

0.45 + 0.03

0.30

28 +__ 1.2

0.37 ___ 0.04

Permeability and flux with NaC1 at pH 3.8. 0.0

39 + 3.1

0

0.15

36 ___ 8.0

0

0.30

43 + 4.8

0.08 ___ 0.02

0.40

23 ___ 3.7

0.37 ___ 0.05

Permeability and flux with CaC12 at pH 3.8. 0.06

65 ___ 5.0

0.18 ___ 0.01

0.08

40 ___ 2.5

0.26 ___ 0.02

0.10

29 ___ 0.7

0.33 + 0.02

0.20

25 ___ 2.0

0.21 + 0.05

Flux

(Jt)

is described by:

Jt --" J1 -

Permeability (Pt) is described by:

b log(t). Pt =

P1 -

S log(t).

Table adapted from Snir et al. (24), with permission

480 Table 2. Permeability, flux, and uronic acid content of PE extract permeated at pH 8.0 followed by permeation at pH 3.8 a pH 8.0 time

P/R, PE

Flux, LMH

UA, R

UA, P

0

1.37

127

0.18 ___0.03

0.09 __. 0.01

5

0.62

57

0.17 ___0.02

0.11 + 0.03

30

0.92

41

0.08 ___0.02

0.06 + 0.01

60

0.56

39

0.21 _ 0.02

0.07 __+0.02

120

0.88

37

0.19 + 0.02

0.09 + 0.02

0.58

168

0.06 _ 0.01

0.03 + 0.01

0.60

137

0.03 __+0.01

0.04 + 0.00

30

0.72

113

0.02 ___0.01

0.03 + 0.01

60

1.18

101

0.02 _ 0.00

0.04 + 0.01

120

1.10

114

0.04 + 0.01

0.06 __+0.01

pH 3.8 0

P/R, PE = ratio of PE permeability; UA, R = uronic acid content in mg/mL in retentate; UA, P = uronic acid content in mg/mL in permeate. a

Table adapted from Snir et al. (24), with permission PE permeability was enhanced by CaCI2 at lower concentrations than effective for NaC1. P18o in 0.1M CaC12 was approximately 0.33, and similar to the permeability in 0.4M NaC1 at pH 3.8. There seems to be a unique cation effect rather than ionic strength effect. At equivalent ionic strength (0.3M NaC1 and 0.1M CaC12, pH 3.8), P18o of PE with CaC12, was approximately four fold higher. The concentration of cations at discrete pH values which resulted in the highest observed PE permeability was obtained at pH 8.0 and 0.15M NaC1, pH 3.8 and 0.4M NaC1, or pH 3.8 and 0.1M CaC12. An increase of pH and/or cation concentration promoted an increase in PE permeability. Conversely, flux decreased with an increase in cation concentration or pH. At similar pH and cation concentrations that stimulate PE activity by competitive displacement, permeation of PE through a 30 kD MWCO UF membrane was observed. At low pH and no salt, the low level of pectin present in the PE extracts bound PE and prevented permeation. To confirm that there was not degradation of pectin, changes in the membrane or changes in PE, UF was conducted at pH 3.8 after initial permeation at pH 8.0 (24). The

481 results are shown in Table 2. At pH 8.0, uronic acids were concentrated in the retentate, permeation of PE was high and flux was low. The pH 8.0 permeate was collected and adjusted to pH 3.8, the membrane was cleaned, and the UF was continued at pH 3.8. The permeation remained high and the flux was typical of previous UF at pH 3.8. These results indicate there is no change in the membrane, PE or pectin as a result of pH, cation concentration or membrane conditions that affects PE permeation. Polyamines act similarly to inorganic cations in UF and allow PE permeation. The addition of 0.8 mM spermidine to pectinesterase (PE) extracts significantly enhanced permeation through UF membrane at pH 8.0 and pH 3.8 (13). Membrane fouling for pH 8.0 with and without spermidine was high during the early stages of UF and remained high throughout UF (Fig 3). At pH 3.8, flux was initially greater than at pH 8.0, With or without spermidine. The initial flux near 100 LMH for pH 3.8 with spermidine was considerably higher than initial observations of 60 to 75 LMH for pH 3.8 or 8.0 alone, or pH 8.0 with spermidine. PE permeability over the UF process time was 0.22 and 0.52 at pH 8.0, and 0.18 and 0.30 at pH 3.8, without and with spermidine, respectively (13). Permeabilities were increased by higher pH and/or spermidine addition. In commercial processes, high temperatures are necessary to inactivate TSPE, which accounts for a low percentage of the overall activity (27). At 0.8 mM, the levels of spermidine are near that present naturally in citrus (3). The ability of naturally present polyamines, such as spermidine, to enhance permeation of PE may allow development of a UF process to separate PE from particulates in juice and avoid high pasteurization temperature. 120 100 80 J 60 40 ~ 20

r------o..._._

---'2---_ _......~1

! 0

50

100 150 Time, rain

200

Figure 3. Change in flux durL,ag ultrafiltration (30 kd MWCO) of PE at pH 8.0 (1"1), pH 8.0 with 0.8 mM spermidine (11), pH 3.8 (A), pH 3.8 with 0.8 mM spermidine (A).

482 An initial, steep decline in flux followed by a plateau is indicative of fouling. The generation of a pectin-pulp deposit on the membrane surface during UF of Mediterranean orange and lemon juices, was proposed to act as a dynamic membrane, becoming a UF process itself (5). Snir et al. (24) suggested a gel layer on the membrane, contributed to resistance to filtration as well as the membrane. They proposed that added cations shield electrostatic repulsion, enhancing hydrophobic interaction and aggregation in a gel layer that lowers flux. Because there is less charge on pectin at pH 3.8, they suggested that with less electrostatic repulsion, less aggregation of pectin occurred, and flux values were higher. 5. CONCLUSIONS The formation of PE-pectate complexes clearly influence PE activity and permeation in UF membrane systems. Particulates, "rag", juice sacs are rich in pectin (21). PE, if not physically entrapped, is at least bound to pectin in juice particulates in a tight electrostatic complex. In these extracts, however, there were no particulates and the pectin level was relatively low. At pH 8.0 and 3.8, a decline in permeability was observed at higher salt concentrations, suggesting a similar mechanism is involved in permeation of PE as in activation. If competitive displacement of PE from a pectate complex is the only mechanism involved in enhancement of PE activity and permeation, then complete PE permeation would be expected at higher cation concentration or higher pH values. Since there was a limit to permeation as for activation suggests that another mechanism is involved. All the cations tested stimulated PE activity to different extents and at different concentrations. A similar effect of NaC1, CaC12, and spermidine at pH 8.0 and pH 3.8 on ultrafiltration was observed as for the activation studies. There was an initial increase, followed by a decline in permeation. These results would suggest that there is a mechanism other than competitive displacement of PE from pectate which influences permeation. If there is a different mechanism of de-esterification depending on the PE isozyme (14) or cation present, then the type of PE-pectate complex that is formed may vary and that in turn will influence permeation, flux and fouling. Acknowledgements. The author gratefully acknowledges the contributions of V. Leiting and R. Snir. This research was partially supported by BARD Project No. US-2222-92R. 6. REFERENCES

.

.

Baker, R.A. 1979. Clarifying properties of pectin fractions separated by ester content. J. Agric. Food Chem. 27: 1387-1389. Balaban, M.O., Arreola, A.G., Marshall, M., Peplow, A., Wei, C.I., and Cornell, J. 1991. Inactivation of pectinesterase in orange juice by supercritical carbon dioxide. J. Food Sci. 56: 743-746, 750. Bardocz, S.; Grant, G.; Brown, D.S.; Ralph, A.; Pusztai, A. 1993. Polyamines in food-Implications for growth and health. J. Nutr. Biochem. 4: 66-71. Blumenkrantz, N.; Asboe-Hansen G. 1973. New method for quantitative determination of uronic acids. Anal. Biochem. 54: 484-489.

483

.

.

,

.

9. 10.

11. 12. 13.

14. 15. 16.

17.

18. 19. 20. 21.

22.

Capannelli, G.; Bottino, A.; Munari, S.; Ballarino, G.; Mirzaian, H.; Rispoli, G.; Lister, D.G.; Maschio, G. 1992. Ultrafiltration of fresh orange and lemon juices. Lebensm. Wiss. Technol. 25:518-522. Castaldo, D., Lovoi, A., Quagliuolo, L., Servillo, L., Balestrieri, C. and Giovane, A. 1991. Orange juices and concentrates stabilization by a proteic inhibitor of pectin methylesterase. J. Food Sci. 56: 1632-1634. Charnay, D.; Nari, J.; Noat, G. 1992. Regulation of plant cell-wall pectin methyl esterase by polyamines -Interactions with the effects of metal ions. Eur. J. Biochem. 205: 711-714. Cheryan, M. Ultrafiltration Handbook. 1986. Technomic Publishing, Lancaster, PA. Evans, R.; McHale, D. 1968. Multiple forms of pectinesterase in limes and oranges. Phytochemistry 17: 1073-1075. Hernandez, E.; Chen, C.H.; Shaw, P.E.; Carter, R.D.; Barros, S. 1992. Ultrafiltration of orange juice: Effect on soluble solids, suspended solids, and aroma. J. Agric. Food Chem. 40: 986-988. Jansen, E.F.; Jang, F.; Bonner, J. 1960. Orange pectinesterase binding and activity. Food Research 25: 64-72. K6seoglu, S.S.; Lawhon, J.T.; Lusas, E.W. 1990. Use of membranes in citrus juice processing. Food Technol. 44: 124-130. Leiting, V.; Wicker, L. 1994. Polyamine interaction with Marsh grapefruit pectinesterase. Abstract No.: 11-7. Institute of Food Technologists, 53th Annual Meeting, Biotechnology Division Graduate Oral Paper Competition, Atlanta, GA, June 23-27, 1994. Macdonald, H.M.; Evans, R.; Spenser, W.J. 1993. Purification and properties of the major pectinesterases in lemon fruits (Citrus limon). J. Sci. Food Agric. 62: 163-168. MacDonnell, L.R.; Jansen, E.F.; Lineweaver, H. 1945. The Properties of Orange Pectinesterase Arch. Biochem. 6: 389-401. Moustacas, A.-M.; Nari, J.; Borel, M.; Noat, G.; Ricard, J. 1991. Pectin methylesterase, metal ions and plant cell-wall extension. The role of metal ions in plant cell-wall extension. Eur. J. Biochem. 279: 351-354. Nari, J.; Noat, G.; Ricard, J. 1991. Pectin methylesterase, metal ions and plant cellwall extension. Hydrolysis of pectin by plant cell-wall pectin methylesterase. Eur. J. Biochem. 279: 343-350. Rouse, A.H. 1951. Effect of insoluble solids and particle size of pulp on the pectinesterase activity in orange juice. F1. State Hort. Soc. Proc. 64: 162-166. Rouse, A.H. 1953. Distribution of pectinesterase and pectin in component parts of citrus fruits. Food Technol. 7: 360-362. Rouse, A.H.; Atkins, C.D.; Huggart, R.L. 1954. Effect of pulp quantity on chemical and physical properties of citrus juices and concentrates. Food Technol. 8:431-435. Rouse, A.H.; Atkins, C.D. 1955. Pectinesterase and pectin in commercial orange juice as determined by methods used at the Citrus Experiment Station. Florida Agric. Exper. Station Bull. 570: 1-19. Seymour, T.A.; Preston, J.F.; Wicker, L.; Lindsay, J.A.; Marshall, M.R. 1991a. Purification and properties of pectinesterases of Marsh white grapefruit pulp. J. Agric. Food Chem. 39: 1080-1085.

484 23.

24.

25.

26.

27.

28.

29. 30.

Seymour, T.A.; Preston, J.F.; Wicker, L.; Lindsay, J.A.; Wei, C.; Marshall, M.R. 1991b. Stability of pectinesterases of Marsh white grapefruit pulp. J. Agric. Food Chem. 39: 1075-1079. Snir, R.; Koehler, P.E.; Sims, K.A.; Wicker, L. 1995. pH and Cations Influence Permeability of Marsh Grapefruit Pectinesterase on Polysulfone Ultrafiltration Membrane. J. Agric. Food Chem. 43: 1157-1162. Versteeg, C. 1979. Pectinesterases from the orange fruit - Their purification, general characteristic and juice cloud destabilizing properties. Ph.D. thesis. Agricultural Univ., The Netherlands. Versteeg, C.; Rombouts, F.M.; Spaansen, C.H.; Pilnik, W. 1980. Thermostability and orange juice cloud destabilizing properties of multiple pectinesterases from orange. J. Food Sci. 45: 969-973. Versteeg, C.; Rombouts, F.M.; Pilnik, W. 1 9 7 8 . Purification and some characteristics of two pectinesterase isoenzymes from orange. Lebensm. Wiss. Technol. 11: 267-274. Wicker, L.; Vassallo, M.; Echeverria, E. 1988. Solubilization of cell-wall-bound, thermostable pectinesterase from Valencia oranges. J. Food Sci. 53:1171-1174 and 1180. Wicker, L. 1992. Selective extraction of thermostable pectinesterase. J. Food Sci. 57: 534-535. Wicker, L.; Leiting, V.A. 1995. Microscale Galacturonic Acid Assay. Anal. Biochem. 157: L14-N.


485

Production, Clmmcted~tion and Application of RhanmoC~acturonase H. Hennink, H. Stam and M~G. van Oort. Quest International, P.O. Box 2, 1400 CA Bussurn, The Netherlands.

Abstract In this paper an overview is given of all the work that is performed on the enzyme RhamnoC~acturonase by different groups (Quest Naarden, URL-Vlaardingen and LUW). The project was focussed on different areas conceming molecular biology, fermentation technology and biochemical characterization of rhamnogalacturonase. Also the functionality of this enzyme in different application areas was studied.

1. INaRODUCIION The use of pectolytic and cellulolytic enzyme has become an indispensable part of fruit juice processing in the last two decades. In this application enzymes improve the extraction yield, resulting in higher yields of juice. These enzymes can also cause viscosity reduction which improves filtration and clarification of the fruit juices and prevent haze formation. Such commercial enzyme preparations comprise a mixture of mainly pectinases together with quantities of other hydrolytic enzymes such as arabinases, cellulases and xylanases. One of the main substrates for these er~mes is pectin, a complex heteropolysaccharide which is a major component of the cell wall of higher plants. It is proposed that pectin consists of highly carboxyl-methylated linear homogalacturonan regions which altemate with the so-called hairy regions consisting of a rhmnnosegalacturonic backbone(RG) with arabinose rich side chains (Voragen 1989) (figure 1). Pectin degradation requires the combined action of various ertzymatic activities. However, evaluation of the contribution of individual pectinases in fruit juice extraction and clarification is rather complicated. Most commercial pectinolytic enzyme preparations are produced by fermentation with filamentous fungi, mostly strains belonging to the genus Aspergillus. Application studies with mixtures of isolated er~mes obtained by fermentation or by means of fractionation of commercial et~me preparations can be used to assess the importance of the various individual enzymes. Subsequently, molecular biology and fermentation technology can be used to enhance specific desirable enzymatic activities.

486

p I=

Ara I Ara I Ara-- Ara--- Ara I EA ~Alra] N

r'-- //P--I~A A[a| IOMe/./ | OMe Gal

RGase~

'smooth region'

'hairy region'

r

Ara I Ara-- Ara

IA~a

IPL

cl/FAE 1 Ara OA Me Oi i

EA:endo-arabinase PE"pectine-esterase PG:polygalacturonase AF:exo- arabinofuranosidase RGase:rhamno-galacturonase PL:pectin-lyase

'smooth region'

AE: pectine-acetylesterase PA:pectate-lyase

Figure 1. Schematic representation of the structure of the smooth and hairy regions of pectin, including the various pectinases. 1.1 RHAMNOGALACH~ONAN AND RHAMNOGALACHSRONASE A cell-wall polysaccharide fraction remaining after treatment with a "classical" pectinase is termed rhamnogalacturonan (RG) or modified hairy regions (MHR). It is characterized by a highly branched rhamnogalacturonan-polymer with some arabinan side chains. In 1990 Schols et al reported the purification of an enzyme which was capable of degradation of the hairy regions prepared from apple juice : RhamnoGalacturonase(RGase). This enzyme can split glycosidic linkages in the rhamnogalacturonan backbone of (apple)pectins producing a range of oligomers composed of galacturonic acid, rhamnose and galactose. Rhamnogalacturonase may be useful in the prevention of haze formation in apple juice concentrates. In combination with other enzymes it might improve liquefaction, resulting in increase juice yield and clarification.

487 1.2. EVALUATION OF THE FUNCIIONAIIIY OF RHAMNOGALACIURONASE In order to test the application of rhamnogalacturonase(RGase), a commercial enzymepreparation was separated into several fractions by ionexchange-chromatography (see figure 2). Fractions were screened on rhamnogalacturonase-activity. The active fractions were tested in apple-application tests. From application trials it was clear that addition of RGase containing fractions when used in combination with other pectinases has a beneficial effect on the apple-juice process.

Separation of commercial preparation

Ion-Exchange Chromatography, OD 280 nm OD 543 nm 0.4 100 L

80 60 40

20 0

i /,\

0.3

i' /"/i / '/

0.2

Protein-pattern RGase-activity

'/ \

0.1

i\

/\

/ Fractions

\ ,,\,,\ 0

Figure 2 Fractionation by ion exchange chromatography of a commercial pectinase preparation.

488 2. MATERIALS AND METHODS

2.1. Detection RhamnoGalacturonase activity. RhamnoGalacturonase activity was determined by following procedures :

Westem blotting Products were analysed by SDS-PAGE followed by Westem blotting (Bumette 1981). RGase antibody production is described by van der Veen et al. 1991 This method can be used to determine the presence of RGase in different enzymepreparations and culture-filtrates. The disadvantage of this method is that only presence, but not activity of the enzyme is monitored.

RhamnoC~actumnase-activity on isolated rhamnogalacturomn. For the isolation of modified hairy regions (see figure 3) the method of Schols et al. (1990a, 1990b) was used. Golden Delicious apples were crushed and treated with a pectinase-preparation without detectable RGase activity. After centrifugation the supematant was ultrafiltered in a microfiltrationsystem (cut off 50.000D) and the residue was dialysed and lyophilized. To determine the presence of RGase in the various preparations isolated RG was treated with these products. After incubation, the formation of reducing end-groups was measured by the DNS-method.

Breakdown products characterization by HPI~-Dionex chmmatogml~. The Dionex system uses a Carbo Pac PA-1 anion exchange column and a CarboPac PA-1 Guard. The column was loaded with 25 pl of the RG solution and eluted with a linear gradient of 0 - 0.5 M NaOAc in 0.1 N NaOH during 50 minutes. The flow rate was 1.0 ml/min and the process was monitored using a PE detector.

Application-trials Apples were chopped and mashed to a free puree. Apple mash was treated with enzyme preparation and incubated for 2 hours at 55~ Viscosity of mash was measured several times using a Brookfield DC3 viscometer with Helipath stand attachment and TD spindle. After two hours of incubation sample was centrifuged for 20 minutes at 10.000 rpm. Volume, clarity, pH and brix of the juices were measured. The pectin level of the juices was assessed by a standard alcohol test.

489

RHAMNO-GALACTURONAN ISOLATION

................................... ("Hairy regions") Apples (Golden Delicious)

I !

Rasp Mill Mash Liquefaction

(Pectinase mix 0.05%, 4 i~r a~ 5C:r',.,_; Centrifugation ...........................,,~- waste putD

l

Ultra Filtration (50kD) ~

t

Dialysis

permeate

LMW

t 1 Lyophilization

Centrifugation

I

Rhamno-Galacturonan (RG)

Figure 3 Isolation-process of rhamnogalacturonan. 2.2. Identification of Rhamnogalactumnase producing Aspergillus straim and lhe development of a RGase production process by lhe wild type strain A.aeuleatus 115.80. Various Aspergillus strains were cultured on rich medium (sugar beet pulp) in shake flasks and examined for RGase production. Westem blots were analysed for crossreactivity with antibodies raised against purified RGase. Various strains of Aspergillus showed 1 distinct band after hybridisation with rhamnogalacturonase antibodies, with molecular weights ranging from 51.000 D - 63.000 D. The highest RGase production as judged by Westem analysis was obtained after cultivation of A. aculeatus and further research was focused on A. aculeatus CBS 115.80.

490 To improve production of rhamnogalacturonase by Aspergillus aculeatus CBS 115.80 shake flask experiments were performed on several substrates. Cross reactivity was found after transfer to rhamnose in combination with galacturonic acid and on apple pectin, citrus pectin, beet pectin and sugar beet pulp. No cross reactivity was found after transfer to media containing simple carbon sources such as sucrose, glucose, fi'uctose, rhamnose or galacturonic acid. The process developed on lab scale was transferred to pilot plant scale. The process is a fed-batch fermentation with a growth-phase (24h) on complex sugars and a production phase (48h). During production phase a linear feed is added containing various carbohydrates. Activity of Rhamnogalactumnase from A. aculeatus CBS 115.80.

Addition of fermentation broth from Aspergillus aculeatus CBS 115.80 to the isolated substrate resulted in an extinction increase measured by DNS-method.. Analysis of degradation products by Dionex (HPAEX) (see figure 4) shows a large peak of galacturonic acid and other free sugars. However the chromatogram described by Schols et a1.(1992) differs from the profile as described in figure 4 in this report. They found various oligomers after incubation of rhamnogalacturonase with RG suggesting a partly degradation of the substrate whereas in this work a predominant peak of galacturonic acid is found indicating a more complete breakdown of the substrate. This suggest that the fermentation broth obtained after cultivation of A. aculeatus on pectins contains beside the known RGase activity other activities capable of breaking down the oligomers formed after RGase treatment. In the following sections we describe the isolation of the rhamnogalacturonase (rhgA) gene from Aspergillus aculeatus and the construction of Aspergillus strains which are capable of producing RGase in larger quantities. 2.3. Isolation of the d g ~ gene from A.aculeatus.

A.aculeatus CBS 115.80 was grown on apple pectin in a fermenter and strong induction of Rgase production could be observed. Total RNA was isolated from such an RCrase producing culture and used for the construction of a eDNA library. In this eDNA library the eDNA is placed under the control of an inducible promoter. After induction of this promoter transcription of eDNA will occur, resulting in the synthesis of proteins. Since the eDNA was synthesised from mRNA isolated fi'om a culture induced for RGase synthesis, the library is "enriched" with eDNA coding for RGase. RGase producing colonies can be detected using the antibodies raised against RGase. In this way eventually 4 colonies that scored positive with RGase-antiseaun were characterized further at DNA level, eDNA inserts from positive colonies were sequenced completely in both directions and were found to comprise the entire coding region of the rhgA gene. The amino acid sequence of the protein was derived from the coding region of the rhgA gene.

491 Regions encoding amino acid sequences corresponding to amino acid sequences of peptide fragments of isolated RGase were encountered within the gene, positively identifying the cloned cDNA as corresponding to A. aculeatus rheA. This clone was used as a probe to screen a genomic library of A. aculeatus, resulting in the isolation of the entire genomic A. aculeattts rhgA gene including the rhgA promoter region (3). Comparison of the genomic DNA sequence of the rhgA gene with the cDNA sequence identified the position and size of three introns. The rhgA gene is encoding a protein comprised of 440 amino acids with a deduced molecular weight of 46kD.

2.4. Ovelpmduclion of RGase In order to construct an A. aculeatus strain which is capable of overproducing RGase, multiple copies of the A. aculeatus rhgA gene were introduced into a suitable acceptor strain by co-transformation with the A. aculeatus pyrA gene (auxotrophic selection marker). A pyrA negative mutant from A. aculeatus, obtained by classical mutagenesis was co-transformed with a DNA fragment consisting of the rhgA gene including the rhgA promoter and a DNA fragment containing the pyrA gene from A.__,. aculeatus. Transformed strains were selected by their ability to grow in the absence of uridine. A selected number of transformants was analysed for co-transformation. Transformants which had incorporated one or more additional copies of the A~ aculeatus rhgA gene were identified by quantitative Westem analysis of the culture supematant after shake flask experiments as described above. In this way overproduction of A.aculeattts RGase could be demonstrated in a number of transformants. Since transformation was performed with DNA fragments instead of entire plasmids, the transformed strains contain only DNA derived from A. aculeatus (no vector material). To further increase the production level of rhamnogalacturonase the rhgA gene from A. aculeatus was fused to the endoxylanase promoter e(e__xlA)from A. awamori which has been isolated during the cloning of the endoxylanase gene (see below). This fragment was used to transform A. awamori strains again making use of the pyrA system (in this case from A. awamori) as selection marker. Transformants, selected by their ability to grow in the absence of uridine, were grown in shake flasks in the presence of xylose. Since xylose acts as an inducer for the endoxylanase promoter, expression of the rhgA gene in strains containing the rhgA gene fused to the exlA promoter will occur. Very high levels of RCrase could be efficiently produced in A. awamori using the exlA promoter.

Activity of Rlmmnogalacturonase produced by mcomi~nant strains. Westem blotting of above mentioned recombinant strains shows a clear increase of rhamnogalacturonase production compared to the wild strain. To check the activity, fermentation broths of the recombinant strains were added to the isolated substrate resulting in a small increase of the extinction. This is due to the fact that rhamnogalacturonase splits the substrate in a range of oligomers which have less effect on the DNS-value compared to the products formed by the wild strain which breaks RG into very small components.

492 Therefore this method can not be used to compare rhamnogalacturonase activity present in the wild strain and the recombinant strains. Dionex analysis of hydrolysis products of RG by these recombinant strains (see figure 4 ) shows the formation of various oligomers as published by Schols et a1.(1992). This proves our observation that the wild strain contains side activities which are responsible for a complete breakdown of RG. From these results it was concluded that the DNS method can be used as a method to optimize and follow the production-process of RGase by the wild strain. Dionex analysis is a technique which provides us to control the production of the fight ertzyme but does not give any indication about activity. Therefore apple-application experiments were performed to detem~e the amount of rhamnogalacturonase which is necessary for an optimal perfonmnce. With these application-experiments an impression could be obtained about the differences in RGase activity of the various strains.

RG BREAKDOWN

ANALYSED

BY DIONEX.

RG BLANCO

tl

15

20

25

t l j t t l i 35

30

40

Minutes

u

r

|

[

!5

i

1

i

I

t

20

t

t

9

i

I

RG+ACCULEATUS

i

i

t

i

25

i

i

30

i

|

i

i

i

i

35

i

|

i

40

Minutes

i 15

RG+AWAMORI 20

25

30

35

Minutes

Figure 4 Dionex patterns of RG treated with different strains.

493 APPLICATION-EXPERIMENt.

Apple application experiments for producing apple juice show a clear benificial effect when rhamnogalacturonase in combination with a pectinase was added to the process. As can be seen t~om figure 5 rhamnogalacturonase has a clear positive influence on viscosity-reduction. Rhamnogalacturonase also prevents formation of haze in juices and therefore gives a stable juice. Other processes wherein rhamnogalacturonase can play an important role are * Other Fruits - Improve yield and clarity. * Beer - Improve clarity and filtration.

All these applications are subject of study at the moment.

APPLE MASH VISCOSITIES VISCOSITY CPS Thousands 40r"

[] pect.mix O pect.mix + RGase WT 9 pect.mix + RGase A.ac 9 pect.mix + RGase A . a w

--,n

10| I 30

. . . . . . .

I ,,, 60

~o

J

120 Time/min

Figure 5 Influence of rhamnogalacturonase of different strains on viscosity-reduction in apple-application-experiments.

494

REFERENCT.S 1 A.G.J. Voragen,(1989) Food enzymes: Prospects and limitations In: Roozen J.P., Rombouts F.M., Voragen A.G.J.(eds): Food science: Basic research for Technological Progress, PUDOC, Wageningen, The Netherlands. 2. H.A. Schols et al., RhamnoGalacturonase: a novel enzyme that degrades the hairy regions of pectins. Carbohydrate Research (206) 105-115. 3. P.van der Veen et a1.,(1991), Induction, purification and characterization of arabinases produced by Aspergillus niger. Arch.Microbiol.(157)23-28. 4. WN. Burnette (1981) "Western Blotting": Electrophoretic transfer of proteins from Sodium Dodecyl Polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal.Biochem. (112): 195-203.

J. Visser and A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996Elsevier Science B.V.All rights reserved.

495

Effect of a new canning process on cell wall pectic substances, calcium retention and texture of canned carrots H. Siliha% W. J a h n b and K. Gierschner b aDepartment of Food Science, Faculty of Agriculture, Zagazig University, E g y p t bDepartment of Fruit and Vegetable Technology, Institute of Food Technology, H o h e n h e i m University, Stuttgart, Germany Abstract

A new process was developed for the canning of carrots. The main features of this process are: filling the cans with carrots and a minimum amount of brine (ca.8.0% of the canned carrots), vacuum sealing, preheating at a certain temperature and time (67~ for 30min) followed directly by sterilization. Quality attributes of the resultant canned carrots were supelior to that conventionally canned in respect to texture, colour, flavour and nutritional value. In this research the effect of preheating time (15, 30 and 60 min) followed by sterilization as well as the comparison between the new and the conventional canned carrots was studied with regard to the texture, Ca *+ retention and cell wall pectic substances. Texture of canned carrots was improved when the time of preheating was increased, while the amount of Ca+* bound in the cell wall was not significantly different from that of fresh carrots. Degree of methylation(DM; measured by HPLC) of total pectin reduced when the time of preheating was increased. This demethylation was reflected on the DM of the oxalate-soluble pectin, while t h a t of the water-soluble pectin remained unchanged. Conventional blanching did not alter the DM of total pectin which was lowered after sterilization. Texture of conventionally canned carrots was considerably lower than t h a t produced by the new process. Addition of CaC12 resulted in similar improvement in texture of carrots canned by both processes. However, the amount of Ca** retained in the cell wall material of conventionally canned carrots was twice as much as that found in carrots canned by the new process. Both canning processes resulted in the conversion of the major part of acid- and alkalisoluble pectin into water- and oxalate-soluble pectin. This conversion was accompanied by alteration in DM, degree of acetylation and in the a m o u n t of individual n e u t r a l sugars. The results suggest that the improvement in texture of canned carrots can be partly ascribed to the activation of the native pectinesterase during preheating, consequently the quantity of free carboxyl groups increased which were cross-hnked with Ca**. Additionally, the improvement in texture was correlated with higher amount of alkali soluble pectin fraction.

496 1. I N T R O D U C T I O N Cell wall and middle lamella polysaccharides determine to a large extent the texture of fresh, stored and processed vegetables and fruits. Their structural modification by cell wall modifying enzymes, either endogenous or exogenous, and by heat treatments during industrial processing have a great impact on the texture of the resultant products. In vegetables and fruits canning i n d u s t r y h e a t processing causes excessive degradation in cell wall and middle lamella polysacchrides which results in an extreme soft texture (Stolle-Smits et a1.,1995). Therefore, informations concerning the course of these modifications are of prime importance for the understanding of the mechanism of the softening reactions and thus for selecting the proper process for retarding them. Cell wall polysaccharides consist of pectins, hemicelluloses and cellulose (Keegstra et a1.,1973 ; Carpita and Gibeaut, 1993) while the middle lamella consists predominantly of pectic polysaccharides cross-linked with Ca ++ (McCann and Roberts, 1991). Due to their high water binding capacity and gel properties pectic polysaccharides have direct correlation with tissue firmness (Jarvis, 1984). Thermal softening of fruits and vegetables appears to occur through two different pectin degrading reactions depending on the pH of the tissue (Van Buren, 1986). As early as 1965, Doesburg proposed that softening below pH 4 is consistent with an acid catalyzed cleavage, while at pH levels over 5, the normal conditions for vegetables, softening is consistent with a pectin depolymerization reaction that has the characteristics of a 13-elimination reaction catalyzed by hydroxyl ions and inhibited by demethylation of the pectin. Several attempts have been made to improve the texture of canned carrots. These include adding calcium (Sterling, 1968), changing the pH during processing (Van Buren and Pitifer, 1992) and modifying the blanching treatment before sterilization (Lee et al., 1979 ; Gierschner and Philippos, 1992 ; QuinteroRamos et al., 1992). These authors found that preheating of carrots at low temperature for long time resulted in firmer texture. The firming effect was attributed to the activation of the native pectinesterase which increases the number of free carboxyl groups on the pectin molecules, thus enabling them to be cross-linked with Ca *+ naturally present in the tissue. Interactions between Ca ++ and cell wall pectin play a key role in stabilizing the wall structure (Demarty et al., 1984) The structural features of cell Wall polysaccharides of carrots have been studied by Stevens and Selvendran (1984) and Massiot et a1.(1988). Plat et a1.(1991), Ben Shalom et a1.(1992) and Massiot et a1.(1992) investigated the changes in pectic substances of carrots after blanching, dehydration and extended heat treatment. Data on the changes in cell wall polysaccharides of canned carrots are lacking. This study aims to investigate the effect of preheating time at low temperature and the addition of CaC12 on texture and on the composition of various pectin fractions of carrots canned by conventional and by a new process.

497

2. M a t e r i a l s a n d M e t h o d s 2.1. C a n n i n g of c a r r o t s : Carrots (Daucus carota, var. carotan) were obtained from Ritz farm (Linkenheim/Baden-Germany). After washing, trimming and peeling the carrots were cut into cubes of 1 cm 3 by means of a cutting machine(Master, Nr. 3695). They were then screened to remove the undersized cubes. The carrot cubes were soaked in distilled water at 40~ for 2 min and allowed to drain. Two h u n d r e d and fourty grams of the cubes and 19.5 ml of distilled water were filled into the can which was then vacuum sealed (0.3 bar). The cans were put in rotary retort (Stock Pilot-Rotor 900, Germany) which was programmed to carry out the preheating and sterilization steps sequentially. Preheating treatments were performed at 65~ for 15, 30 and 60 min. Sterilization was carried out at 12 I~ (Fo=3.5). For investigating the effect of CaC12, the carrot cubes were soaked in and filled with 0.2%CaCI~ .The cans were preheated at 67~ for 30 min and sterilized. As for the conventional canning the carrot cubes were blanched in water at 85~ for 2 min, then 240 g of the cubes and 170 ml distilled water with or without 0.2% CaC12 were filled in the cans and sterilized as described earlier. 2.2. T e x t u r e m e a s u r e m e n t s : Texture of canned carrots was measured using Instron Universal Testing Machine (Model 1011) fitted with Kramer shear cell. Thirty grams of drained carrot cubes were evenly placed in the Kramer shear cell and were compressed, sheared and extruded using a crosshead speed of 100 ram/rain. Each measurement was repeated 10 times and the mean was used to express the firmness of carrot cubes in Newton(N).

2.3. P r e p a r a t i o n of the a l c o h o l i n s o l u b l e solids (AIS): The content of the can was drained and the carrot cubes were immediately frozen in liquid nitrogen, freeze-dried and milled. Carrot powder (ca. 10 g) was mixed with 200 ml 80% ethanol previously heated to 60~ After filtration the residue was extracted with ethanol until the filtrate was colorless (5 times) and gave negative reaction with phenol-sulfuric acid test (Dubois et al., 1956). 2.4 F r a c t i o n a t i o n of cell w a l l p o l y s a c c h a r i d e s : One gram of AIS was suspended in 100 ml distilled water at 40~ for 45 rain. The slurry was centrifuged at 15000xg for 20 rain and s u p e r n a t a n t was decanted. This step was repeated one more time. Both s u p e r n a t a n t s were combined and represented the water- soluble pectin (WSP). The pellet was sequentially suspended in 0.5% ammonium oxalate(at 40~ 0.05N HC1 (at 70~ and 0.05N NaOH (1~ Each extractant(100 ml) was applied two times (each 45 rain) followed by two times washing with distilled water. The extracts and the washing water were combined and represented, respectively, oxalate-soluble pectin (OXSP), acid-soluble pectin (I-ISP) and alkali-soluble pectin (OHSP). The pectin solutions were dialysed, concentrated under reduced pressure at 40~ and freeze-dried. The pellet was extracted with 4M NaOH (containing 10 mg sodium borohydride/ml) for 8 hrs at room temperature, followed by centrifugation.Extraction was repeated two times

498 and the extract was acidified with acetic acid to pH 5.2, dialysed and freeze-dried (hemicellulose). The remaining pellet was dialysed and freeze dried (celluose). 2.5. A n a l y t i c a l m e t h o d s : Anhydrogalacturonic acid (AUA) content was determined by the metahydroxydiphenyl (mHDP) method (Blumenkrantz and Asboe-Hansen, 1973). Degree of methylation (DM%) and degree of acetylation (DAc%) of the pectin fractions were measured by HPLC analysis (Voragen et al., 1986) using a Gynkotek-HPLC system equiped with Aminex HPX 87P column (25cm x 4mm i.d., Bio-Rad) and with refractive index detector (Shodex RI SE61). For the determination of ash and minerals content the AIS was ashed at 550~ The minerals (Ca ++, Na § K § and Mg +§ analyses were performed with PerkinElmer 3030 atomic absorption spectrophotometer. Neutral sugars content of the alcohol insoluble solids and the pectin fractions were determined as their alditol acetates after hydrolysis with sulfuric acid (Seaman et al., 1963) and trifluoroacetic acid, respectively. Derivatization was performed according to Englyst and Cummings (1984). The alditol acetates were separated with GC-14 gas chromatograph (Shimatzu) equiped with DB-225 capillary column 30m x 0.25mm (J & F Scientific)and FID-detector. Molecular size distribution of the pectin fractions was determined by HPLC. The pectin fractions were dissolved in 0.4M acetic acid/sodium acetate buffer (pH 3) containing 0.025M sodium sulfate. The high pressure size exclusion chromatography system (HPSEC)included a Gynkotek M480G pump, GINA 160 injection system, column thermostat at 40~ and Shodex RI SE61 refractive index detector. TSK columns GMPWXL (30cm x 7.8mm) and G6000 PWXL (30cm x 7.8mm)were used in combination with a guard column PWXL. Columns were eluted with 0.4M acetic acid/sodium acetate buffer pH 3 containing 0.025M sodium sulfate at a flow rate of 0.6 ml/min.

3. R E S U L T S AND DISCUSSION 3.1. Effect o f p r e h e a t i n g t i m e on f i r m n e s s a n d p e c t i c p o l y s a c c h a r i d e s Firmness of canned carrots preheated at 65~ for 15, 30 and 60 rain is illustrated in Fig. 1. Preheating for 15 rain showed a firmness of 261.6N. When preheating time was increased higher tissue firmness was observed. Similar observations have been reported by Lee et al., (1979) and Quintero-Ramos et al., (1992) who found that firmness of carrot tissues was increased with increasing the time of blanching at 65~ ' Degree of methylation (DM%) of total pectin of carrot alcohol insoluble sohds (AIS) was decreased from 60.73% for fresh carrots to 48.70, 44.62 and 43.83% for canned carrots preheated at 65~ for 15, 30 and 60 rain, respectively (Fig. 2). Similar levels of demethylation were also reported in potato (Bartolome and Hoff, 1972), in carrots (Lee et al., 1979) and in snap beans (Adams and Robertson, 1987) when they were blanched at low temperature between 65~ and 70~

499 400 35O 300 250 200 150 iZ 100. 50, 0

313 9

~
A A A

l

1

0 [Me2+]t / C

P

1

2 [Me2+]t/C

P

Figure 1. Effect of metal ions binding on the release of protons into the medium for sugar-beet (A) and citrus (B) pectins at 2 mequiv. COO-.1-1, at pH~5.1 in 0.1 M NaNO3 at 25~ (0) Cu2§ (#) Pb2§ (0) Zn2§ (A) Cd2+, (v) Ni2+, (11) Ca2+.

537 As shown in figure 1, the same two groups of metal ions may be distinguished for both pectins. A first one was composed of Ca2+, Ni2+, Cd2+ and Zn2+, for which a small and quasilinear pH-variation was found. Cu2+ and Pb2+, composing the second group, displayed a much higher pH variation, reaching a plateau in the final stages. The same scale of selectivity was therefore obtained whatever the pectins, decreasing as follows: Cu2+ ~ Pb2+ >> Zn2+ > Cd2+-Ni2+ > Ca2+.

3.2 Quantification of the binding The experimental results obtained by measuring ions activity after equilibration with pectins are plotted as binding isotherms [Me2+]b/Cp vs [MeZ+]dCp where [MeZ+]b is the bound cation concentration at equilibrium (equiv.l-1) calculated from measured activity using previously calculated activity coefficients.

[pb2+]b / Cp 1.2 0.81-

/_~0

04L 0

0.4

0.8 1.2 [pb2+]t / Cp

Figure 2. Influence of the ionic strength and the polymer concentration on the binding isotherms of Pbz+ by sugar-beet pectins in water (empty symbols) and in 0.1 M NaNO3 (full symbols) at 25~ (11) 2 mequiv. COO-.1-1, (e) 8 mequiv. COO-.1-1; (--): total binding of added Pb2+. Binding isotherms presented the same features, as shown in figure 2 for Pb2+ and sugarbeet pectins, for the five metal ions and for both pectins. In water, binding isotherms were independent of the pectin concentration, giving rise to a single curve. They followed an ideal "stoechiometric isotherm" for Cu2+ and Pb2+ at least in the early stages. In presence of ionic strength, the binding of metal ions was lower but increased with the polymer concentration.

[Me2+]b / Cp

o o u

r

0.2 0

0

oo

9

f"

0.8 [Me2+]t / Cp 0

9

v

B 0.8 [Me2+]t / Cp 1.6

Figure 3. Influence the metal ion type on the binding isotherms of sugar-beet (A) and citrus (B) pectins at 2 mequiv. COO-.1-1 in 0.1 M NaNO3 and at 25 ~ Symbols as in figure 1.

538 Binding isotherms of the five metal ions, in presence of supporting salt, were compiled (figure 3) either for sugar-beet or citrus pectins. It became possible to differenciate different levels of binding depending on the metal ion, by simple comparison of the binding isotherms. The following order was found for both pectins: Cu2+ ~ Pb2+ >> Zn2+ -- Ni2+ > Ca2+.

3.3 Characterisation of the binding mode The mode of binding was characterised by replotting experimental data obtained from binding isotherms in terms of the Scatchard representation, [Me2+]b /(Cp.[Me2+]f ) VS [Me2+]b/Cp where [MeE+]f corresponds to the final ion concentration at equilibrium. Metal ion concentrations were here exp__ressedin molarity and Cp in number of chain.l-1 (using the weightaverage molecular weights Mw).

[Me2+]b / (Cp.[Me2+]f)

[Mea+]b / (Cp.[Mea+]f) 10 5

2 107

A

v

B V v

Vv~

0

•,Vv~V V~TV~yV~v, 0

9

10 4

1 10 7 v v

100

..

V w-

[Me2+]b / C p

200

0

0

,

I 25

,

[Me2+]b / C p

50

Figure 4. Scatchard representation of binding of Ni2+ to pectins in water (A) and in 0.1 M NaNO3 (B) at 25~ with pectins at 2 mequiv. COO-.1-1: (V) sugar-beet pectins, (V) citrus pectins. In water, Scatchard plots showed clear concave-shaped curves whatever the pectin origin (figure 4A). Nevertheless, differences between sugar-beet and citrus pectins appeared in presence of ionic strength. While citrus pectins exhibited convex-shaped curves whatever the metal ion, sugar-beet pectins display convexe curvature for Cu2+ and Pb2+ but concave-shaped curves for the other three cations (figure 4B, in the case of Ni2+). 4. D I S C U S S I O N

4.1. Selectivity scale In presence of supporting salt, both pectins exhibit the same behaviour towards the two groups of metals consisting of (1) CuE+ and Pba+, the more strongly bound cations, and (2) Zn2+, Ni2+ and Ca2+, which are much less bound. The scale of selectivity, found by a rapid and simple pH method, is in good agreement with the typical sequence found for carboxylates based on Irving-Williams concept (Cotton & Wilkinson, 1976). This order is also in accordance with previously results obtained either for polygalacturonic acid (Jellinek & Chen, 1972; Deiana et al., 1983) or for oligomeric extracts of citrus pectins (Kohn, 1987). The acetylated hydroxyl functions on the galacturonosyl units of sugar-beet pectins (DAc=14%) did not seem to play a significant r61e in the selectivity of pectins for divalent metal ions. 4.2. Quantification of the binding Binding isotherms presented the same characterisitics for sugar-beet and citrus pectins according to the pectin concentration and the conditions of ionic strength. The single case of

539 Pb2§ is shown here (figure 2). In water, where polyelectrolyte effects take place, binding isotherms (figure 2) followed almost a single curve indicating that the level of saturation of the binding sites stayed the same whatever the polymer concentration. Binding was almost stoechiometric in the case of Cu2§ and Pb2+ even in advanced stages of the fixation, as already noticed by Gamier et al. (1994) for low-methoxyl pectins/calcium interactions. In presence of ionic strength, the binding of metal ions was lower than in pure water but increased with the pectin concentration due to increased [Me2+]/[Na+] ratios and competition of both ions for the pectin ionic sites. By comparing the level of the binding isotherms (figure 3) for both metals and pectins, it became possible to set up an affinity order of pectins, whatever their origin, for the five metal ions: Cu2+ ~ Pb2+ >> Zn2§ ~ Ni2+ > Ca2+. This scale, already found by pH-measurements, confirmed that Cu2§ and Pb2§ were more strongly bound than the other three cations with no difference between pectins.

4.3. Characterisation of the binding mode An anticooperative mode of interactions was assumed in case of concave-shaped Scatchard plots, as already proposed by other authors (Mattai & Kwak, 1986; Gamier et al., 1994). A convexe curvature of the plots indicated a cooperative binding process (figure 4). In water, anticooperative interactions were found for both pectins in presence of the five metal ions, as already found by Lips et al. (1991) and Gamier et al. (1994) in the case of calcium. In presence of ionic strength, the type of interactions depended on the metal ion and the pectin origin. Cooperative interactions with both pectins occured with Cu2§ as shown by Schlemmer & Decker (1993), and Pb2+, Which were the two more strongly bound cations. Nevertheless, cooperative and anticooperative binding modes were found for the three weaker bound cations (Zn2+, Ni2+ and Ca2+) with citrus and sugar-beet pectins, respectively. This difference of behaviour, found with sugar-beet pectins, was ascribed to the presence of acetylated functions on the galacturonosyl units, a main chemical difference between the two pectins (Thibault et al., 1993). These functionnal groups could create some specific steric hindrance involving a decreasing of the affinity of ionic sites of the macromolecule towards the more weakly bound ligands. 5. C O N C L U S I O N Interactions studies between some divalents metal ions and pectins from citrus and sugarbeet revealed that the chemical structure of the latter, namely the presence of acetyl functions, induces differences of binding process whereas the scale of selectivity was not affected. Some further studies could be carried out on the correlation between the binding mode and the degree of acetylation. Lastly, pectins showed a clear scale of selectivity towards heavy metals with high capacities of binding which make them suitable to be used in waste-waters depollution.

6. REFERENCES Brady D., Stoll A.D. & Duncan J.R. (1994) Biosorption of heavy metals cations by non-viable yeast biomass. Environ. Technol. 15, 429-438. Cantor C.R. & Schimmel P.R. (1980) Ligand interactions at equilibrium. In Biophysical Chemistry. Part III: The behavior of Biological Macromolecules. W.H. Freeman and Co., San Francisco, chap. 15, 849-886. Cotton F.A. & Wilkinson G. (1976) Basic Inorganic Chemistry, Wiley, New-York, USA. Deiana S., Micera G., Muggiolu G., Gessa C. & Pusino A. (1983) Interaction of transitionmetal ions with polygalacturonic acid: a potentiometric study. Colloids Surf. 6, 17-25.

540 F6rstner U. & Wittmann G.T.W. (1983) Metal Pollution in the Environment, Springer Verlag, Berlin. Gamier C., Axelos M.A.V. & Thibault J.-F. (1994) Selectivity and cooperativity in the binding of calcium ions by pectins. Carbohydr. Res. 256, 71-81. Jellinek H.H.G. & Chen P.A. (1972) Poly(galacturonic acid)-bivalent metal complexes J. Polym. Sci. 10, 287-293. Kerndorff H. & Schnitzer M. (1980) Sorption of metals on humic acid. Geochim. Cosmochim. Acta 44, 1701-1708. Kohn R. (1987) Binding of divalent cations to oligomeric fragments of pectin. Carbohydr. Res. 160, 343-353. Kwak J.C.T. & Joshi Y.M. (1981) The binding of divalent metal ions to polyelectrolytes in mixed counterion systems. I. The Dye Spectrophotometric Method. Biophys. Chem. 13, 5564. Lips A., Clark A.H., Cutler N. & Durand D. (1991) Measurement of cooperativity of binding of calcium to neutral sodium pectate. Food Hydrocolloids 5, 87-99. Marshall W. E., Champagne E.T. & Evans W.J. (1993) Use of rice milling byproducts (Hulls & Bran) to remove metal ions from aqueous solution. J. Environ. Sci. Health A28, 19771992. Mattai J. & Kwak J.C.T. (1986) Divalent metal ion binding to polyelectrolytes with different polyions structure and functionnal groups. Macromolecules 19, 1663-1667. Richardson D.H.S. (1995) Metal uptake in lichens. Symbiosis 18, 119-127. Scatchard G. (1949) The attraction of proteins for small molecules and ions. Ann. N. Y. Acad. Sci. 51, 660-672. Schlemmer U. & Decker H. (1993) On the mechanism of the copper-pectin interaction. In proceedings of Bioavailability '93: Nutritional Chemical and Food Processing Implications of Nutrient Availability. U. Schlemmer (Ed.). Ettlingen, May 9-12, 494-500. Thibault J.-F., Renard C.M.G.C., Axelos M.A.V., Roger P. & Cr6peau M.-J. (1993) Studies of the length of homogalacturonic regions in pectins by acid hydrolysis. Carbohydr. Res. 238, 271-286. Volesky B. (1990) Removal and recovery of heavy metals by biosorption. In Biosorption of Heavy Metals. Volesky B. (Ed.), CRC Press, Boca Raton, chap. 1.2, 7-43.

J. Visserand A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996ElsevierScienceB.V.All rights reserved.

541

Quantitative Vibrational Spectroscopy on Pectins. Prediction of the Degree of Esterification by Chemometrics S. B. Engelsen and L. N~rgaard The Royal Veterinary and Agricultural University, Food Technology, Department of Dairy and Food Science, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark

Abstract

The importance of the degree of esterification (%DE) to the gelation properties of pectins makes it desirable to obtain a fast and robust method to determine (predict) the %DE in pectin powders. Vibrational spectroscopy is a good candidate for the development of such fast methods as spectrometers and quantitative software algorithms (chemometric methods) becomes more reliable and sophisticated. Present poster is a preliminary report on the quantitative performance of different instrumentations, spectral regions, sampling techniques and software algorithms developed within the area of chemometrics.

1. I N T R O D U C T I O N

Pectins are important sugar-based hydrocolloids used in the confectionery industry. In US the sugar confectionery industry is growing approximately 3% per year and the consumption has increased to approximately 11 lb. per capita [1]. Pectin's are widely used in jelly confections often produced using fruit flavors. Pectin gels are characterized by providing a very tender, short texture with excellent clarity and outstanding flavor release properties. For commercial applications pectins are usual extracted from citrus peel, apple pomace and sugar beet and they can be processed to yield two general types of pectin products - high and low methoxyl pectins. Low methoxyl (50 %DE) gel differently. High methoxyl pectins are capable of forming gel networks at acid pH's in the presence of highsoluble salts and the %DE controls their relative speed of gelation. Low ester pectin gelation properties are dependent on the presence of divalent ions such as

542 calcium and are much less pH dependent than that for the high methoxyl pectins. In low ester pectins the %DE controls their calcium reactivity. Finally the presence of amide groups (the degree of amidation, %DA) in low ester pectins provide a third parameter which strongly affects the calcium reactivity and the resulting gelation properties. Although the implications of structural details such as the degree of methoxylation, the distribution of rhamnopyranoses and the molecular weight on functional properties have been demonstrated, it is still not elucidated why such a biopolymer, unlike other similar structures, develops a jelly in the presence of water, sugars and acid [2]. At present our understanding of the 3D structures of pectins and interaction between pectin molecules is highly simplified. However, the still increasing knowledge about pectins and pectin systems combined with the arrival of new modeling tools specifically aimed towards complex carbohydrate structures [3] is likely to provide more profound insight in structural arrangements of pectins. For this spectroscopic investigation 98 amidated pectin samples were provided by Copenhagen Pectin A/S (Hercules Inc.). The samples are spanning a degree of esterification between 20 and 55 per cent and a degree of amidation between 4 and 24 per cent (see Fig. 1). The powder samples were all measured as is without any form of pre-treatment such as drying and dilution.

[

] --e

-'9

a-D-GalpA

-t

a-D-GalpA-6OMe

~o

~o

o~

a-D-GalpAm ~o

o~

o~

Figure 1. A schematic model of a short sequence of the pectin backbone including a-D-Galactopyranuronic Acid, Methyl a-D-Galactopyrnanosiduronate and a-DGalacto-pyranosiduronamide.

543 2. V I B R A T I O N A L S P E C T R A OF P E C T I N S

Five spectral ensembles were collected for the 98 amidated pectin powders (see Table 1 and Fig. 2). Two dispersive NIR reflectance ensembles were collected using a Tecator spectrometer. Two Fourier transform NIR spectral ensembles were collected using a Perkin Elmer System 2000 interferometer. One using a diffuse reflection cell and one using an integrating sphere cell. Finally two spectroscopic ensembles were collected in the region of the fundamental vibrations. One FT-IR ensemble using the diffuse reflection cell and one using the NIR FT-Raman technique (1064 nm).

NIR

Raman

10 00

90b0

80'00

70'00

60'00

50'00

40'00

30'00

20'00

10'00

2(}0

cm-1

Figure 2. The spectra of the amidated pectins (NIR, FT-IR and Raman shift).

Table 1 The spectroscopic data I n s t r u m e n t dispersive dispersive FT

FT

Sampling

integrating diffuse

reflectance reflectance diffuse

FT

FT 180 ~

method

(rot. cell)

reflection sphere

reflection scattering

X-variables 1050

1050

6001

3301

-1

6001 -1

-1

2771 -1

X-units

nm

nm

am

X-min

400

400

4000

4000

700

330

X-max

2500

2500

10000

10000

1750

3100

am

am

am

544 3. C H E M O M E T R I C S

Principal Component Analysis (PCA). Principal component analysis is an extremely important method within the area of chemometrics. By this type of mathematical treatment one finds the main variation in a multidimensional data set by creating new linear combinations of the raw data (e.g. spectral variables) [4]. The method is superior when dealing with highly collinear variables as is the case in most spectroscopic techniques: two neighbor wavelengths show almost the same variation. Partial least squares regression (PLS). Partial least squares regression applies to the simultaneous analysis of two sets of variables on the same objects. It allows for the modeling of inter- and intra-block relationships from an X-block and Y-block of variables in terms of a lower-dimensional table of latent variables [4]. The main purpose of regression is to build a predictive model enabling the prediction of wanted characteristics (y) from measured spectra (X). In matrix notation we have the linear model with regression coefficients b: y=Xb To investigate the variance structure in the raw physical/chemical data material a PCA was performed on the autoscaled Y-data. Figure 3 shows a loading plot of the Y-data as a function of the two first PC's describing together 57 % of the total variance. Loadings for PC-'# 1 versus PC-'# 2

0.8

,

Scores for PC# 1 versus PC# 2

,

2

,

,

,

1.5

ptl

0.6

,

21 22

Trans ~

0.5

~,

.a

0.2 Ca4 Ca3 Ca2

%DE

25 24

z3 24

2~5

24

21~2

20

2021"22

ZZ

-l

26 22

2!

%DFA

-1.5 - 25

22 22 2

-0.4

-2

SAG

.4).6 -0.6

2~176

%DA

Cal -0.2

r

25 2 1 ~ 2 2

, 20

20 2 2~ 2 ~ 12020 .

21

1

0.4

,

g.4

-0.2

F'C#1

o

oJ2

0.4

25

24

-2.5

4

PC#1

Figure 3. Loading (left) and score (right) plots from a PCA on chemical data measured on the pectins (%DE=degree of esterification, %DA=degree of amidation, %DFA=100-%DE-%DA, Transp=transparency, Cal-Ca4 and SAG are gel strength measures).

545 Different product sorts (20-22,24-26) are marked in the score plot of spectral NIR measurements (Fig. 4). Sorts 23 and 31 are in separate classes outside the range of this plot. A gradient is seen in the plot, indicating the chemical differences among the sorts: the %DE are increasing in the 20->25/26 direction while the opposite holds for the Ca-based gel strength measurements. xl0 -4 8

Scores for PC# 1 versus PC# 3 ,

,

xl0 -4

~

,

,

Scores for PL-'# 1 versus PC# 2 ,

,

,

22

4

21

3

t-q 2

1

2:~525 -1 2~ 23 23

3.5

-~

&

0

-2

20~2~ l

-3

2020

~26~

2121 ~.2 2122~2 21 22 21 21 22

-;

-;

-'1

o IK:#1

x 10-3

'1

25

25 2114 24 24 24

-5

015

1

25 2~5 26 ~25 26 2~

22 21 2: "'2122 21 2222

20

-4 2~2 2225

25

2/112~2 22 2,]1 12~

~o20 1.o

313131

,

25

2

25 22

,

22~

5

; xlO 4

Figure 4. Score plots from a PCA of NIR_R spectral data (2nd derivative). Left: PC#1 versus PC#3 shows a clear segregation of sorts 23 and 31. Right: PC#1 versus PC#2 (without sorts 23 and 31) shows a more distinct sort gradient/classification than the one produced by the chemical data. Two PLS-factors

.

.

.

(%DE) . .

/

4O

35 30

15 15

20

25

30

35

40

45

50

55

Measured

Figure 5. Predicted versus measured plot of %DE using the FTNIR_IS ensemble (C=cross validated samples and T= test set samples). The optimal PLS models obtained for the prediction of %DE using the five different spectral sources are listed in Table 2 (see also Fig. 5). The models

546 have been constructed using a conservative approach of systematic cross validation on app. 73 samples (objects) and subsequently tested on an independent test set consisting of 25 samples. Table 2 The optimal spectroscopic PLS models for the pectin powders.

degree of esterification of amidated

derivative

2

1

1

1

1

1

opt. PC's

2

3

3

2

4

4

RMSECV

1.19

1.17

1.54

1.54

1.46

1.90

R

0.98

0.98

0.97

0.97

0.97

0.95

The experimental errors on the %DE measurements are estimated to be between 1 and 2 %, taking into account a relative long time span and the involvement of different lab-workers. As indicated by Table 2 the best models converge to an RMSEP of 1.5 %; to refine the models further the experimental chemical errors have to be thoroughly investigated. Three PLS-factors (%DA)

C

_j T

/ ~

~

1~

1~

~o

~

30

Measured

Figure 6. PLS model for %DA using the NIR_R ensemble. Of the other physico-chemical data available to us we found good predictive correlations between the spectra and the degree of amidation (Fig. 6)

547 and for the pH (Fig. 7). Finally we obtained a somewhat weaker PLS model for the gel strength Ca2 (62 % explained using 3 PC's). Due to the large span in %DE/%DA for the samples it was necessary to use two levels of sugar contents in the Ca2 measurements which perhaps can explain the weak model. It may also be possible that the gelation potential is not "visible" in the spectroscopic ensembles but certainly the weak correlation is worth pursuing with further investigations. 5

,

,

,

Four PLS-factors(pH) ,

,

,

ThreePLS-factors(Ca2) ,

,

4.8

350 ~'42"42 . . . . . r C, ~ T

4.6

3O0

4.4

T

C j"Cc

C

12

4.2 4

200150

IZ 12

, : ~ C

~

C

CC

3.8 100

3.6 3.4 3.2 / 3.2

3.4

T 3.6

3.8

4 4.2 Measured

4.4

4.6

4.8

0

~

1~

1~

2t)0 ~ Measured

3()0

3~

400

Figure 7. PLS model for (left) pH and (right) Ca2 using the FTIR_DR ensemble.

4. CONCLUSIONS

From Table 2 it is observed that the dispersive NIR ensembles (NIR and NIR_R) result in the best cross validated models. The potential advantages of Fourier transform spectroscopy [5] are in practice outnumbered by a more reproducible setup and sampling procedures. When considering the two spectral models in the region of the fundamental vibrations (FTIR_DR and Raman) we observe that more PC's are needed to out-level interferences and describe the Y-variation. On the other hand these ensembles are needed to interpret the most important spectral elements in the models. The lower signal to noise ratio in the Raman spectra caused by the low energies of the scattered light and the poor reproducibility due to scattering effects are responsible for the relative poor performance of this spectral technique. However, the used sampling technique designed for micro analytical purposes (laser focused on a very tiny area, no control of depth focusing and pressure/density of the sample) could play a significant role. We are currently

548 making investigations to improve and control these factors to obtain a more fair picture of the potential of quantitative NIR FT-Raman spectroscopy. Another direction of future research is to investigate the relationships between IR and NIR spectra in the form of PLS2 modeling and 2D correlation spectroscopy [6]. A preliminary 2D correlation spectrum is shown in Fig. 8.

2D NIR/IR correlation spectrum "6 9

*

--~ l l h .

~

~...

~

.m

.,+ t

9

-..,.= P= ~.= = : I n

! m ....

9 l~a

NIR 7000 'gil ~. ~.~ o~N-'-9 " .,~ "'0 . -~=~- . ~ (cm-1) -.. =

6000

"

..'~'. ,=-'.."...m~_":,'-:.. 9~.;, . ~ , , . = , ; ~ = .~

1000

.

~

1500

.,~.,..,

2000

--.,=,, .d.. 2500

l~,ii

", " ~

~

~

~o 3000

3500

4000

IR (cm-1)

Figure 8. 2D NIR/IR correlation spectrum. Only contours for correlation coefficients with numerical values above 0.5 are shown.

7. REFERENCES

[1] J.M. Carr, K. Sufferling and J. Poppe, Food Technology, 7 (1995) 41-44. And references therein. [2] S. Cros, C. Herv~ du Penhoat, N. Bouchemal, H. Ohassan, A. Imberty and S. P~rez, Int. J. Biol. Macromol., 14 (1992) 313-320. And references therein. [3] S.B. Engelsen, S. Cros, W. Mackie and S. P~rez, "A Molecular Builder for Carbohydrates: Application to Polysaccharides and Complex Carbohydrates", submitted (1996). [4] H. Martens and T. Nees, Multivariate Calibration, Wiley, New York, 1993. [5] P.R. Griffiths and J.A. de Haseth, Fourier Transform Infrared Spectroscopy, Chemical Analysis Vol. 83, Wiley - Interscience, 1986. [6] F.E. Barton II, D.S. Himmelsbach, J.H. Duckworth and M.J. Smith, Applied Spectroscopy, 46 (1992) 420-429.


549

POLYSACCHARIDES FROM Chorisia speciosa St. Hil.

E. B. BELESKI-CARNEIROa, b ; M. R. SIERAKOWSKI; J. L. M. S. GANTER; S. F. ZAWADZKI-BAGGIO; F. REICHERa aDepartament of Biochemistry, PO Box 19046. Universidade Federal do Paran~i, 81531-990. Curitiba - Paran~i - Brazil b Departament of Chemistry, Universidade Estadual de Ponta Grossa. Paranh- Brazil

ABSTRACT

The floss silk from Chonsia speciosa fitmished a polysaccharide with a main chain of (1 ~ 4) linked 13-Xylp substituted at O-2 by 5 % of uronic acid. The xylan structure also was interposed with ct-Rhap units in small amounts. The defatted seeds furnished on aqueous extraction a major fraction, (O-acetyl, 10 % and protein, 45 %) wich was hydrolysed and analysed by p.c. and GLC, showing Rha (20 %), Ara (16 %), Gal (64 %) and also uronic acids (45 %). Partial hydrolysis gave rise to a polysaccharide free of arabinose, with 46 % of uronic acids. Methylation analysis (GLC -MS) indicated a chain of (1 -~ 4) - linked Galp (42 % of 2,3,6-Mea-Gal ).

INTRODUCTION

Chorisia speciosa St Hil is a large tree of the family Bombacaceae, native to tropical South America, it grows in Brazil, being abundant in the States of Rio de Janeiro, Minas Gerais and S~o Paulo. Its is frequently found further South in Curitiba as an ornamental tree which flowers, appearing with ten new leaves having five pink, purple petals. The pear-shaped fruits contain an abundance of silk white floss, that is extremely elastic, light in weight and impervious to water, and is used in pillows, sleeping bags, upholstery and life preservers. The genus Chorisia has five species, the most important being C. speciosa, known as floss silk tree or "paineira".

550 Several aspects of this tree have been studied. One is a gum which exudes when the tnmk suffers injury, apparently to heal its wounds. It contains a complex polysaccharide with a backbone composed of glucosyluronic and mannose units [ 1]. Lufrano and Caffmi [2] compared four distinct species of Chorisia by phytochemical analysis, composition of the mucilages from the leaves and suggested a chemotaxonomic approach at genetic and specific levels. The seeds had a high protein content and furnished 22 % of oil. The triglyceride structures have been determined and showing fatty acids predominantely unsaturated [3]. This paper reports the structural features of the silk floss polysaccharide and the partial structure of a viscous acidic polymer obtained from the seeds of Chorisia speciosa. It was of interest to analyze these polysaccarides due to the relationship of Bombacaceae to Sterculiaceae, as well as for its possible commercial uses.

EXPERIMENTAL

General methods. Polysaccharides were hydrolyzed with M trifluoroacetic acid (5 h, 100 oC). Hydrolyzates were reduced with sodium borohydride, then acetylated in 1:1 pyridine-acetic anhydride (16 h, room temperature). The resulting alditol acetates were analyzed by GLC (gas-liquid chromatography), with a model 2440 Varian chromatograph operating at 180 oC, with columns packed with 3 % OV 225 or ECNSS (1.5 mm i.d. x 200 cm. Gas Chrom. Q support). The carrier gas was nitrogen (40 mL./min). Methylated polysaccharides were hydrolyzed with 72 % H2SO 4 (1 h. 0-4 oC) and thereafter water was added to a fmal acid concentration of 1 M (5 h. 100 oC). The solution was neutralized (BaCO3) and the products converted into O-methylalditol acetates that were analyzed by GLC-MS with a model 3980 Hewlett-Packard chromatograph equipped with and HP1 capillary column (0.2mm i.d. X 30 m) linked to an HP 5988 with mass spectrometer unit (electron impact, 70 eV). Injections were carried out at 150 oC and the column programmed to increase at 4 oC/min to 250 oC, then hold. Optical rotations were measured in water at 25 oC using an Acatec automatic polarimeter. I. r. spectra were determined using a Beckman Acculab TM-10 spectrophotometer. Uronic acid was estimated by the m-hydroxyphenyl method [4], O-Acetyl contents by the method of Hestrin [5], carbohydrate by the phenol-sulfuric acid method [6] and protein by the Hartree method [7]. Plant material The fruit of Chorisia speciosa at mature stage was collected from large trees in parks of Curitiba. Polysaccharide isolation. The silk floss (36 g) was cut and extracted with 2:1 benzene-EtOH in a Soxhlet apparatus for 16 h. The residue was extracted with 2 M

551 NaOH in the presence ofNaBH 4 for 6 h at room temperature. The alkaline extraction was repeated twice and the supematant combined the NaOH extract acidified to pH 5 with 50 % acetic acid, exhaustively dialyzed, polysaccharide precipitated with EtOH (2 vol.) and washed with EtOH and MeECO. The seeds (30 g) were ground in a Willey mill (60 mesh) and defatted as described above for 32 h and the residue submitted to sequential aqueous exctractions. The material was stirred in H20 (600 ml) at 5, 20 and 70 oC each for 12 h. Addition of EtOH (2 vol.) precipitated polysaccharide which was isolated via sucessive centrifugation, and washed with EtOH and Me2CO, yielding fractions FI, FII and FIII respectively (Figure I). Chromatoraphyc methods: For gel filtration of polysaccharide fraction PI, a Sephacryl S-300 chromatographyc column (1,1 X 46,7 cm) was calibrated with standard dextrans (molecular mass range 266, 72, 40, and 17 KDa; Sigma Chemicals), and the void volume determined with blue dextran. Polysaccharide sample (0.5 mL; 2 mg/mL) was applied and eluted with 50 mM NaOH, fractions 1 mL being collected and carbohydrate absorbance (phenol-H2SO4) being monitored. Analyses were performed by gel permeation chromatography (GPC) and by high performance liquid chromatography (HPLC). Gel filtration of oligosaccharides was effected in a thermostated (65 oC) column (210 X 1.5 i.d.) filled with polycrylamide gel (Bio-gel P2, 200-400 mesh; Bio Rad-USA), using distilled water as eluent (flow rate 30 ml.h-1). Smith degradation of PI. A sample of FI (50 mg) was dissolved in 0.5 M NaOH (5 mL) and acetic acid added to pH 7, followed by NaIO4 to a final concentration of 50 mM. At the conclusion of the oxidation (7 days) the product was reduced with NaBH 4 and dialyzed. An aliquot was removed and the remainder immediately treated with NaIO 4 and then reduced with NaBH 4. Samples of material subjected to one and two cycles of oxidation-reduction were hydrolyzed and reduced, and the products analyzed by GLC as alditol acetates. Methylation analysis of PI and Flbp. The fractions were methylated according to Ciucanu and Kerek [8]. The procedure was repeated until no absorbance was detected by I.r. at 2500-3500 cm-1 and the per-O-methylated polysaccharides hydrolyzed and analyzed by GLC and GLC-MS of the derived partly O-methylated alditol acetates. Oxidation of PI with chromium trioxide. Fraction P1 was twice acetylated as described above. The peracetylated polysaccharide (75 mg), together with 20 mg of mannitol hexacetate as internal standard was dissolved in 1.5 mL of HCC13, and treated with 1.89 mL of glacial acetic acid and 189 mg of chromium trioxide, at 50oC. Aliquots were removed at zero, 30, 60 and 120 min, water then added, and the material recovered by extraction with chloroform, hydrolyzed and analysed by GLC of derived alditol acetates.

552 F i g u r e 1. F l o w d i a g r a m

o f t h e i s o l a t i o n o f f r a c t i o n f r o m s e e d s o f C. s p e c i o s a

Milled seeds Benzene:EtOH

11 -I

E-I Lipid/pigment . . . . . . . . . . . . . . .

~

_

.~

MeOH

--

E-II Low Mw material

: water

- .. I w a t e r , 4 ~ _

!

.....

R-Ill

E-Ill FI

water, 25*C

R'!iv

centrifugate

water, 70~ E-Ilia Fla

E-Illb Fib NaOH

2 M

,[

PP. HAc 50% . . . . . . . .

. . . . . .

supernatant-I

precipitate Hem. A

EtOH

i

precipitate Hem. B

supernatant-i

i

553

Carboxyl redution. A sample of permethylated PI (5 mg) was carboxylreduced by a modification of the method described by Lindberg and LOnngren [9], as follows. The methylated fraction was solubilized and added a mixture of LiA1H4 (25 mg) in THF (5 mL) at 20 oC for 4 h. and refluxed during 1 h. The excess of reagent was destroyed with ethyl acetate (5-6 drops) and water (10 drops) and the pH of the mixture adjusted to neutrality with acetic acid. The insoluble residue was removed by centrifugation. The reduced fraction was precipitated with EtOH. The reaction was monitored by I.r. specroscopy. Hydrolysis products were analysed by GC-MS as methyl alditol acetates 13C NMR spectroscopy was performed with a Bruker AC-300 spectrometer at 75 MHz in the Fourier-transform mode, with proton decoupling at 30 ~ C, using 5 mm tubes and D20 as solvent. The spectral width was 200 ppm. Chemical shifts are expressed in ppm relative to the resonance of external DSS (sodium 4,4-dimethyl-4silapentane- 1-sulfonate). Partial acid hydrolysis ofFIa. A sample of FIa was treated with TFA at pH 2, 1 h at 100 oC.

