Immobilization of Enzymes and Cells

Immobilization

of Enzymes and Cells

METHODS

IN

BIOTECHNOLOGY”

John M. Walker,

SERIESEDITOR

2. Protocolsin Bioremediation,editedby David Sheehan, 1997 1. Immobilizationof Enzymesand Cells,editedby GordonF, Ihckerstafi 1997

Immobilization of Enzymes and Cells Edited by

Gordon

F. Bickerstaff

University of Paisley, Scotland, UK

Humana Press

Totowa, New Jersey

0 1997 Humana Press Inc 999 Rlvervlew Drive, Smte 208 Totowa, New Jersey 075 12 All rights reserved No part of this book may be reproduced, stored m a retrieval system,or transmitted m any form or by any means, electromc, mechamcal, photocopymg, mlcrotilmmg, recordmg, or otherwlse without written pernusslon from the Pubhsher Methods in BlotechnologyTM is a trademark of The Humana Press Inc All authored papers, comments, opinions, conclusions, or recommendations are those of the author(s), and do not necessarily reflect the views of the pubhsher This publlcatlon ISprinted on acid-free paper a ANSI 239.48-1984 (American Standards Institute) Permanence of Paper for Prmted Library Materials Cover lllustratlon Fig 1 m Chapter 1, “Immobthzatlon of Enzymes and Cells Some Prac~rcal Cowdermom,” by Gordon F Bickerstaff Cover design by Patrlcta F Cleary For additional copies, pricing for bulk purchases, and/or mformatlon about other Humana titles, contact Humana at the above address or at any of the followmg numbers Tel 201-256-1699, Fax 201-256-8341, E-mall humana@mmdsprmg corn Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, 1sgranted by Humana Press Inc , provided that the base fee of US $5 00 per copy, plus US $00 25 per page, IS pald directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923 For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and 1sacceptable to Humana Press Inc The feecode for users of the Transactional Reportmg Service IS [O-89603-386-4/97 $5 00 + $00.25] Printed m the Umted States of America

10 9 8 7 6 5 4 3 2

Library of Congress Cataloging m Pubhcatlon Data Mam entry under title Methods m blotechnologyTM Immoblhzatlon of enzymes and cells/[edlted] by Gordon F Bickerstaff P cm -Methods m biotechnology? 1) Includes mdex ISBN o-89603-386-4 (alk. paper) 1 Immoblhzed enzymes-Biotechnology 2 Immoblhzed cells-Biotechnology I Bickerstaff, Gordon, F II Series [DNLM 1 Enzymes, Immoblhzed 2 Cells, Immoblhzed 3 Biotechnology-methods QU 135 1314 19971 TP248 65 145146 1997 660’ 63Wc20 DNLM/DLC for Library of Congress 96-29281 CIP

Preface Immobihzatron of enzymes, cells, and organelles has expanded greatly in the past 30 years as the advantages of immobilization have been evaluated and utilized in analyttcal, biotransformation, and medical applications. A consequence of this explosion of technology IS that there is now a bewildering array of permutations for the immobilization of biological material. The purpose of Immobilization of Enzymes and Cells is to provrde a basic reference tool for all academic and industrial research workers seeking to start or expand the use of mnnobilization techniques in their work. The book does not aim to provide comprehensive coverage of the vast range of methods available, but will serve as a launch pad for potential users of immobilization techniques. One reason for the vast expanse of mmrobilization technology lies m the subject material to be immobilized. Biological catalysts (enzymes, organelles, and cells) have a high degree of individual variability, and although many tmmobilization techniques have wide applicability, tt is imposstble for one or even a few methods to cater to the great diversity of requirements inherent in biological material. This is especially so when the atm is to produce an optimum system m which the immobihzed biocatalyst will function at high levels of efficrency, stability, and so on. The normal situatton faced by research workers IS the need to try one or more methods of remobilization to reveal the specific requirements dictated by the biological catalyst, then adapt the method to these specific circumstances or try another method when the first approach places too great a restriction on the use or activity of the biocatalyst. This process of discovery can be rewarding, but it is also time-consuming, usually frustrating, and the hardest part is making a successful start. Immobilization of Enzymesand Cells has been designed to provide a wide range of representative examples of immobilization techniques for use by postgraduate, postdoctoral, senior research workers, and technicians throughout academia,industry, government, andmedical researchestabhshments, thereby enabling rapid entry into the world of immobilization. All of the chapters, with the exception of Chapter 1, provide detailed instructions of the materials and methods employed in the particular immobilization procedure described. Each author has used the Notes section to pass on accumulated experience and valuable “trade secrets” that give greater V

Vi

Preface

insight or further practical utility to these materials or methods. A total of 23 research laboratories and over 70 active and extensrvely published exponents of immobilization technology have contributed to the volume and collectively provide a wealth of expertise and knowledge from all over the world. I am indebted to the contributors who, in their enthusiasm for the development and utihzation of unmobilization technology, have produced a volume that I believe ~111be of unusual help to the increasing number of research and process workers seeking to make use of the technology. Safety Note A number of the chemicals used in immobilization technology are potentially harmful, and although some guidance 1sgiven by the authors, the absence of specific reference to harmful chemicals or procedures does not imply that there is no potential hazard. Always seek up-to-date safety data from supphers and manufacturers. Gordon F. Bickerstaff

Contents Preface . . . . .. . .. . . . . . . . . . .. . . .. .. . . .. . .. . ,......... . . . . .. .. . .. . .. . .. . .. . .. . .. . . .. . .. . . .. .. . . .. . .. . . v List of Contributors . .. .. . . .. . . . . .. . .. . . .. . .. . . .. . . .. .. . .. . .. . .. . .. . . . . .. . . . .. . . . .. .. .. . . .. . .XI 1 Immobilization of Enzymes and Cells Some Practical Considerations .,....~,,.~,.,,~............... . . ...*.. .. . .. . .. . .. . . .. .. . ...* .-a.,.-.,*.. 1 Gordon F. Bickerstaff 2 Immobilization of Enzymes by Selective Adsorption on Biotinylaminopropyl Celite or Glass Harold E. Swaisgood, Xiaolin L. Huang, and Marie K. Walsh. . . ,.., 13 3 Immobilization of Proteins on Thionyl Chloride-Activated Controlled-Pore Glass Violeta G. Janolino and Harold E. Swaisgood . . . . . . . . . . . . . .. . . . . .. 21 Enzyme Immobilization on Nylon Pedro Lozano and Jo& L. lborra . . . .. . .. . . . .. .. . .. . .. . .. . . .. . .. . . .. . .. . .. . . .. . . . . . , 27 Visual Assessment of Enzyme Immobilization Arturo Manjdn and J&e L. lborra . . ...--........ .. .. . . .. . .. . . . . . . . .. . . . . . . . 4 1 Immobilization in Carrageenans J&e L. Iborra, Arturo Mani&?, and Manuel Chovas . . .. . . . . .. . . .. . .. . .. 53 Entrapment in Calcium Alginate Jane E. Fraser and Gordon F. Bickerstaff . . . .. . ..a............................, 61 Entrapment of Enzymes and Cells in Poly (2-Hydroxyethyl Methacrylate) Supports Maria Can tare/la, Francesco Alfani, Laura Can tare/la, and Albert0 Gallifuoco ,...,.....,.......,.,....,... ,,,,,,I,.,,..,........ . .. . . . . . . .. . 67 9 Activation of Rayon/Polyester Cloth for Protein Immobilization Hiroshi Yamazaki and C. Scott Boyd . . .. . .. . . . .. . . .-.-.................,...... 77 of Enzymes on Microelectrodes Using Chemical 10 Immobilization Crosslinking Miiena Koudelka-Hep, Nice F. de Rooij, and David J. Strike . . ,..... 83 11 Photolithographic Patterning of Enzyme Membranes for the Modification of Microelectrodes Miiena Koudelka-Hep, Nice F. de Rooij, and David J. Strike. .. . . .. . 87 12 Electrochemical-Based lmmobilizatron of Enzymes David J. Strike, Nice F. de Rooij, and Milena Koudelka-Hep . . .. . . . . 93

vii

vii1 13

14

15

16 17

18 19 20

21

22

23 24

25 26 27

Contents Immobilization of Enzymes on Thermo-Responsive Polymers Kazuhiro Hoshino, Setuko Akakabe, Shoichi Morohashi, and Toshisuke Sasakura .. . .. .. .. . . .. .. . .. . . . . . . . . . . . .. . . . . .. .. . . . . . . 101 Immobilization of Photosynthetic Membranes in an AlbuminGlutaraldehyde Crosslinked Matrix Robert Carpentier . . . . . .. . . .. .. ,.... . . .. .. . . . .. .. . .. . .. . . .. . . . . .. .. . . . . .. . .. . ,... . .. . . . 109 Poly(Ethylene Glycol) Crosslinked to Albumin as a Support for Enzyme Immobilization Guy Fortier, Nicole Demers, Jacques-Jean Franqois, Jean-Charles Gayet, and Edith M. D’Urso . .. . . .. . .. . .. . . . . .. . . ..,.... .. 1 17 Poiy(Carbamoyl Sulfonate) Hydrogels Andreas Muscat and Klaus-Dieter Vorlop. .. . .. . .. . . . . . . .. . . . . . . . . . . . .. 125 Enzyme Immobilization on Polyethyleneimine-Coated Magnetite Partrcles Gilbert Bardeletti. . .. .. . . . .. .. . . , .. .. . . .. . .. . .. . . .. . .. . . . , .. .. . . .. . .. . .. . . . . .. . . .. . . , . ., . . . .. 133 Immobilization of Enzymes and Proteins on Red Blood Cells Laura Chiarantini and Mauro Magnani . . .,. . .. . .. . .. . . . . . . . .. .. . ,..,.. . . .. . . .. . 143 Cellulose Paper Support for immobilization Marion Paterson and John F. Kennedy ..,..... . . . .. . . . . .. . ..,..,. .. . . . . . . . . 753 Immobilization of Cells Using Electrostatic Droplet Generation Mattheus F. A. Goosen, E/tag S. E. Mahmud, Abdullah S. Al-Ghafri, Hamad A. Ai-Hajri, Yousuf S. Al-Sinani, and Branko Bugarski .. . .. . . ...-.. . .. . . . . . . 167 Hepatocyte Immobilization in Agarose and Functional Integrity Testing Hassan Farghali and Sixtus Hynie . . .. . . .. . .. . .. . . .. . . .. . .. . .. . . . .-..... . .. . . .. . 7 75 Immobilized Hepatocytes in Xenobiotic Biotransformation Studies Sixtus Hynie, Ludmila Kamenikova, and Hassan Farghali ,... ..,.....,... .. . .. . .. . .. . . . . .. . .. . .. . .. . .. . . . . .. .. . . . .. .. . . .. 185 Immobilization of Liposomes and Proteoliposomes in Gel Beads Eggert Brekkan, Qing Yang, Gerhard Vie/, and Per Lundahl.... . 193 Cell Immobilization with Phosphorylated Polyvinyl Alcohol (PVA) Gel Kuo-Cheng Chen and Jer-Yiing Houng . ,. , . .. . . .. . . .. . .. . . . .. . .. . . .. . . . 207 Covalent Immobilization of Enzymes to Graphitic Particles Marco F. Cardosi . . . .. . .. . .. . .. .. . . . .. . .. .. . . .. . .. . .. . . .. ..-. .,..,...................... . . .. . . 217 Enzyme Immobilization Using Chitosan-Xanthan Complexes Severian Dumitriu, Pierre Vidal, and Esteban Chornet . ,. . . . .. . . .229 Calcium Alginate Film Formed on a Stainless Steel Mesh Harold E. Swaisgood and FIavia M. L. Passos . . . . . .. . .. . . ., . . . .. . .. . . . .. 237

Contents

ix

Preparation of Immobilized Subunits of a Multisubunit Enzyme Gordon F. Bickerstaff . . .. . .. . .. . .. . .. ,.,,,,,.,,... .. . . . . .. .. . . . .. .. . . . .. . . . . .. . . .. . . 243 29 Characterization of Enzyme Activity, Protein Content, and Thiol Groups in Immobilized Enzymes Gordon F. Bickerstaff . ..~~~.~..~~.~.. .. . . .. . ...-.... . . .. .. . . . .. .. . . . . .. . . . .. . . . . . 253 30 Immobilization of Enzymes Acting on Macromolecular Substrates’ Reduction of Steric Problems Penzol, Pilar Armisen, Jose M. Guissln, Guadalupe Agatha Bastida, Rosa M. Blanco, Roberto Ferntindez-Lafuente, .. . .. . .. . . . .. . . . . .. . . .. . .. . .. . .. . .. . .. . .. -..... 26 7 and Eduardo Garcia-Junceda 31 Immobilization of Enzymes on Glyoxyl-Agarose: Strategies for Enzyme Stabilrzation by Multipoint Attachment Jose M. Guistin, Agatha Bastida, Rosa M. Blanco, Roberto Fern;indez-Lafuente, and Eduardo Garcia-Junceda . .. . . .. . . . . . . .. . .. . .. . .. . . . . .. . . .. . ..-.. . .. . . . . 277 32 Stabilization of immobilized Enzymes by Chemical Modification with Polyfunctional Macromolecules Jo& M. Guistin, Verdnica Rodriguez, Cristina M. Rose/l, Gloria Soler, Agatha Bastida, Rosa M. Blanco, Roberto FernBndez-Lafuente, and Eduardo Garcia-Junceda . .. . .. . . .. .. . .. , . .. . . .. . . . . . . .. . . . . .. . . . . . . . .289 33 Covalent Immobilization of Enzymes Using Commercially Available CDI-Activated Agarose George 3. Piazza and Marjorie 6. Medina . . .. . . .. . . . . . .. . . . .. . . . . .. . . .299 34 lmmobrlization of Cells in Polyelectrolyte Complexes . . . . .. . . ..*. . . .. . . .. . . .. . .. . .. . . .. ,309 Johanna Mansfeid and Horst Dautzenberg 35 Coimmobilization of Enzymes and Cells Johanna Mansfeld and Horst Dautzenberg . ..,.-...... . . . .. .. . . . . . .. -3 19 36 Adsorption of Lipase on Inorganic Supports Jo& V. Sinisterra . . . .. . . .. . .,. . .. . . . .. . . . . .. . .. . .. . .. . . . .. . . .. . .. . . . . .. . . . .,. ,.. 327 37 Immobilization of Enzymes on Inorganic Supports by Covalent Methods Josh V. Sinisterra .. . .. . .. . . .. .. . .. . . .. . ., .. . . . .. .. . . .. . .. . .. . . .. . .. . . . . . .. . ., .,, ,. -, . . .. . . . 33 1 38 Use of Divalent Metal Ions Chelated to Agarose Derivatives for Reversible Immobilization of Proteins Beitle, Jr. and Mohammad M. Ataai . .. . . .. . .. . . .. . . .. . . . . .. . . .. .. 339 Robert R. of Enzymes and Cells 39 Transition Metal Methods for Immobilization John F. Kennedy, Joaquim M. Cabral, Maria R. Kosseva, and Marion Paterson ..*... . . . . . .. .. . . .. . .. . .. . . .. . .. . .. . . .. . . . . .. . . ...*............... 345 Index .. . . . . . . .. . . . .. . . . . . . . . .. . . .. .. . . . .. . . . . .. . . . . . ..*..... .. . . . . ..*....... . .. .. ., . .. . . 361 28

Contributors . Department of Chemical and Btochemical Engtneering, Toyama Untversity, Toyama, Japan FRANCESCO ALFANI Dipartimento Di Chimica Ingegnerta Chimica E Matertali, Untversita De L ‘Aqurla, Italy ABDULLAH S. AL-GHAFRI Department of Bioresource Engineering, College of Agriculture, Sultan Qaboos Untversity, Oman HAMAD A. AL-HAJRI Department of Bioresource Engineering, College of Agriculture, Sultan Qaboos University, Oman YOUSUF S. AL-SINANI Department of Bioresource Engineering, College of Agriculture, Sultan Qaboos University, Oman PILAR ARMISEN 9Laboratorto de Technologia Enzymatica, Campus de Cantoblanco, Instttuto de Catalisis CSIC, Madrid, Spain MOHAMMED M. ATAAI Department of Chemical Engineering, Untverstty of Arkansas, Fayette&e, AR GILBERT BARDELETTI Laboratoite de Biochimie Analytique et Synthese Btoorganique, Untversate C. Bernard-Lyon 1, Villeurbanne Cedex, France AGATHA BASTIDA 9Laboratorio de Technologia Enzymattca, Institute de Catalisis CSIC, Campus de Cantoblanco, Madrid, Spain ROBERT R. BEITLE, JR. 9 Department of Chemical Engineering, University of Arkansas, Fayetteville, AR GORDON F. BICKERSTAFF Department of Biological Sciences, University of Paisley, UK ROSA M. BLANCO Laboratorio de Technologia Enzymatica, Instituto de Catalisis CSIC, Campus de Cantoblanco, Madrid, Spain C. SCOTTBOND Department of Biology, Carleton University, Ottawa, Canada EGGERT BREKKAN Department of Biochemistry, Biomedical Center, Uppsala University, Uppsala, Sweden BRANKO BUGARSKI Department of Bioresource Engtneering, College of Agriculture, Sultan Qaboos Universtty, Oman SETUKO AKAKABE

l

l

l

l

l

l

l

l

l

l

l

xi

Contributors

xii

MANUEL CANOVAS Departamento De Btoquimica Y Biologia Molecular l

Immunologia, Universidad De Murcia, Spain LAURA CANTARELLA Diparttmento Dt Chimica Ingegneria Chimica E Materiab, Universita De L ‘Aquila, Italy MARIA CANTARELLA Dipartimento Di Chimica Ingegneria Chlmica E Materiali, Universtta De L ‘Aquila, Italy MARCO F. CARDOSI Department of Biologxal Sctences, University of Paisley, UK ROBERT CARPENTIER Centre De Recherche en Photobiophysique, Untversite du Quebec, Canada KUO-CHENG CHEN 9Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan LAURA CHIARANTINI Instituto di Chimica Biologica, Universita Degli Stud1 di Urbino, Italy ESTEBAN CHORNET Department de Genie Chimlque, Untversity de Sherbrooke, Canada HORST DAUTZENBERG 9Instttut fur Btotechnologie, Martin-Luther Universittit, Halle, Germany NICOLE DEMERS 9Laboratotre d’Enzymologie Appiqube, Universite du Quebec a Montreal, Canada NICO F. DE ROOIJ Institute of Microtechnology, University of Neuchatel, Switzerland SEVERIAN DUMITRIU Department de Genie Chimtque, Untversity de Sherbrooke, Canada EDITH M. D’URSO Laboratoire d ‘Enzymologie Apptqube, Universtte du Quebec a Montreal, Canada HASSAN FARGHALI First Faculty of Medicine, Institute of Pharmacology, Charels University, Prague, Czech Republic ROBERTO FERNANDEZ-LAFUENTE Laboratorio de Technologia Enzymatica, Instituto de Cataluu CSIC, Campus de Cantoblanco, Madrid, Spain GUY FORTIER Laboratoire d ‘Enzymologie Apptquee, Universtd du Quebec d Montreal, Canada JANE E. FRASER Department of Biological Sciences, University of Paisley, UK ALBERTO GALLIFUOCO Dipartimento Di Chimica Ingegneria Chtmtca E Matertali, Untversita De L ‘Aquila, Italy EDUARDO GARCIA-JIJNCEDA 9Laboratorio de Technologia Enzymhtica, Instttuto de Catalisu CSIC, Campus de Cantoblanco, Madrid, Spain l

l

l

l

l

l

l

l

l

l

l

l

l

l

I..

Contributors

XIII

JEAN-CHARLES GAYET Laboratoire d%nzymologie Appiquee, Universite du l

Quebec d Montreal, Canada MATTHEWS F. A. GOOSEN Department of Bioresource Engineertng, College of Agriculture, Sultan Qaboos University, Oman Josh M. GUISAN Laboratorio de Technologia Enzymatica, Instituto de Catalisis CSIC, Campus de Cantoblanco, Madrid, Spain KAZUH~RO HOSHINO Department of Chemical and Biochemical Engineering, Toyama University, Toyama, Japan JER-YIING HOKJNG Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan XIAOLIN L. HUANG Department of Food Science, College of Agriculture and Life Sciences, North Carolina State University, Raleigh, NC SIXTUS HYNIE First Faculty of Medicine, Institute of Pharmacology, Charels University, Prague, Czech Republic JOSE L. IBORRA Departamento De Bioquimica Y Biologta Molecular Immunologta, Unlversidad De Murcia, Spain VIOLETA G. JANOLINO Department of Food Science, College of Agrtculture and Life Sciences, North Carolina State University, Raletgh, NC JACQUES JEAN-FRANCOIS Laboratoire d ‘Enzymologte Appiquee, Universitk du Quebec h Montreal, Canada LUDMILLA KAMENIKOVA Institute of Pharmacology, First Faculty of Medictne, Charles University, Prague, Czech Republic JOHN F. KENNEDY School of Chemutry, University of Birmingham, UK MILENA KOUDELKA-HEP Institute of Microtechnology, University of Neuchatel, Switzerland PEDRO LOZANO Departamento De Bioquimtca Y Biologia Molecular Immunologia, Universidad De Murcia, Spain PER LUNDAHL Department of Biochemistry, Biomedical Center, Uppsala University, Uppsala, Sweden MAURO MAGNANI Institute di Chimica Biologica, Universith Degli Studi di Urbino, Italy ELTAG S. E. MAHMUD Department of Bioresource Engineering, College of Agrtculture, Sultan Qaboos University, Oman ARTURO MANJ~N Departamento De Bioquimica Y Biologia Molecular Immunologia, Universidad De Murcia, Spain JOHANNA MANSFELD Institutfir Biotechnologie, Martin-Luther Universitat, Halle, Germany l

l

l

l

l

l

l

l

l

l

l

l

l

l

l

l

l

l

Contri6utors

xiv

B. MEDINA North Atlantic Area, Eastern Regional Research Center, US Department of Agriculture, Philadelphia, PA SHOICHI MOROHASHI Department of Chemical and Biochemical Engineering, Toyama University, Toyama, Japan ANDREAS MUSCAT Institut fur Technologie, Bundesforschungsanstalt fir Landwirtschaft (FAL), Braunschweig, Germany FLAVIA M. L. PASSOS Department of Food Science, College of Agrtculture and Life Sciences, North Carolma State Universtty, Raletgh, NC MARION PATERSON School of Chemistry, University of Birmingham, UK GUADALUPE PENZOL Laboratorio de Technologia Enzymattca, Instttuto de Cataluu CSIC, Campus de Cantoblanco, Madrtd, Spain GEORGE J PIAZZA North Atlantic Area, Eastern Regional Research Center, US Department of Agrtculture, Philadelphta, PA VERONICA RODRIGUEZ Laboratorto de Technologta Enzymhtica, Instituto de Cataluu CSIC, Campus de Cantoblanco, Madrid, Spain CRISTINA M. ROSELL Laboratorto de Technologia Enzymcittca, Campus de Cantoblanco, Instituto de Cauilrsu CSIC, Madrid, Spurn TOSHISUKE SASAKURA Department of Chemical and Biochemtcal Engineering, Toyama University, Toyama, Japan JOVE V. SINETERRA . Department of Organic and Pharmaceutical Chemutry, Faculty of Pharmacy, Untversity of Complutense, Madrid, Spain GLORIA SOLER Laboratorio de Technologia Enzymattca, Instttuto de Catalasts CSIC, Campus de Cantoblanco, Madrid, Spain DAVID J. STRIKE Institute of Mtcrotechnology, Untverstty of Neuchatel, Switzerland HAROLD E. SWABGOOD Department of Food Science, College of Agriculture and Ltfe Sciences, North Carolina State University, Raleigh, NC PIERRE VIDAL Department de Genie Chtmique, University de Sherbrooke, Canada GERHARD VIEL Department of Biochemistry, Biomehcal Center, Uppsala Unlverslty, Uppsala, Sweden KLAUS-DIETER VORLOP Institutfur Technologie, Bundesforschungsanstalt fur Landwirtschaft (FAL), Braunschweig, Germany MARIE K. WALSH Department of Food Sctence, College of Agrtculture and Life Sciences, North Carolina State Untversity, Raleigh, NC HIROSHI YAMAZAKI Department of Biology, Carleton Untversity, Ottawa, Canada QING YANG Department of Biochemistry, Biomedical Center, Uppsala University, Uppsala, Sweden MARJORIE

l

l

l

l

l

l

l

l

l

l

l

l

l

l

l

l

l

l

l

1 Immobilization

of Enzymes and Cells

Some Practical Considerations Gordon F. Bickerstaff 1.