RESULTS AND DISCUSSION

In terms of the average from 20 mature fi'uits, floss silk, seeds and fruit coat were isolated, the ratio being 14 %, 15 % and 7 1 % respectively. The filaments of floss silk on submission to microscopy, showed mainly unicellular trichomas, some of them being formed by two cells. The presence of lignin in the filaments was suggested by the characteristic reaction with phloroglucinol hydrochloride. The silk floss was defatted and hydrolyzed by Saeman's method [ 10] giving rise to xylose (73 %) and glucose (27 %), thus indicating the presence of xylan. When the time of hydrolysis was up to 24 h, the presence of rhamnose (5 %) was also evident. However, degradation of xylose was observed after 8 h at 100 oC. The defatted floss silk was submitted to extractions with aqueous NaOH giving rise to a polysaccharide (PI, yield 23 %) composed of xylose as the only neutral sugar. Uronic acid (10 %), was determined by a colorimetric method [4]. When products of acid hydrolysis of PI were fractionated by gel permeation chromatographic (GPC) on Bio-Gel P2, two fractions were obtanined, one being composed of Xyl and the other acid oligosaccharides. The acidic fraction was treated with MeOH-HCI and reduced with NaBH 4. After hydrolysis of the product, analysis by p.c. showed the presence of Xyl, Glc, Rha and 4-O-Me-Glc. These results were confmned by GLC-MS of derived alditol acetates indicating that the xylan contains a small amount of Rha, GlcA and 4-O-Me-GlcA as the acid components. Xylans from wood hemicellulose containing Rha (0.3 to 0.6 %) were related by Fengel and Wegener [11]. Aspinall and McGreth [12] obtained a xylan from

554 lucerne stems containg 11% of 4-O-methyl-D-glucuronic acid residues and 1 % of Rha. Geerdes and Smith [ 13] obtained a hemicellulose from flax straw which gave on hydrolysis, a mixture of aldobiuronic acid, Xyl and a small amount of Rha. Seed hair xylans are present in milkweed (Asclepias synaca) and kapoc (Ceibapentandra) and contain 4-O-Me-GlcA linked (1 ~ 2) to Xyl. The xylan from the silk floss (PI) was homogeneous on gel-column chromatography over Sephacryl S-300, having a molecular mass of z 52000. Methylation analysis yielded 2,3-Me2-Xyl, 3-Me-Xyl and 2,3,4-Me3-Xyl in a molar ratio of 93.0: 5.0: 2.0. When the permethylated product was carboxy reduced and remethylated, 2,3,4,6-Me4-Glc (3.1%) was also characterized (Table I).

Table I. Methylation analysis of products from original fraction PI carboxi reduced and remethylated material by g.l.c-m.s. (C. speciosa). Component

PI

2,3-Me2-Xyl 3-Me-Xyl 2,3,4-Me3-Xyl 2,3,4,6-Me 4 Glc

93.0 5.0 2.0

PI-CR mol % 91.2 4.9 0.8 3.1

Capillar column OV-225 and DB-210.

This results are consistent with a backbone of (1 --> 4) linked D-xylosyl units substituted at O-2 by glucuronic acid and its 4-O-methyl derivatives. Periodate oxidation followed by reduction and acid hydrolysis gave rise to glycerol and Xyl in a molar ratio of 88:12. However, when the polyol was reoxidized with periodate and reduced, acid hydrolysis gave rise to glycerol and Xyl in a molar ratio of 94:6, as expected. Interunit hemiacetal formation must have taken place during the initial periodate treatment inhibiting further oxidation. The anomeric configurations of the sugar residues were determined by chromium trioxide oxidation [ 14]. Oxidation of the fully acetylated polysaccharide and subsequent monosaccharide analysis by GLC indicated that the D-Xyl units are [3-1inked (oxidized more rapidly) and that the D-GlcA are a-linked (Table II).

555 Chromium trioxide oxidation of fraction PI.

Table II.

Monosacharide composition of product a

(mol%) Time (min)

Mannitol

Xylose

0

1.00

1.97

30

1.00

1.33

60

1.00

0.75

90

1.00

0.00

(a): GLC - ECNSS column.

The reaction was monitored also by p.c. which showed clearly on increase in the relative proportion of uronic acids related to xylose. After 90 minutes of oxidation, only uronic acids were observed indicating the a-configuration.

Table III. 13C NMR Data a for the Xylan (PI) from the floss silk of Chonsia

speciosa. Carbon

C-1 C-2 C-3 C-4 C-5 C-6

( 1 --~ 4)-13-D-Xylp

(1 -~ 4)-13-D-Xylp

unbranchedb

branchedb

103.7 74.8 76.1 78.4 65.0

102.8 81.2 78.5 75.9 64.7

a: shifts (5 in ppm); internal DDS reference. b: refers to branched and unbranched at 0-2.

(1 -~ 2)-~-DGlcA 99.4 74.2 75.9 84.2 73.4 178.3

Figura 2. aC NMR spectrum of the PI fraction from the floss silk of Chorkia speciosa, 75 MHz, D20,30"

C5

I

i

i/

ce

OL - D 6 t +

4-0-MI

f

556

I I

180

I

IM,

I

!to

f

f

s

120

1

IK)

1

loo

t

90

1

aa PPM

I

m

I

60

557 The 13C NMR spectrum of PI consisted of 5 large signals at 5 103.7 [C-l], 78.4 [C-4], 76.1 [C-3], 74.8 [C-2] and 65.0 [C-5], corresponding to (1 ---}4) linked 13 -D-Xylp. This polymer showed some residues of (1 ---}4) linked 13-D-Xylp substituted at 0-2 (5 81.2). The signals at 8 99.4 [C-l], 74.2 [C-2], 75.9 [C-3], 84.2 [C-4], 73.4 [C-5], 178.3 [C-6] corresponding to (1 ---} 2) linked o~-D-GIcA. The signal at 8 61.3 corresponding to 4-O-Me in glucuronic acid residues (Table III). The floss silk from Chorisia speciosa which envelops the seeds are important to their dispersion. Each fruit contains from 100 to 200 seeds. The seeds were crushed and defatted (see flow diagram, Fig. I) yielding 20 % of oil. This result is in agreement with Petronici et al [3]. The residual mass was refluxed w~th methanolwater in order to isolate low molecular compounds. Both treatments with organic solvents are efficient in enzyme inactivation [15]. Extractive-free seeds were subjected to aqueous extractions at progressively increasing temperatures (fractions FI to Fill, Table IV). The residue was treated with aqueous NaOH according to Whistler and Feather [ 16], yielding HA and HB (Table IV). All the aqueous extracts showed high viscosities. As FI was not completly soluble in water, it was centrifuged giving rise to FIa (supematant) and Fib (precipitate). The highest yield of polymers was obtained with water at 5 oC (FI) and at room temperature (FII).

Table IV. Yield and monosacharide composition of Fractions obtained by aqueous and alkaline extractions. Fraction

FIa Fib FII Fill Hem. A Hem.

Yield g%

Composition (mol %)a

3.3 15.2 21.6 1.1 3.0

Rha 20.0 13.5 48.0 48.0 13.0

Ara 16.0 16.5 19.0 29.0 4.0

Xyl --4.0 3.0 11.0 82.0

Gal 64.0 66.0 30.0 12.0 1.0

2.2

22.0

8.0

66.0

4.0

B

a: glc ECNSS column. b: Hem. = Hemicellulose

558 Rha, Ara and Gal are the neutral sugar components from all the fractions. Xyl is not present in FIa and is significantly present in the hemiceUulose fractions, indicating that this monosaccharide is component of hemicellulosic polymers. Chemical composition of the water fractions were determined (Table V). High protein contents and the presence of O-acetyl-groups were observed in four aqueous fractions. Neutral sugar and uronic acid composition points to inclusion of these polymers in the class of pectic polysaccharides.

Table V.

Determination of carbohydrates, proteins, uronic acids and O-acetyl of Fractions from aqueous extractions from seeds of C. speciosa.

Fraction

CarbohydrateProtein

FIa Fib FII FIII

33.0 51.0 37.0 24.0

uronic acids*

% content 41.0 48.0 34.0 31.0

45.0 25.0 33.3 23.0

O-Acetyl*

10.5 5.2 8.5 15.6

(*): in relation to carbohydrate.

Fraction FIa was chosen for structural purposes due to its better solubility in water and the absence of Xyl. In order to remove noncovalently associated protein, fraction FIap was submitted to sequential shaking cycles with a mixture of chloroform-buthanol, as indicated by Sevag and described by Staub [17]. The fraction was also treated with trichloroacetic acid. In both procedures, coprecipitation of carbohydrate and protein was observed, suggesting strong linkages and a more complex structure. FIa as was submitted to mild acid hydrolysis yielding FIas and FIap (Table VI).

559

Table VI.

Monosacharide composition of precipitate Flap and supematant Flas produced by partial hydrolysis of fraction Fla. (mol %)

Fraction

Ara*

Rha*

FIa Flap Flas

16 tr 64

20 24 35

Gal* 64 76 tr.

(*): determined by GLC. The removal of arabinose suggested the presence of peripheric ot-Larabinofuranosyl units. Flap was methylated by the method of Ciucanu and Kerek and products of derived O-methyl alditol acetates analysed by GLC-MS The resulting Flap contains uronic acid (46 %), protein (45 %) and was submitted to methylation analysis, GLC-MS of resulting methyl alditol acetates showed the presence of 2,3,6-Me3-Gal (42.6 %), 2,3,4,6-Me4-Gal (19 %), 2,3,4Me3-Rha (19 %), 3-Me-Rha (11.2 %), 3,4-Me2-Rha (4.9 %) and 2,3,4-Me3-Gal (3.3%). These results indicate a complex highly branched glycoprotein whose structure will be published in the future. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

J.L. Di Fabio and G. G. S. Dutton, Carbohydr. Res., 99 (1982) 41-50 N.S.P. Lufrano and N. O. Caffmi, Oiton, 40 (1981) 13-20 C. Petronici et al., La Rcvista Italiana delle Sostanze Grasse, 51 (1974) 11. N. Blumenkrantz and G. Asboe-Hansen, Anal. Biochem., 54 (1973) 484-489 F. Dowins and W. Pigman, Meth. Carbohydr. Chem., 7 (1978) 241-243 M. Dubois et al., Anal. Chem., 28 (1956) 350-356 9 E.F. Hartree, Anal. Biochem., 48 (1972) 422-427 I. Ciucanu and V. Kerek, Carbohydr. Res., 131 (1984) 209-217 B. Lindberg and J. L6nngren, Methods Enzymol., 50 (1978) 3-13 J. F Saeman et al., Tech. Assoc. Pulp Pap. Ind., 37 (1954) 336-343 D. Fengel and G. Wegener, Wood Chemistry, (1989) 108-127 G. O. Aspinall and D. McGrath, J. Chem. Soc. (C), (1966) 2133-2139 J. D. Geerds and F. Smith, J. Am. Chem. Soc., 77(13) (1955) 3569-3572 J. Hoffman and B. Lindberg, Meth. Carbohydr. Chem., 8 (1980) 117-122 F. Reicher et al., Appl. Biochem. Biotech. 34/35 (1992) 349-357 R.L. Whistler and M.S. Feather. Meth. Carbohydr. Chem, V (1965) 144-145 A. M. Staub, Methods Carbohydr. Chem., 5 (1965) 5



561

Rigid and flexible pectic polymers in onion cell walls M.-A. Ha ~, B.W. Evans a, D.C. Apperley h and M.C.

Jarvis a

aChemistry Department, Glasgow University, Glasgow G 12 8QQ, Scotland hEPSRC Solid-state NMR Service, Durham University, Durham DH1 3LE, England.

Abstract The amount of thermal motion occurring in individual polymers of a composite material like a plant cell wall can be determined using NMR relaxation methods. This methodology can be used to indicate of the rigidity of each polymer and its contribution to the rigidity of the cell wall as a whole. We have applied this approach to onion cell walls. The proton T2 is used to identify individual polymer chains differing in rigidity whereas the proton T~p discriminates between spatial locations within which the average amount of motion differs. Through a Tfbased spectral editing procedure we reconstructed sub-spectra corresponding to a mobile, largely pectic fraction of the cell wall and a rigid microfibrillar fraction which also included a pectic component. There was a third extremely mobile pectic component consisting of a of 13(1,4)-linked galactan and highly esterified galacturonan. These highly mobile, hydrated polymers are not represented in a CP-MAS ~3C spectrum obtained under normal conditions. We found, however, that by a combination of a long-contact experiment and a delayed-contact experiment we could reconstruct a ~3C spectrum of the cell-wall components that are normally too mobile to be visible. Through a T~p-based spectral editing procedure we found some pectic material spatially located near cellulose. This included some 'eggbox' pectin. Pectic material was also located more than 2nm away from cellulose. These results show that, within a single cell wall, pectic polymers are very heterogeneous in rigidity as well as in composition, and are not distributed uniformly within the cell wall structure.

1. INTRODUCTION Recent models of the plant cell wall are simplified representations based mainly on what is seen by microscopy but also incorporating information from chemical techniques (1,2). However, electron microscopy is a moisture sensitive

562 technique and the dehydration it requires can affect moisture sensitive polymers such as pectin. Therefore techniques that do not require dehydration of the plant cell wall are needed to give us complementary information on the three dimensional architecture of pectin. Using solid state NMR, Jarvis and Apperley (3) have recently examined the conformation and aggregation of pectic galacturonans forming gels with calcium in vitro and proposed a 'cable' model for the structure of gels. Non-esterified galacturonan chains aggregated by calcium ions adopted both 2~ helical (eggbox) and 3~ helical conformations with some chain segments intermediate between these. Chains that were not aggregated, and were therefore more flexible, adopted a wide range of right-and lefthanded helical forms with the 31 helix as an approximate average conformation. In the cell wall, methyl esterification leads a larger number of chains to take up similar random conformations but there is evidence (4) that some methyl-esterified chain segments can also form aggregates in the 3~ helical form. Data on branched and acetylated chain segments are as yet, inconclusive. In addition to giving conformational information, solid state NMR relaxation experiments can be used to probe the thermal motion of polymers in the hydrated cell wall (5). The motion of the polymers can give us clues as to the environment of the polymer. When there are both rigid and mobile polymers within a composite material, NMR spin-diffusion experiments can be used to find out how far apart they are. We used modifications of the standard solid-state CP-MAS (cross-polarisation, magicangle spinning) experiment to allow the proton relaxation characteristics to be measured for each peak in the ~3C spectrum. It is known that highly mobile, hydrated polymers can not be seen using either usual CP-MAS ~3C spectrum or solution NMR (6). We found, however, that by a combination of a long-contact experiment and a delayed-contact experiment we could reconstruct a ~3C spectrum of the cell-wall components that are normally too mobile to be visible. With these techniques we were able to determine the mobility of pectins and their approximate spatial location in comparison to cellulose.

2. RESULTS AND DISCUSSION Our solid state NMR findings suggest that three distinct groups of pectins differing in mobility on the kHz frequency scale, coexist in at least two different areas of the onion cell wall.

2.1 Very Mobile Pectin The most mobile of the pectins were not visible in conventional solid state CP-MAS ~3C spectra, nor in solution 13C NMR. Because of the high mobility of this material, it was very slow to cross polarise (CP) (7,8). A spectrum of this slow-CP material was obtained by subtracting the signal intensities obtained in a delayed-contact experiment from those obtained in an experiment with long, variable contact times. The method is discussed in detail elsewhere (9). The resulting spectrum (figure 1) showed a highly methyl esterified o~(1,4')-D-galacturonan and a 13(1-4')-D-galactan. Both of these polymers are highly soluble in water. They are the most flexible polymers, identifiable by NMR, in the onion

563 cell wall. Foster et al (6) have arrived at similar conclusions by a quite different NMR approach.

Methyl Pectic C-4

galacturorlan C-6l

Crahctan C-6 Pectic Me

Pectic~C-1 ~ Galactan C- 1

I

180

'

I

'

I

160 "140

'

I

120

'

I

'

100

I

80

'

I

60

'

1

1

40

I

20

'

I

0

Figure 1. Spectrum of very mobile material in onion cell walls.

2.2

Pectins

of Low

and

Intermediate

Mobility

The conventional CP-MAS 13C spectra of hydrated onion cell walls can be seen in figure 2. This spectrum is derived from the low- and intermediate-mobility polymers present in the walls.

564

180

160

140

120

100

80

60

40

20

0

ppm

Figure 2" T2 CP-MAS ~3C spectrum of onion cell walls

Table 1 Solid state CP MAS ~3C signals Chemical Shift Carbon (ppm) . . . . Number 177 C-6 175 C-6 171 C-6 106.4,105.5, C-1 104.5 101 C-1 100-94 90.2, 89.4, 88.5 84.9,83.9 8O 79 77 75,72 69 66.2,65.4 62.5, 61.6 60.5 54 21 .

.

.

.

C-1 C-4 C-4 C-4 C-4 C-4 C-2,C-3 C-6 C-6 C-6 ,, ,,,

for plant cell walls (3,10) Assignment ... Calcium bound pectin Random coil pectic chains bound to monovalent cations Pectic carboxyl, methyl ester, free acid Cellulose Ia and Ib Pectic 3, and 2, helix conformations and other carbohydrates Intermediate pectic conformations Cellulose Io~ and 113 Cellulose Io~ and lib Pectic 3, helix 131-4 galactan Pectic 2, helix General carbohydrate Pectin Cellulose Io~ and 113 Cellulose Io~ and 113 1-4 galactan Pectic methoxyl Acetyl l~roup .....................

565 Peak assignments are given in table 1. A peak can be seen at 80ppm. This corresponds to the resonance from the C-4 of galacturonic acid occurring in a 31 helical conformation (3). The resonance of the C-4 of galacturonic acid in a 21 helical conformation occurring at 77ppm can not be distinguished here because of the large general carbohydrate peak seen at 75-76ppm. Intermediate conformations can be identified from the C-1 resonances (100-94ppm). By measuring the proton relaxation times, T2 and Tip, it is possible to estimate the mobility of polymer chains within the cell wall (11). Proton spin relaxation editing (PSRE) is a method of expressing these results. It separates the components seen in a conventional CP-MAS 13C spectra into low-mobility and intermediate-mobility components. If PSRE is applied to a T2 experiment (12) the mobility of the polysaccharide chains within the cell wall can be identified. Proton T2 increases with increasing mobility. In addition to the highly mobile pectin described in section 2.1, two further mobility classes of pectic galacturonans could be seen within the onion cell wall (figure 3).

Intermediate Mobility Low Mobility

r,

~/v-,.,~-uw,,r.--~/..~/~,,,,

~

I

-i " ~ l l I

180 160 140 120 100

80

I'~hlt_J'v~'vlm4N~'r

60

40

v \ ~ . t ~ "v-"

20

0

ppm

Figure 3: T2 PSRE spectrum of onion cell walls.

The pectic polymers of intermediate mobility included methyl esterified galacturonans and ~(1-4')- linked galactans, similar to those of the most mobile fraction. It is likely that differences in mobility of the galactan chains are a consequence of chain length, with the motion of short chains being more tightly constrained by the attachment to galacturonan at one end (6). The maximum intensity of the galacturonan C-4 resonance was about 79ppm. This may be assigned to a mixture of aggregated chains in the 31 helical

566

conformation and single chains as random coils with intermediate conformations between the 31 and 21 helical conformations. Considerable spectral intensity at around 78ppm suggested that some galacturonan chains of intermediate mobility were in the 21 helical eggbox conformation although overlapping signals from galactan complicate the interpretations. Small quantities of a low mobility galacturonan also appear to be present in both 21 and 31 helical conformations. 2.2 Location of Pectins

If PSRE is applied to the Tip experiment, the spatial location of the wall components can be identified. Within small regions of the cell wall, proton spin diffusion averages the Tip values. Thus the T~p measures the mean mobility within each of these small regions, not the mobility of individual polymer chains (13). It is therefore possible to deduce which polymers are located together in the same region. The proton Tip generally decreases with increasing mobility. Cellulose is the least mobile component of the cell wall. It exists in a crystalline form and there is very little movement of the chains (14). In the hydrated cell wall the Tip experiment averages the movement of the polymers over distances of about 2nm or less in the more mobile parts of the wall, and over 5nm in crystalline cellulose. Therefore any component that is within about 2nm of cellulose will appear in the PSRE spectrum with cellulose (15). It was not possible to work out the location of the highly mobile pectic components. However, there were indications that these pectic components did not exist close to cellulose.

Intermedi ate Mobility Low Mobility

I~1~11 ~ ' V l ~ ' U / ' l f v r g ' W . / - I I ' r ~ l v V r

I i~l~

180 160 140 120 100

I

80

60

40

20

ppm

Figure 4: Tip PSRE spectrum of onion cell walls.

0

567

Both the 21 helix and the 31 helix are represented in the low mobility spectrum of polymers close to cellulose (figure 4), although there was some interference from xyloglucan signals. Both conformational forms of the galacturonans could also be identified from their C4 signals spatially located at a distance from cellulose. Because of the nature of the experiment, we can not tell whether or not pectin from either location is covalently linked to cellulose. If cellulose exists in the cell wall as a network within a pectic matrix, the pectin that is within about 2nm of the cellulose network maybe on or near exposed surfaces of cellulose microfibrils. Both the gel and the eggbox pectins are represented in this low mobility spectrum. There are a number of possible locations within the cell wall for the pectin further away from cellulose. If there are covalent links between pectins and xyloglucans (16), then pectic chain segments close to these links would appear in the region sharing the same mean mobility characteristics as cellulose. The majority of the pectic molecule, diverging from the microfibrils would appear in the region with greater mean mobility. The intermediate-mobility pectin can exist in any space in the cell wall more than 2nm away from cellulose microfibrils. It could therefore be in the middle lamella, cell comers or between layers of microfibrils in addition to the above proposal. The pectin seen in this part of the spectrum are probably a heterogeneous mixture from a number of locations. It has been suggested that in cell walls other than those of onions, different types of pectic matrix are present in different parts of the wall (17). This work clearly demonstrates the existence of at least two spatially separate pectic matrices with polymers having at least two conformational forms and three distinct mobilities. It suggests pectins are more than just 'pore fillers' within the plant cell wall.

3. M E T H O D O L O G Y Onions, c v . Bobosa (100g) were homogenised in Triton-X-100 (2gl 1, 500ml), and cell walls collected on a sintered glass funnel. The walls were washed with water and the excess of liquid removed by suction. The walls were then stirred for 30 min in 15ml of phenol-saturated water. The walls were washed extensively with water, cryomilled in liquid nitrogen and dried to a 3.4:1 wall: water ratio prior to use. The NMR methodology has been previously described in detail (9). Briefly, the NMR experiments were carried out on a Varian VXR-300 spectrometer operating at 75.34 Mhz for '3C. MAS rates varied between 3.5 and 4.3 kHz. The proton decoupling field was nominally 35-36 kHz, and the Hartmann-Hahn match was optimised individually for each sample to allow for radiofrequency energy absorption by mobile water protons. The proton rotating-frame relaxation time Tip and cross-polarisation rate were measured in both variable-contact and delayed-contact experiments. In the variable-contact experiment 12 values of the contact time, in the range 25~ts-18ms, were used. Signal assignments were based on published data (3,10) and are given in table 1.

568 In the delayed-contact experiment a variable delay, during which proton spin-locking was maintained, was inserted prior to a fixed contact time of 0.5 ms. So that the results from the delayed-contact and variable-contact data could be compared, they were normalized to give equal signal intensities at a contact time of 0.5 ms (zero delay in the delayed-contact experiment) after the variable-contact data had been adjusted to allow for the fact that full equilibration of proton and 13C polarisation had not quite been reached after 0.5 ms. The difference in normalised signal intensity between the variable-contact and delayed-contact experiments, at a given time point r, is then a measure of the amount of 13C cross-polarising between 0.5 ms and r. The proton/'2 was estimated by subtracting the signal intensity with 0.5 ms contact after 9.5 ms delay from the signal intensity with 10 ms contact time, as above, but after the insertion of a variable delay (20#s to 2 ms) after the proton preparation pulse. The Tip based PSRE was carried out by taking linear combinations of the spectra at zero and 12 ms delay with the coefficients calculated to make the mean spectral intensity equal to zero between 83 and 85 ppm. The T2 -based PSRE was done in the same way using the spectra at zero and 8 #s delay.


N.C. Carpita and D.M. Gibeaut, Plant J. 3 (1993) 1. 1 M.C. McCann, B. Wells and K. Roberts, J. Cell Sci, 96 (1990) 323. 2 M.C. Jarvis and D.C. Apperley, Carbohydr. Res., 275 (1995) 131. 3 R. Goldberg, C. Morvan, A. Jauneau and M.C. Jarvis, (1996) This volume. 4 K. Fenwick, M.C. Jarvis and D.C. Apperley, unpublished. 5 T.J. Foster, S. Ablett, M.C. McCann and M.J. Giddley, (1996) submitted. 6 x. Wu, S. Zhang and X. Wu, Phys. Rev. B 37 (1988) 9827. 7 L. Muller, A. Kumar, T. Baumann and R.R. Emst, Phys. Rev. Lett., 32 (1974) 1402. 8 M.-A. Ha, B.W. Evans, M.C. Jarvis, D.C. Apperley and A.M. Kenwright, unpublished. 9 10 M.C. Jarvis, Carbohydr. Res., 201 (1990) 327. 11 P.L. Irwin, M.D. Sevilla, W. Chamulitrat, A.E. Hoffman and J. Klein, J. Food Chem. 40 (1992) 2045. 12 P. Tekeley and M.R. Vignon, J. Wood Chem. Technol., 7 (1987) 215. 13 R.H. Newman, A.C.S. Sym. Ser. No. 489, (1992) 311. 14 R.H. Newman, M.-A.. Ha and L.D. Melton, J. Agric. Food Chem., 42 (1994) 1402. 15 R.H. Newman, Holzf0rschung, 46 (1992) 205. 16 C.M.G.C. Renard, A.G.J. Voragen, J.-F. Thibault and W. Pilnik, Carbohydr. Res. 16, (1991) 137. 17 M.-A. Ha, D.M. Gould, R.H. Newman, L.D. Melton and J.I. Mann, Proceedings of the 7th Cell Wall Meeting, (1995) 54.


569

Changes in Pectic Polysaccharides during elaboration of table olives

A. Heredia; R. Guill6n; C. Sfinchez; A. Jim6nez; J. Fernfindez-Bolafios

Departamento de Biotecnologfa de Alimentos, Instituto de la Grasa, Consejo Superior de Investigaciones Cientfficas, P.O. Box 1078, 41012 SeviUa, Spain

Abstract A traditional system for the preparation of table olives, involves a treatment of the fresh fruit with a solution of NaOH to hydrolised the bitter glycoside oleuropein, followed by a lactic fermentation in brine. The modifications that take place on pectic polysaccharides of olives (Manzanilla variety) during this process was studied. Processing induced a net loss of polysaccharides soluble in sodium carbonate and a paralel accumulation of water and Imidazole/HC1 soluble polysaccharides. A general decrease of the apparent molecular weight of water and carbonate soluble polysaccharides was also detected.

1. INTRODUCTION Texture is one of the most important organoleptic characteristics of olives, on the other hand, the physical resistance of the fruit to some manipulations such as depiting is also related to it. Firmnes is, at least in part, related to the composition and structure of cell wall, therefore, from the point of view of controlling the process it is important to know the changes that take place on cell wall polysaccharides through it. On this work the main changes that take place on three pectic fractions, water, imidazole/HC1 and carbonate soluble polysaccharides, of olive cell wall are described and related with modifications of the fruits texture.

2. EXPERIMENTAL Table olives "spanish style" were processed as described by Jim6nez et al. (1). Cell wall were isolated (2) and the pectic polysaccharides were extracted according with their solubility on water (highly esterified polysaccharides), imidazolium (ionically linked polysaccharides) and sodium carbonate (covalent linked polysaccharides), as shows figure 1. Neutral sugars were quantified by trifluoroacetic acid hydrolysis (3) and gas chormatography of alditol acetates (4). Uronic acids were determined by Blumenkrantz method

570 (5). The main components of each fraction were purified by a combination of gel permeation and anionic interchange chromatography and their structure partially elucidated. CELL WALL MATERIAL

Water, 70~ (2X30 min.)

ImidazolelHCI buffer 0.SM 25~

v

WATER

v

IMIDAZOLE-HCI

y

CARBONATE

12 hours

Sodium Carbonate O.05M, 4~

16 hours

Sodium Carbonate O.05M, 25~

3 hours

Figure 1. Fractionation of cell wall

3. RESULTS

When the amounts of the three pectic fractions were determined through the different steps of the process, it was found that it is during the lye treatment and wash where the main changes took place. Neutral sugars increased in the water soluble fraction and d i d n t change on imidazole and carbonate ones. Uronic acids, on the other hand increased in the imidazole fraction and decreased in both water and carbonate ones. Water soluble polysaccharides: Table 1 shows that there was an important increased of neutral sugars associated with neutral polysaccharides on this group, and the glycosyl composition (table 2) reveals that they are basically arabinans (arabinose content about 90%). The acidic fraction in the fresh fruit is constitued by homogalacturonans (90% U.A) together with small amounts of ramnogalacturonans. Processing induced a decreased on the total amount of uronic acids and a parallel increased of neutral sugars, changing the ratio N.S./U.A. from 0.16/1 to

571 0.93/1. It can be concluded that during processing there is an incorporation of arabinans and a loss of homogalacturonans from this fraction. On the other hand a shift on the molecular weight distribution to lower values of the acidic fraction it was found. Table 1 Yield and composition of water soluble polysacchaddes (mg/fruit) NEUTRAL

ACIDIC

FF

PF

FF

PF

Yield (mg/fruit)

1,72

2,22

0,64

1,02

Neutral S.

0,68+0,01

Uronic A.

0

0

0,68+0,01

1,56+0,02

Total S. N.S./UA

1,564-0,02

. . . . . .

0,09a:0,02

0,28u

0,564-0,06

0,30-a:0,01

0,65+0,04

0,58+0,01

0,16/1

0,93/1

Table 2 Glycosyl composition of water soluble polysaccharides (mol%) NEUTRAL

ACIDIC

FF

PF

FF

PF

Rham

0,62

2,00

2,14(16,14)

11,04 (23,03)

Fuc

0,08

0,09

0,18 (1,43)

1,21 (2,51)

Ara

87,94

92,61

8,48 (63,76)

22,89 (47,70)

Xyl

1,15

0,56

0,05 (0,35)

3,54 (7,40)

Man

2,81

0,40

0,22 (1,64)

0,51 (1,10)

Gal

3,10

2,47

1,74(13,17)

7,83 (16,34)

Glc

4,20

1,86

0,46 (3,50)

0,93 (1,95)

Uronic A.

0,00

0,00

86,76

52,03

Ram/U. A.

-.

.

0,025/1

0,21/1

Rham/Ara

. . . . . .

0,25/1

0,48/1

.

.

.

572 Imidazole-HCl soluble polysaccharides: Most of the polysaccharides of this group didn't elute from the Q-Sepharose column with 10 mM buffer (Table 3). The glycosyl composition (Table 4) shows that these polysaccharides are homogalacturonans and ramnogalacturonans with Table 3 Yield and composition of lmidazole-HCl soluble polysaccharides (mg/fruit) i

NEUTRAL

ACIDIC

FF

FP

FF

FP

Yield (mg/fruit)

0,21

0,11

3,78

3,93

Neutral S.

0,04+0,00

0,04+0,00

0,24:L-0,01

0,64+0,09

Uronic A.

0,01

0

1,54+0,01

1,95+0,01

0,04+0,00

0,04+0,004

1,78+0,01

2,59-a:0,36

Total S. N.S/U.A.

-. . . . .

0,16/1

0,33/1

Table 4 Glycosyl composition of lmidazole-HCl soluble polysaccharides (mol%) NEUTRAL

ACIDIC

FF

FF

FF

PF

Rham

0,80

1,98

2,10 (15,50)

6,46 (26,30)

Fuc

0,15

0,60

0,20 (1,40)

0,55 (2,22)

Ara

57,83

66,02

8,84 (65,16)

11,58 (47,10)

Xyl

3,01

1,45

0,18(1,31)

0,91 (3,70)

Man

10,03

4,40

0,19 (1,42)

0,23 (0,92)

Gal

15,03

21,23

0,17 (1,25)

4,34 (17,65)

GIc

13,05

4,32

0,44 (3,28)

0,50(2,01)

Uronic A.

0,00

0,00

86,43

75,40

Rham/U.A.

. . . . . .

0,024/1

0,09/1

Rham/Ara

. . . . . .

0,24/1

0,56/1

573 side chains rich in arabinose, methylation analisis reveals that one of every three arabinose residues are substitued. The ratio Rha/U.A. is similar to that of water soluble polysaccharides meaning that the proportions of ramnogalacturonans are similar in both fractions. Processing induced a decreased on the percentage of uronic acids and an increase on those of galactose and ramnose, the ratio Rha/U.A. went from 0.02/1 to 0.09/1 while that of Rha/Ara from 0.24/1 to 0.56/1. It can be concluded that during processing there is an incorporation of pectic polysaccharides richer in ramnogalacturonans to this fraction. The molecular weight distribution of these polysaccharides d i d n t change as a consequence of processing.

Carbonate soluble polysaccharides: Unlike the other pectic fractions, tile total sugars of this one decreased during processing (from 1,13 to 0,41 mg/frui0, the most prominent decreased taking place on both neutral sugars an uronic acids of acidic polysaccharides (table 5). In the fresh fruit (table 6) this fraction contains high amounts of uronic acids (48 %) and arabinose (34 %), the ratio Rha/U.A. is much higher than that of water and imidazole soluble fractions, meaning that the hairy regions of the pectic polysaccharides are more abundant on this fraction. Methylation analysis shows that one of every four arabinoses is substituted. With processing it was found an increase in the molar ratio of uronic acids together with a decrease in that of arabinose, methylation analisis reveals that there was a decreased in terminal, 3- and 5-1inked arabinose and that the 3,5-1inked arabinose practically dissapeared. It was also found an important decrease in the molecular weight distribution of this group of polysaccharides. Table 5 Yield and composition of carbonate soluble polysaccharides (mg/fi'uit) NEUTRAL FF Yield (nag fruit) Neutral S.

Total S. N. S.FLI. A.

PF

0,35 0,05•

U.A.

0,2 0,02--1:0,01

0 0,05•

ACIDIC

0 0,02• ---

FF

PF

4,27

0,66

0,59•

0,16+0,01

0,54•

0,25•

1,13•

0,41•

1,10/1

0,64/1

574 Table 6 Glycosyl composition of carbonate soluble polysaccharides (mol%) NEUTRAL

ACIDIC

FF

FP

FF

FP

Rham

0,67

7,24

9,53 (18,20)

9,18 (23,00)

Fuc

0,20

0,83

0,72 (1,35)

0,61 (1,62)

Ara

82,55

50,50

34,10 (65,05)

lo8,96 (49,41)

Xyl

3,73

2,05

0,37 (0,70)

1,76 (4,60)

Man

4,04

8,05

0,04 (0,88)

0,44 (1,14)

Gal

5,16

16,20

6,36 (12,13)

6,41 (16,71)

Glc

3,63

15,33

0,87 (1,66)

0,91 (2,36)

Uronic A.

0,00

0,00

47,58

61,61

Rham/U. A.

-.

.

.

.

.

0,20/1

0,14/1

Rham/Ara

. . . . . .

0,28/1

0,48/1

Texture: In the figure 2 the values of firmness along the process for two consecutive seasons is shown, very similar results were found for both seasons. The most important decreases took place during the lye treatment and subsequent wash step, reaching values of 50% of the initial. When the fruits were placed in the brine solution the firmnes recovered to 80% of the initial and, finally, during fermentation there was a new decrease to 60% of the initial.

4. CONCLUSIONS

When the processing liquids were analyzed it wasnt found any important increased of uronic acids (data not shown), this together with the fact that the total amount of pectic polysaccharides quantified in the cell wall didn t change, bring us to the conclusion that there is no important solubilization Of pectic polysaccharides during olives processing and that the main process that take place, is an interchange of polysaccharides betwen different groups. Therefore processing induced a net loss of sodium carbonate soluble polysaccharides and a parallel

575 %

SEASON 1 90

-I- S E A S O N 2

u) uJ Z

:E 80 rr ,,m

u.. u.I >

.~70 60 501 UF

i

1/2LYE

t

LYE

I

WASH

I

EQ.BRINE

FE FIM

Figure 2. Changes in texture during elaboration

accumulation of water and imidazole/HC1 soluble ones. The accumulation of polysaccharides in the water soluble fraction is mostly a consequence of the incorporation of neutral arabinans, probably resulting from losses of pectic polysaccharides side chains. On the other hand, the uronic acid content of this fraction decreased, most likely due to deesterification of the galacturonic acid residues, increasing in this way their ability to linked ionically in the cell wall and becoming imidazole soluble. The fact that the molecular weight distribution decreased shows that together with the deesterification process it must also take place a limited degradation through B-elimination. In the carbonate soluble fraction there is an evident loss of arabinans side chains together with the breakdown of the linkages that held these polysaccharides with the hemiceUulosic matrix (probably phenolic linkages) yielding them imidazole soluble. This is supported by the fact that the imidazole soluble polysaccharides of the processed fruit are of high molecular weight, showing that there wasn Jt an important degradation of the backbone. It is clear that this changes on the cell wall must have important consequences on the firmness of the fruit. The breakdown of linkages between pectic polysaccharides and the matrix is likely to be an important factor on the irreversible loss of firmness. The deesterification of polysaccharides, on the other hand could have an effect of increasisng firmness. There is, however, another factor that should be taken into account, the alcali deesterification of the

576 galacturonic acid units increase the number of free carboxil groups on the pectic backbone this, together with the high pH during lye treatment and wash (between 12 and 10 respectively), creates a net negative charge on the molecule and repulsions between different pectic chains (the carboxil groups at these pHs are completed ionized), wich results in an important loss of texture. When the fruits are placed in brine the pH is corrected to about 7, this decreases the negative charge due to the deionization of carboxil groups and to the neutralization of some charges by sodium cations and deals to a recover of the firmness. The relative importance of the different factors is something that remains to be cleared.

5. REFERENCES

A. Jim6nez; J. Labavich; A. Heredia; J. Agric. Food Chem., 42 (1994) 1194. R.R. Selvendran. Phytochem. 14 (1975) 1011. J.M. Ruiter; J.C. Bums; J. Agric. Food Chem., 35 (1987) 308. H.N. Englyst; J.H. Cumming. Analyst, 109 (1984) 937. N. Blumenkrantz, G. Asboe-Hansen. Anal. Biochem. 54 (1973) 484.


577

Pectins from different tissue zones of apple: characterisation and

enzymatic hydrolysis P. Massiot, A. Baron and J.F. Drilleau

Institut National de la Recherche Agronomique, Station de Recherches Cidricoles, Biotransformation de Fruits et IMgumes, BP 29, 35650 Le Rheu, France

Abstract Pectins from different tissue zones, namely epidermis, the outer parenchyma, the parenchyma of the carpels zone, the carpels and the core line, were isolated from alcoholinsoluble solids (AIS. In both zones of parenchyma, the cell-waU material represented about 80% of the total cell-wall material from the whole fruit. The pectins from the outer parenchyma accounted for 70% of the total. However, there was no change in galacturonic acid concentration. The enzymatic solubilisation of tissues or AIS was higher in the parenchyma zones than in the others. Nevertheless, the depolymerisation of the soluble pectins from parenchyma zones with an endopolygacturonase required the action of pectin methylesterase. The depolymerisation of pectins from the other zones, however, did not.

1. INTRODUCTION

In apple processing, enzymatic treatment of the crushed fruit leads to a lower degree of degradation of the peel and the core than the rest of the fruit. Figure 1 shows the separate tissue zones in diagrammatic form. Their anatomic origins are different: the epidermis and outer parenchyma zones are tissues derived from the fusion of the calyx, corolla and stamens of the flower; the inner zones correspond to tissue derived from ovaries and carpels. The characterisation of the cell-wall material, especially pectins, from the different zones of the fruit may provide additional information on the possibility of finding uses for the discarded fractions.

578

A

Figure 1. Sections of mature apple; A: B: C: D:

epidermis zone, outer parenchyma, parenchyma of the carpels zone, carpels and core line.

2. DISTRIBUTION AND COMPOSITION OF C E L L - W A L L MATERIAL The outer parenchyma (B) is the major tissue zone of the fi'uit, corresponding to more than 80 % of dry matter and the edible zones (B and C) contained 80 % of the cellwall material (Fig.2).

Carpels and core line (D) 10% Parenchyma of the carpels reaion (C) 10%

Epidermis zone (A) 11%

Outer parenchyma (13) 70%

Figure 2. Distribution of alcohol-insoluble solid (AIS) in the different zones of Judeline apple. Total AIS: 12.6 g/100g of apple dry matter

579 The four AIS contained high and equivalent amounts of galacturonic acid (Table 1), indicating that the distribution of pectic substances was similar to the distribution of the AIS in the fruit.

Table 1 Composition of alcohol-insoluble solid (AIS) in different tissue zones of apple Tissue zones Yield (a)

Sugar composition (b)

Rha Ara

Xyl

Man Gal

Glc

Proteins Lipids

Gal.A

(b)

Co)

total

A

274

15

47

34

19

54

154 265

588

57

290

B

107

13

83

74

24

98

296 287

875

27

55

C

160

11

48

88

23

48

301 283

802

44

62

D

308

13

32

135

28

27

344 253

832

55

38

(a) mg/g of d.m. of tissue (b) mg/g of AIS

3. CHARACTERISATION OF PECTIC POLYSACCHARIDES (TABLE 2)

The composition of the pectic fractions confirmed the presence of highly esterified slightly branched rhamnogalacturonan in the CSP fractions and the presence of highly branched rhamnogalacturonan in the HSP fraction.

580 Table 2 Monosaccharides composition of pectic polysaccharides in different tissue zones of apple Tissue

Fraction

zones

Yield

Sugar composition (mole %)

~%AIS

CSP (a)

Rha Ara Xyl Man

Gal

Glc

GalA

DM

Total mg/g

C D

19.2 16.8 16.9 13.0

1.5 1.8 1.9 2.4

9.6 22.7 5.9 7.4

0.8 1.0 0.6 1.3

0.5 0.2 tr 1.0

6.3 11.1 5.4 5.8

1.3 0.4 0.2 1.3

80.0 62.8 86.0 80.8

70 71 62 65

582 722 516 428

A B C D

8.6 12.5 11.1 7.9

4.9 5.7 6.2 7.1

9.7 9.0 10.7 8.4

2.9 4.5 4.6 4.2

0.7 0.4 0.4 0.8

8.2 13.2 8.4 7.3

1.8 2.9 2.3 1.9

71.8 64.3 67.4 70.3

52 41 55 49

650 795 759 692

A B

HSP (b)

(a) CSP Cyclohexane-diamino-tetracetic acid Soluble Pectin. (b) HSP H C1 Soluble Pectin. DM: degree of methoxylation.

4. ENZYME HYDROLYSIS OF C E L L - W A L L POLYSACCHARIDES

When the apple tissues were treated with enzyme preparation for liquefaction (Fig. 3), the cell-wall materials were solubilised with different yields, 95, 86, 66 and 59 % for zones B, C, D and A, respectively. The sequence was the same with the maceration treatment (use of polygalacturonase [PG] only) but the yields were lower.

100

-

m

80-

Liquefaction

,-+.. Maceration

60-

4020-

t

A

C

I

D

A

B

C

Figure 3. SolubilisaUon of cell-wall material (as AIS) when apple tissues were treated with liquefying (SP249) or macerating (UM10) preparations

D

581

With PG [a], the kinetics of pectin solubilisation (Fig.4) were dose to each other, with limits around 40-50 %. A combined action of PG and pectin mcthylcstcrascs (PME) [1:)] or PG, PME and ccllulascs [c] increased these values, especially for the AIS from zone B. The sequence os the zones was D, A, C and B, in rising order of solubilisation.

(a)

100

1

80 60

+ jf

4O

'

20

~ =

A

i i

..---.--c~

B

:

~ : -

C

!

~ - - D

i,

0 0 ,

'qO .,m 0 0 .=. r"

1

2

3

4 ,,

,

,,

,,

100

..r

80

t

5 ,,,

,,,

,

....

OZ

-.--,Z.

_.___._..---~

~ 1 1 ~

A

~ r

C

0

!

60 40

~ t

,,,...,

ilm, 0

,,,

,

(b)

0L 0

,,,,,, ,

20

J

0 0

1

2

3

4

loo I 80 y.........o------c I i

5

~t

:_

(c)

I

-- I '---I"'- A t I

0

O 0

1

2

3

4

5

Time (h) Figure 4. Content of galacturonic acid in the soluble fraction after hydrolysis of AIS with polygalacturonases [a], with polygalacturonases and pectin methylesterase [b], with pectinases and cellulases [c].

582 The fact that the CSP pectic fractions from zones B and C were very little modified by the action of an endopolygalacturonase alone (Fig. 5) suggested that these fractions were structurally different than the others. In particular, the high degree of methoxylation of both fractions and the significant proportion of neutral sugars of the CSP fraction (zone 13) could limit the action of the endopolygalacturonase.

CSP(A)

r ,

~... [CSP(B)i

],': !

. I

& t_

N =

CSP (C~:'

, A

/t;.

I

i

0

-

Kay

i

1

Figure 5. High-Performance Size-Exclusion Chromatography of Cyclohexane-diamino-tetracetic acid-Soluble Pectin (CSP) from different tissue zones of apple (-----) or after hydrolysis with endopolygalacturonase ( - - -) or with endopolygalacturonase and pectin methylesterase (. . . . ).

5. CONCLUSION

The main part (80%) of pectins were in the edible apple zones but no change in galacturonic acid concentration was observed between the different tissue zones. So, carpels and epidermis zones are potentiel sources of highlymethylated pectins.


I n v e s t i g a t i o n s of the influence of v a r i o u s p r o p e r t i e s of h i g h - e s t e r i f i e d pectin gels

583

cations

on

the

rheological

SybiUe Neidhart, Christa Hannak and Karlheinz Gierschner Institute of Food Technology, Department of Fruit and Vegetable Technology, Hohenheim University, Garbenstr. 25, D-70599 Stuttgart, Germany Abstract

The influence of sodium, potassium, calcium and magnesium ions on the gelling behaviour of apple pectins with degrees of methylation between 43% and 73% as well as the effect of these ions on viscoelastic properties and thermal behaviour of the final gels were investigated by means of small amplitude oscillatory shear experiments. The rheological data were obtained for each pectin according to mixture designs for the metal ions with least changes in pH and ionic strength and analyzed by multiple non-linear regression. With each pectin the various rheological properties were shown to be modulated in a complex way. The setting behaviour was always affected in a similar fashion by the ions as the thermal behaviour of the final gel, but not in the same way as the strength or the elasticity of the gels. The rheological results did not reveal any direct dependence on the charge or the size of the cations, suggesting various, more or less specific modes of action to be involved, depending on the nature of the cation and the amount of dissociated carboxylic groups in the sample. 1. INTRODUCTION High-methylated pectins (HMPs), classified as those with a degree of methylation (DMe) higher than about 50%, require high amounts of soluble solid substance, mainly sugar, and low pH for gelation. Besides type and concentration of pectin these parameters strongly determine gelation as well as resulting gel properties. The available results indicate that the junction zones of these gels are mainly stabilized by cooperative sequences of hydrogen bonds besides hydrophobic interactions between the ester methyl groups [1,2]. While low pH is needed to reduce dissociation of the carboxylic groups and hence to diminish repulsive forces between the pectin molecules, the sugars, reducing water activity, are discussed in terms of promoting hydrophobic interactions by their own interaction with water molecules, which depend on their stereochemistry [1]. As low-methylated pectins are able to form gels in the presence of various bivalent cations by chelating them, the influence of numerous metal ions on the physical behaviour of those pectins has been extensively studied [3-5]. As far as gelation of liMPs is concerned, there are somewhat contradictory indications for the influence of metal ions on the gelation behaviour and on the gel properties (e.g. [6-8]). It was felt that a more comprehensive study on this topic, considering various well characterized HMPs with different levels and patterns of methylation as well as different characteristics of the gelation process on basis of an experimental design, which allows for potential interactions between the cations, would be a useful contribution to the understanding of the complex mechanisms involved in HMP gelation.

584 Here data on apple pectins are presented. A comparison of corresponding data on apple and citrus pectins will be given in a future paper. Small amplitude oscillatory shear measurements have been widely used to follow the gelation process of biopolymers [9] because of their non-destroying character and the great variability of procedures, which allow simultaneous investigations of various features of a single sample. 2. EXPERIMENTAL

2.1. Pectin samples Three unstandardized, commercial pectins from apple (Herbstreith & Fox, Germany) were purified by washing six times with acidified ethanol (60%), afterwards with ethanol (60%), and, finally, in the absence of chloride in the filtrate, with ethanol (96%). The residue was dried for 2.5 hours at 105~ and extensively characterized (table 1) [10]. The percentage of anhydogalacturonic acid (AUA) was determined colorimetdcally with m-hydroxydiphenol. Degree of methylation (DMe) and acetylation (DAc) resulted from h.p.l.c, determination of methanol and acetate, while analysis of various neutral sugar contents was realized gas chromatographically. Cation analysis was done by atomic absorption spectrometry. For more details of analytical procedures and pectins, the reader is referred to [10]. The pattern of methylation of each pectin can be considered to be random The content of rhamnose was fairly constant. 2.2. Preparation of the gels The pectin/sucrose gels were characterized as follows (amounts per 100g gel): 0.3 g AUA, 65% soluble solid substance, 0.01 mol sodium acetate / lactic acid buffer, pH 3.0 (20~ The metal ions were added as combinations of chlorides according to a mixture design with constant amount of chloride ions (2.5 mmol / 100g gel). Thus the total amount of metal ions Table 1. Characteristics of the purified pectin samples. Pectin =

A73

A64

A43

AUA [g / 100g dry weight] DMe ~ [%] DAc ~ [%] Total neutral sugars~ [%] Calciumb [%] Magnesium ~ [%]

83.4 73.4 5.6 28.1 0.17

=o

(u 4= 4) rv'

RG+AE

Blank

20

25

30 Time (rain)

35

40

Figure 3. HPSEC results of the cross-linked extract after treatment with RG+AE, EA+AF or PG+PE. Viscosity measurements of the extracted polysaccharides cross-linked at increasing concentrations showed a clear increase in relative viscosity, when the cross-linking reaction took place at concentrations higher than 0.5 % (Fig. 4). At a concentration higher than 1.5 % a gel was formed.

602

~9

6

.~

4

Gel: 1.5%

| n,'

,

0

,

I

I

,

I

1 2 3 Polysaccharide concentration (%)

I--x-Blank

4

4

-~- Cross-linked I

Figure 4. Relative viscosity at increasing polysaccharide concentration in the second autoclave extract before (blank) and after cross-linking.

4. CONCLUSIONS Autoclave extraction of sugar beet pulp yielded pectic polysaccharides, which were rich in 'hairy' regions and ferulic acid. The 'hairy' regions and 'smooth' regions were present in separate populations. Most of the ferulic acid was found in the 'hairy' regions. Oxidative cross-linking Of the autoclave extracted polysaccharides lead to an increase in the molecular weight of particularly those polysaccharides containing the arabinan side chains ('hairy' regions). After cross-linking at higher polysaccharide concentrations the relative viscosity increased and eventually a gel was formed. Enzyme studies confirmed that only 'hairy' regions were responsible for cross-linking. The cross-linked material could be degraded by treatment with endoarabinanase and arabinofuranosidase B, and could only be partially degraded using rhamnogalacturonase and rhamnogalacturonanacetylesterase.


E.L. Pippen, R.M. MeCready, and H.S. Owens, J. Am. Chem. Soc., 72 (1950) 813. J.F. Thibault and F.M. Rombouts, Carbohydr. Res. 154 (1986) 205. F.M. Rombouts and J.F. Thibault, in M.L. Fishman and J.J. Jen (Eds.), Chemistry and Function of Pectins, ACS Symp. Ser. 310, American Chemical Society, Washington, DC, 1986, pp. 49-60. C.C.H. Wang and K.C. Chang, J. Food Sei., 59 (1994) 1153. A. Oosterveld, G. Beldman, H.A. Schols and A.G.J. Voragen, submitted for publication in Carbohydr. Res. (1995). H.N. Englyst and J.H. Cummings, Analyst, 109 (1984) 103. H.A. Sehols, M.A. Posthumus, and A.G.J. Voragen, Carbohydr. Res., 206 (1990) 117. M.-C. Ralet, J.-F. Thibault, C.B. Faulds, and G. Williamson, Carbohydr. Res., 263 (1994) 227. F. Guillon and J.-F. Thibault, Carbohydr. Res., 190 (1989) 85.


603

Pectins in m i l d alkaline conditions: [3-elimination and kinetics of d e m e t h y l a t i o n C.M.G.C. Renard and J.-F. Thibault Laboratoire de Biochimie et Technologie des Glucides, INRA, rue de la G6raudi~re, B.P. 1627, 44316 Nantes cedex 03.

Abstract Pectins were incubated in buffered medium in mild alkaline conditions (pH 8.5 to 11.2) at room temperature, leading to both demethylation and [~-elimination. At higher pHs 13elimination had increased initial speed but soon plateaued. Demethylation was slower but proceeded until completion. It followed a (pseudo)-first order kinetics with respect to concentration of methylesterified carboxyl groups. A rate constant of 27.2 + 9.0 mol ~ 1 min ~ was calculated after correction for the pH variation during the course of the reaction.

1. I N T R O D U C T I O N Loss of functional properties of pectins at high pHs has been recognised for more than 50 years (Kertesz, 1951, and refs cited therein), though the mechanisms in play have been recognised more recently. In alkaline conditions, pectins are degraded by two competitive reactions: 13-elimination, which creates double bonds next to a methoxylated galacturonic moiety, and demethylation by saponification (Neukom & Deuel, 1958; Albersheim et al., 1960). This competition is modulated by pH and temperature conditions: any increase of temperature increases the rate of [~-elimination more than that of demethylation, while an increase of pH increases demethylation more than [~-elimination (Kravtchenko et al., 1992). For exemple, amidated pectins with high molecular weight can be produced at low temperatures (McCready et al., 1944; Joseph et al., 1949), while heating at neutral to slightly acidic pHs (Albersheim, 1959; Kravtchenko et al., 1992) leads to extensive depolymerisation of pectins in solution. At room temperature, in the pH range 9-11, both reactions occur. To better understand these degradations and their competition, we have quantified rate constants of demethylation. 2. MATERIALS AND METHODS

Commercial Rapid Set citrus pectin (864 mg galacturonic acid per g, degree of methylation 73) was from SBI (Beaupte, France). The kinetics of 13-elimination were followed by the increase of the absorbance at 235 nm (Albersheim et al., 1960). The pH of the pectin solution (1 ml, already in the spectrometer) was adjusted at t = 0 by adding 1 ml of 0.2 M sodium hydrogenocarbonate sodium carbonate buffer. This buffer did not have a prohibitively high absorbance at 235 nm. The kinetics of demethylation were followed in hermetically closed vials in a water bath at 25~ Ethanolamine/HC1 buffer, at a final molarity of 0.2 M, was used to vary the pH. Tubes containing 1 ml of pectin solution in distilled water were prepared and at t = 0, 1 ml of ethanolamine buffer was added. One tube in each series was used to read the initial pH of the

604 pectin/ethanolamine buffer mixture, and determine the amount of acetic acid necessary to bring the pH to 4-4.5. In the other tubes, the reaction was stopped after a given time by bringing adding that predetermined amount of acetic acid. Free methanol was measured by gas-liquid chromatography. A sample solution (50 lxl) containing free methanol is mixed with 50 ~tl of a butanol solution (100 lxl in 1 1 of distilled water; internal standard) prior to injection (1 ~tl) on a DBwax column (30 m x 0.32 mm) at 50~ with hydrogen as a carrier gas at 0.85 bar. The injector and the flame ionisation detector were at 150~ After each third sample, the column was cleansed by heating to 150~ Ethanolamine was one of the few buffer systems usable in this pH range that did not give interfering peaks during glc. 3. RESULTS 3.1. B-elimination During 13-elimination of pectic substances, a double bond is created between C-4 and C-5 of the new non-reducing end, leading to absorbance at 235 nm (by conjugation with the carboxyl groups). This spectral property of I]-eliminated pectins was used to follow their degradation (Fig. 1).

0.4 0.3~

O. 1

. ..:..,. O:. ~ ~ O-. - .'. - ~ 2 - O .............

m~

~-

0 0

20

40

60 Time (min)

80

100

120

Fig. 1: Time-course of the 13-elimination reaction of a 2.5 mg/ml pectin solution at 25~ in 0.2 M sodium hydrogenocarbonate carbonate buffer. o : p H 11;O: pH 10.5; I : pH 10;ra: pH 9.5; II : p H 9 . The two most striking features of these time-courses are that, though the initial speed of the reaction increases markedly with the pH, the final level does not, as the reaction stops much earlier. This was not due to a drop of pH below the values suitable for ~-elimination, as the pH change were slight (-0.2 at pH 11, no measurable change at pH 9). In order to check wether this plateau was due to disparition of suitable reaction sites, i.e. methoxylated galacturonic acid residues, we have investigated in detail the deesterification reaction. 3.2. Effect of pH on deesterification The deesterification reaction, or more precisely the demethylation reaction, was followed by measuring the amount of methanol liberated (Fig. 2).

605

0.8

1

A

I

0.6-

I

J

o

f

.,>

0.40.2--

Ratio of residual/initial ester (log scale)

Ratio of free methanol/initial ester

i ,/" ,o v..-=..-

o'"

9 ,,-..D.

-

o-u -- -

"

~-e ~ ~ " o. _- - - -

-~

- ---

-

d,

,,,~.~~--.m" ...................... 0 u u u u u ...... 0 20 40 60 80 100 120 Time (min)

.1

I

100

2d)0 3d0 Time (rain)

Fig. 2: Time-course of demethylation of a pectin solution (-5 mg/ml) at 25~ ethanolamine/HC1 buffer. 9 : pH 11.25; O: pH 10.55; 0: pH 10.05; El : pH 9.53; I1: pH 9.02; O : pH 8.25.

4(~0

in 0.2 M

As for 13-elimination, the higher the pH, the faster the reaction rate: at pH 11.25, half of the methylesters are liberated within one hour, whereas at 8.25, the lowest pH we investigated, less than 20% are liberated after 24 h. The demethylation shows two major differences with 13elimination: the variation of the initial speed of the reaction is less drastic, and the reaction does not plateau so soon, particularly for high initial pHs. 3.3.

Determination

of

the

rate

constants

Classically, saponification reactions can be written as (Connors, 1990): RCOOR' + OH --) RCOO + R'OH, and follow second order kinetics with - d[RCOOR']/dt = d[R'OH]/dt = k [RCOOR'] x [OH]

(1)

with [RCOOR']: concentration of ester (here of methoxylated galacturonate residues); [R'OH]: concentration of free alcohol, here methanol; [OH]: concentration of OH ions; t" time and k: reaction constant. It should therefore be possible, by keeping the concentration of O H ions constant throughout the reaction, e.g. by buffeting the medium, to transform the reaction into a pseudo-first order reaction with respect to the ester. One of the typical features of a (pseudo)-first order reaction is that a plot of the logarithm of the advancement of the reaction versus time (Fig. 2B) should give straight lines. However we observed deviation from linearity before the first half-life, in spite of the fact that another characteristic features of (pseudo)-first order reactions, namely that plots of the extent of reaction vers.us time were independant of the initial concentration (Fig. 3), was verified. We therefore investigated whether variation occured in the reaction conditions as a function of time. We found a slight decrease of pH during reaction (0.1-0.2 pH units in the buffer zone of ethanolamine), which however translated as a decrease of about 20% of the concentration of OH ions. Above pH 10.5, the loss in OH ions reached about 40% of the initial concentration. This variation could be predicted by taking into account the need for replacement of the buffer ions: at any time t ~ 0 eletroneutrality implies that for every carboxylate liberated (i.e. every methoxylated galacturonate saponified), one molecule of ethanolamine is converted from the base form (EtNH2) to the salt form (EtNH3+). The concentration of the base and salt forms at

606 any time t, calculated from the intial pH and the amount of liberated methanol, gave a good prediction of the measured pH variation (Fig. 4). The deviation from the (pseudo)-first order behavior was thus explained by the impossibility to keep the OH ions concentration constant using a buffer. Ratio of residual/initial ester

p H = 10.05 016

-8

8

p H = 9.53 0

0.4 0"20 I

0

I

500 1000 Time (min)

I

0

I

500 1000 Time (min)

1500

Fig. 3" Demethylation of pectin at three different concentrations: 9 95 mg/ml; o: 2.5 mg/ml; ~x 91.25 mg/ml.

o -0.1

o*=.4

[]

;>

r

[]

-0.2

-0.3 0

100

200 300 Time (min)

400

500

Fig. 4: Variation of pH during demethylation of pectin (--5 mg/ml, in 0.2 M ethanolaminePrlC1 buffer). measured pH with initial pH of 10.48 (o) and 9.72 (tJ); pH calculated from the amount of liberated methanol for initial pH of 10.52 (-*-) and 9.75 (--~-). The kinetics of liberation of methanol were therefore recalculated taking into account the pH variations: equation (1) could be rewritten replacing the concentration of OH- ions by its expression as a function of the buffer. At each point the concentration of OH was calculated from the initial pH and the amount of reacted ester:

607 [EtNH3+], = [EtNH3+]0+ [MeOH]~; [EtNH2], = [EtNH2]0- [MeOH]~ with" [EtNH2] + [EtNH3§ = 0.2 M and pH, = pK, + log,0([EtNH2]] [EtNH3*],) (pK~ of ethanolamine/HC1 buffer: 9.5). The new expression of equation (1) is: d[MeOH]

dt

= k' ([RCOOMe]0_ [MeOH]t)

([EtNH2] ~ _

[MeOH],)

(0.2- ([EtNH2]~_ [MeOH]))

with k' = k x 10(PKa -

(2)

14).

Equation 2 can be integrated, giving an expression linear with time. Straight lines were indeed obtained when using this equation to plot the results of demethylation (Fig. 5), confirming that deviation from linearity was due to loss of OH ions during the course of the reaction.

1

Calculated with pH correction

0.8-

II II

0.6II 0.4-

II

Q o

0 0 0

, 1O0

I

I

200 300 Time (min)

I

400

500

Fig. 5: Kinetics of demethylation of pectin expressed from intergration of equation (2). O: pH 10.55; t : pH 10.05; El: pH 9.53; I1: pH 9.02. Twenty-three kinetics have been carried out at 25~ for pH values from 8.25 to 11.25. The rate constant, calculated as the average of all the ks, was of 27.2 + 9.0 mop 1 min ~ . The pH correction according to equation (2) was not perfect, as there was a tendency to obtain higher k values at lower pH values. However, this was specially true for extreme values of our pH range, where the buffer capacity of ethanolamine was limited (higher pHs) or the reaction proceeded very slowly (low pHs), impairing the precision of the data. Another factor that might explain the dispersion of the data is lack of precision of pH measurement (no better than + 0.02 pH units).

608 4. DISCUSSION In mild alkaline conditions, highly methylated pectin was demethylated following a (pseudo)-first order kinetics with respect to the concentration of methoxylated galacturonate moieties. Investigation in this pH range, where the initial concentration of methylesters was higher than the initial concentration of OH ions, was complicated by the necessity to use a buffer. This led to deviations from the theoretical behavior as the concentration of OH ions still varied in proportions which could not be neglected in the equations of the kinetics. However these deviations could be accounted for be the pH variation, and the pH variation itself predicted from the amount of liberated methanol. The constant we found was similar to previously reported data (Scamparini & Bobbio, 1982). 13-elimination had higher initial reaction rates at higher pHs, and the variation in reaction rates appeared more marked than for demethylation (for which the variation was linear with the concentration of OH- ions). Quantification of the demethylation reaction allowed to see that 13elimination reached a plateau long before total demethylation, which could be linked to topological requirements for the 13-elimination, e.g. necessity of two neighbouring methoxylated galacturonate residues. 5. REFERENCES

Albersheim P. (1959) Instability of pectins in neutral solutions. Biochemical and Biophysical Research Communications 1, 253-256. Albersheim P., Neukom H. & Deuel H. (1960) Splitting of pectin chain molecules in neutral solutions. Archives of Biochemistry and Biophysics 90, 46-51. Connors K.A. (1990) Chemical kinetics: The study of reaction rates in solution. VCH Publishers, NY. Joseph G.H., Kieser A.H. & Bryant E.F. (1949) High-polymer ammonia demethylated pectinates and their gelation. Food Technology 3, 85-92 Kertesz Z.I. (1951) The pectic substances, Interscience Publishers NY (specially p. 121-122). Kravtchenko T.P., Arnould I., Voragen A.G.J. & Pilnik W. (1992) Improvement of the selective depolymerization of pectic substances by chemical 13-elimination in aqueous solution. Carbohydrate Polymers 19, 237-242. McCready R.M., Owens H.S. & Maclay W.D. (1944) Alkali-hydrolyzed pectins are potential industrial products, part I. Food Industries Oct. 1944, 69-71, 139-140. Neukom H. & Deuel H. (1958) Alkaline degradation of pectins. Chemistry and Industry June 1958, 683. Scamparini A.R.P. & Bobbio F.O. (1982) Deesterification of citrus pectin. Industri Alimendari Feb.1982, 110-112, 116

J. Visserand A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996Elsevier Science B.V.All fights reserved.

609

P o t e n t i o m e t r i c t i t r a t i o n of p o l y ( a - D ) g a l a c t u r o n i c acid D.Rudan-Tasi5 and C. Klofutar

Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, SI-61000 Ljubljana, SLOVENIA

Abstract A commercially available sample of poly(a-D)galacturonic acid, i.e. pectic acid, was characterized according to the size and shape of its molecule through the volumetric and transport properties of its aqueous solutions. Thus, the average molecular weight of polygalacturonic acid and its average degree of polymerization were estimated on the basis of viscosity measurements. The length-to-diameter ratio, calculated by means of S i m h a ' s equation, strengthened the assumption that pectic acid is a fairly rigid, rod-like molecule. By potentiometric titration of aqueous solutions of poly(a-D)galacturonic acid with several alkaline and tetraalkylammonium hydroxides, the effects of the size and nature of the counterion on the degree and extent of dissociation of the polymeric acid were estimated. In evaluation of the potentiometric curves the treatment proposed by M a n d e l for weak polyacids not exhibiting a conformational transition during titration was used. In addition, the nonelectrostatic character of polyion-counterion interactions was confirmed by the application of the cell m o d e l to the polyelectrolytic solute investigated.

1. INTRODUCTION

In the course of studies on the physicochemical properties of natural polymers in aqueous solution, attention has been drawn to pectic acid, i.e. poly (a-D)galacturonic acid as a potential model of a rigid polysaccharide. Extensive data are given in the literature for the potentiometric titration of polymer acids which may be used to study the behaviour of polyelectrolyte systems under different conditions. For poly(a-D) galacturonic acid there are few data of this kind, especially in connection with the occurrence of a conformational transition induced by pH variations, or with the effect brought about by the addition or the exchange of counterions. Since for a polyacid not exhibiting a conformational transition in the course of titration, p K a ( K a denoting the apparent dissociation constant) increases monotonously with degree

610 of dissociation,a, it is possible to represent this functional dependance by a converging series expansion of pK a in a [1]. The purpose of this study is to consider in more detail the influence of the size and n a t u r e of the counterion on the degree and extent of dissociation of poly(a-D)galacturonic acid already discussed in a previous paper [2], particularly to check the effect of screening the charges on the polyacid in the presence of different counterions.

2. E X P E R I M E N T A L

Solution preparation Commercially available poly(cz-D)galacturonic acid (PGA) was purchased from F l u k a Chemie. To obtain an aqueous solution of the polyacid, insoluble PGA was converted to its soluble sodium salt and then percolated through a cationexchange resin in the H-form [3].

Density measurements Density m e a s u r e m e n t s were carried out using an A. Paar digital densimeter (model DMA 100) at a temperature of 298 K over the mass concentration range 0.75- 6.00kgm-3 -The densimeter was calibrated with water [4] and dry air [5].

Viscosity measurements The viscosities of aqueous solutions of PGA were determined with an Ubbelhode capillary viscometer at 298 K in the same concentration range as the density measurements. The temperature of the water bath was maintained to + 0.05K.

Degree of esterification The degree of esteritication of the methyl ester of PGA was determined acidimetrically after hydrolysing the ester with sodium hydroxide [6].

Potentiometric titration Potentiometric titrations of aqueous solutions of PGd with some alkali hydroxides (LiOH, NaOH, KOH)and tetra-n-alkylammonium hydroxides, e.g.

(CH3)4NOH and (C4H9)4NOH,

were performed in three parallel determinations

in a titration vessel at 298 K using a Radiometer pH meter (type pH M4d) and a combined glass electrode (type GK 2501 C) [2] . The pH meter was standardized with six s t a n d a r d buffers (pH range 3 -10). The end point in the potentiometric titrations was determined using Gran's procedure [7].

611 3. R E S U L T S A N D D I S C U S S I O N

3.1.Transport

properties

of poly(~-D)galacturonic

acid

The mole fraction of polygalacturonate d e t e r m i n e d on the basis of potentiometric h y d r o g e n ion titrations was found to be 0.85. The degree of esterification of the m e t h y l ester of PGA was d e t e r m i n e d to be 5.34 per cent. The density of the investigated solutions is given by d - do - ac~ + bc~

where a a n d fl are empirical constants characteristic of the solute a n d the t e m p e r a t u r e , d e t e r m i n e d by the m e t h o d of least squares on the basis of the d a t a in Table 1. Their values a m o u n t to a = 0.427 andfl = -4.490. The e x p e r i m e n t a l viscosity data were analysed according to the relation 2

3

17-- 17o -- a l c 2 h - a 2 c 2 d - a 3 c 2

where ~ is the absolute viscosity of the solution ( k g m -1 s'l), 1]0 is the absolute viscosity of the solvent a n d a l , a 2 a n d a 3 are empirical coefficients ; their calculated values a m o u n t t o a I - 9.588x10 -5 , a 2 - -3.649x10 -6 and, a 3 - 6.929x10 -7 . The intrinsic viscosity was calculated as

[17] - a___~,= O.10769m3kg -1 r/o The viscosity average molecular weight of PGA was d e t e r m i n e d using the M a r k - H o w n i k - S a k u r a d e equation with the necessary constants from ref. [8] --b

[1"/]- a M v -

4.368xl

0- 7 ~1

Mv

8737

a n d was found to be M v - 30115. Thus, the average degree of polymerization was calculated to be 146. The a p p a r e n t specific volume was d e t e r m i n e d from the density m e a s u r e m e n t s via the relation [9]

1E

qgv = ~o 1

c2

1

where do is the density of the solvent a n d c2 is the m a s s concentration of the

612 solute (gcm 3) and the value of ~Ov*-0.574cm3g-1 was calculated at very low reference concentration, c2 = 3.75x10-4 gcm -3 For comparison, the value of the partial specific volume at infinite dilution for D-galacturonic acid is @o _ 0.554cm 3g-1. The viscosity increment was determined as v - B / v ~ - 172.7 ( ~ 2.5 for spheres) where B is the viscosity coefficient characteristic of a given solue-solvent --o

pair, and amounts to (9.91 + 0.24)x10 -zm3kg -~ for PGA in aqueous solution, v2 is the partial specific volume of the macromolecular component equal to (p: at v a n i s h i n g c2 . The length-to-diameter for P G A was then estimated via S i m h a ' s relation for elongated ellipsoids [ 10] and its value amounts to (a / b) = 49.6.