Introduction

The technology for immobilization of cells and enzymesevolved steadily for the first 25 years of its existence (I), but in recent years it has reached a plateau, if not a slight decline. However, the expansion of biotechnology, and the expected developments that will accrue from advances in genetic technology, has revitalized enthusiasmfor immobilization of enzymesand cells (2). Researchand development work has provided a bewildering array of support materials and methods for immobilization. Much of the expansion may be attributed to developments to provide specific improvements for a given application (3). Surprismgly, there have been few detailed and comprehensive comparative studies on immobilization methods and supports. Therefore, no ideal support material or method of immobtlization hasemerged to provide a standard for each type of immobihzation. Selection of support material and method of immobilization is made by weighing the various characteristicsand required features of the enzyme/cell application against the properties/limitations/characteristics of the combined immobilization/support. A number of practical aspectsshould be considered before embarking on experimental work to ensurethat the final immobilized enzymeand/or cell preparation is tit for the planned purpose or application and will operate at optimum effectiveness (46). This chapter does not aim to provide a review of available methods, but does provide some background to assistin choice evaluation for support and method of immobilization,

2. Choice of Support and Principal Method In solution, soluble enzyme molecules behave as any other solute in that they are readily dispersed in the solution and have complete freedom of movement From

Mefbqds m BmxYmology, Vol 1 Immob~/,zetron of Enzymes and Cells Edited by G F BIckerstaff Humana Press Inc , Totowa, NJ I

Bickerstaff

2 Table 1 Fundamental Considerations in Selecting a Support and Method of immobilization Property

Pomts for consideration

Physlcal

Chemical

Stability

Resistance

Safety

Economic

Reactton

Strength, noncompression of particles, available surface area, shape/form (beads/sheets/fibers), degree of porosity, pore volume, permeability, density, space for increased biomass, flow rate, and pressure drop Hydrophllicity (water bmding by the support), inertness toward enzyme/cell, available functIona groups for modification, and regeneration/reuse of support Storage, residual enzyme activity, cell productivity, regeneration of enzyme activity, mamtenance of cell vlabllity, and mechanical stability of support material BacteriaKmgal attack, disruption by chemicals, pH, temperature, organic solvents, proteases, and cell defense mechanisms (proteins/cells) Blocompatibllity (invokes an immune response), toxrclty of component reagents, health and safety for process workers and endproduct users, specification of immobilized preparation (GRAS list requirements for FDA approval) for food, pharmaceutical, and medical applications Availability and cost of support, chemicals, special equipment, reagents, technical skill required, environmental impact, industrial-scale chemical preparation, feasibility for scale-up, continuous processing, effective working life, reuseable support, and CRL or zero contamination (enzyme/cell-free product) Flow rate, enzyme/cell loading and catalytic productivity, reaction kinetics, side reactions, multiple enzyme and/or cell systems, batch, CSTR, PBR, FBR, ALR, and so on; diffusion limitations on mass transfer of cofactors, substrates, and products

CRL. calculated risk level, CSTR continuous stirred tank reactor, PBR packed bed reactor, FBR fluldlzed bed reactor, ALR air lift reactor

(1). Enzyme immobilization is a technique specifically designed to greatly restrict the freedom of movement of an enzyme. Most cells are naturally nnmobilized

one way or another, so immobilization

provides a physical support for

cells. The first conslderatlon is to decide on the support material, then the main method of nnmobillzation, taking into account the intended use and application. Some of the points to consider when making a decision are listed m Table 1, and

Practical

3

Considerations Table 2 Influence of Immobilization Method on the Biotransformation of Sucrose to lsomaltulose by Cells of Erwinia rhapontici

Activity, g/g wet cells/h Free cells Immobilization method Entrapment in calcium alginate Entrapment in polyacrylamide Adsorption to DEAE-cellulose Crosslinkmg with glutaraldehyde Entrapmentin K-carrageenan Entrapment in agar Adsorption to bone char Reprintedwith permissionfrom ref. 21

Half-life, h

0600

36

0.325 0.130 0.583 0.153 0.263 0.340

8500 570 400 40 38 27 25

0.010

an indication of how different methods of immobilization can influence the activity and half-life of a cell-based biotransformation is presented in Table 2. There are live principal methods for immobilization of enzymes/cells: adsorption, covalent binding, entrapment, encapsulation, and crosslinking (see Fig. 1). The relative merits of each are discussed briefly below. 2.1. Adsorption Immobihzation by adsorption (see Fig. 1) is the simplest method and involves reversible surface interactions between enzyme/cell and support material (7,8). The forces involved are mostly electrostatic, such as van der Waals forces, ionic and hydrogen bonding interacttons, although hydrophobic bonding can be significant. These forces are very weak, but sufficiently large in number to enable reasonable binding, For example, it is known that yeast cells have a surface chemistry that is substantially negatively charged so that use of a positively charged support will enable mnnobihzation. Existing surface chemistry between the enzyme/cells and support is utilized so no chemical activation/modification is required and little damage is normally done to enzymesor cells in this method of immobilization. The procedure consists of mixing together the biological component(s) and a support with adsorption properties, under suitable conditions of pH, ionic strength, and so on, for a period of incubation, followed by collection of the immobtlized material and extensive washing to remove nonbound biological components.

Bickerstaff

ADSORPTION

COVALENT

BINDING

ENCAPSULATION

ENTRAPMENT

CROSS-LINKING

Fig. 1. Principal methods of immobilization.

Among the advantages are: 1. 2. 3. 4.

Little or no damage to enzymes/cells. Simple, cheap, and quick to obtain immobilization. No chemical changes to support or enzyme/cell. Reversible to allow regeneration with fresh enzymes/cells.

Disadvantages include: 1. Leakage of enzymes/cells from the support/contamination

of product.

Practical

Considerations

2. Nonspecific binding. 3. Overloading on the support. 4. Sterrchindrance by the support. The most significant disadvantage is leakage of biocatalyst from the support. Desorption can occur under many circumstances, and envnonmental changes in pH, temperature, and ionic strength will promote desorption. Sometimes a cell/enzyme, firmly adsorbed, is readily desorbed during reaction as a result of substrate binding, binding of contaminants present in the substrate, product production, or other conditions leading to change m protein conformation. Physical factors, such as flow rate, bubble agitation, particle-particle abrasion, and scouring effect of particulate materials on vessel walls, can lead to desorption. Desorption can be turned to advantage if regeneration of support is built into the operational regimen to allow rapid expulsion of exhausted biocatalyst and replacement with fresh biocatalyst. Nonspecific binding can become a problem if substrate, product, and/or residual contaminants are charged and interact with the support. This can lead to diffusion limitations and reaction kinetics problems, with consequent alteration in parameters V,,, and K, (9). Further, binding of protons to the support material can result in an altered pH microenvironment around the support with consequent shift in pH optimum (l-2 pH units), which may be important for enzymes with precise pH optimum requirements (IO,Zl). Unless carefully controlled, overloading the support can lead to low cataltyic activity, and the absence of a suitable spacer between the enzyme molecule and the support can produce problems related to steric hindrance. 2.2. Covalent Binding This method of immobilizatron (see Fig. 1) involves the formation of a covalent bond between the enzyme/cell and a support material (8,12,13). The bond is normally formed between functional groups present on the surface of the support and functional groups belonging to amino acid residues on the surface of the enzyme. A number of amino acid functional groups are suitable for participation in covalent bond formation. Those that are most often involved are the amino group (NH*) of lysine or arginine, the carboxyl group (CO*H) of aspartic acid or glutamrc acid, the hydroxyl group (OH) of serine or threorune, and the sulfydryl group (SH) of cysteine (14). Many varied support materials are available for covalent binding, and the extensive range of supports available reflects the fact that no ideal support exists. Therefore, the advantages and disadvantages of a support must be taken into account when considering possible procedures for a given enzyme immobilization (15,16). Many factors may influence the selection of a particular support, and research work has shown that hydrophihcrty IS the most important

factor for maintaining enzyme activity in a support environment (Z 7). Consequently, polysaccharide polymers, which are very hydrophilic, are popular support materials for enzyme immobilization. For example, cellulose, dextran (Sephadex), starch, and agarose (Sepharose) are used for enzyme imrnobllizatlon. The sugar residues m these polymers contain hydroxyl groups, which are ideal functional groups for chemical activation to provide covalent bond formation. Also, hydroxyl groups form hydrogen bonds with water molecules and thereby create an aqueous (hydrophlllc) environment m the support. The polysaccharlde supports are susceptible to microbial/fungal disintegration, and organic solvents can cause shrinkage of the gels. The supports are usually used in bead form. Other popular supports for enzyme lmmobihzatlon are porous srhca and porous glass. The microarchltecture of these IS shown in Fig. 2. Porous silica consists of small spherical particles of silica fused together in such a way as to form microcavities and small channels. The support IS normally sold m bead form, and is very strong and durable. Sintered borosihcate glass may be tempered to form a system of uniform channels. The diameter of channels depends on the tempering conditions. Porous glass is also durable and resistant to mlcroblal dlsintegratlon or solvent distortion. However, these two supports are less hydrophlltc than the polysaccharide materials. There are many reaction procedures for couplmg an enzyme and a support m a covalent bond (IS). However, most reactions fall into the followmg categories1, 2 3. 4

Formation of an lsourea linkage Formation of a dlazo linkage. Formation of a peptlde bond. An alkylation reactlon.

It is important to choose a method that will not inactivate the enzyme by reacting with amino acids at the active site. So, if an enzyme employs a carboxy1 group at the active site for participation in catalysis, it IS wise to choose a reaction that mvolves amino groups for the covalent bond with the support. Basically, two steps are involved in covalent binding of enzymes to support materials. First, functional groups on the support material are activated by a specific reagent, and second, the enzyme IS added in a coupling reaction to form a covalent bond with the support material. Normally the activation reaction is designed to make the functional groups on the support strongly electrophilic (electron deficient). In the coupling reaction, these groups will react with strong nucleophiles (electron donatmg), such as the ammo (NH*) functional groups of certain amino acids on the surface of the enzyme, to form a covalent bond (I).

Practical

Considera

tlons

POROUS

AGAROSE

GLASS

POROUS SILICA

Fig. 2. Microarchitecture of some support materials used for immobrlrzation of enzymesand cells.

Cyanogen bromide (CNBr) is often used to activate the hydroxyl functional groups in polysaccharrde support materials. In this method, the enzyme and support are joined via an isourea linkage. In the case of carbodiimide activation, the support material should have a carboxyl (C02H) functronal group, and the enzyme and support are joined via a peptide bond. If the support material contains an aromatic amino functional group, it can be drazotized using nitrous acid. Subsequent addition of enzyme leads to the formation of a diazo linkage between the reactive diazo group on the support and the ring structure of an aromatic amino acid, such as tyrosine. It is important to recogmze that no method of immobrhzation 1srestricted to a particular type of support material, and that an extremely large number of

8

Bickerstaff

permutattons are possible between methods of immobillzatton and support material. This is made possible by chemical modification of normal functional groups on a support material to produce a range of derivatives containing different functional groups. For example, the normal functional group in cellulose is the hydroxyl group, and chemical modification of this has produced a range of cellulose derivatives, such as AE-cellulose (ammoethyl), CM-cellulose (carboxymethyl), and DEAE-cellulose (diethylammoethyl). Thus, chemical modification increases the range of immobilization methods that can be used for a given support material. Derivattzation can also be used to modify charges on the surface of a support material to improve binding of biocatalyst. 2.3. Entrapment Immobilization by entrapment (see Fig. I) differs from adsorption and covalent binding in that enzyme molecules are free m solution, but restricted in movement by the lattice structure of a gel (I, 19). The porosity of the gel lattice is controlled to ensure that the structure is tight enough to prevent leakage of enzyme or cells, yet at the same time allow free movement of substrate and product. Inevitably, the support will act as a barrier to mass transfer, and although this can have serious implicattons for reaction kinetics, it can have useful advantages since harmful cells, proteins, and enzymes are prevented from interaction with the immobilized biocatalyst (20,22). There are several major methods of entrapment: 1. 2. 3 4.

Ionotropic gelation of macromoleculeswith multivalent cations (e.g., alginate). Temperature-inducedgelation (e.g , agarose,gelatin). Organicpolymerizationby chemicaVphotochemica1 reaction(e.g , polyacrylamide). Precipitation from an nntniscible solvent (e.g , polystyrene).

Entrapment can be achieved by mixing an enzyme with a polyionic polymer material and then crosslinking the polymer with multivalent cations in an ion-exchange reaction to form a lattice structure that traps the enzymes/cells (ionotropic gelation). Temperature change is a simple method of gelation by phase transition using l-4% solutions of agarose or gelatin. However, the gels formed are soft and unstable. A significant development in this area has been the introduction of tc-carrageenan polymers that can form gels by ionotroptc gelation and by temperature-induced phase transition, which has introduced a greater degree of flexibility in gelation systems for immobiltzation. Alternatively, it is possible to mix the enzyme with chemical monomers that are then polymerized to form a crosslinked polymeric network, trapping the enzyme in the interstitial spacesof the lattice. The latter method is more widely used, and a number of acrylic monomers are available for the formation of hydrophilic copolymers. For example, acrylamide monomer ts polymerized to

Practical Considerations

9

form polyacrylamide and methylacrylate IS polymerized to form polymethacrylate. In addition to the monomer, a crosslinking agent is added during polymerization to form crosslmkages between the polymer chains and help to create a three-dimensional network lattice. The pore size of the gel and its mechanical properties are determined by the relative amounts of monomer and crosslinking agent. It is therefore possible to vary these concentrations to influence the lattice structure. The formed polymer may be broken up into parttcles of a desired size, or polymerization can be arranged to form beads of defined size. Prectpitation occurs by phase separation rather than by chemical reaction, but does bring the cells/enzymes mto contact with a water-miscible organic solvent, and most cells/enzymes are not tolerant of such solvents. Thus, this method is hmited to highly stable/previously stabilized enzymes or nonliving cells. 2.4. Encapsulation Encapsulation (see Fig. 1) of enzymes and or cells can be achieved by enveloping the biological components within various forms of semipermeable membranes (22-24). It is similar to entrapment in that the enzymes/cells are free in solution, but restricted in space. Large proteins or enzymes cannot pass out of or into the capsule, but small substratesand products can pass freely across the semipermeable membrane. Many materials have been used to construct microcapsules varying from IO-100 l.trn in diameter; for example, nylon and cellulose mtrate have proven popular. The problems associated with diffusion are more acute and may result in rupture of the membrane if products from a reaction accumulate rapidly. A further problem is that the immobilized cell or enzyme particle may have a density fairly similar to that of the bulk solution with consequent problems m reactor configuration, flow dynamics, and so on. It is also possible to use biological cells as capsules, and a notable example of this is the use of erythrocytes (red blood cells). The membrane of the erythrocyte is normally only permeable to small molecules. However, when erythrocytes are placed in a hypotonic solution, they swell, stretching the cell membrane and substantially increasing the permeability. In this condition, erythrocyte proteins diffuse out of the cell and enzymes can diffuse into the cell. Returning the swollen erythrocytes to an isotonic solution enables the cell membrane to return to its normal state, and the enzymes trapped inside the cell do not leak out. A distinct advantage of this method is coimmobilization. Cells and/or enzymes may be immobilized in any desired combination to suit particular applications. 2.5. Crosshhng This type of immobilization (see Fig. 1) is support-free and involves joining the cells (or the enzymes) to each other to form a large, three-dimensional

10

Bickers ta ff

complex structure, and can be achteved by chemical or physical methods (25). Chemical methods of crosslinking normally involve covalent bond formation between the cells by means of a bl- or multifunctional reagent, such as glutaraldehyde and toluene diisocyanate. However, the toxicity of such reagents 1s a limitmg factor in applying this method to livmg cells and many enzymes. Both albumin and gelatin have been used to provide additional protein molecules as spacers to minimize the close proximity problems that can be caused by crosslinking a single enzyme. Physical crosslinking of cells by flocculation is well known m the biotechnology industry and does lead to high cell densities. Flocculatmg agents, such as polyamines, polyethyleneimine, polystyrene sulfonates, and various phosphates, have been used extensively and are well characterized. Crosslinkmg IS rarely used as the only means of lmmobillzatlon because the absence of mechanical propertles and poor stability are severe limitations. Crosslinking 1s most often used to enhance other methods of immobilization, normally by reducing cell leakage in other systems. References 1 Bickerstaff, G F. (1995) Impact of genetlc technology on enzyme technology Genet Engineeer Bzotechnologlst 15, 13-30. 2 Bickerstaff, G. F. (1987) Enzymes zn Industry and Meduxze, Edward Arnold, London, UK. 3 Bickerstaff, G. F (1984) Applications of immobilized enzymes to fundamental studies on enzyme structure and fimctlon, m Topccs m Enzyme and Fermentation BZOtechnology, vol. 9 (Wiseman, A., ed.), Ellis Horwood, Chichester, UK, pp. 162-201. 4. Swalsgood, H. E. (1985) Immobilization of enzymes and some applications in the food Industry, m Enzymes and Immobilized Cells zn Bzotechnology (Laskin, A I , ed.) Benjamin Cummmgs, London, pp. l-24. 5. NtiAez, M. J. and Lema, J. M. (1987) Cell immobihzation: application to alcohol production Enzyme Mlcrob Technol. 9,642-65 1 6. Bidley, S. (1985) Immobilized mammalian cells m hormone detection and quantltation, in Immobilized Cells and Enzymes: A Practical Approach (Woodward, J., ed.), IRL, Oxford, UK, pp. 147-171. 7. Messing, R. A. (1976) Adsorption and morgamc bridge formations, m Methods zn Enzymology, vol. XLIV (Mosback, K , ed.), Academic, New York, pp 148- 169 8. Woodward, J. (1985) Immobilized enzymes: adsorption and covalent coupling, m Zmmoblhzed Cells and Enzymes A Practical Approach (Woodward, J , ed ), IRL, Oxford, 3-l 7 9. Goldstein, L (1976) Kinetic behavlour of immoblhzed enzyme systems, in Methods in Enzymology, vol XLIV (Mosbach, K., ed ), Academic, New York, pp 397443.

10 Rudge, J and BIckerstaff, G F. (1984) Thermal stability properties of immoblhzed creatine kinase Blochem. Sot. Trans 12,3 11,3 12.

Practical Considerations

11

11 Toher, J , Kelly, A M., and Bickerstaff, G F (1990) Stability properties of two supports for immobthzation of enzymes. Bzochem Sot Trans 18,3 13,3 14. 12. Porath, J and Axen, R (1976) Immobilization of enzymes to agar, agarose and Sephadex supports, in Methods m Enzymology, vol. XLIV (Mosbach, K., ed.), Academic, New York, pp 1945 13 Cabral, J M S and Kennedy, J. F. (1991) Covalent and coordinatton immobilization of proteins, in Protean Immobilization (Taylor, R F , ed.), Marcel Dekker, New York, 73-138 14. Srere, P. A. and Uyeda, K. (1976) Functional groups on enzymes suitable for bmdmg to matrices, m Methods in Enzymology, vol XLIV (Mosbach, K., ed.), Academic, New York, pp 11-19. 15 White, C A and Kennedy, J F. (1980) Popular matrices for enzyme and other mnnobtlizattons. Enzyme Microb. Technol. 2, 82-90. 16. Taylor, R. F (1991) Commerctally available supports for protein nnmobilization, in Protean Zmmobilzzution (Taylor, R. F., ed.), Marcel Dekker, New York, pp. 139-160 17. Gememer, P. (1992) Materials for enzyme engineering, m Enzyme Engzneerzng (Gememer, P., ed.) Elhs Hot-wood, New York, pp. 13-l 19. 18. Scouten, W. H. (1987) A survey of enzyme coupling techniques, m Methods rn Enzymology, vol. 135 (Mosbach, K., ed.), Academic, New York, pp 19-45. 19. O’Driscoll, K. F. (1976) Techniques of enzyme entrapment m gels, m Methods zn Enzymology, vol. XLIV (Mosbach, K., ed.), Academic, New York, pp. 169-l 83. 20. Brodelius, P. (1985) Immobilized plant cells, in Enzymes and Immobzlzzed Cells zn Biotechnology (Laskin, A. I , ed.) Benjamin Cummings, London, pp. 109-148. 21 Bucke, C. (1983) Immobilized cells. Phil Truns R Sot B 300,369-389 22 Kierstan, M. P. J and Coughlan, M. P. (1991) Immobilization of proteins by noncovalent procedures. principles and applications, in Protew Immobdzzutron (Taylor, R. F., ed.), Marcel Dekker, New York, pp. 13-71. 23. Nilsson, K. (1987) Methods for tmmobilizing animal cells. Trends Blotechnol. 5, 73-78. 24. Grobotllot, A., Boadi, D. K., Poncelot, D., and Neufeld, R. J. (1994) Immobilization of cells for application in the food industry. Crit. Rev. Blotechnol 14,75-107. 25 Broun, G B. (1976) Chemically aggregated enzymes, in Methods zn Enzymology, vol. XLIV (Mosbach, K., ed.), Academic, New York, pp 263-280

Immobilization of Enzymes by Selective Adsorption on Biotinylaminopropyl Celite or Glass Harold E. Swaisgood,

Xiaolin

L. Huang, and Marie K. Walsh

1. Introduction Use of immobilized enzymesin bioprocessesoffers many advantages,includmg greater productivity because the same enzyme molecules can be used over a long period of time, there is more precise control of the extent of reaction, there is the capability of automation and continuous operation, and the elimination of the requirement of a downstream enzyme inactivation step (11. The ease and cost of immobilized enzyme preparation has limited commercial apphcatton of enzyme bioreactor processes. In this chapter a method for mrmobihzation of enzymes usmg the high affinity, bioselective interaction between biotm and avidin is described. Avidin, which is a tetrameric protein, binds four biotin molecules with a dissociation constant of about 10-i5M (2); thus, it represents one of the strongest noncovalent interactions known, Vis-a-vis adsorption by ion-exchange interaction, the avidin-biotin interaction is very specific and much stronger; therefore, enzyme leaking from the support is minimal, resulting in greater operational stability (3-9). Furthermore, the biotin molecule is very robust; consequently, bioreactors can be repeatedly regenerated simply by desorption of inactive enzyme m a chaotropic solvent, followed by selective adsorption of avidin and finally, fresh biotmylated enzyme. 2. Materials 2. I. Preparation of the Biotinylaminopropyl Surface 2.1.1. Derivatization with 3-Aminopropyltriethoxysilane (4) 1. A 10% (v/v) aqueous solution of organosilane. Dilute 3-aminopropyltriethoxysilanewith distilled water and adjust the pH to 4.0. First adjust the pH with concentrated HCl until the pH reaches 5.0, then use 1N HCl From

Methods m Wotechnology, Vol 1 Immob/lrzatlon of Enzymes and Cells Edited by G F BIckerstaff Humana Press Inc , Totowa, NJ

13

14

Swaisgood, Huang, and Walsh

2 Cehte or controlled-pore glass (CPG) beads. 3. Concentrated HN03.

2.7.2. Biotinylation of Surface Amino Groups (3,5) 1 Aminopropyl beads 2 Freshly prepared biotinylation reagent containing 0 5 mg NHS-LC-blotm/mL 50 mM bicarbonate buffer, pH 8 5 NHS-LC-blotin: sulfosuccimmidyl (biotinamido)hexanoate (Pierce, Rockford, IL). 3 50 mM sodium phosphate, pH 6.0, containing 0 02% NaNx

in 6-

2.7.3. Measurement of immobilized Biotin Concentration (6) 1. Avidm solution: 0.5 mg/mL m 50 mphosphate, pH 6.0, containmg 0.9% NaCl 2. HABA reagent. 10 mA4 4’-hydroxyazobenzene-2-carboxyllc acid (Pierce) m 10 mM NaOH.