Table 1 Densities and viscosities (experimental and calculated) of aqueous solutions of P G A at 298 K in the concentration range studied.

C2 / kgm -3

d / kgdm -3

17xl 03 / kgm-ls -1

~L~l~xl 03 / kgm -~s -~

0.75

0.997390

0.9581 __+ 0.0012

0.9581

1.50

0.997700

1.0266 __+ 0.0012

1.0269

3.00

0.998287

1.1657 __+0.0014

1.1646

3.84

0.998629

1.2420 __+0.0015

1.2434

4.80

0.999020

1.3409 __+0.0014

1.3402

6.00

0.999462

1.4835 __+0.0017

1.4836

3.2. P o t e n t i o m e t r i c titration of poly(a-D)galacturonic acid

The degree of dissociation, a, was calculated from the electroneutrality condition

a

= Cp

613 where [BOH 1 is the number of moles of base added per dm a of solution, [H+]and

[OH-] are the molarities of free hydrogen and hydroxyl ions and c; is the concentration of polyacid in monomol dm 3. The apparent dissociation constant of the polyacid, pK a , was calculated by the relation

pica

- pH

+

log (1- a) a

3.2.1. For the systems investigated, the increase of pKawith expressed by a second degree polynomial according to Mandel [1]

pKa = pK ~ + qbla + r where pK~ and r and r

a

could be

2

~ - l i m p K a ) ~ ~ is the intrinsic dissociation constant of the polyacid are the regression coefficients. As an example the dependence of

the apparent dissociation constant pK a for the system PGA +(Call 9)4 NOH at 298 K is given. For an interpretation of the physical meaning of the regression coefficients, the Marcus titration equation for polyelectrolytes was combined with the Poisson-Boltzmann equation for the electrostatic mean potential, determining all charge interactions in a dilute polyelectrolyte solution. In this way it was found thatr depends on the distribution of macromolecular groups in V8 (the total electrically neutral sub-volume assigned to each polymeric ion) for the uncharged polyelectrolyte, and on the mean conformation of the uncharged macromolecule. The coefficients r are mainly determined by the expansion of the dimensions of the polyion in the course of the titration. The data in Table 2 show that 9 the values of pK: decrease with increasing size of the alkali counterion, which may be explained by the formation of contact ion-pairs between counterions and charged carboxylate groups on the chain. The extent of ionpairing depends primarly on the ionic potential, ~o, defined as ( ~ "--

charge ionic radius(A)

9 the positive and almost equal values of coefficients C1 for the investigated systems, C1 =1-616-+0.099, are related to the mean distribution of macromolecular groups for the uncharged polyelectrolyte, i.e. PGA, which is the same irrespective of the nature and size of the counterion. 9 the coefficients r are negative and decrease with increasing size of the alkali counterions, while in the case of tetraalkylammonium ions the sign of the coefficient changes from-0.151 for (CH3)4NOH to + 0.211 for(C4Hg)4NOg.

614 Table 2 Coefficients a n d s t a n d a r d deviation,s, of the l e a s t - s q u a r e s s e c o n d - d e g r e e p o l y n o m i a l r e p r e s e n t i n g t h e t i t r a t i o n curve of PGA with different s t r o n g b a s e s at 298 K.

Base

pK ~

r

r

s

LiOH

3.419 _+ 0.019

1.537 + 0.080

- 0.431 + 0.071

0.019

NaOH

3.413_+ 0.024

1.471_+ 0.099

- 0.439 + 0.091

0.018

KOH

3.327 + 0.034

1.776_+ 0.140

- 0.665 + 0.128

0.024

(CH3) 4 NOH

3.477 _+0.018

1.502 _+0.073

- 0.151 _+0.066

0.013

(C4Hg)4NOH

3.341+0.019

1.792 + 0.086

+ 0.211_+ 0.084

0.017

I

!

I

I

I

I

I

I

I

I

I

I

I

I

I

I

I

5:000

4.500

4.000

3.500

f

I

0.200

0.400 0.600 OK)O Of,

Fig. 1. C h a n g e of t h e a p p a r e n t dissociation c o n s t a n t with a for t h e s y s t e m PGA + (C4H9)4 NOH at 298 K. The curve w a s c a l c u l a t e d from t h e model given by Mendel.

615 3.2.2. The a p p a r e n t dissociation constant p K a is strongly dependent on the electrical potential on the surface of the macroion, v(a)according to the well known equation [ 11] p K a - p K ~ + ApK

where A p K - O . 4 3 4 s v ( a ) / k T . Following the cell model for rod-like ionized polyelectrolyte molecules, a generalized form of the equation which gives ApK in a polyelectrolyte solution of any composition may be written [ 12]

EvJna2h)]

ApK - log (qgpamm+ m~)

+log

1)22J} 2A

where v is the n u m b e r of charges t h a t the polyion carries, h is the effective total length of each molecular cylinder, a is the radius of the rod-like polyion stretched along the axis of a cylindrical cell with radius R, ~op is the osmotic coefficient of the salt-free polyelectrolyte solution, m,, the concentration (monomol cm ~) , m8 the n u m b e r of moles of added salt per cm 3 and 2 is a dimensionless p a r a m e t e r proportional to the n u m b e r of charges per unit length of the macromolecule 2

;~ .

VoC . .

aoc'

2

(h - Zb)

.

DhkT

DjbkT

if every jth monomer carries an ionizable group ( v - a Z / j ) , Z is the degree of polymerization, b the length of the monomeric unit, e the ionic charge, D the dielectric constant, and k T the B o l t z m a n n term. p is an integration constant [ 13] dependent on a and R. Relation 1-fl 2

D

1 + p coth(fly) connects fl with a and y w h e r e yis the concentration parameter, defined as 7 - In --R _ _1In _ _ 1 0 0 0 a

2

rla2bNA

1 In c 2

where c is the monomolar concentration (monomol dm 3) and NA the A v o g a d r o number. For the dimensional p a r a m e t e r s of PGA the values a = 7A and b = 4. 35A" from ref. [14] were used. The relation between 7" and concentration was expressed analytically in the form - 10g c - 0.8687" - 0.394

616 For a univalent counterion the charge density parameter A was found to be ~ = 1 . 5 5 1 a where a value of j = 1.06 was employed because of the degree of esterification of the methyl ester o f P G A ( 5.34 per cent). A comparison of calculated and experimental values of ( p K a - p K ~ at different degrees of dissociation is shown in Fig. 2. For p K ~ the values from Table 2 were used.

2.000

I

I

I

I 0

1.500

cL 1.000

0.500

-

o~176176

0.200 0.400 0.600 OE)O 1.000 et

Fig.2. A p K

of the potentiometric titration of PGA ( 0.011 monomol dm -3) with

strong base as a function of a" ( * ) theoretically calculated curve on the basis of the cell m o d e l , ( + ) LiOH , ( ~ ) (CH 3)4 N O H , and ( O ) (C4H9)4N O H .

4. C O N C L U S I O N S

On the basis of the experimental results, the following conclusions can be made: 9 The length-to diameter ratio strengthens the assumption that pectic acid is a fairly rigid, rod-like molecule and comparable to the structure of cellulose 9 The partial specific volume of the monomeric unit in PGA was found to be approximately 3 per cent higher than the value of D-galacturonic acid 9 For the investigated system the values of p K ~ can be explained by the formation of contact ion pairs between an alkaline ion and the carboxylate group

617 9 The differences in pK ~ values in the case of tetraalkylammonium ions as counterions can be explained by the size of the (CH3)4N§ ion in comparison with the (C4H9)4N+ ion and delocalization of the positive charge on the (CH3)4 N§ ion, which is probably the deciding factor for its stronger interaction with the polyion 9 The values of the coefficients r and r are directly influenced by the size and nature of the counterion 9 The theoretical potential calculated on the basis of the cell model shows that PGA is not suitable for testing a purely electrostatic theory since in this case significant specific binding of counterions to the polyion was detected. For an ion like (C4H9)4N§ this is offset to some extent by the four longer alkyl groups that protect the positive charge of the rigid sphere [ 15].


.

2. 3. 4. .

.

7. 8. .

10. 11.

12. 13. 14. 15.

M. Mandel, Eur. Polym. J., 6 (1970) 807. D. Rudan-TasiS, C. Klofutar, Thermoch. Acta 246 (1994) 11. R. Kohn, B. Larsen, Acta Chem. Scand., 26 (1972) 2455. O.Kratky, H.Leopold, H.Stabinger, Digital Densimeter of Liquids and Gases (A. Paar K. G., A-8054, Graz, Austria). R. C. Weast (Ed.), Handbook of Chemistry and Physics, 62 Edn., CRC Press, Cleveland, 1981-1982. A. Mizote, H. Odagiri, K. T6ei, K. Tanaka, Analyst, 100 (1975) 822. G. Gran, Analyst, 77 (1952) 661. G .Y.M~dy, B. Lakatos, J. J. Kever, N.V.D.Yakonova, Acta Aliment., 21 (1992) 337. H. Durschlag, Specific Volumes of Biological Macromolecules and Some other Molecules of Biological Interest, in: Thermodynamic data for Biochemistry and Biotechnology, HansJurgen Hinz, Ed. (Springer-Verlag, Berlin, 1986) p. 45. R. Simha, J. Phys. Chem., 44 (1940) 25. A. Katchalsky, Z. Alexandrowicz, O. Kedem, Polyelectrolyte Solution, in: Chemical Physics of Ionic Solutions, Ed. 03. E. Conway, R. G. Barradas, J. Wiley, New York, i966), p.295. Z. Alexandrowicz, A. Katchalsky, J. Polym. Sci. Part A,1 (1963) 3231. S. Lifson, A. Katchalsky, J. Polym. Sci. 13 (1954) 43. J. C. T. Kwak, G. Murphy, E. J. Spiro, Biophs. Chem., 7 (1978) 379. J. Nagano, H. Mizuno, M. Sakiyama, J. Phys. Chem., 95 (1991) 2536.


J. Visserand A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996Elsevier Science B.V.All fights reserved.

619

Structural studies of a pectic polysaccharide from

Plantago major L. A.B. Samuelsen, Ellen Hanne Cohen, Berit Smestad Paulsen and Jens Kristian Wold. Institute of Pharmacy, Department of Pharmacognosy, University of Oslo, P.O.Box 1068 Blindern, N-0316 Oslo, Norway

Abstract

The leaves of large plantain (Plantago major L.) are used for wound healing in the traditional medicine. The effect might be due to biologically active polysaccharides. A pectin, PMII with anti-complementary activity has been isolated from the leaves by water extraction and ion exchange chromatography

(i).

Oligosaccharides were isolated from PMII by weak acid hydrolysis and separation by SEC and HPAEC-PAD. The isolated oligosaccharides were desalted, reduced and methylated. GC-MS analysis of the partially methylated alditol acetates has been used to reveal the structure of the oligosaccharides. Oligosaccharides consisting of galacturonic acid and rhamnose with dp 3-5 and a series of 1,4 linked galacturonic acid oligosaccharides of dp 4-10 were isolated.

1. M E T H O D S

1.1 W e a k a c i d h y d r o l y s i s PMII was dissolved in 0.5M TFA and incubated at 100oc under N2 for 2.5h. 1.2 SEC

The partially hydrolysed material was fractionated by size exclusion chromatography on a Bio Gel P10 (Bio Rad) column (2,6 x 90 cm) and eluted with 50 mM ammonium hydrogen carbonate at 20 ml/h and fractions of 3.2 ml each were collected. Fractions 45-70 were pooled and subjected to HPAEC-PAD for further separation.

620 1.3 HPAEC-PAD Oligosaccharides were isolated preparatively by high-pH anion exchange chromatography carried out on a LC-system (Dionex Corporation, Sunnyvale CA) equipped with a CarboPac PA-1 column (9 x 250 mm), coupled to a Spectra System AS 3500 auto sampler. The detection was carried out using Pulsed Amperometric Detection (PAD-II). Eluents: Gradient: Flow rate:

100mM NaOH (El) and 100mM NaOH/1000 mM NaOAc (E2). 0-5min 20mM NaOAc, 5-60 min 20-600 mM NaOAc. 1 ml/min.

100mM NaOH was prepared from a 50% solution NaOH (Baker) to minimise the carbonate content in the final eluent. 1000 mM NaOAc was prepared using NaOAc from Riedel-deHaan, Germany and the distilled water used was filtered in a Waters Milli-Q System. E1 and E2 were degassed by flushing with helium and pressurised continuously with the eluent degas module from Dionex.

1.4 Desalting Sodium ions were removed from the oligosaccharide fractions by treatment with Dowex H +. Acetate was evaporated by washing with methanol.

1.5 Reduction Galacturonic acid was methylesterified by shaking with 0.08M HC1 in methanol for 24h. Then water was added, methanol was evaporated and the remaining solution was neutralised. NaBD4 was added and the mixture was left over night. Sodium ions were removed by treatment with Dowex H + and borate was evaporated by washing with acetic acid in methanol followed by addition of methanol several times.

1.6 Methylation Oligosaccharides were methylated using NaOH in DMSO for sugar alkoxide formation(2).

621

2. RESULTS AND DISCUSSION 2.1 Separation of oligosaccharides by HPAEC-PAD Separation of oligosaccharides by HPAEC is shown in Figure 1. 16 fractions were collected (upper chromatogram). None of the oligosaccharides are identical with any of the standards available (lower chromatogram). Standard GalAI-~4 GalA was eluted before the oligosaccharides of the sample. Standard GalAI-,4GalAI-~4 GalA was eluted between fractions 3 and 4 of the sample. Fractions 1-4 and 6-12 were desalted, reduced and methylated for identification.

i,~le:olqqooolo~ol i r a . ~ 1 ~ .,~to 1200

100%

1000

7

000 PAD

"1

I

I

1.--z ....

...............

illtlYI ~

...........

t2

2100

'... .....

. . . .

i

!

:

10

'

;"'

'

~'~

20

....

"1"'

30

.

.

.

.

40

''

"

~

(

60

~

' ' " i

80

~

' '

~--'

70

"

9 9 1

80

"~

"

'

i

0%

N

Mlnutr

1800

~-

lOO%

-

eo~

1600 1400 1200 1000

PAD

60%

800 000

-

4O%

-

30%

4OO 200 0 0

'

' '"

I r ", t0

,',

I " ~'~ ~"1' ' 2O :10

'

"'

!'" 4O

;"~'1 ~' 80

'

'-] ;' 6O

""

~ ' 70

'

~ '

~ '"' 8O

' e0

M i r a

Figure 1. Separation of oligosaccharides from PMII by HPAEC-PAD.

622 2 . 2 0 l i g o s a c c h a r i d e structures The monosaccharide residues identified in fractions 1-4 are:

1-1inked GalA, 1,4-1inked GalA 1,2- linked Rha.

The amount of each monomer found in the different oligosaccharides could not be determined because of the small quantities that were isolated. The dp of the oligosaccharides are about 3-5 with 2-4 galacturonic acid residues and one or more rhamnose residues of each. The methyl glycoside formed at position 1 by methylation of reducing sugars will be hydrolysed off and appear as a l-linkage. Rhamnose is at the reducing end of each oligosaccharide and is also present between two galacturonic acid residues. Only small amounts of this type of sequences are found in the molecule since only minor amounts of each oligosaccharide were isolated. The monosaccharide residues identified in fractions 6-12 are:

1-1inked GalA 1,4-1inked GalA

Fraction 6 has the same retention time as a previously run sample of tetragalacturonic acid (not shown). The oligosaccharides consist of unbranched 1.4 linked galacturonic acid of dp 4-10. GalA 1~4 GalAI(-~4 GalA 1)n~4 GalA n=l-7

3. A C K N O W L E D G E M E N T S This work has been supported by the Norwegian Research Council, project no 100594/410. The authors are indebted to Finn Tonnesen for recording the GC-MS data.

4. R E F E R E N C E S 1. 2.

A.B. Samuelsen, B.S. Paulsen, J.K. Wold, H. Otsuka, H. Yamada and T.Espevik Phytother. Res. 9 (1995) 211 M.J. McConville, S.W. Homans, J.E. Thomas-Oates, A. Dell and A. Bacic J. Biol. Chem. 265 (1990) 7385

J. Visserand A.G.J. Voragen(Editors),Pectinsand Pectinases 9 1996ElsevierScienceB.V.All rightsreserved.

623

Structural characterization of a novel rhamnogalacturonan II with macrophage Fc receptor expression enhancing activity from the leaves of Panax ginseng C.A. Meyer K.-S. Shin, H. Kiyohara, T. Matsumoto and H. Yamada Oriental Medicine Research Center, the Kitasato Institute, Shirokane 5-9-1, Minato-ku, Tokyol08, Japan Abstract A complex pectic polysaccharide (GL-4IIb2') with macrophage Fc receptor expression enhancing activity has been isolated from the leaves of Panax ginseng C.A. Meyer. GL-4IIb2' consisted of at least 15 different component sugars which included the rarely-observed sugars such as 2-Me-Fuc, 2-Me-Xyl, apiose (Api), 3-C-carboxy-5-deoxy-L-xylose (Aceric acid, AceA), 3-deoxy-D-manno-2-octurosonic acid (Kdo) and 3-deoxy-D-lyxo-2-heptulosaric acid (Dha). Sequential degradation experiment provided evidence that GL-4IIb2' comprised of a highly branched a-(1-~4)-linked galacturono-oligosaccharide backbone with side chains such as aRhap-(1-~5)-Kdo, Ar~-(1-~5)-Dha, AceA-containing oligosaccharide and uronic acid rich-oligosaccharide. These results suggest that GL-4IIb2' resembles rhamnogalacturonan II (RG-II). However, structure of GL-4IIb2' possessed several differences from that of RG-II: rich in a-Rhap-(1 ~5)-Kdo side chain, some additional glycosyl linkages, and a relatively higher molecular weight. It was also found that GL-4IIb2' was directly purified from hot water exracts of ginseng leaves without treatment of endo-a-( 1 -+4)-polygalacturonase.


The roots of Panax ginseng C.A. Meyer are a well known Chinese component herb widely used clinically for the treatment of gastrointestinal disorders as well as an erythropoietic and a tonicl Several pharmacologically active saponins and polysaccharides have been found in the roots of P. ginseng as active ingredients. The roots of P. ginseng is valuable because it takes 4 - 6 years for growing from the seed whereas the leaves of P. ginseng can be harvested every year. Therefore, if the leaves have a similar activity as the roots, the leaves will be available as well as the roots. As the results of investigation for the clinical value of the leaves of P. ginseng, we have previously reported that the leaves polysaccharides showed more potent anti-complementary [1] and anti-ulcer activities [2]. Recently, We have found that the crude polysaccharide fraction (GL-2) from the leaves and the purified polysaccharide (GL-4IIb2') from GL-2, showed potent

624 immune complex clearance enhancing activity of macrophages, and this activity was due to increment of de nove synthesis of Fc receptor (FcR) [3]. In the prsent sudy, GL-4IIb2' was found to be a complex pectic polysaccharide consisting of 2-Me-Fuc, 2-Me-Xyl, Api, AceA, Dha and Kdo which are characteristic in RG-II [4] of plant cell wall polysaccharides, therefore we describe structural characterization of a macrophage FcR expression enhancing polysaccharide (GL4IIb2') from the leaves of Panax ginseng C.A. Meyer.

2. Isolation of GL-4IIb2' Crude polysaccharide fraction (GL-2) was prepared from the leaves of P. ginseng by hot water extraction, ethanol precipitation and dialyw and GL-2 was fractionated by Cetavlon precipitation and weakly acidic polysaccharide fraction (GL-4) was obtained[3]. GL-4IIb2 was purified from GL-4 by DEAESepharose CL-6B as described previousely [3]. In order to remove the colormaterials, GL-4IIb2 was further purified by Q-Sepharose (CI form), and the major fraction, eluted with 0.3M NaC1, was repurified by gel filtration on Bio-gel P-30 column to obtain purified active polysaccharide, GL-4IIb2'.

3. Property of GL-4IIb2' GL-4IIb2' was eluted from Bio-Gel P-10 as a single peak and also gave a single peak in HPLC on Asahi-pak GS-510 + GS-320 and GS-320 + GS-220. It contained hexose (64.1%), uronic acid (33.7%) and acetyl (0.82%) and had a molecular weight of 8,000 - 10,000. Component sugar analysis indicated that GL-4IIb2' consisted of at least 15 kinds of sugars such as 2-Me-Fuc, 2-Me-Xyl, Api, AceA, Kdo and Dha which observed as rare sugars in RG-II [4] in addition to Fuc, Ara, Xyl, Rha, Man, Gal, Glc, GlcA and GalA. GL-4IIb2' did not react with ~-glucosylYariv antigen on single radial gel diffusion. Digestion with endo-a-(1-->4)polygalacturonase (endo-PGase) gave no oligogalacturonides from GL-4IIb2' and its molecular weight was not changed. Methylation analysis indicated that GL4IIb2' consisted of at least 32 kinds of sugar residues observed in RG-II. The results suggested that GL-4IIb2' has a similar structural feature as RG-II [4], but the proportion of Kdo and branching frequency were higher than those of RG-II.

4. Structural. analysis of GL-4IIb2' by sequential degradation 4.1. Characterization of fragments derived by procedure 1 In order to elucidate the differences between structures of GL-4IIb2' and RG-II, sequential degradation including partial acid hydrolysis was used for generation of characteristic oligosaccharides from GL-4IIb2' as shown in Scheme 1. After GL-4IIb2' was partially hydrolyzed with 0.1M TFA (60~ 30min), the products

625 GL-4IIb2' Procedure

1

I I

0.1 M TFA, 60~

30 rain

Bio-gel P-10 I

I

PA-1

PA-2 ]

0.1 M TFA, 40~

Procedure 2

PA-2'

24 hr

a-Rha-(l~5)-Kdo

Bio-gel P-6

I

I

PA-I-I

PA-I-III

PA-I-II AceA-containin~g oligosaccharide (Figure 2.)

Procedure 3 --

0.1 M TFA, 40~

--

Bio-gel P-6

PA-I-Ia

84 hr

i

I

PA-I-Ib

PA-I-Ic Rha-(~5)-Kdo

Endo-polygalacturonase

Procedure 4

Rha-(l~5)-Kdo Ara-(1-~5)-Dha

I

PA-I-Id Monosacc h andes

Bio-gel P-6

I PG-2

PG-1

Procedure 5

0.1 M TFA, 50~

I PG-3

48 hr

Bio-gel P-6

PG-1A

I

I

PG-1B •

PG-1C

Uronic acid r~ch oligosaccharide Scheme 1. Sequential degradation of GL-4IIb2'. were fractionated on Bio-gel P-10, and about 35% of TBA-positive materials (PA-2) was eluted in the small oligosaccharide fraction. PA-2 was further purified on QAE-Sephadex by using linear gradient of HCOONH 4, and it gave a single peak (PA-2') containing TBA-positive material. PA-2' consisted mainly of Rha (43.2%) and Kdo (40.8%). Methylation analysis indicated that PA-2' was composed mainly of terminal Rhap and 5-substituted Kdo (ratio 1:1). The methylated oligosaccharide-alditol derived from PA-2' gave only a single peak in the region of disaccharide-alditol in GC-MS. EI-MS of PA-2' contained prominant fragment

626 ions at m/z 189 (Rha) and m/z 308 (Kdo). The presence of a fragment ion at m/z 162 and the absence of m/z 177 showed that Kdo was substituted at C5 but not at C4. PMR of PA-2' exhibited a signal due to an anormeric proton (at 5.09 ppm), indicating that Rha is to be a-configuration. Therefore, it was indicated that PA-2' mainly contained a-Rha-( 1-~ 5)-Kdo.

4.2. Structural characterization of oligosaccharides obtained from PA-1 by procedure 2 PA-1 was partially hydrolyzed with 0.1M TFA (40~ 24 h) and the three fractions (PA-I-I, PA-I-II and PA-I-III) were obtained by Bio-gel P-6. Especially, about 50% of TBA-positive material (PA-1-III) was eluted in the fraction of small oligosaccharide. Permethylated oligosaccharide-alditols from PA-I-III were analyzed by GC-EIMS, and three disaccharide-alditols (1P, 2P and 3P) were detected. EI-MS and component sugar analysis suggested that the major peak, 1P was Rha-(l~5)-Kdo-ol and the minor peaks, 2P and 3P were two epimers of Araf-(1-~5)-Dha. The intermediate size fraction, PA-I-II consisted mainly of 2-Me-Fuc, Rha, Ara, Api, AceA and Gal in molar ratios of 19.7 935.1 913.6 97.6 94.1 914.7 and the glycosyl composition was similar to that of the AceA-containing oligosaccharide isolated from RG-II [5]. The glycosyl linkage composition of neutral glycosyl residues in PA-I-II was similar as that in AceA-containing oligosaccharide derived from RG-II, but PA-I-II contained the relatively large amounts of terminal Araf and 2-1inked Rha. It was also found that PA-I-II also consisted of 2,3-branched Arap which was not concluded the presence in AceA-containing oligosaccharide of RG-II. The presence of AceA-containing oligosaccharide in PA-I-II was confirmed by FAB-MS. Negative FAB-MS of PA-I-II gave pseudo-molecular ions [ (M-H) ] at m/z 1055, 1187 and 1201 which were suggested to be due to non-acetylated hepta- and octasaccharides, respectively. PA-I-II also gave (M-H) of monoacetylated heptasaccharide (m/z 1097) and octasaccharides (m/z 1229 and m/z 1243), and

1097

-132 205

-"

-(162,160) 337

~"

-160

'

... 659

/'

-146 " '' ; 8 1,9 :

....

965

,,,., ""]

.....

.J ~

I

rn/z

Figure 1. Negative FAB/CAD spectrum of the AceA containing oligosaccharide

627 diacetylated heptasaccharide (m/z 1137)and octasaccharides (m/z 1271 and 1285). CAD spectrum using B/E-linked scan of monoacetylated heptasaccharide (m/z 1097, as the highest ion peak) gave fragment ions at m/z 965, 819, 659, 337 and 205 due to fragments derived from the heptasaccharide by successive elimination of Api, Rha, AceA, (Gal + 2-Me-Fuc) and Ara, respectively (Figure 1). Recently, Whitcombe et al. have reported the presence of AceA-containing oligosaccharide having an octasaccharide unit in which non-reducing Araf was attached to position 2 of Rha of heptasaccharide unit [6]. Methylation analysis indicated that terminal Ara and 2-1inked Rha were present in PA-I-II, and it was assumed that the Ara was attached to 2-1inked Rha. These proposed that GL-4IIb2' comprised the same side chain of AceA-containing octasaccharide unit as RG-II. However it was also shown that GL-4IIb2' consisted of 2,3-branched Ara. Negative FAB-MS gave other ions at m/z 1333, 1375 and 1417. By comparison with (M-H) of hepta- and octasaccharides, it was suggested that (M-H) of hepta- and octasaccharides were produced by elimination of pentosyl or deoxyhexosyl units from the ions at m/z 1333, 1375 and 1417. From the results of methylation and FAB-MS analysis, it was proposed that GL-4IIb2' comprised AceA-containing oligosaccharide chains possessing nonasaccharide unit in which terminal Rha was attached to position 3 of 2-1inked Arap of octasaccharide chain (Figure 2). There was also a possibility that this structural unit is probably not unique for GL-4IIb2' because 2,3-branched Arap has been detected in RG-IIs from sycamore, and red wine in relatively large proportions [6,7]. From above results, it is inferred that the AceA containing oligosaccharide from GL-4-IIb2' has the following structure (see Figure 2.). Rha 1

$ 3 Araf-(l~2)-Rha-(l~2)-Arap-(1 ~4)-Galp-(1-~2)-AceA-(1-~3)-Rha-(l~3')-Api-(1-~ 2 1 2-O-Me-Fuc Figure 2. The possible structure of the AceA cotaining oligosaccharide obtained after procedure 2.

4.3. Characterization of fractions obtained by procedure 3 PA-I-I was further treated with 0.1 M TFA at 40~ for 84 h, and gave four subfractions (PA-I-Ia, PA-I-Ib, PA-I-Ic and PA-I-Id) by gel filtration on Bio-gel P-6. About 50% of remaining TBA-positive material in PA-I-I was eluted in the small oligosaccharide fractions (PA-I-Ic and PA-I-Id). Glycosyl sugar composition

628 analysis of PA-I-Ic revealed Rha and Kdo as the major constituents. GC-MS analysis of methylated oligosaccharide-alditols indicated that PA-I-Ic was consisted mainly of Rha-(l~5)-Kdo as same as PA-2'. The high-molecular-weight and major fraction, PA-I-Ia consisted mainly of Rha, Fuc, 2-Me-Xyl, Ara, Api, Gal, Dha, GalA and GlcA in molar ratio of 12.1 : 7.8 : 6.4 : 10.1 : 3.2 : 6.7 : 1.2 : 31.2 : 17.6. Methylation analysis showed that PA-I-Ia mainly contained terminal Ara, terminal 2-Me-Xyl, 3,4-branched Fuc, 2,3,4,-fully branched Rha, terminal Gal, 3'-linked Api, 2-1inked GlcA and terminal-, 4-liked, 2,4-branched and 3,4branched GalA. From these results, it was assumed that PA-I-Ia might be comprised uronic acid rich octasaccharide and Araf-(l~5)-Dha, which have been found in RG-II [4, 8], and branched galacturono-oligosaccharide.

4.4. Characterization of fractions obtained by procedures 4 and 5 After PA-1-Ia was digested with endo-PGase, and the product was fractionated by Bio-gel P-6. A large proportion (PG-1) of the products was eluted in the void volume and the product also gave an intermediate fraction (PG-2) and a considerable proportion of the lowest-molecular-weight fraction (PG-3). PG-2 contained a large proportion of GalA in addition to a small proportion of Rha, Fuc, 2-Me-Xyl, Ara, Gal, GalA and GlcA. Whereas PG-3 contained GalA only. These results suggested that the several side chains such as AceA-containing oligosaccharide, Araf-(1-~5)-Dha and Rha-(1-~5)-Kdo which were attached to the a - ( l ~ 4 ) galacturonan core in GL-4-IIb2'. In order to release uronic acid-rich oligosaccharide, PG-1 was further treated with 0.1 M TFA (in according to the procedure of Thomas et al. [9], Scheme 1, Procedure 5). The products gave PG-1A ~ PG-1C and PG-1A was shown to comprise similar component sugars as PG-1. Among the fractions, PG-1B was shown to contain Api, Rha, Fuc, 2-Me-Xyl, Gal, GalA and GlcA (5.4 : 15.1 : 12.0 : 10.0 : 9.3 : 26.4 : 14.7 in molar ratios), and the composition was almost consistent with ~he uronic acid-rich octasaccharide in RG-II [8]. Therefore it was assumed t h a t GL-4-IIb2' also comprised similar structural unit of uronic acid-rich octasaccharide side chains in RG-II. 5. CONCLUSION The primary structure of a complex pectic polysaccharide (GL-4IIb2') with macrophage Fc receptor expression enhancing activity, isolated from the leaves of P. ginseng C.A. Meyer, was elucidated (Figure 3.). The present results suggested that GL-4IIb2' resembles, in many respects, with a typical RG-II. However, RG-II from ginseng leaves presented discrete differences from previously reported RG-II [4] as shown in Table 1. Especially, GL-4-IIb2' consisted of 1) AceA containing oligosaccharide side chains possessing higher branched nonasaccharide unit than RG-II, 2) higher molecular weight than RG-II, and 3) greater richness of aRhap-(l~5)-Kdo side chains than RG-II. Because the pharmacological funtion of RG-II has not ever been reported up to now, present finding may be the first case for a pharmacological activity of RG-II. These postulated the possibility that the

629 aRhap-(1--S)-KDO-(2--2 or3) aRhap-(1--S)-KDO-(2--2 or3) aRhap-(1--S)-KDO-(2--2 or3) Uronlc acid-rich octasacchadde

I--

Uronlc acid-rich octasacchadde Ara-(1--S)-DHA-(2--2

or 3

Uronlc acid-rich octasacchadde Rha 1 i 3 Ara-(1---2)-Rha-(1---2)-Ara-(1-.4)-GaI-(I -,.2)-AceA-(1--3)-Rha-(1-.3')-Apl-(1--2 or 3)t 1 2-O-Methyl-Fire

aRhap-(1-*5)-KDO-(2---2 or3) aRhap-(1--5)-KDO-(2--2 or3).

Figure 3. The proposed partial structure of GL'4-IIb2' from the leaves of P.

ginseng C.A. Meyer.

Table 1. The decrete differences between GL-4-IIb2' and RG-II

GL-411b2 ~

RG-II

Preparation

Obtained w i t h o u t t r e a t m e n t of endo-PGase

dp

60

Pharmacological activity

Structural feature

Released f r o m p r i m a r y cell walls u p o n d i g e s t i o n with endo-PGase 60 - 30

Relatively s t r o n g m a c r o p h a g e FcR e x p r e s s i o n e n h a n c i n g activity [ Rha-(1-~5)-KDO

Not reported

Ara-(1-~5)-DHA 9 ]

5-1

1 -1

[ AceA containing oligosaccharide ]

:

R

i

,

3

,

Ara-(1 -, 2)-Rh a-(1 -:-)2)-Ara-(1 ~4)-Gal-(1 -)2)-AceA-(1 -~3)- Rha-(1 -)3')-A pl ,.. . . . . . . . . _, 1 2-O-MethyI-Fuc R = Rha

R = OH

630 phamacological activity may be related to some structural differences of GL-4IIb2' from RG-II. On the other hands, GL-4IIb2' was directly isolated from the hot water extracts of the leaves of P. ginseng without treatment of endo-PGase although RG-IIs have been purified from plant cell walls by endo-PGase digestion [10] or from crude preparation of the enzyme [11]. These facts suggest the possibility that GL-4IIb2' exists as a free form of RG-II. 6. R E F E R E N C E S

1. Q.-P. Gao, H. Kiyohara, J.-C. Cyong and H. Yamada, Plant Med., 57 (1991) 132. 2. X.-B. Sun, T. Matsumoto and H. Yamada, Plant Med., 58 (1992) 445. 3. X.-B. Sun, T. Matsumoto and H. Yamada, Phytomedicine, 1 (1995) 225. 4. V. Puvanesarayah, A.G. Darvill and P. Albersheim, Carbohydr. Res., 218 (1991) 211. 5. M.W. Spellman, M. McNeil, A.G. Darvill and P. Albersheim, Carbohydr. Res., 122 (1983) 131. 6. A.J. Whitcombe, M.A. O'Neill, W. Steffan, P. Albersheim and A.G. Darvill, Carbohydr. Res., 271 (1995) 15. 7. T. Doco and J.M. Brillouet, Carbohydr. Res., 243 (1993) 333. 8. L.D. Melton, M. McNeil, A.G. Darvill P. Albersheim and A. Dell, Carbohydr. Res., 146 (1986) 179. 9. J.R. Thomas, A.G. Darvill and P. Albersheim, Carbohydr. Res., 185 (1989) 261. 10. A.G. Darvill, M. McNeil and P. Albersheim, Plant Physiol., 62 (1978) 418. 11. Y.S. York, A.G. Darvill, M. McNeil and P. Albersheim, Carbohydr. Res., 138 (1985) 109.

J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V.All fights reserved.

631

Structural features of pectic polysaccharides of red beet (Beta

vulgaris conditiva) Georg R. Strasser, Daniel E. Wechsler, Renato Amadb Swiss Federal Institute of Technology, Institute of Food Science, ETH-Zentrum, CH-8092 Zurich, Switzerland

Abstract Cell-wall material from ripe red beet was extracted as alcohol-insoluble residue (AIR). The CDTA-soluble extract from AIR was fractionated by anion-exchange chromatography. Four fractions were isolated by a step-wise increase in the ionic strength of the elution buffer. The main fraction was further fractionated by gel filtration chromatography. This chromatogram showed one regular broad peak, which was divided into three parts and pooled. All fractions isolated from both chromatographic systems were freeze-dried and their neutral sugar compositions as well as uronic acid contents were determined. Furthermore methylation analysis of these fractions were performed prior and after reduction of the pectic polysacchaddes with NaBD4.

1. INTRODUCTION Pectins are a group of polysaccharides from the primary cell wall and the intercellular regions of higher plants [1]. They have been investigated for their structural features and their functions within the plant cell wall for many years, because changes in the texture of fruits and vegetables and in the properties of their products are related to changes in the pectic components [2]. From literature it is known, that the pectic backbone consists of ~t-(1---~)linked D-galacturonic acid units, interrupted by the insertion of o~-(1---)2)-linked L-rhamnosyl residues in adjacent or alternate positions [3]. Side chains consisting essentially of arabinans, galactans, arabinogalactans and single xylose residues are attached to this backbone. Other sugars have been found less frequently. In addition some non-sugar substituents, mainly methanol, acetic acid and phenolic acids are known to be present in pectins. Although the main structural elements of pectins are known, the complexity of these polymers has prevented a complete understanding of their fine structure so far. The aim of this project is to get additional information about the fine structure of pectic polysaccharides. Therefore pectins from red beet were isolated and fractionated by chromatographic methods. Some results obtained by methylation analysis of these pectin-rich fractions are presented.

632 2. MATERIALS AND METHODS

Extraction and fractionation of pectins (Figure 1): Red beets of the variety Red Ace F~ were purchased from a local store. Preparation of the AIR was done according to Selvendran and O'Neil [4]. Extraction with trans-l,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) was performed according to Selvendran et al. [5]. The CDTA-soluble extract was dialysed first against running tap water and subsequently against distilled water. Then it was fractionated by anion-exchange chromatography on DEAE Sepharose CL-6B using 0.04M phosphate buffer (pH 6.5) as eluent with a step-wise increase in the ionic strength (0, 0.1, 0.2, 1.0M NaC1). Related fractions were pooled. The main fraction (IE 0.2M) was further fractionated by gel filtration chromatography on two coupled columns, one packed with Sephacryl S-500 and the other one with Sephacryl S-200 using 0.04M phosphate buffer (pH 6.0) as eluent. The peak on the chromatogram was divided into three parts, which were separately pooled. All samples were dialysed and freeze-dried before further analysis. Analytical techniques: Fractions from anion-exchange chromatography were assayed for uronic acid and neutral sugar contents using an automated segmented flow analyser [6]. Neutral sugars (NS) of all samples were analysed by GC as alditol-acetates [7]. Uronic acids (UA) were determined by the m-hydroxy-diphenyl method [8]. Methylation analysis [9,10] was performed with polysaccharides prior and after carbodiimide-activated reduction with NaBD4. Peaks were identified by GC-MS, whereas the quantification was done by GC (FID) using calculated relative response factors on an effective carbon response (e.c.r.) basis [ 11]. 3. RESULTS AND DISCUSSION The results are summarised in Table 1. As expected, for the IE samples higher amounts of uronic acid are found with increasing ionic strength of the elution buffer. For the GF samples larger molecules (GF1) contain half as much NS compared to smaller molecules (GF3). The sum of differently linked NS residues determined by methylation analysis is calculated to 100%. 1,4-Galp is difficult to determine because of the high amount of 1,4GalAp present in most samples. Therefore 1,4-Galp was determined by methylation analysis without prior reduction with NaBD4. Whereas the percentage of some NS residues remain nearly unchanged in the different samples (e.g. T-Araf, 1,5-Araf and 1,6-Galp), others differ considerably. The relative amounts of 1,3-Araf, 1,3,5-Araf, 1,2,3,5-Araf, 1,4-Galp, 1,2-Rhap and 1,2,4-Rhap increase at higher ionic strength elution on anion exchange chromatography and decrease for smaller molecules on gel filtration chromatography. 1,3-Galp and 1,3,6-Galp show the opposite behaviour. This indicates that pectins eluted at high ionic strength and larger pectic molecules of the IE 0.2M fraction contain on average smaller side chains because of the relatively high amount of 1,2,4-Rhap compared to the total amount of NS residues (1,2,4-Rhap is the main branching residue in the pectic backbone). Furthermore it can be assumed that the side chains of these pectins mainly consist of structures similar to arabinans and arabinogalactans type I, because of the differently linked arabinose and galactose residues present. Pectins eluted at low ionic strength and smaller pectin molecules of the IE 0.2M fraction contain more galactose, which is mainly 1,3- and 1,3,6-1inked. These residues are believed to be part of structures, which are known to be present in arabinogalactans type II.

633 Red Beet Alcohol soluble extract

Extraction with ethanol ,,~ Alcohol insoluble residue (AIR)

CDTA insoluble residue

Extraction with CDTA =~ CDTA soluble material (CDTAS)

Anion exchange chromatography on DEAE-Sepharose CL-6B 1200 T --o- Uronic acids ~ --.- Neutral sugars ~ ~~ 800 + .... 0.04M phosphate buffer ~o~.= containing NaC1 a~ ~=

1 I/ .ll

400

............

q- 1.0 ] ] "=~=~ 0.5

,

0 i ,

I

20 ~ IEOM

i I '

I

40 i 60 I 1 ' 1 IE0.1M IE0.2M

i

=o

80 (I, IE1.0M

Fraction number

Gel filtration chromatography on Sephacryl S-500 and on Sephacryl S-200

~

Mw

(Pullulan)

6

]

i

L60

80

rlE 0.2M GF1 [IE 0.2M GF2] IE 0.2M GF3"[ Figure 1. Extraction and fractionation of pectins from ripe red beets

Fraction number

634

Table 1 Neutral sugar, uronic acid and methylation analysis (molS) of fractionated pectins from ripe red beets Chromatographic fractions

NS to UA ratio

NS residues

CDTAS

OM

0.1M

IE 0.2M

E 0.2M 1.OM

GFl

GF2

GF3

UA

25 75

99 1

92 8

17 83

18 82

10 90

16 84

20 80

Arabinose

T-Araf T-h a p 1,2-Araf 1,3-&af 1,5-Araf 1,3,5-Araf 1,2,3,5-Araf

22.9 0.9 1.2 2.1 17.4 12.0 2.0

26.2 1.1 1.3 3.3 22.4 1.2 0.5

22.6 0.4 0.8 0.9 19.0 0.6 0.0

23.4 1.0 1.6 2.1 17.4 14.1 2.2

21.8 0.8 1.3 2.9 17.3 14.9 3.1

22.1 1.3 0.8 2.6 16.9 17.9 2.9

23.7 0.8 1.6 1.9 16.3 13.2 2.7

22.3 1.2 0.9 1.6 16.0 10.1 1.8

Fucose

T-FUCP 1,3,4-Fucp

0.8 0.2

0.0 0.0

0.1

0.0

1.1 0.2

0.8 0.1

1.5 0.1

1.1 0.2

1.1 0.2

Galactose

T-G@ 1,3-G@ 1,4-Galp 1,6-Galp 1,2,4-Galp 1,3,4-Galp 1,3,6-Galp

2.8 6.8 n.d. 2.4 0.9 0.8 13.0

1.1 11.6 0.6 2.5 0.1 0.1 23.6

1.7 14.6 n.d. 2.3 0.0 0.2 33.8

2.4 5.1 n.d. 2.2 1.2 0.9 10.7

3.6 6.0 n.d. 3.0 0.9 0.7 5.6

3.7 3.7 n.d. 1.7 1.3 0.9 1.8

2.8 6.1 n.d. 2.1 0.9 0.7 11.7

2.0 7.8 n.d. 2.3 1.5 1.0 17.2

Glucose

T-GlCp 1,4-Glcp 1,6-Manp

0.9 3.3 0.1

0.2 3.1 0.1

0.0 1.5 0.1

0.5 2.8 0.0

0.0 4.4 0.0

2.8 5.2 0.3

0.0 3.7 0.4

0.0 2.2 0.4

NS

after reduction with

NaBD4

Mannose

Rhamnose

Xylose

T-Rhap 1,2-Rhap 1,3-Rhap 1,2,3-Rhap 1,2,4-Rh~

1.4 1.7 0.9 0.2 2.8

0.3 0.1 0.2 0.0 0.0

0.7 0.1 0.1 0.0 0.3

1.5 2.0 1.3 0.3 2.6

1.4 2.5 0.9 0.3 5.1

1.5 3.1 1.2 0.4 3.2

0.9 0.3 2.5

1.7 1.3 1.4 0.2 1.9

T-Xylp 1,CXylp

0.8 1.7

0.3 0.0

0.2 0.1

1.2 2.2

0.9 1.5

1.6 1.4

1.2 1.8

1.2 2.7

100

100

100

100

100

100

100

100

3.5

0.3

n.d.

3.6

6.6

4.7

4.2

2.2

2.5 0.3

100 0.0

49.4 8.6

2.1 0.0

2.8 0.0

0.7 0.0

2.7 0.3

2.8 0.4

0.9 93.3 0.6 1.4 1.0

0.0 0.0 0.0 0.0 0.0

0.0 41.7 0.0 0.3 0.0

1.0 93.9 0.7 1.2 1.2

0.6 92.8 0.6 2.0 1.2

0.4 95.9 0.6 1.1 1.3

0.8 93.2 0.7 1.2 1.1

1.2 92.7 0.7 1.4 0.8

100

100

100

100

100

100

100

100

Total NS

NS residues

Galactose without prior reduction

1,4-Galp

UA residues

Glucuronic acid T-GlcAp after reduction with 1,CGlcAp NaBD4 Galacturonic acid T-GalAp 1,4-GalAp 1,2,CGalAp 1,3,4-GalAp 1,4,6-GalAp

Total UA

1.9 1.7

Ratio branchinglterminal

after reduction with NaBD4 without prior reduction

1.1 1.5

0.9 0.7

1.2 ad.

1.1 1.1

1.1 1.1

1.1 1.1

1.1 1.1

1.1 1.1

Ratio Iinearhranchinp:

after reduction with NaBD4 without prior reduction

5.4 1.o

1.7 2.0

1.3

6.6 1.1

6.0 1.2

9.6 1.2

5.5

5.5

n.d.

1.2

1.0

n.d. not determined 635

636 The ratios between linear and branched residues are presented as well. All values obtained without prior reduction (NS residues only), are in the range between one and two. From this it can be assumed, that side chains contain only slightly more linear than branched residues. After prior reduction (NS and UA residues) much more linear than branched residues are found. This surplus of linear residues originates from the 1,4-1inked galacturonic acid backbone, which is also known as smooth region. The results presented allow the following assumptions to be made: CDTA-soluble material eluted at low ionic strength (IE 0M) contains hardly any uronic acids and consists mainly of neutral sugar residues, which indicate the presence of arabinans and arabinogalactans type II. Pectins eluted at high ionic strength (IE 1.0M) contain large amounts of uronic acids. The side chains consist mainly of arabinans, galactans and mixtures of them. High molecular weight pectins eluted at intermediate ionic strength (IE 0.2M GF1) contain large smooth regions of 1,4-1inked galacturonic acid and relatively small side chains. These side chains consist of sugar residues, which probably belong to arabinans, arabinogalactans type I and mixtures of them. Low molecular weight pectins of the same ionic strength fraction (IE 0.2M GF3) contain larger side chains, consisting of sugar residues, which indicate the presence of arabinogalactan type II similar structures. Additionally other sugar residues (e.g. T-Galp, T-Xylp, T-GlcAp) and non-sugar residues (e.g. methanol and acetic acid; results are not shown) are attached to these pectic polysaccharides, but further investigations are needed to clarify the fine structure in detail.

4. REFERENCES

8

9 10 11

Voragen, A.G.J., Pilnik, W., Thibault, J-F., Axelos, M.A.V., Renard, C.M.G.C. (1995). Food polysaccharides and their applications (Stephen A.M., ed.), Marcel Dekker, Inc., 287-339. Carpita, N.C., Gibeaut D.M. (1993). Plant J. 3, 1-30. McNeil, M., Darvill, A.G., Fry, S.C., Albersheim, P. (1984) Ann. Rev. Biochem. 53, 625-663. Selvendran, R.R., O'Neil, M.A. (1987). Meth. Biochem. Anal. 32, 25-153. Selvendran, R.R., Stevens, B.J.H., O'Neil M.A. (1985). Biochemistry of plant cell walls (Brett, C.T., Hillman, J.R., eds.) Cambridge University Press, Cambridge, 39-75. Thibault, J.F. (1979). Lebensnt-Wiss. u. Technol. 12, 247-251. Blakeney, A.B., Harris, P.J., Henry, R.J., Stone, B.A. (1983). Carbohydr. Res. 113, 291-299. Blumenkrantz, N., Asboe-Hansen, G. (1973). Anal Biochent 54, 484-489. Kvemheim, A.L. (1987). Acta Chent Scand. Ser. B41, 150-152. Harris, P.J., Henry, R.J., Blakeney, A.B., Stone, B.A. (1984). Carbohydr. Res. 127, 5973. Sweet, D.P., Shapiro, R.H., Albersheim, P. (1975). Carbohydr. Res. 40, 217-225.


637

Isolation and Physicochemical Characterisation of Xylose-rich Pectic Polysaccharide from Wheat Straw

Runcang Sun*, a, J. Mark Lawthera and W. B. Banksb aThe BioComposites Centre, bSchool of Agricultural & Forest Sciences University of Wales Bangor, Gwynedd LL57 2UW, United Kingdom

ABSTRACT Xylose-rich pectic polysaccharide (XRPP) was extracted from defatted and protein-free wheat straw with a solution adjusted to pH 1.6 using hydrochloric acid (HC1). The yield of XRPP obtained was 1.1% of the dried wheat straw. The isolated XRPP contained 44.8% galacturonic acid released by pectolyase treatment and 32.1% released by 2 N trifluoroacetic acid hydrolysis. The XRPP also contained 17.1% neutral sugars released by pectinase reaction and 28.4% released by the acid hydrolysis. A comparison of the FT-IR spectroscopic data of citrus pectin and XRPP showed that the extracted XRPP belongs to pectic substances and differs from hemicellulose with an intensive absorption band at 1740 cm -1.

Key words: xylose-rich pectic polysaccharide, pectin, wheat straw, extraction, sugars, lignin, FT-IR, phenolic acids and aldehydes.

INTRODUCTION There is growing interest in the use of cereal straws such as wheat straw for animal feed after increasing its digestibility by various methods, or as a raw material for paper and board production. This is particularly important in areas with limited forest resources (1). For all these purposes a good physicochemical characterisation of cereal straw is necessary. To date, the structural features of pectic polysaccharides and plant cell walls have been studied extensiv.ely using chemical analysis and enzymatic degradation. In addition, research on isolation and physicochemical characterisation of pectin from citrus peels, apple peels, sunflower head residues and sugar beet pulp has been reported (2). However, the pectic polysaccharides extracted from wheat straw have only previously been reported by Przeszlakowska (3). The author extracted 0.44% pectic substances from * Author to whom correspondence should be addressed.

638 wheat stem. In addition, an outline study of wheat straw pectin has been described by Harbers and co-workers (4) using scanning electron microscopy. The study indicated that wheat straw cell walls possess relatively small amounts of pectin. The objectives of the present study, which is part of an ongoing research program on the multiuse approach to cereal straw fractionation using thermomechanical pulping, were to isolate the pectic polysaccharides from wheat straw and to study the physicochemical properties of the polymers.

M A T E R I A L S AND M E T H O D S Materials Wheat straw was obtained from Silsoe Research Institute (Silsoe, Bedfordshire, UK), and was dried in a cabinet oven with air circulation at 60~ for 16 h. The dried wheat straw was then ground using a Christie Laboratory mill to pass a 60-mesh size screen and stored at 5~ until use. All chemicals were of analytical or regent grade. All experiments were performed in duplicate and yield is given on a dry wheat straw weight basis.

Isolation and analysis of wheat straw XRPP Xylose-rich pectic polysaccharide was extracted from defatted and protein-free cell wall preparation (5) using HC1 solution (pH 1.6) at 85~ for 4 h. The extract was adjusted to pH 5.0 with ammonia, concentrated on a rotary evaporator under reduced pressure at 40~ and precipitated with 5 volumes of 96% ethanol. After washing twice with 80% ethanol and drying in an air circulated oven at 40~ for 2 h, the pellet was redissolved with distilled water and then precipitated with 4 vols 96% ethanol. Before the pellet was gently ground, the precipitated pellet was washed twice with 70% ethanol and dried at 40 o in an air circulated oven for 16 h. The resultant white powder was labelled "xylose-rich pectic polysaccharide" and stored in a refrigerator. For measurement of the neutral sugars and galacturonic acid in extracted XRPP, both acid hydrolysis and pectinase digestion methods were used. During acid hydrolysis, XRPP (60 mg) was hydrolysed using 15 mL of 2 N trifluoroacetic acid (120~ for 2 h) in sealed pressure tubes. For pectinase digestion, 60 mg XRPP was dissolved in 15 mL of KH2PO4-NaOH buffer, pH 5.6 and 10 mg pectolyase (p-3026, 3.4 units/mg solid, Sigma) was then added. The mixture was incubated for 7 h at 35~ After filtration, the filtrate was evaporated to dryness at 40~ under reduced pressure. The released sugars arabinose, xylose, mannose, galactose and glucose were determined by gas chromatography after conversion to trimethylsilyl ether derivatives (5). myo-Inositol was used as an internal standard. The amounts of rhamnose released by acid hydrolysis and pectinase reaction were determined by the quantitative colorimetric procedure of Gibbons (6). The galacturonic acid released in both cases was assayed colorimetrically as anhydrogalacturonic acid using 3-phenylphenol colour reagent, according to the procedure

639 outlined by Blumenkrantz and Asboe-Hanson (7). Methyl ester content was determined using the method described by Wood and Siddiqui (8) whilst acetic acid was determined using the transesterification method described by Browing (9). The weight-average molecular weights of the XRPP were determined using gel permeation chromatography

(5). Viscosity was measured using a Brookfield Synchro-Lectric Viscometer (Model LV). A citrus pectin was used as a reference. XRPP samples (2%, w/v) were prepared in 0.1 M sodium phosphate buffer, pH 7.0, allowed to hydrate at 4~ for 16 h (10, 11). Viscosity was then estimated (cps) at 25~ Optical rotation was determined on a polarimeter (Perkin Elmer, type 108) according to the methods described by Phatak et al. (10) and McCready et al. (12). XRPP samples (1.0%, w/v) were prepared in double distilled water, and solutions were centrifuged before measurement. A citrus pectin was again used as a reference. The gelling properties of XRPP samples were tested according to the procedures of Phatak et al. (10), and Chang and Miyamoto (13). XRPP samples were prepared in distilled water at a concentration of 1.0% (w/v). IR spectra were obtained on an FTIR spectrophotometer (Mattson Cygnus 100), using KBr discs containing 1% finely ground samples. For the method of determination of lignin remaining attached to/associated with XRPP fraction see our previous report (5).

R E S U L T S AND D I S C U S S I O N

Composition of X R P P Extraction of wheat straw at 85~ for 4 h yielded an XRPP value of 1.1% for the given regime. The anhydrogalacturonic acid released by pectinase reaction and acid hydrolysis of XRPP were found to be 44.8% and 32.1%, respectively. This result indicated that pectolyase p-3026 treatment XRPP under the conditions chosen is more effective for release of galacturonic acid than 2 N trifluoroacetic acid hydrolysis (120 ~ for 2 h) which only released 71.7% of total galacturonic acid . Further hydrolysis with 2 N trifluoroacetic acid at 120~ or increases in trifluoroacetic acid concentration/hydrolysis temperature are necessary for release all of the galacturonic acid present in extracted XRPP. The methoxy content was low, 5.8%, indicating that wheat straw XRPP is a lowmethoxy XRPP. The data also shows that extracted XRPP possesses acetyl groups in its structure. The acetyl content of XRPP was 6.0%. Partial acid hydrolysis of the acetyl groupsrestored the gelation power of the pectin (14). The HC1 extracted XRPP contained a low amount of ash, 6.9%. This accords the study of Phatak and co-workers (10) on sugar-beet pulp pectin. Summarised in Table 1 is the neutral sugar compositions and anhydrogalacturonic acid content of XRPP released by pectinase reaction and acid hydrolysis, respectively. In both cases, XRPP were found to be rich in xylose and galactose content, but low in mannose content. The total neutral sugar content in XRPP released by acid hydrolysis was 28.4%,

640 which dropped to 17.1% for pectinase treatment. In contrast to the greater release of rhamnose during pectinase treatment, values obtained for the other sugars, arabinose, xylose, mannose, galactose and glucose, were higher in XRPP released by the acid hydrolysis. The greater release of rhamnose and galacturonic acid during pectinase treatment suggests that rhamnose coexists with galacturonic acid in the main chain of XRPP, and arabinose galactose and xylose are found in the side chains. Aspinall et al. (15), in discussions about pectin in soybean cotyledons, suggest the possibility that most of the xylose residues occur as xylosyl short side chains branched on the rhamnogalacturonan backbones. .Hence in wheat straw XRPP it is possible that a proportion of the xylose is present as an integral component of the acidic pectic fraction. In addition, as XRPP was readily degraded by pectinase, the material must contain homogalacturonan regions of the molecular chains. It is therefore concluded that XRPP belongs to a group of pectic substances. Although XRPP is a minor constituent of the polysaccharides in wheat straw, it probably has a distinct functional role in the cell walls.

Table 1. The composition of neutral sugars and content of anhydrogalacturonic acid (%) in XRPP extracted with pH 1.6 HC1 solution at 85~ for 4 h (1 g wheat straw/100 mL extractant) from defatted, protein-free wheat straw.

Sugars/anhydrogalacturonic acid

A(%)

B(%)

Rhamnose Arabinose Xylose Mannose Galactose Glucose Anhydrogalacturonic acid

1.2 0.9 8.2 0.1 5.8 0.9 44.8

1.1 3.5 14.0 0.2 6.8 2.8 32.1

Total

61.9

60.8

Areleased by pectinase reaction, Breleased by acid hydrolysis.

Matsuura and Hatanaka (16) observed that xylose-rich acidic polysaccharide having high mannose content was present in appreciable amounts in Japanese radish. This contained large amounts of neutral sugars, the galacturonic acid contents being only 11-25%. Xylose, arabinose and galactose were found to be the major constituents with xylose comprising more than 50% of the sugars in each sample, with the exception of one isolated from the leaves. Ray and co-workers (17) have also isolated a type of xylose-rich acidic polysaccharide, extracted with an aqueous 10% trichloroacetic acid, from the seeds of Acacia auriculaeformis. The composition of monosaccharides in this material were arabinose 13.5, xylose 18.0, galactose 23.0, glucose 10.5, and glucuronic acid 35.0%, respectively. Also, since pectin represents the material found in the primary cell wall of

641 plants, it is probable that the qualitative nature as well as quantity of various pectic polysaccharides found in pectin may vary with the degree of maturity/differentiation of the plant source (18).

Physicochemical characterisation of XRPP Due to degradation during acid (HC1, pH 1.6) extraction, the extract possessed a relatively low weight-average molecular weight: 8000. The viscosity (2%, w/v) was determined at 3.10 cps and was much lower than that normally observed for citrus pectin (93.50 cps). The pH, molecular size, degree of methylation, and temperature significantly affect the viscosity of wheat straw pectin. However, this low viscosity property of wheat straw pectin, which is similar to sugar beet pulp pectin, indicates a high potential for application in low-caloric, high fibre beverages (10). The optical rotation of XRPP (1.0%, w/v) was +60 ~ which was also low compared to that exhibited by citrus pectin (+ 162~ Because of the presence of acetyl groups, low viscosity and low molecular weight in the extracted wheat straw XRPP, no gel formation was observed at 1% levels of addition to water. Citrus pectin at 1.0% formed a firm gel. The FTIR spectra of citrus pectin and wheat straw XRPP (Figure 1) appeared to be similar. Both of the spectra have absorptions at 1740, 1608, 1430, 1360, 1244, 1080, 1060, 1035, 890 and 524 cm -1. The pectic substances belong to a class of carboxypolysaccharides which differ from neutral polysaccharides, with an intense band in the region 1740 cm -1 (for salts 1608 cm -1) related to vibrations of the carboxyl group (19). FTIR spectra of extracted wheat straw hemicellulose and cellulose do not exhibit this band (spectra not shown). From this point, the extracted wheat straw XRPP is also assigned to pectic substances. The intensity ratio of the bands uas(CO0-) at 1608 cm -1 and p(C:O) ester at 1740 cm -1 corresponds to fully deesterified pectin and Me pectate (20). Due to much stronger absorptions at 1608 cm -1 than that at 1740 cm -1 in wheat straw XRPP, it is also clear that wheat straw XRPP is a low-methoxy XRPP, which is in accordance with the results obtained by colorimetry. However, on closer examination of the spectrum of the citrus pectin, it can be seen that there is a specific feature in the 9501140 cm -1 region, where a group of six bands is observed at 950, 1008, 1035, 1060, 1080 and 1140 cm -1, whereas that of XRPP in this region has two weak absorption at 1008 and 1140 cm -1 and two very strong absorptions around 1060 and 1035 cm -1. This can be ascribed to the neutral polysaccharides present in the extracted wheat straw XRPP. The very weak absorption at 1510 cm -1 in extracted wheat straw XRPP is due to aromatic skeleton vibrations in wheat straw lignin. These data indicated that the extracted XRPP fraction contained small amounts of neutral polysaccharides and residual lignin. The total phenolic content in XRPP was 1.10%. The major components were found to be p-hydroxybenzoic acid (0.44%), vanillin (0.19%), syringic acid (0.13%), and syringaldehyde (0.13 %). The contents of p-hydroybenzaldehyde, vanillic acid and ferulic acid were 0.032, 0.015 and 0.020%, respectively. Gallic acid, protocatechuic acid and cinnamic acid were detected in trace amounts.

642

2.6 _

I

2.8 I

3.0 I

3.5 !

4.0 !

Microns 4.5 5.0 I I

6.0 I

7.0 I

8

!

tO I

t5 !

20 I

1 e,,

._o m

E c

IU

I-

000

''''

I ' ' ' ' 1 ' ' ' ' 1 ' ' ' ' 1 ' ' ' ' 1 , , , , I , , , , i

3500

3000

2500

2000

Wavenumber

1500

t000

500

(cm -1 )

Figure 1. FT-IR spectra of citrus pectin (a) and wheat straw xylose-rich pectic polysaccharide (b).

SUMMARY Our results have indicated that wheat straw XRPP contains 44.8% galacturonic acid and 28.4% neutral sugars and is particularly rich in xylose. The XRPP also contains 5.8% methoxyl ester content and 6.0% acetyl ester groups. The viscosity of XRPP was very low. The isolated wheat straw XRPP did not form gels under the experimental conditions. The XRPP extracted under acidic conditions, such as pH 1.6 HC1 solution, gave low molecular weight. In this work, wheat straw XRPP can be assigned to a group of polysaccharides termed as xylose-rich pectic polysaccharide. The fractional and structural characterisation of wheat straw XRPP is currently the subject of detailed further study in our laboratory.

643 ACKNOWLEDGEMENTS We acknowledge the financial support for the research from LINK Collaborative Programme in Crops for Industrial Use and Dr. James Bolton, Director of The BioComposites Centre. This study was supported by IlK. Ministry of Agriculture, Fisheries and Food for the LINK Collaborative Programme(Multi-use Approach to Cereal Straw Fractionation Using Thermomechanical Pulping) in Crops for Industrial Use under Agreement CSA 2054.

REFERENCES

1 0 . Theander and P. Aman, Swedish J. Agric. Res., 8 (1978) 189. 2 A. Miyamoto and K. C. Chang, J. Food Sci., 57 (1992) 1439. 3 M. Przeszlakowska, Acta Agrobot, 26 (1973) 115. 4 L. H. Harbers, G. L. Kreitner, G. V. Davis, M. A. Rasmussen and L. R. Corah, J. Ani. Sci., 54 (1982) 1309. 5 J. M. Lawther, R.-C. Sun and W. B. Banks, J. Agric. Food Chem., 43 (1995) 667. 6 M. N. Gibbons, Analyst, 80 (1955) 268. 7 N. Blumenkrantz and G. Asboe-Hanson, Anal. Biochem., 54 (1973) 484. 8 D. J. Wood and I. R. Siddiqui, Analytical Biochemistry, 39 (1971) 418. 9 B. L. Browing, (ed.) Methods of Wood Chemistry, New York, 1967. 10 L. Phatak, K. C. Chang and G. Brown, J. Food Sci., 53 (1988) 830. 11 S. A. Andon, Food Technol., 41 (1987) 74. 12 R. M. McCready, A. D. Shepherd, H. A. Swenson, R. F. Erlandsen and W. D. Maclay, Analytical Chemistry, 23 (1951)975. 13 K. C. Chang and A. Miyamoto, J. Food Sci., 57 (1992)1435. 14 E. L. Pippen, R. M. McCready and H. S. Owens, J. Am. Chem. Soc., 72 (1950) 813. 15 G. O. AspinaU, I. W. Cottrell, S. V. Egan, I. M. Morrison and J. N. C. Whyte, J. Chem. Soc., C (1967) 107. 16 Y. Matsuura and C. Hatanaka, Agric. Biol. Chem., 52 (1988) 2583. 17 B. Ray, P. K. Ghosal, S. Thakur and S. G. Maiumdar, Carbohydrate Research, 185 (1989) 105. 18 M. M. Baig, C. W. Burgin and J. J. Cerda, J. Agric. Food Chem., 30 (1982) 768. 19 M. P. Filippov, Food Hydrocolloids, 6 (1992) 115. 20 M. P. Filippov, G. A. Shkolenko and R. Kohn, Chem. Zvesti., 32 (1978) 218.



645

Chemical synthesis of oligosaccharides related to arabinogalactan-proteins (AGPs) J.F. Valdor and W.Mackie Department of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.

Abstract

Synthetic routes to two terminal arabinose-containing oligosaccharide fragment.s related to arabinogalactan (AGP) polysaccharides have been devised using standard protecting group and coupling strategies. These new compounds represent possible carbohydrate epitopes of arabinogalactan polysaccharides that may be involved in developmental processes of plant cell walls. They may also be related to some of the observed pharmacological activities of AGPs.

INTRODUCTION Arabinogalactan-proteins (AGPs) are an important class of glycoproteins widely distributed in plant tissues and exudates ~. Their precise biological functions in plants remain unknown but, using a panel of monoclonal antibodies specific for carbohydrate structures, it has been demonstrated that the presence of certain AGP epitopes are closely related to cell development in plant morphogenetic processes 2'3. These monoclonal antibodies have been shown to cross-react with the type of AGP typified by Lolium multiforum (ryegrass) arabinogalactan-protein (Fig.l). This polysaccharide is characteristic of type II arabinogalactans and has a linear 13-(1-3) galactan backbonecarrying short 13-(1-6) galactan side branches substituted with arabinofuranose units4. More recently, oligosaccharide fragments from structurally similar arabinogalactans isolated from the Chinese herbs, Angelica acutiloba and Bupleurum falcatum have been shown to have various potential pharmacological activities5.

-~3.~.o.Galp.( 1-~3)-~-D.Galp-(1-~3)-~-o-Galp-( 1-~3)-~-o.Galp-(1-~3)-~-D-Galp-(1-~3)-I~-o-Galp-(1-~ 6 6 6 t t t 1 1 1 ~-t.-~a f-(1-~3)-o-Galp ~-t.-~a f-(1-~3)-o-Galp ~-L-Ara f-(1-~3)-9-Galp 6 6 6 t t t 1 1 1 ~-L-Araf-(l-~3)-o-Galp ~-L-PSaf-(1-~3)-o-Galp ~-t.-Ara f-(1-~3)-D-Galp 6 6 6 t t t 1 1 1 ~-L-~a f ~-I.-Araf ~-L-~V'af

Fig.l: Structure of ryegrass (Lolium multoflorum) endospermarabino-galactan-proteirf

646 At present there is little information concerning the detailed composition and number of sugar units that constitute the carbohydrate epitopes in the antibody interactions and in the pharmacological activities and very few well-defined arabinose-containing oligosaccharides are available for biological studies. Accordingly, to provide model examples of these putative bioactive oligosaccharides, we have undertaken the synthesis of some AGP fragments of welldefined composition. Initially these will be utilised to provide structural parameters that may be relevant to biological activity and will also be used in immunochemical studies as potential hapten inhibitors of anti-AGP monoclonal antibodies.

SYNTHETIC STRATEGY Possible oligosaccharide fragments considered to be potential bioactive epitopes or hapten inhibitors are trisacchafide A and tetrasaccharide B (Fig.2). To provide these target oligosaccharides, synthetic schemes using a stepwise approach have been devised. Routes to the oligosaccharides A and B and intermediate disaccharides are based upon standard procedures involving 1,2-transglycosidic linkage formation which requires the use of a donor glycosyl halide with participating-group assistance in the 2-position, a suitably protected glycosyl acceptor and the presence of silver salts as promoter. The (1-3) linked disaccharides 8 and 9 were prepared with high stereoselectivity and good yield from a single glycosyl acceptor, methyl-2,4,6-tri-O-benzyl-~-D-galactopyranoside 1 the synthesis of which involved selective 3-crotylation of methyl-13-D-galactopyranoside via an alkyl-stannylation reaction6. The optimum conditions to yield the desired products involved the coupling of I with either 2,3,5-tri-O-benzoyl-ct-L-arabinofuranosyl bromide 2 or 2,3,4,6-tetraO-acetyl-t~-D-galactopyranosyl bromide 3 in dichloromethane with silver triflate as promoter 7. As in Kochetkov's polycondensation which included glycosylation of trityl ethers with cyanoethylidene derivatives of sugars s, the preparation of the (1-6) linked disaccharide 10 required a triphenylmethylated acceptor, methyl-2,3,4-tri-O-acetyl-6-O-trityl-f3-Dgalactopyranoside 4. The glycosyl halide 3 was used without recourse to the usual corresponding orthoester and the coupling reaction was carried out rapidly under reflux in dichloromethane in the presence of a co-activator, silver cyanide, to trap the tritylium ion liberated during the reaction9. To form the internal galactosyl residues in the synthesis of the target oligosaccharides A and B, the selective and readily removable bromoacetyl group 1~was employed according to a previous study on the stereospecifity of the coupling reaction of galactosyl halides beating different substituents 1~. In the trisaccharide synthesis, this was introduced in the 3-position of the protected glycosyl donor 6, made available by pmethoxybenzylation of the dibutylstannylene complex of methyl-13-D-galactopyranoside, followed by selective oxidative removal with 2,3-dichloro-5,6-dicyano-benzoquinone (DDQ) in neutral conditions 12 after blocking the remaining hydroxyl groups. In the tetrasaccharide synthesis, the 6-position of the glycosyl donor 7 was bromoacetylated from the methyl-2,3,4tri-O-benzoyl-13-D-galactopyranoside 5 obtained after a temporary protection of the primary hydroxyl group as a trityl ether and followed by a perbenzoylation step and a selective detritylation. In both cases, bromoacetyl derivatives were converted to their corresponding txglycosyl chlorides, which were easier to purify than their bromo analogues, by using 1,1dichloromethylmethylether (DCMME) in the presence of zinc chloride as catalyst ~3. In this

647

-

Fig.2 (a): Synthetic routes to carbohydrate fragments from AGPs

\

mo

648

L..G OQ

Fig. 2 (b): Synthetic routes to carbohydrate fragments from AGPs

649 way, the monosaccharide glycosyl donors 6 and 7 provided the advantage of a substituent at 0-2 capable of neighbouring-group participation with the anomeric center and a protecting group at 0-3 or 0-6 that could be selectively removed. The coupling reactions were performed under base-deficient conditions 14 (taking care to minimize acid-catalyzed migration of acyl groups in the nucleophilic acceptor) in the presence of silver triflate and sym-collidine. Although good stereoselectivities were achieved yielding mainly the desired 1,2-trans products, there is scope for improvement and optimization of the coupling yields. It is possible that the reduced yields were due to competing formation of orthoesters as co-products in the formation of the required 1,2-trans compounds. Removal of the bromoacetyl group TM was achieved in the presence of other ester groups by treatment with thiourea in dichloromethane/methanol to afford a new glycosyl acceptor having a free hydroxyl group for further glycosylation. The above strategy has been applied successfully to obtain trisaccharide A of which the 13C NMR spectrum was fully consistent with the expected structure. Similar approaches leading to tetrasaccharide B are currently in progress. Although none of the synthetic oligosaccharides described above have yet shown any biological activity 15, it is intended in future work to use these oligosaccharides for the formation of synthetic antigens and the development of new monoclonal antibodies as molecular probes of plant cell wall development. In future syntheses of oligosaccharides, it is planned to utilise the application of triphenylmethylated glycosyl acceptors as used suceessfuUy in the synthesis of the (1-6) linked galactobioses. This should allow coupling and detritylation to proceed in one step avoiding the need for hydrolytic removal of trityl groups. In addition, since trisaccharide A and tetrasaccharide B have a common saccharidic unit, it is planned to utilise the synthesis of disaccharide glycosyl donors in block syntheses of these and higher oligosaccharides.

ACKNOWLEDGEMENTS The authors are grateful to the BBSRC for financial support (JFV).

REFERENCES

5 6 7 8 9 10

G.B. Fincher, B. Stone and A.E. Clarke, Ann. Rev. Plant Physiol., 34 (1983) 47. R.I. Pennel, J.P. Knox, G.N. Schofield and K. Roberts, J. Cell. Biol., 108 (1989) 1967. J.P. Knox, P.J. Linstead, J. Peart, C. Cooper and K. Roberts, The Plant Journal, 1 (1991) 317. R.L. Anderson, A.E. Clarke, M.A. Jermyn, R.B. Knox and B.A. Stone, Aust. J. Plant Physiol., 4 (1977) 143. H. Yamada, Carbohydr. Polymers, 25 (1994) 269. P. Kovac and C.P.J. Glaudemans, Carbohydr. Res., 138 (1985) C10. A. Rashid and W. Maekie, Carbohydr. Res., 223 (1992) 145. N.K. Kochetkov, Tetrahedron 43 (1987)2389. C. Bliard, G. Massiot and S. Nazabadioko, Tetrahedr. Lett., 34 (1993) 5083. P. Kovac, Carbohydr. Res., 153 (1986) 237.

650 11 12 13 14 15

T. Ziegler, B. Adams, P. Kovac and C.P.J. Glaudemans, J. Carbohydr. Chem., 9 (1990) 135. K. Horita, T. Yoshioka, T. Tanaka, Y. Oikawa and O. Yonemitsu, Tetrahedron, 42 (1986) 3021. P. Kovac, Carbohydr. Res., 144 (1985), C12. P. Kovac, C.P.J. Glaudemans, Carbohydr. Res., 142 (1985) 158. E.A. Yates, J.F. Valdor, S. Haslam, A. Dell, W. Mackie and J.P. Knox, Glycobiology, (1996) in press.


651

Structural features of pectic substances during growth and ripening of apples Daniel E. Wechsler, Georg R. Strasser, Renato Amadb

Swiss Federal Institute of Technology Zurich, Institute of Food Science ETH-Zentrum, CH-8092 Zurich, Switzerland

Abstract

Pectic fractions, extracted with the chelating agent trans-l,2-diaminocyclohexaneN,N,N',N'-tetraacetic acid (CDTA) and with dilute sodium carbonate solutions from the alcohol-insoluble residues (AIR), were investigated by methylation analyses. To characterise structural changes related to growth and ripening, pectic fractions of unripe, mature and stored apples were analysed. Pectic fractions isolated from unripe and mature apples contained various amounts of starch. Therefore an amyloglucosidase treatment followed by a dialysis was carried out prior to further analyses. Linkage analyses were performed after a twofold reduction of uronic acids to the corresponding neutral sugar residues with NaBD4. Results of neutral sugar and uronic acid determinations revealed a loss of galactose during the first part of the ripening process. The linkage patterns of the investigated pectic fractions indicated that the loss of galactose is mainly due to a decrease in linear 1,4-1inked galactose residues.

1. INTRODUCTION Pectic substances make up about one third of the cell wall dry matter of dicotyledoneous plants, where they fulfill different functions. Pectins located in the middle lamella are important for the adhesion between adjacent cells, whereas pectins of the primary cell wall contribute to the water binding capacity by forming gels [8,11]. During plant development pectins undergo remarkable changes which lead to textural changes of plant tissues. As food additive with gelling, thickening and stabilising properties or as source of dietary fibre, pectins are also of technological and nutritional interest [9]. For a comprehensive understanding of the manifold functions of pectins detailed insights into the fine structure of this complex group of polysaccharides are needed. The ripening process has been subject of many studies [2,6,8]. Due to the large variety of plant tissues investigated, the results of these studies are quite heterogeneous. In general an increase in water-soluble pectins is observed, which is related to the combined action of

652 pectin methylesterases and polygalacturonases. In addition to the solubilisation of protopectin, losses of non-cellulosic neutral sugar residues also occur during the ripening of several fruits. The decrease in neutral sugar residues is not uniform for different species but is generally due to losses in galactose and/or arabinose residues [3]. L-arabinose and Dgalactose are known to be the major sugars in side chains (arabinans, galactans, arabinogalactans) of pectins. Losses of these residues during the softening process could therefore be related to the action of fruit endogeneous enzymes on pectic side chains. The degradation of these side chains might also affect the solubilisation of pectins by decreasing the entanglement with other cell wall constituents. The aim of the present investigation was to characterise the changes found in sugar composition of pectic fractions during growth, ripening and storage of apples. In order to determine structural changes, linkage analyses were performed on pectic fractions, extracted at different periods of development.

2. EXPERIMENTAL

Sampling: Golden delicious apples were sampled in intervals of three weeks over a hole season. Sampling started at the so-called "June-drop", which corresponds to the end of the cell division phase (= week 0). After harvest (week 14), the mature apples were stored over a period of 20 weeks at 4~ and 95% relative humidity. Preparation of AIR and extraction of pectic fractions: For the preparation of the alcoholinsoluble residue (AIR) the apples were peeled, cut into small pieces and boiled in 96% ethanol for 10min. After this enzyme inactivation step, the sample material was blended, homogenised and filtered through a G3 sintered glass filter funnel. The residue was washed with 96% ethanol, followed by acetone and diethyl-ether, dried overnight at 40~ under vacuum and stored at-20~ in the dark. Portions of about 10g of AIR were fractionated according to the method of Selvendran et ai. [ 10] as shown in figure 1. Removal of starch impurities: About 100mg of the extracted pectic fractions were dissolved in 30ml of NaOH 0.05M and stirred for 20h at 0~ The solutions were neutralised by the addition of 0.18ml glacial acetic acid and the pH was adjusted to 4.6. After the addition of 1 ml of an amyloglucosidase solution (60U/ml) without any detectable side activities, the samples were incubated at 60~ for 3h. Finally, the samples were cooled to room temperature, dialysed (cut-off: 12000D) against deionised water (4~ for 72h and freeze-dried. Reduction of uronic acids: Uronic acids (UA) were converted to the corresponding neutral sugars (NS) by carbodiimide activation of the carboxyl groups followed by a reduction with NaBD 4 according to the method of Kim and Carpita [5]. In order to achieve a complete reduction of the uronic acids the procedure was repeated once. Methylation analysis: Permethylation of the sample material was carded out according to Kvemheim [7]. After the first methylation excess of methyl iodide was evaporated with a stream of nitrogen and the methylation was repeated. Extraction, hydrolysis, reduction and acetylation was carded out according to Harris et al. [4]. The partially methylated alditol acetates (PMAA) were analysed by GLC-MS, using a Fisons GC 8065 gas chromatograph (Carlo Erba, Milano, Italy) coupled to a Finnigan MAT SSQ 710 mass spectrometer (Finnigan MAT, San Jose, CA, USA). For the gas chromatographic separation a DB-225

653 column (30m x 0.25mm id. 0.251am film thickness, J&W Scientific, Folsom, CA, USA) was used with helium (30cm s-1) as carder gas. The injector was hold at 220~ employing a splitless injection of 15s. The temperature programm was: 160~ for lmin, increasing by 2~ min -1 up to 220~ (holding time 19rain).

AIR

1

Stir with 1000ml 50mM CDTA at pH 6.5 for 6h at 20-22~ Filter on G3 glass filter, wash residue with water, centrifuge filtrate at 8000rpm for 20min, dialyse, concentrate. Reextract the residue under the same conditions and freeze-dry the combined filtrates CDTA-Fraction

Residue

Stir with 1000ml 50mM Na2CO 3 + 20raM NaBH4 (pH t0.8) for 16h at 1~ Filter on G3 glass filter, wash residue with water, bring f'tltrate to pH 5 with 2M acetic acid, dialyse, concentrate, freeze-dry Fraction N1

Residue

Stir with 1000ml 50mM Na2CO 3 + 20mM NaBH4 (pH 10.8) for 3h at 20-22~ Filter on G3 glass filter, wash residue with water, bring filtrate to pH 5 with 2M acetic acid, dialyse, concentrate, freeze-dry Fraction N2

l

Depectinated residue (DR)

Fig. 1: Extraction scheme for pectic fractions

3. RESULTS AND DISCUSSION The results of the linkage analyses indicated remarkable changes in the sugar composition as shown for fraction N1 in table 1. The decreasing ratio of neutral sugars to uronic acids is mainly due to a increase in galacturonic acid and to a loss of galactose residues during ripening. This trend was found in all the pectic fractions of golden delicious apples (data not shown) and is in good agreement with the results obtained by Gross and Sams [3] and Fischer et al. [2], respectively. Table 1 Ratio of PMAA derivatives of uronic acids and neutral sugars in fraction N1 Stage of development Weeks after "June drop" Neutral sugars Uronic acids

unripe 3

mature 15

stored 33

74.3 25.7

31.4 68.6

30.1 69.9

654 Methylation analyses of fractions N1 indicated that the linkage pattern of uronic acid residues (table 2) remains unchanged during ripening whereas the glycosidic linkage composition of neutral sugar residues showed different alterations. Table 2 Glycosidic linkage composition of the uronic acid residues in fraction N1 Stage of development Galacturonic acid 1,4-GalpA 1,2,4-GalpA 1,3,4-GalpA Glucuronic acid Total UA-PMAA

1,4-GlupA

unripe

mature

stored

94.9 1.2 3.4 99.6 0.4

97.0 1.0 1.9 99.9 0.1

96.4 0.8 2.7 99.9 0.1

100.0

100.0

100.0

The most important change in the glycosidic linkage pattern of the neutral sugars (table 3) was the decrease in linear 1,4-1inked galactose residues. Linear, (1--->4)-linked galactans have been isolated from pectic material of different sources [1,8]. Arabinogalactan type I has also a backbone of (1--->4)-linked I$-D-galactopyranosyl residues but contains short side chains of (1--->5)-linked a-L-arabinofuranosyl residues linked to position 0-3. The low amount of 1,3,4-branched galactose residues and the fact that the net amount of arabinose remains unaffected by the loss of (1--->4)-linked ~-D-galactopyranosyl residues indicate that the decreasein galactose might be related to a degradation of an unsubstituted galactan associated with pectins. The linkage pattern for arabinose residues showed an increasing relative amount of 1,51inked residues during ripening. Since the net amount of arabinose remains nearly unchanged during ripening, the observed changes indicate a slight linearisation of arabinan side chains. The detected rhamnose residues were typical for the backbone of pectin. Some of the (1--->2)-linked a-L-rhamnopyranosyl residues were branched, having side chains attached to 0-4 and to 0-3. Additionally terminal and double branched rhamnose residues were found in small amounts. Finally typical fucose and xylose residues were detected in all the analysed fractions. The starch content in apples reaches a maximum during growth and starts to decrease towards harvest. Therefore the AIR contained up to 40% starch for unripe apples and 10% for mature apples, respectively. During the extraction of pectic substances with CDTA and dilute sodium carbonate solutions starch was co-extracted. Although an amyloglucosidase treatment was carded out prior to methylation analysis, the fractions of all development stages still contained glucose residues. Assuming a complete starch degradation, the remaining glucose residues can not be considered as starch impurities but could be part of a xyloglucan. On the other hand most of the mannose residues must be considered as impurities, since they were found to originate from the commercial amyloglucosidase preparation.

655 Table 3 Glycosidic linkage composition of the neutral sugar residues in fraction N1 Stage of development Rhamnose T-Rhap 1,2-Rhap 1,3-Rhap 1,2,4-Rhap 1,2,3,4-Rhap Fucose

T-Fucp 1,3,4-Fucp

Arabinose

T-Araf T-Arap 1,2-Araf 1,3-Araf 1,5-Afar 1,2,5-Araf 1,3,5-Araf 1,2,3,5-Araf

Xylose

T-Xylp 1,4-Xylp

Galactose

T-Galp 1,3-Galp 1,4-Galp 1,6-Galp 1,2,4-Galp !,3,4-Galp 1,3,6-Galp 1,4,6-Galp

Glucose

T-Glup 1,4-Glup 1,3,4-Glup 1,4,6-Glup

Mannose

T-Manp 1,2-Manp 1,4-Manp 1,3,6-Manp

Total NS-PMAA

unripe 0.1 2.3 0.1 1.8 0.1

mature 0.3 2.8 0.3 2.8 0.3

stored 0.4 4.5 0.4 5.8 0.3

4.4

6.5

11.4

0.1 0.1

0.2 0.3

0.3 0.0

0.2

0.4

0.3

6.6 0.3 0.2 0.8 10.7 1.3 6.9 5.5

7.0 0.3 0.3 1.1 12.6 1.4 7.7 6.0

9.1 0.3 0.5 1.7 20.2 2.0 8.9 5.8

32.2

36.5

48.5

0.2 0.5

0.6 0.4

1.3 0.7

0.8

1.1

2.1

0.1 1.3 49.3 0.2 0.6 0.4 1.2 2.3

0.3 0.9 43.0 0.4 0.7 0.0 1.2 1.6

0.4 1.3 23.0 0.5 0.7 0.6 1.6 0.7

55.3

48.1

28.9

0.5 3.6 0.0 0.4

0.9 2.2 0.4 0.3

1.0 3.8 0.0 0.5

4.5

3.9

5.3

1.2 0.3 0.8 0.3

1.6 0.4 1.1 0.4

2.1 0.6 1.0 0.0

2.6 100.0

3.5 100.0

3.6 100.0

656 4. CONCLUSIONS -Losses of galactose residues occufing in pectic fractions during growth and ripening of apples are mainly due to a loss of (1--->4)-linked [~-D-galactopyranosyl residues. The low amount of branched galactose residues indicate the presence of a linear galactan associated with the pectic fraction of apple cell walls. -Branched arabinans with a backbone of (1--->5)-linked ot-L-arabinofuranosyl residues are present in pectic fractions of apples. During ripening a slow linearisation of the arabinans ocCurS.

5. REFERENCES [1] Eda, S., Kato, K. (1978). Galactan isolated from the midrib of the leaves of Nicotiana tabacum. Agric. Biol. Chem. 42, 2253-2257. [2] Fischer, M., Arrigoni, E., Amad6, R. (1994). Changes in the pectic substances of apples during development and postharvest ripening. 2. Analysis of the pectic fractions. Carbohyd. Polym. 25, 167-175. [3] Gross, K.C., Sams, C.E. (1984). Changes in cell wall neutral sugar composition during fruit ripening: a species survey. Phytochemistry 23, 2457-2461. [4] Harris, P.J., Henry, R.J., Blakeney, A.B., Stone, B.A. (1984). An improved procedure for the methylation analysis of oligosaccharides and polysaccharides. Carbohydr. Res. 127, 59-73. [5] Kim, J-B., Carpita, N.C. (1992). Changes in esterification of the uronic acid groups of cell wall polysaccharides during elongation of maize coleoptiles. Plant Physiol. 98, 646653. [6] Knee, M., Bartley, I.M. (1981). Composition and metabolism of cell wall polysaccharides in ripening fruits. In: Friend, J., Rhodes;, M.J.C. (eds.). Recent advances in the biochemistry of fruits and vegetables. Academic Press, New York, 133-148. [7] Kvernheim, A.L. (1987). Methylation analysis of polysaccharides with butyllithium in dimethyl sulfoxide. Acta Chem. Scand. Ser. B41,150-152. [8] Melford, A.J., Dey, P.M. (1986). Postharvest changes in fruit cell wall. Adv. Food Res. 30, 139-193. [9] Pilnik, W. (1990). Pectin - a many splendoured thing. In: Phillips, G.O., Wedlock, D.J., Williams, P.A. (eds.). Gums and stabilizers for the food industry. Elsevier, London, 209221. [ 10] Selvendran, R.R., Stevens, B.J.H., O'Neill, M.A. (1985). Developments in the isolation and analysis of cell walls from edible plants. In: Brett, C.T., Hillman, J.R. (eds.). Biochemistry of plant cell walls. Cambridge University Press, Cambridge, 39-78. [11]Van Buren, J.P. (1991). Function of pectin in plant tissue structure and firmness. In: Walter, R. (ed.). The chemistry and technology of pectin. Academic Press Inc., San Diego, 1-22.

PECTIN BIOSYNTHESIS AND BIOLOGICAL EFFECTS OF (DEGRADED) PECTIN



659

Metabolism of pectin in the gastrointestinal tract G. Dongowski and H. Anger

German Institute of Human Nutrition Potsdam-Rehbr[icke, Department of Food Chemistry and Preventive Nutrition, D-14 558 Bergholz-Rehbriicke, Germany

Abstract Pectin was depolymerized and de-esterified only to a small extent after treatment under conditions simulating the gastrointestinal lumen. The degree of esterification was slightly decreased in the upper part of gastrointestinal tract of germfree and conventional rats and additionally in caecum and colon of germfree rats. Pectin passes the small intestine as a macromolecule. The molecular weight distribution of pectins measured by gel chromatography with viscosity detection from faeces of germfree rats remained nearly unchanged. No or very low amounts of galacturonan were found in contents of caecum, colon and in faeces of most of the conventional rats. Oligogalacturonic acids were not detected in faeces of these animals. Diand trigalacturonic acid which could then be absorbed by the host were present in the colon of a few conventional rats. However during in vitro fermentation of pectin with human faecal flora unsaturated oligogalacturonic acids were found as intermediate products in variable concentration and composition within ca. 8 hours using thin-layer chromatography and HPLC (PAD, chiraliser and UV detection). Low-esterified pectin was fermented faster than highesterified pectins.

1. INTRODUCTION

The physiological importance of pectin as a soluble dietary fiber in the small intestine is closely related with its macromolecular properties (interactions with bile acids, lowering of serum cholesterol, effects on the postprandial lipemia etc.). Pectin is not depolymerized by intestinal enzymes. A partial degradation seems to be possible under the conditions of stomach and small intestine. Pectin is completely fermented in the (caecum and) colon by the microflora [1-4]. In order to be absorbed, pectin has to be degraded to mono- or oligomers having a low degree of polymerisation (DP). It is unclear whether oligogalacturonic acids (OligoGalA) as metabolites of pectin degradation are formed in detectable concentrations in the colon. OligoGalA intravenously applied or injected directly in the caecum were found in the urine of rats [5]. Pectin influences the absorption, incorporation and the renal excretion of lead [6].

660 This study reports on investigations of the metabolism of pectin by in vitro and in vivo experiments using chromatographic methods.

2. MATERIAL AND METHODS

2.1. Treatment of pectin under conditions of gastrointestinal tract (GI) Pectin solutions were treated 2 h at pH 1-2 and each then for 2 h at pH 6.0, 7.0 and 8.0.

2.2 Animal experiments 5 groups of 10 conventional rats and 4 groups of 6 germfree rats were fed ad iibitum over 21 days. The diet had the following composition: 6.5 % or 0 % pectin (galacturonan) with different degree of esterification (DE), 5.0 % cellulose, 63.0 % or ca. 54 % wheat starch, 20.0 % casein, 5.0 % sunflower oil, 5.0 % mineral mixture and 2.0 % vitamin mixture. The diets for the experiments with germfree rats were sterilized by T-irradiation (20 kGy). This resulted in a partial depolymerization of pectin (Table l). Faeces were collected in two periods (3 d during weeks 2 and 3). At the end of the experiments, the contents of ileum, caecum and colon were also investigated.

Table l Pectins used in experiments with rats Original pectins DE [ 11 ] (%) (mFg AG) 92.6 328 K 70.8 455 K'~; 34.4 294 K

7-irradiated pectins DE [ 11 ] (%) (ml/g AG) 92.6 208 c; 70.7 292 c 34.4 211G

K = conventional rats; G = germfree rats; AG - anhydrogalacturonic acid (galacturonan).

The lyophililized intestinal contents or faeces were treated for enzyme inactivation in 5 ml 96 % EtOH for 20 min at 75-80 ~ After addition of 5 ml water the mixture was stirred 30 rain and centrifuged at 6000*g also for 30 min. In the supernatant galacturonan was estimated by the m-hydroxydiphenyl (MHDP) reaction [7] and OligoGalA were determined using HPTLC. In the dried residues, the content of galacturonan and the DE were estimated after extraction with 0.5 % EDTA. Molecular weight distribution was analysed by gel chromatography (0.5 % AG) equipped with a Shodex Ohpak B 805 column (500*8 mm) with phosphate buffer (pH 6.5) using differential-refractometer/viscometer detection (Knauer). For calibration a pectin series from vibration milling was applied.

661 2.3. In vitro incubation 150 ml pectin media (0.5 % AG) were incubated with 4 g human faecal flora at 37 ~ without aeration. The contents of macr.omolecular pectin and OligoGalA were estimated in the culture after different periods. OligoGaiA were determined using the Camag HPTLC system on silica gel 60 developed with n-propanol-water (7+3.75) and (7+2.75). The spots were detected by measuring at 235 nm and after dipping in the MHDP reagent at 525 nm. Further a Kontron HPLC system equipped with UV (250 nm), chiralyser and PAD detectors was used for estimation of OligoGalA. The column was CarboPac PAl (250*9 mm) with a precolumn and the gradient of 40 to 100 % solvent B (0.15 M NaOH, 1 M Na-acetate) and of 0.15 M NaOH (A) was applied. The system was calibrated with a mixture of OligoGalA (DP 2-15) prepared from pectic acid using pectate lyase from Erwinia carotovora.

3. RESULTS AND DISCUSSION

Milieu conditions in gastrointestinal tract can influence the pectin structure and properties. Under the acid conditions of the stomach (pH 2-4) extraction of pectin from plant cell walls and hydrolysis of side chains can occur. In small intestine (pH 5-6) I]-elimination of main chains or de-esterification seems to be possible. In caecum and colon (pH 6-8) a strong fermentation of pectin takes place causing depolymerization to oligomers and leading to formation of short chain fatty acids and gases. The presence of OligoGalA is not yet clarified. After treatment under the conditions simulating the gastrointestinal lumen pectin was depolymerized only to a small extent. The DE of pectin is slightly decreased in the upper part of GI and as well as in caecum and colon of germfree rats (Table 2).

Table 2 DE of pectin Original pectin 92.6 70.8 34.4

(%) isolated from intestinal contents of conventional (K) and germfree rats (G) Ileum Ileum Caecum Colon K G G G 91.2 90.4 90.0-~.6 89.8_+0.3 70.6 70.4 69.9_+0.8 70.7_+0.6 34.1 34.5_+0.4 34.0-Z-O.5 34.6_+0.3

In intestinal contents and faeces from germfree rats quite high amounts of galacturonan were found, especially in the case of the pectin with the highest DE (Table 3). The isolated pectins were depolymerized to a small extent. The molecular weight distribution of pectins from intestinal contents and faeces remained relatively unchanged (Figures 1 and 2).

662 Table 3 Pectin in intestinal contents and faeces of germfree rats DE Ileum Caecum Colon (%) (mg) (mg) (mg) 92.6 87-+10 1599-+ 87 143-+59 70.8 42_+ 7* 655_+124'* 167_+28 34.4 55+ 0* 421+102"* 115+28 Soluble part in 50 % EtOH: 1.4-6.0 %; * P < 0.05; ** P
4)-cx-D-GalA-(1-->2)-~-L-Rha-(1--)4)-cx-D-GalA had a similar partial structure as the rhamnogalacturonan core, however this tetrasaccharide did not show anti-complementary and mitogenic activities. When uronic acids in PG-Ic were degraded by lithium, the products (which contained neutral oligosaccharide-aiditols and degradation products from uronic acids) showed decreased anti-complementary and mitogenic activities compared to PG-lc (Figures 2A and 3). However, the products still showed weak but significant activities, and it was concluded that neutral carbohydrate chains in PG-lc might contribute to expression of these activities.

9..\\\\\\\'< control GR-2IIc PG-lc ("ramified region) PG-2c (RGII like region)

0%

PG-3c (oligoGalA) o~5

o.~5

Mitogenic activity (OD540)

o

0%

o

!

!

50

~oo

Anti-complementary activity (%)

Figure 1 Immunomodulating activity of fractions derived from GR-211c by endo-cx(1--~4)-polygalacturonase digestion

676

100-

PG-lc

A w

PG-Ic

J A w

I,

A v

v

.I

PG-Ic-4

t_

"~ 50 a_

s0 ,i

PG-Ic-2

Lithium-degraded PG-lc

i

o-

PG-Ic-5

~

Pen-~deoxyHex-oi, Pen-~Hex ~deoxyHex-oi, Hex ~Hex-~Hex-ol, Pen (~Pen)2-~Pen-ol, Hex (~Hex)2~deoxyHex-ol and Hex (--)Hex)~-~Hex-ol. Since PG-lc-4 consisted mainly of Ara as pentose, it was assumed that Pen in the

678 oligosaccharide-alditois might be Ara. PG-lc-4 also mainly comprised Man, Gal and Glc as hexose, however Hex units and glycosidic linkages in the oligosaccharidealditois could not be deduced in the present study. Since PG-lc-4 contained various kinds of neutral oligosaccharide-alditols, some standard oligosaccharide and oligosaccharide-alditols were measured for anticomplementary and/or mitogenic activities in order to investigate whether other oligosaccharide-alditols have such activities. However, all oligosaccharides and oligosaccharide-alditols tested, such as maltose, maltohexaose, isomatohexaose, laminarihexaose, maltitol, maltotriitol and maltoheptitol, did not show any activities. These results suggested that certain neutral oligosaccharide-alditols in PG-Ic-I and 4 might be responsible for expression of the activities.

5.

CONCLUSION

The present results proposed that the certain neutral carbohydrate chains in the "ramified" region of GR-2IIc plays important roles for expression of anticomplementary and mitogenic activities. However the activities of the neutral carbohydtae chains were weaker than those of the intact "ramified" region. It was assumed that substitution of the neutral carbohydrate chains to the rhamnogalacturonan core might be enhance the activities of the chains, and there might be a possibility to exist a minimum molecular seqeunce consisting of neutral carbohydrate chains and the acidic core for expression of the potent activities.

6.

ACKNOWLEDGEMENTS

We thank Dr. V. Pozsgay (NIH, U.S.A.) for his kind gift of methylglycoside of o~-L-Rha-(1-->4)-o~-D-GalA-(1--->2)-o~-L-Rha-(l-->4)-o~-D-GalA.

7. 1 2 3 4 5 6

REFERENCES H. Kiyohara, T. Matsumoto, N. Takemoto, H. Kawamura, Y. Komatsu, H. Yamada Planta Med., 61 (1995) 429. H. Kiyohara, T. Matsumoto, Y. Komatsu, H. Yamada, Planta Med., in press. H. Yamada, Asia Pacific J. Pharmacology, 9 (1994) 209. H. Yamada, H. Kiyohara, N. Takemoto, J.-F. Zhao, H. Kawamura, Y. Komatsu, J.-C. Cyong, M. Aburada, E. Hosoya, Planta Med., 58 (1992) 166. J.-F. Zhao, H. Kiyohara, X.-B. Sun, T. Matsumoto, J.-C. Cyong, H. Yamada, N. Takemoto, H. Kawamura, Phytotherapy Res., 5 (1991) 206. J.-F. Zhao, H. Kiyohara, H. Yamada, N. Takemoto, H. Kawamura, Carbohydr. Res., 219 (1991) 149.


Immunologically active

679

polysaccharides from cell suspension of H e l i a n t h u s a n n u u s

1805

M. Kratchanova a, M. Ilievab, E. Pavlova a, A. Pavlovb,N. Markova b

a Bulgarian Academy of Sciences, Institute of Organic Chemistry with Phytochemistry, 95 V. Aprilov Str., P. O. Box 27, LBAS - Plovdiv, Bulgaria 4002

b Bulgarian Academy of Sciences, Institute of Microbiology, Sofia, Bulgaria 1113

acad. G. Bontchev Str. 26,

Abstract Cell suspension cultures from H.annuus 1805 which release large amounts of extracellular polysaccharides were investigated. It was established that the main amount of polysaccharides was secreted during the logaritmic phase of the growth cycle. Three extracellular polysaccharides of pectic type were isolated from the spent culture medium of H.annuus 1805 alternately by 96 % ethanol precipitation and by freezing at -18~ The polysaccharide isolated by freezing manifested a higher immunostimulating activity. Three GelC fractions were obtained after purification on Sephadex G100. The fraction with the highest molecular weight and the highest content of D-galacturonic acid was the carrier of the biological activity.

Introduction

A considerable amount of extracellular polysaccharides is produced in the process of cultivation of certain plant suspension cultures and the spent culture medium has proved to be an accessible source for their production (1-3). The interest in investigating these extracellular polysaccharides has been quite strong over the past 10 - 15 years, motivated by their biological activity (4,5). Plants of the Asteraceae family, as well as their cell cultures, have been established to contain polysaccharides with immunostimulating activity (1-6). The object of our research was Helianthus annuus 1805 cell culture (Asteraceae), which according to the preliminary investigation produces a considerable amount of exopolysaccharides. The purpose of this research was to study the time course of growth of the cell culture, the production of extraceUular polysaccharides, and their characteristics.

680 Materials and methods

Cell culture and biosynthesis of polysaccharides. The Helianthus annuus 1805 cell culture was initiated according to a previously described method (7), using germs of Helianthus annuus as an explant. A medium of Linsmayer Skoog (8) supplemented with 0.2 m g ~ 2.4dichlorophenoxyacetic acid as a growth regulator was used for the growth of cell suspension. The cultivation was carried out in Erlenmayer flasks with 1/5 net volume on a shaker (11.6 rad/s) in the dark, at 26 - 28~ In compliance with the nature of the experiment, flasks of different size were used: 100 - 1000 cm 3, and the duration of the cultivation was 5 days for growing the inoculum and 12 days for studying the time course of growth. Experiments were carried out by varying the amount of inoculum (10, 15 and 20 % v/v) to determine the optimal quantity which ensures a steady growth. The time course of growth of the cell suspensions, inoculated with the corresponding amount of inoculum was traced by day-to-day determining the yield of dry cell biomass (7), while the time course of biosynthesis of extracellular polysaccharides was followed by their daily determination, using the carbazole method (9). Isolation ofpolysaccharide fractions: After a growth period of 8 days the cell biomass was separated by filtration and the spent culture medium was used for the isolation of polysaccharides. The following procedures were examined: Procedure 1:96 % ethanol was added to the culture medium (3:1), while continuously stirring. The precipitated polysaccharide was kept overnight at -4~ and then separated by filtration. It was washed with 70 % hydrochloric acid ethanol, then with 70 % ethanol to a neutral pH. In the end it was washed with 96 % ethanol and dried at 50~ (Sample 1). Procedure 2: The culture medium was frozen at -18~ for 24 hr's. The precipitated polysaccharide was filtered through a cloth, washed as in Procedure 1 and dried (Sample 2). The filtrate was concentrated 10 fold on a rotation vacuum- evaporator at 45~ Then 96 % ethanol was added to the concentrated filtrate and the sample was kept overnight at 4~ The precipitated polysaccharide was filtered, washed as in procedure 1, and dried (Sample 3). Gel chromatography on Sephadex G100 (2.8x50cm) of polysaccharide fraction I (Sample 2). The polysaccharide fraction was dissolved in a phosphate buffer at pH 6. After centrifugation, the supernatant was applied to the column at a flow rate of 0.8 ml/min. The elution was performed with a phosphate buffer and fractions of 10 ml each were collected. The refraction of each fraction was measured interferometrically. Fractions with coincident peaks were collected and analyzed for content of galacrturonic acid, neutral sugars and protein. Immunological tests were performed for studying the reactive of peritoneal-exudative cells, especially peritonial macrophages, which are the main effector cells involved in natural resistance (host defence system) against bacterial infection.

681 Tests:

1.Determination of number ofperitoneal-exudative cells after i.p. (intraperitoneal) application of polysaccharide fractions and during infection with Y. pseudotuberculosis in experimental animals. 2.Bioassay for "killing" ability (in vivo and in vitro against bacteria) of peritonial macrophages after treatment with polysaccharide fractions. 3.Bioassay for metabolic activity (glycolytic and acid phosphatase activity) of peritonealexudative cells after treatment with polysaccharide practions. Methods of analysis. The polyuronic acid content (PUAC) and the degree of esterification (DE) were determined according to Owens et al (10). The specific viscosity rl for different values of the concentration (C) of the analyzed polysaccharide solutions in 0.15 M NaC1 was determined by means of a capillary viscometer (Ubbelode) at 25~ The intrinsic viscosity [~1] and Huggins constant K H were calculated according to Huggins equation (11). The average molecular mass M M was determined by solving the equation, following the methods in (12). The content of neutral monosaccharides was determined after an acid hydrolysis, performed as follows: with 72 % sulphuric acid for 1 hr at 30~ and then, after dilution to 1M sulphuric acid, for another 3 hr's at 100~ The determination was performed by GC analysis of the prepared alditol acetates (13,14) The uronic acids in polysaccharide fraction I (Table 2) were determined colorimetrically with m-hydroxybiphenyl (15). The content of galacturonic acid in the fractions, obtained by gel chromatography, was determined by the carbazole method (9), and the content of neutral monosaccharides was determined by the anthron reaction (16). The protein content of the polysaccharide fractions was determined by the method of Lowry.

Results

Growth of the Helianthus annuus 1805 cell suspension and biosynthesis of extracellular polysaccharides. A particular characteristic of plant cell suspensions is the requirement for a high inoculation density in order to initiate growth. This is due to one of their special features: in order that their growth be initiated when transferred into the new medium, they need certain growth factors which are released and secreted into the medium by the cells themselves. Consequently, to ensure the growth of plant cell suspensions, a certain volume (in which plant cells have to be present at above certain densities) has to be used to import the necessary quantity of these substances (17). On the other hand, with cell suspensions synthesizing a considerable amount of polysaccharides, the excessive quantity of inoculum can lead to intensive aggregation before the maximum in the biomass synthesis is reached.

682 18

2.2

16-

2.0

~1~4 -

"~ .4

~O-

~9 .2

E 0

"~8-

4

ao.4

2

0.2

0 0

I

i

i

1

i

i

I

I

1

2

3

4

5

6

7

8

0.0 9

10

0

Time, days

Fig. 1. Growth of Helianthus annuus 1805 cell suspension inoculated with different amounts of inoculum

.

.

1

2

.

. 3

. 4

5

6

7

8

9 10

Time, days

Fig. 2. Time course of biosynthesis of polysaccharides from a cell suspension of Helianthus annuus 1805.

=

10% - i n o c u l u m

-"

15% - i n o c u l u m

=

15% - inoculum

"--

20% - i n o c u l u m

-"

20% -inoculum

=

1 0 % - inoculum

The time courses of growth of H.annnuus 1805 cell suspension for 10,15 and 20% (v/v) inoculum used, were followed (fig. 1). In all three cases the maximum amount of synthesised dry biomass was 15 - 15.5 g/dm 3, and it was attained on the 6th day, on the 8th and on the 10th day for a 20, 15 and 10 % inoculum used respectively. After the maximum was reached, the amount of biomass remained constant for 1 - 2 days, and then slight lysis was observed. The preliminary chromatographic analyses of the polysaccharides indicated that the Dgalacturonic acid is their major component. Thus the amounts the extracellular polysacchafides in the culture medium was examined by determining the quantity of D-galacturonic acid using the carbazole method. The time course of biosynthesis of extracellular polysaccharides from H.annuus 1805 cell suspension, when 10, 15 and 20 % v/v inocula were used, indicated that in all cases the maximum in the amount of extraceUular polysaccharide was achieved on the 8th day of cultivation. The use of a 10 % inoculum was inexpedient and inasmuch as the polysaccharide synthesis was considerably lower (0.95 mg/ml galacturonic acid), compared with the other two cases. The use of a 20 % (v/v) inoculum was most expedient. Maximum extracellular polysaccharides was the 8th day of cultivation. The increase of inoculum over 20% leads to problems, connected with the higher viscosity at the later stages of cultivation. Isolation and characteristics of the polysaccharides. It is known (1,3,4,6) that the polysaccharides from a culture medium can be precipitated by adding different volumes of ethanol (1:2, 1:3). Our experiments with precipitation with ethanol at a ratio of 1:3 led to the isolation of crude polysaccharides from the culture medium (Sample 1).

683 Table 1 Obtaining and characteristics of the polysaccharides from culture annuus 1805 cell suspensions Sam- Methodfor obtaining Polysaccharide Polyuronides pie yield g/L culture DE, PUAC, Nr medium, g % % Polysaccharide, isolated 1,6 56,0 61,0 by precipitation with ethanol (1:3) Fraction I 2. Polysaccharide,isolated 1,2 36,5 74,3 by freezing the culture medium at t=- 18~ for 24 hr's Fraction II 3. Polysaccharide,isolated 0,3 52;0 18,52 by precipitation of concentrated filtrate with ethanol (1:3) (after the removal of fraction I)

medium of the Helianthus Molecular Intrinsic Huggins mass viscosity constant dlxs- 1 KH 100 000

6,99

0,6

150 000

912,1

0,7

17 000

0.67

0.58

A new possibility for isolation of the exopolysaccharides in deep freezing of the culture medium (-18~ was arrived at in the course of our research (Sample 2, Polysaccharide fraction I). A second polysaccharide fraction (Sample 3) was isolated from the filtrates by concentration and following precipitation with ethanol (1:3). The yield and characteristics of the obtained polysaccharides (Samples 2 and 3) are given in Table 1. It is evident that the sum of the yields for Samples 2 and 3 is almost equal to the yield for Sample 1. The polyuronic content data are also well balanced. This fact indicates that the suggested method is suited for fractional isolation of the polysaccharides from the spent culture medium of H.annuus 1805 cell suspension. As can be seen from Table 1, the main part of the exopolysaccharide was in fraction I. The data on the molecular mass and on the intrinsic viscosity confirmed the expectation that the fraction, precipitated under freezing, had a higher molecular mass (Table 1). The values of Huggins constant were close and indicated that there was an increased tendency towards aggregation for the dissolved macromolecules. The results of the biological investigation showed that polysaccharide fraction I had a distinct immunostimulating activity. Characteristics of fraction 1. The carbohydrate content of Fraction I was 84.4 %, in which the main component is D-galacturonic acid (71%). Consequently, the polysaccharide is of a pectic type. The neutral sugars accounted for 13.4 % and according to their qualitative composition (Table 2) they correspond to the composition of pectin, isolated from sunflower heads (18). It is worth noting the high content of L-arabinose and D-galactose, compared with the other mon0saccharides. The protein content was 7.8 %.

684 Table 2 Composition of polysaccharide fraction I, isolated from the culture medium of H. annuus 1805

Nr 1

Compounds D-Galacturonic acid Neutral sugars: Rhamnose Fucose Arabinose

% n/w 71.005 13.4 0.5137 0 1177 6 258 0.3685 1 023 3.3231 1 7941 7.87 11

Xylose Mannose Galactose Glucose Protein by Lowry Ash

3 4

mol % 81.28 0.72 0.17 9.59 0.56 1.28

4.16 2.24

The investigated polysaccharide was further applied to gel chromatography. Three fractions were elued from a Sephadex G100 column (Table 3). The uronic acid content and the protein content were different for the three fractions. The uronic content was especially high in fraction I (4.5 times higher than both the level of neutral sugars and the level of protein). Fraction II had a higher protein content, and the ratio of the D-galacturonic acid to the neutral sugars was (1.1:1). In fraction III, whose molecular weight was the lowest, the content of neutral sugars was 4 times higher than that of D-galacturonic acid. Table 3 Composition of the fractions, obtained by gel chromatography of Fraction I on Sephadex G100 Eluate composition Composition of the fractions

Fractions

1

2 3

Total D-galactuvolume/eluate, ronicacid, ml mg

100 110 80

12,5 1,2 0,3

neutral sugars, mg D-glucose

2,8 1,1 1,1

protein, D-galactumg ronic acid %

2,8 0,7 -

69,1 40,0 21,4

neutral sugars %

protein content, %

15,5 36,7 78,6

15,5 23,3 -

Immunological tests indicated that fraction 1, obtaned by gel chromatography had an immunostimulating activity. It induced migration of peritoneal-exudative cells, respectively peritoneal macrophages into the peritoneal cavity of experimental animals. These cells are with high bactericidic metabolitic activity. The selective stimulation of these cells is of importance because they are the most active effector cells in host defense mechanisms against bacterial and viral infections.

685 The results suggest that polysaccharide fraction I may be thought as unspecific modulators of immune responsiveness.

Discussion

Immunoactive polysaccharides, containing galacturonic acid have been isolated from different plants and cell suspension culture liquids (2,4,19-22). Generalising the research into polysaccharides isolated from chinese herbs Yamada (22)concludes that most biological activities are observed in the case of pectic polysaccharides. Besides, results prove that the complementarily active power of these pectins is represented mainly through the branched regions and the activity is regulated by the polygalacturonic regions. Our studies showed that the exopolysaccharides from the H.annuus 1805 (Asteraceae) cell suspension culture were also of pectic type because the content of D-galacturonic acid varied from 22 to 69-70 %. Part of the crude polysaccharide (Sample 2, Table 1) was waterinsoluble, and the soluble part allowed the GelC isolation of three heteropolysaccharides. Polysaccharide fraction 1 had the highest molecular mass and the highest galacturonic acid content (69.1%). This fraction contained 15.5 % neutral sugars. It is worth noting that this polysaccharide of pectic type was different from the pectins isolated from sunflower plants (18) in that it contained a considerable amount of proteins. Consequently, this fraction is to be classified as a glycoprotein; it also has the highest biological activity compared to the other two fractions. Polysaccharide fraction 3 also manifested biological activity. It contained the highest percentage of neutral sugars (78.6%): mainly L-arabinose and D-galactose. Most probably, this is a polysaccharide of pectinoarabinogalactan type.

ACKNOWLEDGEMENT

The autors thank the National Research Foundation of Bulgaria for the financial support of this work. The valuable contribution of Prof. Dr. A. Voragen and his staff (Agricultural University, Wageningen, The Netherlands) on the Chromatographic analysis is gratefully acknowledged. Abbreviations: PUAC, polyuronic acid content; DE, concentration; I"1,specific viscosity; MM, molecular mass

degree of esterification; C,

686 References

1.Wagner H., Stuppner H., Schafer W and Zenk M. Phytochemistry 27: (1988) 119 2.Puhlmann J., Wagner H. Planta medica 55:(1989) 99 3.Uchiyama T., Numata M., Tereda S., Hosino T. Plant cell Tissue and Organ Culture 32: (1993) 153 4.Skvastava R., Kulshreshtha D. Phytochemistry 28: (1989) 2877 5.Labadie R. Immunomodulatory compounds Chapter in Bioactive Natural Products CRC Press London: 280 (1993) 6.Proksch A., Wanger H. Phytochemistry 26:(1987) 1989 7.Dixon K. Isolation of callus and suspension cultures in Plant cell culture a practical approach IRL Press (1985) 8.Linsmayer E., Skoog F. Physiol Plant. 18: (1965) 100 9.Bitter T., Murr H. Anal. Biochem. 4: (1962) 330 10.Owens H., Cready R., Shepheral A., Shultz T., Pippen E., Swenson N., Miers J., Erlander F., Maclay W AIC Report 340, Western Regional Research Laboratory, Albany, CA (1952) 11 .Moravettz H. Interscience Publishers, New York Macromolecules in solution: 254 (1967) 12.Anger H., Berth G. Carbohyrate Polymers 6:(1986) 193 13.Albersheim P., Nevins D., English P., Karr A. Carbohydr. 5:(1967) 340 14.Brakeney A., Harris P., Henry R., Stone B. Carbohydr. Res. 113: (1983) 291 15.Thiboult J. Lebensm. Wiss Technol. 12: (1979) 247 16.Briskorn C. Lebensm. Unters- Forsch. 108: (1958) 170 17.Kratchanov Hr. Helia 5:(1982) 49 18.Miyamoto A., Chang K J. Food Science 57: (1992) 1439 19.Kiyohara H., Cyong J., Yamada H Carbohydr. Res. 193: (1989) 201 20.Yamada H., Ra S., Kiyahara H., Cyahg C., Yang C., Otsuka Y. Phytochemistry 27: (1988) 3163 21.Yamada H., Ra S., Kiyahara H., Cyahg C., Otsuka Y. Carbohydr. Res. 189: (1988)209 22.Yamada H., Carbohydr. Polymers 25: (1994)269


687

Pectins and pectinases in stem rust-infected wheat M. Mierau a, B. Grael3ner a, A. J. Mort b and B. M. Moerschbacher c Institut for Biologie III, RWTH Aachen, Worringer Weg 1, D-52056 Aachen, Germany

a

b Department of Biochemistry and Molecular Biology, OSU Stillwater, 246B Noble Research Center, OK, USA c current address" Institut for Biochemie und Biotechnologie der Pflanzen, WWU MOnster, Hindenburgplatz 55, D-48143 MOnster, Germany

Abstract The dimer and trimer of galacturonic acid are able to suppress the hypersensitive defence reaction of wheat against the stem rust fungus. Our results suggest that the fungus produces an endopolygalacturonase during host tissue colonization and that thus, depending on the methylation patterns of the cell wall pectins which appear to be different in susceptible and resistant wheat plants, the suppressor active oligogalacturonides may or may not be formed. The rate of suppressor production during host cell wall penetration and the amount of suppressor accumulating around the growing fungal haustorium might then decide on compatibility or incompatibility of the host/pathogen-interaction.

1. I N T R O D U C T I O N The interaction between wheat plants (Triticum aestivum) and a biotrophic pathogen of wheat, the wheat stem rust fungus (Puccinia graminis f. sp. tritici), can range from full susceptibility to complete resistance. In highly resistant host plants, fungal growth is arrested by the rapid and active death of all host cells penetrated by a fungal haustorium, thus depriving the obligately biotropic parasite of its nutritional basis. The suicide of a few host cells only efficiently protects the plant from fungal colonization and the development of disease symptoms. The triggering of the hypersensitive response of penetrated host cells appears to involve a range of different molecular interactions. The defence reaction can be elicited by a range of fungal surface constituents, such as a glycoproteogalactan isolated from rust germ tube cell walls [Moerschbacher et al. 1986] or chitin-oligomers and chitosanpolymers [Vander & Moerschbacher, 1993]. Interestingly, these different elicitors

688 appear to induce differem aspects of the resistance reaction, and only their timely interplay may effectively trigger cell death. We assume that the host cell takes a well balanced decision based o n the recognition of several indicators of the presence of a pathogen before undergoing hypersensitive suicide. All of the isolated elicitors investigated so far were able to induce resistance reactions in both resistant and susceptible host plants. It has been speculated that the specific difference between resistance and susceptibility may be caused by the action of suppressor molecules in the susceptible plants only [Bushnell & Rowell, 1981]. We have shown that oligomers of galacturonic acid can suppress elicitor-induced resistance reactions [Moerschbacher et al., 1990]. The most active molecules turned out to be the dimer and trimer of galacturonic acid, known to be the main degradation products of pectins hydrolyzed by endopolygalacturonases [Collmer & Keen, 1986]. Our hypothesis is that the wheat stem rust fungus, like many other fungi [Cooper, 1983] including rusts [Deising et al., 1995], produces such an endopolygalacturonase to degrade the wheat cell wall locally when forming haustoria, thus producing these oligosaccharins which we call endogenous suppressors [Moerschbacher et al., 1990]. Though the pectins of grasses, in comrast to those of dicots, account for only around 5 % of their cell wall dry weight [Carpita & Gibeaut, 1993], a role of pectic fragments acting as oligosaccharins can still easily be imagined since any intercellularly growing fungus, upon penetration of a host cell wall, will invariably first meet the pectin-rich middle lamella which it will have to degrade. In this paper we present some evidence for the above proposed role of pectic fragments and of an endopolygalacturonase in the rust-infected wheat leaves. Firstly, we have chemically analyzed and compared the methylation patterns of the pectins isolated from cell walls of resistant and susceptible wheat plants. We have secondly chosen a molecular genetic approach in the search for an endopolygalacturonase of the stem rust fungus. In addition, we will summarize the effects of some pectic samples on the triggering of the hypersensitive response of wheat to the stem rust fungus or elicitors isolated from that pathogen.

2. MATERIALS AND METHODS 2.1 Biotests To test for suppressor activity, solutions of pectic substances in a concentration of usually 1 mg/ml were injected together with the glycoproteogalactan-elicitor isolated from germ tubes of the rust fungus (concentration 40 ~tg/ml glucose-equivalents) into the intercellular spaces of primary leaves of 7-day-old wheat plants, using a hypodermic syringe [Moerschbacher et al., 1989]. Control leaves were injected either with the

689 elicitor alone (positive control) or with distilled water (negative control). Further controls involved the injection of pectic fractions alone, to exclude possible elicitor activities. The leaves were harvested 24 h after injection, immediately frozen in liquid nitrogen. For spectrophotometric determinations of phenylalanine ammonium lyase (PAL) and peroxidases (POD), crude enzyme extracts were prepared [Moerschbacher et al., 1988], and the protein concentration of the extracts was estimated with the Biuret reagem [Gornall et al., 1949]. In additional tests, the suppressor activities of pectins in the intact host/pathogeninteraction were investigated by injecting genetically resistant plants with pectic substances prior to inoculation with the rust fungus. Infected leaves were harvested, cleared, and stained with Calcofluor one week after inoculation, and fungal growth was assessed under the UV-epifluorescence microscope.

2.2 Pectic samples Oligomers of galacturonic acid were produced chemically by autolyzing polygalacturonic acid in an autoclave for 20 min at 121~ followed by separation and fractionation using anion exchange chromatography [Robertsen, 1986]. Pectins were extracted from isolated cell walls of 5-week-old wheat plants using different methods. Enzymic digestions of the cell walls involved pectinases such as a commercial pectolayse or recombinant endopolygalacturonase [Maness & Mort, 1989]. Chemical extractions involved the chelating agent imidazole [Mort et al., 1991] or solvolysis with anhydrous HF at 0 ~ in a closed teflon line [Mort et al., 1989] followed by imidazole extraction.

2.3 Anion exchange chromatography Oligalacturonides were separated on a Dionex Bio LC with a CarboPac PA 1 column and pulsed amp~rometric detection using a linear Na-acetate-gradient in 0.1 M NaOH.

2.4 Analysis of methylation patterns [Mort et al., 1993] Pectins were isolated from cell walls of susceptible (Prelude-srx) and resistant nearisogenic wheat plants (Prelude-Sr5) by solvolysis in anhydrous HF at -23 ~ and subsequent imidazole extraction. The methyl-esterified galacturonic acid residues were quantitatively reduced to galactose by treatment with sodium borohydride [Maness et al., 1990]. The galactosyl bonds were then selectively cleaved by HF-solvolysis ( 1 % distilled water) at -15 ~ The oligomers formed were labelled by coupling of 2aminopyridine groups to their reducing ends [Maness & Mort, 1989], separated by anion exchange chromatography using a potassium oxalate gradient, and quantitated by fluorescence detection [Maness et al., 1991].

690 2.5 DNA-isolation and PCR DNA was isolated [Schillberg, 1994] from axenically grown mycelium of the wheat stem rust fungus [Fasters et al., 1993] and used as a template in PCR with degenerate primers, designed from highly conserved regions of known endopolygalacturonase genes from other fungi. PCR conditions were 55 ~ annealing temperature, 2.5 mM MgC12-concentration, and 40 cycles. The primers were kindly provided by H. Kusserow and W. Sch/ifer (AMP III, Hamburg, Germany).

3. R E S U L T S A N D D I S C U S S I O N Solutions of different pectic substances were injected into healthy wheat plants, with or without the glycoproteogalactan elicitor, and the activities of the enzymes PAL and POD were determined. These enzymes are involved in the hypersensitive reaction of wheat against the rust fungus, and increased activities can be expected after elicitation, whereas suppressor active substances will cause a reduction of the elicitor-induced enzyme activities. Oligomers of galacturonic acid with degrees of polymerisation ranging from 1-6 were produced chemically and tested for elicitor or suppressor activity. Injected alone, none of them were active as elicitors of PAL or POD activities, and only the dimer and trimer were active as a suppressor when co-injected with the elicitor. Using enzymic and chemical methods for the isolation of pectins from wheat cell walls, we produced fractions with different contents of galacturonic acid. We found that the higher the galA content in these fractions, the higher was their suppressor activity. This points to a possible role of the cell wall pectins, particularly the homogalacturonan, in the suppression of hypersensitive resistance in the wheat/stem rust-system. The suppressor activities of pectins isolated from wheat cell walls by HF-solvolysis and subsequent imidazole extraction increased from about 35 % to 74 % reduction of elicitor-induced PAL and POD activities, when the pectins were pretreated with a bacterial endopolygalacturonase. The production of small oligogalacturonides during this digestion was monitored by means of HPLC analysis. The suppressor active trimer of galacturonic acid was formed predominantly. This experiment further supports the influence of the length of the galacturonan oligomers on their suppressor activity towards the defence reaction. In additional tests, the suppressor activities of pectins in the intact host/pathogeninteraction were demonstrated. Injection of suppressors rendered genetically highly resistant plants more susceptible, i. e., we observed increased growth of the fungus, and in some cases sporulation occured. Obviously, oligogalacturonides do not only have an

691 effect on isolated elicitors in an in vitro-bioassay, but they can in fact play an important role in vivo in the intact wheat/rust-system. Analyzing the methylation patterns of wheat pectins, we found roughly equal degrees of methylation in resistant and susceptible plants (15 % and 19 %, resp.), but there were clearly different distributions of the methyl-esterified galacturonic acid residues along the linear pectin molecules. In the pectins of resistant plants, we found a presumed random distribution of methyl-esterifications, whereas in susceptible plants, these methylations appeared to be grouped blockwise. Consequently, an endopolygalacturonase secreted by the rust fungus would meet longer sections of nonmethyl-esterified galacturonic acid residues during the cell wall penetration in a susceptible plant than in a resistant plant. Accordingly, the enzyme might produce larger amounts of the suppressor-active molecules in susceptible plants, which in turn might be responsible for their susceptibility. Thus, the difference in the methylation patterns of cell wall pectins in susceptible and resistant wheat plants may result in a compatible or incompatible host/pathogen-interaction, respectively. It might be speculated that this difference may be involved in the expression of race/cultivarspecific resistance in the wheat/stem rust-interaction. In an attempt to investigate whether the stem rust fungus is in fact able to produce an endopolygalacturonase, we analyzed its DNA by PCR with degenerate primers designed from highly conserved regions of known endopolygalacturonase genes of other fungi. A ca. 650 bp long fragment was amplified. The size of this fragment tends to indicate that the fragment might in fact be part of an endopolygalacturonase gene, as a 630 bp fragment was amplified with these primers from PeniciUium olsonii DNA [Kusserow & Sch/ifer, personal communication]. Cloning and sequencing of this fragment are currently in progress. The results obtained so far suggest that the wheat stem rust fungus does possess a gene for an endopolygalacturonase. If the enzyme is synthesized during the infection of wheat plants, the production of suppressor-active oligogalacturonides will depend on the methyl-esterification of the host pectins, and will thus differ in resistant and susceptible plants. The amount of suppressor produced might then decide on compatibility or incompatibility of the host/pathogen-interaction.

692

5. Acknowledgements We like to thank Dr. H. Kusserow and Dr. W. Sch/ifer for providing the PCR-primers and Dr. M. Zimmermann for help with the molecular genetic experiments. Financial support from the German Research Council DFG, from the German Academic Exchange Service DAAD, and from the Land Nordrhein-Westfalen is gratefully acknowledged.

6. References Bushnell WR & Rowell JB (1981). Phytopathology 71:1012-1014 Carpita NC & Gibeaut DM (1993). Plant J 3" 1-30 Collmer A & Keen NT (1986). Annu Rev Phytopathol 24:383-409 Cooper RM (1983). in" Biochemical Plant Pathology, Callow JA ed; Wiley, New York: 101-135 Deising H, Frittrang AK, Kunz S, Mendgen K (1995) Microbiology 141"561-571 Fasters MK, Daniels U, Moerschbacher BM (1993). Physiol Molec Plant Pathol 42: 259-265 Gornall AG, Bardawill CJ, David MM (1949). J Biol Chem 177:751-766 Maness NO, Miranda ET, Mort AJ (1991). J Chromatogr 587" 177-183 Manees NO & Mort AJ (1989). Anal Biochem 178:248-254 Maness NO, Ryan JD, Mort AJ (1990). Anal Biochem 185:346-352 Moerschbacher BM, Flott BE, Noll U, Reisener HJ (1989). Plant Physiol Biochem 27: 305-314 Moerschbacher BM, Heck B, Kogel KH, Noll U, Reisener HJ (1986). Z Naturforsch 41c: 830-838 Moerschbacher BM, Noll UM, Flott BE, Reisener HJ (1988). Physiol Molec Plant Pathol 33:33-46 Moerschbacher BM, Schrenk F, Grael~ner B, Noll U, Reisener HJ (1990). J Plant Physiol 136:761-764 Mort AJ, Komalavilas P, Rorrer GL, Lamport DTA (1989). in: Modern Methods of Plant Analysis Vol 10 Plant fibers, Linskens HF, Jackson JF eds; Springer, Berlin Heidelberg New York: 37-69 Mort AJ, Moerschbacher BM, Pierce ML, Maness NO (1991). Carbohydr Res 215: 219-227 Mort AJ, Qui F, Maness NO (1993). Carbohydr Res 247:21-35 Robertsen, B (1986). Physiol Molec Plant Pathol 28:137-148 Schillberg S (1994). Ph-D-thesis, RWTH Aachen, Germany Vander P & Moerschbacher BM (1993). in: Chitin Enzymology, Muzzarelli RAA ed; EUCHIS, Ancona: 437-440

J. Visserand A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996ElsevierScienceB.V.All rights reserved.

693

Bioactive fragments from pea pectin O. Zabotina a, N. Ibragimova a, D. Ayupova a, O. Gurjanov a, V. Lozovaya a, G. Beldman b, A. Voragen b aInstitute of Biology, P.O.Box 30, 420503 Kazan, Russia bWageningen Agricultural University, The Netherlands.

Abstract Several bioactive fractions from pea stem cell wall pectin have been separated. The fractions contained mainly galacturonides inhibited the process of root formation in thin cell-layer explants, while the fractions contained only neutral sugars stimulated this process to different extend. Analysis of the last fractions showed that they mainly consisted of galactan and arabinogalactan fragments. INTRODUCTION The plant cell wall is a source of polysaccharide fragments named oligosaccharins which show several kinds of biological activity [1]. These activities have been demonstrated by some pectic and hemicellulose oligofragments produced by various means. Pectins are the major polysaccharides of the primary cell wall (especially in dicots) and of the middle lamella. The main structural components of the primary cell wall pectin of higher plants are homogalacturonan, composed of 1,4-1inked D-galactosyluronic acid residues and highly branched rhamnogalacturonans I and II, composed of DGalactose, L-Arabinose, L-Rhamnose, L-Fucose, Xylose, Apios, KDO, etc. Pectic oligosaccharides were the first oligosaccharins of higher plants to be detected [2]. Partial digestion of homogalacturonan generates oligogalacturonides that exhibit different regulatory effects in plants such as the elicitation of defense responses [2], the regulation of growth and development [3], the induction of rapid responses at the cell surface [4]. These oligomers can act as a trigger for hypersensitive responses [5] and as wound signals [6], can inhibit protein synthesis [7], induce lignification [8] and ethylene synthesis [9]. To date only homooligogalacturonides from plant pectin have been shown to exhibit the regulatory effects. Meanwhile pectin polysaccharides consist of neutral highly branched blocks, so it was of interest to search for possible biological activity of oligomers released from such structures.

694 This paper reports on the separation of some fragments obtained by acid hydrolyses of pectin from pea shoot cell walls, which had effect on thin cell-layer explant rhizogenesis. M A T E R I A L AND M E T H O D S

Plant material. Oligosaccharide fractions were isolated from 10 - 12 dayold pea shoots (Pisum sativum L.), cv. Bulat, grown at 25 C in light (12-h light period, 20 W/m 2 ). Plant material fractionation. The detailed steps of isolation and separation of oligosaccharide fractions were described earlier [10]. Pectin was separated by boiling the cell walls in 0.5 M ammonium-oxalate buffer, pH 5.2 at 100 o C for lh. The dialyzed solution of pectin was hydrolyzed with 0.15M HC1 for 3h at 100 C. Neutralized and desalted hydrolysate was loaded to the column (lx90cm) filled with biogel TSK HW-40 (Toyo Soda, Japan) equilibrated and eluted with 50mM sodium acetate (pH 5.2) at a rate of 0.3ml]min. In all fractions (lml) the sugars were determined by o-toluidine method (Resnikov et al., 1982) and fraction IP was collected as shown on Figure 1.

200

1P

-

-

I

I

100

0

0

10

20

.30

40

50

60

Fraction number

Figure 1. Size-exclusion chromatography on TSK HW-40 of pectin hydrolysate from pea shoot cell wall. The fractions (1 ml each) were pooled as indicated. o - Uronic acids, A- pentoses, x - hexoses.

695 This fraction was desalted and separated on a column (1X8cm) with DEAEcellulose (Serva, Germany) in 10raM phosphate buffer, pH 7.0. The separation was conducted using NaC1 gradient (0- 0.5M) at a rate of elution 0.3 ml/min, and 2 ml fractions were collected. In each fraction, the content of sugars was asseied by o-toluidine method. The fraction IPN was collected (see Figure 2), desalted and than separated on the column (2.5xl00cm) filled with Bio-Gel P-4 (Bio-Rad, USA).

300 1

1PN ,

1PK1 I

1PK2 I

1PK3 I

I

1PK4

~-0.6

I

0.5 "~ 200

0.4

o =

z

03

~} 1 0 0

F, i,

0.2

~

-0.1 O

i~i . . . . . .

0

~T--~;--T-~;TT i

10

u ; I;

20

"u" ~,-7 i

....

I i I i" i ' } ~ - i

30 Fraction number

i I i ~ i "31~. . . .

40

1 ~. . . . . . .

50

~ill~qlWiliU 0 . 0

60

Figure 2. Anion-exchange chromatography on DEAE Cellulose of fraction IP obtained after size-exclusion separation. Fractions (2 ml each) were pooled as indicated, o - Uronic acids, A- pentoses, x - hexoses.

Elution was conducted at 60 C with distilled water at a rate of 0.3ml/min and the fractions (2.5ml) were combined as shown on Figure 3. Analysis of pectic fractions. Qualitative analyses of pectic fractions obtained after separation on Bio-Gel P-4 was performed by HPAEC using a BioLC GPM-II q u a t e r n a r y gradient module equipped with a Dionex CarboPac PAl00 column (250x4mm, 20 C) (Dionex, Sunnyvale, CA). The eluate (lml/min) was monitored using a Dionex pulsed electrochemical detection detector in the PAD mode. The fractions studied and standard digests of arabinogalactan were analyzed by application of the following gradient: 0 to 30 min, linear gradient of 0 to 0.2M sodium acetate in 0.1M NaOH; 30 to 40 min, linear gradient of 0.2 to 0.5M NaOAc in 0.1M NaOH; 40 to 45 min, linear gradient of 0.5 to 1M NaOAc in 0.1M NaOH. After each analyses the column was rinsed for 5 rain with 1M NaOAc in 0.1M NaOH and equilibrated in 0.1M NaOH for 15min.

696

Sugar composition. Desalted fractions (IPN1-IPN14) were hydrolyzed using 2N TFA for 1.5h at 121 ~ C. The released neutral sugars were converted to their alditol acetates and analysed by GC as described [12]. 200

I !

II1~i 2

160~o

,.~

120-

.o ~

;/

3

4

I I

5

I

6

I

7

I

8

9 10 11 12

I II

13

I I

I

I

14

1

i/

vo I / 80-

0

o

40-

i

r~

,,

I

29

glucose

\ I

58

I

87

I

116

145

Fraction number

Figure 3. Size-exclusion chromatography on Biogel P4 of fraction IPN obtained after anion-exchange chromatography. Fractions (2.5 ml each) were pooled as i n d i c a t e d . - - neutral sugars, ---- uronic acid. Fractions 1, 2, 3 ... were named later as fractions IPN 1, IPN 2, IPN 3 ... r~spectively.

Enzymic hydrolysis. 2rag of each oligosaccharide fraction were incubated at 35 o C for 24h in lml 5raM sodium acetate buffer (pH 4.0) containing 0.01% (w/v) Na azide and 0.03 U/ml of endo-[3-galactanase type F (EC 3.2.1.89) [13] or a-Larabinofuranosidase type B (EC 3.2.1.55 ) [14]. Test for biological activity. The thin cell-layer explants approximately 5 mm long and 2-5 mm wide were cut from hypocotyls of buckwheat seedlings grown aseptically and cultured individually in 2 ml liquid RX medium in the absence of phytohormones as described earlier [15]. Oligosaccharide fractions were added to the medium at a concentration of 0.1 ~g/ml. Throughout the cultivation, the number of explants with roots was counted. After 20 days of cultivation, the number and weight of roots in each explant were asseied.

697

RESULTS AND DISCUSSION After multistep fractionation of cell wall pectin hydrolysate several bioactive fractions were obtained. They exhibited various influence on process of root development in buckwheat thin cell-layer (BTCL) explants (Table 1).

Table 1. Effect of oligosaccharide fractions on root formation in BTCL explants after 20 day culture period. Root fresh weight Number of Day when 50% of /explant, (mg) roots/explant explant form roots Control + lpg/ml IPN + l~g/ml IPA1 +l~g/ml IPA2 +l~g/ml IPA3 +lp~/ml IPA4

25_+6 45• 20• 11• 22• 18•

7+1 14• 5• 2• 4• 3•

9 6 15 19 16 18

Control +0. lpg/ml IPN1 +0. lpg/ml IPN2 +0. l~g/ml IPN3 +0. l~g/ml IPN4 +0.1pg/ml IPN5 +0.1~g/ml IPN6 +0. l~g/ml IPN7 +0. lpg/ml IPN8 +0. lpg/ml IPN9 +0. lpg/ml IPN10 +0. l~g/ml IPN11 +0. l~g/ml IPN12 +0. l~g/ml IPN13 +0.1p~/ml IPN14

20• 19• 17+3 21+3 35• 38+8 75+33 70• 61+14 61+15 58+9 66+20 59+25 59+10 25+10

5+1 6+1 4+1 5+1 7+2 7+1 16+3 15+3 14• 14• 15+2 15+4 13+3 10• 8•

9 10 11 11 7 7 6 6 6 6 6 6 7 7 10

Among them fractions IPA1, IPA2, IPA3, IPA4 contained mostly galacturonic acid (Table 2) and apparently consisted of oligogalacturonides. These fractions inhibited root development in BTCL explants, the same effect of such fragments has been observed in tobacco TCL explants [16]. Addition of other fractions IPN4-IPN14 to the culture medium resulted in activation of root development in BTCL explants to different extent (Table 1).

698 More r a p i d induction of roots on t h e e x p l a n t s in c o m p a r i s o n w i t h t h e control w a s observed. Oligosaccharides of t h e s e fractions i n d u c e d root f o r m a t i o n on t h e e x p l a n t s in n u m b e r s g r e a t e r t h a n in control v a r i a n t s , a n d the n u m b e r of roots f o r m e d on each e x p l a n t of such v a r i a n t s were h i g h e r t h a n on control. At the s a m e time t h e s e fractions p r o m o t e d g r o w t h of roots, w h i c h r e s u l t e d in h i g h e r for different e x t e n t dry w e i g h of t h e roots c o m p a r i n g w i t h the control. T h e y s t i m u l a t e d root i n i t i a t i o n as well (decreasing the time before t h e 50% of e x p l a n t s will form the roots). All t h e s e fractions c o n t a i n e d only n e u t r a l r e s i d u e s (Table 2).

Table 2. M o n o s a c c h a r i d e composition of bioactive fractions. Fucose R h a m n A r a b i n Galactose (mol%) ose ose (mol%) (mol%) (mol%) IPN IPA1 IPA2 IPA3 IPA4 IPN1 IPN2 IPN3 IPN4 IPN5 IPN6 IPN7 IPN8 IPN9 IPN10 IPNll IPN12 IPN13 IPN14

0.5 1.1 0.6 1.5 -

-

2.0 5.0 2.0 2.4 1.8 13.2 7.8 5.9 4.1 4.4 4.7 9.6 -

7.0 4.5 2.5 2.0 1.6 0.9 0.4 1.5 2.6 1.8 10.9 3.3 3.1 2.5 1.2 3.4 0.6 -

60.5 1.5 2.2 1.5 2.0 47.1 19.3 61.8 85.8 91.5 76.3 92.6 93.4 96.2 85.2 92.7 89.9 84.7 94.7

Glucose (mol%)

Xylose (mol%)

Uronic acids (mol%)

4.5 4.0 0.8 1.5 1.4 6.9 4.0 9.2 5.1 1.5 4.1 2.5 2.0 0.8 1.8 2.2 4.1 6.8 5.3

1.0 1.0 2.3 3.2 6.3 2.4 0.7 4.0 1.6 1.4 0.5 2.2 1.7 5.4 8.5 -

24.0 84.0 92.5 92.6 93.2 28.5 65.3 13.8

A n o t h e r fractions I P N I - I P N 3 did not exhibit a n y s t r o n g activity in t h e b i o a s s a y t e s t e d (Table 1). T h e s e fractions consisted both n e u t r a l a n d acidic r e s i d u e s (Table 2), so it m i g h t be t h a t the p r e s e n c e of oligomers consisted g a l a c t u r o n i c acid w i t h opposite effect on root f o r m a t i o n w a s the r e a s o n of inactivity of fractions I P N I - I P N 3 . F u r t h e r s e p a r a t i o n a n d i n v e s t i g a t i o n of t h e s e fractions will be done in the future. Thus, the hydrolysis of pectic p o l y s a c c h a r i d e s r e v e a l e d t h e f r a g m e n t s w i t h different effect on T C L e x p l a n t root formation. The n e u t r a l oligomers s t i m u l a t e d

699 this process were the subject of the next investigations. The fractions IPN4IPN14 were analyzed by HPAEC (Figure 4) and retention times of the products were compared with those of standard digests obtained after degradation of soybean arabinogalactan, apple xyloglucan and rhamnogalacturonan (data not shown). It seemed that elution patterns of all fractions represented the pools of the similar oligomers with decreasing molecular sizes. Monosaccharide analysis showed (Table 2), that all these fractions contained mainly galactose and some arabinose.

!

,

1 3

"

-

]:PN

12

]:PN

11

]:PI~T 1 0

i, :

L I

IPN~

i

I

0,00

,

5,00

,

10,00

,

15,00 Retention

,

,

20,00 time

25,00

,

30,00

35,00

(mizz)

Figure 4. Elution profile on HPAEC of pea shoot pectin fractions obtained after Biogel P4 separation.