2.2. Selective Adsorption of Avidin 2.2.1. Preparation and Adsorption of the Avidin Solution (5) 1. Avldm solution: 2 5 mg/mL in 50 mM phosphate, pH 6 0, containing 0 9% NaCl and 0 02% NaN3. 2 Biotinylated beads. 3 Phosphate buffer: 50 mMphosphate, pH 6.0, containing 0 02% NaN3.

2.2.2. Assay of the Number of Biotin-Binding Sites (6) 1 Avidin-HABA solution: Mix 1 0 mL of 0.5 mg/mL avidin in 50 mM phosphate, pH 6.0, containmg 0.9% NaCl with 0 05 mL of 10 mMHABA in 10 mMNaOH 2 Brotin solution. 0.5 mM blotm in 50 mM phosphate, pH 6.0, containing 0.9% NaCl

2.3. Biotinylation

of the Enzyme

2.3.1. Biotinylation Procedure: Reaction with Lysyl &-Amino Groups (5) 1 Biotinylation reagent. Prepare a fresh solution of 2 mg/mL NHS-LC-blotin in 50 mM bicarbonate buffer, pH 8.5. 2. Enzyme solution: 4-10 mg/mL m 50 n&f bicarbonate buffer, pH 8.5. This solutlon should be freshly prepared and held at 4OC 3 Washmg and storage buffer: 50 ntiTrls-HCI, pH 7.0, containing 0.02% NaN,. Note: This buffer should be chosen to be compatible with the stability of the enzyme

2.3.2. Determination

of the Extent of Biotinylation (5,6)

1 The extent of biotinylation is determined using the HABA dye-binding method similar to that previously described. An avidin solution (0.5 mg/mL) 1sprepared m 50 mM phosphate buffer, pH 6.0, containing 0.9% NaCl

Adsorption on CeMe or Glass

15

2. HABA reagent: 10 mM 4’-hydroxyazobenzene-2-carboxyltc NaOH

acid m 10 mM

2.4. Adsorption of Bio tiny/a ted Transglu taminase 1. Biotinylated transglutaminase solution (2.5 mg/mL). 2. 50 mMTris-HCl, pH 7 0, containing 2 mMDTT, 3 mMEDTA,

and 0.02% NaN3

2.5. Adsorption of Biotinylated /3=Galactosidase 1 Btotinylated P-galactostdase solutton (10 mg/mL). 2. 50 mMTrrs-HCI, pH 7.0, containing 0.02% NaNs. 3 10 mA4 Trrs-HCl, pH 7 4, containing 0.5M NaCl, 10 mJ4 MgCl2, and 1 mA4 EDTA.

2.6. Assay of Immobilized

Transglutaminase

1. 2. 3 4. 5 6 7. 8. 9

1.OMTrts-acetate buffer, pH 6.0. 0.2M (Benzyloxycarbonyl)~L-glutaminylglycine m the Tris buffer. 4.OM Hydroxylamine-HCl, pH 6.0. 0 02MEthylenediaminetetra-acetic acid (EDTA) 15% Trichloroacetic acid (TCA). 5% FeCls in O.lNHCl. 2.5N HCI. O.lMCaClz 2.OM Hydroxylamme-HCl: Dtlute stock reagent 1:l with water, prepare fresh dally. 10. Color developing reagent: equal volumes of 15% TCA, 5% FeC13, and 2.5NHCl (prepare fresh daily). 11. Substrate solution (prepare fresh daily): 1.O mL Tris buffer, 0.5 mL substrate, 0.25 mL CaC12, 0.25 mL hydroxylamine, 0.25 mL EDTA, 2.5 mL water

2.7. Assay of Immobilized /I-Galactosidase 1. O.lM sodium phosphate, pH 7.5, containing 3 mA4 MgClz. 2. 14 mM o-nitrophenyl-P-o-galactoside (ONPG) in the phosphate buffer. 3. 1.OM mercaptoethanol m water.

2.8. Regeneration of the Surface 1 Desorption solution: 6M guanidinium chloride, pH 1.5. 2. 10 miI4 Trts-HCl buffer, pH 7.5, containing 0 02% NaNs.

3. Methods

3.1. Preparation of the Bio tinylaminopropyl 3.1.1. Derivatization

Surface

with 3-Aminopropyltriethoxysilane

(4)

1. When using Celite, the beads are cleaned by washmg extensively with distilled water. However, CPG beads are cleaned with concentrated HN03 (1.4, v/v) by

16

Swaisgood, Huang, and Walsh

heating at 95-100°C for 1 h. After cleaning, the CPG beads are washed with distilled water until the pH IS neutral (see Note 1). 2. The 10% solution of organosilane IS added to the cleaned beads at a ratro of 10.1 (v/v). After checking, and readmsting the pH if necessary, the mixture is mcubated at 70°C Following a 3-h reactron, the hqutd IS decanted and the beads are dried at 1 IO’C overnight. 3 The 3-aminopropyl beads can be stored at room temperature (see Note 2)

3.1.2, Biotinylation of Surface Am/no Groups (3,5) 1 Place 0 5 g of ammopropyl beads m a IO-mL column and add 4 mL of the precooled (4°C) blotinylatron reagent solutton. 2 Recirculate this solution through the beads for 2 h at 4°C using a peristaltic pump. 3 Dram the NHS-LC-biotm reagent from the beads and wash the beads thoroughly with phosphate buffer. 4. Store the biotmylated beads m the phosphate buffer at 4°C

3.1.3. Measurement of Immobilized Biotin Concentration

(6)

1. Mix 25 pL of the HABA reagent with 1.O mL of the avidin solutron m a 1-mL cuvet 2. Measure the absorbance of this solution at 500 nm and record the reading (A,). 3. Add 50 yL of the biotinylated beads to the solution and mix well. After standing for 5 min at room temperature to allow settling, the absorbance of the supernatant 1smeasured at 500 nm and recorded (AZ). 4 The difference between the two absorbancies (A, - AZ) reflects the displacement of HABA from avrdm by the rmmobrhzed brotm; thus, the amount of rmmobrhzed brotm can be calculated by. pmol btotin/mL beads = [(A, - AZ) x 1 025]/34 x 0.05 mL of beads assayed = 0.603 x (Al -AZ)

(1)

(see Notes 3 and 4)

3.2. Selective Adsorption of Avidin 3.2.1. Preparation and Adsorption of the Avidin Solution (5) 1. Place 0 5 mL of biotmylated beads and 10 mL of avldin solution m a 16mL column and recirculate the avldm sohmon over the beads at 4’C using a pertstaltic pump. 2. After reaction for 12-24 h (a shorter time may be chosen but rt is important to ensure maximum adsorption), the beads are washed extensively with the phosphate buffer Wash until avrdin IS no longer detected in the washing. 3. Store the avldm-biotm beads m the phosphate buffer at 4°C.

3.2.2. Assay of the Number of Biotin-Binding Sites (6) 1. Measure the absorbance of 1.O mL of the avidin-HABA in a I-mL cuvet.

solutton at 500 nm (A,)

Adsorption on Celite or Glass

17

2. Incubate 0.5 mL of biotm solution with 0.1 mL of avidin-biotm beads m a test tube for 5 mm at room temperature (2O-24°C). 3. Add 50 yL of supematant from the bead mixture to the avtdm-HABA solutton m the 1-mL cuvet and mix by inversion. 4. After a 5 min reaction at room temperature, record the absorbance at 500 mn (A&. 5. The absorbance difference [(A, - 1.05 AZ)]/34 reflects the concentration of biotin m the bead supematant (see Note 5).

3.3. Biotinylatlon of the Enzyme 3.3.1. Biotinylation Procedure: Reaction with Lysyl &-Amino Groups (5) 1. Mix the biotmylation reagent with the enzyme solution (see Note 6) 2. Incubate the reaction mixture for 2 h at 4°C. 3. Unreacted NHS-LC-Biotin is removed by dialysis or by ultrafiltration Centricon 30 microconcentrator using the Tris buffer (see Note 7). 4. Store the btotinylated enzyme in the Tris buffer at 4°C.

3.3.2. Determination

with a

of the Extent of Biotinylation (5,6)

1. Mix 20 uL of HABA reagent with 1.O mL of avtdm solution in a 1-mL cuvet and measure the absorbance at 500 nm (A,). 2. Add 50 pL of brotinylated enzyme solutton to the avidin-HABA mixture and mix by inversion of the cuvet 3. Measure the absorbance at 500 nm (AZ). 4. The extent of biotinylation is determined by calculation (see Note 8).

3.4. Adsorption of Biotlnylated (see Notes 9-12)

Transglutaminase

1. Four milliliters of biotmylated transglutaminase solutton (2.5 mg/mL) 1srectrculated through 0.5 mL of beads for 2 h at 4°C 2. The immobilized enzyme is extensively washed with 50 mM Tris-HCl, pH 7.0, containing 2 mM DTT, 3 mA4 EDTA, and 0.02% NaNs and stored in the buffer at 4°C.

3.5. Adsorption

of Biotinylated

P-Galactosidase

(see Notes 9-12)

1. Ten milliliters of biotinylated P-galactosidase solution (10 mg/mL) in 50 mA4 Tris-HCl, pH 7.0, containing 0.02% NaNs is recirculated through 2 mL of beads for 12 h at 4°C. 2. The immobilized enzyme IS washed with 10 vol of 10 mit4 Tris-HCl, pH 7.4, containing 0.5MNaC1, 10 mM MgC12, and 1 mMEDTA and stored in the pH 7.0 Tris buffer.

3.6. Assay of Immobilized

Transglutaminase

1. Set up a mtcrorecirculatton bioreactor system (9). 2. Pipet 3.0 mL of substrate solution into the bioreactor reservoir.

Swaisgood, Huang, and Walsh

78

3. Place 20-50 pL of immobilized enzyme beads in the btoreactor 4 Recirculate the substrate solution through the beads for 30 mm at 37°C. 5 Stop the pump, remove 0.5 mL of the assay mixture, and mix with 0 5 mL of color developing reagent 6. Measure the absorbance at 525 nm. One micromole hydroxamate/mL gives an absorbance at 525 nm of 0.29 (IO). 7. A control is assayed in the same manner, except the substrate solution is not recirculated through tmmobdized enzyme beads.

3.7. Assay of immobilized

p=Galacfosidase

1 Ptpet 1.0 mL phosphate buffer, 0 3 mL stock mercaptoethanol, 0.5 mL ONPG solution, and 1.2 mL deionized water into the broreactor reservoir. 2. Recrrculate the assay substrate solution through the tubing and spectrophotometer cuvet for 1 min. 3. Stop the pump and insert the bloreactor containing 10-20 uL of immobilized enzyme beads. 4. Recirculate the substrate solutron through the broreactor and the spectrophotometer cuvet and record the increase in absorbance at 405 nm 5. Calculate AA,,, from the initial slope of the trace (see Note 13)

3.8. Regeneration of the Surface 1. Place the beads m a column and rinse with 5 column volumes of deionized water 2. Wash the beads with 2 bead volumes of desorption solution and stop the column outlet 3. Add 3 bead volumes of desorption solution and mcubate for 20 min at room temperature 4. Open the column outlet and wash with 6 bead volumes of desorptton solutton. 5. Rinse the beads with deionized water and finally with the Tris buffer. 6. Store the beads in the Tris buffer.

4. Notes 1. In all operations with either Celite or CPG beads, stirring wrth a magnetic stirrer should be avoided because this will result in breakage and production of fines. 2. Removal of tines is Important if a high flow rate through the bioreactor is required. Fines can be removed by adding a large volume of distilled water and decanting. The beads are then washed and collected on a fretted-glass filter and drted overnight at 80°C. 3. Note that 34 is the milhmolar absorptivity of the avidin-HABA complex 4. For this and other procedures throughout this chapter, bead volume can be conveniently and accurately measured using a modified graduated plastic syringe. By cuttmg off the tip of the syrmge, beads can be pulled mto the syrmge usmg the plunger, the syringe can be inverted allowing the beads to settle and pack on top of the plunger, the volume of beads adjusted by movement of the plunger, and finally, the beads can be delivered using the plunger.

19

Adsorption on Celite or Glass

5. The absorbance difference [(A, - 1.05 AZ)]/34 reflects the concentration of btotm in the bead supematant (see Note 3), and from the decrease in biotin concentration of the brotm solution added to the beads, the amount of biotm bound can be calculated; pmol biotin/ mL beads: = ((0.25 pmol biotin added) - [(A, - 1.05 A2)/34][0.5/0.05]}/0.1 = 2.5 - [2.94](A, - 1 05 AZ) 6. The molar ratio of the reagent to enzyme should be chosen on the basis characteristics of the enzyme. We have used mole ratios ranging from 1.0-l reagent/m01 enzyme. The obJective is to incorporate at least one biotinyl per molecule. 7. The membrane size should be chosen on the basis of the molecular size enzyme 8

AA=[1.02/(1 pmol biotin/mL pmol btotin/mol

02+0.05)]

xAr-AZ

(2)

of the 6 mol group of the (3)

of reaction mixture = AA/34

(4)

enzyme = [(AA/34) x (1.07/0.05)1/M enzyme used = 0.629 x AAIM enzyme used

(5)

9. Btotmylated enzyme 1s bioselectively adsorbed on the avrdm-biotm beads by recnculatmg the btotmylated enzyme solutton through a column of the beads for 2 h at 4°C using a peristaltic pump. Nonspecifically adsorbed protein IS removed by washing with an appropriate buffer. 10. Nonselectrvely bound protein can be removed by washing with the appropriate buffer containing 0.5M NaCl or, if the enzyme is stable in low concentrations of urea, for example, 2-4h4 urea, inclusion of urea in the buffer is very effective in removal of nonselectively bound protein. 11 Determination of the enzyme immobilized (3J). The amount of enzyme bound can be estimated from amino acid analyses. Hydrolysates of weighed amounts of pure enzyme, avidin, a 1: 1 molar ratio mixture of avidin and pure enzyme, and unmobilized enzyme-avidin beads are prepared by heating m 6N HCl for 24 h at 110°C. The hydrolysates are dried under Nz and the amino acids are analyzed using standard procedures. Choosing two or more amino acids whose values are most different in avidm and the enzyme, the percentage of immobthzed avidm and enzyme can be determmed using a regression equation developed with values from the standards. 12. Determine the amount of protein immobilized (7,8). The total amount of protein immobrhzed can be calculated from a determination of the concentratton of prrmary amino groups, which can be measured by reaction with o-phthalaldehyde (OPA). Measurement of the amino group concentration m an acid hydrolysate allows calculation of the protein concentration in a manner similar to the Kjeldahl procedure. We have developed a spectrophotometric OPA method, using the maximum absorbance at 340 nm for the OPA adducts of ammo acids, to determine protein concentration from acid hydrolysates. Using a variety of proteins as

20

Swaisgood, Huang, and Walsh

standards, the specific absorptivity of OPA adducts of hydrolysates was EmL,,,,s = 50 + 3.4. The molar ratio of avldin/enzyme can be calculated from the percentages determined above and the total protein nnmoblhzed. 13. The umts of actlvlty, in pmol/mm, 1s given by units/ml of beads = (3 x AAdo5/ min)/(3 1 x mL of beads), where 3.1 1s the mllllmolar absorptivity of o-nitrophenol.

References 1. Swalsgood, H. E. (199 1) Immobihzed enzymes: apphcatlons to bloprocessmg of food, mFoodEnzymology, vol. 2 (Fox, P. F., ed.), Elsevler, London, pp. 309-341. 2. Green, N. M. (1970) Spectrophotometric determination of avidm and blotm Methods Enzymol 18A, 4 18-424. 3. Walsh, M. K. and Swalsgood, H. E. (1993) Characterlzatlon of a chemically coqugated P-galactosidase bioreactor. J Food Biochem 17,283-292. 4. Janolino, V G. and Swaisgood, H E (1982) Analysis and optimization of methods using water-soluble carbodiimide for immobilization of biochemlcals to porous glass. Biotechnol Bioeng 24, 1069-1080. 5 Huang, X. L , Catlgnam, G L , and Swalsgood, H E (1995) Immoblllzatlon of blotmylated transglutammase by bloselectlve adsorption to unmobilized avldin and characterization of the immobilized activity. J Agrzc Food Chem 43,895-901 6. Janolino, V. G., Fontecha, J., and Swaisgood, H. E. (1995) A spectrophotometric assay for blotm-bmdmg sites of munobilized avldm. Appl Blochem Btotechnol. 56, l-7. 7 Goodno, C C , Swalsgood, H. E , and Catlgnam, G L (198 1) A flourlmetrlc assay for available lysine m proteins. Anal Bzochem 115,203-211 8. Thresher, W. C. (1989) Characterization of macromolecular interactions by high performance analytical affinity chromatography. Ph D. Dissertation, North Carolina State Umverslty, Raleigh, NC. 9. Taylor, J. B. and Swaisgood, H. E. (1980) Microrecrrculation reactor system for characterization of immobilized enzymes Blotechnol Bloeng 22,26 17-263 1 10. Folk, J. E. (197 1) Transglutammase. Methods Enzymol. 17,889-894.

3 Immobilization of Proteins on Thionyl Chloride-Activated Controlled-Pore Glass Violeta G. Janolino

and Harold E. Swaisgood

1. Introduction Procedures for covalent immobilization should be capable of reacting with a limited number of surface residues of the protein under very mild conditions, preferably those that are optimal for the stability of the protein being immobilized. Sometimesit is also desirableto use amethod of covalent immobilization that 1s reversible under mild condittons. We have developed the thionyl chlondeactivated succinamidopropyl-glass asa matrix that provides thesedesirable properties (Z-3). The activated support will react with only primary amino groups or sulfhydryl groups on the surface of the protein m physiological buffers in the pH range of 4.0-8.0 and in the cold room or at room temperature. Although the exact chemistry of the reaction still eludes definition, the required conditions and the reacting groups are well established. The thionyl chloride-activated succinamidopropyl surface can be stored for long periods of time (perhaps more than one year), and protein can be immobihzed simply by incubating it with the matrix under the conditions chosen. The immobilized protein remains covalently bound even in the presence of strong denaturants, such as 6Mguanidinium chloride; however, if desired, it can be partially removed by reaction with dilute solutions of hydroxylamine at room temperature (2). If the reaction was through amino groups, up to 80% of the protein can be removed. We have used this procedure both as a reversible method of covalent immobilization (2,4) and as a convenient as well as effective means for preparation of affinity matrices (.5,6),

2. Materials 2.1. C/caning of fhe Glass Surface 1 Controlled-pore glass(CPG beads). 2. ConcentratedHN03. From

Methods m Blofechnology, Vol 1 Immob~hzarron of Enzymes and Cells E&ted by G F BIckerstaff Humana Press Inc , Totowa, NJ

21

Janolino and Swaisgood

22 2.2. Synthesis 1 2. 3. 4

of Aminopropyl

Cleaned @INO,-treated) CPG beads 3-Ammopropyltriethoxysilane. 2,4,6-Trmttrobenzenesulfonate. Sodium borate

2.3. Succinylation 1. 2 3 4 5.

of Aminopropyl

Glass

Ammopropyl-CPG beads. Acetone. Trtethylamme Succmtc anhydrtde. TNBS reagent. 1% TNBS m saturated sodium borate.

2.4. Thionyl Chloride

Activation

1 Succmamidopropyl-CPG 2. Methylene chloride. 3. Thionyl chloride.

2.5. Washing/Drying

beads

the Beads

1 Thionyl chloride-activated 2 Methylene chloride. 3 Acetone.

2.6. Immobilization 1 2. 3. 4.

Glass

succmamidopropyl-CPG

beads

of Glutamate Dehydrogenase

Thionyl chloride-activated succinamidopropyl-CPG Bovine liver glutamate dehydrogenase. 50 mA4 sodium phosphate, pH 7.0. 4.OMurea in 50 mM sodium phosphate, pH 7.0.

beads

2.7. lmmobillza tion of Antibodies 1. 2. 3. 4 5. 6.

Thionyl chloride-activated succmamidopropyl-CPG beads. PBS: 0.02M sodium phosphate, 0.15MNaC1, pH 7 3. Antibovine IgG 1 OM glycine methyl ester 2Murea in PBS. 0.2M NaCl in 0.02M sodium phosphate buffer, pH 7.3.

2.8. Removal

of Protein with Hydroxylamine

1. Immobilized protein on thionyl chloride-activated CPG beads. 2. 50 mA4 sodium phosphate, pH 7.0. 3. 2.M.OMurea in phosphate buffer, pH 7.0; and/or 0.2-l.OMNaCl buffer, pH 7.0. 4. 0. l-l .OM hydroxylamine in phosphate buffer, pH 7.0.

in phosphate

Thionyl Chloride-Activated 3. Methods 3.7. Cleaning

23

CPG

of fhe Glass Surface

1. Clean (see Note 1) the CPG surface by heating in a boiling water bath with 200 mL concentrated mtrrc acid/100 mL of CPG beads for 60 mm (see Note 2). 2. Wash the beads extensively in a large, coarse fritted-glass filter under suction with 2 L distilled water/100 mL beads until the pH of the washings is neutral (7,8)

3.2. Synthesis 1. 2. 3. 4. 5 6. 7. 8. 9 10.

of Aminopropyl

Glass (see Note 3)

Prepare a 10% (v/v) aqueous solution of 3-aminopropyltriethoxysilane. Adjust the pH to 4 0 with 6N HCI Add the cleaned CPG beads to 3 vol of the silane solution. Degas at reduced pressure to ensure that all of the pore volume is filled with the reagent. Incubate at 70°C for 3 h with occasional mixing Decant excess reagent and place the wet beads in an oven at 100°C overnight for direct polymerization. Remove the fines generated by mixing the beads wrth a large volume of distilled water, let the normal sized beads settle out, and decant the suspended fines. Wash the beads with 1 L of distilled water/100 mL of beads on a fritted-glass filter under suction. Dry the beads overnight in an oven at 8O“C. Add to an aliquot of the beads, 2,4,6-trinitrobenzenesulfonate (TNBS) (1% m saturated sodium borate solution) to qualitatively detect the presence of amino groups (see Note 4).

3.3. Succinylation

of Aminopropyl

Glass

1 Rinse the dry aminopropyl-CPG beads with acetone (see Note 5). 2 Add to the beads 3 vol of a solution containing 1% (v/v) triethylamine and 10% (v/v) succinic anhydride m acetone. 3. Degas the mixture briefly under reduced pressure to ensure complete exposure of the pore surfaces. 4. After reaction is complete (10-20 min at room temperature), rinse the succinamidopropyl glass thoroughly with acetone 5. Use the TNBS procedure (see Section 3.2., step 10) to test an aliquot of the beads for completion of succinylation reaction (see Note 6) 6. Store the dried beads at room temperature until required.

3.4. Thionyl Chloride

Activation

1. Place the dry succmamidopropyl-glass beads m a round-bottom flask and thoroughly rinse by swirling with methylene chloride (see Note 7). 2. After decanting the methylene chloride, add enough thionyl chloride to cover the beads and allow the mixture to react on a heating mantle for 1 h at 60°C (see Note 8).