The main peaks on their HPAEC elution profiles were corresponded to the peaks of unsubstituted galactan oligomers and some peaks to galactan oligomers

700 containing side chains of arabinose (data not shown). During the acid hydrolysis used, most of the arabinoses were cleaved from arabinogalactan fragments, so the result mixture could contain mostly different galactan oligomers and some arabinogalactan oligomers with the lower amount of side arabinoses. Incubation of fractions IPN4-IPN14 with galactanase resulted in disappearance of the main peaks corresponding to the peaks of the arabinogalactan digest. Meanwhile some new peaks with lower molecular sizes appeared and peaks of mono- and dimers increased (Figure 5: a,b). The elution profiles of HPAEC of the most active fraction (PN6) is shown on Figure 5 as the example. Very similar results have been obtained for all fractions analysed after their incubation with hydrolases. Some of the new products obtained after galactanase treatment disappeared after subsequent digestion of fractions by arabinofuranosidase (Figure 5: c).

rJl

o

0,00

5,00

10,00

15,00

20,00

25,00

30,00

35,00

Retention time (min) Fig.5 Elution profile on HPAEC of fraction IPN 6 before (a) and after treatment with galactonase (b) and arabinofuranosidase (c).

Results reported testify the possibility of galactan or arabinogalactan oligomers to be that active fragments which stimulate root formation on BTCL explants. The detailed structural analysis of the individual active oligosaccharide is currently in progress, meanwhile the data presented revealed new type of bioactive oligomers with new regulatory effect in plant tissues. Acknowledgment: This research was supported in part by NWO (The Netherlands, project N299.780 ) and ISF (USA, grant RH8000). We wish to thank Dr.Henk Schols for helpful discussion of HPAEC results.

701 REFERENCES

1.

2. 3. 4. 5. 6. 7. ~

9. 10.

11. 12. 13. 14. 15. 16.

P. Albersheim, A.G. Darvill, M. McNeil, B.S. Valent, J.K. Sharp, E.A. Nothnagel, K.R. Davis, N. Yamazaki. In "Structure and Function of Plant Genomes", O. Ciferri and L. Dure (eds), Plenum, New York 1983, 293312. M.G.Hahn, A.G. Darvill, P. Albersheim, Plant Physiol, 68 (1981) 11611169. S. Eberhard, N. Doubrava, V. Marfa, D. Mohnen, A. Southwick, A.G. Darvill, P. Albersheim, Plant Cell, 1 (1989) 747-755. C.A. Ryan, E.E. Farmer, Annu. Rev.Plant Physiol.Plant Mol.Biol., 42 (1991) 651-674. S.Aldington, S.C.Fry Oligosaccharins. Adv.Bot.Res., 19 (1993), 1-101 D. Bowles, Current Biology, 1 (1991) 165-167. N. Yamazaki, S.C. Fry, A.G. Darvill, P. Albersheim, Plant Physiol., 72 (1983) 864-869. R.J. Bruce, C.A. West, Plant Physiol., 91 (1989) 889-897. D. Roby, A. Toppan, M.T. Esquerre-Tugaye, Plant Physiol., 81 (1985) 228233. O.A. Zabotina, O.P. Gurjyanov, R.G. Malikhov, D.A. Ayupova, G. Beldman, A.J.G. Voragen, V.V. Lozovaya, Russian J. of Plant Physiol., 42 (1995) 366371. V.M. Reznikov, T.G. Matusevich, T.S. Selivestrova, Khimiya Drevesiny, 7 (1982) 109 H.N. Englyst, J.H. Cummings, Analyst, 109 (1984) 937. J.W. Van de Vis, M.J.F. Searle-Van Leeuwen, H.A.Siliha, F.J.M. Kormelink, A.G.J. Voragen, Carbohydr. Polym., 16 (1991) 167. F.M. Rombouts, A.G.J. Voragen, M.J.F. Searle-Van Leeuwen, C.C.J.M. Geraeds, H.A. Schols, W. Pilnik, Carbohydr. Polym., 9 (1988) 25. V.V. Lozovaya, O . A . Zabotina, N.I. Rumyantseva, R.G. Malihov, M.V. Zihareva, Plant Cell Rep., 12 (1993), 530-533. D. Bellincampi D, G. Salvi, G. De Lorenzo, F. Cervone, V. Marfa, S. Eberhard, A.G. Darvill, P. Albersheim, Plant J., 4 (1993), 207.


IDENTIFICATION, MODE OF ACTION AND 3-D STRUCTURE OF PECTINASES



705

Stereo chemistry of hydrolysis of glycosidic linkage by three AspergiUus polygalacturonases Biely, P.*, Benen, J.A.E.**, Kester, H.C.M.**, Heinrichova, K.* and Visser, J.** * Institute of Chemistry, Slovak Academy of Sciences, 84238 Bratislava, Slovakia. ** Section Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural University, Dreyenlaan 2, 6703 HA Wageningen, The Netherlands.

Abstract The stereochemistry of the hydrolytic action of endopolygalacturonases I and II (PGI and PGII, respectively) from Aspergillus niger and of an exopolygalacturonase (PGX) from A. tubingensis was investigated by ~H-NMR spectroscopy by following the configuration of the reducing ends in the products formed in D20 reaction mixtures. It has been shown that all three polygalacturonases are inverting enzymes; the newly formed reducing ends all showed the B-configuration.

Introduction Enzymic hydrolysis of glycosidic linkages either proceeds via a single chemical step, the so-called single displacement mechanism, or via a two step chemical process, the so-called double displacement [ 1]. Enzymes functioning according the single displacement mechanism generate newly formed reducing ends which show inversion of the anomeric configuration and are therefore called 'inverting'. Hydrolases using the two-step chemical process always generate reducing ends which have exactly the same anomeric configuration as found in the glycosidic linkage. Such enzymes are called retaining. It has been suggested that endoglycanases are retaining enzymes while exoglycanases are inverting enzymes [2]. This generalization is not completely valid however. Several exoglycanases have been shown to be retaining enzymes [3]. There is one generalization that is st~ll valid. Retaining enzymes are capable to catalyse glycosyl transfer reaction at high substrate concentrations while inverting glycanases have not been reported to be able to do this. For pectinolytic enzymes no glycosyl transfer reaction have been reported except for Dgalacturonan digalacturonohydrolase (EC 3.2.1.82) from Selenomonas ruminantium [4]

706 suggesting that this enzyme is a retaining hydrolase. For other pectinases the stereochemistry of the hydrolytic activity is unknown. In this report the stereochemistry of the catalytic reaction of two endo- and one exopolygalacturonase from fungal origin are described.

Materials and Methods Endopolygalacturonases PGI and PGII isolated from a recombinant Aspergillus. niger and exopolygalacturonase (PGX) isolated from A. tubingensis are described elsewhere in this volume (see Benen et al. and Kester et al., respectively). For NMR spectroscopy the enzymes were lyophilized three times from D20. Reduced galacturonic acid and oligogalacturonides (GalU-ol, diGalU-ol, triGalU-ol and pentaGalU-ol) were prepared by NaBH4 reduction of the corresponding alduronic acids. For NMR experiments the compounds were lyophilized three times from D20. PentaGalU-ol was used as a substrate for PGI and PGII and triGalU-ol was used for PGX. Substrates were used at 20 mM final concentration in 0.5 ml 100 mM D20 buffer pD 4.5. The amount of enzyme used was such that the rate of hydrolysis was much higher than the rate of mutarotation. Time courses of the reaction mixtures were recorded on a Brucker AM 400 spectrometer at 25 ~ The assignments of relevant resonances was based on data published by Hricovini et al. [5].

Results and Discussion PGI PentaGalU-ol is primarily hydrolyzed to diGalU-ol and triGalUA, by PGI with subsequent hydrolysis of triGalUA to diGalUA and GalUA. This is reflected in the 1H-NMR spectra of the reaction mixture recorded after time intervals. The signals of pentaGalU-ol (Fig. 1, Table 1) were replaced by the signals of the 13-anomer of triGalUA (H-113, 4.64 ppm, J1,2 7.8 Hz) (Table 2). Due to the further hydrolysis of triGalUA at a rate exceeding mutarotation, the ot-anomer of the trimer (H-lo~, 5.35 ppm, J1,2 3.8) was barely detectable. Instead the 13anomer of the diGalUA and GalUA appeared quite strongly at the expense of the 13-anomer of the primary product triGalUA. As a result of mutarotation the H-lot signals of diGalUA and GalUA appear later. PGII The cleavage mode of pentaGalU-ol by PGII is essentially the same as found for PGI. Only the secondary hydrolysis reaction of the primary product triGalUA proceeds much more slowly. Spectra are not shown. The time course of the relevant resonances depicted in Fig. 2 demonstrates that the 13-anomer of the triGalUA is initially formed. Thus, like PGI, PGII is an inverting enzyme.

707

PGX PGX releases GalUA from the nonreducing end of oligogalacturonides. TriGalU-ol is the smallest reduced substrate for PGX (See Kester et al., elsewhere in this volume). Therefore triGalU-ol which is hydrolyzed into GalUA and diGalU-ol by PGX was used in this study. The GalUA released immediately after hydrolysis appears to have the configuration of the 13anomer (H-113 4.60 ppm, J1,2 7.9 Hz, Table I) as is a proof for the inverting character of PGX (Fig. 3). Again, the ot-anomer appears much later as a result of mutarotation.

Table 1. ~H-NMR data (400 Hz, D20) for the anomeric and C-1 protons in galacturonic acid (GalUA), diGalUA, triGalUA, reduced diGalUA (diGalU-ol), triGalU-ol and pentaGalU-ol. 8 ppm values are centred for doublets and are relative to the D20 resonance (4.80 ppm)

Compound

Chemical shift 8 ppm

J1,2(Hz)

Coupling constant

Proton

GalUA

5.31 4.60

3.8 7.9

H- 1ot H-113

diGalUA

5.35 4.65 5.13

3.8 7.8 3.8

H-lot H-113 H'-I

diGalU-ol

5.16

4.0

H'-I

triGalUA

5.35 4.65 5.14 5.09

3.8 7.8 3.7 3.8

H-lot H-113 H'-I H"-I

triGalU-ol

5.18 5.10

4.0 3.9

H'-I H"-I

pentaGalU-ol

5.17 5.12 5.11 4.87

4.0 4.3 4.3 3.8

H'-I H"-I H'"-I H .... -1

708

A

---o

-

g

i" m

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9

9

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9

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.

.

.

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"

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A '~ P i l m

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5.5

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Fig. 1. 1H-NMRspectra (400 MHz, D20 ) of pentaGalU-ol (A) and of its hydrolysis products with A. niger PGI for 3 rain (B), 11 min (C) and 3 h (D). The assignment of crucial signals (Several doublets and multiplet of H-213) is indicated.

709 Table 2. IH-NMRdata (400 Hz, D20) for the anomeric and C-1 protons in products formed in the initial stages of reaction by endopolygalacturonase I (PGI) from A. niger from reduced pentagalacturonic acid (pentaGalU-ol).

Enzyme

Chemical shift 5 ppm

Coupling constant Assignment J1,2(Hz)

PGI

4.64 5.09 5.14 5.15

7.8 3.9 4.2 4.9

>,, .,-, "~ t-

6

--

5

-

._~ o0 ._>9

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-

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9

O

t-

!

1 0

I

I

I

I

I

I

5

10

15

20

25

30

T i m e (min.)

Fig. 2. Changes in relative intensity of anomeric proton resonances during hydrolysis of pentaGalU-ol by A. niger PGII: H- 113of triGalUA, O; H- 1ot of trigalUA, O.

710

_.=

8

~> 4

z,

o 0

10

20

30

40

50

60

Time (min.)

Fig. 3. Changes in relative intensity of anomeric proton resonances during hydrolysis of triGalU-ol by A. tubingensis PGX: H- 113of GalUA, O; H- 1ot of GalUA, O.

Conclusions The lack of glycosyl transfer reaction is the class of pectinolytic hydrolases is in agreement with the observed inversion of the anomeric configuration of the newly formed reducing ends of the products. All three polygalacturonases studied here utilize the single displacement mechanism of hydrolysis.

Acknowledgement This work was partly funded by the European Community grant no. AIR2-CT-941345.

References 1) Koshland, D.E. Jr. (1953) Biol. Rev. 28 416-436. 2) Reese, E.T. (1977) in Recent Advances in Phytochemistry (Loewus, F.A. and Runeckles V.C. eds.) Plenum Publishing Corporation, New York, vol. 11.311-367. 3) Svensson, B. (1994) Plant Mol. Biol. 25, 141-157. 4) Heinrichov~i, K., Dzurovh, M. and Rexov~-Benkov~, L. (1994) Carbohydr. Res. 235, 269280. 5) Hricovini, M., Bystrick~,, S. and Malovikovh, A. (1991) Carbohydr. Res. 220, 23-31.


711

PECTIN METHYLTRANSFERASES FROM SUSPENSION-CULTURED CELLS AND SEEDLINGS OF FLAX (Linum usitatissimum L. ).

Bruyant P., Bruyant-Vannier M.P., Bourlard T., Gaudinet-Schaumann A., Thoiron B., and Morvan C. Universitd de Rouen, SCUEOR, URA 203, F-76 821 Mont Saint Aignan cedex. Tel: (33) 35 14 67 50, Fax: (33) 35 70 55 20. E-mail: [email protected].

Abstract

Pectin methyltransferases solubilized from endomembranes of flax cells (Linum usitatissimun) (E.C. 2.1.1.1.18) consisted of several polypeptides, the molecular relative mass of which varied from very low (Mr < 5000) to very high (Mr ca 200,000) values. Several isoforms were detected from acidic to very basic pH with two main forms, 1) a basic form (pI 8-8.5) and 2) a neutral form (pI 6.5-7.5). The activity of pectin methykransferase measured in vitro was optimal at pH 7 in the presence of high-methylated pectins and at pH 5.5 when low-methylated pectins had been added. Besides, the activity increased significantly throughout the seedlings growth, but differently according to the organs and pH. Key Words: Plant cell-wall; Pectin biosynthesis; Methylester; Golgi apparatus. I. INTRODUCTION The methylation of pectin is an important process which limits ionic interactions between charged polymers in the wall, and hence, it might be involved in the control of cell-wall behaviour and cell elongation. Goldberg et al [ 1], while investigating the growth gradient exhibited by the mung bean hypocotyl, observed that highly esterified pectins were located in all the wall area of young cells and in the primary cell-walls of mature cells. For a better comprehension of the methylation of pectins, it is necessary to get information on the enzymes involved in the process. The methylation of pectins has been demonstrated to occur in endomembrane systems [2]. In Golgi-enriched preparation of flax, enzymes have been shown to catalyze the transfer of methylgroup onto endogenous pectic polysaccharides [3,4]. Pectin methyltransferase complex was easily solubilized from the endomembranes of flax cells (Linum usitatissimum L.), when using Triton X-100 detergent [5]. In this paper, first we will consider some properties of the complex. Then, we will report on the variations of these activities over the culture duration.

712 2. MATERIAL AND METHODS 2.1 Plant material Flax seeds were placed for germination on moist paper for three days at 22~ and in the dark; then, the plantlets were transferred under continuous white light on a liquid culture medium, as previously described [6]. Suspension-cultured cells of flax were obtained from hypocotyl-derived calli as described by Schaumann et al. [4] and cultured on a medium described by Murashige and Skoog [7] containing kinetin (0.75 mg 1-1)and 2-4 D (0.2 mg l-l). 2.2. Microsome preparation Flax cells or cut organs were plasmolysed for 15 min in 50 mM Tris-HC1 (pH 7.5) containing 12% (w/w) sucrose, 1 mM ethylen glycol-bis-(b aminoethylether)N,N,N',N' tetra acetic acid (EGTA), 1 mM dithiothreitol (DTT) and 0.1 mM MgC12 (Buffer E). The cells were ground in a tenbroeck glass Potter homogenizer in a minimum volume; the homogenate was strained through nylon cloth (30 ktm) and the filtrate was centrifuged at 11,000 g and supematant at 200,000. The pellet, resuspended in 4 ml of the buffer E, constituted the crude microsomal fraction (CM). All these operations were carried out at 4~ in less than 2 h. 2.3. Pectin methyltransferase assays PMT assays were performed as described by Vannier et al. [3] by adding an equal volume of an enzyme preparation to a 0.1 M Tris-HC1 buffer containing 3.36 ~tM of [14C]SAM (1.8 GBq mmol~, 740 kBq ml-~, NEN), 1% (W/V) BSA and 12% sucrose, with or without 0.2% pectic acceptor. The incubation was run at 28~ for 12 h. After precipitation of the reaction product in 70% ethanol, the methylated polymers were selectively extracted with 0.5% ammonium oxalate and radioactivity was measured in a Tricarb 2250 CA Packard scintillation counter.

3. RESULTS AND DISCUSSION 3.1. Characterization of pectin methyltransferases of flax cells. Figure 1 indicates that pectin methyltransferase (PMT) activity from freeze-thawed microsomes measured without exogenous substrate was maximal at neutral pH (6.5 to 7.5). When exogenous pectic substrates of various DE had been added, similar optimal neutral pH was observed, and the activity was slightly stimulated (1.2 to 1.8 times). A second optimal pH occured at pH 5.5, but in the presence of low methylated pectin (DE: 0.1). As suggested by Lineweaver and Ballou [8] to explain the behaviour of another pectic enzyme -i.e. pectin methylesterase (PME), the mobility and the activity of PMT might be influenced by the presence of polyanionic substrates. On the other hand, the existence of several forms of pectin methyltransferase in flax microsomes might be responsible for such variations of the activity.

Bruyant-Vannier et al [5] have shown that pectin methyltransferases solubilized from endomembranes of flax cells consisted of several polypeptides, the molecular relative mass of which varied from very low (Mr _ 9. The amino acid composition of the purified AE is shown in table 1.

Table 1.

Amino acid composition of AE obtained after 24 hrs. hydrolysis. ND: not determined AE

Asp Thr Ser Glu Pro Gly Ala Cys Val Met Ile Leu Tyr Phe His Lys Arg Trp

43.8 20.9 29.3 27.2 18.3 33.8 36.5 16.0 24.0 9.9 16.4 26.3 12.1 23.1 11.3 21.5 14.6 ND

No. of residue MW (D)

385 42,000

Contrary to AE from orange peels described by Williamson (2) this AE has a higher molecular mass and pI > 9. AE isolated from mung bean, however, has similar MW and pI in agreement with the present findings (1). These differences could indicate the presence of several AE isoformes in different varieties of orange fruits.

727 The AE activity was strongly dependent of the pH. When measured with 1% sugar beet pectin and 80 mM triacetin an optimum was found at pH 5 - 5.5.

LU

AU 2.0

MW kD

O,.

~9 9 2 '~9 6 7 AE

1.5

30

1.0

21

14

0.5 0.0 ~ 0.0

~9 4 6

20.0

lie 40.0

60.0

Figure 1. Elution profile of PME and AE on the Mono S column.

min.

Figure 2. SDS-PAGE of acetyl esterase.

The affinity for sugar beet pectin was determined using a Lineweaver-Burk plot. The KM was calculated to be 26 mg/ml for sugar beet pectin whereas the KM for triacetin was 39 mM. This showed a very low affinity for sugar beet pectin and triacetin. Substrate specifity of purified AE is summarized in table 2.

Table 2. Substrate specificity of AE. In each experiment 16 #g enzyme was used. Substrate

Concentration

Sugar beet pectin Apple pectin Citrus pectin

10 mg/ml 10 mg/ml 10 mg/ml

Triacetin p-Nitrophenyl acetate ct-Naphthyl acetate

80 mM 2 mM 2 mM

Enzyme activity /~mol/hr/mg 2 0 0 816 472 0

728 AE hydrolyses acetyl groups from sugar beet pectin, triacetin and p-nitrophenyl acetate but no reaction was observed with et-naphthyl acetate. The specific activity of AE for sugar beet pectin is much lower compared to the activity with triacetin and p-nitrophenyl acetate. This could be due to the higher complexity of pectin. The enzyme showed no affinity with apple and lime pectin as substrates. In apple pectin the acetyl groups are mainly bound to galacturonic acid residues in the hairy region of pectin (7). It has to be further investigated whether AE can deacetylate isolated modified hairy regions. A rhamnogalacturonan acetyl esterase from A.aculeatus has been isolated which is specific for deacetylation of hairy regions of pectin, but this enzyme has no specificity for sugar beet pectin (8).

Immuno localization of acetyl esterase Immuno localizations of AE in sections of orange fruits are shown in Fig. 3. The most intensive depositions of acetyl esterase were found in the outermost parts of the peel (exocarp or outermost albedo and the flavedo) and in the segments (juice vesicles), although quite high levels of acetyl esterase were found in most other tissues as well. The acetyl esterase depositions were all intracellular. In the peel strong immunological depositions of acetyl esterase were found in epidermis, the small cells of the exocarp and in the oil cavities (Fig. 3 A,B,C). In the mesocarp and endocarp the immunological depositions were more moderate (Fig. 3 D), but strong immunological depositions were found in the vascular bundles, especially in xylem. The immunological depositions in the peel seem to be correlated with cell size or cell age. The small cytoplasma rich cells have a higher content of acetyl esterase. In the segments strong immunological deposition was found throughout the tissue. Again the results indicate a slight correlation of cell size and the amount of acetyl esterase. In the small cells in the periphery of the juice vesicles, acetyl esterase is clearly intracellular (Fig 3 D,E), whereas the acetyl esterase was found on the cell walls of the large inner juice cells. This

Figure 3" Immuno localization of acetyl esterase. Sections were incubated with antibodies raised against the acetyl esterase, followed by visualization with alkaline phosphatase conjugated secondary antibodies and staining with Fast Red. A: Overview of the acetyl esterase immuno localizations in the peel (40x) (Ex: exocarp, M: mesocarp, OC: oil cavity). B: Immuno localizations of acetyl esterase in the exocarp (Ex) and oil cavity (OC) (294x). The most intensive acetyl esterase depositions are found in the small sized exocarp cells and in the oil cavity. C: Immuno control with preimmune serum on the following section used in B (294x). D: Immuno localization of acetyl esterase in endocarp (En) and juice vesicle (JV) (94x). Acetyl esterase depositions in the juice vesicles are more intensive than those observed in the endocarp. No acetyl esterase was detected in the innermost cell layer of the endocarp (see arrows). E: Immuno localization of acetyl esterase in lamella (L) and juice vesicle (JV) (294x). Acetyl esterase depositions in the juice vesicles are more intensive than in lamella. Acetyl esterase was absent from the outermost cell layer of lamella (see arrows). F: Immuno localization of acetyl esterase in core, where intensive acetyl esterase deposition was found in the xylem (94x).

729

~

~

i

~.

~~i!~iii!!!~~,,~

~~,:~,~;~

.. ii!-'?q

~-

~-

~

~

~$~

- ~,-

-

_ .

730 is most likely a fixation artefact. Controls stained with PAS (Periodic Acid Schiff) and ABB (Aniline Blue Black) revealed that proteins form aggregates with the cell walls of the large inner juice cells (not shown). PAS (9) and ABB (10) stain carbohydrates red and proteins blue, respectively. In lamella and core the strongest immunological depositions were found in the vascular bundles (Fig. 3 F), whereas acetyl esterase was present in moderate amounts in all other cells. No acetyl esterase was found in the outermost parts of the tissues, cuticula of epidermis, innermost cell layer of endocarp, outer walls of juice vesicles and outer cell layer of lamella.

A high yield of acetyl esterase and the strong immunological reaction in situ indicate that AE playes an important role in the orange fruit. These findings are, however, in contrast to the low reactivity observed for AE when the substrate is acetylated pectin. This could be due to the steric hindrance exerted by the methyl groups linked to the galacturonic acid residues. For AE from mung bean (1) and orange peel (2) it has been found that demethylated sugar beet pectin increases the AE activity considerably. However, the distribution of AE in peel and lamella is consistent with the pectin distribution in orange fruits but significantly high levels of AE were found in the fruit segments. This could indicate that pectin deacetylation is not the main activity of AE and that a hitherto unidentified substrate is present in the orange fruit. To clarify this suggestion the principal activity of AE has to be found by testing the enzyme for different activities.

Acknowledgements The excellent assistance of Jytte Rasmussen, Bo Lindberg and Clive Phipps Walter is gratefully acknowledged.

REFERENCE (1) (2) (3) (4)

(5) (6) (7) (8)

(9) (10)

M. Bordenave, R. Goldberg, J.C. Huet and J.C. Pernollet, Phytochemistry 38 (1995) 315-319. G.Willliamson, Phytochemistry 30 (1991) 445-449. J.A. Matthew, S.J. Howson, M.H. Keenan and P.S. Belton, Carbohydr. Polymer 12 (1990) 295-306. C. Versteeg, F.M. Rombouts and W. Pilnik, Lebensmittel.-Wiss. u. Technol. 11 (1978) 267-274 M. M. Bradford (1976) Anal. Chem 72:248-254 V. Barkholt and A.L. Jensen, Anal. Biochem. 177 (1989) 318-322. H.A. Schols, M.A. Posthumus and A.G.J. Voragen, Carbohydr. Res. 206 (1990) 105-115. M.J.F. Searle-van Leeuwen, L.A.M. van der Broek, H.A. Schols, G. Beldman and A.G.J. Voragen, Appl. Microbiol. Biotechnol 38 (1992) 347-349. N. Feder and T.P. O'Brien, Amer. J. Bot. 55 (1968) 123-142. D.B. Fisher, Histochemie 16 (1968) 92-96.


731

Enzyme-Mediated Substrate Immunolocalization of Polygalacturonic Acid Within Barley Epidermal Cell Walls Utilizing Endopolygalacturonase of Coddiobolus sativus and a Monoclonal Antibody Specific for the Enzyme

Clay R. P.~, Bergmann C. W. ~ and Fuller M. S. 2

Complex Carbohydrate Research Center, University of Georgia, 220 Riverbend Rd., Athens, GA 30602, USA.

2

Darling Marine Center, University of Maine, Walpole, ME 04573, USA.

Abstract The fungal pathogen Cochliobolus sativus is known to penetrate its barley host via the anticlinal cell wall junctions between leaf epidermal cells. Microscopic and cytochemical evidence that this area is rich in pectic polysaccharides led us to investigate the contribution of endopolygalacturonase to the penetration process. Accordingly, an indirect method was developed for localization of the substrate of Cochliobolus sativus Ito and Kuribay (strain SB85) endopolygalacturonase in barley epidermal cell walls at the electron microscope level. The localization of the polygalacturonic acid substrate was accomplished by exposing thin sections of the walls to purified Cochliobolus sativus endopolygalacturonase, followed by exposure to a murine monoclonal antibody specific to the enzyme and finally goat-anti-mouse conjugated to colloidal gold. The resulting substrate localization pattern was identical to that obtained directly with JIM 5, a monoclonal antibody specific for tmesterified pectin. We refer to this indirect localization method as EMSIL (Enzyme-Mediated Substrate Immunolocalization). Potential advantages of this indirect method relative to direct substrate localization methods are discussed.

1. INTRODUCTION

It is generally accepted that cell wall degrading enzymes (CWDEs) produced by plant pathogens during penetration and subsequent infection of their hosts, are crucial to pathogenesis (Misaghi, 1984; Cooper, 1983; and Hahn et al., 1989). Activities attributed to these CWDEs include not only the enzymatic cleavage of structural cell wall polysaccharides, thus compromising the integrity of the cell wall-barrier to pathogen entry, but also more

732 complex roles within the sphere of host-pathogen interactions such as the release of oligosaccharides that function as "elicitors" of plant defense responses (Hahn et al., 1989). In spite of current evidence and consensus as to the importance of CWDEs in pathogenesis, unequivocal evidence for the sole responsibility of any particular enzyme for virulence in a given system has yet to be presented. Rather, it appears that multiple enzymes act in concert or succession to achieve the state of successful pathogenesis (Bateman and Basham, 1976). In addition, much of the research concerned with the importance or requirement of specific enzymes during pathogenesis fails to address the specific roles of these enzymes in cell wall degradation. A better understanding of the roles of CWDEs during normal pathogenesis would likely render interpretation of these enzyme studies more meaningful. Such understanding would include a thorough knowledge of the composition and distribution of components within the host cell wall. Although the general .composition and distribution of plant cell wall polymers are known (Roberts, 1989), the specific distributions of cell wall components in areas traversed by penetrating pathogens are unknown for most systems studied. Without this information, meaningful conclusions concerning enzymatic penetration are limited. One method for determining the distribution of cell wall components is by application of cytochemical probes for the various wall components at the electron microscope level. Colloidal gold particles have been utilized as cytochemical markers at the electron microscope level since the early 1970's. During this time, molecules of several categories appropriate for investigating plant cell walls cytochemically have been conjugated to gold particles for probe production, including immunoglobulins, lectins, enzymes and various other proteins not included in these catagories (see Handley, 1989 for a historical reveiw). These probes exploit the affinity of the conjugated ligand for specific sites in the subject material. For most of the probe catagories mentioned above, the labeling mechanism is easily understood; antibodies bind respective antigens strongly, lectins bind specific polysaccharides with high affinity, etc. The mechanism by which enzyme-gold probes label their respective substrates in situ is less easily understood. The interaction between an enzyme and its respective substrate is typified by substrate recognition, substrate binding, catalysis and dissociation. Thus, the in situ recognition of substrate by an enzyme-gold probe is understandable but why such a probe would remain associated with its substrate in lieu of subsequent catalysis and dissociation is not fully understood. Not withstanding, the fact remains that numerous enzyme-gold probes have been applied successfully for many years (Bendayan, 1989). During a preliminary study of the early host/pathogen interaction between C. sativus (Ito and Kuribayashi) and H. vulgare L., we obtained microscopic and cytochemical evidence that pectin degrading enzymes are utilized by the fungus during the early penetration of the barley host (Clay, 1995). This evidence plus findings from the C. carbonum endopolygalacturonase (EPG) gene-disruption experiment by Scott-Craig et.al. (1990) led us to investigate the contribution of fungal EPG to the early infection process operative in the H. vulgare/C, sativus system by application of cytochemical techniques at the electron microscope level. A particular objective was to utilize the monoclonal antibody JIM 5 (Knox etal., 1990) specific for non-esterified pectin to visualize the progressive alteration or loss of EPG substrate from the host plant cell wall during penetration by the fungal pathogen. Although unesterified pectin (polygalacturonic acid) is the apparent substrate of the EPG, there was a need to confirm that the pectic polymer recognized by JIM 5 was indeed the substrate recognized by the EPG. Thus, we produced, purified and characterized an

733 extracellular EPG of C. sativus and subsequently generated both polyclonal and monoclonal antibodies to this EPG. The pectic substrate of the EPG was then localized at the typical penetration site of barley leaf epidermis by using a novel application of purified EPG and one of the anti-EPG monoclonal antibodies. The labeling pattern obtained with the purified EPG was identical to that obtained with JIM 5, thus supporting the singularity of the JIM 5 antigen and the EPG substrate. Subsequently, the loss of EPG substrate from the host plant cell walls during progressive stages of pathogen ingress was visualized cytochemically at the electron microscope level (Clay, 1995). The method by which the purified EPG was utilized to indirectly localize the substrate of the enzyme in situ constitutes a novel cytochemical method and is the subject of the present work.

2. M E T H O D S

Cytochemical localization of pectin in barley leaf epidermal cell walls was performed at the electron microscope level by both direct and indirect means. Direct immunocytochemical localization of pectin was accomplished using a monoclonal antibody (JIM 5) specific for non-esterified polygalacturonic acid (Knox et.al., 1990), followed by a secondary antibody (goat anti-rat IgG, whole molecule, Sigma Cat. # R-5130) coupled to colloidal gold. Antibody-gold conjugates were made and immunolabeling was performed as described in Freshour et.al. (1995). Tissue to which the direct immunolocalization was applied was processed for electron microscopy according to Knox et.al. (1990) using LR White resin as the embedding medium. Sections 100 nm thick were cut with a diamond knife on an RMC (Tucson, AZ) ultramicrotome, picked up on gilded slot-grids and placed on formvar bridges (Rowley and Moran, 1975) to dry. After immunolabeling, sections were post-stained with 4% (w/v) uranyl acetate and lead citrate (Reynolds, 1963) and observed with a Zeiss EM10 or EM902 electron microscope. Experimental controls for the specificity of the direct immunocytochemical localizations with JIM 5 included omission of the primary antibody, pre-incubation of primary antibody with polygalacturonic acid, and substitution of the primary antibody with an extraneous monoclonal antibody derived from rat. Indirect immunolocalization of pectin was accomplished using 70 nm thick sections of host tissue processed and embedded in Quetol 651 according to the resin manufacturer's recomendations. For labeling, sections were exposed to purified EPG as outlined in Bendayan (1989) for enzyme-gold probes, followed by exposure to a primary antibody (EPG-4) specific for the EPG and finally to a secondary antibody (goat anti-mouse IgG, whole molecule, Sigma Cat. # M-8642) coupled to gold. Antibodies were applied as outlined in Freshour et.al. (1995) and sections post-stained as described above. The authors have coined the term EMSIL (Enzyme-Mediated Substrate ImmunoLocalization) for this indirect localization technique. Controls applied to EMSILs included omission of the enzyme, omission of primary antibody, and substitution of the primary antibody with an extraneous murine monoclonal antibody of the same type-class. Cellulase-gold was made and applied according to (Berg et al., 1988) with sections from material embedded in Queto1651. Chromatographically purified cellulase complex from Trichoderma reesei was obtained from Worthington Enzymes (Cat. # CEL).

734

3. RESULTS

The EMSIL obtained with the purified EPG on transverse sections of barley leaf epidermal cells taken pependicular to the long axis of the cells and anticlinal to the leaf surface, revealed that EPG substrate is localized primarily in the cell comers and middle lamella of these cells (Fig. 1).

~ .-~~i-i.~i!ii~!~-.

..-..

: ?:-.. .":21

,,^

~,

..-,

,

..~

-..

Figure 1. Transverse section of barley leaf epidermal cells taken perpendicular to the long axis of the cells and anticlinal to the leaf surface. The section has been labeled by the EMSIL technique (see Methods) utilizing purified C. sativus endopolygalacturonase and monoclonal antibody EPG-4, which is specific for this enzyme, in order to localize the substrate of the enzyme at the typical site penetrated by the fungal pathogen. Bar = 1 lam. Inset: Comparable cell wall region as in Fig. 1 but labeled with monoclonal antibody JIM 5 to localize non-esterified pectin. Bar = 1 lam. Note the identical labeling patterns obtained with either method.

735 Immunogold labeling with JIM 5 exhibited an identical labeling distribution for polygalacturonic acid as was obtained indirectly with the EPG EMSIL (inset of Fig. 1). Control experiments for labeling specificities obtained by the direct or indirect methods resulted in total elimination of specific labeling. The cellulase-gold probe heavily labeled the epidermal cell walls (Fig. 2).

Figure 2.

Advanced stage of barley leaf penetration by C. sativus. The pathogen has penetrated the anticlinal cell wall junction between two host epidermal cells (e). The fungal appressorium (a) is visible above the cell comer. The host cell comer matrix has been displaced by an enlarged hyphal element (h) situated between the thin cell walls of the host epidermal cells. The host epidermal cell walls have been densely labeled with the cellulase-gold probe. An intercellullar hyphal element (ih) is present within the penetrated host cell. Bar = 1 laM.

Advanced stages of penetration by the fungus were characterized by displacement of the pectin-rich cell comer regions with concurrent stretching of the cellulose-rich primary cell walls (Fig. 2).

4. DISCUSSION

The EMSIL method may prove useful in cytochemical applications where lack of substrate immunogenicity precludes antibody generation, or for which enzymes specific to the

736 substrate cannot be labeled for use in enzyme-gold localizations due to insufficient size of the enzyme molecule, instability of the enzyme, inactivity of gold-bound enzyme, lack of enzyme purity, etc. EMSIL would also be appropriate when an enzyme is extremely difficult or costly to purify, rendering it unfeasible to obtain quantities of purified enzyme sufficient for coupling directly to gold. With the EMSIL technique, it may be possible to utilize a defined mixture of enzymes as a primary cytochemical reagent, followed by a complimentary mixture of enzyme-specific antibodies, each coupled to a different-sized gold colloid, thus labeling several different substrates in one step, on one thin-section. Accordingly, it may be be possible to simultaneously visualize the degradation or loss of multiple substrates at a given location within the host-pathogen arena.

5. REFERENCES

Bateman D. F. & Basham H. G. (1976). Degradation of plant cell walls and membranes by microbial enzymes. In: Heitefuss R. & Williams P. H., ed.; Encyclopedia of Plant Physiology, Vol. 4. New York: Springer-Verlag, 316-355. Bendayan M. 1989. The enzyme-gold approach: a review. In: M. A. Hayat, ed. Colloidal Gold: Pnnciples, Methods and A pplications. Vol. 2. Academic Press, Inc., New York, 118-145. Berg R. H., G. W. Erdos, M. Gritzali and R. D. Brown. (1988). Enzyme-gold affinity labeling of cellulose. Journal of Electron Microscopy Techniques 8:371-379. Clay R. P. (1995). Studies of the mechanism of host penetration during the infection of Hordeum vulgare by Cochliobolus sativus. Ph.D. Dissertation, University of Georgia, Athens, GA, USA. Cooper M. R. (1983). The mechanisms and significance of enzymatic degradation of host cell walls by parasites. In: Callow J. A., ed. Biochemical Plant Pathology. John Wiley & Sons Ltd., 101-135. Freshour G., R. P. Clay, M. S. Fuller, P. Albersheim, A. Darvill and M. G. Hahn. (1995). Developmental and tissue-specific structural alterations of the cell wall poysaccharides of A rabidopsis thaliana roots. Plant Physiology 110:1413-1429. Hahn M. G., Bueheli P., Cervone F., Doares S. H., O'Neill R. A., Darvill A. & Albersheim P. (1989). Roles of cell wall constituents in plant-pathogen interactions. In: Nester E. & Kosuge T., ed. Plant Microbe Interactions, Vol. 4. McGraw-Hill Publishing Co., 131-181.

737

Handley D. A. (1989). The development and application of colloidal gold as a microscopic probe. In: M. A. Hayat, ed. Colloidal Gold: Principles, Methods and Applications. Vol. 1. Academic Press, Inc., New York, 1-12. Knox J. P., Linstead P. J., King J., Cooper C. & Roberts K. (1990). Pectin esterification is spacially regulated both within cell walls and between developing tissues of root apices. Planta 181:512-521. Misaghi I. J. (1982). The role of pathogen-produced cell-wall-degrading enzymes in pathogenesis. In: Physiology and Biochemistry of Plant Pathogen Interactions. Plenum Press, 17-34. Reynolds E. S. (1963). The use of lead citrate at high pH as an electron opaque stain in electron microscopy. Journal of Cell B iology 17:208-212. Roberts, K. (1989). The plant extracellular matrix. Current Opinion in Cell Biology 1:10201027. Rowley J. C. III and D. T. Moran. (1975). A simple procedure for mounting wrinkle-free sections on formvar coated slot grids. Ultramicroscopy 1:151-155. Scott-Craig J. S., Panaccione D. G., Cervone F. & Walton J. D. (1990). Endopolygalacturonase is not required for pathogenicity of Cochliobolus carbonum on maize. The Plant Cell 2:1191-1200.


J. Visserand A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996ElsevierScienceB.V.All rightsreserved.

739

I n f l u e n c e of glucose a n d p o l y g a l a c t u r o n i c acid on t h e s y n t h e s i s a n d a c t i v i t y of t h e p o l y g a l a c t u r o n a s e f r o m t h e y e a s t s t r a i n S C P P A. Gainvors and A. Belarbi Laboratoire de Microbiologie G~n~rale et Mol~culaire, Facult~ des Sciences, Europor Agro, B.P. 1039, 51687 Reims cedex 2, France. Abstract The yeast strain SCPP exhibits all of the panoply required for the degradation of pectins from various sources. This enzymatic machinery is secreted into the growth medium. Hence, we developped interest on the regulatory mechanisms controlling the synthesis of the main activity produced by the SCPP strain, i.e. the polygalacturonase. We observed that this activity is maximum five days after culture inoculation. Its biosynthesis is influenced by the glucose concentration. It is also stimulated by pectin concentrations similar to the ones seen in fruit juices.

1. INTRODUCTION Pectinases find industrial application in the food-industry during the extraction and stabilization ~f juices. Grape must contains pectins that are frequently removed by the ac:~ion of fungal pectinases. It would, however, be preferable to use Saccharomyces cerevisiae yeast strains that could produce pectinases. The catalytic capacity of several excreting pectolytic enzymes obtained from various yeast strains was examined using in vivo and biochemical techniques. Of the 33 yeast strains studied 30 were isolated from champagne wine during alcoholic fermentation. Only one yeast strain was found to excrete pectolytic enzymes and was identified as Saccharomyces cerevisiae designated SCPP. Three types of pectolytic enzymes were found to be excreted by SCPP : polygalacturonase (PG), pectin-lyase (PL) and pectin-esterase (PE) [1]. The polygalacturonase was studied and this enzyme was secreted constitutively both under anaerobic or aerobic conditions. A few physico-chemical properties of the secreted PG have been determined (PM, PI) [2]. The results obtained regarding its stability and pH-optimum are encouraging for its potential industrial usage. In effect, the SCPP strain is able to degrade pectins with varying esterification levels, thus participating in the clarification phenomena of fruit juices. The clarification potential of that strain was compared with the one of Several commercially available pectolytic mixes[3]. Recently, Gainvors and Belarbi [4] have set up a screening method for the selection of Saccharomyces cerevisiae yeast strains exhibiting pectinolytic

740 activities. This method is based on a particular physiological characteristic of this type of yeast. PG representing most of the pectinolytic power of the SCPP strain, we focussed on the role of glucose and polygalacturonic acid on the synthesis of this enzyme. 2. P O L Y G A I ~ C T U R O N A S E :ACTIVITY-SYNTHESIS R E L A T I O N S H I P Our study deals with the polygalacturonase activity in its globality and does not concern individual isoforms [2]. Gainvors et al. [1] have shown that the release of reducing groups in Pg glc growth culture medium (].% polygalacturonic acid, 1% glucose, 6.7 g/1YNB Base, 50 mM phosphate buffer pH 5.5) by the SCPP strain is significant after only 24 hours of culture. Even though it is very weak at first, it rises over the first four days of culture before reaching a plateau. Two distinct phases can be distinguished in the kinetic of release of reducing groups in order to study "~he regultion and synthesis of PG by the SCPP strain. Thus, we decided to estimate the effect of glucose on the PG activity at the start of the culture and define the cause of the stabilization phase in the release of the reducing groups Three hypothesis can be made in order to explain the existance of such a plateau: - The stable concentration of reducing groups may suggest that PG is absent from the culture medium on the fourth day. In the case PG is present, the plateau can be obtained by an inhibition of the enzymatic activity. - or by the enhanced breakdown of the reducing groups freed by the SCPP strain. -

In order to choose among these various hypothesis, we followed the kinetic of secretion of the polygalacturonase in the Pg glc medium. Hence, the proteins present in daily aliquots have been acetone precipitated [1]. The protein extracts were then either placed oil a reaction medium to assay for their PG activity [1] or studied by zymogram [5].

741 0,8

0,7

m

0,6 f~

0,5 SCPP 0,4 --O--- X2180-1A

~ 0,3 ~ 0,2 =L 0,I

2

3

4

5

6

Days

F i g u r e 1 9PG activity m e a s u r e m e n t of crude protein e x t r a c t of the S C P P s t r a i n grown on Pg glc m e d i u m C r u d e p r o t e i n e x t r a c t s a n d r e a c t i o n m e d i a w e r e m a d e as d e s c r i b e d by G a i n v o r s et al. [1].

Measurements were made on the reaction medium as described by Milner et Avigad [6].

W h e n studied on the PG glc m e d i u m , the PG e n z y m e of the S C P P s t r a i n is a l r e a d y p r e s e n t on the first day of culture and keeps on being secreted until the sixth day of culture (figure 1). On the other hand, the X2180-1A s t r a i n does not display and polygalacturonase activity during this period of time.

F i g u r e 2 9PG activity detection by zymogram[ 5] of the S C P P s t r a i n cultivated on Pg glc m e d i u m for two a n d five days Each deposited volume contains all ot' the proteins secreted by 5. 105 cells. 1 9Culture medium of SCPP cultivated on Pg glc medium for 5 days 2 9Culture medium of SCPP cultivat(.d on Pg glc medium for 2 days 3 9Culture medium of X2180-1A cultivated on Pg glc medium for 5 days

742 This results were confirmed by zymogram where a greater PG activity was observed on the fifth day over the second day (figure 2). Both techniques agree with each other and suggest a gradual enhancement of PG over time. At this point of our study, the stabilization in the release of reducing groups cannot be attributed to a disappearance of enzymatic activity beyong the fourth day. This phenomenom can thus only be explained on two ways : the first one is based on the idea t h a t galacturonic acid molecules are not metabolized by the SCPP strain. In the case, the reducing groups would accumulate to a given value which inhibits PG activity. Since the enzyme is still present in the medium, its inhibition would lead to a reduction in reducing group formation. the second one m a k e s the hypothesis t h a t the reducing groups are metabolized by the yeast cells, thus leading to an equilibrium between reducing groups biosynthesis by 1;he enzyme (still present in the medium) and their degradation by the SCPP strain. Earlier results obtained in our laboratory showed t h a t the concentrations of galacturonic acid found in the culture medium after four days of culture do not inhibit PG activity [2]. We, thus, favored the last hypothesis. This one is actually supported by an observation made during a search on PG activity of a dry active yeast. In the case of this strain, the quantity of reducing groups is m a x i m u m on the third day of culture and sharply decreases thereafter (figure 3), indeed suggesting that galacturonic acid molecules are utilized by this yeast strain and t h a t its basal PG activity is strong enough to lead to a fast enough renewal of the reducing groups and this maintain the equilibrium. Moreover, we have shown t h a t the SCPP s t r a i n is able to metabolize galacturonic acid [4]. -

-

0,8 0,6 O

--a-- SCPP .2 0,4

LSA X2180-1A

"6 0,2 -i

0,0

9

o

1

2

.

.

3

4

days

-

.

5

6

F i g u r e 3 9Comparison o:? the kinetics of release of reducing groups by various yeast strains grown on Pg glc medium Strains were grown at 30~ as non agitated cultures. Measurements were made on 300 ILl of the culture supernatants as described by Milner et Avigad [6].

743 This study has allowed us to demonstrate the existance of polygalacturonase after four days of culture, its activity being m a x i m u m on the fifth day. We have also observed t h a t the release of reducing groups stabilizes on the fourth day. The most plausible h y p o t h e s i s b e h i n d this p h e n o m e n o m is b a s e d on the existance of an equilibrium b e t w e e n the freeing of r e d u c i n g groups after polygalacturonic acid degradation and their utilization by the SCPP strain. 2. E F F E C T O F G L U C O S E O N T H E S Y N T H E S I S A N D E N Z Y M A T I C ACTMTY OF POLYGAI~CTURONASE

2.1. Effect of g l u c o s e on p o l y g a l a c t u r o n a s e b i o s y n t h e s i s In order to estimate the effect of glucose on the release of reducing groups in the culture medium, the SCPF' strain has been inoculated on Pg glc media with glucose concentrations ranging from 0.1% to 1%. The g r o w t h of the S C P P s t r a i n d e p a n d e d on t h e s e different glucose concentrations in the culture medium. It was raised by increasing glucose concentrations. Hence, in order to compare the release reducing groups in these media, we have expressed our results in terms of nmol of reducing groups freed per 0.5 unit of adsorbency at 600 nm which corresponds to 5.106 cells, i.e., the weakest cellular concentration obtained with 0.1% glucose. Figure 4 shows t h a t the highest release in reducing groups can be obtained with 0.25% glucose. This result r e m a i n s valid on the first and third day of culture.

120 m~ 110 100 ~ g 90

......

-

80

~N 70

"~a~~ 60 ~. 50

"iii~ii. . . . . ~ .............. .._.|

10 0

II !~1 11 P"A

1% glucose 0,5%glucose 0,25%glucose 0,1%glucose

~ 1

days

3

F i g u r e 4" Effect of glucose on reducing groups release by SCPP Measurements were achieved after growing the strain in the presence of 1% polygalacturonic acid and varying concentrations of glucose. They were done on the culture supernatants as described by Milner et Avigad [6].

744 It is difficult to interprete the result obtained when the SCPP strain was cultived in the presence of 0.1% glucose because its growth on such a medium was severely reduced. In the light of these results, it seems that PG activity is minimal at an initial glucose concentration of 1% in the presence of 1% Pg. It is maximal at an initial glucose c o n c e n t r a t i o n of 0.25% with the same polygalacturonic acid concentration. The best g r o w t h - P G activity ratio is t hus obtained with this l at er concentration. 2.2. Effect of g l u c o s e on PG activity A crude protein extract has been prepared by acetone precipitation on a three days old culture supernatant of the SCPP strain on Pg glc medium. In order to estimate the effect of glucose on PG activity, these protein extracts were deposited as dots on solid Pg glc medium. On this medium, the protein extracts of the SCPP strain exhibited PG activity similar to the one obtained on 1% Pg medium (data not shown). Hence, it seems t h at at this concentration (1%) glucose does not inhibit the PG activity of the SCPP strain. 3. E F F E C T OF P O L Y G A L A C T U R O N I C ACID C O N C E N T R A T I O N ON THE ACTIVITY AND SYNTHESIS OF PG The PG activity of the SCPP strain is expresssed on a constitutive basis. We have, however, observed that it rises in the presence of 1% Pg [1]. In order to determine the minimal substrate concentration capable of stimulating PG activity, we constructed Pg glc media containing 1% glucose and increasing concentration',5 of polygalacturonic acid (ranging from 0 to 10 g/l). These media were inoculated with 5.105 cells per ml and incubated at 30~ for three days in full tubes without stirring. The yeast cells were removed by centrifugation. Then, 500,~1 of the supernatants were acetone precipated, placed in reaction media and assayed for PG activity. Yeast growth was similar on all culture media whatever the polygalacturonic acid concentration used. Cell density was about 3.5.107 cells/ml. These results depicted on figure 5 are expressed in terms of nmol of reducing group per ml and min.

745

~ 0,5

-

,Phase 1

~ 0,4 ~o

E

Phase

3

-

S

0,3

. . 0,2 o

Phase 2

9

0

I

'

1

I

2

'

I

I

3

4

'

I

5

'

I

6

'

I

I

7

8

'

I

'

9

I

10

g/1 polygalacturonic acid

F i g u r e 5 : Detection of PG activity of crude protein extracts of the SCPP strain in the presence of 1% glucose and varying concentrations of polygalacturonic acid. Crude protein extracts and reaction media were made as described by Gainvors et al. [1]. Measurements were made on the reaction medium as described by Milner et Avigad [6].

These curve exhibits four distinct phases depending on the polygalacturonic acid concentration in the culture medium. Phase 1 (0 to 1.25 g/l) : PG activity corresponds to basal levels. The speed of substrate hydrolysis is 0.28 nmol/ml/min. Phase 2 (1.8 to 3.5 g/l) : rises in PG activity correspond to enhancements in polygalacturonic acid concentration in the culture medium. It is stimulated by a factor of 1.35 at a polygalacturonic acid concentration of 350 mg/1. Phase 3 (5 to 7.5 g/l) : At these concentrations, a drop in PG activity is observed. This phenomenom is reprocible and can be attributed to pectin-protein interactions. These interactions only occur between specific pectin-protein couples after a first depolymerization action of PG [7]. Depolymerized pectins associate with proteins to yield aggregates which can easily sediment. Their removal during medium centrifugation would explain the lowering in PG activity in the reaction medium over this polygalacturonic acid concentration range. Phase 4 (7.5 to 10 g/l) : A phenomenom similar to the one observed during phase 2 occurs, i.e., PG activity rises with the polygalacturonic acid concentration. Highest PG synthesis is actually obtained with 1% pectin, yielding a total stimulation factors of 1.7. PG induction levels are admittedly very weak. We would have been better off if we had taken the effect of glucose concentration on PG synthesis into account and had carried our experiment at a glucose concentration of 0.25%. 4. C O N C L U S I O N AND D I S C U S S I O N We display evidences showing t h a t glucose has inhibitory effects on PG

746 organisms [8-9]. Some of them are plant pathogens, PG enzymes playing a major role during cell penetration and break-down. It seems that the presence of large quantities of glucose in severals plants leads to the inhibition of the proliferation of the pathogenic micro-organisms [10-13]. Additionnally, we show that the PG activity of the SCPP strain is regulated. It is stimulated by polyglacturonic acid concentrations similar to the ones seen in various fruit juices. On an industrial point of view, this induction would be diminished by the elevated glucose concentrations found in these fruit juices when compared to the pectin concentrations. Basal activities would be sufficient to eliminate all pectins and obtain juice stabilization. It would also have been i n t e r e s t i n g to study the effect of glucose concentrations of 15% and 20% (concentrations found in fruit juices, beers and ciders) on the PG activity of the SCPP strain. The lack inhibition of enzymatic activity by glucose still leaves the PG enzymes produced on a constitutive basis free to hydrolyze their substrate. Glucose and polygalacturonic acid concentrations would have to be taken into account if an optimum production of polygalacturonases is to be obtained for its purification.

5. REFERENCES 1 A. Gainvors, V. Fr6zier, H. Lemaresquier, C. Lequart, M. Aigle and A. Belarbi. Yeast, 10 (1994a) 1311. 2 C. Lequart, A. Gainvors and A. Belarbi. Enzyme Microb. Technol., (Submitted) 3 A. Gainvors, N. Karam, C. Lequart and A. Belarbi. Biotechnol. Letters, 16 (1994b) 1329. 4 A. Gainvors and A. Belarbi. Yeast, 11 (1995)in press. 5 R.H. Cruickshank and G.C. Wade. Anal. Biochem, 107 (1980) 177. 6 Y. Milner and G. Avigad. Carbohyd. Res, 4 (1967) 359. 7 B. Perez. M6moire National d'(Enologue. Univ. Reims Champagne-Ardenne (1990). 8 F. Federici. Antonie van Leeuwennoek, 51(1985) 139. 9 V.E. Shevchik, A.N. Evtushenkov, H.V. Babitskaya and Y.K. Formichev. World J. Microbiol. Biotechnol, 8 (1992) 115. 10 J.C. Horton and N.T. Keen. Phytopathology, 56 (1966) 908. 11 S.S. Patil and A.E. Dimond. Phytopathology, 58 (1968) 676. 12 G. Holz and P.S. Knox-Davies: Physiol. Mol. Plant Pathol., 28 (1986a) 403. 13 G. Holz and P.S. Knox-Davies. Physiol. Mol. Plant Pathol., 28 (1986b) 411.

Acknowledgements We t h a n k L. Legendre for the translation of this manuscript. This work has been supported by Pascal Biotech sarl Paris, AEB Spindal and Europol'Agro.


747

Pectin lyase from Fusarium oxysporum f. sp. radicis lycopersici" purification and characterization M.A. Guevara a, M.T. Gonz~ilez-Ja6n b and P. Est6vez a aDepartamento de Biologia Vegetal, Facultad de Biologia, Universidad Complutense, 28040 Madrid, Espafia. bDepartamento de Gen6tica, Facultad de Biologia, Universidad Complutense, 28040 Madrid, Espafia.

Abstract A pectin lyase has been purified from Fusanum oxysporum f. sp. radicislycopersici. Proteins from cultures of 4 days on pectin, were precipitated with ammonium sulphate and separated with a Superdex 75HR1030 column and by preparative isoelectric focusing (LKB column of 110 ml capacity). A single band, with isoelectric point of 9.20, was detected by silver staining on analytical isoelectric focusing. The molecular mass, calculated from its partition coefficient on the Superdex column, was 18 kDa. The highest activity of this enzyme was attained at pH 9.5 and 50 ~ C. The pectin lyase showed high specificity for pectin, an "endo" mode of action and calcium dependence.

1. INTRODUCTION Fusarium oxysporum f.sp. radicis-lycopersici Jarvis and Shoemaker (FORL) (Jarvis and Shoemaker, 1978) [1] is a pathogen of tomato which, with the arrival of intensive tomato culture under glass, has developed to serious proportions [2]. This forma specialis of F. oxysporum affects largely the root and crown tissues of tomato and the symptoms occur as foot and root rot. FORL isolates are pathogenic on tomato plants with genes for resistance to races 1 and 2 of Fusarium oxysporum Schlecht. f.sp. lycopersici (Sacc.) Snyd. & Hans (FOL), that cause the common Fusarium wilt of the tomato. However, although resistance to FORL has been found and incorporated into commercial cultivars, the disease is a severe problem in wide areas of the North Hemisphere [3-9]. Colonization of tomato root tissues by FORL is associated with striking modifications of host cell walls, as it has been shown by ultrastructural studies which have been carried out describing the penetration of the fungus through

748

infected root tissues [4, 10-19]. The pattern of penetration, with disruption or even loss of middle lamella matrices [16], implicates production of pectic enzymes by FORL [11]. In fact, pectic enzymes have been considered to play a critical role in parasitism involving pathogens of dicotyledons, in which rhamnogalacturonan has a key role in wall structure [20]. The r2 isolate of Fusanum oxysporum f. sp. radicis-lycopersici (FORL) produced several pectic enzymes that differ in substrate preference, reaction mechanism, and action pattern. We have detected three forms that have lyase activity, an absolute requirement for calcium, and pIs of 9.20, 9.00 and 8.65. The two most alkaline forms had a weak preference for pectin whereas the other was more active on pectate. The three lyases were produced when the fungus grew on pectin and on restricted galacturonic acid (data presented in the "XV Congreso National de Microbiologia" [21] and sent for publication). The objective of this work has been the purification of the pectin lyase with pI 9.20, which is the most abundant.

2. MATERIALS AND METHODS

Fungus The isolate of F. oxysporum f.sp. radicis-lycopersici used in this study was strain r2, supplied by Dr. J. Tello (Instituto Naeional de Investigaciones Agrarias, Madrid). It was isolated as a single-spore culture, from an infected tomato plant (Lycopersicon esculentum Mill.) and grown on potato sucrose agar (PSA) at 22 ~ C. The ability of the isolate to the infect tomato was periodically checked as described by S~nehez et al. [22]. Stock cultures were maintained in Petri plates on potato sucrose agar at 5~ (transferred every month) and in soil for long time preservation.

Enzyme production FORL cultures were grown in 250 ml Edenmeyer flasks containing a carbon source in 100 ml salts medium shaken (in)on a rotary incubator (150 rev.min 1) at 22~ The medium contained easamino acids 0.46, KHzPO 4 0.1, MgSO4.7H20 0.05g/100ml, FeSO4.7H20 0.2, ZnSO4.7H20 1.0, NazMoO4.2H20 0.02, CuSO4.5H20 0.02, MnC12.4H20 0.02 ~tg m1-1. Flasks were inoculated with 1 ml of distilled water containing 1 x 10 6 conidia obtained by flooding fungal colonies on potato sucrose agar. The carbon source (pectin, galacturonic acid or glucose) to shake cultures, was added at 0.5% (w/v) or supplied from diffusion capsules for restricted supply [23, 24]. The capsules, containing D-galacturonic acid or glucose (30g/100ml), were provided with membrane layers allowing

749 linear rates of release of sugar over 20 to 24h, so that capsules needed to be changed only once daily. Capsules with membrane layers were sterilized at 120~ for 10 min and filled with the carbon source (sterilized by filtration) before placing in cultures. The pH of cultures was adjusted at 5.5 with NaOH before sterilization. Cultures with restricted supply of the carbon source were first grown on 0.5% (w/v) glucose for three days on a rotary incubator (150 rev.min ~) at 22~ Then, cells were removed from culture fluid by centrifugation (1800 g, 30 min), and grown for one day on 100 ml fresh medium supplied with the capsule containing glucose. After one day with restricted supply of glucose, cultures were grown for three more days with restricted supply of galacturonic acid or with restricted supply of glucose. For cultures on pectin, 1 ml of grown cells for three days on glucose, were transfered to fresh inorganic salts medium with 0.5% (w/v) pectin (apple pectin, Fluka) and grown for six days. Cultures from different times of growth were collected. Culture fluids were cleared by passing through glass fibre filter. After dialysis for 16-18 h against distilled water at 5~ filtrates were assayed for enzyme activities and proteins. Assay Method Pectin lyase (PNL) activity was measured spectrophotometrically by the increase in absorbance at 235 nm of the 4,5-unsaturated reaction products. Reaction mixtures containing 0.25 ml of culture filtrate, 0.25 ml of distilled water and 2.0 ml of 0.24% pectin from apple (Fluka) in 0.05M tris-HC1 buffer (pH 8.0) with lmM CaC12, were incubated at 37~ for 10 minutes. One unit of enzyme is defined as the amount of enzyme which forms l~tmol of 4,5unsaturated product per minute under the conditions of the assay. The molar extinction coefficients of the unsaturated products is 5550 M-~cm-~ [25]. Also viscosity measurements were made using Cannon-Fenske viscometers or Ostwald micro-viscosimeter, at 37~ Reaction mixtures consisted of enzyme solution and 0.75% pectin in 0.05 M tris-HC1 buffer (pH 8.0) with 0.5 mM CaC12. One unit is defined as the amount of enzyme required to change the inverse specific viscosity by 0.001 min -1 under the conditions of reaction. Specific viscosity (n~p) is (t/t0)-l, where t is the flow time (sec) of the reaction mixture and t o is the flow time of the buffer. The inverse~specific viscosity (n~p-~) is proportional to the incubation time and the amOunt of enzyme used [26]. Units of enzyme activity were determined for 10 min of reaction. Protein determ ination. Protein was determined by Lowry's method [27], using bovine serum

750

albumin (Sigma) as a standar.

Purification of Pectin Lyase Preparation of enzyme. Culture fluids of three days on glucose 0.5% (w/v) and then four days on pectin 0.5% (w/v), cleared by passing through glass fibre filter, were used for the purification of PNL. A small quantity was remainder, dialyzed, and assayed for enzyme actitity and the remained was precipitated. Ammonium Sulfate Precipitation. The extract was made up to 40% saturation with the slow addition, with stimng, of ammonium sulfate at 4~ After several hours, the precipitate was removed by centrifugation at 30,000 g for 30 min and the supematant retained. It was brought to 100% saturation in similar conditions, the precipitate was collected by centrifugation, dissolved in the minimum of distilled water, dialyzed against water and then against 1% glycine, and lyophilized. Gel Filtration. The lyophilized protein was redissolved in 50 mM phosphate buffer, pH 7.4; 0.15 m NaC1; 0.013 % sodium azide and loaded on a Superdex 75HR1030 column equilibrated with the same buffer. Elution was downward flow (0.15 ml/min) and 0.25 ml fractions were collected. Fractions with pectin lyase activity were combined, dialyzed against distilled water and used in the next step. To estimate the molecular mass of PNL, the column was calibrated with standard proteins (Sigma MW-GF-70: Albumin, 66,000 Da; Carbonic Anhidrase, 29,00; Cytochrome, 12,400; and Aprotinin, 6,500). The proteins were eluted in the conditions described above and their volumes (Vo) were calculated from the peak maximum of the absorbance at 280 nm. The partition coefficient was obtained from the relationship K ~ - (Vo-Vo)-(V~-Vo) where Vt represents the bed volume of column and Vo the void volume (which was calculated using blue dextran, Sigma). The molecular mass was determined using a standard curve of K,v vs the logarithm of the molecular masses of the standards [28, 29] Preparative Isoelectnc Focusing. The PNL eluted from gel filtration was subjected to isoelectric focusing using a column of 110 ml capacity (LKB). The density gradient was formed with sorbitol [0-50% (w/v)]. Enzyme extract was distributed equally between the two gradient component solutions prior to the establishement of the gradient using a gradient former. The concentration of the career ampholytes, Servalyte 7-9 and 9-11 (Serva), was 1,2% (w/v) and the catode was placed at the botton of the column. The experiment was performed at 7~ with constant power (9.6 W) giving a maximum voltage of about 1600 V. After 48 h, fractions of 3 ml were removed from the bottom of the column. The pH values of the fraction were inmediately measured at 7~

751

Ultrathin-layer analytical isoelectric focusing Proteins were separated according their pI by isoelectric focusing (IEF) at 7~ on a LKB 2117 Multiphor II apparatus. Ultrathin layers (0.4 mm) of polyacrylamide gels with ampholytes pH 2-11 were cast for isoelectric focusing as recommended by the manufacturer. Polyacrylamide solutions containing 5.2 % acrylamide (Pharmacia), 0.17 % N,N'-methylenebisacrylamide (Pharmacia), 1.1 ml of Servalyte career ampholytes (Serva), 0.6 ml of 1 % ammonium persulphate (Pharmacia) and 20 lal TEMED (Pharmacia), per 12.72 ml of the total volume, were cast on a glass as support using an Ultro Thin Layer Casting Tray (Bio Rad). Electrode wicks for the anode and catode were soaked in 1 M H3PO4 and 0.5 M NaOH respectively. Gels were preelectrofocused for 30 minutes at a constant 5.0 W. Samples of 10 ~g of protein in 10 lal were applied onto the gel via a small tab of glass fibre paper. Subsequent electrofocusing was earned out for 60 min at a constant 15 W with a maximum of 1400 V; sample application tabs were removed 30 min after focusing began. Broad pI Calibration Kit standards (Pharmacia) were used for pI estimation. Agarose overlays (2 mm thickness) for enzymes detection, containing pectin, were cast by capillary action between two glass plates separated by spacers. On one of these glass plates, a GelBond support film (LKB) was affixed by a thin film of water. The agarose solution was heated to 95~ and the gel mold was heated to 50~ before casting. The agarose solutions contained 1 % agarose, 0 . 1 % of pectin in 0.05M tris-HC1 buffer (pH 8.0) with lmM CaC12 [25]. After focusing, gels were incubated in the appropriate buffer for 5 min. Then agarose overlays were placed on the surface of the isoelectricfocusing gels, incubated at 37 ~ for 20 min and the overlays stained with 0.05% ruthenium red. Afterwards, isoelectricfocusing gels were stained with silver (Bio-Rad kit) for protein detection.

Substrate specificity and mode of action Mode of action and substrate specificity of the purified enzyme were determined by following the decrease in viscosity and the increase in absorbance at 235 nm in reaction mixtures in the presence of 0.187 % substrate (pectin or pectate) at pH 8.0.

Optimum pH Optimum pH was determined by following the decrease in viscosity of the reaction mixture using 0.187 % pectin as a substrate in 0.05 M tris-HC1 buffer (pH 7.0-9.0) or glycine buffer (pH 9-10). Controls were run without enzyme preparation.

752

Effect of temperature Optimum temperature was determined at pH 8.0 by following the decrease in viscosity of reaction mixtures containing 0.187 % pectin, at temperatures between 30 ~ and 55~ Controls were run without enzyme preparation. Requirem ent of calcium The effect of C a 2+ w a s assayed by viscosimetry in reaction mixtures containing 0.187 % pectin, buffered at pH 8.0, and ethylene diaminetetraacetic acid (EDTA) (0.005 M) or GaG12 (0-0.02 M).

3. RESULTS

Production of pectin enzymes on restricted galacturonic acid and on pectin FORL was grown on restricted galacturonic acid and on pectin in order to ascertain the production of lyases by FORL and if different forms were produced. Figure 1 shows the time course of PNL activity during growth of the fungus in the two culture conditions experienced: pectin lyase was produced both (1)

4 1

E ctO ('O Cq

< (D o

200 o 3

0.02 restricted restricted galacturonic/ /' glucose acid ? // /

< .-- 0.01 t~

6

e-

1

2

3

4

5

'~ ~, '\\

6

100

7

Days of culture

,'7"

0.09 E t-

tO

O4

._= it) GI

(2)

40 ~-

0.06

""

p tin

0.03

7

r

i

1

2

3

20 '~

"""

,~

5

6

Days of culture

7

i

i

8

9

Figure 1. Time-course of pectin lyase activity in cultures of F. oxysporum f. sp. radici s - l y c o p e rsici. Fungus was first grown on unrestricted glucose for three days, then the biomass was: or shifted to restricted glucose and, after one day, to restricted galacturonic acid (1); or shifted to pectin (2). Enzyme activity was determined as increase in A235 nm (-*-) and by viscosimetry (-o-) and determined for 10 min.

753

on galacturonic acid as well as on pectin. Maximum of activity was obtained at 60 h on galacturonic acid (1) and 106 h on pectin (2).

Purification of Pectin liase Culture fluids of four days on pectin were cleared by passing through glass fibre filter and fractionated by ammonium sulfate, gel filtration and preparative isoelectric focusing: Table 1 summarizes the purification steps. A peak with PNL activity was eluted from the Superdex 75HR1030 column (figure 2) and subjected to preparative isoelectric focusing (figure 3). Table 1 Step

Protein

Total activity

Specific activity

Purification

Yield

mg

U

U/mg

fold

%

Extract

35.96

995.92

27.69

-

100

(NH4)2SO4

2.50

62.76

25.10

0.91

6.30

Gel Filtration

0.24

36.99

154.12

5.56

3.71

IEF

0.03

23.59

899.35

32.48

2.36

"13 0

!

~0

0.06

5"

:r,. .

E o~ o

ro

c~

m

o o3

5

0.03 ,-,

I

>

-

0

10

20

_

30

| | |

.

40

50

60

70

80

i

_

_

90

Fraction no.

Figure 2. Gel filtration. The dry residue obtained after ammonium sulfate precipitation was redissolved in 50 mM phosphate buffer, pH 7.4; 0.15 M NaC1; 0.013 % sodium azide, which was loaded on a Superdex 75HR1030 column equilibrated with the same buffer. Elution was downward flow (0.15 ml/min) and 0.25 ml fractions were collected. The fractions were assayed for protein content ( - - ) and PNL activity (-r

754

12

\

8"1" \

I

0

\\

r

A

0

5

10

_ _ _ I . . . .

i

20

25

15

. . . .

I

30

m

_

e

Fraction no.

Figure 3. Preparative isoelectric focusing. The PNL eluted from gel filtration was subjected to isoelectric focusing using a column of 110 ml capacity (LKB) with ampholytes pH 7-11. After 48 h (9.6 W constant power), fractions of 3 ml were removed and assayed for PNL activity ( § and pH (- -).

Molecular Weight The PNL, eluted from the Superdex column, showed a molecular weight of around 18 kDa (figure 4). lsoelectric Point Figure 5 shows the pattem of lyase isoenzymes along the purification process: at first, three bands with lyase activity (pls 9.20, 9.00 and 8.65) were detected in the ammonium sulfate precipitate (B 1); in the peak eluted from the Superdex 75HR1030 column, only one band with lyase activity was detected, that correspond to the PNL with pI 9.20 (B 2), but more proteins were detected by silver staining (A 2).

121 r

0

~

PNL

40

L.. m

~

1

80

2010-

~ -

4

4 1

0

0.1

I

I

I

0.2

0.3

0.4

K av

Figure 4. Estimation of molecular weight by calibration of Superdex 75HR1030. Standard proteins: 1, Albumin (66,000 Da); 2, Carbonic Anhidrase (29,000 Da); 3, Cytochrome c (12,400 Da); 4, Aprotinin, (6,500 Da). The line has been drawn using the equation lg m = 5.01930882 2.757789171 * kay; r = - 0.9965.

755

However, after the preparative isoelectric focusing column, the PNL was the only band detected both by lyase activity staining (B 3) and by protein staining (a 3).

A 9.30 8,65 8.45 8.15 7.35 6.85 6.55 --5.85

M 1

2

3

B 1

2

3

9 ~''~i

9.20 9.00 8.65

5.20 4.55 3.50

Figure 5. Analytical isoelectric focusing. Ultrathin layers (0.4 mm) of polyacrylamide with ampholytes pH 2-11 were used. Samples of 10 lag of protein in 10 lal of 1% glycine were applied. A.- Silver staining. B.- Stain for activity on overlays containing pectin in tris/HC1 buffer at pH 8.0 with CaC12. M.- Broad pI Calibration Kit protein (Pharmacia), samples of 5 lag of protein were applied. 1.Ammonium sulphate precipitated proteins from cultures on pectin. 2.- Fractions with PNL activity eluted from the Superdex 75HR1030 column. 3.- Purified PNL.