24

Janolino and Swaisgood

3.5. Washing and Drying of the Activated

Beads

1 Following reaction, decant the thronyl chlortde and thoroughly rinse the activated beads with methylene chlortde 2 Rinse the thionyl chloride-activated beads successively with acetone and water 3. Dry the beads at 110°C and store desiccated at room temperature until required for immobrhzation (see Note 9)

3.6. lmmobiliza tion of Glutamate Dehydrogenase 1. Degas the thionyl-activated beads in 50 rnM sodium phosphate, pH 7.0. 2 Transfer the beads to a column and wash them with a small volume of sodium phosphate buffer 3. Allow the phosphate buffer to drain, then recirculate a solution (30 mL) of glutamate dehydrogenase (2-6 mg/mL) m 50 mM sodium phosphate, pH 7 0, through the column consisting of -1 g of beads in a fluidized-bed configuration at room temperature (24-26”(Z) for 3 h 4. Wash the column with 400 mL of 4 OMurea in 50 Mphosphate buffer, pH 7.0, in a fixed bed configuration at room temperature and a flow rate of 150 mL/h (see Note 10)

3.7. Immobilization

of Antibodies

1 Degas the thionyl chloride-activated beads in PBS 2. Transfer the beads to a column and wash with a small volume of PBS 3 Allow the PBS to drain away, then recirculate through the column a solution of anttbovme IgG (at least 1 mg/mL) at a flow rate of 0.2 mL/mm, overnight at 4°C 4. Followmg IgG immobihzatron, recirculate 1.OMglycine methyl ester in PBS for 2 h at room temperature to block unreacted thionyl chloride-activated sites. 5 Wash the column with 2Murea in PBS and with 0.2MNaCl in 0.02Mphosphate buffer, pH 7.3.

3.8. Removal

of Protein with Hydroxylamine

1. Wash the column of immobilized protein with 2.0-4.OM urea and/or 0.2-l OM NaCl m phosphate buffer, pH 7.0, at room temperature and with a flow rate of 150 mL/h. 2 Release the immobihzed protein with O.l-l.OM hydroxylamine in phosphate buffer, pH 7.0 (see Note 11).

4. Notes 1. In preparation for derivattzatron, it is important to clean the CPG beads to generate the maximum number of srlanol groups. 2 If the glass beads have been used previously, heat the beads at 600°C for 24 h before incubation with nitric acid. 3. Silanization produces a stable, covalently attached surface-functional group, the amino group, onto which peptrde chains can be synthesized to produce a given

Thionyl Chloride-Activated

4 5. 6. 7. 8

9

10.

11.

CPG

25

microenvironment. After hydrolysis of silane’s ethoxy groups to hydroxyls in an aqueous medium, the reaction between the hydroxyls and the glass surface silanols can take place (7-9). The beads should turn yellow and remain yellow after several washes wtth distilled water. The aminopropyl-CPG beads can be conveniently succinylated by reaction with succinic anhydride dissolved in acetone (2,9). No yellow color on the ahquot of beads indicates that all of the ammo groups were derivatized The methylene chloride should be free of water and should be pretreated with molecular sieves to remove any traces of water The reaction of succinamidopropyl glass with nonaqueous thionyl chloride followed by washing with distilled water produces an activated surface that reacts with ammo and thiol groups under nondenaturing conditions (2,6). Washing the beads after reaction with thionyl chloride results m removal of the yellowish color Only molecules that possess accessible primary ammo groups or thiol groups (such as amino acid derivatives and proteins) react rapidly to give a covalent bond, which prevents their subsequent removal on extensive washing with 8M urea or 6M guanidmmm chloride. The dry activated beads are stable for long periods and immobilization can be accomplished simply by addition of an aqueous protein solution at pH of 4.0-8.0, and a temperature of 4-40°C The amount of protein that can be unmobilized by this method is equivalent to that which can be unmobihzed onto succmamidopropyl-glass beads that have been activated using water-soluble carbodumides (2,3). Immobilization is essentially limited to amino and thiol groups on protein, and subsequent release by mild treatment with hydroxylamme is greater if attachment is through ammo groups. The wash with urea and salt solution prior to hydroxylamine is to remove noncovalently bound protein. Percentage release of protem is determined as the difference m matrix-bound protein before and after release with hydroxylamine (l-3)

References 1. Brown, R. J., Swaisgood, H. E., and Horton, H. R. (1979) A procedure for covalently immobilizmg enzymes which permits subsequent release Bzochemwy 18, 49014906. 2. DuVal, G , Swaisgood, H. E., and Horton, H R. (1984) Preparation and characterization of thionyl chloride-activated succinamidopropyl-glass as a covalent mnnobilization matrix. J Appl. Biochem 6,240-250 3. Horton, H. R. and Swaisgood, H. E (1987) Covalent immobilization of proteins by techniques which permit subsequent release Methods Enzymol 135, 130-14 1. 4. Heth, A A. and Swatsgood, H E. (1982) Examination of casem micelle structure by a method for reversible covalent immobtlrzation J. Dazry Ser. 65, 2047-2054.

Janolino and Swaisgood

26

5. Swaisgood, H E. and Chatken, I. M (1986) Analytical high performance affinity chromatography: evaluation by studies of neurophysm self-associatton and neurophysin peptide hormone interaction using glass matrices. Blochemutry 25, 4148-4155 6. Stabel, T. G., Casale, E. S , Swarsgood, H. E., and Horton, H. R. (1992) Anti-IgG nnmobilized controlled-pore glass. Thionyl chloride-activated succmamidopropylglass as a covalent mnnobthzation matrix. Appl Blochem Bzotechnol. 36, 87-96 7 Janolmo, V. G. and Swaisgood, H E. (1982) Analysis and optimization of methods using water-soluble carbodumide for munobilization of btochemicals to porous glass. Bzotechnol Bloeng. 24, 1069-1080 8 Taylor, J B. (1979) A general study of the effects of microenvironment and partttionmg on the kinetic parameters of immobilized enzymes. Ph.D. Dissertation, North Carolina State University, Raleigh, NC 9 Swaisgood, H E and Horton, H. R (1987) Sulfhydryl oxtdase from milk. Methods Enzymol 143,504-5

10

Enzyme Immobilization Pedro Lozano and Jo&

on Nylon

L. lborra

1. Introduction Nylons are a family of lmear condensation polymers, involvmg the repeated alkane segments bound by secondary amide linkages. The different types of nylons available commercially are named and classified according to the number of carbon atoms in their monomeric units (i.e., nylon 6,6 is obtained from adipic acid and 1,6-diamme hexane). Chemtcally, nylon is a thermoplastic polymer with high mechanical strength (Le., limiting tension to flexion = 500-l 500 kp/cm2), superficial hardness, and resistance to abrasive conditions caused by the mtermolecular hydrogen bond interactions established between the amide groups of parallel chains. This is a useful charactertstic for immobilized enzymes because it gives a favorable hydrophilm microenvironment to support both catalytic activity and a stable enzyme structure. Additionally, nylon is readily available in many physical forms, such as powder, pellets, tubes, membranes, and more, which offer vanous surface/weight ratios and have low economic costs. However, as a support for enzyme mnnobilization, nylon is nonporous and provides advantages and disadvantages with respect to the porous supports (see Chapter 1). It is particularly advisable for use with high-molecular-weight substrates. Nylon has few free end groups for covalent attachment of enzyme molecules, so it must be pretreated to generate potentially reactive centers. There are three different approachesto produce thesereactive centers(1). The first approach deals with a partial cleavage of the secondary amide groups by acidic means to yield free amino and carboxyl groups on the support surface (Fig. 1). In the second approach, production of the reactive centersmvolves an 0-alkylation process of the amide bonds, giving imidoester groups on the support surface (2,3). The third approach generates reactive centers using the partially hydrolyzed nylon From

Methods m EOotechnology, Vat 7 lmmobrlrzatron of Enzymes and Cells Edlted by G F BIckerstaff Humana Press Inc , Totowa, NJ

27

Lozano and lborra

OCHS

Fig. 1. Scheme of methods for activation of nylon.

for a subsequent reconstttution of the amide bonds by a four-component condensation process based on the Ugr’s reaction (N-alkylation). This process mvolves both the free amino and carboxyl groups of the nylon surface, and an aldehyde and a diisocyanide reagent, respectively, to obtain a nylon-isonitrile derivative. In both the N- and 0-alkylatlon cases,the mechanical strength of the nylon backbone is not modified, whereas m the hydrolytic cleavage case,a clear disruption on the nylon superficial structure can be observed (1,4). Both the carboxyl and amino groups of the partially hydrolyzed nylon can be used to attach the enzyme molecules directly, involving the a-ammo groups of lysine residues by using carbodiimide or dialdehyde (i.e., glutaraldehyde), respectively, as crosslinking reagents. The use of carbodiimide reagent provides attachment of the enzymeby amide linkages, but results in a lower yield of immobilized enzyme than with the glutaraldehyde, because carboditmide is involved m the reconstitution of amide linkages into the nylon backbone. The imidoester groups of the 0-alkylated nylon are highly reactive toward free amine groups, and are probably the best activation method for direct immobilization of protein onto the nylon surface by amidme groups, with high protein mnnobrhzation yields (.5,6). However, imidoester groups are highly sensitive toward hydrolysis in aqueous media, losing their reactivity. This fact could be minimized by avordmg contact of the 0-alkylated support with water and by using alkaline conditions in the obligatory aqueous step, such as the enzyme attachment. In N-alkylated nylon, the enzyme molecules can be immobilized directly to the isonitrile groups by a new four component condensation reaction involving the amino or carboxy1 groups of the protein, and in the presence of acetaldehyde and carboxyl (i.e., acetate) or amme (i.e., Tris) reagents, respectively (Z).

Immobilization on Nylon

f! 1 1

6cH2

NYLON IMIOOESTER

Y

1 Ill

!I n

/+-JHd

/G+H-J\

NH YH (CH2)2 AH

4” 60 (4”2,6

F0 NH AH2

Fig. 2. Severalmethods for chemical modification of 0-alkylated nylon. Additionally, different bifunctional reagents, such as aliphatic diamine spacers (2,3), polyethyleneimine (.5,7-9) or aromatic diamines (Z,4), can be attached onto the activated nylon surface to give a desired chemical modification or to increase the enzyme-support distance (i.e., hexamethylenediamme), surface hydrophobicity (Le., p,p’-diaminodiphenylmethane), or the number of potentially reactive centers (i.e., polyethyleneimine). Also, a desired amino acid residue, such as tyrosme (i.e., p-phenylenediamme) or cysteine residues (i.e., 2-mercaptoethylamine), could be involved in the protein attachment to improve the immobilization yield (Fig. 2). Sometimes the enzyme-attachment step onto the derivatized nylon may require exposure of enzyme to potential enzyme inactivators, e.g., coupling agents (i.e., Fe(CN)63- for the nylon-thtol derivative) or by previous treatment with crosslinking reagents (i.e., glutaraldehyde or dimethyl adipimidate in nylon-amine derivatives). In each case, the best conditrons to maintain the enzyme activity (i.e., a weak alkaline pH, cold room) should be selected (ZO) (Fig. 3). The simultaneous presence of the crosslmking reagents during the enzyme attachment step can yield inactive derivatives resulting from intermolecular crosslinking of the enzyme molecules. Thus, reactivation of the modified support by these reagents should be carried out prior to enzyme attachment to avoid enzyme deactivation. The aim of this chapter is to show all the methodologies for enzyme immobilization on nylon and to outline a strategy to obtain a desired immobilized

Lozano and lborra

1

ENZYME-SH

NH

Ibp=im~

ENZYME-NH2 ENZYME-lY&OH [wmel

0 H 8 =N0 B EN?ZWE

Fig. 3. Methods for covalent nnrnobrhzatron of enzyme to drfferent derrvatized

nylons

enzyme derivative with the highest activity and stability. The high reactivity of the imldoester groups have shown the 0-alkylatlon methods to be the best approach for nylon activation, and they can be further improved by coating the activated support with the soluble polymer polyethyleneimine, then attaching the enzyme by Schiff’s bases with glutaraldehyde (4-9). This improvement multiplies the number of reactive centers on the support surface and provides a highly hydrophilic environment for the enzyme. Coupling mvolves an ammo acid residue (1~s) that IS available on the surface of all proteins.

2. Materials

2.1. Preparation of Nylon Powder (see Note I) 1. Nylon-6,6 pellets: 0 1-l nun particle size 2 Nylon membrane* 500-5 mesh count/in., 0.04-l mm thread diameter, 35-400 g/m* weight 3. Nylon tube: l-5 mm external diameter, 0 2-2 mm internal diameter. 4. Nylon powder: O-2-0.7 pm particle size. 5 Nylon dissolving reagent. 20% (w/v) anhydrous CaC12 solution m methanol 6. Washing solutrons: water, ethanol, ether.

31

Immobilization on Nylon 2.2. Nylon Activation 2.2. I. Nylon Activation by Partial Hydrolysis

1. Support surface-treatment reagent: 18.6% (w/v) CaClz, 18.6% (v/v) water in methanol. 2. 3.65M HCl solutton 3. Washing solutions. water, ether.

2.2.2. Nylon Activation by O-Alkylation 1. 0-alkylation reagent: 100% dimethyl tetrafluoroborate in dtchloromethane. 2. Washing solutton: ice-cold methanol.

sulfate or 0. IA4 triethyloxonium

2.2.3. Nylon Activation by N-Alkylation 1 Partially hydrolyzed nylon. 2 Reagents for Ugi’s condensation: acetaldehyde, isopropanol, 1,6-dnsocyanohexane 3, Washing solutions* isopropanol, ether.

2.3. Direct Enzyme Immobilization onto Activated Nylon 2.3. I. By Amide Bonds onto Partially Hydrolyzed Nylon 1. Partially hydrolyzed nylon. 2. Coupling reagent: 1% (w/v) 1-ethyl-3-(3-dimethylammopropyl) in 0. 1M sodium phosphate buffer, pH 5.0. 3. 2% (w/v) enzyme solution m O.lM sodium phosphate buffer, pH 5.0. 4 Washing solutton: 0.M sodium phosphate buffer, pH 7.8.

2.3.2. By SCM’S Bases onto Partially Hydrolyzed NY/on 1. Partially hydrolyzed nylon 2. Crosslmkmg agent: 10% (w/v) glutaraldehyde in 0.2Msodmm bicarbonate buffer, pH 9.2. 3. 1% (w/v) enzyme solution in 0.M sodmm phosphate buffer, pH 7.8 4. Washing solutions: 0.2Msodium bicarbonate buffer, pH 9.2,O. 1M sodium phosphate buffer, pH 7 8.

2.3.3. By Amidine Groups onto Partially Hydrolyzed Nylon 1. Partially hydrolyzed nylon. 2. Crosslinkmg agent: 5% (w/v) dimethyl adtpimidate in 30% (v/v) N-ethylmorpholine in methanol. 3. 1% (w/v) enzyme solution in 0. 1M sodium phosphate buffer, pH 7.8 4. Washing solutions: ice-cold methanol, O.lM sodium phosphate buffer, pH 7.8.

2.3.4. By Amidine Groups onto 0-Alkylated Nylon 1, 0-alkylated nylon, 2. 1% (w/v) enzyme solution in 0 1M sodium phosphate buffer, pH 7.8 3. Washing solution. 0.M sodium phosphate buffer, pH 7.8

32 2.3.5. By Ugi’s Condensation

Lozano and lborra onto N-Alkylated Nylon

2.3.5 1. THROUGH AMINO GROUPS ON THE PROTEIN 1 Nylon-isonitrile derivative. 2. Cold 0. IM sodmm phosphate in 0 5M sodium acetate, pH 7 0 3. Acetaldehyde. 4 1% (w/v) enzyme solution in water. 5. Washing solution O.lM sodmm phosphate buffer, pH 7.0 2.3.5.2. THROUGH CARBOXYL GROUPS ON THE PROTEIN 1. Nylon rsomtrtle derivative 2 Cold 0 1M Trrs-HCI buffer, pH 7 0 3. Acetaldehyde. 4. 1% (w/v) enzyme solutron m 0 IM sodium phosphate buffer, pH 7 0. 5 Washmg solution. O.lM sodmm phosphate buffer, pH 7.0 2.4. Chemical Modification of Activated Nylon 2.4.1. Introduction of Spacers on the Free Carboxyl Groups 1. Partially hydrolyzed nylon, 2 Coupling reagent: 1% (w/v) 1-ethyl-3-(3-dimethylammopropyl) carbodiimrde m 0. IM sodium phosphate buffer, pH 5.0. 3. Spacer: IMethylenediamme, 1,6-hexanedramine, p-phenylenedtamine, or 2-mercaptoethylamme m 0. 1M sodium phosphate buffer, pH 5.0. 4. Washing solutron O.lM sodium phosphate buffer, pH 5.0

2.4.2. lntroductlon of Spacers on the Free Amino Groups 1 Partially hydrolyzed nylon. 2. Crosslmkmg agent: 10% (w/v) glutaraldehyde m 0 2Msodium bicarbonate buffer, pH 9.2. 3 Spacer: 0.5M ethylenediamine, 1,6-hexanediamine, p-phenylenedtamine, or 2-mercaptoethylamme m 0.2M sodium bicarbonate buffer, pH 9.2. 4 Washing solution* 0 2M sodium bicarbonate buffer, pH 9 2

2.4.3. Introduction of Spacers on the 0-Alkylated Nylon 1. 0-alkylated nylon. 2. Spacer: 0.5M ethylenedramme, 1,6-hexanediamine, p-phenylenediamme, or polyethylenermme m 0 2M sodium bicarbonate buffer, pH 9 2, or 3% (w/v) adrpm acid drhydrazme m 30% (v/v) N-ethylmorpholme m methanol. 3. Washing solution: 0. 1M sodmm phosphate buffer, pH 7.8. 2.4.4. Introduction of Spacers on the N-Alkylated Nylon 1. Nylon-isonitrile derivative. 2. Coupling reagents: methanol, isobutyraldehyde, and glacial acetic acid. 3. Spacer p-phenylenediamme or p,p’-dtammodiphenylmethane. 4. Washing solution: methanol.

Immobilization on Nylon

2.5. Enzyme Immobilization

33

onto Chemically Modified Nylon

2.51. By Schiff ‘s Base 1. Nylon-amine dertvattve. 2 Crosslinking reagent. 10% (w/v) glutaraldehyde solutton m 0.2M sodmm bicarbonate buffer, pH 9.2. 3. 1% (w/v) enzyme solutron in O.lMsodium phosphate buffer, pH 7 8. 4. Washing solution: 0.M sodmm phosphate buffer, pH 7.8

2.5.2. By Amidine Group 1. Nylon-amine derivative. 2. Crosslmkmg reagent: 5% (w/v) dimethyl adtptmidate in 30% (v/v) N-ethylmorpholme m methanol 3. 1% (w/v) enzyme solution in 0.M sodium phosphate buffer, pH 7.8 4. Washing solutrons: ice-cold methanol, O.lM sodium phosphate buffer, pH 7.8.

2.5.3. By Azo Group 1. 2. 3. 4.

4% (w/v) sodium nitrite solution. 2M HCl solution 1% (w/v) enzyme solution in 0.M sodium phosphate buffer, pH 7.8 Washing solution: O.lM sodium phosphate buffer, pH 7.8.

2.54. By Disulfide Bridge 1. Coupling reagents. 55 rnM K,Fe(CN),; 0.9M NH&l; 0.9M NH40H. 2. 1% (w/v) enzyme solution in 0. 1M sodium phosphate buffer, pH 7 8 3. Washing solutron: O.lM sodium phosphate buffer, pH 7 8

3. Methods

3.1. Preparation of Nylon Powder 1. Suspend 15 g of commercially available nylon (tube, membrane, or pellets) in 500 mL of nylon drssolving reagent and stir at room temperature until a homogeneous and viscous solution is obtained (see Note 2). 2. Add (dropwise) the nylon solution into a large excess of water with strong stirring. A white aqueous suspension of nylon powder will be formed. 3. Separate the nylon powder thus obtained on a suctron filter, wash with water, and then suspend m fresh water. 4. Separate the nylon powder again on the suction filter. Wash wtth fresh water, then ethanol, then with ether, and finally dry by air. Remove the traces of solvent and moisture overnight in a vacuum desiccator over phosphorous pentoxrde.

3.2. Nylon Activation 3.2.1. By Partial Hydrolysis 1. Add 1 g of nylon pellets, 10 cm of nylon tube (1 -mm internal drameter), or 15 cm2 of nylon membrane (0.2-mm thread diameter) into a screw-capped test tube

34

2

3. 4 5

Lozano and lborra containmg 15 mL of support surface treatment reagent (see Sectton 2.1.1.), and shake the mtxture for 30 min at room temperature (see Note 3) Separate the treated nylon by filtration or centrtfugatton and then wash several times with excess of distilled water Add 15 mL of 3.65MHCl to the test tube containmg the treated nylon or to 100 mg of nylon powder and mechanically shake the mixture for 24 h at room temperature. Eliminate the HCl solution on a suctton filter and wash again with excess of distilled water several times (see Note 4). For long-term storage, wash the partially hydrolyzed nylon by ether and dry by an Remove traces of solvent and moisture overnight m a vacuum desiccator over phosphorus pentoxide and store at room temperature until use.

32.2. By O-Alkylation 1 Add 1 g of nylon pellets, 100 mg of nylon powder, 10 cm of nylon tube (l-mm internal diameter), or 15 cm2 of nylon membrane (0.2-n-m thread diameter) mto a screw-capped test tube. Then add the 0-alkylation reagent to cover up the support and close the test tube Immediately immerse the test tube in either a boiling water bath for the dimethyl sulfate reagent or m a 22°C water bath for the triethyloxomum reagent for exactly 4 mm without stirring, after which tt should be plunged mto an ice bath to stop the reaction (see Note 5) 2. Separate the excess of alkylatmg reagent on a suctton filter and then wash the 0-alkylated nylon several times with ice-cold methanol. Separate the washing solution each time by filtration (see Note 6). 3. Eliminate the washing solution by filtration and place the test tube containmg the 0-alkylated nylon mto the ice-bath. The activated support should be immediately used for the enzyme attachment (see Section 3.3.4.) or subsequent chemical modtfication (see Section 3 4.3 ).

3.2.3. By N-Alkylation 1. Into a screw-capped test tube containing partially hydrolyzed nylon (0 5 g of nylon pellets, 100 mg of nylon powder, 10 cm of nylon tube, or 15 cm2 of nylon membrane, obtained as described m Section 3.2.1.), add 10 mL of tsopropanol, 2.5 mL of acetaldehyde, and 1 mL of 1,6-diisocyanohexane Mechanically shake the mixture for 24 h at room temperature. 2. Separate the excess of reagents on a suction filter and then wash the N-alkylated nylon with several portions of isopropanol and finally with ether, separating the washing solution each time. 3 Dry the nylon-tsonitrile product by an and store m darkness at 5°C m a desrccator over silica gel.

3.3. Direct Enzyme /mmobi/izafion onto Activated Nylon 3.3.1. By Amide Bonds onto Part/ally Hydrolyzed Nylon 1. Into a screw-capped test tube containing the partially hydrolyzed nylon (0 5 g of nylon pellets, 100 mg of nylon powder, 10 cm of nylon tube, or 15 cm2 of nylon

Immobilization on Nylon

35

membrane, obtained as described in Section 3.2.1.), add 5 mL of the couplmg reagent. Mechanically shake the mixture for 1 h at room temperature 2. Add 5 mL of the enzyme and mechanically shake the mixture overnight at 4°C 3 Separate the excess enzyme solution by filtration and then wash the rmmobilrzed derivative consecutively with two portions of 10 mL of washing solutlon 4. Suspend the rmmobrlized derivative m 10 mL of washing solution and store at 4°C until use

3.3.2. By Schiff’s Bases onto Partially Hydroiyzed Nylon 1. Into a screw-capped test tube containing the partially hydrolyzed nylon (0.5 g of nylon pellets, 100 mg of nylon powder, 10 cm of nylon tube, or 15 cm2 of nylon membrane, obtained as described in Section 3.2. I.), add 10 mL of crosslmkmg agent and mechanically shake the mtxture for 30 mm at room temperature 2. Separate the excess glutaraldehyde by vacuum filtration and then wash the nylon-aldehyde derivative with several portions of 0.2M sodium bicarbonate buffer, pH 9.2. 3. Add 10 mL of enzyme solution and mechanically shake the mixture overnight at 4°C (see Note 7). 4 Separate the excess enzyme solution by filtration and then wash the immobilized derivative consecutively with two portrons of 10 mL of O.lM sodium phosphate buffer, pH 7.8. 5 Suspend the immobtlized derivative in 0 1M sodium phosphate buffer, pH 7 8, and store at 4°C until use

3.3.3. By Amidine Groups onto Partially Hydrolyzed Nylon 1. Into a screw-capped test tube containing the partially hydrolyzed nylon (0.5 g of nylon pellets, 100 mg of nylon powder, 10 cm of nylon tube, or 15 cm2 of nylon membrane, obtained as described in Section 3.2.1.), add 10 mL of the crosslinkmg agent and mechanically shake the mixture for 1 h at room temperature 2. Separate the excess reagent by vacuum filtration and wash the nylon-imidate derivative with several portrons of ice-cold methanol and then with 0. 1Msodmm phosphate buffer, pH 7.8. 3 Add 10 mL of the enzyme solutron to the test tube containmg the nylon-imidate derivative and mechanically shake the mixture overmght at 4°C. 4. Separate the enzyme solution by filtration and then wash the immobilized derivative consecutively with two portions of 10 mL of 0 1Msodmm phosphate buffer, pH 7.8. 5. Suspend the immobilized derivative m 0. 1M sodium phosphate buffer, pH 7.8, and store at 4°C until use.

3.3.4. By Amidine Groups onto 0-Alkylated Nylon 1. Into a screw-capped test tube containing the 0-alkylated nylon (0.5 g of nylon pellets, 100 mg of nylon powder, 10 cm of nylon tube, or 15 cm2 of nylon membrane, obtained as described m Section 3.2.2.), add 10 mL of the enzyme solution

Lozano and lborra

36

to the test tube contammg the nylon-tmtdate denvatrve and mechamcally shake the mixture overnight at 4°C. 2. Separate the enzyme solution by filtration and then wash the immobilized derivative consecuttvely with two porttons of 10 mL of the washing solutton 3. Suspend the immobilized derivative in the washing solution and store at 4OC until use.

3.3.5. l3y Ugi’s Condensation onto N-Alkylated Nylon 3.3.5.1. THROUGH AMINO GROUPS OF THE PROTEIN I. Into a screw-capped test tube containing the nylon-isonitrile derivative (0.5 g of nylon pellets, 100 mg of nylon powder, 10 cm of nylon tube, or 15 cm2 of nylon membrane, obtained as described in Section 3.2.3.), add 6 mL of cold 0 1M sodium phosphate m 0.5Msodium acetate, pH 7.0, and 3 mL of the enzyme solution followed by 0.3 mL of acetaldehyde. Mechanically shake the mixture overnight at 4’C 2 Separate the excess reagents by vacuum filtration and then wash the immobilized derivative consecuttvely with two portions of 10 mL of the washing solutton 3. Suspend the munobthzed derivative in the washing solutton and store at 4°C until tt will be used 3.3.5.2.

THROUGH CARBOXYL GROUPS OF THE PROTEIN

1 Into a screw-capped test tube containing the nylon-isonitrile derivative (0.5 g of nylon pellets, 100 mg of nylon powder, 10 cm of nylon tube, or 15 cm2 of nylon membrane, obtamed as described m Sectton 3.2 3.), add 6 mL of cold O.lM Tris-HCI and 3 mL of the enzyme solution followed by 0 3 mL of acetaldehyde Mechanically shake the mixture overnight at 4°C 2. Separate the excess reagents by vacuum filtration and then wash the unmobtlized derivative with two portions of 10 mL of the washing solution 3. Suspend the mmrobihzed derivative m the washing solution and store at 4°C until use.

3.4. Chemical Modification of Activated Nylon 3.4.1. Introduction of Spacers on the Free Carboxyl Groups 1. Into a screw-capped test tube containing the partially hydrolyzed nylon support (0.5 g of nylon pellets, 100 mg of nylon powder, 10 cm of nylon tube, or 15 cm2 of nylon membrane, obtained as descrrbed m Section 3.2.1.), add 5 mL of the coupling reagent and mechanically shake the mixture for 1 h at room temperature. 2 Add 5 mL of the spacer solution mto the test tube and mechamcally shake the mixture for two additional hours at room temperature. In each case, nylon-amme, ammoaryl, or thiol is obtained (see Note 9) 3. Separate the excess reagents by filtration and then wash the modified hydrolyzed nylon with several portions of the washing solution. The modified support could also be stored m dryness as described m Section 3.1. (see Note 10).

Immobilization on Nylon

37

3.4.2. Introduction of Spacers on the Free Amino Groups 1. Into a screw-capped test tube containing partially hydrolyzed nylon (0.5 g of nylon pellets, 100 mg of nylon powder, 10 cm of nylon tube, or 15 cm2 of nylon membrane, obtained as described m Section 3.2.1.), add 10 mL ofthe crosslinkmg agent and mechanically shake the mixture for 30 min at room temperature. 2. Separate the excess of glutaraldehyde by vacuum filtratton and then wash the modified hydrolyzed nylon with several porttons of the washing solutton 3. Add 10 mL of the spacer solution and shake for two additional hours at room temperature. 4 Separate the excess reagents by vacuum filtration and then wash the modified hydrolyzed nylon with several portions of washing solutton (see Note 10).

3.4.3. Introduction of Spacers on the O-Alkylated Nylon 1. Into a screw-capped test tube containing the 0-alkylated nylon (0.5 g of nylon pellets, 100 mg of nylon powder, 10 cm of nylon tube, or 15 cm2 of nylon membrane, obtained as described m Section 3.2.2.), add 10 mL of the spacer solution and shake the mixture for 1 h at room temperature. 2. Separate the excess reagents by vacuum filtration and then wash the modified 0-alkylated nylon with several portrons of washing solution.

3.4.4. Introduction of Spacers on the N-Alkylated Nylon 1. Into a screw-capped test tube, add 0.1 g of spacer dtssolved in 10 mL of methanol and 0.05 mL of tsobutyraldehyde 2. Add the nylon-isomtrile derivative (0.5 g of nylon pellets, 100 mg of nylon powder, 10 cm of nylon tube, or 15 cm2 of nylon membrane, obtained as described m Section 3.2.3.), mto the solution and add 0.3 mL of glacial acetic acid mto the mixture Close the test tube and mechamcally shake for 24 h at room temperature. 3. Separate the excess reagents by filtration and then wash the nylon-ammoaryl obtained, first with methanol, then with ether. Finally, dry with air. 4. Store the obtained nylon-ammoaryl in darkness at 5°C m a desrccator over phosphorous pentoxtde.

3.5. Enzyme Immobilization 3.5.1. By Schiff ‘s Base

onto Chemically

Modified

Nylon

1. Add the nylon-amine derivative (0.5 g of nylon pellets, 100 mg of nylon powder, 10 cm of nylon tube, or 15 cm2 of nylon membrane, obtained as described in Section 3.4.) into a screw-capped test tube and add 10 mL of crosslinking reagent. Shake the mixture for 1 h at room temperature 2. Separate the excess reagent by filtration and then wash the nylon-aldehyde with several porttons of the washing solution. 3. Add 10 mL of the enzyme solution and mechanically shake for 12 h at 4°C. 4. Separate the excess enzyme solution by filtration and then wash the immobtlized derivative with two portions of 10 mL of the washing solution. 5. Suspend the immobthzed derivative in washing solutton and store at 4’C until use.

38

Lozano and lborra

3.5.2. By Amidine Group 1. Add the nylon-amine derivative (0.5 g of nylon pellets, 100 mg of nylon powder, 10 cm of nylon tube, or 15 cm2 of nylon membrane, obtained as described in Section 3.4 ), mto a screw-capped test tube, add 10 mL of the crosslinking reagent, and mechanically shake the mixture for 1 h at room temperature 2 Separate the excess reagent by vacuum filtration and wash the nylon-imidate, first with several portions of me-cold methanol and then with 0.M sodium phosphate buffer, pH 7.8, at 4°C (see Note 8). 3. Add 10 mL of the enzyme solution to the test tube containing the nylon-imidoester and shake for 12 h at 4°C. 4 Separate the enzyme solution by filtration and then wash the immobihzed dertvattve with two portions of 10 mL of 0.M sodium phosphate buffer, pH 7.8. 5 Suspend the immobilized derivative m O.lM sodium phosphate buffer, pH 7 8, and store at 4°C until use.

3.5.3. By Azo Group 1. Add the nylon-ammoaryl or nylon-hydrazme derivatives (0 5 g of nylon pellets, 100 mg of nylon powder, 10 cm of nylon tube, or 15 cm2 of nylon membrane, obtained as described m Section 3.4.), mto a screw-capped test tube. Add 4 mL of 4% (w/v) sodium nitrite, then 10 mL of 2M HCl, and mechanically shake the mixture for 1 h at 4’C. 2. Separate the excess reagents by filtration and then wash the nylon-azo derivative with several portions of the washing solution at 4°C 3 Add 10 mL of the enzyme solutton to the test tube containing the nylon-azo derivative and shake for 48 h at 4°C. 4. Separate the excess enzyme solution by tiltratton and then wash the immobilized derivative with two porttons of 10 mL of the washing solution 5 Suspend the immobihzed derivative in washing solution and store at 4°C until use.

3.5.4. By Disulfide Bridges 1. Into a screw-capped test tube containing the nylon-thiol dertvatlve (0.5 g of nylon pellets, 100 mg of nylon powder, 10 cm of nylon tube, or 15 cm2 of nylon membrane, obtained as described in Section 3.4.), add 10 mL of the enzyme solution, then 1 mL of coupling reagent, and shake the mixture for 12 h at 4°C. 2 Separate the excess of the enzyme solution by filtration and then wash the mrnobilized derivative with two portions of 10 mL of the washing solution. 3 Suspend the immobthzed derivative in washing solution until use.

4. Notes 1. All the protocols have been designed for use with nylon pellets, membrane, tube, or powder as support by employmg an excess of reagents m each of the steps. Nevertheless, the total external surface of the nylon support will depend on the chosen form, which increases as follows: powder > pellets > membrane > tube

Immobilization on Nylon

2. 3.

4.

5.

6.

7.

8.

9.

39

Similarly, the increase in quantity of irnmobrhzed protein/g of support follows the same pattern. The support form should be selected to best suit the particular reactor type in which the immobilized derivative will be applied (see Chapter 1). In the case of nylon tube, the enzyme immobilization process is directed onto its internal surface, and may be carried out by recycling the different reagents and enzyme solution with a peristaltic pump. Methanol is a potent poison and should be manipulated with caution The reagent solution will become white during this step, because it dissolves the regions of amorphous nylon, increasing both the available surface area and its wettability. If nylon powder is selected it is not necessary to treat the powder with the surface treatment reagent, because it has the highest available surface area. With nylon pellets or powder, it is very important to maintain the initial amount of nylon support constant during all the wash steps. The high density of nylon pellets allows its direct precipitation, whereas for nylon powder it will be necessary to centrifuge the test tube at 25OOg for 10 min. In both cases, separation of the different reagents can be easily made by a Pasteur’s pipet having a filter placed at the tip to avoid a loss of support. Always weigh the amount of nylon after each step and, particularly, prior to the final enzyme-attachment step. Dimethyl sulfate is a potent poison and a carcmogemc agent, and should be manipulated m an extraction fume cupboard with extreme caution, using gloves and glasses. The triethyloxonium tetrafluoroborate is a toxic, but nonpoisonous reagent As general rule, all the organic solvents and reagents should be manipulated with extreme caution, using the extraction fume cupboard, gloves, glasses, and so on. After treatment wrth dimethyl sulfate, the nylon pellet will compact as a ball, allowing an easy separation of the alkylating reagent. Then, by a vigorous agitation wtth ice-cold methanol in the washing step, the nylon pellets become particulate. Additionally, if the 0-alkylation step has been excessive, the external shell of nylon can be dissolved, extracting nylon powder (visible as a white solution) during this washing step. In this way, the washing step should be continued until white powder is observed m the methanol phase. The optimal pH for the carbonyl-amine reaction is in alkaline conditions (pH 9.2). However, at this pH some enzymes become inactive, and it may be necessary to carry out the enzyme attachment step at a lower alkaline pH (pH 7.8). Thus, enzyme attachment will depend on a compromise between enzyme deactivation and loss of yield in protein immobilized. The property of imidoesters to hydrolyze in aqueous media requires developing the reactivation step in organic medra. However, the enzyme attachment step should be made in aqueous media to minimize the enzyme deactivation, which may result in a reduction in the yield of enzyme immobilized as a consequence of the loss of potentially reactive centers to attach protein onto the support surface The preparation of the p-phenylenediamine solution as well as the chemical modrfication steps with this reagent should be carried out in darkness, because of the high light sensitivity of this compound and rts tendency to polymerize in light.

Lozano and lborra

40

10. All the chemically modttied nylons (i.e., nylon-amine, nylon-ammoaryl, and so on), can be dried or lyophrlized and then stored at room temperature m a desrccator. Nylon-aminoaryl should also be stored m darkness

References 1 Hornby, W. E and Goldstein, L. (1976) Immobilization of enzymes on nylon Met/rods Enzymol 44, 118-l 34. 2. Campbell, J., Hornby, W. N., and Morris, D L. (1975) The preparation of several new nylons tube-glucose oxrdase derivatives and their incorporation into the “reagentless” automated analysis of glucose Brochlm Bzophys Acta 384, 307316 3. Thompson, R. Q,, Mandoke, C S., and Womack, J P. (1985) A procedure for unmobil~zing enzymes on nylon. Anal Lett 18,93-107 4 Canales, I., ManJon, A , and Iborra, J L. (1991) Immoblhzation of P-glucuromdase on pelhcular nylon. Blocatalysls 4, 277-290 5. Lozano, P , Manjon, A., RomoJaro, F., and Iborra, J L (1989) Pectolytic enzymes covalently bound to derivatized nylon for juices clarification, in Advances zn Food Technology, vol. 3 (Pnmo, E and Flto P., eds ), Reproval, Valencia, Spain, pp 2169-2178 6 Lozano, P., ManJon, A., Romojaro, F., and Iborra, J L (1987) Activity of pectolytic enzymes unmobrl~zed to nylon for vrscous juices clarrflcatron. Dependence on covalent attachment method, m Proceedmgs of the 4th European Congress on Blotechnology, vol. 2 (Neijssel, 0. M., van derMeer, R. R., and Luyben, K. Ch A. M., eds.), Elsevier, Amsterdam, pp 52-55 7. Sundaram, I? V. (1976) Coupled and multienzyme-nylon tube reactors, in Enzyme Engzneenng, vol. 3 (Pye, E K. and Wingard, L D., eds.), Plenum, New York, pp 133-138. 8. Iborra, J. L , Castellar, M. R., Canovas, M., and Man@. A. (1992) Prcocrocm hydrolysis by immoblhzed P-glucosidase Biotechnol Lett 14,475-480 9. Obon, J. M., ManJon, A., Canovas, M., and Iborra, J. L. (1990) Amon exchange nylon laminated membranes for enzyme immoblhzation Bzotechnol Tech 4, 357-362.

IO. Onyzrli, F. N. (1987) Glutaraldehyde activation step m enzyme munobilization nylon. Biotechnol Bioeng. 29,399-402

on

Visual Assessment of Enzyme Immobilization Arturo Manjdn and Josh L. lborra

1. Introduction Immobilization of enzymes is a basic technique that is well established in very different fields of both research and industry. The actual knowledge of the basic principles for immobilizing enzymes is so broad that one can design the immobilized enzyme to accomplish a given task or to show properties that are the most suitable for a given application (1,2). However, for most circumstances,the successor failure of an immobilization process can only be checked once the immobilization process is complete, because the physical appearance of the support material does not change during the immobthzation process. Thus, it is very difficult (unless appropriate chemical or physicochemical methods are used at every intermediate step) to know if the support has been adequately activated, the spacer arm was successfully attached to the support, or the protein was bound to the spacer arm. Therefore, a method for enzyme immobilization in which the different steps of the process can be assessedby changes in color of the derivatives would be a useful check, in the first instance, of the successor failure of the immobilization procedure (3). The supports used have different chemical natures, although all are basically hydrophilic-based. The characteristics, properties, and derivattzation methods of the supports used here have been discussed in Chapters l-4 of this book. The activation steps used in this chapter were chosen to enable simple and reproducible methods that allow the generation of a chemical function capable of reacting wrth an amino group from p-phenylenediamine. Controlled-pore glass (CPG) is composed of nearly pure quartz glass. Silanization of glass renders an amino derivative (4,5), and glutaraldehyde is used to establish a bridge between the amino groups of the amino-glass and of From

Methods m Botechnology, Vol 1 Immoblzatron of Enzymes and Cells Edlted by G F BIckerstaff Humana Press Inc , Totowa, NJ

41

42

Manjh

and lborra

p-phenylenedramine. The labile Schiff’s base bonds formed are reduced with borohydride and an aminoaryl-glass is obtained. Bare glass has been reported to have a negative influence on the activity of some immobilized enzymes because of the interaction between enzyme active site amino acids and functional groups on the glass surface. To mmlmaze these influences, the bare glass 1s coated with a hydrophilic nomomc carbohydrate (glyceryl groups) monolayer that effectrvely reduces the mteractron of the support with the protem, leaving unchanged the basic properties of the glass as a support (6). Glycophase-coated CPG, Sephadex, Sepharose, and cellulose are all acttvated by a partial oxtdatton of vicmal hydroxyl groups to form aldehyde groups (3,612). These can form a SchtfYs base lmkage with an amino group ofp-phenylenediamine, and the free reversibility of this interaction is blocked by reducing the formed linkage with borohydride

(3,942).

Nylon is a linear polymer consisting of repeating assemblies of methylene groups joined by secondary amrde lmkages. The number of methylene groups in the repeating alkane segments and the occurrence of amrde groups decides the type of nylon and its hydrophilrc/hydrophobic balance. Nylon can be acttvated by partial hydrolysis of its amide groups. This renders both free carboxylate and ammo groups that can be used forp-phenylenedlamine coupling. When the amino groups are used, the process for p-phenylenedramme attachment IS the same as that used for the amino-glass (then, negatively charged ammoarylnylon is obtained). Alternatively, carboxylate groups can be easily activated by a water-soluble carbodrimide, and the resulting activated carboxylate IS able to form an amide linkage with one of the amino groups ofp-phenylenedtamine (and, thus, positively charged aminoaryl-nylon is obtained) (23,14). Weak cation exchangers are a group of supports having m common carboxylate groups in a basic structure that chemtcally IS either a polyacrylic (Lewattt CNP 80) or a polysaccharide (CM-cellulose, CM-Sephadex) support. All weak cation exchange resins can be derivatized to acrylamine dertvatrves by following the same reaction scheme as for nylon when forming amide linkages between the support carboxylates and the amino group on the p-phenylenedramine, i.e., carboxylate activation by a water-soluble carbodnmtde and amide bond formation (7,8). Eupergit C is a sphertcal macroporous bead polymer of methacrylamtde, NJV-methylenebis(methacrylamide), glycidyl methacrylate, and/or ally1 glycrdyl ether. The oxirane groups of the glycidyl radical are chemically reactive and form covalent bonds with compounds containing amino, mercapto, or hydroxy groups, m which there is no change in the electrochemical character of the bound ligand. The chemical nature of thts materral ensures a htgh degree of chemical stability of the matrix and the bound hgands. Eupergrt C surface chemistry allows direct converston of the support to an ammoaryl derivative

Assessment of lmmobilrza tion

43

by simply placmg the support m contact withp-phenylenediamine. The 0x1rane groups react spontaneously with the amino groups ofp-phenylenediamine, resulting in a stable secondary amine bond (25-18). The formation of ammoaryl derivatives on a colorless support provide a change m the original color of the support to a red-brick color when p-phenylenediamine 1scovalently coupled to the support. The intensity of the color depends on the amount ofp-phenylenediamine coupled, so a visual assessment(with a bit of practice) can be made on the level of derivatization of the support (Fig. 1). Enzymes can be munobihzed to aminoaryl-derivatized supports by a variety of methods. However, to enable a color change during the attachment process, activation of the aminoaryl groups to a diazonium salt 1s undertaken, and the activation is accomplished by treatment with nitrous acid. Hydrochloric acid is added first, and the derivative color changes to yellow, then to dark red when sodium nitrite 1s added and the formation of the dlazonium salt takes place. Enzyme coupling takes place mainly through the tyrosyl residues of the protein (1,19,20), thus extending the coqugation of the aryl group and resulting in a strengthening of the ammoaryl derivative color. The intensity of the red-brick color is related to the amount of coupled enzyme.Again, with some experience, It IS possible to make an assessmentof the enzyme loading. Finally, the dark red-brick color becomes more intense, even dark brown or nearly black, when the unreacted diazo groups are blocked with phenol (3,20-12) (Fig. 1). 2. Materials

2.1. Formation of an Aminoaryl Derivative 2.1-l. From Glass 1. CPG. 2. y-aminopropyltnethoxysilane: 1% (v/v) in double-dtstilled water. 3. 6N HCI solution. 4. O.lM sodium bicarbonatebuffer, pH 9.0. 5. Glutaraldehyde:2.5% in O.lMsodium bicarbonatebuffer, pH 9.0. 6 p-Phenylenediamine-buffered solution: 20 mg/mL in 0. 1M sodium bicarbonate buffer, pH 9.0. 7. Sodium borohydride (solid). 2.1.2. From Glycophase-Coated and Celullose

CPG; Sephadex, Sepharose,

1. Glycophase-coatedporous glass (Pierce [Rockford, IL] or Sigma [St. LOUIS, MO]) and cellulose (Sigma). 2 Sephadexand Sepharose(Pharmacla,Uppsala, Sweden). 3 6 mA4sodium periodate solution. 4. 0.M

sodmm bicarbonate buffer, pH 9.0.

Manjdn and lborra SUPPORT

Colo~~~Iess or white

Activatron

i

Red brick Yellow

HCI

I

SUPPORT

Rea brick

OH

c HO

Dark red buck / Brown

HO

Brown / Dark brown

Fig. 1. Scheme to produce an ammoaryl-derivative support for enzyme unmobllization with indication of color changes associated with each step in the process

45

Assessment of Immobilization 5. p-Phenylenediamine-buffered buffer, pH 9 0. 6 Sodium borohydride (solid)

solution: 20 mg/mL in 0. IA4 sodium bicarbonate

2.1.3. From Nylon 1. 2 3. 4. 5. 6 7

Nylon (Zurrcher Beuteltuchfabrrk, Zurich, Switzerland). 18 6% (w/v) CaCl*, 18 6% (v/v) double-distilled water m methanol Methanol. HCl solutions: 0. lM, 4N. Glutaraldehyde: 2.5% in 0. 1M sodium bicarbonate buffer, pH 9.0. 0 1M sodium bicarbonate buffer, pH 9.0. p-Phenylenedramine-buffered solution: 20 mg/mL m O.lM sodium bicarbonate buffer, pH 9.0. 8. p-Phenylenediamine aqueous solution: 50 mg/mL in double-distilled water, 9 Sodium borohydride (solid). 10. 1-Ethyl-3(3-dimethylaminopropyl)carbodumide hydrochloride. 2 mg/mL m double-distilled water.

2.1.4. From Cation Exchange Resins 1 2. 3 4. 5 6.

CM-Sephadex (Pharmacia). CM-Cellulose (Sigma). Lewatit CNP 80 (Bayer, Switzerland). 0.05MNaOH solution. 0.05M HCl solution. hydrochlorrde: 2 mg/mL 1-Ethyl-3-(3-dimethylammopropyl)carbodiimrde double-distilled water. 7. p-Phenylenediamme aqueous solution: 50 mg/mL in double-distilled water.

in

2.1.5. From Oxirane-Acrylic Beads Containing Epoxy Groups 1. Eupergit C (Riedel-de Haen, Seelze, Germany) 2. O.lM Sodium phosphate buffer, pH 7.0. 3. p-Phenylenediamine-buffered solution: 20 mg/mL in O.lA4phosphate pH 7.0.

2.2. Activation of the Aminoaryl and Enzyme Immobilization 1. 2. 3. 4.

buffer,

Derivative

Ice-cold 2M HCl solution. Ice-cold NaNOz solution: 4% (w/v) in double-distilled water Enzyme: usually 5-l 0 mL of a 1 mg/mL solution in coupling buffer. Coupling buffer: buffer in which the enzyme to be coupled 1s active and stable. 5. 0.01% (w/v) phenol m 10% (w/v) aqueous sodium acetate solution.