Properties of purified enzyme Substrate specificity and mode of action. Previous information, which we had obtained from FORL crude culture filtrates, showed that the pectin lyase (characterized by an isoelectric point of 9.2) had a predominantly "endo" way of action. This fact has been confirmed with the purified protein: it decreased the viscosity of reaction mixtures with pectin, but no increase in absorbance was detected in standard conditions. Moreover, the enzyme showed a great specificity for the substrate, as no activity was detected when the decrease in viscosity of pectate was tried. So, properties of the purified enzyme were studied by using pectin as substrate and following the decrease in viscosity of the reaction mixtures.

756

Effect of pH on the activity of PNL. The enzyme exhibited maximum activity at pH 9.5 (figure 6). Effect of temperature. The optimum temperature for the PNL activity was 50~ (figure 7). Effect of Ca2+. The addition of 0.005 M EDTA to the reaction mixtures, resulted in complete loss of activity, whereas the addition of CaC12 increased the activity (figure 8). Calcium concentrations of 0.001 M and lower were without effect on PNL activity, the optimum concentration being in the range of 5 to 15 M, and higher concentration resulted in a decrease in activity.

~

100

@9 80 ._> < 6o (!;)

.>-

40

"~ nr'

20 0

I

I

I

I

I

I

I

7

7.5

8

8.5

9

9.5

10

pH

Figure 6. Effect of pH on the activity. Reaction mixtures, buffered at different pH values: 7-9 (tris/HC1), 9-10 (glycine), were incubated under standard conditions. Both buffers were 0.05M of final concentration in the reaction mixture.

/ ~" 100 ~-~9 80

~> 40 ~.

20

o/

~I~

~1 ~

30

40

45

50

55

Temperature (~

Figure 7. Effect of temperature on the PNL. The optimum temperature was determined using temperatures between 30 ~ and 55~

under standard conditions.

757

"r

100 80

60

~: eo 0

EDTA 0

0.1

0.5

1

5

10

15

20

CaCI 2Concentration (mM)

Figure 8. Effect of CaC12 and EDTA on the PNL. The addition of EDTA (0.005 M) and CaC12 (0-0.02 M) to the reaction mixtures were assayed under standard conditions.

4. DISCUSSION It has been generally believed that, among plant pathogens promoting pectolysis, bacteria produce predominantly pectate lyase while fungi usually secrete pectin lyase (Phoma medicaginis var. pinodella synthesizes a pectin lyase [25 ). However, both types of lyase activity are frequently present in an organism: Fusarium solani f. sp. phaseoli produces a calcium-dependent lyase that degrades both pectin and pectic acid under alkaline conditions [30]. Fusanum solam f. sp. pisi produces an endopectate lyase that seems to be involved in pathogenesis [31 ]. It has been suggested that both types of enzyme should be considered as pectin lyase, and that they be distinguished according to their preference for highly esterified and low-esterified pectin [32]. As other pectolytic microorganisms the r2 isolate of Fusarium oxysporum f. sp. radicis-lycopersici produces a battery of pectic enzymes differing in substrate preference, reaction mechanism, and action pattern. When separated by isoelectric focusing and stained for activities, we have detected three forms that have lyase activity and pIs of 9.20, 9.00 and 8.65 (figure 5). Phoma medicaginis var. pinodella synthesizes a pectin lyase that has a pI of 7.9 [25], the pectate lyase from Fusarium solani f. sp. pisi has a pI of 8.3 [31], the multiple endopectate lyases from Hypomyces solani f. sp. cucurbitae obtained from culture and from infected tissue possess isoelectric points in the range of 10.2-10.3 and 10.510.6 respectively [33].

758

The more abundant lyase produced by FORL has been purified to homogeneity as it is shown by analytical isoelectric focusing (figure 5). The data in Table 1 show that a 32.48-fold increase in specific activity is achieved with a recovery of approximately 2.36%. The enzyme showed an "endo" type of action and a great specificity for pectin. The PNL exhibits an optimum pH of 9.5 (figure 6) and an optimum temperature of 55 ~ C (figure 7). Lyases catalyze the reaction in an alkaline or in a neutral medium at high temperatures [32]: pectin lyase from Phoma medicaginis var. pinodella showed an optimum pH of 7.5 [25], endopectate lyase from Fusarium solam f. sp. pisi showed an optimum pH of 9.4 [31 ], and pectate lyase from Rhizoctonia solani showed an optimum pH of 8.0 [34]. The molecular weight calculated by Superdex chromatography was 18 kDa (figure 2). Endopectate lyases from Hypomyces solani f. sp. cucurbitae from culture and infected tissue have molecular sizes between 32 and 42 kDa [33]. Fusarium solani f. sp. pisi possess an endopectate lyase of 26 kDa [31] and Erwinia aroideae possess one of 67 kDa [25]. Phoma medicaginis var. pinodella has two forms of the pectin lyase with molecular weight of 29.5 and 118 kDa which suggested the existence of monomeric and tetrameric components [25]. The enzyme had a requirement for calcium. The addition of EDTA to the reaction mixtures, resulted in complete loss of activity, whereas the addition of CaC12 increased the activity (figure 8). Presumably, sufficient contaminating calcium ions were present in the dialyzed enzyme and substrate mixture to permit the limited activity of the controls, but apparently these were removed by chelation with EDTA. The optimum concentration was in the range of 5 to 15 M, and higher concentration resulted in a decrease in activity. Phoma m edicaginis var. pinodella synthesizes a pectin lyase that lacked an absolute requirement for calcium ions but maximum enzyme activity required the presence of 1 mM C a 2+ [25]. The lyase from Fusarium solani f. sp. phaseoli, that is active on pectin and pectic acid, is calcium-dependent [30]. Most of the pectate lyases characterized are calcium-dependent: the pectate lyase from Rhizoctoma solam [34] and the endopectate lyase from Fusanum solam f. sp. pisi [31 ]. Two characteristics of the lyase that we have purified may be significant. First, the small molecular size of the protein may confer it a high mobility that could be helpful to its movement through host cell walls. In second place, it is an endo-type enzyme, fact that has been considered essential for maceration of plant tissues [35]. In this sense it is noteworthy that between the battery of pectic enzymes produced by FORL, this pectin lyase is the only protein that behaves as an endo-type.

759 REFERENCES

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

W.R. Jarvis and R.A. Shoemaker, Phytopathology, 68 (1978) 1679. E.C. Tjamos & C.H. Beckman (ed.), Vascular wilt diseases of plants, Springer-Verlag, Berlin, 1989. C.H. Beckman, The Nature of Wilt Diseases of Plants. APS Press St. Paul, Minnesota (1987). R.A. Brammall, and V.J. Higgins, Canadian Journal of Botany, 66 (1988) 1547-1555. T. Katan, D. Zamir, M. Sarfatti and J. Katan, Phytopathology, 1 (3) (1991) 255. S. Kuninaga and R. Yokosawa, Annals of the Phytopathological Society of Japan, 57 (1991) 9. R.C. Rowe, J.D. Farley and D.L. Coplin, Phytopathology, 67 (1977) 1513. J.C. Tello and A. Lacasa, Boletin de Sanidada Vegetal. Plagas, 14 (1988) 307. D.J. Vakalounakis, Plant Pathology, 37 (1988) 71. N. Benhamou, H. Chamberland, G.B. Ouellette and F.J. Pauz6, Physiological and Molecular Plant Pathology, 32 (1988) 249. N. Benhamou, H. Chamberland and F.J. Pauz6, Plant Physiology, 92 (1990) 995. N. Benhamou, J. Grenier, A. Asselin and M. Legrand, The Plant Cell, 1 (1989) 1209-1221. N. Benhamou, M.H.A.J. Joosten and J.G.M. De Wit, Plant Physiology, 92 (1990) 1108-1120. N. Benhamou, D. Mazau, and M.-T. Esquerre-Tugaye, Molecular Plant Pathology, 80 (2) (1990) 163-173. N. Benhamou, D. Mazau, J. Grenier and M.-T Esquerre-Tugaye, Planta, 184 (1991) 196-208. R.A Brammall and V.J. Higgins, Canadian Journal of Botany, 66 (1988) 915. H. Chamberland, N. Benhamou, G.B. Ouellette and F.J. Pauz6, Physiological and Molecular Plant Pathology, 34 (1989) 131. H. Chamberland, P.M. Charest, G.B. Ouellette and F.J. Pauz6, Histochemical Journal, 17 (1985) 313. P.M. Charest, G.B. Ouellette and F.J. Pauz6, Canadian Journal of Botany, 62 (1984) 1232. J. Callow (ed.), Biochemical Plant Pathology, pp. 101-35. John Wiley & Sons, New York (1983). M.A. Guevara, M.T. Gonz~ilez-Ja6n and P. Est6vez, XV Congreso de la SEM. Madrid (1995), L.E. Sanchez, R.M. Endo and J.V. Leary, Phytopathology, 65 (1975) 726.

760 23 24 25 26 27 28 29 30 31 32 33 34 35

R.M. Cooper and R.K.S. Wood, Nature, 246 (1975) 309. R.M. Cooper and R.K.S. Wood, Physiological Plant Pathology, 5 (1975) 135. W.A. Wood and S.T. Kellogg, Methods in Enzymology, vol. 161, Academic Press Inc., London, 1988. W.A. Wood & S.T. Kellogg, Methods in Enzymology, vol. 160, Academic Press Inc., London, 1988. O.H. Lowry, N.J. Rosebrought, A.L. Farr, and R.J. Randall, Journal of Biological Chemistry, 193 (1951) 265. J. Bodenmann, U. Heininger and H.R. Hohl, Can. J. Microbiol., 31 (1985) 75. P. Prasertsan and H.W. Doelle, Appl. Microbiol. Biotechnol., 24 (1986) 326. D.F. Bateman, Phytopathology, 56 (1966) 238. M.S. Crawford and P.E. Kolattukudy, Archives of Biochemistry and Biophysics, 258 (1987) 196. L. Rexob~i-Benkov~i and O. Markovic, Adv. in Carb. Chem. And Biochem., 33 (1976) 323. J.G. Hancock, Phytopathology, 66 (1976) 40. W.A. Ayers, G.C. Papavizas and A.F. Diem, Phytopathology, 56 (1966) 1006. R.M. Cooper, B. Rankin and R.K.S. Wood, Physiological Plant Pathology, 13 (1978) 101.


761

Enzymic release of ferulic acid from sugar beet pulp using a specific esterase from Aspergillus niger P.A. Kroon, C.B. Faulds, C. Br6zillon & G. Williamson Food Molecular Biochemistry Department, Institute of Food Research, Norwich Laboratory, Colney Lane, Norwich, NR4 7UA. U.K.

Abstract We have purified and characterised a novel esterase (CinnAE) from Aspergillus niger. The enzyme demonstrated activity towards various soluble feruloylated oligosaccharides derived from sugar beet pulp (SBP) but, when acting alone, the esterase released only 0.9% of the alkali-extractable ferulic acid from SBP. However, when incubated with a mixture of endo-arabinanase and o~-L-arabinofuranosidase, there was a 14-fold increase in ferulic acid release, demonstrating a strong synergy between these three enzymes. No increase in ferulic acid release was observed when SBP was incubated with CinnAE plus endo-(1,4)-13-Dgalactanase and /3-D-galactosidase. Hence, feruloylated arabinans in SBP are readily available for hydrolysis by arabinan-degrading enzymes, whereas feruloylated galactans ;are not available for hydrolysis by galactan-degrading enzymes.

INTRODUCTION Ferulic acid is a constituent of many plants and, in some, is accumulated to significant levels in the cell walls. Ferulic acid is found esterified to sugars in the cell wall polysaccharides [1-3] and/or etherified to components of the lignin [4]. Sugar beet pulp (SBP), a by-product of the sugar-refining industry, is a rich source of ferulic acid containing some 1% (w/w) [5]. Ferulic acid is associated almost exclusively with the pectic side chains in sugar beet [6]. Sugar beet pectins, which comprise some 25 % (w/w) of the whole pulp, are complex heteropolysaccharides containing galacturonic acid, rhamnose, arabinose and galactose as the major sugar constituents. The pectins are themselves composed of "smooth" regions comprising a backbone of (1-,4)-linked o~-D-galacturonic acid residues, and "hairy" regions where there is a 1:1 ratio of D-galacturonic acid and L-rhamnose residues in the backbone and a high degree of substitution of the rhamnogalacturonan. The pectic side chains in the hairy regions are comprised of highly branched (1--,5)-linked a-L-arabinans and linear (1--,4)-linked/3-D-galactans [6,7]. It has been shown that ferulic acid can be esterlinked to either C-2 of arabinofuranose residues or C-6 of galactopyranose residues in the pectic side chains [8]. Ferulic acid is distributed roughly equally between the arabinan and galactan components of the pectic side chains [9,10]. The European Economic Community have shown considerable interest in releasing ferulic acid from low value agricultural waste residues such as SBP, with subsequent bioconversion of the free acid to vanillin. Previous studies with commercial enzyme preparations have shown a high degree of solubilisation of ferulic acid from SBP is possible, giving rise to a mixture of free and esterified forms [9,10]. However, it is not known which

762 enzymes specifically are important in removing esterified ferulic acid from SBP. A major problem to date has been the isolation of a suitable esterase capable of cleaving the ferulic acid-sugar linkages present in sugar beet. Although a ferulic acid esterase (FAE-III; [11]) isolated from Aspergillus niger CBS 120.49 grown on oat spelts xylan (OSX) was able to release FA from wheat bran [11,12], it was not active on the FA-sugar ester linkages present in SBP [13]. The objectives of this study were (a) to isolate an esterase which is active on the ferulic acid-sugar ester linkages present in SBP, and (b) determine the enzymes required and the relationships between the various enzymes required, in the solubilisation of ferulic acid from SBP.

MATERIALS AND METHODS Source of Aspergillus niger strains, enzymes and substrates The source of A. niger strain CS 180 has been described [14]. The source and growth of A. niger CBS 120.49, and purification of FAE-III have been described previously [11]. Sugar beet pulp (0.02-0.8 mm particle size) was prepared as described previously [10]. Cinnamoyl esterase (CinnAE) was purified from culture filtrates of A. niger CS 180 grown with 1.5 % (w/v) SBP as carbon source [5]. Feruloylated oligosaccharides were purified from SBP as described elsewhere [9,10]. endo-Polygalacturonase, endo-arabinanase, o~-Larabinofuranosidase, endo-(1,4)-j3-D-galactanase and cellulase, were purchased from Megazyme Pty. (Australia). /3-D-galactosidase was purchased from Sigma. All the commercial enzyme preparations were of A. niger origin except for endo-cellulase which was from Trichoderma spp. Each enzyme was desalted (dialysis against water) and triplicate portions (20/xg) of the desalted enzyme assayed for esterase activity (see below). Enzyme assays Esterase activity was assayed by either a continuous photometric method [ 11] or using HPLC with detection at 310 nm [5]. Methyl esters of caffeic (MCA), p-coumaric (MpCA), ferulic (MFA) and sinapinic (MSA) acids, were used as substrates. The activity of purified CinnAE was determined on a range of SBP-derived feruloylated oligosaccharides as described previously [15]. Extracellular esterase and acetyl esterase activities were measured by a method similar to that described by Donnelly & Crawford [16] using p-nitrophenyl butyrate and p-nitrophenyl acetate, respectively (0.9 mM final concentration in 100 mM MOPS), at 30~ and pH 6.0. All p-nitrophenyl derivatives were purchased from Sigma. c~Glucosidase, ot-galactosidase, a-rhamnosidase, /3-xylosidase, o~-arabinosidase, xylanase, cellulase, polygalacturonase and arabinanase activities were assayed as described previously [5]. All enzyme assays were performed at least in duplicate, and concomitant with appropriate blanks to allow correction for any background reactions. Total protein was estimated using the Coomassie Protein Assay Reagent (Pierce). For all assays performed, one unit (U) of activity was defined as the amount of enzyme releasing 1 #mol of product min -~ under the assay conditions described. Electrophoretic methods SDS-PAGE was performed by the method of Laemmli [17]. The methods for native PAGE, isoelectric focussing, detection of esterase activity in electrophoresis gels, and assays for protein glycosylation have been described elsewhere [5].

763 Release of ferulic acid from SBP Portions (50 mU MCA-hydrolsing activity) of purified CinnAE were incubated at 37~ with SBP (10 mg), both in the presence and absence of other carbohydrases, in 100 mM MOPS (pH 6.0) in a final volume of 1 mL. Incubations containing boiled enzyme were performed as controls. Reactions were terminated by boiling (3 min) and the amount of free ferulic acid determined using a method described previously for de-starched wheat bran [18]. The total amount of alkali-extractable ferulic acid present in the SBP was 0.87 %

[51. RESULTS Induction of esterase activity Although A. niger CBS 120.49 produces high levels of ferulic acid esterase (FAE) activity when grown with oat spelts xylan as the major carbon source, the major esterase produced [11] is not active on the ferulic acid-sugar linkages present in SBP [13]. Another strain of A. niger (CS 180) is known to produce pectin-degrading enzymes and degrade sugar beet pectins [14,19]. We grew these two strains with 1% glucose, or with 0.1% glucose plus either 1% oat spelts xylan or 1% SBP as the carbon source. After 4 days, culture filtrates were assayed for FAE activity using methyl ferulate (MFA) as substrate. Both strains gave high activities when grown on OSX [180 and 80 U (L media) -1 for CBS 120.49 and CS 180, respectively]. Growth of CBS 120.49 on SBP yielded low levels of FAE activity [0.7 U (L media)-1], whereas a similar growth with CS 180 gave a ten-fold higher FAE activity. Hence, when grown on SBP, A. niger CS 180 gave highest FAE activity. Flasks containing basic media and either glucose (1% w/v) or glucose (0.1% w/v) plus SBP (1% w/v), were inoculated with A. niger CS 180 and incubated for 10 days, and portions removed after the first two days for measurement of esterase activity (Fig. 1). No esterase activity was detected in any of the flasks containing glucose as the sole carbon source. However, activity against all four of the simple phenolic methyl esters used as substrates was detected in SBP-grown cultures. The ratio of activites against the four substrates changed during the course of the experiment indicating that more than one esterase was produced. FAE-III was shown to be absent from SBP- and glucose-grown cultures using immunodetection with specific anti-FAE-III antibodies. Purification of the esterase A. niger CS 180 was cultured in shake flasks with 1.5 % SBP as carbon source for 108 h at 25~ Cultures were harvested by filtration through a single layer of muslin, clarified by centrifugation and concentrated prior to purification. Esterase activity was purified from the concentrated culture filtrate using (NH4)2SO4 precipitation, hydrophobic interaction chromatography and anion-exchange chromatography [5]. In total, 260 ~g of pure enzyme with a specific activity of 96.9 U (mg protein) -1 was purified form 22.5 L of crude culture filtrate. The purified protein gave a single, darkly-stained band on SDS-PAGE corresponding to a molecular weight of 75,800 Da (Fig. 2). Gel filtration chromatography (Superdex 200, Pharmacia) gave a native molecular weight of 145,000 Da, indicating the native enzyme is probably a dimer. A 1.1 ~g sample of partially pure enzyme gave a very darkly stained band corresponding to CinnAE when tested for glycosylation. IEF of the pure protein gave a single band after staining with a pI of 4.8, and this was shown to coincide with the esterase activity.

764 Figure 1 Changes in extracellular esterase activity with incubation time. Esterase activities were assayed using the following methyl esters; MCA (l--l), MpCA (m), M S A ( 9 and MFA (zx).

l

30

I

I

I

1

,-.25

I m~

3

E '~" 20 -

/

[]

/

-

/ >'15

o

o

uJ

5

0d

0

2

4 Time a f t e r

6 Inoculation

8

10

(days)

The pure enzyme was tested for activity against several methylated phenolic and cinnamic acids (Table 2). The enzyme was active on methyl esters of cinnamic acids : caffeic > p-coumaric > ferulic, and is therefore termed a cinnamoyl esterase (CinnAE). Assays using p-nitrophenol-acetate (pNPA) and butyrate (pNPB) confirmed the esterase activity of the purified enzyme was not due to the action of a "general" extracellular esterase since there was a decrease in specific activity between the crude culture filtrate and the pure enzyme, and the specific activity (for both pNPA and pNPB) of CinnAE is two orders of magnitude below that reported for general esterases, which possess activities in the region 220 U (mg protein) -1 [20]. The enzyme showed no detectable activity in assays for c~-glucosidase, c~-galactosidase, c~-rhamnosidase, /3-xylosidase, o~-arabinosidase, xylanase, cellulase, polygalacturonase and arabinanase activities.

Figure 2. SDS-PAGE of purified CinnAE (10% polyacrylamide gel). Samples in lanes as follows: Lane 1; 6/xg CinnAE after anion-exchange chromatography; lane 2, high molecular weight markers from Sigma.

LANE 1

2

MR ~205

116 ~---97.4

Imw

------------6s

~ 4 5

~

~ 2 9

765 Table 2 Properties of CinnAE purified from Aspergillus niger CS-180. (n.d. = not detected).

Substrate

Specific activity [U (mg protein) -1]

Methyl caffeate (MCA) Methyl p-coumarate (MpCA) Methyl ferulate (MFA) Methyl sinapinate (MSA) Methyl vanillate MVA) Methyl syringate (MSyA Methyl-3,5-dimethoxycinnamate (MDMCA)

96.9 84.2 22.4 n.d. n.d. n.d. n.d.

AEBSF, an irreversible inhibitor of serine proteases, was found to completely inhibit MCA-hydrolysing actMty in the concentrated crude culture filtrate at a concentration of 1 mM. We studied AEBSF inhibition of CinnAE at concentrations of 1 and 5 mM AEBSF and found activity was reduced to less than 1% of that found in the uninhibited sample within 18 h of treatment. These results indicate tlaat CinnAE has an active site serine residue. Activity of CinnAE on SBP and SBP-derived feruloylated oligosaccharides Several ferulolyated oligosaccharides have been isolated from SBP [9] and their structures determined by NMR [8]. CinnAE was able to release free ferulic from all the feruloylated oligosaccharides tested (Table 3). The enzyme therefore shows a different specificity compared to A. niger FAE-III [13] since, unlike FAE-III, it is able to release ferulic acid from feruloylated arabinose oligosaccharides [FA-(1--,2)-arabinose] and feruloylated galactose oligosaccharides [FA-(1-,6)-galactopyranose]. Further, the enzyme is active whether the primary arabinose is in the furanose or pyranose form. The enzyme was most active on the feruloylated arabinose trisaccharide (Ara3F) and disaccharide (Ara2F), and least active on the feruloylated arabinose monomer (Ara~F).

Table 3. Activity of CinnAE for a range of feruloylated oligosaccharides derived from SBP.

Feruloylated oligosaccharide

2-O-(trans- feru lo yl)-L-arap O-[2-O-(trans-ferulo yl)-a-L-araf]-(1-,5)-L-araf O-[6-O-(trans-feruloyl)-fl-D-galp]-(1--,4)-D-galp

Specific Activity

O-c~-L-araf-(1--,3)-[2-O-(trans-feruloyl)-~-L-araf]-(1--,5)-L-araf Feruloylated arabinose hexasaccharide

0.39 1.36 0.64 3.54 0.45

Feruloylated arabinose heptasaccharide Feruloylated arabinose octasaccharide

0.56 0.67

766 The sample of SBP contained 0.87% (w/w) of alkali-extractable FA and CinnAE was able to release only a fraction of this from the whole pulp when acting alone. In a 30 rain incubation, CinnAE released FA with a specific activity of 0.24 U (mg protein) -~. In a 24 h incubation, CinnAE was able to release 0.91% of the alkali-extractable FA. Synergy with other carbohydrases SBP was incubated in the presence of carbohydrases either individually, or in pairs, and in the absence or presence of esterase, and the soluble incubation products assayed for feruloyl groups by HPLC. None of the carbohydrases used contained FAE activity. Incubation of SBP with CinnAE alone gave a single peak of absorbance at 310 nm corresponding to FA (0.91% of the alkali-extractable ferulic acid was released). No feruloylated material was released when SBP was incubated with a mixture of endo-(1,4)-13D-galactanase and/3-D-galactosidase. There was no increase in FA release when CinnAE was supplemented with these two enzymes. A mixture of endo-arabinanase and c~-L-arabinofuranosidase gave three peaks of absorbance at 310 nm which corresponded to 4.78 (peak a), 0.84 (peak b) and 0.12 % (peak c) of the feruloyl groups in the pulp, while incubations containing these two enzymes and CinnAE gave no peak a, a reduced peak b, and a large increase in peak c (Fig. 3). Peak c corresponded to 12 % of the alkali-extractable ferulic acid in the SBP sample, indicating that in the presence of the endo-arabinanase and a-L-arabinofuranosidase, CinnAE was able to release 14-fold more ferulic acid than when acting alone. These results are consistent with a CinnAE-mediated hydrolysis of the (soluble) feruloylated oligosaccharide esters produced by the action of the endo-arabinanase and c~-L-arabinofuranosidase leading to release of free FA. Clearly, CinnAE is more active on some of the feruloylated arabinose oligosaccharide esters (e.g. peak a) than others (e.g. peak b), which was also seen in assays with isolated SBP-derived feruloylated oligosaccharides (Table 3). CinnAE requires ferulic acid to be in a readily accessible form to allow hydrolysis of the ester bond.

Figure 3. HPLC chromatogram illustrating the release of feruloylated material and free ferulic acid when SBP (10 mg) was incubated with a mixture of endo-arabinanase (2 U) and a-L-arabinofuranosidase (2 U), either in the presence ( ~ ) or absence ( ........) of CinnAE (0.5/xg). FA=ferulic acid. 1.0-

FA

r c

0.5a -

esterase

';'

~

+ esterase

i

0.0

o

!

1;

30 R e t e n t i o n t i m e (min)

767 DISCUSSION Growth of A. niger CS 180 with SBP as the major carbon source induces production of at least two extracellular esterase activities (Fig. 1), neither of which is due to a known A. niger esterase, FAE-III [11]. At least one of the novel esterases demonstrates activity towards methyl caffeate (MCA) which is not a substrate for FAE-III. We purified one of the induced esterases from culture supernatants of A. niger CS 180 grown on SBP using MCA as substrate, and obtained an electrophoretically homogeneous enzyme with a molecular weight of 145,000 (dimer), a pI of 4.80, and pH and temperature optima of 6.0 and 50~ respectively. The enzyme is one of several esterases produced by A. niger, but possesses both physical and catalytic properties that distinguish it from several others purified previously [21,11]. The enzyme demonstrated activity towards the methyl esters of several cinnamic acids : caffeic > p-coumaric > ferulic, and is therefore termed a cinnamoyl esterase (CinnAE). CinnAE demonstrated activity for all the SBP-derived feruloylated oligosaccharides tested (Table 3), releasing free ferulic acid from both feruloylated-arabinose and-galactose oligosaccharides. Thus, CinnAE is active on the ferulic acid-sugar linkages present in SBP, which distinguishes this enzyme from another A. niger esterase (FAE-III), which is not active on SBP-derived feruloylated oligosaccharides. However, when acting alone, CinnAE demonstrated only limited activity on SBP (0.91% of alkali-extractable ferulic acid released in 24 h). This indicated that physical rather than chemical factors were inhibiting the action of the enzyme on a complex cell wall substrate such as SBP. Incubation of SBP with individual carbohydrases including polygalacturonase, endoarabinanase and endo-(1-,4)-13-D-galactanase, failed to solubilise more than 1% of the feruloyl groups in the pulp. However, we observed a strong synergy between endoarabinanase and o~-L-arabinofuranosidase in solubilising feruloyl groups from SBP. These results indicate that th endo-arabinanase has only limited activity on (1--,5)-linked arabinan main chains in SBP due to a high degree of substitution with arabinofuranose residues or short (1->3)-linked arabinofuranose side chains [6,7]. The o~-L-arabinofuranosidase is able to cleave these arabinose substitutions, leaving the (1--,5)-linked arabinan main chains susceptible to hydrolysis by endo-arabinanase. The action of the endo-arabinanase on the debranched arabinan releases small, soluble feruloylated oligosaccharides which are good substrates for CinnAE. Ferulic acid is distributed roughly equally between the arabinan and galactan components of sugar beet pectins [9], and in the linear (1--,4)-/3-D-galactans, ferulic acid is linked to C6 of galactopyranose residues [8]. However, a mixture of endo(1--,4)-/3-D-galactanase and/3-D-galactosidase failed to solubilise feruloylated material from SBP, and did not increase the amount of ferulic acid released by CinnAE. Hence, although it has been demonstrated that treatment of SBP with more complex carbohydrase mixtures such as Driselase [9], or sequential treatment of isolated sugar beet pectins with endogalactanase and /3-D-galactosidase [22], leads to solubilisation of significant quantities of feruloylated galactose oligosaccharides, the results presented here show that the feruloylated galactans in SBP are not readily accessible for enzymic degredation by a simple mixture of endo-galactanase and/3-D-galactosidase.

The authors thank the Biological and Biotechnolgical Sciences Research Council and the European Commission (Grant No PL 920026)for funding. We would also like to thank David Archer for donation of the Asoergillus niger strain, lan Colquhoun for NMR analysis, and Marie-Christine Ralet for help in purifying the feruloylated oligosaccharides.

768 REFERENCES

10 11 12 13 14 15 16 17 18 19 20 21 22

M.M. Smith and R.D. Hartley, Carbohydr. Res., 118 (1983) 65. Y. Kato and D.J. Nevins, Carbohydr. Res., 137 (1985) 139. F.M. Rombouts and J.-F. Thibault, Carbohydr. Res., 154(1986) 189. A. Scalbert, B. Monties, J.-Y. Lallemand, E. Guittet and C. Rolondo, Phytochem., 24 (1985) 1359. P.A. Kroon, C.B. Faulds and G. Williamson, Biotechnol. Appl. Biochem. (In Press). F. Guillon and J.-F. Thibault, Carbohydr. Res., 190 (1989) 85. F. Guillon, J.-F. Thibault, F.M. Rombouts, A.G.J. Voragen and W. Pilnik, Carbohydr. Res., 190 (1989) 97. I.J. Colquhoun, M.-C. Ralet, J.-F. Thibault, C.B. Faulds and G. Williamson, Carbohydr. Res., 263 (1994) 243. M.-C. Ralet, J.-F. Thibault, C.B. Faulds and G. Williamson, Carbohydr. Res., 263 (1994) 227. V. Micard, C.M.C. Renard and J.-F. Thibault, Lebensm.-Wiss. U Technol., 27 (1994) 59. C.B. Faulds and G. Williamson G, Microbiol., 140 (1994) 779. C.B. Faulds and G. Williamson G, Appl. Microbiol. Biotechnol., (In Press). M.-C. Ralet, J.-F. Thibault, C.B. Faulds and G. Williamson, Carbohydr. Res., 263 (1994) 257. J.A. Matthew, S.J. Howson, M.H.J. Keenan and P.S. Belton, Carbohydr. Polym. 12 (1990) 295. P.A. Kroon and G. Williamson, Biotechnol. Appl. Biochem. (In Press). P.K. Donnelly and D.L. Crawford, Appl. Environ. Microbiol. 54 (1988) 2237. U.K. Laemmli, Nature 227 (1970) 680. C.B. Faulds and G. William son , J. Gen. Microbiol. 137 (1991) 2339. J.A. Matthew, G.A. Wyatt, D.A. Archer and M.R.A. Morgan, Carbohydr. Polym. 16 (1991) 381. M. Sundberg, K. Poutanen, P. Markkanen and M. Linko, Biotechnol. Appl. Biochem. 12 (1990) 670. C.B. Faulds G. Williamson, Biotechnol. Appl. Biochem. 17 (1993) 349. F. Guillon and J.-F. Thibault, Carbohydr. Polym. 12 (1990) 353.


Characterization

of some e n d o - p o l y g a l a c t u r o n a s e s

769

f r o m Sclerotinia

sclerotiorum M. B. Martel, R. IAtoublon and M. FSvre Laboratoire de Biologie Cellulaire Fongique, CGMC, CNRS UMR 106, Universit6 Lyon I, 43 Blvd 11 Novembre 1918, 69622, Villeurbanne, France.

Abstract The isolation and characterisation of an endo-polygalacturonase from S. sclerotiorum is reported. The purified glycoprotein has a molecular mass of 42 KDa and a pI of 4.8 and shows the enzymatic characteristics an endo-polygalacturonase. Large amounts of the purified endopolygalacturonase have been prepared in order to raise antibodies which are found to be cross reactive with all isolated endo-polygalacturonases. The purification scheme shows at least fifteen chromatographic fractions enzymatically active. The four endo-polygalacturonases so far isolated and purified have the same molecular mass but differ by their charge. 1. I N T R O D U C T I O N Among the economically important group of plant pathogens, Sclerotinia sclerotiorum is an ubiquitous phytopathogenic fungus which attacks a wide range of plants. The fungus secretes a complete set of enzymes[ 1] that are able to degrade cell wall components, to macerate plant tissues and cause cell death. From all the pectinolytic enzymes secreted by S. sclerotiorum special attention is paid to the polygalacturonases frequently produced in several molecular forms, and considered as important in the pathogenesis and virulence [2, 3]. Previous works on S. sclerotiorum have shown the occurence of both exo and endo-polygalacturonases [4-6] but the number of isoenzymes is still in debate since seven endopolygalacturonase genes have been cloned [7]. The answer should be given by the isolation and characterization of a foremost endopolygalacturonase which preludes the overall knowledge of the secreted pectinolytic enzymes of S. sclerotiorum. 2. MATERIALS AND METHODS

2.1. Culture conditions Sclerotinia sclerotiorum (strain ssl3) was grown for 10 days under constant stirring at 22 ~ on a liquid minimal medium supplemented with 0.5 % of polygalacturonic acid (wt/vol) as carbone source. The minimal medium contained per liter NH4NO3 (2 g), KH2PO4 (0.1 g), MgSO4 (1 g), DL malic acid (3 g) and yeast extract (0.5 g) and the pH was ajusted to 6 with NaOH. Cultures were maintained on potato dextrose agar (PDA). For enzyme production, 2 liter cultures were inoculated with 4-days-old colonies removed from the growing edges of 2 Petri plates.

2.2. Enzyme purification The mycelium was harvested by centrifugation and the supernatant was dialyzed overnight at 4~ against distilled water and freeze-dried. The lyophilized filtrate containing the secreted

770 enzymes was solubilized in 50 ml of distilled water and brought to 50 % ammonium sulfate saturation. The precipitate was collected by centrifugation (30 min, 20,000 g) and the pellet discarded. The resulting supernatant was brought to 85 % ammonium sulfate saturation. The final pellet obtained after centrifugating the solution at 20,000 g for 30 min was the starting material for polygalacturonases purification.

2.3. Enzyme assays The polygalacturonase activity was determined by measuring the amount of reducing sugar released from polygalacturonic acid according to the 2-cyanoacetamide assay [8]. The standard reaction mixture (0.5 ml) was composed of 0.5 mg of polygalacturonic acid dissolved in 50 mM acetate buffer (pH 3-5). The reaction was initiated by addition of 1 to 20 Ixl of the enzymatic fraction and incubated at 45 ~ for 20 min. The reaction was stopped by addition of 1.2 ml of TBC reagent (100 mM sodium tetraborate, 100 mM boric acid and 0.1% 2-cyanoacetamide). After boiling for 10 min and cooling, the coloration was determined spectrophotometrically at 270 nm. A standard curve, 0-0.4 lamol of galacturonic acid, was prepared for each experiment. The unit of activity was defined as the amount of enzyme required to liberate 1 i~mol, of reducing group per minute at 45~ The exo-polygalacturonase activity was assayed in the same conditions using digalacturonic acid as the substrate. The endo-polygalacturonase activity was also assayed by measuring the decreasing viscosity of a 2% polygalacturonase solution according to [9]. 2.4. Chromatography Liquid chromatography was monitored by a Gilson HPLC system. Gel filtration was performed on a column (0.75 x 30 cm) of AcA 54 (IBF) equilibrated in 20 mM Tris-HC1 (pH 7.5), 25 mM NaC1. Ion exchange chromatographies were carded out first on a Macro-Prep High Q (IBF) column (10 ml) in Tris-HC120 mM (pH 8), then on a 5 ml Econo-Pac Q cartridge (BioRad) equilibrated in ammonium carbonate 20 mM buffer (pH 5) and on a 5 ml Econo-Pac S cartridge (BioRad) equilibrated in ammonium carbonate 20 mM buffer (pH 5). Hydroxy-apatite chromatography was carded out on a 1.7 ml HA-Ultrogel (IBF) column equilibrated in 10 mM phosphate buffer (pH 6). Elution was performed with a linear 10-300 mM phosphate buffer (pH 6) gradient. Chromatography on immobilized reactive dyes (1-4 ml) was performed in 10 mM ammonium acetate buffer (pH 4 or 5) and elution followed with a linear 0-1 M NaCI gradient in the same buffer. 2.5. Electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins was carded out according to Laemmli [10] in a 5% stacking and 10 % resolving gel. Analytical isoelectric focusing (IEF) gel electrophoresis was carried out on ready precoated gels (Serva) containing 5 % ampholine, pH range" 3 to 10. 2.6. Immunological detection of polygalacturonases on nitrocellulose Transfer of the proteins, from the polyacrylamide gel facing the anode, to the nitrocellulose sheet was performed in a Hoefer Semiphor semi-dry transfer unit, in Towbin buffer ( 25 mM Tris-HCl, 192 mM glycine buffer (pH 8.3), 1.3 mM SDS and methanol 15% ) for 1 hour at 0.8 mA/cm 2. The proteins from the IEF were blotted in the same apparatus, for 30 min at 0.8 mA/cm 2, with the following anode buffers : 30 and 300mM "Iris, 20% methanol and 25 mM "Iris, 40 mM 6-aminocaproic acid (pH 9), 20% methanol for the cathode buffer. The nitrocellulose sheets were incubated overnight in 5% non fat dry bovine milk in 0.15 M NaC1, 50 mM Tris-HC1 buffer ( TBS, pH 7.4) at 4~ then rinsed and incubated 1 hour at 20~ with a 1:1000 dilution of rabbit polyclonal antibodies raised against the acidic endopolygalacturonase (T) from S. sclerotiorum. The nitrocellulose sheet was washed three times

771 with the milk saline buffer and then incubated, for 1 hour at 20~ with a 1:1000 dilution of a HRP-conjugated secondary antibody (Pierce Goat anti-Rabbit IgG, (H+L) Horseradish peroxidase conjugated). After three washings in the former TBS saline buffer, the nitrocellulose sheet was soaked in a solution of 50 mg of D A B , 50 ml of 30% H202, in 100 ml TBS. The reaction was stopped by washing with distilled water and the blots were dried. 3. RESULTS AND DISCUSSION Treatment of the culture filtrate with increasing ammonium sulfate amounts showed that the bulk of polygalacturonase activity precipitates between 45 and 85 % ammonium sulfate saturation (Table 1). Table 1 Ammonium sulfate precipitation N ~ fraction (pH) P25 P45 P65 P85 P100 P100 S100

(5) (") (") (") (") (7) (")

% saturation S04(NH4)2

prot (mg)

vol (ml)

25 45 65 85 100 100 100

1.4 10 14.5 4.2 1.4 0.8 2

2.5 8.5 24 19.5 4.8 30 280

Polygalacturonase activity SA TA % (103U/mg) (106 U) 0 4.5 13 180 74 0 0

0 0.4 4.6 14.8 0.5 0 0

0 2 22 73 2.5 0 0

The filtrate supplied with a 2 liter culture, was treated with increasing amounts of ammonium sulfate. At each concentration step, the precipitate was checked for the polygalacturonase activity. SA : specific activity; TA : total activity. The purification of an endo-polygalacturonase secreted by S. sclerotiorum deals with the use of several chromatographic steps. The first one was an anion exchange chromatography at pH 8. The polygalacturonase acivity was essentially recovered in 2 fractions; the greatest specific activity was recovered in the fraction eluted with 500 mM NaC1. This last fraction referred to R1 was chromatographied on the same anion exchange medium at pH 5. The polygalacturonase activity was resolved in the four protein peaks separated on the column. The first fraction which possesses the highest specific activity was dialysed against 10 mM phosphate buffer pH 6 and then chromatographied on a HA Ultrogel column equilibrated in the same buffer. Two fractions were separated, one which did not bind to the column and a retained fraction eluted with 100 mM phosphate. The first fraction named NR2'A was lyophilized and subjected to gel permeation on AcA 54. Two active fractions were separated corresponding to proteins of >70 kDa and 45 kDa. The second peak with the highest specific activity was chromatographed on a Brown-10 column. A first fraction (S) was eluted with the equilibrium buffer and a second fraction, named T, was eluted with 700 mM NaCI. This last fraction was pure as judged by SDS-PAGE and IEF of Fig. 1. The purified polygalacturonase (T) is an acidic (pI : 4.8) glycoprotein with a molecular mass of about 42 kDa which is in the range of most fungal endo-polygalacturonases [2, 11-14]. The molecular mass is however slightly higher than those observed for the endo-polygalacturonases isolated from A. niger [ 15, 16], from G. candidum [ 17] and even from soybean hypocotyls

772

..~_ 97 9

..,,_.. 66

5.9

..~__45 ~__ 4.6

..~. 31

I .,~._ 4.2

4._.21 .,....14 1

2

3

MWkDa

1 2 3

4

pl

Figure 1. a - SDS-PAGE of fraction T (silver staining), lane 1 : fraction T, lane 2 : ovalbumine and lane 3 : molecular weight standards. b - IEF of fraction T (Coomassie blue staining), lane 1 : Soybean trypsin inhibitor, lane 2 : glucose oxidase, lane 3 : carbonic anhydrase (bovine) and lane 4 : fraction T. infected by S. sclerotiorum [ 18]. Acidic plfor polygalacturonases have already been described in saprophytic fungi like A. niger [19], in a mycorrhizal ericoid fungus [11] and in the same pathogenic strain [5, 6, 20]. The N-terminal sequence (A-T-X-X-T-F-S-G-X-X-G-A-A) is similar to the endo-polygalacturonases isolated from S. sclerotiorum by Waksmann et al [5] and is in good agrement with the sequence deduced from the genes pgl-3 [7]. This is not surprising since the deduced amino acid sequences of most of the fungal endo-polygalacturonases so far studied show a great similarity [21 ]. The enzymatic parameters of the purified polygalacturonase are 9an optimal temperature around 45 ~ and an optimal pH between 3.8 and 4.2. Under these conditions the kinetic is linear for one hour and the activity is linear with respect to the enzyme concentration up to 150 ng. All activity is abolished after boiling for 5 minutes and after proteolytic digestion with trypsin. From the differences observed during the release of reducing groups from polygalacturonate or from digalactm'onate ( less than 1% of the former activity) we conclude that the enzyme is an endo-polygalacturonase. This conclusion was corroborated by viscosimetry experiments. The isolated endo-polygalacturonase (T) is by its physical properties thus very similar to the enzymes named PG2 and PG3 by Waksmann [5]. However the enzyme which is a true endo-polygalacturonase has a Km of 0.4 mg/ml which is 2 times lower than the Km already allowed to S. sclerotiorum PG2 and PG3 and a Vm of 80 mmol/min/mg far much lower than the values attributed to the above mentioned enzymes [5]. These kinetic properties are much closer to those found for the polygalacturonase isozymes of Botrytis cinerea by 22]. The isolated endo-polygalacturonase (T) has been purified in great amount in order to raise polyclonal antibodies. The antibodies were used in assessing the purity and the enzymatic content of the various chromatographic fractions isolated according to the purification scheme (Fig. 2). The scheme is divided in two sections emerging from the first chromatographic step. The left part contains acidic, strongly charged polygalacturonases referred to as S to X. The fight part contains less acidic and less charged polygalacturonases named A to J. All these fractions are enzymatically active, some have a true endo-polygalacturonase activity and others like fractions E and I possess also an exo-polygalacturonase activity. The SDS-PAGE and IEF analyses (not shown) of the purified enzymes S, T, U and C show a similar mass but a different charge which indicates some variability among the charged aminoacid composition. This situation is corroborated by the analysis of the three genes already

773

Figure 2. Purification scheme of the polygalacturonases from Sclerotinia sclerotiorum Ammonium sulfate orecipitation

I

MacmPreD High 0 DH 8 I

I

Rl(100)

I

Econo Pac 0 D H ~ I1 I

NR2A(25)

I WB(30)

1

N R 2 q 10)

I

W30)

k(33)

I

AcA 54 + Green-19 DH

NR :not refined ,R : retained. , (number) : polygalacturonaseactivity in units, * : exo + endo-polygalacturonaseactivities.

774 sequenced (pgl, 2 and 3) which have more than 98% homology and slightly differ from their calculated pI[7]. We can assume that the herein purified enzymes are part of a secreted multienzymatic pattern composed of numerous endo-polygalacturonases which have almost the same molecular weight but differ slightly by their isoelectric point. This situation could be somehow similar to the four to nine closely grouped isoenzymes of the anaerobic fermentative yeast Kluyveromyces marxianus [23]. Anyhow this multiplicity must confer flexibility to the hydrolytic complex and increases its efficiency. Using different chromatography matrices, our results show that the endo-polygalacturonase activities could be resolved in 8 endo enzymes and 2 exo enzymes were also detected. This equipment appears much more complex than previously reported (5, 18). The reappraisal of the polygalacturonase equipment of S. sclerotiorum is in agrement with the molecular studies which reveals 7 homologous genes. One cannot exclude that this multiplicity is also due to posttraductional modifications of a limited number of gene products. However glycosylation is probably not involved in this process as separation of the enzymes was based on the charge of the proteins which is not affected by the extend and (or) the mode of glycosylation. 4. REFERENCES

1 C. Riou, G. Freyssinet and M. F~vre, Appl. Environ. Microbiol., 57 (1991)1478. 2 F. Cervone, G. De Lorenzo, G Salvi and L. Camardella, Ed.NATO ASI series. H vol 1. B Berlin: Springer-Verlag, (1986) 385. 3 P. Alghisi and F. Favaron, Eur. J. Plant Pathol., 101 (1995) 365. 4 C. Riou, G. Freyssinet and M. F~vre, Appl. Environ. Microbiol., 58 (1991) 578. 5 G. Waksman, J. P. Keon and G. Turner, Biochim. Biophys. Acta, 1073 (1991) 43. 6 F. Favaron, P. Alghisi, P. Marciano and P. Magro, Physiol. Molec. Plant Pathol., 33 (1988) 385. 7 P. Reymond, G. Deltage, C. Rascle and M. F~vre, Gene, 146 (1994) 233. 8 S. Honda, Y. Nishimura, M. Takahashi, H. Chiba and K. Kakehi, Anal. Biochem., 119 (1982) 194. 9 D. Bateman and H. Basham H., Physiol. Plant Pathol., Ed. R. Heitefuss & P. Williams Berlin : Springer-Verlag, (1976) 316. 10 U.K. Laemmli, Nature (London), 222 (1970) 680. 11 R. Peretto, V. Bettini and P. Bonfante, FEMS Microbiol.Lett., 114 (1993) 85. 12 M. Gupta, D. Guoqiang and B. Mattiasson, Biotechnol. Appl Biochem., 18 (1993) 321. 13 C. Caprari, C. Bergmann, Q. Micheli, C. Salvi, P. Albersheim, A. Darvill, F. Cervone and G. De Lorenzo, Physiol. Molec. Plant Pathol., 43 (1993) 453. 14 V. Hugouvieux, S. Centis, C. Lafitte and M. T. Esquerrt-Tugayt, C. R. Acad. Sci., 318 (1995) 113-. 15 E. Stratilova, O. Markovic, D. Strovinova, L. Rexova-Benkova and H. Jornvall, J. Chem., 12 (1993) 15. 16 C. Morvan, A. Jauneau, A. Flaman A, J. Millet and M. Demarty, Carbohyd. Polymers, 13 (1990) 149. 17 A. Golubev, A. Nuradieva, N. Rodionova and K. Neustroev, Biokhim., 57 (1992) 1855. 18 F. Favaron, P. Alghisi and P. Marciano, Plant Sci., 83 (1992) 7. 19 H. Kesler and J. Visser, Biotechnol. Appl. Biochem., 12 (1990) 150. 20 C. Riou, L. Fraissinet-Tachet, G. Freyssinet and M. F~vre, FEMS Microbiol. Lett., 91 (1992) 231. 21 H. Bussink, F. Buxton, B. Fraaye, L. de Graaff and J. Visser, Eur. J. Biochem., 208 (1992) 83. 22 R. Tobias, W. Conway, C. Sams, Molec. Biol. Intern., 30 (1993) 829. 23 S. Harsa, C. Zaror and D. Pyle, Enzyme Microbiol., 15 (1993) 906.

J. Visser and A.G.J. Voragen (Editors), Pectins and Pectinases

9 1996 Elsevier Science B.V. All rights reserved.

775

Analysis of the interaction between PGIP from Phaseolus vulgaris L. and fungal endopolygalacturonases using biosensor technology B.Mattei a , G. Salvib, C. Caprari b, G. De Lorenzo b, V. Crescenzia, and F. Cervoneb. a Dipartimento di Chimica, Universita' di Roma "La Sapienza", P.le A. Moro, 00185 Roma. b Dipartimento di Biologia Vegetale, Universita' di Roma "La Sapienza", P.le A. Moro, 00185 Roma.

Abstract.

The interaction between endopolygalacturonase t~om Fusarium moniliforme and PGIP from Phaseolus vulgaris L. was investigated using a biosensor technique based on surface plasmon resonance (BIAlite). This new analytical system provides information on the strength and the kinetics ofbiomolecular interactions. PGIP (the ligand) was covalently attached to the dextran matrix of a sensor chip while endopolygahcturonase (the analyte) was introduced in a flow passing over the surface. Results from real-time BIA are presented as a sensorgram, which is a plot of changes in the resonance signal as a function of time. The experimental data were evaluated with an appropriate software to derive the values of kinetic rate constants and to compare the affinities of PGIP for several variant endopolygalacturonases obtained by site-directed mutagenesis of the Fusarium

moniliforme enzyme. 1. INTRODUCTION Polygalacturonase-inhibiting protein (PGIP) is a cell wall protein that specifically binds to and inhibits the activity of fungal endopolygalacturonases (PG). It has been shown that formation of the P G - PGIP complex at pH 5.0 and at low salt concentrations results in nearly complete inhibition of PG activity; the activity is restored upon dissociation of the complex at high salt concentration and pH values lower than 4.5 and higher than 6.0 [1]. As a first step to study the structure-fimction relationship of fimgal PGs and PGIPs, we have employed a biosensor technology based on smface plasmon resonance (SPR) [2-3] in order to characterize the strength of the interaction between the two proteins in terms of the kinetic and equilibrium binding constants. This new powerful technique has shown a considerable potential for the characterization ofbiospecific interactions such as those between antigens and antibodies [4-5] biologically active ligands and receptors [6] and (oligo)saccharides and lectins [7]. As model proteins the endopolygalacturonase of the phytopathogenic fimgus Fusarium moniliforme and the PGIP of Phaseolus vulgaris L. were used. A systematic study of the relationship between the structural features of these proteins and their implications on interaction dynamics has been undertaken in our laboratory. The sequence encoding the endopolygalacturonase ofFusarium moniliforme [8] was cloned into the E. coli yeast shuttle vector Yepsecl for secretion in yeast [9]. The recombinant plasmid (pCC6) was used to transform Saccharomyces cerevisiae strain S150-2B. Three residues in a highly conserved

776 region were subjected to site-directed mutagenesis [10]" His 234 was mutated into Lys, Ser 237 and Ser 240, respectively into Gly. Both the wild type and the mutated enzymes were purified and characterized with respect to their enzymatic activity and the binding to PGIP.

2. EXPERIMENTAL PROCEDURES

Equipment and materials BIAlite* system, Sensor Chip CM5, HBS buffer (10mM Hepes, pH 7.4, 150 mM NaC1, 0.005% v/v surfactant p20 in distilled water), amine coupling kit were from Pharmacia Biosensor (Uppsala, Sweden).

Preparation of Sensor Surface PGIP, purified from P. vulgaris hypocotyls [11], was immobilized to the sensor chip via amine coupling. A continuous flow of HBS buffer (5 lxl/min) was mantained over the sensor surface. The carboxylated dextran matrix of the sensor sin-face was first activated by a 6-min injection of a mixture of N-hydroxy-succinimide and N-ethyl-W- (3-diethylaminopropyl) carbodiimide, followed by a 7-rain injection ofPGIP (10ng/lxl in 10 mM acetate, pH 5.0). The immobilization procedure was completed by a 7-min injection of 1 M ethanolamine hydrochloride to block the remaining ester groups.

Binding of endopolygalacturonases to immobilized PGIP Solutions of endopolygalacturonases in acetate buffer, pH 5.0, were injected into the flow cell and passed over the PGIP surface at the flow rate of 10 ~min. The interaction was followed in real time at different analyte concentrations. The binding was monitored as a mass change in the vicinity of the sensor surface, reflecting the progress of the interaction.

Surface plasmon resonance Surface plasmon resonance (SPR) is an optical phenomenon associated with total internal reflection that occurs at the boundary between substances of different ret~active index, e.g. glass and aqueous solutions. Normally, fight travelling through the material of higher refractive index (the glass) is totally reflected back when reaching an interface to the optically less dense medium (aqueous solution), provided that the angle of incidence is larger than the critical angle. Importantly, although the fight is totally reflected, a component of the electromagnetic field called the evanescent wave penetrates a short distance (of the order of one wavelength) into the solution. If the interface between the media is coated with a thin layer of metal, and the fight is monochromatic and p-polarized, the evanescent wave can interact with free oscillating electrons (plasmons) in the metal film surface, and the intensity of the reflected light is markedly reduced at a specific incident angle, producing a sharp "shadow". The angle at which the shadow is observed, called the SPR angle, is dependent on the refractive index in the solution close to the surface. Changes in the refractive index out to about 300 nm from the metal film surface can thus be followed by continuous monitoring of the resonance angle. All proteins, independent on their aminoacid composition, alter the refractive index of water by a similar amount per unit mass, and thus there is a linear correlation between the surface concentration of protein and the resonance angle shift. 1000 resonance units (RU) correspond to a 0.1 ~ shitt in the SPK angle and this is equivalent to a sm'face concentration change of about 1 ng mm-1.

777 Instrumentation

The instrument consists of a processing unit, reagents for ligand immobilization, exchangeable sensor chips and a personal computer for control and evah~tion. The sensor chip con~sts of a glass slide on to which a 50-nm thick gold film has been deposited. The gold film is then covered with a linker-layer to which a matrix of carboxylated dextran is attached. The dextran, which extends typically 100 nm out from the surface, provides a hydrophilic, activatable and flexible polymer to which biomolecules can be coupled through amine, sulphydryl, carboxyl and other groups. The sensor chip is held in contact with the prism of the optical system by a microttuidic cartridge that controls the delivery of sample plugs into a transport buffer that passes continuously over the sensor chip surface. By continuously monitoring the SPR response, expressed in resonance units (RU), in the detected vohnne and plotting this value against time a sensorgram is obtained. The sensorgram can be divided into three phases: association during sample injection, steady-state where the rate of analyte binding is balanced by dissociation from the complex and dissociation from the surface during buffer flow at the end of sample injection (Fig. 1).

E~ill'b~ Association

o

constants

Dissociation

?

2DO0

I I I I

0 1800-

1000-

Concentration

f

lzg~O-

I I

12D0-

$ 1000

800

0

'

I

100

"

I

200

'

I

300

'

I

400

'

I

600

'

I

600

Time (s) Fig. 1. Schematic sensorgram, showing association, equilibrium and dissociation phases.

Sensorgrams were analyzed by nonlinear least squares curve fitting using BIAevaluation 2.0 software (Pharmacia). A single-site binding model (A + B = AB) was used for the analysis of the interactions. The association rate and the dissociation rate can be expressed with the following equations, respectively:

778 dR/dt = -(konc + koff)R + koncRmax

(1)

dR/dt = - koff R

(2)

where Rmax is the maximum analyte binding capacity (in RU) of the PGIP surface and R is the SPR signal in RU at time t. The association phase was analyzed fitting the integrated form of equation (1)" R t = Req (1- exp( -ks (t-t o )))

(3)

where ~ = koncRmax/(konc + koff) was the amount of ligand bound in RU at equilibrium, to was the time the injection started and k s = konc + koff, where c was the concentration of the protein injected over the sensor surface. The association rate constant, kon, was determined from the slope of a plot ofk s versus c. The dissociation rate can be determined fitting the integrated form of equation (1)

gt = ~ exp(-kofr (t-to))

(4)

by non-linear least square analysis : R t is the amount of ligand (in RU) remaining bound at time t and to was the be~nning of the dissociation phase. The equilibrium association constant, KA,, was then calculated from kon/koff.

3. RESULTS AND DISCUSSION The study of the structure and function of fungal endopolygalacturonases and their plantderived interacting PGIPs is an essential starting point to understand some of the recognition phenomena occurring between plants and microorganisms [12]. We have undertaken a systematic study of the structure and function of PGIP from P. vulgaris and of endopolygalacturonase from F. moniliforme. The gene encoding this enzyme was introduced into S. cerevisiae via a shuttle vector and an active endopolygalacturonase was secreted into the medium by the transformed yeast cells. Point mutations were introduced in the amino acid sequence of the endopolygalacturonase ofF. moniliforme. Target sequence of these mutations was a region which is highly conserved in all endopolygalacturonases so far characterized [8]. Each mutated sequence was expressed in S. cerevisiae. His 234 was mutated into Lys, Set 237 and Ser 240 were mutated, respectively, into Gly. Both the wild type and the mutated enzymes were purified and characterized with respect to their enzymatic activity and the binding to PGIP. The replacement of His 234 with Lys abolished the enzymatic activity, confirming the biochemical evidence obtained by Cooke et al. [13] and by Rexovfi-Benkovfi et al. [14] that a histidine residue is critical for the activity of the enzyme. Replacement of either Set 237 or Ser 240 with Gly reduced the enzyme activity to 48% and 6% respectively, indicating that Set residues are also important for the activity. The interaction between PGIP of Phaseolus vulgaris and the different endopolygalacturonases was studied using a biosensor based on SPI~ PGIP was immobilized

779 as a ligand on the sensor surface, while endopolygalacturonase was passed in solution as an analyte on the surface. Sensorgrams for the interaction of PGIP with different amounts of F. moniliforme PG are shown in fig. 2. The increase in RU t~om the initial baseline represents the binding of the PG to the surface-bound PGIP. The plateau line represents the steady state phase of the PGPGIP interaction while the decrease in RU at the end of the injection represents the dissociation phase.

(RU) 9O3

i IO0

-lOa

,, -100

0

........

, IO0

,,

,

I

2DO

3OO

Time (=)

----; 4OO

.........

; ~00

........ : 6(7O

(,)

Fig. 2. Sensorgrams of Fusarium moniliforme endoPG injected over a PGIP surface at different concentrations.

The different interactions with either the wild type and the mutated polygalacturonases were analyzed kinetically as described in the experimental procedures. In fig. 3 , the slope of the plot ofk s versus c represent the association rate constant Icon for different endoPGs.

780 0.1 0.09 0.138 0.07 0.06 0.05 0.04

9 FmPG

0.03

D S240->G-.PG

0.02

y H234->K-PG 0.01

I Y,OG .,

O

0

8o-8

1.6e-7 Z4e-7

3.3s-7 4 e - 7 4.8e-7 5.6e..7 6.4e-7 7.2e-7 Concenb'atlo. (M)

8e-7

Fig. 3. Plots ofk s (or konc + koff) versus concentration for different endoPGs.

The kinetic parameters of each interaction are reported in Table 1. These data show that the values of the equilibrium association constant I ~ for the interaction of PGIP with the different yeast-expressed polygalacturonases do not differ si~ificantly, but in each case they are 2 + 5 times lower when compared to that of the F. moniliforme enzyme. This is probably due to the steric hindrance caused by hyperglycosylation of the yeast enzymes. Since the modification that causes loss of activity in the enzyme H - , K 234 does not interfere with the formation of the PG-PGIP complex, the site responsible for PGIP recognition may reside in a domain different from the active site. Studies are now under way to establish which site(s) and amino acid residues of the endopolygalacturonase are critical for interaction with PGIP.

4. ACKNOWI.J~DGEMENTS This work was supported in part by the National Research Council of Italy, Special Project RAISA, subproject N. 2 by the Ministero delle Risorse Agricole e Fores'taft (MIRAAF), and by the European Community Grant R 3 - C T 9 4 - 2 2 1 5 .

781 Table 1. Kinetic constants of the interaction between wild type and variant endoPGs with immobilized PGIP t~om P. vulgaris. PG cone

kon

koff

K A = kon/koff

(riM)

(105 M "1 s"1 )

(10.3 s"1 )

(107 M "1 )

Fusarium PG

25---400

1.17 + 0.11

1.2 + 0.2

9.7

S. cerevisiae PCC6 -PG

80---600

0.60 + 0.08

3.0 + 0.2

2.0

H 234 - , K PG

22--180

1.9 + 0.09

3.5 + 0.2

5.4

S 237-~ G PG (48 % activity)

10--70

1.1 __ 0.4

3.0 +_ 0.2

3.6

S 240--, G PG (6 % activity)

27--340

0.94 + 0.09

3.3 + 0.2

2.8

(0 % activity)

5. REFERENCES

1 Cervone, F., De Lorenzo, G., Degr~, L., Salvi, G. and Bergami, M., Plant PhysioL, 85 (1987) 631-637. 2

R. Granzow and R. Reed, Bio/Technology, 10 (1992) 390.

3 S.C. Schuster, 1LV. Swanson, L.A. Alex, ILB. Bourret and M.I. Simon, Nature, 365" (1993) 343. 4 G. Zeder-Lutz, D. Altschuh, I-I.M. Geysen, E. Trififiet~ G. Sommermeyer and M.H.V. Van Regenmortel, MoL lmmtmol. 30 (1993) 145. 5 D.J. O'Shannessy, M. Brigham-Burke, K.I~ Soneson, P. Hensley and I. Brooks, Anal. Biochem 212 (1993) 457.

782 6 L.D. Ward, G.J. Howlett, A. I-Iammacher, J. Weinstock, K. Yasukawa, 1LJ. Simpson and D.J. Winzor, Biochemistry, 34 (1995) 2901. 7 Y. Shinohara, H. Sota, F. Kim~ M. Shimizu, M. Gotoh, M. Tosu and Y. J. Hasegawa, Biochem (Tokyo) 117 (1995) 1076-1082. 8 C. Caprari, A. Richter, C. Bergmmm, S. Lo Cicero, G. Salvi, F. Cervone and G. De Lorenzo, Mycol. Res., 97 (1993) 497. 9 C. Baldari, J.A.H. Murray, P. Ghiara, G. Cesareni and C.L. Galeotti, EMBO J., 6 (1987) 229. 10 C. Caprad, B. Mattei, M.L. Basile, G. Satvi, V. Crescenzi, G. De Lorenzo and F. Cervone, submitted. 11 P. Toubart, A. Desiderio, G. Salvi, F. Cervone, L. Daroda, G. De Lorenzo, C. Ber~mann, A.G. Darvill, and P. Albersheim, Plant J. 2 (1992) 367. 12 G. De Lorenzo, F. Cervone, D. Bellincampi, C. Caprari, A.J. Clark, A. Desiderio, A. Devoto, 1L Forrest, F. Leekie, L. Nuss, and G. Salvi, Biochem_ Soc. Trans., 22 (1994) 396. 13 Cooke, 1LD., Ferber, C.E.M., and Kanagasabapathy, L., Biochim Biophys. Acta, 452 (1976) 440. 14

L. Rexov~-Benkov~, and M. Mrackovfi, Biochim Biophys. Aeta, 523 (1978) 162.


783

Rhamnogalacturonan r pyranosyluronide iyase, a new enzyme able to cleave RG regions of pectin ~ M. Mutter', I.J. Colquhoun b, G. Beldman', H.A. Schols', A.G.J. Voragen" 'Department of Food Science, Biotechnion, Bomenweg 2, 6703 HD Wageningen, The Netherlands, fax +31 317 484893 blnstitute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA United Kingdom ~Financial support was from Novo Nordisk A/S (Copenhagen, Denmark)

Abstract The recently described rhamnogalacturonase B (RGase B), which is able to degrade hairy regions of pectin, was found to be a rhamnogalacturonan (RG) ot-L-rhamnopyranosyl(1-->4)-c~-D-galactopyranosyluronide lyase. The cleavage site and mechanism are different from that of the previously described rhamnogalacturonase A (RGase A), which is a hydrolase, and can now be termed RG a-D-galactopyranosyluronide-(1--->2)-ot-L-rhamnopyranosyl hydrolase.

1. INTRODUCTION The classical pectolytic enzymes, active toward the homogalacturonan or "smooth" regions of pectin, have been shown not to be active toward the RG or "hairy" regions of pectin. Schols et al. (1990a) were the first to discover an enzyme, rhamnogalacturonase (RGase), able to degrade the backbone of hairy regions. Since then a specific RG acetylesterase (Searle-van Leeuwen et al., 1992) and a rhamnohydrolase (Mutter et al., 1994), specific for the terminal nonreducing Rha unit in RG regions, have been found by our group. Recently, in our laboratory, another enzyme able to degrade the backbone of hairy regions was discovered (referred to by Kofod et al., 1994), named RGase B. Both RGase A (Schols et al., 1990a) and RGase B have been cloned and expressed in Aspergillus oryzae. In their study, Kofod et al. (1994) could not give evidence that RGase B was an RGase. In the present study we prove that RGase B is indeed an RGase. Furthermore we show that the two RGases are different, since RGase B was shown to be lyase (RG-lyase) while RGase A is a hydrolase (RG-hydrolase). A more specific nomenclature for the two enzymes is suggested (Mutter et al., 1996).

784

2. RESULTS AND DISCUSSION RG-lyase was purified from Pectinex Ultra SP-L, produced by Aspergillus aculeatus, using anion- and cation-exchange chromatography. The purified RG-lyase differed from RGhydrolase in pI and pH optimum and stability (Table I). Table I Characteristics o f RG-lyase compared with those o f RG-hydrolase (Schols et al., .19..9oq.).................................................................................................................................. .........................................

.............................

Mw 51 kD ~ pl 4.1-4.5 p H optimum 3-4 ~ p H stability below p H 6* T optimum 40-50 ~ Ts(qb(t!tY ..............UP tO5OOff* . . . . . . . . . . . * according to Schols et al, 1990a

R

e ........................

57 kD 5.1-5.3 6 p H 6 and higher 50-60 ~ up to 40 ~ ....

Saponified Modified Hairy Regions (MHR) of apple pectin (produced and saponified according to Schols et al., 1990b) could be degraded by RG-lyase, as observed using highperformance size-exclusion chromatography (HPSEC), producing a slightly different degradation pattern as compared with RG-hydrolase (Fig. 1). More markedly was the difference in elution behaviour of the oligomeric reaction products upon high-performance anionexchange chromatography (I-IPAEC) (see elsewhere in these proceedings, Beldman et al.). The oligomers as produced from saponified MHR were isolated using Sephadex G50 and preparative HPAEC. 1D and 2D NMR experiments (COSY and ROESY) were used to determine the structure of the smallest oligosaccharide, eluting at 23 min upon HPAEC. The chemical shifts of the assigned peaks in the ~H NMR spectrum are summarised in Table II. 1

f.................................................................................................................................................................................................. ~b/e lg ...................H Chem~c~t ~h!f:s f o r the smattest Rq-teq~e ot:ige~fehqrJde ............. ....v...~.!t.....................................................C...h..e.m...!.~t...~h~..(~)............................................................................................ H-1 H-2 H-3 H-4 H-5 H-6 Rha

GalA Rha

us-GalA Gaff'

A~ Ap B C D

5.22 4.94 5.08, 5.16 ~ 5.32 5.13 4.63

3.97 4.06 3.94, 3.98 ~ 4.32 3.80 3.50

4.09 n.d. 4.13, 4.15 ~ 4.08 4.34 3.66

3.71 n.d. 4.43

3.95 n.d. 4.63

1.34 n.d. -

3.62 5.81 3.90

3.85 n.d.

1.29 n.d.

n.d., not determined; a, Two values are for unit B linked to a- and fl-forms o f the reducing end unit, respectively; b, Two residues, 8 values differ by < 0.01 ppm.

The most important difference in the spectrum as compared with RGs released by RGhydrolase action (Colquhoun et al., 1990) was a doublet at 5 5.81 (J = 3.4 Hz). From the COSY experiments this doublet was found to belong to a four-proton spin-coupling network

785 that had chemical shifts and coupling constants characteristic of an a-linked A-4,5-unsaturated GalA residue at the nonreducing terminus (Tjan et al., 1974). For this residue the anomeric signal was at/5 5.13, and the doublet at 8 5.81 was assigned to the olefinic proton. Finally, the structure deduced for the oligosaccharide was: D

C

B

A

o~-D-us-GaleA-(1-->2)--tx-L-Rhap-(1-~4)-tx-D-GaleA-(1-~2)-L-Rhap. 4 4

1'

1'

1

1

l~-D-Galp

l~-D-Cralp.

The oligomer contained an alternating RG chain, similar to the previously published structure of the RG oligomers liberated by RG-hydrolase from saponified MHR (Colquhoun et al., 1990). However, the reducing and nonreducing ends had interchanged. Most surprising was the presence of the unsaturated bond in the nonreducing GalA unit. This showed that the new enzyme cleaved the backbone by 13-elimination (Mutter et al., 1996).

Absorbance

/~

~

RG-lyase RG-hydrolase ......~----~--'*~'~-~.---. Blank

16

20

24

28

32


36

16

L

20

24

28

32

Retentiontime(min)

36

Figure 1 HPSEC chromatograms of saponified A,fftR (bottom chromatograms); saponified MHR after degradation by RG-hydrolase (middle chromatograms); and saponified MHR after degradation by RG-lyase (top chromatograms). Left chrornatograms give the RI signal, right chromatograms the UV absorbance at 235 nm. After cleavage by [3-elimination, conjugation of the double bond with the carboxyl group at C5 of the 4,5-unsaturated GalA occurs. In homogalacturonan chains, the absorption maximum is at 235 nm. The absorption maximum for the 4,5-unsaturated GalA in RG chains was found to be the same. Figure 1 shows the HPSEC chromatograms of saponified MHR before and after degradation by RG-hydrolase and RG-lyase, using simultaneous detection by refractive index and absorption at 235 nm. In the region where oligosaccharides eluted from the column (32 min), upon UV-detection a large peak appeared only in case of the RG-lyase. Activity of RG-lyase toward various substrates was measured from the increase in the A:35 (Table III).

786 Table 111

Activity o f RG-lyase toward various substrates (0. 02-0.1%w/v) (U rag-l), determined

Substrate 6~ od' ............................................................................................ P_,H .............................. P_:H ...................... -Ca

MHR-S 8.8 Linear RG oligomers 3.9 pectin D M 92.3% 0 PGA 0 "~i%'iJO"~H::61:i'i:::20~p:H:8

+Ca c

-Ca

+Ca

9.8 n.d. 0 0

10.6 n.d. 0 0

11.8 n.d. 0 0 ; ~, 1 mM CaCI2;

RG-lyase was not active toward polygalacturonic acid or highly methoxylated pectin, also not with additional calcium ions. Considering the specificity of the enzyme for RGs, and given the type of linkage cleaved and the cleavage mechanism, the appropriate name is RG otL-rhamnopyranosyl-(1--->4)-a-D-galactopyranosyluronide lyase, abbreviated as RG-lyase. To our knowledge, the existence of an RG-lyase has not been reported before.