Manj6n and lborra

46

3. Methods (see Notes 1-5) 3.1. Formation of an Aminoaryl 3.1.1. From Glass (see Note 6)

Derivative

1 Weigh 200 mg of CPG and mtx tt wtth 20 mL y-aminopropyltriethoxystlane

solution. 2 Adjust the pH of the mixture to 3.0-4.0 with 6N HCl. 3 Let the reaction to proceed for 2 h m a water bath at 75°C (4) 4. Wash the derivattzed support with 500 mL double-distilled water and 100 mL 0.M sodium btcarbonate buffer, pH 9.0. 5 Add 20 mL of the glutaraldehyde solution in sodium bicarbonate buffer and allow It to react for 30 mm at room temperature 6. Wash the aidehyde-glass with sodium bicarbonate buffer, 7 Add 10 mL of the p-phenylenedlamme-buffered

solution and allow the mixture

to react 8. At 20 and 40 mm of reactron, add 0 5 mg of NaBH4 and then stop the reaction after 1 h. 9 Wash the aminoaryl-glass derivative exhaustively wtth water.

3.7.2. From Glycophase-Coated and Cellulose (see Notes 7,8)

CPG, Sephadex, Sepharose,

1. Weigh 150 mg of the support and place tt in a 15 mL screw-capped test tube. 2. Add 10 mL of 6 mA4NaI04 and apply vacuum for several minutes to remove the au trapped within the matrix 3 Rotate the tube end-over-end on a mechanical stu-rer for 1 h at room temperature 4. Wash the aldehyde derivative so obtained wtth 500 mL double-dtsttlled water and 100 mL 0 1Msodmm bicarbonate buffer, pH 9.0. 5. Add 10 mL of the p-phenylenediamme-buffered solutron and allow the mixture to react. 8. At 20 and 40 mm of reaction, add 0.5 mg of NaB& and stop the reaction after 1 h. 9 Wash the aminoaryl derivative exhaustively

with water.

3.1.3. From Nylon (see Notes 9-l 1) 3.1.3.1 NEGATIVELY CHARGED AMINOARYL NYLON 1. Treat the support first with a mtxture of 18.6% CaCl, + 18 6% HZ0 in methanol for 20 min at 50°C 2. Wash the support with 100 mL of 0 1M HCl. 3 Treat the nylon support with 10 mL of 4N HCl for 30 mm at 40°C

4 Wash thoroughly the partially hydrolyzed nylon with double-distilled water. 5. Add 20 mL of the glutaraldehyde solutron in sodium btcarbonate buffer and allow to react for 30 min at room temperature. 6 Wash the aldehyde nylon with 0. 1M sodium bicarbonate buffer, pH 9 0. 7 Add 10 mL of the p-phenylenedtamme-buffered solution and allow the mixture to react.

Assessment of Immobilization

47

8. At 20 and 40 min of reaction, add 0.5 mg ofNaBH,, and stop the reaction after 1 h 9. Wash the ammoaryl nylon derivative extensively with water. 3.1.3.2.

POSITIVELY CHARGED AMINOARYL-NYLON (SEE NOTE 12)

1. Treat the support first with a mixture of 18.6% CaC12 + 18.6% Hz0 in methanol for 20 mm at 50°C 2. Wash the support with 100 mL of O.lMHCI. 3. Treat the nylon support with 10 mL of 4N HCl for 30 min at 40°C. 4. Thoroughly wash the partially hydrolyzed nylon wrth double-distilled water 5. Add 2 mL of the carbodiimide solution, mix, add 5 mL of thep-phenylenediamine aqueous solution after 5 mm. and allow the reaction to proceed for 3 h at room temperature or overnight in a cold room (always use end-over-end agitation). 6. Wash the ammoaryl derivative formed exhaustively with water.

3.1.4. From Weak Cation Exchange Resins (CM-Cellulose, CM-Sephadex, Lewatit CNP-80; see Note 12) 1. Weigh 200 mg of support. 2 Wash thoroughly with 0.05M NaOH, then 0 05M HCI, and finally with doubledrstrlled water. 3. Add 2 mL of the carbodumrde solution, mix, add 5 mL of thep-phenylenediamme aqueous solution after 5 min. and allow the reaction to proceed for 3 h at room temperature or overnight in a cold room (always use end-over-end agttatton) 4 Wash the aminoaryl derivative formed thoroughly with water.

3.1.5. From Oxyrane-Acrylic Beads Containing Epoxy Groups (Eupergit C; see Note 13) 1. Weigh a quantity of 200 mg of Eupergit C and place it in a screw-capped test tube. 2. Add 2 mL of the p-phenylenediamine-buffered solution and mcubate at room temperature with end-over-end mechanical stirring for 1 h 3. Add another 2 mL of the p-phenylenediamme solution and continue the incubation for a further 2 h. 4. Wash thoroughly the aminoaryl derivative with double-distilled water and coupling buffer 3.2. Activation of the Aminoaryl Derivative and Enzyme Immobilization (see Notes 14-21) 1. Add 10 mL of ice-cold 2M HCI solution to the aminoaryl derivative in a screw-capped test tube. Allow the components to mrx for 5 min. 2. Add 4 mL of ice-cold 4% NaNO* solution, close the tube, mix carefully, and allow the mixture to react for 20-40 mm in a cold room (3-5°C) with end-overend mechanical agitatron or m an Ice bath with frequent end-over-end stu-ring. 3. Wash exhaustively the drazo derivative with double-distilled water and couplmg buffer.

48

Manjch and lborra

4 Add S-10 mL of the enzyme solution and allow the reactlon to proceed for up to 48 h m a cold room under end-over-end mechanical stlrrmg 5. Recover the supernatant (keep it for protem/activlty measurements). 6 Wash the derivative with three IO-mL portions of coupling buffer (keep these washings for protein/activity determinations). 7 Block the unreacted diazo groups by reaction with 10 mL of phenol m aqueous sodium acetate solution for 15 mm at room temperature. 8 Wash the nnmoblhzed enzyme derivative extensively with couplmg buffer. 9 Store the nnrnobilized enzyme derivative suspended m couplmg buffer m a cold room.

4. Notes 1. The method has been applied to supports that were mltlally colorless or white, although it would be applicable to any other type of support in which the origmal color would not mask the yellow and different tones of red and brown colors that are developed along the proposed protocols, 2 Lewatlt CNP 80 has a cream color, and it is possible to follow the color changes caused by derivatlzation, activation, couplmg, and blocking steps 3 The process steps to provide color changes for monitoring a given sequence of support derlvatlzation to an ammoaryl derivative, and activation of this later to a diazonium salt m enzyme coupling do, however, llmlt the range of methods used to immobilize enzymes covalently to the support. This means chemical modification of the enzyme involves (mainly) the tyrosyl residues of the enzyme, which are the least frequently available of the amino acid residues on the surface of proteins. 4 The supports chosen and support derlvatlzatlon schemes followed are simple processes to achieve an ammoaryl-denvatlzed support able to couple an enzyme through dlazo covalent bonds. Several other white or colorless supports allow for a chemical derivatlzation to an ammoaryl derivative Additionally, the denvatlzatlon steps can follow a different scheme to obtain llgand (aminoaryl group) arms, the length and chemical nature of which can be adapted for the reqmrements of any particular enzyme that would be unmoblllzed. 5 Magnetic stirring with Teflon-coated magnetic bars 1snot allowed for all supports, except nylon, since the support 1sground by the movement of the magnetic bar. 6. Bare glass can be, alternatively, derlvatized to an ammo-glass by refluxing the glass support overnight m benzene-containing y-ammopropyltriethoxysilane. Periodic acid (m double-distilled water), mstead sodium periodate, can also be 7. used for glycophase-CPG, Sephadex, Sepharose, and cellulose activation to an aldehyde derivative. 8. The couplmg of amine-containmg ligands to the aldehyde supports can be performed in the pH range of 7.5-9.0, depending on the pH stability of the hgands 9. Nylon is treated first with CaC12 plus Hz0 in methanol. This process etches the surface of the nylon by dlssolvmg out the regions of amorphous nylon, thereby increasing both the available surface area and its wettablhty.

Assessment of Immobilization

49

10. The extent of this treatment on nylon determines the degree of roughness of the nylon surface and the capacity of the nylon to immobilize enzyme. More etching will result in more enzyme immobilized. However, more etching of the nylon surface means also less mechanical resistance of the nylon support. Thus, it may be necessary to attain a compromise between immobilized enzyme capacity and mechanical strength of the support 11. The level of nylon hydrolysis by HCl also influences the relationship between amount of enzyme bound/mechanical strength of the support. More hydrolysis provides more immobilized enzyme capacity, but less mechanical strength. 12. p-Phenylenediamine coupling to a carbodiimide-activated carboxylate group can be done under a wade set of condttions. Various buffers, typically 0. IM concentration and a range of pH values between 6.0 and 9.0, have been successfully used in place of double-distilled water. 13. p-Phenylenedtamme bmding to Eupergtt C can be done under very easy conditions, since the value of pH and the ionic strength of the buffer solution for binding can be varted over a wide range (values of pH 1.0-12.0, concentratron 0.01-l ,OA4)to obtain the optimal conditions for the binding of a particular ltgand. A temperature between 5 and 30°C can be selected accordmg to the stability and reactivity of the ligand. 14. Diazomum compounds react extremely well with tyrosyl residues of proteins, although lysyl, argmyl, and histidinyl residues can also be involved m the linkage. 15. Nitrous acid (HCl + NaN02) 1sused to activate ammoaryl derivatives However, the activation process works better if HCl is added first (this additionally origtnates a color change of the ammoaryl derivative to yellow) and NaN02 is added about 5 min after HCl addition. 16 NaNOz must be freshly prepared for each activation. Additionally, the solid NaNOz must be stored m a desstcator to avoid decompositton. 17. Aminoaryl derrvattve activation by nitrous acid is accompanied by high evolution of bubbles. This is indicative that the activation process is proceeding properly. 18. Both HCl and sodium nitrite solutions must be ice-cold before use and the activation reaction must proceed under cold conditions; otherwise, poor results will be obtained 19. It is recommended that the ammoatyl derivatives be used within a short time or lyophtlized In the latter state they will be stable for months. However, if the ammoaryl derivative is kept suspended m buffer, it will decompose (with accompanying inability to be activated and to link proteins), and the original color will change to violet, the intensity of this color change being more noticeable with time. 20. Data for tyrosinase and P-galactosidase showed that enzyme coupling after 8-10 h of reaction reached approx 85% of the enzyme coupled after 40 h of coupling reaction. Thus, enzyme coupling reaction lasting overnight m a cold room would result in an immobilized enzyme derivative with activny usually adequate for charactertzatton.

Manjh

50

and lborra

21. When enzyme 1s immobihzed on the inner surface of nylon tubing having a l-mm wall thickness or thicker, shces of the tubing can be cut and observed under a stereo magnifier or microscope. This would allow the depth of support etching/chemical modification to be assessed. 22. Safety precautions: p-Phenylenediamme IS toxic and may produce eczematold contact dermatitis or bronchial asthma. Avoid skm contact and inhalation Sodium borohydride is toxic and may cause inJury to liver, kidneys, and the central nervous system. Phenol 1s poisonous and caustic. Do not handle with bare hands; wear gloves. I-Ethyl-3-(3-dimethylammopropyl)carbodnmide is harmful by inhalation, in contact with skin, and if swallowed. It is also nritatmg to eyes, respiratory systems, and the skin.

References 1. Scouten, W. H (1987) A survey of couplmg techniques. Methods Enzymol 135, 30-65. 2. Katchalski-Katzir, E. (1993) Immobilized enzymes-learning from past successes and failures. Trends Biotechnol 11,47 L-478. 3. ManJon, A., Bonete, M. J., Llorca, F. I., Jimeno, A., and Iborra, J. L. (1987) A visual-practical method for following the immobilization of biomolecules. Bzochem Educ 15, 85,86.

4 Weetall, H. H (1970) Storage stability of water-insoluble enzymes covalently coupled to organic and morgamc carriers. Biochzm. Bzophys Acta 212, l-7 5. Weetall, H. H. (1976) Covalent coupling methods for inorganic support materials Methods Enzymol 44, 134-148. 6. Pierce General Catalog, Rockford, Illmois (1976) Glycophase GTM 7. Porath, J. and Axen, R. (1976) Immobilizatton of enzymes to agar, agarose, and Sephadex supports. Methods Enzymol. 44, 19-45. 8. Lilly, M. D. (1976) Enzymes immobihzed to cellulose. Methods Enzymol 44,46-53 9. Vilanova, E., Manjon A., and Iborra, J. L. (1984) Tyrosine hydroxylase activity of immobilized tyrosinase on Enzacryl AA and CPG-AA supports. Stabilization and properties. Biotechnol Bzoeng 26,1306-l 3 12. 10. Manjon, A., Llorca, F. I., Bonete, M. J., Bastida, J., and Iborra, J L. (1985) Properties of P-galactosidase covalently nnmobilized to glycophase-coated porous glass Process Bzochem 20, 17-22. 11. Manjon, A , Bastida, J., Romero, C., Juneno, A., and Iborra, J. L. (1985) Lmmobihzation of naringinase on glycophase-coated porous glass. Biotechnol. Lett. 7,477-482. 12. Borrego, F., Tart, M., Manjon, A., and Iborra, J. L. (1989) Properties of pectmesterase munobthzed on giycophase-coated controlled-pore glass. Appl. Bzochem Biotechnol 22, I-10 13. Hornby, W. E. and Goldstein, L. (1976) Immobilization of enzymes on nylon. Methods Enzymol. 44, 118-134. 14. Berenguer, J. J., Manjon, A., and Iborra, J. L. (1989)ApH-tyrosinase biosensor for ammoacids, catecholammes and adrenergic drugs determination. Biotechnol Tech 3,211-216.

Assessment of Immobilization

51

15. Kramer, D. M., Lehmann, K., Pennewis, A., and Plainer, H. (1978) Oxirane-acrylic beads, Preparation 2878-C. Enzyme Eng. 4, 153,154. 16. Burg, K , Mauz, 0 , Noetzel, S., and Harnish, K. (1988) New synthetic carriers for enzyme couplmg, Die Angewandte Makromolekulare Chemle 157, 105-l 2 1. 17 Wehnert, G., Sauerbrei,A , and Schtigerl, K. (1985) Glucose oxidase immobihzed on Eupergit C and CPG-10. A comparison Biotechnol Lett 7, 827-830. 18. Canales, Y., ManJon, A., and Iborra, J. L (1990) Immobilization of P-glucuronidase on an epoxy-activated polyacrylic matrix. Blotechnol. Tech 4,205-2 10. 19. Goldman, R Goldstein, L., and Katchalski, E. (1971) in Biochemical Aspects of Reactions on Solid Supports (Stark, G. R., ed.), Academic, New York, pp. 1-78 20 Srere, P and Uyeda, K. (1976) Functional groups on enzymes suitable for bmding to matrices Methods Enzymol 44, 1l-l 9

Immobilization

in Carrageenans

Josh L. Iborra, Arturo Manjbn, and Manuel Ctinovas 1. Introduction Immobilization in carrageenans is amethod of gel entrapment. It is one of the most widely used methods for cell immobilization because it is cheap, simple, and reproducible with mild conditions during the immobilization. The major advantages of the entrapment method are high cell density, mild immobilization conditions, and low risk of loss of cells from the support (see Chapter 1). Immobilization in carrageenans is carried out in a similar way to that for alginate (see Chapter 7). The principle of gel entrapment is that biocatalysts are mixed with a pregel solution, and after gelification the biocatalysts are enclosed in the gel material. The carrageenan concentration depends on the type of carrageenan used, but has to be sufficient to produce a firm gel. The mixture of biocatalyst and carrageenan solution is extruded dropwise through an orifice (extrusion method) or hollow needle (dripping method), or is dispersed in liquid or air (dispersion method). Various shapes(cubes, beads, or membranes) of immobilized biocatalysts can be tailored for particular applications, e.g., spherical beds shown good flow properties in a packed-bed reactor. Gelification of carrageenantakes place when the solution is cooled down to the appropriate gehfication temperature, but only in the presence of gel-inducing agents, usually cations, such as K+. Generally, enzyme activities and yields of immobilized cells are relatively high. Gel formation is thermally reversible and gels may soften or disintegrate at elevated temperatures, and consequently are not well suited to applications mvolvmg higher temperature reactions, such as those employing thermophilic microorganisms (I). Another disadvantage of carrageenan 1sthat when a gel-inducing reagent is not present in the reaction mixture, dissolution of the gel will occur and biocatalysts are released.Also, the gel-inducing reagent may inhibit desirable enzyme activity (2). From

Methods m k?~otechnotogy, vat 1 tmmobrluafron of Enzymes and Ce//.s Edlted by G F Bfckerstaff Humana Press Inc , Totowa, NJ

53

54

Iborra, Manjdn, and Cinovas

The dripping method of immoblhzation is the easiest procedure for bead formation at laboratory scale, is used routinely for cell immobilization, and will be used m the work described m this chapter. The aqueous gel solution is pressed through a syrmge at a low flow rate and droplets are formed at the tip of the needle. Droplets of a more uniform size can be obtained when an air flow around the needle is applied. By thts procedure, a large number of cells are homogeneously munobilized in carrageenan gel. The cells may be growing, resting, or in an autolyzed state. The pore size of the gel matrix is small enough to prevent enzymes from leaking out from the gel lattice, whereas substrates and products easily pass through the gel wall. Figure 1 shows a scanmng electron microscopy picture of the surface of a carrageenan bead before (A) and after (B) cell immobilization (3). The large-scale production of gel beads is achieved by two mam procedures, i.e., extrusion techniques and dispersion of the gel solutlon in liquid or air. In the extrusion technique, the aqueous gel solution is pressed at such a high flow rate through a small orifice that aJet is formed (4,.5). With a membrane, a sinusoidal vibration of a certain frequency is transferred to the liquid. This signal will cause the break-up of the jet in uniform droplets. A loo-fold increase m flow rate compared to the dripping method can thus be obtained, with a much larger production for immobilized biocatalyst, and another benefit is the production of uniform droplets. In the dispersion method, an aqueous K-carrageenan solution is dispersed in soybean oil and 1shardened by lowering the temperature and subsequently soaking the beads in potassium chloride (6). The reproducibllity of the droplet formation and the uniformity in size attained by the dlspersion method are considerably lower than those for the extrusion technique. 2. Materials 1. K-Carrageenancan be of commercialgradeblendedfrom varrous seaweeds,contaming low amountsof h-carrageenen,or pure gradefrom a single speciesforming very rigld gels (seeNotes 1,2) 2. 0.3M potassiumchloride solution in distilled water 3 0.18M calcium chloride solution in distilled water (seeNote 3) 4. 0.9% (w/v) NaCl physlologlcal saline solution. 5. Simple equtpment for the dripping method includes a peristaltic pump, two thermostatedstirred reservoirs,andanHPLC conventional needle*length 51mm, outer diameter 0.47 mm, inner diameter 0 13 mm (seeNotes 4-6) 3. Methods 3.1. Preparation of the K-Carrageef?an 1. Dissolve completely the K-carrageenanin deionized water (3 g/100 mL) at the desired concentrationat 50°C (seeNotes 7-9) 2. Centrifuge the solution or apply vacuum to remove an bubbles.

Entrapment in Carrageenans

Fig. 1. Scanning electron microscopy pictures of the surface of a K-carrageenan bead (A) without cells and (B) with immobilized Rhodococcusfascians cells after 23 d of continuous operation in nutrient broth. Cells were immobilized into 2% K-carrageenan and the resulting beads had a biomass concentration of 1.4 g/L and an average diameter of 2 mm. The bar shown on the pictures represents 10 p.

Iborra, Manjh,

56

and Cdnovas

3. Sterilize at 110-12 1°C for 1 h if the solution is to be used for cell immobihzation. 4 Cool the solution to lO.O. More recently,

we have shown that there are several advantages for tosyl chloride activation over the CD1 and CNBr methods (3), As compared to CNBr activatton, tosyl chloride activation does not require vigorous mixing and it is much safer. The tosyl chloride-activated rayon/polyester cloth has the capacity to immobilize at least three trmes more bovine serum albumm (BSA) than CNBr or CDIactivated cloths, and is able to efficiently immobilize low concentrations of proteins. Furthermore, the linkage formed is stable under alkaline condttlons

(e.g., pH 11.O). It has been shown that antibodies and enzymes (e.g., glucose oxidase, glucoamylase, and invertase) may be immobilized onto tosyl chloride-activated rayon/polyester cloth with no apparent loss of their activities. Tosyl chloride activation is considerably less expensive than CNBr and CD1 activation and much less expensive than tresyl chloride acttvatton (5).

This chapter describes the optimized procedure for tosyl chloride activation of rayon/polyester

cloth and its subsequent use for protein immobilization.

2. Materials 1. Nonwoven rayon/polyester (70/30%) blend cloth (Sontara 8423, DuPont, Boston, MA). 2 10% (w/v) NaOH solution made fresh each day. 3. 10% (w/v) p-toluenesulfonyl chloride (tosyl chloride) (Sigma, St. Lotus, MO) m acetone. The tosyl chloride solution may be stored for long pertods of time provided that it is kept sealed and free from moisture. Care: tosyl chloride is corrosive and is a lachrymator. Be sure to wear gloves and a face mask when handling tosyl chloride 4. Vacuum filter apparatus. 5. PBS: O.OlM sodium phosphate buffer, pH 7.3, containing 0.85% NaCI. 6. PBST: Prepare PBS containing 0 05% (v/v) Tween 20. 7. Blocking buffer Prepare PBS contaming 0.5% (w/v) nonfat skim milk and 1% (w/v) glycine m PBS.

Immobilization on Rayon/Polyester

79

3. Method 1 Cut rayon/polyester cloth mto sizes (squares or disks) appropriate for the application in mind (see Note 1). Calculate the area of the segments in cm2 2 Place the rayon/polyester cloth segments in the 10% NaOH solution (use about 1 mL/cm2 of cloth) and mix gently for 10 min at room temperature to ensure even mixing (see Note 2). 3. Wash the rayon/polyester cloths three times with distilled water followed by five times with acetone under suction (see Note 3). Be sure that the cloths are mtxed in each wash solution prior to the removal of the solution via suction, 4. Immediately transfer the acetone-washed cloths while still wet (see Note 4) to the 10% tosyl chloride solution (use approx 1 mL/cm2 of cloth) and mix gently for 10 mm at room temperature for tosyl chloride activation 5. Wash the tosyl chloride-activated rayon/polyester cloths five times with acetone followed by three times with distilled water under suction. 6. The tosyl chloride-activated rayon/polyester cloths are now ready for protein immobilization. The immobilization procedure may be carried out immediately or the cloths may be stored for immobilization at a later time (see Note 5). 7. Dissolve the protein to be immobilized in a suitable buffer (e.g., PBS). Incubate the tosyl chloride-activated rayon/polyester cloths in the protein solution for 20 min at room temperature (see Note 6). 8 Wash the cloths five times with PBST to remove noncovalently bound protein. 9. If the tosyl chloride-activated rayon/polyester cloth is to be used for immunoassays or imrnunoaffrmty purification, it should be treated to prevent nonspecific bmding following the nnmobihzation of the protein ligand (see Note 7). 10. After washing the cloths five times with PBST, the cloths are ready for use The mrmobilized protem-rayon/polyester cloth may be stored for extended periods of time in PBS containing a suitable preservative (e.g., 0.01% [w/v] benzalkomum chloride) that does not affect the immobilized protein The stability of the irnmobillzed protein is the limitmg factor in the potential storage time.

4. Notes 1, If the nnmobilized protein is to be used in a packed column, then it may be useful to cut the rayon/polyester cloth into disks that match the inside diameter of the column. For batchwise use, tt may be preferable to cut the cloth into sizes that can be easily mixed within the reaction chamber. 2. Alkaline treatment of the rayon/polyester cloth is required to disrupt hydrogen bonding between the hydroxyl groups of the rayon, thereby permitting the subsequent activation reaction between the rayon’s hydroxyl groups and the tosyl chloride. If the NaOH treatment is not performed there is a significant decrease m protein immobilization (e.g , a sixfold decrease in BSA immobilizatton was observed when the alkali treatment was omitted). 3. Prior to tosyl chloride actrvation, it is essential that the cloths be thoroughly dehydrated wrth acetone since the presence of water can severely inhabit the reaction between the hydroxyl groups of rayon and the tosyl chloride. Dehydration of

80

4

5.

6.

7.

Yamazaki and Boyd tradttional supports for protein tmmobiltzatton (e.g., crosslmked agarose) are performed using a sequential washing with increasing ratios of acetone:water mixtures to ensure the stability of the support (6). With rayon/polyester cloth (segment approx 1 cm2), direct transfer of the cloth from water to acetone is possible with httle adverse effect on the structure of the cloth. Drying of the acetone-washed cloths prior to their transfer to the tosyl chloride solutton will be accompanied by a substantial drop in protein immobilization (e.g., a sixfold decrease for BSA). This probably results from the reformation of hydrogen bonds wtthm the rayon fibers during drying. Prior to the addition of the protein solution to the tosyl chlortde-activated rayon/ polyester cloth, the cloths may be partially dried via blottmg or vacuum suction Because the tosyl groups are covalently lurked to the rayon/polyester cloth it is possible for the activated cloths to be stored for extended periods of time at 4°C in an appropriate buffer (e.g., PBS). If the tosyl chloride-activated rayompolyester cloth is allowed to completely dry before protein immobilization, there will be a significant decrease in the binding capacity of the cloth. To saturate the tosyl chloride-activated rayon/polyester cloth with protein, yet mimmize the amount of excess protein lost, a preliminary experiment may be necessary to calculate the loading capacity of the cloth for that particular protein As a guideline, we found that at least 25 mg of BSA could be immobilized onto 1 g of tosyl chloride-activated rayon/polyester cloth. If only a small number of cloth segments are being treated with the protein solution it may be most convenient to apply the protein solution to the cloth with the use of a pipet (try adding approx 100 yL of the protein solution/cm2 of cloth). For large numbers of cloth segments and/or if the cloth segments are of a large size, incubation of the cloths in the protein solution within a beaker or some reaction vessel may be more practical, If the molecule to be purified is primarily hydrophobic m nature (e.g , unmunoglobulins), then it IS possible to block nonspecific binding efftctently by mcubatmg the cloths in a protein blocking solution containing glycme (e.g., PBS contammg 0.5 % [w/v] nonfat skin milk and 1% [w/v] glycme) for 1 h at room temperature. The nonfat skim milk will prevent the hydrophobic interaction of the molecule to be purified with the tosyl chloride-activated rayon/polyester cloth as well as to block, in conjunction with glycme, any unreacted tosyl sites on the cloth. When purifying molecules that have a strong hydrophthc character, such as glycoprotems (e.g., horseradish peroxidase [HRP]), high degrees of nonspecific adsorptton may occur as a result of hydrogen bonding between the molecules to be captured and rayon fibers. Typical protein blocking agents seem unable to prevent thts nonspecific adsorption. If the cloth is allowed to completely an dry prior to being subjected to the hydrophilic molecules to be purified, then nonspecific adsorption IS dramatically reduced (e.g., there was a greater than lo-fold decrease in the nonspecific adsorption of HRP followmg drying of rayonlpolyester cloth to which anti-HRP antibody had been immobilized). During drymg, the hydroxyl groups of the rayon fibers resume their interaction with one another through hyurogen bonding, thereby mhibttmg their ability to hydrogen bond with

Immobilization on Rayon/f o/yes ter

81

the molecule to be captured Once hydrogen bonding between the rayon fibers has resumed, the cloths may be rehydrated in PBS and then blocked with the nonfat skim milk and glycme mixture m PUBS (this blocking condition is unable to disrupt the hydrogen bonding between the rayon fibers). The drying of tosyl chloride-activated rayon/polyester cloth has shown no adverse effects on the abtlity of various tmmobrlized ligands (e.g., different immunoglobulms and protein antigens) to react to then complementary molecule. A trial experiment will probably be necessary to determine whether drying of the cloths prior to the addrtron of the molecule to be captured is required.

References 1. Janson, J (1984) Large-scale affinity purification-state of the art and mture prospects. Trends Blotechnol 3,23-26. 2 Howlett, J. R., Armstrong, D. W, and Yamazaki, H. (1991) Carbonyldtimidazole acttvatron of a rayon/polyester cloth for covalent nnmobilrzation of protems. Btotechnol

Techn. 5,395-400.

3. Boyd, S. and Yamazaki, H. (1993) Tosyl chloride activation of a rayon/polyester cloth for protein immobilizatron. Btotechnol Techn. 7,277-282. 4. Yarmush, M. L. and Colton, C. K. (1985) Affrmty chromatography, m Comprehensive Biotechnology (Moo-Yang, M., ed.), Pergamon, Toronto, pp. 507-52 1. 5. Nilsson, K. and Mosbach, K. (1980) p-Toluenesulfonyl chloride as an activating agent of agarose for the preparation of immobilized affinity ltgands and proteins. Eur. J Biochem 112,397-402.

6. Harlow, E and Lane, D. (1988) Antzbodles-A Laborutoiy Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 535,536.

10 Immobilization of Enzymes on Microelectrodes Using Chemical Crosslinking Milena Koudelka-Hep,

Nice F. de Rooij, and David J. Strike

1. Introduction

Chemical crosslinking is perhaps the most commonly used method for immobilizing enzymes onto microelectrodes. It is rapid, simple to perform, and has wide applicability. It may be ideal for situations in which surfaces of a few hundred micrometers are to be modified. In principle, the procedure is simple: A drop of solution containing the enzymeto be immobilized, a bifunctional reagent, and frequently a lysine-rich auxiliary protein, such as bovine serum albumin (BSA), is placed over the microelectrode; the bifunctional reagent crosslmks the protein molecules together, finally leading to the formation of an insoluble gel, There are a number of blfunctional reagents that can be used, of which glutaraldehyde appears to be the most satisfactory (1). Although the procedure appears simple, the situation 1sin reality far more complex, stemming from the involved chemistry of glutaraldehyde (2,3), and care must be taken to ensure that reproducible results are obtained. In this chapter, we shall illustrate this technique with the immobilization of glucose oxidase in a gel of glucose oxidase and BSA. 2. Materials 1. Glucose oxidase (GOx) type VII from Aspergzllus lzzger (250 U/mg, Sigma, St. Louis, MO). 2. BSA (fraction V, Calbiochem, La Jolla, CA). 3. Glutaraldehyde: 25% solution in water, diluted to 2.5%, stored at 4°C (see Notes 1,4). 4. Phosphate-buffered saline (PBS): 5.3 tipotassium phosphate, pH 7.2, contammg 2.5 mM potassium chloride and 0.15M sodium chloride (see Note 2).

From

Methods m Biotechnology, Vol. 1’ Immobrlizatfon of Enzymes and Cells Edited by G F Bickerstaff Humana Press Inc , Totowa, NJ

83

Koudelka-Hep,

84

de Rooji, and Strike

3. Method 1, Carefully dissolve 50 mg of GOx in 1 mL of PBS (see Note 3) 2 Carefully dissolve 80 mg of BSA m 1 mL of PBS (see Note 5) 3. Mix 50 PL of the GOx solution with 250 pL of the BSA solution and carefully add, with stlrrmg, 100 PL of 2.5% glutaraldehyde (see Note 6) 4. Deposit 10 pL of this solution on the transducer surface (1 mm*) using a microplpet (see Note 7) 5. Leave for 3 h at room temperature for the gel to form. 6 After gently rinsing with PBS, the Immobilized enzyme electrode 1s ready for use. 7. Store in 10 &phosphate buffer, pH 7.2, at 4°C.

4. Notes 1 Contrary to some mdlcatlons m the hterature, the diluted 2.5% stock glutaraldehyde solution can be used over a 4 mo period when stored at 4°C Although high purity grades of glutaraldehyde can be obtamed, m our experience, less pure and older stock solutions can give better results. 2. Attention should be paid to the nature of the buffer since some, notably Tns, contam amme groups that react with glutaraldehyde, preventing satisfactory gel formation 3 Protein solutions have to be freshly prepared prior to use 4 It IS well known that the use of glutaraldehyde 1sprone to serious reproduclblllty problems. This is partly because of large differences m the compositions of commerclal aqueous glutaraldehyde that consist of a mixture of ohgomerlc and even polymeric material Moreover, glutaraldehyde tends to polymerize at room temperature, thus changing Its composltion with time The simphclty of the procedure, however, explains the wide use of the glutaraldehyde, notwlthstandmg the practical problems, and the lack of a complete understanding of the mechanism. 5 Although it 1snot strictly necessary for crosslinkmg, BSA serves several lmportant functions since it can increase the total protein concentration, allowing gel formation from solutions that would otherwise give only soluble oligomers, and it reduces the excess crosslmkmg that can occur when only the active enzyme 1s present. When BSA is used at concentrations of 50 mg/mL or higher, gel formatlon 1s rapld at any pH between 5.0 and 7.0 6. The stirring during mixing should be gentle enough to avold the formation of a protein foam and denaturatlon of the proteins 7. A 10 pL drop of the protein solution results in a gel membrane approx 30 pm thick

References 1. Broun, G. B. (1976) Chemically aggregated enzymes, in Methods in Enzymology vol XLIV Immobihzed Enzymes (Mosbach, K , ed.), Academic, New York, pp 263-280

Cross-Linking Enzymes onto Microelectrodes

85

2. Quiocho, F. A. (1976) Immobilized mology vol. XLIVImmobilrzed

proteins in single crystals, m Methods in EnzyEnzymes (Mosbach, K., ed.), Academic, New York,

pp. 546-558. 3. Walt, D. R. and Agayn, V I. (1994) The chemistry of enzyme and protein immobilization with glutaraldehyde Trends Anal Chem 13,425430.

11 Photolithographic Patterning of Enzyme Membranes for the Modification of Microelectrodes Milena Koudelka-Hep,

Nice F. de Rooij, and David J. Strike

1. Introduction There are several situations in which conventional crosslinking-based nnmobilization is inadequate in the construction of microelectrodes, for example, when on-wafer deposition (I.e., immobilization on the whole wafer before it is diced up mto mdividual devices) is required, leading to many localized imrnobilizations, or during the fabrication of multianalyte sensors needing several distinct enzyme membranes. The three main types of immobiltzation developed to overcome these problems are based on photochemistry, electrochemistry, or printing. In this chapter we shall deal with the first, photochemical-based immobilization. Two mam types of photochemical rmmobilization have emerged, chemical crosslinking followed by lift-off (A-3) and entrapment in photopolymerlzed gels (4-9). We will rllustrate both approaches with the immobilization of glucose oxidase on a silicon wafer. The lift-off technique is composed of three main parts: 1 DeposItion andphotolithographic patterning of aphotoresistlayer, giving a layer of photoresist in those areasin which enzymeis not required. 2. Deposition of enzymeand crosslinkerover the whole surface. 3. Removal of the photoresist,which takesoff the portion of the enzymaticlayer that was above it, leaving enzymeonly where required. The various steps are Illustrated in Fig. 1, Lift-off allows enzyme nnrnobilization and patterning to be performed at the on-wafer level and gives adherent films of thickness 1-2 pm with much higher levels of reproducibility than with conventional crosslinking. There are, however, a number of drawbacks. First, it requires several pieces of apparatus that, although common in semiconductor fabrication, are less common in a biochemistry laboratory. Next, the From

Methods m Biotechnology, Vol 1 Immobil,ratlon of Enzymes end Cells Edited by G F Bakerstaff Humana Press Inc , Totowa, NJ

87

88

Koudelka-Hep, ,Reyons

y

de Rooij, and Strike

ified initial substrate

Positi/ve ptotoresist Deposition of photoresist

UV-exposure

Development of photoresist Enzyme layer

//

\ :‘:‘,!::..:y;:,: .,.., z.:.:/ ‘1: /“‘!,.j;.:,;:5:j.I‘::‘!‘j:y ..“‘.,,../>//:..::.. s:::/,.: :. ,:,:.:,,,s ;,.::./.:. .::..:..;/:y. :I’,.

.:/

*.

‘.:;;,..:.//

,:.:

:

Deposition of enzyme

Lift-off

Fig. 1. The stepsof the lift-off technique.

enzyme layer is exposed to organic solvents during the development of the photoresist layer. Furthermore, a certain amount of enzyme is lost during the initial deposition and subsequently during the lift-off. In this chapter we shall describe the use of lift-off to prepare localized membranes of glucose oxidase and bovine serum albumin (BSA) on a silicon wafer. The entrapment of enzymes in a photopolymerized gel begins with the spreading of a solution of enzyme, monomer, crosslinker, and initiator over the

89

Photochemical Immobilization of Enzymes Regions to be modified //

\ Substrate

Prepolymeric mixtura /

\

Deposition of prepolymeric mixture

UV-exposure

After removal of unpolymerised material

Fig. 2. The stagesfor

entrapmentof enzymein a photopolymerizedgel.

surface of the wafer. This is exposed through a mask to polymerize certain areas of the film. The excessunpolymerized material is removed to leave discrete areas of the wafer covered with photopolymer containing entrapped enzyme. The various stages are shown in Fig. 2. It is compatible with standard integrated circuit (IC) fabrication techniques and allows the immobilization to be performed at the on-wafer level. It shares several disadvantages with the lift-off technique described above; it requires some highly specialized apparatus and exposes the enzyme to organic solvents. Furthermore, adhesion between the deposited membrane and the transducer surface may be problematic and require chemical pretreatment of the surface. Also, the enzyme is exposed to UV irradiation, which may lead to some loss of activity. Despite these problems, entrapment in photopolymerized gels has been successfully used for the modification of both potentiometric and amperometric electrodes.

90

Koudelka-Hep,

de Rooij, and Strike

The main polymers described m the literature are poly-2-hydroxyethylmethacrylate (poly-HEMA) (46), polyacrylamide (7), and polyvinylalcohol (8,9). Here we shall illustrate the technique with the wafer-level entrapment of glucose oxldase in poly-HEMA. 2. Materials

2.7. Lift-Off Technique 1 2. 3 4 5 6. 7 8 9.

Mask aligner (Karl Stiss). Photoresist spinner (Convac ST 145) Ultrasomc bath. Glucose oxidase (GOx) type VII from Aspergrllus nzger (250 U/mg, Calbiochem, La Jolla, CA) BSA (fraction V, Calbiochem) Glutaraldehyde. 25% solution in water, diluted to 2 5% with water, stored at 4°C. Shipley Microposit S 1650 positive photoresist. 10 mM potassium phosphate buffer, pH 7 1. Acetone

2.2. Entrapment in Photopolymerized

Gels (see Note 7)

1. Glucose oxidase (GOx) type VII from A niger (250 U/mg, Calbiochem) 2. TMSM solution. toluene containing 0.6 wt% water and 11.6 wt% (trimethoxysilyl)propyl methacrylate (TMSM, Aldrich, Milwaukee, WI) 3 Prepolymeric mixture: 54% (w/v) HEMA, 37.7% (w/v) ethyleneglycol, 4 3% (w/v) tetraethyleneglycol dimethacrylate, 1.5% (w/v) dimethoxyphenylacetophenone, and 2.5% (w/v) polyvinylpyrolidone (HEMA, Aldrich; ethylene glycol, Fluka [Buchs, Switzerland]; tetraethyleneglycol dimethacrylate, Fluka, polyvmylpyrolidone, Aldrich). 4. Mask aligner (Karl Suss). 5. Mylar sheet, 12 pm thick (DuPont, Boston MA)

3. Methods

3.7. Lift-Off Technique 3.1.1, Preparation of the Enzyme Solution 1. Carefully dissolve 50 mg of GOx m 1 mL of IO mM potassium phosphate buffer 2. Carefully dissolve 80 mg of BSA m 1 mL of 10 mMpotassmm phosphate buffer 3. Mix 50 pL of the GOx solution with 250 pL of the BSA solution and carefully add with stirring 100 pL of 2.5% glutaraldehyde (see Note 2).

3.12. Immobilization of GOx and BSA 1. Spin-coat the silicon wafer with photoresist at 6000 rpm for 4 min to give a 4 pmthick layer. 2 UV pattern and develop the photoresist according to the manufacturer’s recommendations.

Photochemical lmmob~lization of Enzymes

91

3. Using a brush, spread 100 PL of the enzyme solution over the surface of the wafer, and then spin the wafer at 1400 rpm to give a uniform thickness (see Notes 3,4). 4 Leave the wafer for 1 h in air at room temperature to allow the membrane to crosslink. 5. Place the wafer in a beaker of acetone and agitate ultrasonically for 1.5 s to remove the photoresist. 6. Carefully rinse the wafer with buffer.

3.2. Entrapment in Photopolymerized 3.2.1. Surface Pretreatment

Gels

Prior to membrane deposition, the surface of the silicon wafer is functionalized with methacrylic groups by treating the wafer in the TMSM solution for 10 min at 60°C.

3.2.2. Immobilization 1. Dissolve 50 mg of GOx m 100 pL of water and add to 1 mL of the prepolymerlc mixture with gentle stirring (see Note 5). 2 Using a micropipet, place an appropriate volume (see Note 6) of the mixture on the pretreated surface of the wafer. 3. Cover the liquid film with the mylar sheet (see Note 7) 4. Level the solution using a glass plate so that the whole surface IS uniformly covered 5. Expose to UV irradiation through a mask for 2-4 min depending on the film thickness (see Note 8). 6. Carefully remove the mylar sheet. 7 Remove the unpolymerized material with ethanol.

4. Notes 1. All solutions are prepared immediately prior to use. 2 Because the thickness of the enzyme layer is of critical importance, the viscosity of the enzyme solution must be carefully controlled. This can readily be performed by applying the solution to the wafer a fixed time after preparation. We have found that a delay of 15 min between the addition of the glutaraldehyde and application to the wafer surface gives good results. 3. To achieve rapid lift-off and defined enzymatic membranes, the ratio of photoresist to membrane thickness should be at least 3. 4. The best results from the point of view of membrane geometry definition, reproducibility, and enzyme activity were obtained with a membrane thickness of 1 pm. 5. It is critical to ensure that the enzyme 1svery finely dispersed in the prepolymeric mixture since it is light scattering during the photopolymerization that determines the resolution of the resulting pattern. For a membrane of 50 pm thickness a resolution of 10 pm could be obtained.

9.2

Koudelka-Hep,

de Rooij, and Strike

6 The relattonship between volume of solution placed on the wafer and the resultmg membrane thickness was 150 uL for 10 pm. 7 The mylar sheet prevents oxygen quenching the photopolymertzatron reactton Because of the excess of photomitiator present no degassing is required. 8 Membrane thicknesses from 10-100 pm can be obtained, although the latter may have long response ttmes

References 1. Murakamt, T., Nakamoto, S., Ktmura, J., Kuriyama, T., and Karube, I. (1986) A micro planar amperometrtc glucose sensor using an ISFET as a reference electrode Anal Lett. 19,1973-1986 2 Nakamoto, S., Ito, T , Kuriyama, T., and Kimura, A. (1988) A lift-off method for patterning enzyme-immobilized membranes m multi-biosensors. Sensors Actuators 13, 165-I 72. 3 Gernet, S., Koudelka, M., and de ROOIJ, N. F. (1989) A planar glucose enzyme electrode. Sensors Actuators 17, 537-540 4 Arica, Y. and Hastrci, V.N (1987) Innnobiltzatron of glucose oxtdase in poly(2hydroxyethyl methacrylate) membranes. Biomaterzals 8,489-495. 5. Hmberg, I., Kapoulas, A., Korus, R., and O’Driscoll, K. (1973) Gel entrapment of enzymes: kinetic studies of nnrnoblhzed glucose oxldase. Bzotechnol Bzoeng 16, 159-168. 6. Strike, D. J., van den Berg, A., de ROOIJ, N. F., and Koudelka-Hep, M. (1994) Spatially controlled on-wafer and on-chip enzyme nnmobihzatton using photochemical and electrochemical techniques, in Dzagnostic Biosensor Polymers (UsmaniA. M. and Akmal N., eds.), ACS Symposium Series 556, American Chemical Society, Washmgton DC, pp. 298-306. 7. Guilbault, G G. and Lubrano, G. J. (1973) An enzyme electrode for the amperometric determination of glucose. Anal. Chzm. Acta. 64,439-455. 8. Takatsu, I. and Morilzuml, T (1987) Solid state biosensors using thin-film electrodes. Sensors Actuators 11,309-317. 9. Martly, J.-L., Mtonetto, N., and Rouillon, R. (1992) Entrapped enzymes m photocrosslinkable gel for enzyme electrodes. Anal. Lett. 25, 1389-1398.

12 Electrochemical-Based

Immobilization

of Enzymes

David J. Strike, Nice F. de Rooij, and Milena Koudelka-Hep I. Introduction

Electrochemical-based enzyme immobilization methods are a convenient way of unmobihzmg enzymes on microelectrodes, albeit one restricted only to amperometric sensors. They enable the immobilization to be localized at one electrode (the working electrode), frequently offer some control of the thicknessof the resultant film, and may offer significant interference rejection properties. Furthermore, the immobilization can usually be performed from aqueous solution near neutral pH and can coat complex or otherwise inaccesible surfaces, such as in situ detectors. Although in principle they could be used in mass production to perform on-wafer level modificattons (i.e., to unmobthze enzyme in separate regions of a whole silicon wafer, prior to dicing it into separate devices) they are usually used only in the final stageson sensor fabrication to modify individual devices. As with the photochemical-based immobilization (see Chapter 11), electrochemical techniques do require some specialized equipment. In this chapter, we shall describe two depositions, one entrapment in an electrochemically grown polymer, the other electrochemitally aided crosslinking. The literature describes the entrapment of enzymes in several different electrochemically grown polymers. These can be separated into two broad classes, conducting polymers (i.e., polypyrrole [2,2], poly-N-methylpyrrole [3], and polyaniline [4,5-l), and nonconducting polymers (i.e., poly[o-phenylenediamine] [6,8/ and polyphenol[9j). Both types of polymer can be grown from an aqueous solution of monomer and enzyme, buffered near neutral pH, by polarizing the working electrode at a potential at which the monomer will be oxidized resulting in the formation of a polymeric film containing the entrapped enzyme on the electrode surface, m effect a one step immobilization proceFrom

Methods m Bmtechnology, Vol 1 lmmobrkabon of Enzymes and Cells Edlted by G F BIckerstaff Humana Press Inc , Totowa, NJ

93

94

Strike, de Rooij and Koudelka-Hep

dure. Both classesof polymer can function as simple entrapment matrlces that offer significant protection from interfermg substances in the sample (6,7,9). Conducting polymers allow conslderable flexibility since: 1, They can be readily chemically modified, either by functlonalizmg the monomer, with, for example, a pendant electron-transfer mediator, or after deposltlon (1045). 2 They can be doped with useful amonic species, such as electron transfer medlators (I 6). 3 The thickness of the deposit can be regulated via the chargepassed. 4. They can be used to form multllayer structures allowing the fabrication of complex devices (I 7) 5 They are electrochemlcally active giving the possibility of direct electron transfer between enzyme and electrode (18)

Whereas the nonconductmg polymers do not offer the same flexibility, they have found important apphcatlons as immobilization matrices in sensorsrequirmg fast response times and good interference rqectlon. Although the literature deals mainly with glucose oxidase, the nnmoblhzation of other enzymes has been reported, including alcohol oxldase (19), cholesterol oxldase and esterase (20), flavocyctochrome b2 (22), galactose oxrdase (22,23), glucose dehydrogenase (24), horseradish peroxidase (25), invertase (26), lactate oxldase (27), mutarotase (26), nitrate reductase (15), tyrosinase (19), and urease (22). Here we will Illustrate thesetechniques with the simple one-step immobilization of glucose oxidase in polypyrrole. Electrochemlcally aided crosslinkmg, which does not involve entrapment tn a polymeric support, offers a viable alternative to entrapment in electrochemltally grown polymers for those situations in which the special properties of the electrochemically grown polymers are not required, particularly for amperometric sensors based on hydrogen peroxide detection. In our experience, the technique gives larger responses than entrapment in polypyrrole. It has most frequently been used for the coimmobllization of glucose oxidase with a metal (28-3U), but has also been used for the deposition of biological species alone, such as avldm (31), glucose oxldase (32-34),

sarcosme oxldase, galactose 0x1-

dase, choline oxidase and alcohol oxidase (34), and urease (33). In Section 3.2., we shall Illustrate the technique with the codeposltion of glucose oxldase and bovine serum albumin (BSA). In this illustration, the deposition is made from a solution of glucose oxldase, BSA, and glutaraldehyde

that IS sufficiently

dilute for crosslinking not to form an insoluble gel. By polarizing the working electrode so that it has a charge opposite to the proteins, the protein concentratlon at the working electrode is increased, leading to crosslinking and the deposition of a gel on the electrode surface.