3. R E F E R E N C E S Colquhoun IJ, de Ruiter GA, Schols HA, Voragen AGJ (1990) Carbohydr Res 206:131-144 Kofod LV, Kauppinen S, Christgau S, Andersen LN, Heldt-Hansen HP, D6rreich K, Dalboge (1994) J Biol Chem 269:29182-29189 Muttter M, Beldman G, Schols HA, Voragen AGJ (1994) Plant Physiol 106:241-250

Mutter M, Colquhoun IJ, Schols HA, Beldman G, Voragen AGJ (1996) Plant Physiol 110: 7377 Schols HA, Geraeds CCJM, Searle-van Leeuwen MF, Kormelink FJM, Voragen AGJ (1990a) Carbohydr Res 206:105-115 Schols HA, Posthumus MA, Voragen AGJ (1990b) Carbohydr Res 206:117-129 Searle-van Leeuwen MJF, van den Brock LAM, Schols HA, Beldman G, Voragen AGJ (1992) Appl Microbiol Biotechnol 38:347-349 Tjan SB, Voragen AGJ, Pilnik W (1974) Carbohydr Res 34:15-32


787

Purification and characterisation of galactose-induced pectinases from the exo-1 mutant strain of Neurospora crassa L.B. Crotti, J.A. Jorge, H.F. Terenzi and M.L.T.M. Polizeli Departamento de Biologia - Faculdade de Filosofia, CiEncias e Letras de Ribeir~o Preto, Universidade de S~o Paulo, Av. Bandeirantes 3900, 14040-901 - Ribeir~o Preto, S~o Paulo, Brasil Abstract Pectinases produced by the exo-1 mutant of N. crassa in galactose plus glucose supplemented medium, were separated by ion-exchange chromatography into two pools. Pool I contained pectate and pectin lyases, and variable polygalacturonase activity. Pool 2 contained polygalacturonase activity only. Gel filtration indicated a MWapp of 80 kDa (higher than those of separate enzymes) for all activities in the first pool, suggesting a complex. Polygalacturonase, pectin and pectate lyases were purified 39-fold, 22-fold and 33fold, respectively. Optima of temperature and pH were 45~ and 5.5 for polygalacturonase activity and 50~ and 9.5 for lyase activities. Km and Vmax values for polygalacturonase were 0.023 mg polypectate/ml and 2.08 ~moles (reducing sugar)/min/mg protein.

1. INTRODUCTION Previous studies from our laboratory [1,2] demonstrate that the filamentous fungus Neurospora crassa produces pectic enzymes as effectively as other hydrolases such as

cellulases [3], amylases [4], and xylanases [5]. The production of polygalacturonase was studied using the mutant strain exo-1. This interesting strain exhibits a rather exaggerated synthesis and secretion of several exoenzymes, among others amylase and invertase [6]. We demonstrated that this strain, when cultivated in the presence of pectin as the sole carbon source, secretes five to six times more than the wild type a glucose-repressible endopolygalacturonase [2]. Interestingly, the production of polygalacturonase was also induced by galactose, four times more efficiently than by pectin. The inducing effect of galactose, different of that of pectin, was not counteracted by glucose. Thus, we decided to investigate in more detail the effect of galactose as inducer of pectolytic activities and to biochemically characterise the pectolytic complex produced by the N. crassa exo-1 strain in the presence of galactose and glucose. 2. METHODS Culture conditions: The exo-1 strain was cultivated in two-stages: (I) pre-cultivation for 24 hours in Vogel's medium [7] supplemented with 2% glucose, and (II) transfer of the mycelial

788 mass to fresh medium supplemented with 2% glucose plus 2% galactose or other carbon sources, for 48 or 72 hours, according with the experiment, at 30oc, with agitation.

Enzymatic assays: Polygalacturonase was assayed: (a) by measuring the amount of reducing sugar released from sodium polypectate as a substrate. An enzyme unit is the amount which releases reducing sugar at an initial rate of l~mol/min at 30oc, using galacturonic acid as the standard [8]. Co) By the decrease in relative viscosity of a 0.2% pectin solution using an Ostwald viscometer. One activity unit was expressed as a percentage (50%) change in viscosity [9]. Lyase activities were measured by the increase in A232 nm of the unsaturated products of degradation of pectin or sodium polypectate. One activity unit was the amount of enzyme which released 1 lxmol of unsaturated product per minute [10]. Protein was determined by the Lowry method using bovine serum albumin as standard [11 ]. Separation of pectic enzymes: The crude filtrate was precipitated with 2 volumes of ethanol for 2 hours at -20oc and then centrifuged at 15,900g for 10 minutes. The precipitate was dissolved in 10 ml of Tris-HC1 buffer 10mM, pH 7,5 (buffer A) and applied to a DEAEcellulose column (1,6 x 20cm) equilibrated and eluted with buffer A. The flow-through protein was dialysed against 10raM sodium acetate buffer, pH 5,0 (buffer B) and applied to a CM-cellulose column (1,6 x 25cm). The column was eluted with a NaC1 gradient (0 500mM) in buffer B. Fractions (10ml) were collected at a flow rate of 33.5 ml/h. Determination of molecular mass of pectic enzymes: The molecular mass were determined by gel filtration in a Sepharose CL-6B column (1,8 x 88cm) equilibrated and eluted with TrisHC150 mM, pH 7,5 buffer, plus 100 mM KC1. Fractions (3,3 ml) were collected at a flow rate of 10 ml/h. Molecular mass markers were: tyroglobulin (660 kDa); apoferritin (440 kDa); 13amylase (200 kDa); alcohol dehydrogenase (150 kDa); bovine serum albumin (66 kDa) and carbonic anhydrase (29 kDa). Urea-SDS-PAGE (7%) was carried out according to Swank and Munkres [12]. Molecular mass markers were: myosin (205 kDa); [3-galactosidase (116 kDa); phosphorylase b (97 kDa); bovine serum albumin (66 kDa), ovalbumin (45 kDa) and carbonic anhydrase (29 kDa). Determination of neutral carbohydrate: Total neutral carbohydrate in protein samples was estimated by the phenol/sulphuric acid method of Dubois [13] using mannose as standard. Chromatographic characterisation of hydrolysis products: Hydrolysis products from sodium polypectate were analysed by thin-layer chromatography on silica gel G-60, using ethyl acetate / acetic acid / formic acid / water (9:3:1:4, by volume) as the mobile phase system. Sugars were detected with 0,2% orcinol in sulphuric acid-methanol (10:90ml) [14]. 3. RESULTS AND DISCUSSION. The effects of galactose and pectin as inducers of polygalacturonase activity in the exo-1 N. crassa strain is shown in figure 1. Both substances were efficient inducers, but in the presence of galactose the enzyme production was about four-fold higher than with pectin. A remarkable difference between induction with pectin or with galactose, was that the former was severely repressed by glucose, whereas galactose induction was not repressed by addition of glucose.

789

.~ > .i

1,0

ci.

t~

0,8

19 U) t-

0,6

/ / / / / /

/

/

/

/

. , . . , / / , ,

.• ,\/• ?

2

./

/

'

./

/

/

/

/

/

,/

/

/

/

I

,,

,,

/

/

/

/

/

/

/.,

/

~

i

i

0,4

i

I:~ i. 0

/

0,2

.

.

gal + glu

0,0

no carbon pect pect + glu

gal

Figure 1. Effect of 1% (w/v) pectin (pect), 1% galactose (gal), and of the simultaneous presence of 2% glucose (glu) on the production of extracellular polygalacturonase activity. Two-stages cultures were prepared as described under methods. Polygalacturonase was assayed in the culture filtrate as reducing sugar-releasing activity using sodium polypectate as a substrate. These results prompted us to examine the characteristics of the extracellular pectolytic enzymes secreted in medium supplemented with glucose and galactose. Figure 2 shows the profile of elution of pectolytic activities recovered from the flow-through of a DEAEcellulose column chromatographed on a CMC-cellulose column.

~~ NaCI0~'_ l:: 12

m

8

~"

0,25

v

0,20

t~ C

0,15

"~

2

I

im

6

0,10

19

0,05

0

E

0,30

19

10

t~ >,,

0,35

0

20

40

60

fractionnumber

9

80

I~ 0 Q.

0,00 100

Figure 2. CM-cellulose chromatography of pectolytic enzymes. The activity peaks of the flow-through of a DEAE-cellulose chromatography was applied to a CM-cellulose column. The column was eluted with a NaC1 (0-0.5M) continuous gradient at a flow rate of 34 ml/h. 10 ml fractions were collected and assayed for pectolytic activities Symbols: (O) pectate lyase; ($) polygalacturonase (reducing sugar-releasing activity); (x) protein. Other details in Methods.

790 Polygalacturonase activity eluted into two main fractions, the first coeluting with pectate lyase (and pectin lyase, not shown) activities, and the second free of other activities. When the first peak was rechromatographed under the same conditions, identical result was obtained. The distribution of polygalacturonase in the two peaks varied with the experiment. In other cases, lyases and polygalacturonase activities separated completely into two peaks, one containing the two lyases and the other containing polygalacturonase activity only. Interestingly, at this stage of separation all pectolytic activities had reached a considerable degree of purification: polygalacturonase was purified about 39-fold, while pectate and pectin lyases were purified 33-fold and 22-fold, respectively. Gel filtration of the peak showing associated lyases and polygalacturonase activity (Figure 3A) gave a single activity peak eluting with a MWapp of approximately 79.4 kDa, suggesting the existence of a multienzyme complex. On the other hand, the same peak run under denaturing urea-SDS-PAGE (Figure 3B) was resolved into two bands, one of pectate/pectin lyase activity (MWapp 56.2 kDa) and a second band with polygalacturonase activity (MWapp 44.7 kDa). 6,5 6,0 r

E _~

5,4 5,2

5,0 4,5

pectin lyases

4,8

pectate/pectin lyase

+ polygalacturonase

4,0

4,6

3,5 310

2 3

5,0

0

~

B

5,5

(D

O E

1

A 1 2

4,4 i

1,6

,

i

1,8

i

VeNo

i

2,0

,

i

2,2

0,0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

Rf

Figure 3. (A) Determination of molecular mass of pectic enzymes by gel filtration in Sepharose 6B. Molecular mass markers:l- tyroglobulin, 2- apoferritin, 3- 13-amylase, 4alcohol dehydrogenase, 5- bovine serum albumin, 6- carbonic anhydrase. (B) SDS-PAGE of pectolytic activities. Molecular mass markers: 1- myosin, 2- 13-galactosidase, 3- phosphorylase b, 4- bovine serum albumin, 5- ovalbumin, 6- carbonic anhydrase.

Table i shows some biochemical properties of the pectolytic enzymes present in pool 1. The pectin lyase/pectate lyase activities (pool I) and polygalacturonase activity (pool II) were not significantly affected by NH4+, Na + and K + (0,25 - 2,5mM), while A13+, 13-mercaptoethanol, Hg 2+, EDTA, Ba 2+ and Zn +2 (2,5mM) inhibited 30-100% these activities. On the other hand, Ca2+, Mg 2+ and Mn 2+ at 2,5mM concentration activated 20-100% pectin/pectate lyases but Ca 2+ and Cu 2+ (2,5mM) inhibited polygalacturonase activity about 42 - 70%.

791 Viscosimetric assays and analysis of hydrolysis products by thin layer chromatography (TLC) were used to determine the mechanism of action of the polygalacturonase on sodium polypectate. The time required for 50% decrease in viscosity of a 2.0% (w/v) substrate solution at 45~ was approximately 105 min, at which time about 9% of total galacturonide bonds had been hydrolysed. The products of hydrolysis, analysed by TLC, demonstrated that oligogalacturonates accumulated initially, but the monomer was found after 24 h of reaction (not shown). These results suggested that the polygalacturonase of the mutant exo-1 of N.crassa induced by galactose in the medium exhibited a random mechanism of hydrolysis of sodium polypectate, suggesting that is was an endopolygacturonase.

Table 1 Kinetic constants and others intrinsic properties of pectolytic activities Parameters

Polygalacturonase

pectate/pectin lyases

reducing sugar

viscosity

pectin

pectate

KM (mg/ml)

0.023

n.d.

0.076

0.50

Vmax (U/min/mg protein)

2.08

n.d.

363.4

273.2

neutral carbohydrate

38.8%

38.0% (*)

optimal temperature

45~

45~

50~

500C

thermostability 60~ (T50 -min)

5

30

1.5

3

optimal pH

5.5

4.5

10

9

stability pH

5.0

4.0-5.5

9.5

10

(*) the sugar content of lyases was determined in a fraction containing both activities.

792

References

5 6 7 8 9 10 11 12 13 14

M.L.T.M. Polizeli, R.C.L.R. Pietro, J.A. Jorge and H.F. Terenzi, J. Gen. Microbiol., 136 (1990) 1463. M.L.T.M. Polizeli, J.A. Jorge and H.F. Terenzi, J. Gen. Microbiol., 137 (1991) 1815. B.M. Eberhart, R.S. Beck and K.M. Goolsby, J. Bacteriol., 130 (1977) 181. R.D. Sigmund, M.T. McNally, D.B. Lee and S.J. Free, Biochem. Genet., 23 (1975) 89. C. Mishra, S. Keskar and M. Rao, Appl. Environ. Microbiol., 48 (1984) 224. H.G. Gratzner and D.N. Sheehan, J. Bacteriol., 97 (1969) 544. H.J. Vogel, Am. Nat., 98 (1964) 435. G.L. Miller, Anal. Biochem., 31 (1959) 426. R. Tuttobello and P.J. Mill, Biochem. J., 79 (1961) 51. C.W. Nagel and M.M. Anderson, Arch. Biochem. Biophys., 112 (1965) 322. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. R.W. Swank and K.D. Munkres, Anal. Biochem., 39 (1971) 462. M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers and F. Smith, Anal. Chem., 28 (1956) 350. J.D. Fontana, M. Gebara, M. Blumel, H. Schneider, C.R. Mackenzie and K.G. Johnson, Methods Enzymol., 160 (1988) 560.


Acetyl esterases of Aspergillus purification and mode of action

793

niger: on

pectins

M.J.F. Searle-van te, euwen, J.-P. Vincken, D. Schipper*, A.G.J. Voragen, G. Beldman. Dept. of Food Science, Agricultural University, Bomenweg 2, 6703 HD Wageningen, The Netherlands *Gist- brocades, P.O. Box 1, 2600 MA, Delft, The Netherlands

Abstract Acetyl esterases with different specificity occur in one AspergiUus niger preparation. Three acetyl esterases were purified and characterised: pectin acetyl esterase (PAE), feruloyl acetyl esterase (FAE) and rhamnogalacturonan acetyl esterase (RGAE). Only PAE, a novel acetyl esterase, could remove acetyl from beet pectin, to a maximum of 30%. This was shown to be one specific acetyl group in the,homogalacturonan chain of pectin (smooth region) by NMR spectroscopy. PAE activity was influenced by buffer salts and the addition of bivalent cations. PAE worked cooperatively with pectolytic enzymes. Contrary to PAE, RGAE removed at random the acetyl esters from apple pectic hairy ramified regions (MHR), to a maximum of 70 %. RGAE was essential for the activity of rhamnogalacturonase (RG), and as such comparable with A. aculeatus RGAE. FAE was specific for esterified xylan-oligomers, but did not show selectivity towards a specific ester. This enzyme could release ferulic acid as well as acetyl groups from esterified arabinoxylans in the presence of an endoxylanase.

1. INTRODUCTION Pectins are an important part of plant cell walls. Two structurally different regions can be distinguished in pectins: a "smooth" homogalacturonan region and a highly branched "hairy" or ramified region with side chains composed of arabinose and galactose [1]. The hairy regions contain a high amount of rhamnose in the main chain. The galacturonic acid of the two regions can be esterified with both methyl and/or acetyl groups. Also feruloyl esters occur in pectins, and these are found linked to the side chains. All the above mentioned substitutions influence the gelation properties of pectins [2], and consequently their use in the food industry as gelling agents.

794 Enzymes can be used to specifically modify the pectins. Pectin methyl esterase is already widely used to adjust the gelling properties of commercially available pectins. The acetyl esters also strongly affect the gelation [2,3] and removal is important for the upgrading of sugar beet pectin, extractable from a by-product of the sugar industry. This study deals with the purification and characterisation of acetyl esterases from A.niger with different specificity.

2. METHODS

2.1. Enzyme purification Rapidase C-80 (Gist-brocades) was used as enzyme source. The fractionation procedure of the crude preparation included chromatography on Bio-Gel P10 (100-200 mesh), DEAEBio-Gel A, and Bio-Gel HTP (Bio-Rad, Richmond, CA, USA). Other column materials used were cross-linked alginate (degree of cross-linking 2.34, prepared in our laboratory), Phenyl Superose HR 5/5 and Mono Q HR 5/5 (Pharmacia Biotech, Uppsala, Sweden).

2.2. Enzyme activities Acetyl esterase activities were determined toward either a solution of sugarbeet pectin (1% w/v), or one of several acetylated polysaccharides and synthetic substrates using equivalent acetyl concentrations. Incubations were carried out in piperazine buffer at pH 6.0 and 30 ~ for 1-24 hours. The reaction was stopped by heating for 5 min at 100~ Acetyl release was determined using HPLC [4] or using an enzymatic assay (BoehringerMannheim, Germany). Enzyme activities were expressed as units: one unit (U) corresponds to the release of one/~mol acetyl group per minute under the above standard conditions. KP pectin from sugar beet pulp was from Kobenhavns Pectinfabrik (Lille Skensved, Denmark). G-pectin was extracted from the whole sugar beet by Grindsted Products, Denmark. A preparation of modified hairy regions (MHR) was isolated from apple [5]. The non pectic acetylated substrates are described elsewhere [6,7,8]. Other enzyme activities, and protein content were determined as reported previously [8]. 600 MHz 1H NMR spectra were measured on solutions of ca 25 mg pectin/0.5 ml D20 on a Bruker AMX600 NMR spectrometer. The temperature was 350 K to diminish the viscosity of these solutions and 32 scans were measured. Solvent suppression was performed using presaturation during the recycle delay. 3. RESULTS AND DISCUSSION

3.1. Enzyme purification and characterisation Acetyl esterases were isolated from a Rapidase C-80 preparation according to the scheme shown in Figure 1. The purified acetyl esterases were devoid of relevant side activities, and showed great differences in their specificity towards the different acetylated substrates. The characteristics of the purified enzymes are summarised in Table 1. PAE was the only enzyme able to remove acetyl from beet pectin, FAE showed the highest activity towards

795 triacetin and xylo-oligomers, while RGAE was specific for MHR.The specificity of the enzymes will be discussed further for each enzyme separately

I crude A. niger prep]

11

i,, Bio..Gei P 10 i

]

.....

I,. D EAE Bio-GeI,A

lcrosslalginate I

IBio-Gel HTP

I FPLC

HTP-I

PAE

i ii

,l,

' Bio'-' Gel ,

iii

i

i

P 100

I

Mono Q i

AE I!1

,,,

HTP-un i

AE !1

IAEI

',''

ii

I

pectin acetyl eMerase

FAE

RGAE

feruloyl AE

rhamnogalacturonan AE

Figure 1. Purification scheme of acetyl esterases.

Table 1 Characterisation

of the acetyl esterases

activity in mU/mg protein G beet pectin Triacetin max acetyl release (% of total) KP beet pectin G beet pectin MHR ac xylan oligomers ac xylan polymers ac galacto gluco mannan Mol weight (Bio-Gel P-100, kDa) pI pH optimum temp optimum ( ~C) n.d., not determined

PAE

FAE

RGAE

6000 275

0 16358

0 0

28 30 10 9 0 0.5

0 0 0 31 4 0

0.6 0.4 70 0 0 0

60 4.1 5.5 50

>100 n.d. n.d. n.d.

42 4.5-6 5.5 50

796 3.2. PAE

This novel enzyme was the only esterase able to release acetyl from sugar beet pectin and removed about 30% of the total acetyl groups present. It also caused the release of acetyl groups from a range of other acetylated substrates, either synthetic or extracted from plants, in small amounts. PAE had an apparent molecular weight of 60 kDa and showed optimal activity at pH 5.5 and a temperature of 50 ~ The enzyme is sensitive to buffer composition and requires a bivalent cation for optimal activity and stability. In purified form this enzyme proved unstable, especially in phosphate buffers. Pectolytic enzymes appeared to have an influence on the initial rate of deacetylation of beetpectin by PAE, but not on the total amount of acetyl groups released. This remained a maximum of about 30 %. Pretreatment of the highly esterified G-pectin with pectin methyl esterase (PME) or pectin lyase (PL) resulted in an increase in the activity of PAE. As can be seen from Figure 2 removal of about 50 % of the original methyl esters resulted in a decrease of PL activity to almost zero, while PG and PAE activity increased at this point. At higher degrees of demethylation, the measurement of PG and PAE, with a strong requirement for bivalent ions is hampered by a tendency of the deesterified pectin to gelate. The actual increase of activity could therefore be higher than indicated. A decrease in the pectic molecular weight by pectin lyase resulted in an increase in the activity of PAE (Figure 2). This could be caused by easier access of the enzyme to the substrate, but PAE is not limited in its action by the substrate size as was found for FAE.

250 c Ot

250

PG

200

PAE

~= 200 E~ "E:

o 150

o 150

.=.. 0

0

z~

100

--- 100 z',

~

~ 5o

"~

50

........................................................................

PL 0

20

40 60 % demethylation

80

1oo

0

200 .. 400 600 800 1000 mint~es preincubation PL (200 mu/ml)

1200

Figure 2. The effect of enzymatic demethylation of G-pectin on the activity of PAE, PG and PL (left) The effect of pretreatment of G-pectin with PL on the activity of PAE (right) 'H NMR spectra analysis of KP-pectin and G-pectin revealed not only a substrate, but also a location specificity of PAE. The ~H NMR spectra of these pectines show two major types of acetyl groups; at 2.10 and 1.95 ppm. Upon treatment with PAE the resonance at 1.95 ppm disappears completely whereas the resonance at 2.10 ppm is only slightly reduced. The origin of these two acetyl groups is not completely clear. The signals might be related to substitutions on C-2 or C-3 of the galacturonic acid moieties, or even with single or double

797 substitutions on the same galacturonic acid. Due to the high molecular weight of these molecules and the high viscosity of the pectin solutions, even at high temperature, more sophisticated NMR techniques to locate the exact position of substitution failed as yet. 3.3. RGAE As this enzyme proved specific for the release of acetyl groups from MHR, to a maximum of 70 %, and is essential for the degradation of MHR by RG ( Figure 3), it was concluded to be comparable to that isolated previously from A. aculeatus[8,9]. The importance of this enzyme in the application of tailormade commercial pectinases is discussed elsewhere in these proceedings as well (H.P. Heldt-Hansen et al.).

(~ x (i)

,,

6

i

c-

,

I ~

.~..~,

,,

0

(I) '

o

;o

0 i

O ~

O

: ,,'/

','V O

O / O

',v

t.

,.'~:

:,,,q.'"

"~-,... _,.," ~".

d)

cc

:,/'

~-~, o o o

'qi

. ~-~-~

"8

20

I9

22

I-o- blank

I

24

I.

I

26

28

Time (min.) --,,,-- RGAE --~- RG

I

30

I

32

Comb. I

Figure 3. Degradation of MHR by RGAE, RG and the combination of these enzymes, as determined by HPSEC.

1H NMR spectra of MHR showed a more complex pattem of resonances in the acetyl group region. Although there appears to be two main groups of acetyl substituents, upon careful analysis after resolution enhancement both main signals consist of at least three different acetyl resonances. After treatment with RGAE (70% of acetyl groups removed) the overaU pattern remains unchanged, indicating that there is no preference of this enzyme for an acetyl group at a specific position. So RGAE differs from PAE in this aspect as well.

798 3.4. FAE This enzyme was shown to be specific for xylan oligomers and small acetylated synthetic substrates. Many characteristics have been published recently about this type of enzyme, purified from Trichoderma reesei, and A. oryzae [6], and a different A. niger preparation[7] FAE was identical to these enzymes in requiring an endoxylanase for activity on xylans. Under these circumstances ferulic acid could be released too. It has been observed that for the complete breakdown of different xylans, different enzyme combinations are required, as the substitutions on the xylan chain can vary widely [7]. No activity could be shown on pectins, neither in combination with other AE's and/or pectolytic enzymes.

4. CONCLUSIONS The A. niger preparation investigated in this study contains at least three different acetyl esterases, each with its own specificity. The activities of RGAE and FAE are comparable to those of similar enzymes isolated previously from A. aculeatus and a different A. niger preparation. PAE appears to be a new enzyme, with an activity specific towards one type of acetyl ester in the homogalacturonan chain of beet pectin.

5. LITERATURE J.A. De Vries, F.M. Rombouts, A.G.J. Voragen, and W. Pilnik, Carbohydr. Polym., 2 (1982) 25. G.L. Pippen, R.M. McCready, and H.S. Owens, J. Amer. Chem. Soc., 72 (1950) 813. J.A. Matthew, S.J. Howson, M.H.J. Keenan and P.S. Belton, Carbohydr. Pol., 12 (1990) 295. A.G.J. Voragen, H.A. Schols and W. Pilnik, Food Hydrocoll., 1 (1986) 65. H.A. Schols, M.A. Posthumus and A.G.J. Voragen, Carbohydr. Res., 206 (1990) 117. M. Tenkanen, PhD thesis Helsinki University of Technology (Espoo, Finland), VIT publications 242 (1995). F.J.M. Kormelink and A.G.J. Voragen, Appl. Microbiol. Biotechnol., 38 (1993) 688. M.J.F. Searle-van Leeuwen, L.A.M. van den Broek, H.A. Schols, G. Beldman and A.G.J. Voragen, Appl. Microbiol. Biotechnol., 38 (1992) 347. S. Kauppinen, S. Christgau, L.V. Kofod, T. Halkier, K. Dorreich and H. Dalboge, J. Biol. Chem., 270 (1995) 27172.


799

Polygalacturonase inhibitor of Dieffenbachia maculata Archana Chitre and N.V. Shastri University Department of Biochemistry, Amravati Road, Nagpur, 440010, India.

ABSTRACT Stem juice of Dieffenbachia maculata contains an inhibitor of fungal polygalacturonase. The inhibitor is non dializable and heat stable. The double reciprocal plot indicates that the inhibitor causes a mixed type of inhibition. The paper also describes the distribution of the inhibitor in different varieties of Dieffenbachia, and some of its properties.

INTRODUCTION Naturally occuring enzyme inhibitors are often found as major components of the cytoplasm, secretions and intercellular fluids of many organs and tissues. Although a large number of reports are available on the presence of various enzyme inhibitors, both in animal and plant systems, comparatively very little work appears to have been done on inhibitors of pectolytic enzymes (Fielding, 1981; Resova et al., 1981; Akira and Murakawa, 1993; Mueller and Gersler, 1993). During the course of our investigations, a potent inhibitor of polygalacturonase (PG) was detected in the stem extract of Dieffenbachia, a plant known for its therapeutic effects (Barnes and Fox, 1955; Walter and Khanna, 1972; Ardditti and Rodriguez, 1982), which may, in part, explain the resistance of this plant to fungal attack. Polygalacturonase inhibitors have now gained increased importance in biotechnology, as the transfer of a PG inhibitor gene has yielded a transgenic tomato variety of much longer shelf life. This chapter describes investigations conducted on the PG inhibitor of Dieffenbachia maculata .

MATERIALS Polygalacturonase used in the present investigations was a A. niger pectinase (E.C. 3.2.1.15) purchased from Sigma Chemical Company (St. Louis, Missouri, USA). Polygalacturonic acid (PGA) sodium salt, polyvinylpolypyrolidone (PVPP), galacturonic acid (monohydrate) were also purchased from Sigma Chemical Company. 3,5-Dinitrosalicylic acid was the product of Loba Chemicals, Bombay, India. All other reagents were of analytical grade.

METHODS The A. niger PG preparation was suitably diluted with distilled water and was used as the enzyme source. Polygalacturonic acid was prepared as a one percent solution of in distilled water.

800

Preparation of inhibitor'. All the operations were carried out at 0-4~ unless otherwise mentioned. For the preparation of stem extract fresh stem was cut into small pieces and crushed mechanically. The pulp obtained was squeezed through two layers of muslin cloth. The juice was made 1% with PVPP to remove polyphenols, stirred gently for 10 minutes and cintrifuged at 1 ~ xg for 30 minutes 0-4~ The supernatant was dialysed against water. The precipitate formed was removed by centrifugation and the supernatent was used as the partially purified inhibitor. For the preparation of leaf and petiole extracts respective parts were homogenised with 1% PVPP using acid-washed sand and suspended in distilled water to get a 25 % homogenate, which was centrifuged at 10000 xg for 30 minutes. The supernatent obtained was dialysed and used as the inhibitor. Polygalacturonase assay: Polygalacturonase activity was assayed by measuring the liberated reducing groups by DNS reagent. The reaction mixture in a final volume of 2 ml contained 0.2 ml of a suitable aliquot of PG solution, 40 mM acetate buffer (pH 4.0) and 0.5% polygalacturonic acid. After 30 minutes incubation at 37~ the reaction was terminated by adding 1 ml of DNS reagent. In control tubes, the reaction was terminated prior to the addition of the substrate. The reaction mixture was then kept in a boiling water bath for 10 minutes, cooled and diluted to 13 ml with distilled water. The colour intensity was measured at 540 nm. A calibration curve established with galacturonic acid monohydrate was used to calculate the polygalacturonase activity. One unit of PG activity is defined as that amount of enzyme which liberates 1 mg galacturonic acid under the given assay conditions (30 min, 37~ Specific activity is expressed as units per mg of protein. Inhibitor assay: A suitable amount of inhibitor was preincubated with 0.2 ml of polygalacturonase and buffer in a total volume of 1 ml for 10 minutes at 37~ Control without inhibitor was run simultaneously. The enzyme reaction was initiated by the addition of 1 ml of substrate solution (1% polygalacturonic acid). The decrease in PG activity was a measure of the inhibitory activity. Proper controls containing only Dieffenbachia extract and no fungal PG in the assay mixture were also run to account for the inherent PG activity, if any, of Dieffenbachia extract. One unit of inhibitor activity is defined as the amount of inhibitor that reduces the polygalacturonase activity under the assay conditions by one unit. Specific activity of the inhibitor is expressed as units per mg protein. Protein estimation: Proteins were determined by the method of Lowry et al. (1951), using bovine serum albumin as standard.

RESULTS Each experiment was carried out several times and representative values are given.

PG inhibitor in different varieties of Dieffenbachia: Polygalacturonase inhibitor activity in whole stem was examined in different varieties of

Dieffenbachia. The data presented in Table 1 shows that the level of PG inhibitor in different species of Dieffenbachia is not significantly different. PG inhibitor activity in various parts of D. maculata: PG inhibitor was extracted from various parts of D. maculata as well as from different sections of young and mature stem by method described above. Table 2 shows that, although highest specific activity of PG inhibitor was observed in the petiole, stem tissue showed much higher concentration of inhibitor units per ml. Leaf showed the least levels of inhibitor activity.

801 Table 1 PG inhibitor in stem of different Dieffenbachia varieties.

Variety

]aahibitor units/ml

Dieffenbachia Dieffenbachia Dieffenbachia Dieffenbachia

maculata exotica amoena picta

2.00 2.87 2.62 2.17

Table 2 PG inhibitor in different parts of D. maculata.

Plant Part Units/ml

Leaf Petiole Stem (whole)

0.37 0.25 1.58

PG Inhibitor Activity Proteins/ml Specific activity (U/mg protein) 1.80 0.24 2.43

0.21 1.04 0.65

PG inhibitor in various parts of D. maculata stem: Various parts of the stem, in turn, showed variable inhibitor activity, the middle (1/3rd of the total stem length) section showing highest inhibition followed by the apical and the basal sections (Table 3). The age of the plant does not seem to have much significant effect on the level of PG inhibitor.

Table 3 PG inhibitor in different sections of D. maculata stem.

Stem Section Units/ml

9PG Inhibitor Activity Proteins/ml Specific activity (U/mg protein)

Young Stem Apical Portion Middle Portion Basal Portion

1.65 2.00 1.45

2.80 3.00 2.40

0.59 0.66 0.60

Mature Stem Apical Portion Middle Portion Basal Portion

1.62 1.87 1.25

2.56 2.64 2.08

0.63 0.71 0.60

802

Properties of D. maculata stem PG inhibitor: Effect of dialysis: Stem juice dialysed against distilled water for 16 hours. PG inhibitor activity was examined in the dialysate after 16 hours after removal of precipitate by centrifugation. Table 4 shows that the inhibitor is more or less non-dialysable although a part of its activity is lost during dialysis. Dialysis results in about 3 fold purification of the inhibitor. Dialysed inhibitor was used in subsquent studies.

TaMe 4 Effect of dialysis on PG inhibitor of D. maculata stem.

Fraction

Total Inhibitor Units/ml

Proteins mg/ml

Specific activity

Purification fold

Crude inhibitor

1.86

2.12

0.88

1.00

Dialysed inhibitor

1.38

0.56

2.46

2.78

Effect of heat treatment on PG inhibitor activity: Suitable aliquots of D. maculata stem inhibitor were kept in a boiling water bath for various periods of time, cooled quickly and assayed for residual PG inhibitory activities.

Table 5 Effect of heat treatment on PG inhibitor of D. maculata stem.

Time of exposure in boiling water bath (min) 0 10 20 30 40 50 60

Percent PG inhibitor activity remaining 100 100 93 93 93 83 83

As shown in Table 5, the PG inhibitor is relatively heat stable, retaining about 83 % of its activity after one hour at boiling water bath temperature.

Effect of preincubation of PG inhibitor with PG: A suitable aliquot of the inhibitor was incubated with 0.2 ml of polygalacturonase and buffer in a total volume of 1 ml for various periods of time at 37~ The reaction was initiated by addition of 1 ml of 1% polygalacturonic acid solution. It is apparent from Table 6 that the magnitude of inhibitory

803 activity was dependent on the preincubation of the inhibitor with polygalacturonase. Maximum inhibition was observed with preincubation for 10 minutes at 37~ Table 6 Effect of preincubation of PG inhibitor of D. maculata with PG.

Preincubation mixture Inhibitor + Polygalacturonase

Period (min)

Inhibitor activity (units/ml)

0 5 10 15

1.38 2.13 2.87 2.59

Effect of preincubation of PG inhibitor with polygalacturonic acid: One ml of 1% polygalacturonic acid was incubated with the inhibitor along with buffer in a total volume of 1.8 ml for various periods of time at 37~ The enzyme reaction was initiated by addition of 0.2 ml polygalacturonase. Results presented in Table 7 indicate that preincubation of the inhibitor with substrate for various time periods does not alter the inhibitor activity significantly. This probably indicates a minimal direct interaction involved between the substrate and the inhibitor during the course of reaction.

Table 7 Effect of preincubation of PG inhibitor of D. maculata stem with polygalacturonic acid.

Preincubation mixture Inhibitor + Polygalacturonic acid

Period (min)

Inhibitor activity (units/ml)

0 5 10 15

1.5 1.5 1.5 1.3

Effect of PG inhibitor concentration on PG activity: Various amounts of inhibitor were added in the assay mixture and the degree of inhibition of polygalacturonase activity was measured which showed a linear relationship between inhibitor concentration and percent inhibition (data not given). Effect of substrate concentration on PG inhibitor: Polygalacturonase activity was measured, with or without inhibitor, at different concentrations of the substrate in the reaction mixture to understand the mode of inhibition of polygalacturonase by D. maculata stem inhibitor. The data presented in Fig. 1 indicate that the nature of inhibition is mixed-type. The inhibitor affects both Km as well as Vmax of polygalacturonase.

804

DISCUSSION The results obtained in the present studies indicate for the first time that Dieffenbachia plants contain a potent inhibitor of polygalacturonase; the stem showing significantly higher activity than either leaf or petiole. Polygalacturonase inhibitor of D. maculata was observed to be comparatively heat-stable. Similar heat stable inhibitors of other enzymes such as amylases and proteinases have been reported by other workers also (Gudiseva et al., 1981; Shivaraj and Pattabiraman, 1980; Cinco et al., 1985; Brecher and Pugatch, 1969). Akira and Murakawa (1993) isolated a polygalacturonase inhibitor from a culture broth of Pezizales, which was completely heat stable at 100~ for 60 minutes. The magnitude of inhibition of polygalacturonase was found to be dependent on preincubation of inhibitor with the enzyme. Similar observations have been reported for other enzyme inhibitors (Shivaraj and Pattabiraman, 1980; Sharma and Pattabiraman, 1980; Padmanabhan and Shrasti, 1990). However, preincubation of the inhibitor with substrate did not show any effect on inhibitor activity. In contrast, Shivaraj and Pattabiraman (1980) and Buonocore et al. (1977), have observed inactivation of amylase inhibitor activity on pretreatment with starch. Lineweaver-Burke plot of polygalacturonase in the presence and in absence of inhibitor, suggests that the inhibition by Dieffenbachia PG inhibitor is of mixed type. Km as well as Vmax value of polygalacturonase changes in the presence of the inhibitor. Similar mixed inhibition has been reported by Mueller and Gersler (1993) for a PG inhibitor isolated from apple leaves. A heat stable, noncompetitive glycoprotein inhibitor (MW 1-2 x 106) of fungal polygalacturonase has been recently reported from Pezizales (Akira and Murakawa, 1993).

0.6

0.4 1/v 0.2

0.0

i

I

-2.0

-1.5

-1.0

-0.5

0.0

I

I

0.5

1.0

1/S Figure 1: Lineweaver - Burke plot of fungal polygalacturonase activity in the presence and in the absence of D. maculata stem PG inhibitor. S: Substrate concentration (mg/ml); V: Velocity of reaction (U/ml), o - o: Without inhibitor; o -o:With inhibitor.

805 REFERENCES

Akira, E. and Murakawa, S., Chem. Abstr., 118 (25) (1993). Abst. No. 250690n. Ardditti, J., and Rodriguez, E., J. Ethnopharmacol., 5 (1982) 293. Barnes, B.A., The Pharmacology and Toxicology of Certain Species of Dieffenbachia, Masters thesis, University of Hawaii, Gaines Ville 1 (1953). Brecher, A.S., and Pugatch, R.R., Experimentia (Basel), 25 (1969) 251. Buonocore, V., Petrucci, T. and Silano, V., Phytochem. 16 (1977) 811. Cino, F.J., Frels, J.M., Holt, D.L. and Rubnow, J.H., J. Food Sci, 50 (1985) 533. Fielding, A.H., J. Gen. Microbiol, 123 (2) (1981) 377. Gudiseva, C., Suryaprasad Raju, D. and Pattabiraman, T.N., J. Sci. Food Agr, 32 (1981) 9. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., J. Biol. Chem., 193 (1951) 265. Mueller, M. and Gersler, C., J. Dev. Plant. Pathol., 2 (1993) 68. Padmanabhan, S. and Shastri, N.V., J. Sci. Food Agr., 52 (1990) 68. Rexova, B.L., Heinrichova, K., Goebel, H. and Bock, W., Chem. Abstr., 95 (1981) Abstr. No. 92890c. Sharma, K.K. and Pattabiraman, T.N., J. Sc. Food. Agric., 31 (1980) 981. Shivaraj, B. and Pattabiraman, T.N., Ind. J. Biochem. Biophys., 17 (1980) 181. Walter, W.G. and Khanna, P.N., Eco. Bot., 26 (1972) 364.



807

Multiple forms of carrot exopolygalacturonase. Eva Stratilov~i, M~iria Dztirov~i, and Danica Mislovi6ov~i

Institute of Chemistry of SAS, Dtibravsk~i cesta 9, 842 38 Bratislava, Slovakia

Abstract

Four forms of exopolygalacturonase were found in carrot juice and one additive form more in carrot roots pulp. They differ in pH optima (3.6; 3.8; 4.7; 5.0 and 5.4), in molecular masses (about 50 000 for all excluding that with pH optimum 3.6, which has about 30 000), and in isoelectric points varying from 4.0 to 6.5. The action pattern on substrates with different degree of polymerization was different, too. The form with pH optimum 3.8 preferred oligogalactosiduronates, while the others the substrates with higher degree of polymerization. Exopolygalacturonase with pH optimum 3.8 was able to cleave the dyed D-galacturonan (DP 10). The D-galactopyranuronic acid inhibited the individual forms to various extend and with various types of inhibition. All forms showed an affinity to ConA-cellulose indicating the presence of Nglycosylation.


Exopolygalacturonases [poly(1,4-ot-D-galacturonate)galacturonohydrolase (EC 3.2.1.67)] are exo-hydrolases catalyzing the hydrolytic cleavage of glycosidic ot-l,4-bonds of D-galactopyranuronic acid units situated at the nonreducing end of D-galacturonans and releasing D-galactopyranuronic acid as a sole reaction product. Substrates for these enzymes are polygalacturonic and oligogalacturonic acids and, in contrast to polygalacturonases, also digalacturonic acid [1]; the particular enzymes differ from each other by the range and rate of the effects on substrate in relation to the chain lenght [2]. Exopolygalacturonases are produced by microorganisms and by higher plants. The enzyme in plant tissue was supposed to be bound to cell wall materials via ionic interactions [3], what required hight salt concentrations for their solubilization. The most suitable substrates for exopolygalaeturonases of vegetal origin are D-galacturonan or partly degraded D-galacturonan of degree of polymerization 20 [4]. The common feature of all known exopolygalacturonases is that they do not fully degrade the substrate with

808 higher degree of polymerization [4]; the limit degradations being proportionally different with the content of neutral saccharides in the pectin molecule [2]. Therefore, it appears that these enzymes serve in cell wall metabolism, for instance in the partial degradation of pectic polysaccharides during cell growth and/or differentiation [3]. From this reason great interest is given to exopolygalacturonases from pollen in the last time [5 - 9] and to exopolygalacturonases at all [10]. Although the first study of exopolygalacturonases from carrots [11] indicated the presence of multiple forms of this enzyme based on the three present pH optima, the latter studies supported the idea of one form of exopolygalacturonase [2 - 4]. The present study deals with the whole spectrum of multiple forms of exopolygalacturonase, with forms described sooner and with forms found only now.

2. E X P E R I M E N T A L

Purification of exopolygalacturonaseforms. Juice from 10 kg of carrot roots (Daucus carota L. cv Zino) were extracted on juice extractor ES-3551 (Severin, Germany), submitted to two precipitation steps, with ammonium sulfate (std.) and ethanol (1:4), and desalted / equilibrated on Sephadex G-25 Medium (Pharmacia, Sweden) in 0.05 M acetate buffer pH 3.8. CM-Sephadex C-50 (Pharmacia, Sweden) stepwise elutions were utilized, with 0.05 M acetate buffer, pH 3.8, 0.10 M acetate buffer, pH 4.8, 0.15 M acetate buffer, pH 5.6, and finally this buffer containing 1.0 M NaC1. FPLC (Pharmacia, Sweden) utilized Superose 12 HR 10/30 in 0.05 M phosphate, pH 5.6, 0.15 M NaC1. Flow rate was 0.5 ml.min -1. Concanavalin A - cellulose chromatography [ 12] was performed in 0.1 M acetate buffer, p H 4.7 using 0.1 M m-methyl-Dmannopyranoside as an eluting agent. The enzyme forms strongly bound on cell walls were extracted by 1.0 M NaC1 solution from dry carrot roots pulp and handled as described for the multiple forms in carrot juice. Substrates. Sodium pectate (D-galacturonan content 89.8%, average M r determined viscosimetrically 27 000) was prepared by repeated alkaline deesterification of citrus pectin (Genu Pectin, Kobenhavns Pektinfabrik, Denmark) followed by precipitation with hydrochloric acid at pH 2.5 and neutralization with NaOH. Di(D-galactosiduronic) acid and penta(D-galactosiduronie ) acid were isolated from enzymatic hydrolysate [ 13] using gel filtration on Sephadex G-25 Fine in 0.05 M phosphate buffer pH 7.0 and desalting on Sephadex G-15 (both Pharmacia, Sweden) [14]. Enzymatic properties. Exopolygalacturonase activity was assayed at 30 ~ p H optimum, in 0.1 M acetate buffer, by measurement of the increase of reducing groups [15], using sodium pectate (0.5%) , di(D-galactosiduronic) acid or penta(D-galaetosiduronic) acid (1 mM solutions) as substrates and Dgalactopyranuronic acid as standard. Initial velocities were determined at five penta(D-galactosiduronic) acid concentrations, ranging from 0.1 - 1.0 mM and calculated by nonlinear regression. Products of the catalytic action on

809 oligogalactosiduronates were analyzed by thin-layer silica gel chromatography [16]. Relative molecular masses of individual exopolygalacturonase forms were approximately estimated by FPLC gel exclusion chromatography on Superose 12 utilizing low molecular weight protein markers (Pharmacia, Sweden). Ultrathin-layer isoelectric focusing in polyacrylamide gels on polyester films was performed as described [17]. Proteins were stained with Serva Violet 49 (Germany). Polygalacturonase activity was determined by the print technique with a dyed substrate (Ostazin Brilliant Red-D-galacturonan DP 10) [18] or by the print technique with colouress D-galacturonan DP 10 with following use of ruthenium red. The enzyme forms were washed out by water from freezed gel segments obtained by preparative izoelectric focusing. Activities of so separated enzyme forms were detected by pH optima values.

3. RESULTS AND DISCUSSION

Crude exopolygalaeturonases from carrot juice and carrot pulp were purified in two stages; to produce first an intermediate product and then partially separated exopolygalacturonases (Fractions A, B, C). The first stage utilized two precipitations and Sephadex step. Purification of resulting lyophilisate from juice by ion-exchange chromatography on CM-Sephadex C-50 gave a separation into two active fractions (Fig. 1).

0.8

~,b

fc

O r

~d

A

.

1.0 O

) 0.4

- 0.5 )

|

3O

9O 6O Fraction, No.

120

0

150

Fig. 1. Separation of two exopolygalacturonase groups (Fraction A, Fraction B) on CM-Sephadex C-50. Column size, 20x250 mm. Stepwise elution with 0.05 M acetate buffer, pH 3.8 (starting at arrow marked a), 0.10 M acetate buffer, pH 4.8 (at arrow marked b), 0.15 M acetate, pH 5.6 (at arrow marked c) and the latter buffer plus 1.0 M NaC1 (at arrow marked d). Fraction size 6 ml per half hr. Exopolygalacturonase activity determined with sodium pectate, pH 5.0 ( O - - O ) and expressed as As~o.

810 The course of separation of the lyophilisate from pulp by this method was very similar to separation of the juice lyophilisate but led to only one activity peak. The pH optima determination (Fig. 2) and gel chromatography on Superose 12 (Fig. 3 a,b,c) showed these fractions to be partially purified, complex mixture of more exopolygalacturonase forms with different molecular masses and pH optima. 100!

# :~_ 50 u

o

OBz

OH

I

"~

l

6

-'-, '~o AgN03

~

HO 3~~20~

7

8 Figure 3

2

(R=Me)

848 SYNTHESIS OF 5 - E P I - K D G METHYL ESTER 13 To obtain this compound the key step consisted in the epimerization of the C-5 in compound 6. This was acomplished by triflation 3 of the alcohol 6 and nucleophilic substitution of the triflate by a large excess of tetrabutylammonium acetate in diehloromethane. A controlled (4 ~ 3 h) basic methanolysis of the enol benzoate led to the keto-ester 114 whose hydroxyl functions at C-4 and C-6 were simultaneously deprotected under acidic conditions to furnish 12. Finally a Zemplen deprotection of the 5-acetoxy group led to 13 obtained in five steps and 11% overall yield from 6 (figure 4).

o-

AcO- Bu4N+

~ ~',~~COOMo

6

3

/ ~ 0

2"OBz

~

CO0 e

~10Ol~l

OBz M

'N/~~

O~ 9 x ~ ~ ~ C OOBz OMe

MeONa MeOH

O 11

AcOHO

PPTS /COOMe MeOH Ill

~

AcO O

O

OH

/

COOMe

~COOMe A_cO O 12

HO 6 4 I

O

OH

0

5

COOMe 3

1

I OH

.OH 2

HO 6 ~ 4 ~ C O O M e HO

OH

5-epi-KDG 13 Figure 4

CH2C12

MeONa MeOH

849 SYNTHESIS OF 5-DEOXY-KDG METHYL ESTER 20

As for the synthesis of 5-epi-KDG,compound 6 seemed to be a suitable precursor of the methyl ester of 5-deoxy-KDG 20 since only the C-5 hydroxyl was unprotected. In this case the key step was not the epimerization but the removal of that hydroxyl. Our attempts of radicalar deoxygenation of 6 were unsuccessful because the intermediate radical was intramolecularly trapped by the C-2,C-3 double bound. Therefore we first reduced the double bond and then converted the resulting diastereoisomeric alcohols 14 into the corresponding tdflates 15 which were submitted to the action of sodium iodide. Finally the iodides 16 thus obtained were hydrogenolyzed in the presence of diisopropylethylamine5 to give 17.

L /~

H2/Pd/C ~ ''O ~-~. \6 5.~OH = "0f ~ 4 ~~,,~,..,~COOMe / Et3N 6

3

= ~ O H (CF3SO2)20 O-- -,~.~COOMe tN,,~/x

2\O8z

14

NaI~ ~ ' ~ q 15

17

18

PPTS ~_ I ~ COOMe MeOH HO

/,

o 6

,--o

OH

5-deoxy-KDG

f

H2/Pd/C COOMe -~ "~OBz Et3N -

.COOMe

MeOH

"~OBz

0 ~ ~ 19

16

"a~om

.COOMe

~ O

-'OSz

20

Figure 5

"~OH

....~COOMe O

PDC AcOH

850 We checked that in the absence of hydrogen, compounds 16 did not undergo any elimination of the axial iodine. If such an elimination had occured (involving axial proton at C-4) the further hydrogenation of the enol ether thus formed would have led to a partially, or totally, racemised 17. The methanolysis of the benzoate ester in diastereomers 17 followed by an oxydation of the resulting alcohols 18 using Czemecki et aL conditions 5 led to the keto-ester 19 which was then deprotected to give the target comtxxmd 20.

SYNTHESIS OF 4-O-Me-KDG METHYL ESTER 23

The synthesis of this derivative of KDG was accomplished following the sequence depicted in figure 6. Methylation of the intermediate 8 with methyl triflate afforded compound 21. Subsequent removal of the enol benzoate group at C-2 and of the silyl ether at C-6 provided the target compound 23 in 11% overall yield based on 8. This low yield could be due to the unstability of compounds 8, 21 and 22 in a basic medium. However 24 was obtained in quantity sufficient for testing. I

-I--Si-O----!6

I

-I-si-o---1 ,

s,L.-o

CH3COOK MeOH

o

HO~ O B z

21

8 I

OBz

I

-I-si-

-I-~i-o-~

I

o

~

O

OMeCOOMe O~~tH

= 22

O

I

-I-~i-~

o

oH

Ho-~

//,,

' OMe

6 __

0

OH

1\4 / (COOMe HO I 3 1 OMe Figure 6

o

oH

" coo o

OMe

851

SYNTHESIS OF 4-DEOXY-KDG METHYL ESTER 29

In order to prepare this compound, since we anticipated that the intermediate 8 would not survive deoxygenation conditions, we decided to start from D-glyceraldehyde 26 having the required configuration (figure 7). The latter compound was prepared by oxydative cleavage of 1,2:5,6 di-O-isopropylidene-D-mannitol 257 and was reacted with phosphorane 248 to furnish the keto-ester 27. After hydrogenation of the C-3,C-4 double bond leading to 28 and, finally, methanolysis of the dioxolane group, the target compoud 29 was obtained in three steps and 42% overall yield based on 26.

COOEt

1 COOMe

0

2

0

3 H~PPh 3 Wittig

24

H

"

NalO~ OH

H20 ~

----

o,,,

Lo'/. 25 6

26 6

O O

0

coo.o

1143 /"'

0.15

o I

-4

,

II

E

cO

r(x)

, o

0.05

-

-1

0 20

, 25

30

35

40

45

50

55

0 60

F r a c t i o n no.

Figure 1. Separation profile on Sephacryl S 100 column of extracellular PG. 2.7 ml fractions were analysed for protein ([3) and reducing sugars released (A). Peaks I, II, III and IV correspond to PG activity expressed as lamol galacturonic acid released rain-1.

3.3. Mechanism of enzyme action A viscometric assay and identification of hydrolysis products were used to determine the mechanism of action of PG. An endo-PG is characterized by a strong reduction in viscosity (e.g. 50%) with a concomitantly low (e.g. 1-3%) release of reducing groups [9]. The time required for 50% decrease in viscosity of a 3.0% (w/v) sodium polypectate solution at 25~ was approximately 10 min, at which time about 1.5% of the total galacturonide bonds had been hydrolysed (data not shown). These results reveal a random mechanism of hydrolysis of sodium polypectate and the enzyme was a poly oc(1,4)-D-galacturonide glycanohydrolase (EC 3.2.1.15) or endo-PG. 3.4. Hydrolysis of pectin from plant tissue by endo-polygalacturonase Endo-polygalacturonase was tested against pectin from cocoa seed pulp by measuring decrease of viscosity and against pectin present in potato and cucumber by loss of coherence of tissue (maceration activity). Cocoa pulp was removed from the seeds and centrifuged to remove solid material and the supematant was then used as a substrate for the viscometric assay. A sharp decrease in viscosity was observed after 10 min of incubation and 50% decrease of viscosity was achieved within 18 min (Figure 3). Endo-PG produced by K. marxianus had very strong maceration activity on potato and cucumber. Maceration of tissue from both species was extremely rapid with softening apparent even after only 5 min and complete cell separation occurring within one hour.

864

Mr

( X 10 3)

A

'~176 I

_.= >

E 75

.--

o

To L

~.~

~.

se si

.

s4 lO t4

1

l

a

4

~"

Figure 2. SDS-PAGE of culture supernatants. Freeze-dried samples were resuspended in distilled water, mixed with an equal volume of sampling buffer and heated to 100~ for 5 min. 101al aliquots were applied to the gel. The right lane contains standards of Mr 14 - 66 kDa. Lanes 2, 3, 4 and 1 are increasing dilutions of the supernatant respectively.

o 4., 6 0 E

o L

Q.

>25 " 0 0 > 0--

o

10

I

I

I

I

I

I

20

30

40

50

60

70

Time (rain)

Figure 3. Decrease in viscosity of cocoa pulp by PG secreted by K. marxianus.

3.5. Regulation of endo-polygalacturonase production PG production was constitutive and not subject to carbon catabolite repression. Highest yields were on glucose and fructose (upto 10% w/v) (Table 1). Inclusion of pectic compounds had no effect on growth or PG production (data not shown). 3.6. Distribution of endo-polygalacturonase in batch cultures The distribution ofPG (PG) as culture medium supernatant, cell-wall associated and cell-bound enzyme was observed in K. marxianus during the time course of growth in 5% glucose medium (Table 2). PG secretion started between 8 and 12 h alter inoculation and approximately 90% of total PG was secreted in early stationary phase. PG was not detected intracellularly alter 24 h of growth. 3.7. Intracellular location of endo-polygalacturonase The subcellular location of PG was studied in cells disrupted by osmotic lysis through formation and disruption of sphaeroplasts from self-induced anaerobically-grown cells. A discontinuous sucrose-density gradient produced four bands labelled I, II, III and IV. Band I included many vesicles and a peak of alkaline phosphatase activity (a vacuolar marker in yeasts), NADPH cytochrome c oxidoreductase activity, an endoplasmic reticulum marker, and

865

Table 1 Growth, enzyme production and enzyme yield Carbon Source

Growth (mg dry wt ml-~)

PG Activity (RVU m1-1 Jig protein -1)

PG Yield (RVU mg biomass -~ ml-~)

D-Glucose Sucrose D-Fructose D-Galactose Lactose D-Xylose

0.70+0.04 0.73+0.03 0.68+0.05 0.69+0.06 0.63+0.06 0.31+0.05

48 9 + 1 . 2 46 9+0.3 27 2+0.9 19 0+1.0 14 3+0.5 2.7+0.9

69.8 64.2 40.0 27.5 22.7 8.7

All cultures were grown with 10g 1-1 sugar under self-induced anaerobic conditions. Growth and PG activity were measured after 16 hrs. There was no growth on galacturonic acid, pectins, cellulose or rhamnose. • indicates standard deviation.

Table 2 Distribution of PG in sphaeroplasts and sub-cellular fractions. Cellular Fraction Time (hrs)** Sphaeroplasts Low-density vesicles Vacuoles * ~ ER*** p, . *** lasma-memorane

PG activity* 8

10

12

14

16

673 350 84 160 59

715 314 115 50 246

516 210 88 48 170

356 81 32 38 205

212 57 0 0 148

* Expressed as RVU. ** Hours after inoculation of self-induced anaerobic culture. *** Organelle identified by peak of marker enzyme activity. transmission electron microscopy revealed the presence of lipid-vesicles, identified by their affinity for osmic acid. Over 85% of the vanadate-sensitive Mg++ ATPase activity, marking yeast plasma membranes, was detected in bands II, III and IV and confirmed by TEM. Vesicles were also detected in these bands, but they appeared to be part of the plasma membrane and had not resulted from fraction contamination. Sphaeroplast lysates fractionated on sucrose-density gradients showed sequential movement of enzyme from the ER via low density vesicles to the plasma membrane (Table 2). PG activity was not found in fractions of cells harvested after 24 h. of growth.

866 3.8. Mutants

One hundred and thirty eight mutants produced clearing zones of repeatable and different diameter compared to the parent strain (diameter of 30 + 2 mm). PG activity of each mutant was then measured in the supernatant from liquid cultures. Only five mutants showed increased extracellular PG activity compared with the parental strain. The highest activity was 23.4 PG units, which is 25% above the wild type level. The great majority of the mutants (107) showed reduced PG activity ranging from 0 to 15 PG units. One hundred and thirty mutants showed similar or lower intracellular PG levels compared to the wild type; these comprised both over-and under-producers. Three mutants with higher intracellular activity secreted the enzyme later than wild type; five mutants with reduced levels of secretion had Very high intracellular PG activity, possibly indicating a defective secretory pathway. However, the total PG activity remained below wild type levels. Sub-cellular fractionation of five strains revealed the same numbers of bands. The distribution of PG activity in sub-cellular organelles was broadly similar in these five strains. PG activity was detected in low-density vesicles, vacuoles and ER fractions in samples harvested during the early exponential phase of growth. However, PG levels were always lower (at least 1.5 fold) than those found in wild type. Cells of the mutants harvested during stationary phase of growth showed that 84% of total intracellular PG activity was located in the vesicle fraction. No intracellular PG activity was found in stationary phase wild type cells.

pl 9.30

-

8.75

_

7.35

_

6.85_

5.85

-

5.20-

,d

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~fl/t

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4

3

6

7

27

30

31

32

35

Figure 4. Isoenzyme profiles of wild type and mutant strains. Detection of PG activity was by ruthenium red staining on pectate-agarose overlay gels after IEF. WT indicates wild type and the numbers refer to specific mutant isolates.

867

3.9 Isoenzyme profile of polygalacturonase in wild type and mutant strains Isoelectric focusing showed up to nine grouped apparent PG isoenzymes; three major bands (estimated pI's 5.9, 5.6 and 5.3) and six minor bands of apparent isoforms range from 6.4 to 5.0 (Figure 4). The time-course of growth showed that all nine isoforms were present in the culture filtrate aider only 24 h of incubation. Extracellular PG activities of 18 representative mutants showed a common pattern of at least three major isoenzymes, and five minor apparent isoforms. Profiles of PG from wild type and the very low under-producers (mutants secreting less than 1~ of the normal PG) were equivalent in band number (eight), but the intensity of all bands from the mutants was generally weaker. However, some mutants which are also under-producers (3 and 4) had almost identical profiles to the wild type, ie, they showed the three major and the five minor isoenzymes. 4. D I S C U S S I O N PG secreted by K. marxianus CCT 3172 showed activity from pH 4 to 6, with an optimum at pH 5 typical of PG secreted by yeasts. Unlike some pectinases, the activity of PG from K. marxianus CCT 3172 was not affected by buffers used across the pH range studied. The effect of temperature on the activity of PG from K. marxianus was similar to that reported for PGs from yeasts [eg 2]. The relative molecular masses of the four proteins revealed by gel filtration were in close agreement with previous estimates [ 16,17]. Activity-stained, IEF gels revealed nine multiple forms that could be divided into two acidic groups in relation to intensity of bands: the first one consisting of three major bands and the second with six minor isoforms showing weaker band intensities. The pI values in the present study are similar to those previously obtained [ 17]. Numbers of apparent isoenzymes are sometimes caused by variations in glycosylation or by degradation due to the use of old cultures. However, the presence of all nine PG isoforms in the growth medium from young cultures might suggest that they are not artefacts from IEF. None of the under-producer mutants had lost a single isoenzyme indicating that none of the mutations were in structural genes. Those under-producer mutants showing 8 isoenzymes with similar intensities of each band, seem to have had a mutation in the regulatory gene(s) affecting a subset of the isoenzymes. Differences were found in isoenzyme profile, growth rate and total excreted proteins when mutants showed about 20% of wild type extracellular PG activity, a phenotypically pleiotropic effect in whiizh various enzyme systems were modified (unpublished data). The endo-action of the K. marxianus PG was demonstrated by a extremely rapid attack on plant tissue. This activity appears to be at least equivalent to that of several commercial preparations used for separating plant cells for protoplast preparation (RMC, unpublished data). Most of the endo-PGs produced by plant pathogens and saprophytes have so far been reported to possess macerating activity. PG secreted by K. marxianus CCT 3172 also had a strong activity in reducing the viscosity of cocoa pulp. Cocoa pulp generally contains 1 - 1.5% (w/w) of pectin consisting of 68% esterification and 11.6% methoxyl content [ 18]. The distribution of PG activity, among supernatant, cell-wall and cell-bound fractions changed throughout the time-course of growth and it is likely that PG activity in cell-wall fractions was enzyme in the process of being released, rather than located in the periplasmic space. Studies on the subcellular location of PG using sucrose density gradient suggested that PG was synthesized and secreted by the classic yeast secretory pathway [ 19]. This conclusion is consistent with the results from the five mutants which synthesized PG but were unable to release it into the medium. 90% of intracellular PG activity was located at the vesicle fraction, indicating a specific transfer block at the stage from vesicles to plasma-membrane. This is the first study of subcellular location of PG in K. marxianus.

868 The screening method based on diameter of halos yielded only five strains with at least 20% more PG than wild type. Most attempts to enhance pectinase production have been made in filamentous fungi, in which enzyme production is regulated by induction and catabolite repression mechanisms [8]. By contrast, PG produced by K. marxianus is constitutive and not subject to catabolite repression [6]. Failure to find greatly enhanced secretion levels is consistent with the idea of a constitutive gene that is capable of only modest further induction. PG was found in this study to be already the most prolific secreted protein ofK. marxianus. This strain ofK. marxianus was found during the first 36 h of cocoa fermentation when the pulp is degraded which suggests that PG from K. marxianus may be utilized directly on cocoa beans to speed up the fermentation process and also to obtain higher quality fermented beans. No other pectinolytic enzymes, apart from PG, were secreted by K. marxianus into culture medium. This should facilitate PG purification. PG from K. marxianus may in future, be utilized directly on cocoa beans for the extraction of cocoa juice. The pulp obtained with the aid of pectinolytic enzymes has a lower viscosity and this aids processing of pulp for pasteurized juice and soft drinks from cacao. Another potential industrial application for PG produced by yeasts, is in the manufacture of fruit nectars or in softening of vegetables for preparation of baby foods. Advantages of this enzyme over PGs from filamentous fungi, mainly Aspergillus niger, appear manifold, and include no requirement for pectin inducers, absence of numerous contaminating enzymes, absence of PME which releases toxic methanol, and much faster growth rates of K. marxianus.


6 7 8 9 10 11 12 13 14 15 16 17 18 19

Schwan, R.F., Rose, A.H. and Board, R.G. (1995) In: Fermentation: Foods, Feeds and Condiments. R.G. Board, D. Jones and B. Jarvis. (Eds) Supplement to J. Appl. Bacteriol. Symposium 79, pp 96S-107S. F.M. Barnby, F.F. Morpheth and D.L. Pyle (1990) Enzyme Microb. Technol. 12, 891. A.P. Espinoza, E. Barzana, M. Garcia-Garibay and L. Gomez-Ruiz, (1992) Biotech. Letts 14, 1053. S. Harsa, C.A. Zaror, and D.L. Pyle (1993) Process Biochem. 28, 187. L. Hankin and G.H. Lacy (1984) In Compendium of Methodsfor the Microbiological Examination of Foods, 2nd edition, ed. M.L. Speck. American Public Health Association, Washington, D.C. R.F. Schwan & A.H. Rose (1994) J. Appl. Bacteriol. 76, 62. G.L. Miller (1959) Anal. (7hem. 31,426. R.M. Cooper and R.K.S. Wood (1975)Physiol. Plant Path. 5, 135. R.M. Cooper, B. Rankin and R.K.S. Wood (1978) Physiol. Plant Path. 13, 101. P.K. Durrands and R.M. Cooper (1988) Physiol. Molec. Plant Path. 32, 343. U.K. Laemmli (1970) Nature 227, 680. F. Altherthum and A.H. Rose (1973) J. Gen. Microbiol. 77, 371. T.G. Cartledge, A.H. Rose, D. Belk and A.A. Goodall (1977) J. Bacteriol. 132, 426. P.A. Henschke, D.S. Thomas, A.H. Rose, and F.J. Veazey (1983) J. Gen. Microbiol. 129, 2927. C.W. Lawrence (1991) In Guide to Yeast Genetics and Molecular Biology, C. Guthrie and G. Fink. (eds)Methods in Enzymology 194. Academic Press, New York. S. Inoue, Y. Nagamatsu, and C. Hatanaka, C. (1984) Agric. Biol. Chem. 48, 633. A.B. Smith and D.L. Pyle (1990) J. Food Biochem. 14, 273. P.R.F. Berbet (1979) Revista Theobroma 9, 55. R. Schekman (1985) Ann. Rev. Cell. Biol. 1, 115.


Polygalacturonase and pectinmethylesterase activities during growth of 1805 cell suspension

869

Helianthus annuus

M. Ilievaa, M. Kratchanova b, E. Pavlovab,T. Dimova a, A. Pavlov a

a Bulgarian Academy of Sciences, Institute of Microbiology, acad. G. Bontchev Str. 26, Sofia, Bulgaria 1113

b Bulgarian Academy of Sciences, Institute of Organic Chemistry with Phytochemistry, 95 V. Aprilov Str., P. O. Box 27, LBAS - Plovdiv, Bulgaria 4002

Abstract Time courses of growth of Helianthus annuus 1805, changes of the amount of cell walls, as well as changes in polygalacturonase and pectinmethylesterase and related changes in the degree of esterification and polyuronic content of the cell walls were investigated. Dependences between the cell enzyme activities and the cell wall changes, as well as between the cellular and extracellular enzyme activities, were established. Growth of the cell suspensions and the related remodelling of the cell walls were considered a process in which the enzymes under study were active both in the cell and in the culture medium.

Introduction

The growth of plant cell suspensions is connected with changes in the mechanical and structural elements of the cell wall (1). This process may involve changes in the amount and/or the structure of the pectic polysaccharides (2). The loss of the native strength of the wall and the secretion of structural fragments from the latter is facilitated by the activity of endogenous hydrolytic enzymes during growth (2-4). Recent studies have followed their changes and have related them to the process of development (5-8). The objective of this research was to follow the course of growth of H.annuus 1805 cell suspension in parallel with the changes in the amount of cell walls, as well as the course of biosynthesis and secretion of the enzymes polygalacturonase and pectinmethylesterase and the

870 related changes in the polyuronic content and the degree of esterification of the cell polysaccharides.

Materials and methods

Cell culture. The Helianthus annuus 1805 cell culture was grown in Linsmayer-Skoog medium (9), supplemented with 0.2 mg/L 2.4 - dichlorphenoxyacetic acid and 3% sucrose. The callus cultures were kept in an agar medium of the same composition. They were grown in a thermostat in the dark at 26-28 ~ for two weeks and could be stored up to two months in a refrigerator. H.annuus 1805 cell suspension was cultivated on a shaker (11.6 rad/s) at 26-28 ~ in the dark in 1/5 net volume flasks. Duration of growth was different: from 5 days for obtaining the inoculum to 10 days for studying the course of growth, the course of changes in the cell walls and the courses of biosynthesis and secretion of the enzymes polygalacturonase and pectinmethylesterase as well. For inoculation 20 % (v/v) five-day cell suspension, containing 9 g/L dry cell biomass, was used. By daily taking samples during the 10-day cultivation, changes in certain parameters were followed, using the relevant methods: Course of growth. The course of growth of H.annuus 1805 cell suspension was followed by measuring the amount of dry cell biomass/DB/(10). Polygalacturonase /PG/ and Pectinmethylesterase /PME/ A ctivities Cellular enzyme activities. The cell PG and PME were determined after their extraction from the cell biomass. For that purpose the cell biomasses were frozen for 12 hours, then, after defreezing, 0.2 M acetate buffer (pH 7.9) was added at a ratio of 1:2.5 and the mixture was homogenized for 10 minutes using Polytron homogenizer. The sample stayed in the refrigerator for 24 hours, after which it was centrifuged for 40 minutes at 6,000 xg. The supernatant was separated and ammonium sulphate was added to 40 % concentration and the precipitated interfering proteins were removed by centrifugation (30 min, 4500 xg). Once again, ammonium sulphate was added to the separated supematant until 70 % saturation was reached which led to precipitation of the proteins carriers of both enzyme activities. The precipitate was separated by centrifugation (30 min, 4500 xg) and diluted with 5 ml 0.1 M phosphate buffer (pH 7.0). The solution was dialyzed for 12 hours against distilled water and after dilution to the required volume with the same phosphate buffer, polygalacturonase and pectinmethylesterase were determined. Extracellular Enzyme Activities. The protein, carrier of the polygalacturonase and pectinmethylesterase activities, was salted out from the H.annuus 1805 culture medium by adding ammonium sulphate to 70 % saturation. The precipitate was separated by centrifugation (30 min, 4500 xg), diluted with 0.1 M phosphate buffer (pH 7.0) and dialyzed for 12 hours against distilled water. After dilution to the required volume with the same buffer, both enzyme activities under study were determined in the solution.

871 Polygalacturonase was determined by the viscosimetric method (11), and pectinmethylesterase - according to the titrametric method (12). Cell Walls (CW). After the extraction of the cellular enzymes from the biomass, the residue of homogenized cells was repeatedly washed with water and finally with 96 % ethanol, dried and the dry weight of cell walls was determined (10). The polyuronic content (PUC) and the degree of esterification (DE) of the dry cell walls were determined according to the method of Gee (13). The results for these two parameters are given in terms of per cent of the total amount of cell walls. The presented results are average values from three independent experiments.

Results and discussion

Course of growth of H. a n n u u s 1 8 0 5 cell suspension and of changes in the cell walls. The plant cell wall contains different types of polysaccharides, proteins (structural glycoproteins and enzymes), lignin and water, as well as some inorganic components (1, 14-16). The plant cell suspensions, however, grow as a population of cells with a primary cell wall(17). The main components of these walls are cellulose-free polysaccharides and pectic polysaccharides in particular, which constitute 1/3 of their dry weight. (18). Some fragments, e.g. methanol, acetic, ferulic and p-cumaric acids, are connected with the pectic polysaccharides by ester bonds with the carboxylic and hydroxylic groups. Besides, it is known that the culture medium acts as a common external sink like a lamella (15) or a vacuole (19), in which polysaccharides, enzymes and other metabolites are secreted during growth. Consequently, the growth of plant cell suspensions is a complex process, connected with structural and metabolite changes both in the cell wall and in the culture medium, involving a complex of hydrolytic enzymes. Our results, regarding the course of growth of H.annuus 1805 and the course of changes

in the cell walls show that the cell suspension underwent an intensive growth from the 3rd to the 7th day of cultivation (Fig 1). The changes in the amount of the cell walls followed the course of growth, i.e. by the 6th day the maximum was reached both in the amount of cell biomass and in the amount of cell walls. It is worth mentioning that after the 6th day of cultivation of H.annuus 1805 the synthesized biomass was preserved until the 8th/9th day, while the amount of cell walls decreased considerably. As it is known (2), at the beginning of the stationary phase of growth the cell mass does not change substantially, but the cell wall thickness, as well as the number of the cells decrease as a result of the processes of decomposition of the cell walls. The latter reflects more tangibly on the total amount of cell walls, than on the total amount of biomass.

872 60 55 5O

~s

16 14 12

4

_1

8 2

3O 1

25 2O

1

i

i

i

i

I

i

i

i

2

3

4

5

6

7

8

9

Time,

10

days

Fig. 1 Time course of growth ofH. a n n u u s 1 8 0 5 cell suspension, variations in the cell walls, their polyuronic content and degree of esterification. =

DB

"--

CW

-" "~

DE PUC

Course of biosynthesis and secretion of PME and PG, and related changes in the degree of esterification /DE/ and in the polyuronic content/PUC/of the cell walls. During cultivation the polyuronic content of the cell walls varied within narrow limits (Fig. 1). It is known that polygalacturonase is the enzyme, included in the decomposition of acid pectic polymers (3). As follows from the course of biosynthesis and secretion of polygalacturonase

(Fig 2), its amount at the end of the lag phase (4 th day) was considerable. With the beginning of the intensive growth of the cell suspension, PG gradually decreased. Part of it was secreted into the culture medium and the main peak in the extracellular enzyme activity was observed on the 8th day, preceded by a smaller one on the 5th day. It is known that the polygalacturonase activity is manifested along with other hydrolytic enzymes (3) and, as it was mentioned above, its chief role is to decompose the acid pectic polymers to polysaccharide chains of certain structure and molecular mass and also to ensure a certain polyuronic content of the cellular polysaccharides. Consequently,' its activity is probably regulated by relevant inhibitors. On the other hand, the polyuronic fragments, separated from the cell wall, are secreted into the culture medium. This is a potential substrate for the polygacturonase and when they are present in a considerable amount, then, by certain mechanism, part of the enzyme activity is secreted into the culture medium. It is probable that here it takes part in the remodelling of the secreted polysaccharide fragments to molecular sizes and structures with certain physiological functions for the cell. These functions are similar to those of the polysaccharides from the central lamella (15) and are related primarily to intercellular interactions and more precisely to information transfer regarding the development of the cells and their protection (20). It is also probable that the extracellular polygalacturonase takes part in carbon regeneration by decomposing the soluble acid polymers to compounds that can be

873 assimilated by the cell once again to provide for the growth and construction of new cell walls (3,19). Fig.1 follows the course of change of another important characteristic of cellular polysaccharides - their degree of esterification, which is regulated by the pectinmethylesterase (6). A sharp change in the degree of esterification of the cell walls of H . a n n u u s 1 8 0 5 was observed from the 3rd to the 6th day of cultivation (from 35 % to 58 %, Fig 1). The same period of growth marked an increase in the PME activity as well (Fig 3) and coincided with the period of intensive growth (3 rd - 7th day, Fig 1). The highly esterified polysaccharide fragments were more loosely connected and more water soluble, consequently they were more easily secreted into the culture medium, which is an important peculiarity of the process of suspension growth (6). After the 7th day of cultivation, as a result of secretion of a considerable amount of highly esterified watersoluble polysaccharide fragments, the degree of esterification in the cell walls was estimated to be 50 % by the 8th day. For the period between the 3 rd and the 7th day part of the cellular PME was secreted into the culture mediumand peaks of extracellular PME were observed on the 6th and

120

40

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3o ~

20

25 ~

-~

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35

-~

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~

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-5

~0

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10

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0 20

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4

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6

7

8

9

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C eUular PG

-

Eztrac ellular PG

m

5

6

7

8

9

10

0

]]me, days

Time, days

Fig. 2 Time course of biosynthesis and secretion of polygalacturonase from H a n n u u s 1805.

4

Fig. 3 Time course of biosynthesis and secretion of pectinmethylesterase from H. a n n u u s 1805.

v~

Cellular PIvEE

"

Extrac ellular PIVIE

.

the 8th days of cultivation (Fig 3). They can be related to the sharp decrease (consequently secretion) of intracellular activity between the 6th and the 8th day.

874 Apart from PME, pectinmethyltransferase (6) and some other enzymes, connected with polysaccharide esterification, were involved in the process of regulating DE of the pectic polysaccharides. Therefore, a more detailed interpretation is difficult to make, but the data from this research confirm the fact that in the case of H. annuus 1805 cell suspension the processes of esterification and deesterification in the cells and in the culture medium are in a certain equilibrium and should be considered in correlation. Pectinmethylesterase, like polygalacturonase, is not limited in its involvement to remodelling the cell wall during suspension growth. When the highly esterified pectic fragments accumulate in the culture medium in considerable amounts, the enzyme is secreted and takes part in their deesterification, which allows the formation of acid regions in the polysaccharide fragments. These acid regions link the polysaccharide fragments with other components until polysaccharide structures with certain functions are established (17).

Acknowledgement The authors gratefully acknowledge the financial support for this work from the National Research Foundation of Bulgaria.

References 1. Sakuzai N., Bot. Mag. Tokyo 104 (1991) 235 2. Konno H., Y.Yamasaki, K. Katoh, Physiol. Plantarum, 69 (1987) 405. 3. Konno H., Y.Yamasaki, K. Katoh, Physiol. Plantarum 76 (1989) 514. 4. Konno H., Y.Yamasaki, K. Katoh, Physiol. Plantarum,68 (1986) 46. 5. Uchiyama T., M. Numata, S.Terada, T. Hosino, Plant Cell Tissue and Organ Culture 32 (1993) 153. 6.Schaumann A., M. P. Bruyant, V. F. Ogubet, C. Mowan, Plant cell Physiol. 34 (6) (1993) 891. 7. Amino Sh. Z., Naturforsch. 44C, (1989) 754. 8. Mc Camm, M.C., K. Roberts, Y. of Exp. Botany 45 (1994) 24. 9. Linsmayer E. M., F. Skoog, Physiol. Plantarum 18 (1965) 100. 10. Dixon R. A. (Ed.) Plant cell culture- a practical approach IRL Press (1985) 15. 11. Pherr D. M., P. B. Dickinson, Plant Physiol. 51 (1973) 577. 12. Kertesz Z. I. - Methods in Enzymology, New York, 1 (1955) 159. 13. Gee M., Mc Comb E. A., Mc Cready R. M., J. Food Sci. 23 (1958) 72. 14. Van Cutsen R., J. Messiaen, Acta Bot. Neerl 43 (1994) 231. 15. Ficher G. B., B. A. Stone, Ann Rev Plant Physiol. 34 (1983) 47. 16. Mutaftschiev S., A. Macaya, R. Prat, P. Devillers, R. Golberg, Plant Physiol. Biochem. 31 (4) (1993)459. 17. Heredia A., A. Jimenez, R. Guillen, Lebensm. Unters. Fosch., 200 (1995) 24. 18. Buwn J. B., S. C. Fry, Plant Physiol. 103 (1993) 993. 19.Wink M, Plant Cell Tissue and Organ Culture 38 (1994) 307. 20. Mohnen D., M. G. Hahn, Seminars 4 (1993) 93.


875

Differential expression of Erwinia chrysanthemi strain 3937 pectate lyases in pathogenesis of African violets: importance of low iron environmental conditions C. Masclaux, a N. Hugouvieux-Cotte-Pattat, b and D. Expert a aLaboratoire de Pathologie V6g6tale, INA P-G / INRA, 16 rue Claude Bernard, F-75 231 Paris, France bLaboratoire de G6n6tique Mol6culaire des Microorganismes, URA CNRS 1486, Biochimie 406, INSA de Lyon, 20 Avenue Albert Einstein, 69 621 ViUeurbanne, France

Abstract

Multiplication of Erwinia chrysanthemi pel mutants and pel gene expression (via the use of GUS fusions) were studied during the first hours following plant inoculation. The expression of pel::uidA mutants affected in iron assimilation was compared to the wild type background. The results showed that low iron condition encountered by bacterial cells during pathogenesis modulate the e• of pel genes. Moreover, the expression of peID::uidA was stimulated by the presence of iron chelators in the growth medium and the absence of functional chrysobactin mediated iron uptake.

1. Introduction The pathogenicity of Erwinia chrysanthemi 3937 on African violets involves at least five pectate lyases (PelA to PelE), one methyl pectin esterase (Pem), encoded by pelA to pelE, pem genes and an iron assimilation system mediated by chiTsobactin [ 1], (Fig. 1). Soft rot symptoms produced by E. chrysanthemi consist of a disorganisation of parenchymatous tissues following the release of bacterial pectinolytic enzymes. The diverse enzymes do not contribute equally to the virulence on a given host and their implication may vary according to the host considered. For instance, inactivation of pelE, pelD, pelA or pem in strain 3937 considerably reduces the virulence on African violets while mutations in pelB or pelC remain ineffective [2]. Pectinolysis is regulated by the transcriptional repressor KdgR, inactive in the presence of pectic inducers. Soft rot spreading depends on the efficiency of the iron uptake pathway mediated by the siderophore chrysobactin. Biosynthesis of the ferrichrysobactin outer membrane receptor (Fct) and of the chrysobactin precursor, i.e. the activated form of 2,3-dihydroxybenzoic acid, are encoded by an operon,fct cbsCEBA [3]. Furthermore, the occurence in strain 3937 of a second iron acquisition zystem, dependent of a siderophore designated achromobactin, has been recently demonstrated. The structure of achromobactin is still unknown and it is likely to be an iron ligand less competitive than chrysobactin. The ascA gene is involved in achromobactin synthesis. Ferriachromobactin uptake pathway utilises a specific ABC transporter referred to as the Cbr permease. Mutations in cbr locus, interrupting the transport of ferriachromobactin into the cytosolic compartment give rise to derepression of chrysobactin production [4-5].

876 KdgR is a well knownrepressor of pectate lyase genes. In kdgR mutants, pectate lyases are still inducible in the presence of pectin derivatives, suggesting the existence of other regulatory factors [6]. Pectinolyticenzymes ~'(~lant cellwalldegradation.) insaturated oligogalacturonides f

. - -

o

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0

t~. .m

o

rm

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2.5 3.5 5.5 6.5

2.5 3.5 5.5 6.5

2.5 3.5 5.5 6.5

pH

pH

pH

o

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Figure 2. Specific Exo (A and B)and Endo (C) pectinase activities produced by FP-180 (open bars) and Aspergillus niger N402 (dashed bars) at different pH values growing on 1% pectin at: 30~ (A) and 37~ (B, C).

Aspergillus

Electrophoreses of samples from the different pH values showed a very interesting differences in the band pattern of secreted proteins, both, between pH values for each strain and between strains itself (Fig.3). From SDS-PAGE it was observed, for both strains, a reduction in the number of band as the pHi diminished. At 2.5 pHi very few proteins were found to be secreted, particularly with Aspergillus FP-180. With Aspergillus niger N402 the effect on secretion was also observed at this pH, although, the growth was just slightly affected at this pH. The in situ activity overlays exhibit, for N402 strain, clearing and darker zones when polygalacturonic acid (PGA) and pectin, respectively, were used as substrates. These zones correspond to a proteins with a molecular mass of around 63-65 kDa

919

for clearing bands, probably due to a lyase activity, and to a proteins in the range of 48-52 kDa for darker zones in pectin overlay. This later being a pectinesterase. It could be observed that the clearing zones on PGA were very weak at 2.5 and 3.5 Pectin overlay

SDS-PAGE

PGA overlay

6.5

5.5

3.5

2.5

a

b

c

d

2.5

3.5

5.5

6.5

6.5

5.5

3.5

2.5

a

b

c

d

2.5

3.5

5.5

6.5

Figure 3. SDS-PAGE and in situ pectinase activity on pectin and polygalacturonic acid-agarose overlays of culture filtrates of Aspergillus niger N-402 (upper panel) and Aspergillus FP-180 (lower panel) at 2.5, 3.5, 5.5 and 6.5 pHi (Lanes a, b, c, and d, respectively). Electrophoresis on 10% acrylamide slab gel (14 X 8 cm) in the presence of SDS was according to Laemmli (6), run at 30 mA constant current for 2 hours. Crude cell-free samples were concentrated by lyophilization, dialyzed, boiled with sample buffer by 60 sec. and applied to each well. Polyacrylamide gel and overlays were incubated overnight with 0.17 acetate buffer at room temperature. pHi and of high intensity for samples from 5.5 and 6.5 pH which appeared also on pectin overlay (Fig. 3). On the other hand, FP-180 strain showed only clearing zones in overlays with both substrates in a range of molecular mass of 48-60 kDa (Fig. 3). In no case a pectinesterase activity was found for Aspergillus FP-180. However, the

920

clearing zones were of higher intensity for this strain than for Aspergillus niger N402. It is interesting to notice that Aspergillus FP-180 produced very high level of exo and endo activities as compared to A. niger under the conditions used in this work. It is possible that the complementation of both strains could improve the production of a complex and more efficient pectinolytic system. The above results showed that stress induced by the pH of culture medium affects the growth of Aspergillus FP-180 and to a lesser extent that of Aspergillus niger N 402. However pH not only affects growth, but also modified the pattern of pectinase production and/or secretion. It is believed that the reduced number of protein bands produced at 2.5 pH must be essential enzymes to degrade the outer substrate during fungal growth and as the pH turns less stressing factor the microorganisms could be able to produce a more complex mixture of extracellular enzymes. 4. ACKNOWLEDGMENTS Part of this work was supported by DGAPA/UNAM Project IN 209194 5. REFERENCES

1) E. Nahas, H.F. Terenzi and A. Rosi. J. Gen. Microbiol., 128 (1982) 1017-1021. 2) A. Rossi and H.N. Arst. FEMS Microbiology Lett. 66 (1990) 51-54. 3) G. Leone and J. van den Heuvel. Can. J. Bot. 65 (1986) 2133-2145. 4) G. Aguilar, B.A. Trejo, J.M. Garcia and C. Huitron. Can. J. Microbiol. 37 (1991) 912-917. 5) C. Dijkema, R.P. Rijken, H.C.M. Kester and J. Visser. FEMS Microbiology. Lett. 33 (1986) 125-131. 6) U.K. Laemmli. Nature 227 (1970) 680-685.


S e l e c t i o n of a c o n s t i t u t i v e P e n i c i l l i u m strain

hyper-pectinolytic

921

mutant

from

a

Noomen Hadj-Taieb, Malika Ayadi and Ali Gargouri Centre de Biotechnologie de Sfax; BP "W"3038. Sfax-TUNISIA

ABSTRACT: The production of pectinases was studied in fungi. One strain, Penicillium occitanis, was chosen for nitrous acid mutagenesis. Based on Hexadecyl -trimethyl-ammonium bromide (CTAB) staining and after only one round of mutagenesis, an interesting mutant, CT1, was selected. It secretes about 20 times more pectinases than the CL100 wild-type strain but not cellulases or other hydrolases. In comparison with another already known mutant of the same strain, CT1 is, not only genetically stable and sporulating, but also able to secrete high amounts of pectinases on local substrates such as "orange peel". The most interesting feature of this mutant is its constitutivity: it produces the same specific activity of pectinases on citrus pectin as on glycerol, which is a potent repressor of pectinolytic activities in Penicillium and many other fungi.

1-INTRODUCTION: In biomass degradation, the biological decomposition of pectin is an important process which is accomplished by enzymes produced by a wide variety of saprophytic and phytopathogenic microorganisms (Rombouts and Pilnik, 1980). The enzymatic systems occur during the fruit ripening process causing softening. Pectinases hydrolyse pectins by different mechanisms, those of which act on principal chain of galacturonics and those acting on lateral chain or "hairy regions" (Schols et al. 1994). Pectinases play an important role within food industry since they improve the extraction, filtration and clarification of fruit juices (Pilnik and Rombouts, 1985). Their use in the olive oil and wine sectors has recently been reported (Servili et al., 1992; Gainvors et al.,1994). The synthesis of pectinases is a property shared by many different microorganisms, especially the Aspergillus genera. Many plant pectinases have been characterized and some of them cloned and now, in order to shed lights on the "plant-fungus" interaction, attention is turned on fungal enzymes. Many Erwinia and Aspergillus pectinases and the corresponding genes have been isolated and studied (Laing et a1.1993, Bussink et al, 1992). In this context, the selection of novel producer strains is an important step for phylogenetic comparisons and for biotechnological applications. One can even expect that strains producing "hairy regions" hydrolytic enzymes should be useful for some specific applications. It is also of a great benefit to search mutant strains producing enzymes at a much lower cost. Penicillium occitanis produces a wide range of extracellular enzymes such as cellulases and pectinases (Jain et al, 1990-a ). After multiple rounds of mutagenesis, a mutant named Po16 was selected from the CL100 wild strain and was shown to secrete very high amounts of pectinases and cellulases enzymes (Jain et al, 1990-b). Unfortunately, the fact that this mutant was poorly sporulating (Jain et al, 1990-a) raised serious problems for an industrial use and for an eventual continuation in the genetic improvement program. In the present study, we report the isolation of a new hyper-pectinolytic mutant from P.occitanis after a single round of nitrous acid mutagenesis. We present here comparisons with CL100 and Po16 strains and data indicating that our mutant is fully constitutive and that it can grow on cheap and local substrates.

922 2. M A T E R I A L S AND M E T H O D S

2.1 Microorganisms" Some of them were originally isolated in our laboratory and others kindly provided by others research laboratories. These strains were maintained on potato dextrose agar (PDA) at 4~ or as spores in 20% glycerol at -20~ Pol6 is a mutant of the wild type strain Penicillium occitanis CL100, both kindly provided by Professor G. Tiraby, Toulouse University. Three strains, two Aspergillus niger : A1 and F38 (Hamdi et al, 1991) and one Trichoderma inhamatum:: OL1J, have been isolated in our laboratory.

2.2 Mutagenesis and Screening conditions" The strain was grown on PDA plates for 7 days and spores were resuspended in water containing 0.1% triton. 0.4 ml (108 sp./ml ) are suspended in 5 ml of citrate buffer pH 3, in ice. 501.tl of Na NO 2 0.2 M were added at time "zero". 0.5 ml were taken at various time and were neutralized with an equal volume of K2HPO4 (0.2M). Spores are then diluted and plated on PDA in order to plot the viability curve. A survival rates of 10 % andl % were obtained for 40 and 80 mn of treatment respectively and were considered to be suitables for the screening of mutants.The basic screening medium was Hankin (Hankin et al. 1971) and BSM (2.5 g/1 (NH4)2SO4, 1 g/1 KH2PO 4, 0.3 g/1 MgSO4, 0.2 g/l CaCL2, 5 mg FeSO 4, 2 mid Tween 80 and 1 ml/1 oligoelement solution, pH 5.5) supplemented with 0.5% of the appropriate carbon source and 0.1% v/v Triton X 100 to restrict growth. CTAB 1% or iodine solution were added on colonies. The selection of pectinase-producing strains was based on the detection of clearing halo surrounding the colonies. Total pectinase, cellulase and lipase activities secreted by colonies were detected on BSM plates containing respectively 1% of citrus pectin, 2% Walseth cellulose and 1% olive oil + rhodamine. After few days at 30~ pectin plates were covered by 1% CTAB for lhour, positive colonies became surrounded by a clear halo; walseth plates are not stained: the halo is visible directly on positive clones; lipase activity is revealed under UV on oil-rhodamine plates.

2.3 Production of enzymes in flasks: The basal medium of Mandels (Mandels et al., 1976) was used with the following modifications: it was buffered with 3 g/l of sodium nitrate to pH 5.5 and supplemented with 1% w/v citrus pectin " Sigma" or other carbon sources. For enzyme production, 50 ml medium in 250 ml erlemneyer flasks were inoculatedwith spores (106 spores/ml ) exept for the non sporulating Pol 6 strain, where mycelium was used. The culture were incubated at 30 ~ C on a rotary shaker (150 rev mn -1) for 5 days. The culture broth was filtered (Millipore 0.45 ktm ) and the supernatant was analysed for pectinolytic activities, reducing sugars and proteins.

923

2.4 Enzyme Assays : Reducing sugars in the culture filtrate were determined using the DNS method ( Miller, 1959) using the galacturonic acid as standard. Exo and endo-pectinolytic activities were determined by measuring the formation of reducing sugars and by the relative change in viscosity, respectively. Exo-pectinase and exo-polygalacturonase activities were determined as follows: 0.5ml of culture filtrate were added to 0.5 ml of 0.9% citrus pectin 75% DM (Sigma) or 1% Polygalacturonic acid respectively in 50 mM citrate buffer pH4.8. After lh incubation at 45~ the reducing sugars liberated by the enzyme were determined using the DNS method. One unit is defined as the amount of enzyme liberating one ktmole galacturonic acid per minute. Endo-polygalacturonase and Endo-pectinase activities were determined via the reduction in viscosity of 0.5 % polygalacturonic acid solution and 0.25% citrus pectin solution respectively. The relative change in viscosity was measured using an Ostwald viscosimeter as described by Sakai T. (1988) : 6 ml of 0.25% citrus pectin or 0.5% polygalacturonic acid in 50 mM citrate buffer pH 4.8 was incubated at 37 o C for 3 mn and then 1 ml of culture filtrate was added, the mixture was incubated at 37 ~ C for 5 mn. The rate of viscosity reduction (A) is calculated using the equation: A = (Ta - T / Ta - To ) x 100, where T is the flow time (sec) of the reaction mixture, Ta is the flow time (sec) of polygalacturonic acid solution or pectin citrus solution added to the heat-inactivated enzyme, and To is the flow time (sec) of water added to the heat-inactived enzyme. One unit of enzyme activity is defined as the activity reducing the viscosity by 50%. Proteins concentration was estimated with the Biorad reagent by the method of Bradford (Bradford, 1976), with crystallin bovine serum albumin as standard.

3. R E S U L T S

3.1 Penicillium occitanis as a pectinases producer strain: Among more than 20 fungal strains tested on agar plates, we selected some of them to be tested on liquid cultures: two Aspergillus niger (A1 and F38), one Trichoderma inhamatum (OL1J), one Penicillium occitanis (CL100) and its hyperproducer mutant Po16 (Jain et al, 1990-b). As it is shown in table 1, the Po16 mutant is the best producer of pectinases. Moreover, this mutant secretes a wide variety of hydrolytic enzymes such as cellulases, hemicellulases and even lipases. The major disavantage of Po16 is that it can not sporulate. We shall note that this mutant was selected after 8 rounds of mutagenesis and selection ( Jain et al 1990-b): it should have accumulated many mutations responsibles of this particular state. Table 1 Qualitative comparison between fungal strains Strains(a)

Pectinases (b)

Cellulases (b)

Lipases (b)

Sporulation

P.occitanis (CL100) w.t P.occitanis (Po16) mutant A.niger (A1) w.t A.niger (F38) w.t T.inhamatum (OL1J) w.t

+ +++ + + +

+ +++ +++-

+++ +++-

++ -++ ++ ++

(a): the name of the strain is indicated between (b): enzymatic activities were determined qualitatively on plates as in Materials and Methods with +++>++>+>+->--. Sporulation was estimated on PDA plates.

924

3.2 Mutagenesis strategy CTI mutant:

of P. occitanis

parental strain and selection of the

In order to select a sporulafing hyperproducer mutants from P.occitanis, we planned to mutagenize the wild type strain by Nitrous Acid. After two times of treatment, 40 and 80 mn, leading to 10% and 1% of viability, mutagenized spores were plated on either PDA or Hankin medium containing 1% of citrus pectin. Two ways of revealing were adopted: 1) Indirect staining with iodine: colonies were arranged in duplicate grids one on PDA and the second on BSM+pectin, the latter ones were stained by iodine. 2) Direct staining by CTAB (Hexadecyl -trimethyl- ammonium bromide ): the colonies, which arose from mutagenized spores on Hankin medium, were directly stained by CTAB and the interesting mutants having a big halo were picked up and sub-cloned on a fresch plate. All the primary clones were then subcloned giving a set of secondary clones which were tested again by the same staining procedure. From the interesting ones, three independant colonies were cultured in liquid and their enzymatic capacities were assessed. From 800 colonies tested by the "indirect iodine procedure", only one interesting mutant was selected but after sub-cloning, it was discarded due to its instability. From 2000 colonies tested by the "CTAB procedure", one mutant named CT1 was selected. Surprisingly, it exhibited, as a primary clone, a big precipitate or white halo but not a clear halo. After sub-cloning on plate to purify it from the potential contaminating spores and after multiple round of growth and sporulation, it became clear that the CT1 mutant was pure, still sporulating and genetically stable.

3.3 In comparison to the Po16 mutant, CT1

hypersecretes only pectinases.

The wild type CL100 and the mutant CT1 strains were cultured in liquid Mandels media containing 1% citrus pectin. It was already known that the wild strain CL 100 produces very low amounts of both pectinases and cellulases and that the mutant Po16 secretes very high amounts of both cellulases and pectinases (Jain et al, 1990 b) but the CT1 mutant exhibits a particular feature in sense that it hypersecretes only pectinases. In fact, CT1 hyper-produces all classical pectinolytic activities such as endo-polygalcturonase (endoPG), endo-pectinase (endoPC), exopolygalacturonase (exoPG), exo-pectinase (exoPC), pectin methyl-esterase (PE), polygalacturonic acid lyases (PGlyases) and pectin lyases (PClyases); these two latter activities are not shown here. We don't yet know if CT1 secretes also debranching pectinases. Cultured grown on pectin or on cellulose substrate, the CT1 mutant didn't produce more cellulases than the wild strain, compare on table2 the fp (filter paper) activity of both strains. Table 2 Enzymes produced by parental (CL100) and mutant (CT1) strains cultured on citrus pectin Strain

Prot(a)

ExoPC(b)

EndoPC(b)

CT1 CL100

55 23

47 2.3

100 2.1

ExoPG(b) 250 6.5

EndoPG(b) 80 1.5

PE(b)

fp(C)

4 0.6

0.22 0.05

The strains were cultured on Mandels medium + 1% citrus pectin for 5 days and the enzymatic activities of culture filtrates were determined on three substrates: citrus pectin, polygalacturonic acid and filter paper. (a): extracellular proteins are in ktg/ml. (b): pectinolytic activities on pectin (PC) and on polygalacturonic acid (PG) and Pectin esterase (PE) are in units/ml. (c): total cellulolytic activity (filter paper, fp) are in mg of liberated reducing sugars/ml.

925

3.4 C T I h y p e r p r o d u c e s peel" local substrate.

pectinases on "citrus pectin "

as well as on "orange

One of the important criteria taken into account for the choice of an industrial producer strain is its ability to secrete enzymes on cheap and local substrate. Thus, we cultured our mutant as well as the Po16 mutant on a local substrate: milled "orange peel", at the same concentration as citrus pectin in the liquid medium. The results summarized on table 3 show a net difference between both strains: the CT1 mutant is able to produce high amounts of endo and exopectinases on both substrates whereas Po16 is unable to hyper-produce both pectinases on "orange peel". Table 3 Comparison of Enzymes production by CT1 and Po16 strains cultured on citrus pectin and orange peel Citrus Pectin Strain CT1 Po16

Prot 55 76

Orange peel

ExoPC 47 25

EndoPC 100 23

Prot 145 118

ExoPC

EndoPC

54 3.5

100 2.8

Proteins are in gg/ml and pectinases are in U/ml. 3.5 CT1 is a constitutive mutant"

In order to better characterize our mutant, we compared its pectinases production (endo and exoPG, endo and exoPC) on different carbon sources with that of the CL100 mother strain as well as the Po16 mutant and the Aspergillus niger F38 strain. We cultured these strains on Mandels medium contaning 1% of one of the following substrates: citrus pectin, orange peel, polygalacturonic acid, glucose and glycerol. Figure 1 summarizes all the results and shows clearly that the CT1 mutant is a constitutive one. Indeed, it produced the same amounts of specific activites of all pectinolytic activities on inducer substrates as well as on the repressor ones (glucose and glycerol) although it was more derepressed on glycerol than on glucose. Note that the scale of ordinates on figure 1 is very different between CT1 and the other strains and strengthens the fact that even on glucose the CT1 mutant produced relatively more pectinases than CL100 and Po16 strains. On the other hand, the pectinase production of all three remaining strains are repressed by glucose and glycerol, what is currently known about Aspergillus (Solis et a1.1990; Aguilar et al., 1990). Other hydrolytic enzymes, such cellulases, are still repressed by glucose and glycerol in the CT1 mutant (data not shown). Figure 1 Extracellular pectinases (expressed as specific activities) produced by strains grown on different substrates: The substrates used were: PC: citrus pectin; E: orange peel; G: glucose, Gly: gycerol, APG: polygalacturonic acid. Results of two cultures (1 and 2) are presented. On ordinates, specific activities (Activity Units par gg of extracellular proteins) are presented. Note: the ordinates scale differs from one histogramme to another.

926

CT1 10 o

8 =L

1 ExoPC m EndoPC I! ExoPG !~ EndoPG rIPE

6 ,,m

2

om

O, 9

PC1

PC2

E1

E2

G1

,

9

|

62 GLY1GLY2 APG1 APG2

Carbon source

CLIO0 0,3

0,2

,H

~

0,1

,m

o

0,0 PC1

PC2

E1

E2

G1

n

n

,-.,~n...r

G2 GLY1 GLY2 APG1 APG2

Carbon source

1 ExoPC [I EndoPC Im ExoPG IrA EndoPG rIPE

927

Po16 ~

0,8

0

0,6

"~.

0,4

m m m Q

0,2"

ExoPC EndoPC ExoPG PE

O,

0,0 PC1

PC2

E1

E2

G1

G2 GLY1 GLY2APG1 APG2

Carbon source

F38 0,3 o

0,2" o~

1 m m D

o~

o_,

0,1"

5

ExoPC EndoPC ExoPG EndoPG

OPE 0,0

. . PC1 PC2

. E1

. E2

. . . . . G1 G2 GLY1 GLY2 APG1 APG2

Carbon source

928 4. DISCUSSION We have tested various fungal strains for their secretion of pectinases in order to apply these extracellular juices in the improvement of olive oil extraction. The hypercellulolytic mutant strain Po16 secreted high amounts of pectinases (Jain et al, 1990-a,b) but didn't sporulate. Therefore, we decided to select a novel mutant from the wild type mother strain CL100. Indeed, after Nitrous acid treatment and selection on pectin plates stained with CTAB, we selected a very interesting mutant named CT1. It has the same enhanced capacity to secrete pectinolytic enzymes as the Po16 mutant but it sporulates very well and does not hyperproduce other hydrolases such as cellulases or lipases. The most interesting result concerns its ability to produce high specific activities of pectinases on glycerol and glucose whereas the mother (CL100) and the mutant (Po16) strains have to be cultured on pectin based substrates to produce pectinolytic enzymes. This result suggests that CT1 should be a regulation mutant and henceforth a constitutive one. The fact that the CT1 mutant was selected after only one round of mutagenesis suggests that it has been touched by a single mutation, which is an ideal situation for fondamental studies to discover the nature of this mutation. This mutant is deregulated for more than one pectinolytic enzyme ,but not for the other hydrolytic enzymes such as cellulases, subsequently we believe that the responsible mutation should probably lie in a regulatory gene encoding a trans-acting factor involved in the specific expression of pectinolytic enzymes. The CT1 mutant can produce high amounts of pectinases on local "orange peel" substrate whereas Po16 hyper-secretes pectinases only on citrus pectin or polygalacturonic acid. Concerning the mother strain, even if it produces much lower amounts of pectinases, its behaviour resembles that of Po16. This result suggests that the mutation in CT1 would have turned on the expression of a gene whose product is involved in the degradation of orange peel and/or in the uptake of some nutrients. Whatever the explanation is, since the CT1 mutant secretes high amounts of pectinases on cheap local substrates, it should be very useful for industrial applications. We have already successfully tested it in the improvement of oil extraction from olives. In this work, we focussed our attention on some pectinolytic enzymes in order to compare the various strains studied. Other activities such as lyases and "hairy regions" specific enzymes have to be assessed in order to get more detailed on the capacities of our mutant and the other strains. 5. R E F E R E N C E S

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Rombouts F.M. and Pilnik W. Microbial Enzymes and Bioconversions, (1980) 227-281 Schols H.A., Voragen A.G.J. and Colquhoun I.J. Carbohydr. Res. 256 (1994) 97-111 Pilnik W. and Rombouts F.M. Carbohydr. Res., 142 (1985) 93-105 Servili M., Begliomini A.L.and Montedoro G. J. Sci. Food Agric. 58, (1992) 253-260 Gainvors A., Karam N., Lequart C. and Belarbi A. Biotechnol. Letters 16, 12 (1994) 1329-1334 Laing E., Pretorius I.S. Appl. Microbiol. Biotechnol. 39, (1993) 181-188 Bussink H.J.D., van den Hombergh J.P.T.W., van den Ijssel P.R.L.A. and Visser J.. Appil Microbiol Biotechnol 37 (1992) 324-329 Jain S., Parriche M., Durand H. and Tiraby G. Enzyme Microbiol. Technol., 12 (1990-a) 691-696 Jain S. Durand H. and Tiraby G. Appl. Microbiol. Biotechnol., 34 (1990-b) 308-312 Hamdi M., Bouhamed H. and Ellouz R. Appl. Microbiol. Biotechnol.36,(1991) 285-288 Hankin L., Zucker M. and Sands D.C. Applied Microbiology, Aug.(1971) 205-209 Mandels M., Andreoti R. and Roche C. Biotechnol. Bioeng., 6 (1976) 21-23 Miller G.L. Anal. Chem. 31 (1959) 426-428 Sakai T. Methods in Enzymology 161 (1988) 335-350 Bradford M. Anal. Biochem. 72 (1976) 248 Solis S., Flores M.E. and Huitron C. Biotechnol.Letters, 10 (1990) 751-756 Aguilar G. and Huitron C. Biotechnol. LetterS 12, 9, (1990) 655-660

APPLICATIONS.

A) DEVELOPMENTS IN PECTIN MANUFACTURING AND APPLICATIONS



Production pectin

931

of hypocaloric jellies of grape juice with

sunflower

M.L. Alarc&o-Silva a, H. Gil Azinheira b, M. I. N. Janu&rio c, M.C.A.Leit~o b and T. C. Curado d

a Centro de Microbiologia e IndQstrias Agricolas, Instituto Superior de Agronomia, Tapada da Ajuda, 1399 Lisboa, Portugal. b Centro de Estudos de Produg&o e Tecnologia Agricolas, Instituto de Investigas Tapada da Ajuda, 1301 Lisboa, Portugal.

Cientifica Tropical,

c Secs Aut6noma de Agronomia Tropical e Sub-Tropical, Instituto Superior de Agronomia, Tapada da Ajuda, 1399 Lisboa, Portugal. d Estas Nacional de Tecnologia de Produtos Agrarios, Instituto Nacional de Investiga;&o Agraria, Quinta do Marquis, 2780 Oeiras, Portugal

Abstract The increasing interest of low-caloric foods in diet is well known. This fact has led to the study of Iow-methoxyl pectins that allow to obtain jellified products (such as jams, jellies, marmalade) by using very small quantities of sugar. Sunflower head residues are one of the richest sources of Iow-methoxyl pectin, their most important property being the ability to form gels, if correct proportions of divalent ions (usually calcium) are available. Following earlier studies about physico-chemical characterization of sunflower pectin (Alarc~o-Silva, 1990; Leit~o et a/, 1995) and technological utilization in the manufacture of low-caloric gels (Alarc~o-Silva eta/, 1992), we intend with this contribution to study the behaviour of this pectin in the confection of grape juice jellies and the evaluation of their organoleptic characteristics.

Keywords: hypocaloric jelly; Iow-methoxyl pectin; sunflower; texture profile analysis; sensory evaluation. 1. INTRODUCTION

The importance of some pectic substances (pectin or pectins) either due to the great amounts that certain raw materials contain, or the existence of any specific food use, as well as its hypocholesterolemic effect (based in in vitro and in vivo studies) would explain the increasing interest on deepening physico-chemical studies of pectic substances.

932

Sunflower plant (Helianthus annuus L.)is an important crop in Portugal and its head residues, remaining on soil after the seeds have been removed for oil industry, are one of the richest sources of Iow-methoxyl pectin (ca 19% original dry matter), the most important property being the ability to form gels even without sugar addition, if correct amounts of divalent ions (usually calcium) are present. In our country, as well as in other regions of the European Union where vineyard and wine consumption are traditional there is a great interest in diversifying grapederivated products, especially those varieties of less enological potential. The manufacture of reduced calorie jellies and/or jams is one of the possible items to be considered. Today such products are gaining importance as an interesting segment of food market, because people are more and more aware of their beneficial effects on health. Following previous works on physico-chemical characterisation of sunflower Iowmethoxyl pectins (Alarc~o-Silva, 1990, Leit~o et al., 1995) and technological utilisation in the manufacture of low calorie gels (Alarc~o-Silva et aL, 1992), this investigation was carried out to test the suitability of that pectin to the confection of grape juice reduced calorie jellies in comparison with two types of commercial pectin. Aiming at the optimisation of low-calorie jelly formula, based on consumers' preferences, the jellies were submitted to a sensory panel test judgement and instrumental texture-analysis.

2. MATERIALS AND METHODS 2.1. Sample composition Grape juice was obtained from a 1:1 mixture of two red cultivars, "Benfica" and "Piriquita". After harvest the grapes were washed, sorted out, crushed, treated with SO2, pressed and kept at 7~ for three days. The juice was filtered and stored at -3 to 0 ~ using 0.06% potassium sorbate as preservative. The soluble solids content was 22 ~ the pH 3.3, calcium, potassium and magnesium contents were 125, 1170 and 240 mg dm 3, respectively. Experimental Iow-methoxyl pectin was obtained from dry heads (without seeds) of sunflower (Helianthus annus L.). The extraction of pectin was carried out according to the method of Lin eta/. (1975) with slight modifications. Only oxalate-soluble fraction which was submitted to consecutive treatments of purification as described previously was considered (Leit~o et a/., 1995). The standard Iow-methoxyl commercial pectins used were Violettband D-075 (amidated pectin) and Violettband Rein (non-amidated pectin) provided by OBIPEKTIN AG (Switzerland). 2.2. Jellies preparation The grape jellies were made using the pectin concentrations of 0.5, 0.75, and 1% for the three types of essayed pectin.

933

The formula (Table 1) and the general procedure used in the preparation of the low-calorie jellies was as follows: the pectin was blended with 25% its weight in sugar, dispersed in hot water with magnetic stirring and added to the grape juice previously heated at 60-70 ~ The mixture juice-pectin was heated with continuous stirring to ensure full suspension of pectin. The remaining sugar was incorporated when temperature approaches 70 ~ and heating stopped after the mixture reached 39 ~ (refractometer reading). Then it was poured into glass containers, kept at18~ for 15 minutes and stored at 4~ until evaluation of jellies quality characteristics. Table 1 Jellies experimental Ingredients Pectin Sugar Water Grape juice

formula Amounts (g) 1; 1.5; 2 52 50 100

2.3. Sensory evaluation

The jellies (20 sets) were submitted to a sensory panel (ten panellists from the laboratory staff with some experience in sensory evaluation) requested to give a score (from /ow to high in a non-structured 10 cm scale) to each of the following characteristics: aroma (intensity), taste (sweet, acid and intensity), texture (hardness, spreadability) and overall acceptance. Tests took place in a standardised test room provided with individual booths and the trials assessed in four sessions with five randomly grouped samples at each time.

2.4. Texture-profile analysis

The textural characterisation of the jellies was made by using the empirical technique of Texture-Profile Analysis (TPA) that allows the evaluation of the following parameters: fracturability, hardness, cohesiveness, adhesiveness, springiness, gumminess and chewiness. The samples were analysed on a TAX T2 Texture Analyser programmed for the following conditions: a cylindrical plunger with 1 in. diameter; contact force of 5 g, contact area of 284.88 mm2, speed of 2 mms 1 and a 5 s interval between the first and second bites. The experiments made in triplicate were performed at room temperature.

934

3. RESULTS AND DISCUSSION 3.1. Physical characteristics of low-calorie jellies In this study we intend to investigate if a correlation between sensory evaluation and instrumental measurements of the Iow-methoxyl pectin jellies could be established. Texture profile analysis is an empirical technique of double-penetration that simulates two bites of the jaw action. Data obtained from the Force-time plots enable the evaluation of seven texture parameters (Figure 1). Hardness is an estimation of the required force to penetrate jelly (peak force during the first bite). The results showed that jellies prepared with non-amidated pectin had such a low hardness that values could not be measured in the used instrumental conditions. Therefore the non-amidated pectin will not be considered in the other parameters interpretation. This fact agrees with the general information that non-amidated pectins usually require more calcium ions than those already present in the juice for a good gelation (Pedersen, 1980; Pilgrim et al, 1991). As far as amidated pectin and the sunflower pectin for range concentration under study (0.5, 0.75 and 1%) are concerned, the behaviour of jellies was similar, hardness values increasing for higher concentration levels (Figure 2). The lowest value was registered for 0.5% amidated pectin and a maximum was reached at 1% of the same pectin. The cohesiveness that represents the work required to overcome the internal bonds of the sample (jelly) shows an increase with the pectin content of jelly and there are no apparent differences between the two types of pectin considered (Figure 2). Similar behaviour was observed for springiness (elasticity) which is given by the time that the material spends to recover its non-deformed condition after the first bite (Figure 2). Adhesiveness, defined as the work necessary to overcome the attractive forces between the surface of the sample and the surface of other materials with which the food comes into contact, e.g. tongue, teeth, palate, etc. (Szczesniak, 1963), is given on the texturometer curve by the negative force area, representing the work needed to pull out the plunger from the sample. This parameter's value may be considered an evaluation of stickiness of jelly. Fracturability, also called brittleness, is given by the measure (%) of the plunger path into the jelly when it breaks. The jellies analysed had no stickiness at all and were non-brittle for the levels tested. This finding, especially as far as sunflower pectin is concerned is quite different from what could be expected, based on Chang's results (1992). The evaluation of two other parameters results from simple arithmetics: gumminess is the product of hardness x cohesiveness and chewiness is given by the product of hardness x cohesiveness x springiness. The results obtained for the analysed jellies are shown in Figure 3. As one would expect, both parameters increase with pectin concentrations as it was likewise observed with hardness, cohesiveness and springiness.

935 M1 /~eas (g s) 1. 16.509 2. 14.SS2 3. N/A

!

2

Peaks (4~) 1, 15.6 2. 14.7 3. N/A

I

-0.0

S~'oJn : N/A

ll~ ~ |llVintJPllill II

,+0

0.

l

,Ill 32 +0

,,.

I n ~ l l Stress : 3.997E§ lll/n.+ Ploduot Height : N/A Compression : 2.000 mm Resilienoe : N/A

Springiness : -0.900

Gumminess :

Cohesiveness :0.877

Adhesiveness : N/A

Chewiness :

In~ad Modulus : N/A

12.314

13.683

Fraoturabilil~ : N/A

N/A

Hardness:

N/A

IS.6 g

Figure 1. TPA profile (Sunflower pectin 1%)

18,000

Q Springiness

] i El Cohesiveness 1 G Hardness

16,000 14,000 12,000 10,000 8,000 6,000 4,000 2,000 0,000

-o

~

~

of)

(f)

c/)

Figure 2 Hardness, Springiness and Cohesiveness vs. pectin concentration

936

16,000

EIChewiness I

14,000

i OGumminess

12,00010,0008,000

6,000 4,0002,000-

0,000-

e

9 ~n

. . . . . . . .

o ~ ,~.-

Figure 3 Gumminess and Chewiness

0,8

IA50

I,~

Ln

pectinconcentration

vs.

l

i

o,6 +

! I

I 0,4 4

I

0,2 !

IIA75 9

XS50 X AC

,

/t

/

"

..

XTI

$100

'

/ / ,,

/ '

/ ,, -0,2 '-:-, "~ { .

..04•

-/ " sXw X F l a

~,

$75

'

~ ~

".

'

~"OHad

".

~ x

".

~ '

06"I"

A_~I100

"

XHS ~ "xOA

I

i

Figure 4. Plotting of jellies (6) and variables (12) on a plane formed by the first and second principal components, F1F2. Had= Hardness; Spr.= Springiness; Che= Chewiness; Gum= Gumminess; Coe= Cohesiveness; HS= Sensory hardness; SP= Spreadability; Ac= Acid; Sw= Sweet; Fla= Flavour; TI= Taste intensity; OA= Overall acceptance.

t

937

3.2. Sensory analysis The evaluation of some parameters was quite discrepant probably due to the resort to a non-trained panel. Nevertheless all the answers were considered (maximum value for standard deviation being ___3.0,results not shown). The data from sensory evaluation and texture profile analysis of the jellies made with amidated pectin and sunflower pectin were subjected to Principal component analysis (PC) using the statistical software based on Jacobi method (Univac, 1973). The results of PC analysis are shown in figure 7. The plane of two principal components (F1,F2) explain 89,75 % of the variance contained in the original data. The attributes related with textural evaluation are highly correlated with the first principal component (Had.=0.95, Spr.=0.97, Che.=0.98, Gum.=0.95, Coe=0.98, HS=0.82 and SP=-0.93). As it could be expected, spreadability increases along the negative side of the axis unlike other textural parameters. The overlapping of textural attributes suggests that characterisation of this kind of jellies could be based on the evaluation of a single parameter. The concept of hardness being the easiest to apprehend and due to its close relation with the same sensory attribute, we believe that when jellies are to be appreciated from a textural point of view, hardness may be measured on its own. Overall acceptance is an attribute that seems to be influenced either by textural attributes or the flavour ones. However it is possible to establish correlations between overall acceptance and hardness (Figures 5, 6, 7, 8). The jellies made with sunflower pectin and amidated pectin (level 1%) are very similar (in so far as texture is concerned) but as the polynomial correlation (degree 2) suggests, above 0.7% of sunflower pectin the overall acceptance decreases probably due to the appearance of perceived in-mouth sensations described as "greasy" and "clammy" and leading to an unfavourable appreciation. A decrease in the perception of taste and aroma of the jellies has been noted when hardness increases, which agrees with Chai eta/. (1991) who refer a reduction in the perception of flavour intensity with an increase of gel rigidity which may be related with the available surface area of gel exposed on chewing.

4. CONCLUSION From the results of this study it appears that commercial amidated and experimental sunflower pectins have similar behaviours and from a consumer's point of view there are only small differences mainly related with a visual evaluation of the jellies, and "greasy" and "clammy" tastes. Jellies made with sunflower pectin show small air bubbles that could be responsible for the slight opacity observed; with amidated pectin jellies are very transparent as it is also referred by several authors.

I

y = - 0 , 0 8 9 9 ~+ ~2 , 3 3 8 9 ~ - 9,7961

I

R2 = 1

4 9 -4 B -4.7 ~46 0

5.4

,

I I

4

r

b

938

5.5

/h e o u e l d e o o e IleJeAO

eoum, d e o o e IleJeAO

5.5 5.4 5,3 5.2 --

y = -0,742+ ~ ~8 , 2 0 0 8 ~ - 17,203 R2 = 1

m 5

10

15

20

4,7

Hardness

46

2

0

Figure 5 Correlation between overall acceptance and hardness (jellies made with sunflower pectin)

a

4 6 Sensory hardness

Figure 6. Correlation between overall acceptance and sensory hardness (jellies made with sunflower pectin)

6

3.-

N

y = 0.781+ ~ 0,759 R2= 0,9998

O

2.-

1

-

y = 0 . 4 5 2 7 ~ 2.0352 R2 = 0,9912

IIRJeA

e0ue%de00e IleJeA O

eoue%deooe

5

~~

0

0-

Figure 7. Correlation between overall acceptance and hardness. (Jellies made with amidated pectin)

2

4

6

8

Sensory hardness

Figure 8. Correlation between overall acceptance and sensory hardness. (Jellies made with amidated pectin)

939

The good results obtained in the production of jellies with experimental sunflower pectin are all the more interesting as this pectin has not suffered any standardisation process like the commercial ones. Such a process could eventually overcome the undesirable characteristics mentioned above. Although this study needs further work it is thought to give some contribution to a better understanding of the behaviour of Iow-methoxyl sunflower pectin in food technology namely in reduced calorie jellies or jams. The results obtained corroborate the interest, already suggested by the authors, in using sunflower agricultural wastes as an important source of natural Iow-methoxyl pectin for these purposes.

5. REFERENCES

Alarc~lo-Silva, M. L. Characterization of a pectin from sunflower head residues. Acta Alimentaria, 19, 1 (1990) 19-26. Alarc~.o-Silva, M.L.; Curado, T.C.; Sousa, I.M.N. Gelificados hipocal6ricos de um sumo de uva por incorpora~o de pectina com baixo teor de metoxilo extraida de residuos de girassol. In: Actas das I Jornadas das Ind0strias Agro-Alimentares, ISA, Lisboa, 1992. Chai, E.; Oakenfull, D.G.; McBride, R.L.; Lane, A.G. Sensory perception and rheology of flavoured gels. Food Australia, 43, 6 (1991) 256-257. Chang, K.C.; Miyamoto, A. Gelling characteristics of pectin from sunflower head residues. J. Food Sci., 57(1992)1435-1443. Leit,~o, M. C. A.; Alarc&o-Silva, M. L.; Janu&rio, M. I. N.; Azinheira, H. G. Galacturonic acid in pectic substances of sunflower head residues: quantitative determination by HPLC. Carbohydr. Polym.,26(1995)165-169. Pedersen, J.K.. Carrageenan, pectin and xanthan/Iocust bean gum gels. Trends in their food uses.Food Chem. 6 (1980) 77-88. Pilgrim, G.W.; Walter, R. H.; Oakenfull, D.G. Jams, jellies and preserves. In: The chemistry and technology of pectin . Reginald H. Walter (ed.) Academic Press, London, 1991, 23-50. Szczesniak, A. S. Classification of textural characteristics. J. Food Sci., 28, 4 (1963) 385-389. Univac. Large scale systems, STAT-PACK, FACTAN - Factor and principal component analysis (1973) 33-39.


941

Influence of microwave pretreatment of fresh orange peels on pectin extraction

M.Kratchanova a, E.Pavlova a, I.Panchev b, Chr.Kratchanov b aBulgarian Academy of Sciences, Institute of Organic Chemistry, with Centre of Phytochemistry, 95 V.Aprilov Str, P.O.Box 27, LBAS - Plovdiv, 4002, Bulgaria bHigher Institute of Food and Flavour Industries, 26 Maritza Blvd., Plovdiv, 4002 Bulgaria

Abstract Laboratory studies on the extraction of pectin from orange peels, pretreated in an electromagnetic field of hyper frequency, were carried out. The influence of intensity of microwave treatment (Pw) and time on pectin yield and pectin quality was investigated. It was established that the increase of Pw and time lead to increase in the pectin yield with 180 240 % in comparison with the control. Apparently, the microwave treatment leads to a considerable increase in the soluble form of pectin, characterized by increase in the jelly strenght and in the polyuronic content.

Introduction In a previous publication (1) we reported that the pretreatment of fresh fruit waste using microwave heating ensured a better extraction of pectin, resulting in an increase in the yield of pectin from 10 to 50 %. It was established that this microwave pretreatment ensured retention of the degree of esterification of the extracted pectin, better expressed in citrus peels. The aim of the present investigation is to study the influence of microwave pretreatment of fresh orange peels on the extraction time of orange pectin and the influence of the intensity and duration of microwave heating on pectin yield. Materials and Methods Two lots of oranges of the Navel I and II type imported from Greece, were used in this study. The peels were removed, then finely cut and processed following two procedures: part of them were dried in the laboratory drier at 60~ while the rest were pretreated in a microwave oven and then dried in a laboratory drier at 60 ~ Microwave heating of the fresh orange peels The orange peels (200g) were placed in a glass vessel and heated in a microwave oven Samsung with different duration of exposure: 2,5,10,15 and 20 min and with different intensity: 0.45, 0.63 and 0.9 kW.

942 Extraction of pectin The dry mass, obtained atter drying 200 g of fresh orange peels, was subjected to extraction by adding 2.5 L water. By applying 0.5 N hydrochloric acid, pH was adjusted to 1.5. The mixture was then heated to 80 - 82 ~ and extraction was carried out with continuously stirring in a laboratory stirrer for 1 hr (Table 3). The experiments quoted in Table 2 were conducted with 30, 60, 90 and 120 min duration of extraction. The hot mass was filtered through cloth. After cooling, the filtrate was coagulated using an equal volume of 96 % ethanol and let~ for an hour. The coagulated pectin was separated by filtration, washed once with 70 % hydrochloric acid ethanol, then with70 % ethanol to neutral reaction and finally with 96 % ethanol. It was dried at 60 ~ in a laboratory drier. Methods of Analysis The anhydrouronic acid content (AUAC) of the initial material was determined by .the method of Gee (2). Analysis for pectin was implemented by the method of Owens et al. (3). The gel strength was determined by the Tarr-Baker method according to the procedure described by Bender (4). Intrinsic viscosity [~1] and Huggins' constant KH were calculated according to Huggins' equation qsp/C = [vi] + K'H [q]2c according to (5). The average molecular mass Mv was determined by solving the equation following the methods in (6). Results and discussion Data on the pectin content of the initial material (AUAC and DE) is presented in Table 1. It is evident that the main difference between the two lots of oranges was in their anhydrouronic acid content. Observations from previous research (1) were confirmed that microwave heating of the fresh material ensured better drying conditions by inactivating pectolitic enzymes, particularly pectinesterase. Thus a better retention of the degree of esterification of pectin was ensured. Table 1 Analysis of initial materials Kind of materials AUAC, % DE, % Orange peels Navel I 4.8 76,9 Fresh 12.1 68.8 Dry 12.7 72.7 Dried a~er 10-min microwave heating Orange peels Navel II 3.4 73.8 Fresh 13.5 71.7 Dry 14.0 72.8 Dried after 10-min microwave heating

In the first series of experiments (Table 2) the influence of microwave pretreatment on extraction of pectin was followed. Microwave pretreatment had a slight positive effect on the

rable 2 Influence of extraction time on the yield and characteristics of pectin from fresh Navel I orange peels pretreated by microwave heating Sample Nr

Kind of initial material for pectin extraction

1

Dried material - control sample Dried after 10 min microwave heating Dried material - control sample Dried after 10 min microwave heating Dried material - control sample Dried after 10 min microwave heating Dried material - control sample Dried afker 10 min microwave

2

3 4 5 6 7 8

Yield of pectin, g per 200gfresh material

AUAC, %

30 30

3.5 8.7

60.6 68.9

60

3.9 9.4

90 120 120

Extraction time min

60 90

DE, %

Molecul mass

Huggins constant

Gel. strength

M V

KH

Intrinsic viscosity [q]d1.g''

63.3 70.3

72 000 65 000

0.5 0.5

4.5 3.9

175 200

68.6 66.3

64.6 69.4

64000 59 000

1.o 1.1

3.8 3.4

195 215

4.8 9.2

65.8 66.4

63.1 67.8

66 000 60000

0.7 0.6

4.0 3.5

200 225

4.3 8.6

64.5 73.2

60.1 68.2

53 000 57000

1.0 1.o

3.0 3.3

190 205

~~

"TB

943

944

Table 3 Influence of intensity and duration of microwave heating of fresh Navel I1 orange peels on pectin yield and characteristics Time of Yield of pectin, g AUAC, DE, % Molecul Huggins Intrinsic Intensity of viscosity constant KH mass microwave % per 200 g fresh microwave treatment K, heating min material M" [q]d1.g-l 61 600 3.1 3.7 64.3 64.0 2.8 control sample 0.45 0.45 0.45 0.63 0.63 0.63 0.63 0.90 0.90 0.90 0.90

10 15 20 2 5 10 20 2 5 10 15

7.1 7.5 8.5 6.1 7.4 8.4

Gel. strength

"TB 155

63.4 70.7 70.2 67.3 68.9 69.1

70.6 71.0 68.7 67.0 71.3 72.4

60 000 65 000 61 000 60 000 66 000 61 000

3.6 1.4 1.3 2.8 1.2 1.7

3.5 3.9 3.6 3.5 4.0 3.6

165 185 185 165 185 185

70.4 71.8 72.0

70.4 72.6 72.7

54 600 54 500 55 000

3.5 2.5 3.3

3.1 3.1 3.2

190 180 175

material bruns 7.3 8.0 8.7

material bruns

945 duration of extraction - maximal yield of pectin was achieved for ca 60 min; 90 min for the control samples.It may be concluded that duration of extraction has a stronger degradation effect on pectin molecules than microwave treatment of the fresh material. It is worth noting the considerable difference between the yield of pectin in the experimental samples and in the control samples. Apparently, microwave heating of the fruit tissue affects mainly the state of the protopectin - it destroys the bonds of the pectin macromolecules with the other polymers and thus protopectin turns into a water-soluble form. At the same time there is a slight positive effect on the capillary permeability of the fruit tissue and hence on the following process of penetration of the molecules of the solvent. It is worth noting the data on the intrinsic viscosity and the molecular mass. Data for experiments 1-6 show that microwave treatment rather leads to disintegration of the association bonds than to distinct depolymerization effect on the pectin macromolecules. This is observed in terms of a slight decrease in the molecular mass and a distinct increase in the gel strength force of pectin. Data from experiments 7 and 8 (long-lasting extraction) are a deviation from this dependence which is in support of the conclusion that long-lasting extraction in an acid medium has a strong negative effect on the quality of pectin. A sharp decreased yield of pectin was also observed in the control samples. The next series of experiments was dedicated to studying the effect of the intensity of the microwave field and duration of microwave exposure on the yield and quality of pectin. Data from oranges of the Navel II type (Table 3) show there is an inverse correlation between the field intensity and duration of exposure mainly expressed for 0.45 and 0.63 kW intensity. In case of a weaker field, longer microwave treatment is needed. According to expectation, a stronger destructive effect of microwave heating was observed at the top intensity of 0.90 kW: the molecular mass of pectin decreased by 10 %, accompanied by a slight increase in the yield. Duration of acceptable microwave heating was reduced for the higher values of the field intensity because of burn of material. The results presented confirmed previous observations by our team (1) and other authors (7) about the favourable influence of microwave heating of pectin row material on the yield and quality of extracted pectin. Data from the present experiments show that microwave destruction processes on biopolymers in the flesh fruit tissue can be overcome by choosing the intensity of the field and duration of exposure. Microwave effect is rather in terms of a denaturation process on the protein molecules, polysaccharide associates and protopectin, than a depolymerization process. An evidence that speaks for itself is the increase in the gel strength of the pectin, obtained from microwave pretreated materials, compared to that for the control sample: a sharp increase in the gel strength at the same or slightly lower molecular mass. On the other hand, comparison with data from Manabe's publication (7) shows that a more favourable microwave effect is accomplished when the raw material is treated before extraction instead of during pectin extraction. This conclusion is more favourable for industrial production of pectin- the orange raw materials should be. first subjected to microwave heating and then dried. Acknowledgement: The authors thank the Research Foundation of Bulgaria for the financial support of this work (project CC-457).

946 REFERENCES 1. M.Kratchanova, I.Panchev, E.Pavlova, L.Shtereva. Carbohydrate Polymers, 25 (1994) 141. 2. M.Gee, E.A.Mc Comb, R.M.Mc Cready. J.Food Sci., 23 (1958) 72. 3. H.S.Owens, R.Credy, A.Chepheral, T.Shultz, E.Pippen, N.Swenson, J.Miers, F.Erlander, W.Maclay. AIC Report 340, Western Regional Research Laboratory, Albany, CA, 1958. 4. W.A.Bender, Analyt.Chem., 21 (1949) 408. 5. H.Moravettz, Macromolecules in solution Interscierice Publishers, New Yormk, (1967) 254. 6. H.Anger, G.Berth. Carbohydrate Polymers, 6 (1986) 193. 7. M.Manabe, I.Naohara, T.Sato, J.Okada. Nippon Shokuhun Kogyo Gakkaishi, 35 (1988) 497.

Visser and A.G.J. Voragen(Editors), Pectins and Pectinases 9 1996 Elsevier Science B.V. All rights reserved.

J.

P r o p e r t i e s of p e c t i n e s t e r a s e from Penicillium new developments in p e c t i n a p p l i c a t i o n s

947 fellutanum

V.L. Aizenber 9, S.A. Syrchin, S.A. Sedina, V.N. Vasil'ev, Demchenko, P.N.Vitte

Biourge

L.N. Shinkarenko,

and

P.I.

Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine, Kiev, Ukraine Abstract Some properties of Penicillium fellutanum pectinesterase were studied. The optimum of pectinesterase action was detected at pH 5 and 45 ~ The enzyme was stable at pH 4 - 5 and 40 ~ (pH 5)"for 240 min. and was specific towards lemon pectin. An enzyme preparation composed mainly of pectinesterase was partially purified by gel filtration. Pectinesterase activity was accumulated in one of the obtained fractions. Molecular weights of fraction determined were found to be 46,000 and 1,200. Disk electrophoresis in polyacrilamide gel of the purified preparation revealed two protein bonds with one active component. The partially purified enzyme had the kinetic characteristics: Vm = 14.7"10 -5 M*min -1', Km = 5.56"10 -3 M; Ks = 0,22 M. New preparation of pectinesterase is recommended for production of lowmetoxilated pectin example for medicine. 1. I N T R O D U C T I O N

Pectinesterases are formed by numerous fungi but knowledge about fungal pectinesterase is limited [1], Capacity to synthesize extracellular enzymes depolymerising pectin was studied in 340 fungi isolates of the genera Penicillia belonging to 38 species of five sections (according to classification of Pidoplichko} [2]. There are a few data in the literature concernin 9 ability of Penicillia to split pectin substances in c o m p a r a t i v e - t a x o n o m i c aspect. The species, which were not described previously (such as P. fellutanum, P. thomii and P. multicolor) have been found among these possessing pectolytic activity. Such species of fungi as P. wortmanii and P. velutinum were found to be potentially low active. New n o n - t o x i c strain P. fellutanum Biourge from the soil of Kiev region was selected, It was characterized by the predominant pectinesterase synthesis [3]. Subject of the present message is study of the conditions for Penicillium fellutanum cultivation (that is a producer of pectinesterase) and analysis of some properties of pectinesterase enzymatic preparation. Miller and Macmillan [4] carried out purification of pectinesterase from Fusarium oxysporum f. sp. vasinfectum culture fluid (fivefold degree of purification). According to the obtained data the purified enzyme possessed very low polygalacturonatlyase one. Disk electrophoresis at pH 4.3 revealed two protein components. The authors did not study distribution of pectinesterase activity in these components. Molecular weight of fungal pectinesterase determined using gel filtration on Sefadex G - 75 was found to be 35,000. It was reported [5] that 4 active components with molecular weight of 56, 000, 30,000, 10,000 and 1,600 were obtained by separation of complex enzyme preparation from Penicillium citronum usin 9 a Sephadex G - 7 5 column. Three components revealed both pectinmethylesterase and pectinlyase activities, and the last one mentioned revealed only pectinlyase activity.

948 2. MATERIALS AND METHODS In the study culture of p. fellutanum, strain 57599 was used, which was grown by d e e p - g r o w t h method in flasks, dia 750 ml on Chapec medium with addition of beet pectin (1%) as a source of carbon or beet pulp (4%). It was grown on the rockers (220- 240 rev/min.} at 2 6 - 28 ~ For study of substrate specificity pectin with various degrees of metoxilation (expressed as a percentage) were used: beet substrate-37.8, apple s u b s t r a t e - 7 0 , l e m o n - 8 2 . Specificity of pectinesterase action was analyzed under optimum temperature and acidity of the medium using beet, apple and lemon pectin according to the speed of methanol formation (M 910 .5 ' rain.Enzymatic preparation with predominant content of pectinesterase (obtained from Penicillium fellutanum culture liquid by isolation by acetone was purified. Primary enzymatic preparation was r e - p r e c i p i t a t e d by three volumes of ethyl alcohol and centrifuged (6000 rev/min.} during 20 rain. The obtained precipitate of partially purified pectinesterase preparation was dried in v a c u u m - d e s i c c a t o r . Sephadexes G 50, G - 7 5 , G - 1 0 0 , G - 2 0 0 "LKB" (Sweden) and Toyopearl H W - 5 5 (Japan) were used for separation of enzymatic complex by gel-filtration. The specimens were analyzed at spectrophotometer at 280 nm. Preparations were purified from salts by dialysis. Protein concentrations in the initial and purified enzyme preparations were determined by Lowry method [6]. Molecular weight of the components of the enzymatic complex was determined using a Sephadex G - 7 5 column after its calibration by dextrans with molecular weight equal to 10,000, 40,000 and 70,000 and rafinose with molecular weight of 504. Fractions were also analyzed by the disk-electrophoresis method in PAAG [7] using 7.5% polyacrilamide gel (pH 4.3). Activity of pectinesterase was determined by titrometric method [8]. The enzymatically released methanol analyzed by g a s - l i q u i d chromatography [9]. 1% solution of highlymetoxilated beet pectin (made by "Biochimreactiv" co.) was used as a substrate for enzymatic activity determination (degree of metoxilation is equal to 37.8%}. Pectinesterase activity expressed as a unit corresponding to the microequivalent of ester bonds of pectin molecule, which were hydrolyzed during 1 rain. at 45 ~ and pH 5.0 under the conditions, which were optimum for these enzymes. Endopolygalacturonase and exopolygalacturonase activities were determined using a technique determined by Lifshitz [8]. Activity of pectintranseliminase was determined by procedure [ 10]. Speed of pectin hydrolysis catalyzed by pectinesterase was measured according to the quantity of alkali, used for titration of the free carboxyl groups during one minute (M'min-1} at 45 ~ Acidity of the reaction mixes after incubation increased as the activity of the probe r o - s e during determination of pectinesterase activity of the samples.It was caused by the f o r - m a t i o n of carboxyl groups as a result of pectin ester bonds hydrolysis under pectinesterase a c - t i o n . T h a t is why kinetic characteristics of substrate hydrolysis were measured according to the speed of pectin hydrolysis by continuously recorded titration of the free carboxyl groups [11]. After Michaelic constant (Kin} determination pectin concentration with known esterification degree was expressed in m o l / M according to the content of anhydrogalacturoni c acid. Kinetic parameters of the reaction of pectin hydrolysis (catalyzed by analyzed pectinesterase) were obtained by Z i n e w e a v e r - B e r k method [12].

949 3. RESULTS AND DISCUSSION Peak of relative activity was observed at pH 5.0 when it was determined pH optimum of P. bellutanum pectinesterase action. Under pH 4 and 6 the above mentioned activity accounted for 60% of its maximal value. Study of the temperature optimum of pectinesterase activity showed, that peak of pectinesterase activity was observed at the temperature equal to 45 ~ It is shown on Figure 1 that pectinesterase was stable at pH 4 - 5 . At pH 2 activity of the enzyme reduced by 25% in 60 min., at pH 3 and pH 6 it decreased by 6 - 8 % . At pH 8 the activity decreased by 90.7% during the same time. At pH 9 the enzyme activity was inactivated during 15 min. Fig. 1

Determination of pH-stability pectinesterase from P.fellutanum

100 90

pH 2

80 >

pH 3

7o

---)le--

pH6 ,...,

o >

pH 7

E:

pH8

10

,

0

.

10

.

.

.

20 30 40 Incubation time, min

60

.

.

.

.

.

,It was found out that P. fellutanum pectinesterase was sensitive to the temperature of 5 0 - 6 0 ~ At this temperature gradual decrease of the activity was observed. For example at 50 ~ the initial activity of the enzyme decreased by app. 43% in 60 min., at 60 ~ it decreased by 94% during the same time. At 65 ~ the enzyme was nearly totally deactivated yet in 20 min. The obtained data show kinetics of enzymatic activity at various temperatures within various time intervals. Pectinesterase was stable at the temperature of 40 ~ (pH 5) during 240 min. (Figure 2}.

950 Fig 2.

Determination of termostability of pectinesterase from P.fellutanum

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0 50

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0

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10

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30

40

50

60

90

120

240

Time, minutes

Data given on Figure 3 show that optimal concentration of studied pectin varied from 0.75 to 1%. Under this concentration maximal speed of released methanol (caused by pectinesterase) was observed. It was equal to 1 , 8 2 - 2 . 0 2 M*I0 - s * rain.-1. Both low (0.25?/o) and high (3%) concentrations hindered methanol release. Fig 3,

Speed of methanol formation during enzyme action on various substrate ._c E 2.2! / u~"/ '0 2"

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;eet '~le

951 Degree of pectin metoxylation is the main factor that determines the pectinesterase action. Lemon pectin was hydrolyzed most easily among all pectin studies: the speed of methanol release was 2.02 M* 10 -5 * min.-1. This is accounted by the fact, that the degree of the lemon pectin metoxylation is higher than in case of beet and apple pectin, All above mentioned shows, that pectinesterase from P. fellutanum possesses specificity towards lemon pectin (Figure 3}. P. fellutanum 57699 is characterized by the predominant synthesis of pectinesterase. Enzymatic complex produced by the fungus is distinguished from known industrial producers for increased content of extracellular pectinesterase of pectolytical complex. After purification of the enzyme preparation both at Sephadexes of various types and at Toyopearl H W - 5 6 two fractions were obtained (fractions 1 - 2}. One of them (fraction 1} possessed pectinesterase activity under the condition of absence of the activity of other pectolytic enzymes. Molecular weights of fractions I - 2 determined using a Sephadex G - 7 5 column (after its' calibration by various dextranes} were found to be 46,000 and 1,200. The results obtained after purification of pectinesterase preparation using columns with various gels certified that the active fraction (fraction 1 has greater molecular weight} was not subjected to further separation on the tested gels. Components of fraction 1 increased an activity of elute. That can be explained by their acid properties. Pectinesterase activity was accumulated in fraction 1. Activity of the other components of pectolytic complex was not found in the other studied probes of fraction 1. Disk electrophoresis o f pectinesterase enzyme preparation in polyacrilamide gel revealed 4 protein bands in the fraction 1 after its g e l - f i l t r a t i o n through Toyopearl H W - 5 5 . One of the two obtained bands (a wider one} possessed pectinesterase activity, while the second one did not reveal it. Partial purification of pectinesterase preparation enlarged to some extent its specific activity. A number of kinetic characteristics were obtained with preparation of the fraction 1. It was found out that reaction of the hydrolysis of highlymetoxilated beet pectin (catalyzed by P. fellutanum pectinesterase} obeyed M i c h a e l i s - M e n t e n equation only under low substrate concentrations (up to 1.2%}, when graph of the dependence of reaction speed was hyperbolic in form. In case of t w o - s t a g e enzymatic reactions, which did not obey M i c h a e l i s Menten e q u - a t i o n reaction speed was a t its maximum and then decreased.Graph of speed of substrate h y d - r o l y s i s against In concentration acquired a shape of symmetric or asymmetric bell (Figure 4}.

952 Fig 4

10g 8 7 5

ZnZopt,

q -6

-5

-q

-3

Is] Figure 4. Initial speed of the pectin hydrolysis reaction catalyzed by pectinesterase: 1 - theoretically calculated symmetric bell, 2 - e x p e r i m e n t a l l y obtained curve.

In our study the traditional pattern was changed when the concentration of substrate was enlarged. Figure 4 shows, that speed of the reaction of hydrolysis plotted against concentration of substrate yields a line in the shape of asymmetric bell. R i g h t p a r t of the plot has the smaller slope at great concentrations of substrate as against calculated one for t w o - s t a g e reaction. All above stated is the evidence of the formation of the triple e n z y m e substrate complex. It possesses some activity but its activity is lower than that of e n z y m e - s u b s t r a t e complex, i.e. B is other than zero (0

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