Electrochemical-Based

2. Materials 2.7. Entrapment

Immobilization

in EIectrochemically

95

Grown Polymers

1. A potentiostat, preferably one able to perform cyclic voltammetry and with a current integrator to allow the charge passed to be measured. In our laboratory, we use an EG&G 273A potentiostat/galvanostat (Princeton, NJ) 2. A chart recorder. 3 An electrochemical cell, preferably of small volume. 4 A reference electrode, such as a saturated calomel electrode (SCE) 5. A counter electrode 6 Glucose oxidase (GOx) type VII (Sigma, St. Louis, MO). 7. BSA (fraction V, Calbiochem, La Jolla, CA) 8. Pyrrole (98% pure, Aldrich, Milwaukee, WI). 9. Storage buffer: 10 mMpotassium phosphate, pH 7.2 IO. Phosphate-buffered perchlorate (PBP): O.lM potassium phosphate, pH 7.0, containing 10 rnA4 sodium perchlorate 11. Nitrogen gas (oxygen-free). 12. H,PtCl, (Fluka, Buchs, Switzerland)

2.2 Electrochemically

Aided Cross/inking

1. A potentiostat capable of controlled current (galvanostatic) techniques 1srequired. In our laboratory we use an EG&G273A potentlostat/galvanostat 2. A chart recorder. 3. An electrochemical cell, preferably of small volume (see Note 7) 4 A reference electrode. We have found that a silver chloride-coated silver wire (Ag/AgCl) is quite sufficient. 5. A counter electrode. A short piece of platmum wire IS sufficient, providmg It has an area several times that of the electrode to be modified. 6. Glucose oxidase (GOx) type VII (Sigma). 7. BSA (fraction V, Calbiochem). 8 Glutaraldehyde, practical grade (Fluka): 50% in water. 9. Phosphate-buffered saline (PBS): 5.3 nU4potassium phosphate, pH 7.2, containing 2.5 mA4 potassium chloride and 0.15M sodium chloride 10. Storage buffer: 10 mMpotassium phosphate, pH 7.2.

3. Methods 3.1. Entrapment

in EIecfrochemically

Grown Polymers (see Note 1)

1. Fill the electrochemical cell with the required volume of PBP, insert the reference and counter electrodes, and bubble the solution for 30 min with oxygen-free nitrogen gas (see Note 2). 2. Add purified pyrrole to give a final concentration of O.lM and 2.4 mg/mL of glucose oxidase, and gently mix to give a homogeneous solution (see Note 3) 3. Insert the electrode to be modified, pretreated if necessary (see Note 4 and Section 3 1.1.).

96

Strike, de Rooij, and Koudelka-Hep

4 Set the potential of the electrode to 800 mV (vs SCE) and allow a total of 70 mC/ cm2 of charge to pass. During this step, gently blow oxygen-free mtrogen gas over the solution.This completes the immobilizatton (see Notes 5 and 6 and Section 3 1.2.).

3.1. I. Pretreatment of Microelectrodes

Prior to Deposition

1. Saturate a solutton of 0 5M H2S04 with nitrogen or argon gas 2 Perform five consecutive cyclic voltammetric sweeps, at a speed of 50 mV/s, as follows: a -0 25-1.2 V (vs SCE). b. -0 25-l .2 V (vs SCE). c. -0 25-2 0 V (vs SCE). d. -0 25-1.2 V (vs SCE). e -0 25-O 2 V (vs SCE). During the third sweep there will be electrolysts, and gas bubbles may become attached to the electrodes. For this reason it is important to gently tap the workmg electrode during the second part of this sweep to remove any attached bubbles. 3. Rinse the electrode m deionized water and dry with a nitrogen stream. The treated electrode should be used within a few hours of this treatment.

3.1.2. Improving the Adhesion of the Polypyrrole Film 1 Saturate a solution of 50 mM KC1 contaming 0.04 wt% H,PtCl, wrth nitrogen gas 2 Depostt Pt on the working electrode by applying a potential of -0.2 V (vs SCE) until a charge corresponding to ca. 0 12 C/cm2 has been passed. 3. Rinse the electrode with deionized water

3.2. Electrochemical/y

Aided Crosslinking (see Notes 7, 8,ll)

1 Usmg a Pasteur pipet, carefully add 950 pL of PBS to 5 mg of glucose oxidase. Carefully agitate the solution to dissolve the protein (see Notes 9, IO) 2 Using a Pasteur pipet, add this to 5 mg of BSA and dissolve the second protein (see Notes 9, IO). 3 Add, with gentle mixing, 50 pL of 50% glutaraldehyde. 4. Carefully transfer this to the electrochemical cell. 5. Place the electrodes m the solution. 6. Apply a train of 0 5 s pulses of 2 mA/cm2 separated by 5 s at open circuit potential for 10 min (see Note 11). 7. Remove the electrode and gently rinse with PBS. 8. Store at 4°C in IO mA4potasstum phosphate buffer, pH 7.4.

4. Notes I. We have found that this procedure, based on the report of Yon Hin et al. (22) is both sample and reliable. 2. It 1simportant to both saturate the PBS with oxygen-free nitrogen or argon and to gently blow this over the surface during the deposmon. Care must be taken that

Electrochemical-Based

3.

4.

5.

6.

7. 8. 9. 10. 11.

immobilization

97

the gas flow does not agitate the solution during the actual deposition since this ~111 result m irreproducible depositions. It is essential that the pyrrole be purified before use either by distillation or by passage down a short alumina column. Freshly prepared pyrrole is a clear, almost colorless liquid. After purification, the pyrrole must be stored m the dark at 4°C for a maximum of a few hours prior to being used, although with storage under nitrogen, longer storage periods may be used. The polypyrrole film will be red-brown to black m color dependmg on its thickness. If only thin, irregular films are obtained it may be necessary to pretreat the electrode surface prior to attempting deposition. The ease of polypyrrole growth may be strongly affected by the quality of the electrode surface prior to deposition, We have found the protocol given in Section 3.1.1. to be a simple way of rapidly preparing a surface suitable for deposition. Although adhesion of the polypyrrole is not normally a problem, with thicker films it may become so. We have found that the following electrochemical deposition of platinum described in Section 3.1.2. greatly reduces this problem, as well as increasing the effective surface area of the electrode. The modified electrode must usually be conditroned prior to use. This allows both the removal of loosely bound enzyme and, depending on the intended use of the film, can give a stable and small background current. This is particularly important in applications m which the hydrogen peroxide produced by the enzyme is measured, when it is normally observed that there is a large background current when the sensor is first used. This current rapidly decreases to a small value after several hours of polarization If possible, a good method of condittoning is to leave the sensors polarized overnight in stirred buffer. Rather than an electrochemical cell, a small glass vial may be used. It is not necessary to saturate the solutions with nitrogen. It 1s essential that care 1s taken to avoid introducing bubbles into the protein solution during transfer and agitation. To agitate the solutions for dissolving the proteins, careful but sharp tapping on the bench top has been found to be effective. When using different enzyme preparations, it is frequently necessary to modify the concentrations or pulse sequence used. Below we give some simple gmdelines to surmounting the problems that we have most frequently encountered. If the deposition solution rapidly goes cloudy or forms a gel, then reduce the concentration of either protein or glutaraldehyde. If the electrode is covered by a large plume of protein: a. Lower the current during deposition; b. Reduce the length of the on portion of the pulse; or c. Reduce the protein concentration. If no deposit is formed* a. Increase the current, taking care to avoid bubble formation; b. Reduce the time between pulses or increase the on pulse length; c. Increase the glutaraldehyde or protein concentration, if necessary using BSA,

Strike, de Rootj, and Koudeka-Hep

98

which can greatly enhance deposition, but take care to avoid the problems noted above; or d. Allow the deposition more time. This is frequently a solution, although care must be taken since some enzymes show a rapid loss of activity under these conditions.

References 1. Foulds, N. C. and Lowe, C R (1986) Enzyme entrapment m electrically conducting polymers. J Chem. Sot., Faraday Trans 182,1259-1264. 2 Umana, M. J. and Waller, J (1986) Protein modified electrodes. The glucose oxidasel polypyrrole system. Anal. Chem. 58,2979-2983 3 Bartlett, P. N. and Whnaker, R C J. (1987) Electrochemical nnmobihzatton of enzymes. II Glucose oxidase immobilized in poly-N-methylpyrrole. J Electroanal. Chem 224,37-48. 4. Shinohara, H., Chiba, T , and Azawa, M. (1988) Enzyme microsensor for glucose with an electrochemically synthesized enzyme-polyarnhne filrm Sensors Actuators 13, 79-86 5. Cooper, J. C. and Hall, E. A. H. (1992) Electrochemical response of an enzyme-loaded polyaniline film. Bzosensors Bzoelectronics 7,473+85. 6. Sasso, S V., Pierce, R. J., Walla, R., and Yacnych, A. M. (1990) Electropolymerized 1,2-diammobenzine as a means to prevent interferences and fouling and to stabilize mnnobilized enzymes in electrochemical biosensors. Anal Chem 62, 1111-1117. 7 Malitesta, C., Palmisano, F., Torso, L., and Zambonm, P. G. (1990) Glucose fastresponse amperometric sensor based on glucose oxidase immobilized m an electropolymerized poly(o-phenylenediamme) film. Anal Chem 62,2735-2740 8. Bartlett, P. N., Tebbutt, P., and Tyrrell, C H. (1992) Electrochemical immobihzation of enzymes. 3 Immobilization of glucose oxidase in thin films of electrochemically polymerized phenols. Anal Chem. 64, 138-142. 9. Centonze, D , Guerneri, A , Mahtesta, C , Pahnisano, F., and Zambonin, P. G (1992) Interference-free glucose sensor based on glucose-oxidase immobihzed m an overoxtdized nonconducting polypyrrole film Fresenzus J. Anal Chem 342, 729-733.

10. Foulds, N. C and Lowe, C. R. (1988) Immobilization of glucose oxidase m ferrocenemodified pyrrole polymers. Anal. Chem 60,2473-2478 11. Schuhmann, W., Kranz, C., Huber, J., and Wohlschlager, H. (1993) Conductmg polymer-based amperometric enzyme electrodes. Towards the development of miniaturized reagentless btosensors Synth. Metal. 61,3 l-35. 12 Yon-Hm, B F. Y and Lowe, C R (1994) An investigation of 3-functionalized pyrrole-modified glucose oxidase for the covalent electropolymerization of enzyme films. J Electroanal Chem 374, 167-172. 13. Schuhmann, W., Lammert, R., Uhe, B., and Schnudt, H.-L. (1990) Polypyrrole, a new possibility for covalent binding of oxidoreductases to electrode surfaces as a base for stable biosensors. Sensors Actuators Bl, 537-541.

Electrochemical-Based

Immobilization

99

14. Schallchammer, T., Mann-Buxbaum, E., Pittner, F , and Urban, G. (1991) Electrochemical glucose sensors on permselective nonconducting substnuted pyrrole polymers. Sensors Actuators B4,273-28 1. 15. Cosnier, S., Innocent, C., and Jouanneau, Y. (1994) Amperometrtc detection of nitrate vta a nitrate reductase immobilized and electrically wired at the electrode surface. Anal Chem 66,3198-3201 16. Kajiya, Y., Sugai, H., Iwakura, C., and Yoneyama, H. (1991) Glucose sensitivity of polypyrrole films containing immobilized glucose oxidase and hydroquinonesulfonate ions. Anal. Chem. 63,49-54. 17. Bartlett P. N. and Birkin P. R. (1993) The application of conducting polymers in biosensors. S’nth. Metal. 61, 15-21. 18 Koopal, C. G J., Feiters, M C., Nolte , R. J. M , de Ruiter, B , and Schasfoort, R. B. M. (1992) Glucose sensor utilizing polypyrrole mcorporated in track-etch membranes as the mediator. Bzosensors Bloelectronics 7,46 l-47 1. 19. Wang, J , Neser, N., and Renschler, C. (1993) Enzyme nanoband electrodes Anal Lett 26,1333-1346. 20. Yon Hin, B. E Y and Lowe, C. R. (1992) Amperometric response of polypyrrole entrapped bienzyme films. Sensors Actuators B7,339-342. 21. Bartlett, P N. and Caruana, D. J. (1994) Electrochemical immobilizatton of enzymes. VI. Mtcroelectrodes for the detection of L-lactate based on flavocytochrome b2 munobd~zed m a poly(pheno1) film. Anal. Chem 119, 175-180. 22. Yon Hin, B F. Y, Sethi, R. S., and Lowe, C. R (1990) Multi-analyte biosensors. Sensors Actuators Bl, 550-554 23. Cosnier, S. and Innocent, C (1992) A novel brosensor elaboration by electropolymerrzation of an adsorbed amphiphilic pyrrole-tyrosmase enzyme layer J. Electroanal. Chem. 328,36 l-366. 24. Kajiya, Y., Matsumoto, H , and Yoneyama, H (1991) Glucose sensivtty of poly(pyrrole) films contammg tmmobilized glucose dehydrogenase, mcotmamide adenine dinucleotide and P-naphtholqumonesulphonate ions J Electroanai. Chem 319,185-194. 25. Tatsuma, T , Gondana, M., and Watanabe, T (1992) Peroxidase-incorporated polypyrrole membrane electrodes. Anal. Chem. 64, 1183-l 187 26. Slater, J M. and Watt, E J. (1989) Use of the conducting polymer, polypyrrole, as a sensor. Anal Proc. 26,397-399. 27. Palmisano, F., Centronze, D., and Zambomn, P. G. (1994) An In situ electrosynthesized amperometric biosensor based on lactate oxidase immobilized in a poly-o-phenylenedtamine film. determmatton of lactate m serum by how mJection analysts. Blosensors Bioelectronlcs 9,47 l-479. 28. Ikariyama, Y., Yamauchi, S., Yakiasht, T , and Ushioda, H. (1989) Electrochemtcal fabrication of amperometric microenzyme sensor. J Electrochem Sot. 136, 702-706. 29. Wang, J. and Angnes, L. (1992) Miniaturized glucose sensors based on electrochemical codeposition of rhodmm and glucose oxidase on to carbon fibre electrodes Anal. Chem 64,456-459.

100

Strike, de f?oo$ and Koudelka-Hep

30. Wang, J. and Chen, Q (1994) Enzyme microelectrode array strips for glucose and lactate. Anal Chem 66, 1007-1011 3 1 Anzai, J.-I., Tomonori, T., and Osa, T (1993). Electrochemical preparation of active avtdm tilms for enzyme sensor applicattons. Chem Lett. 1993, 123 1-1234 32. Johnnson, K W (199 1) Reproducible electrodeposmon of biomolecules for the fabrrcatton of miniature electroenzymatic brosensors Sensors Actuators B5, 85-89. 33. Strike, D. J., van den Berg, A., de Rooij, N. F., and Koudelka-Hep, M. (1994) Spatially controlled on-wafer and on-chip enzyme tmmobilization using photochemical and electrochemical techniques, m Dlagnostlc Bzosensor Polymers (Usmani A. M. and Akmal N., eds.), ACS Symposium Senes 556, American Chemtcal Society, Washmgton, DC, pp. 298-306 34. Strike, D. J., de Rooij, N F., and Koudelka-Hep, M. (1995) Electrochemical techniques for the modification of microelectrodes. Bzosensors Bioelectromcs 10, 61-66

13 Immobilization of Enzymes on Thermo-Responsive Polymers Kazuhiro Hoshino, Setuko Akakabe, Shoichi Morohashi, and Toshisuke Sasakura

1, Introduction

Immobilization of enzymes on supports is a valuable technique for increasing total productivity and life-span of biocatalysts. Conventionally, enzymes are immobilized on water-insoluble supports, such as polysaccharide derivatives, ion exchange resin, and so on. However, in a heterogeneous reaction system involving solid substrate and/or reactant, the enzyme immobilized on such supports can cause problems of substrate diffusion within the support (I), enzyme leakage from the support (2,3), and separation of the immobrlized enzyme from unreacted solid residue and/or solid reactant m the reaction mixture (see Chapter 1) (5). A potential method of solving the problems inherent in such heterogeneous systemsis the use of enzymesimmobilized on a reversibly soluble-insoluble support that can change solubility depending on the temperature of the reaction mixture. Recently these mnnobilized enzymes have been studied because they not only act effectively in a soluble state with soluble high-molecular-weight substrate and solid substrate,but also can be recovered in an insoluble state from the reaction mixture by a slight shift in temperature (56). However, the selection of thermoresponsive polymer must be made carefully becausethe characteristics of each enzyme are likely to be different and must be considered. In this chapter we present a strategy for preparation of a highly efficient thermo-responsive imrnobilized enzyme taking the characteristics of the enzyme mto consideration (Fig. 1). This strategyinvolves selectionof onemonomer with thermo-responsive properties and one monomer with a suitable functional group for the subsequent From. Methods m Btofechnology, Vol 7 Immobrhzation of Enzymes and Cells Edited by G F Btckerstaff Humana Press Inc , Totowa, NJ

101

Hoshino et al.

102

Selection of N-Alkyl Acrylannde (NAA) Monomer LCST N-propyl acrylannde (NPAM) 22°C N-isopropyl acrylanude(NIPAM) 32°C N-isopropyl methacrylamide (NIPMAM) 40°C N-acyloyl pyrrolidme (NAL,P) 52°C

1

IMMOBILIZED

ENZYME PREPARATION

1

Fig. 1. A protocol for preparation of therrno-responsive immobilized

enzyme.

coupling reaction with the enzyme,polymerization of the two monomers, and coupling of the enzyme to the thermo-responsivepolymer. In general, N-alkyl acrylamides (mainly N-isopropyl acrylamtde) have been utrhzed as monomers for preparatton of thermo-responsive polymers. It is known that the lower critical solutron temperature (LCST) of the polymer prepared with iv-isopropyl acrylamide is about 32°C (7,s). However, to effectively

Thermo-Responswe

Polymers

103

use the enzyme immobilized on thermo-responsive polymer, the enzyme should be soluble at the reaction temperature. Recently, several workers have found that the LCSTs of the polymer N-alkylamide can be adjusted from 4 to 70°C by synthesis of various N-alkylamines with acryloyl chloride (9), and it is now possible to select the correct monomer to suit the temperature/solubility characteristics of the enzyme. For efficient immobilization, it is important to consider the selection of a monomer with free functional groups, such as amino, carboxyl, epoxy, and so on, that will be involved in interaction with groups on the enzyme. For example, if the optimum pH of the enzyme is C5.0, then selection of a monomer with a free carboxyl group, such as acrylic acid (AAc) or methacrylic acid (MAAc), would be suitable, and the enzyme could be immobilized by the EDC method. Although immobilized enzymes prepared in this way are less thermo-responsive at pH values >5.0, they have the advantage that higher loading of enzyme IS possible than can be achieved with other monomers. Conversely, when enzymes with pH optimum >5.0 are immobilized, it 1sdesirable to use monomers with epoxy or succinimide groups, and to have immobilization carried out by the alkaline solution method. Although greater flexibihty in thermoresponsive behavior is possible, the efficiency of enzyme immobilization is lower. By taking account of the characteristics of the enzyme and the reaction conditions, a suitable thermo-responsive immobilized enzyme can be produced. A working strategy is described in Fig. 1. 2. Materials 2.1. Preparation of Thermo-Responsive Polymer 1 Monomer with thermo-responsive properties, such as N-alkyl acrylamrde (NAA), for example, N-isopropyl acrylamide (NIPAM) (see Note 1). 2. Monomer with a carboxyl group, such as AAc or MAAc, and monomer with epoxy or succinimide group, such as glycidyl methacrylate (GMA) or N-acryloxy succinimide (NASI) (see Note 2). 3. n-Hexane (reagent grade). 4. Polymerization initiator: 1 mL of 0.5% (w/v) ammonium persulfate and 1 mL of 0.5% (w/v) sodium bisulfate in water (see Note 3). 5. Solution for recovering thermo-responsive polymer: 500 mL of 2% (w/v) salme water. 6. Ice water

7. Dialysis tubing (seamlesscellulose tubing, size2.4 nm, Viscose,SaksCo., Chtcage, IL) (seeNote 4). 8. Separatingflask equippedwith agitator andjacket. 9. Nitrogen gas. 10. Fume cupboard. 11. Refrigerated centrifuge.

Hoshino et al.

104 2.2. Immobilization of Enzyme (pH Active ~5.0) on Thermo-Responsive Polymer

1. Thermo-responsive polymer, NAA-AAc or NAA-MAAc copolymer. 2 Enzyme(s), e.g., amylase, cellulase (see Note 5) 3. Reagent for peptlde synthesis between enzyme and polymer: l-ethyl-3-(3dimethylaminopropyl)-carbodrimrde hydrochloride (EDC) (see Note 6) 4. Buffer O.lM acetate buffer, pH 4 O-6.5 (pH varied as required for the given enzyme). 5 Dialysis tubing (seamless cellulose tubing, size 2 4 nm, Viscose) (see Note 4) 6. Refrigerated centrifuge. 7. Recovery buffer 1sthe normal buffer and pH used m the given enzyme reaction, typically O.lM glycine-HCl buffer, pH 3.0, or 0 1M sodium acetate buffer, pH45 8. Magnetic stirrer.

2.3. Immobilization of Enzyme (pH Active >5.0) on Thermo-Responsive Polymer 1 2. 3. 4 5 6

Thermo-responstve polymer, NAA-GMA or NAA-NASI copolymer Enzyme(s), e.g , protease Buffer: 0.2M sodium tetraborate, pH 8 O-10 0 (see Note 7). Dialysis tubing (seamless cellulose tubing, size 2.4 nm, Viscose) (see Note 4) Refrigerated centrifuge. Recovery buffer is the normal buffer and pH used m the given enzyme reaction typically 0. lMglycine-NaOH, pH 9.0, or 0 1M Tris-HCi, pH 7.5 7. Magnetrc stirrer

3. Methods 3.1. Preparation

of the Thermo-Responsive

Polymer

1. Dissolve the N-alkyl acrylamide monomer, such as NIPAM, in 100 mL of dlstilled water. 2. Seprarately dissolve the selected monomer bearing required functronal groups (e.g., AAc, MAAc, GMA, NASI) in 25 mL of distilled water. The total weight of monomers is adjusted to 5.0 g (see Note 8). 3. Extract the polymerlzatron inhibitor @-methoxyphenol) from the N-alkyl acrylamide solution with 100 mL of n-hexane usmg a separating funnel (250 mL). 4. Recover the aqueous solution containing N-alkyl acrylamrde and pour out the extraction solvent (n-hexane) into a waste container. 5 Extract the polymerization inhibitor @-methoxyphenol) from the other monomer solution (AAC, MAAc, GMA, NASI) with 25 mL of n-hexane using a separatmg funnel (50 mL). 6 Recover the aqueous solution containing the monomer and pour out the extraction solvent into a waste container 7. Place both monomer solutions together in a separating funnel (500 mL).

Thermo-Responsive

Polymers

105

8. Adjust the total volume of the mixture to 500 mL by adding distilled water At this point, the concentration of total monomers is 10 g/L (see Note 9) 9. Maintain the temperature of the separatmg flask at 65°C using a hot-water jacket (see Note 10). 10. Bubble nitrogen gas through the separating funnel to deaerate the solution, then keep the solution under a mtrogen atmosphere during the polymertzation (see Note 11). 11. Agitate the solution at 3 5g. 12 Add the redox catalysts into the flask, i.e., 1 mL of 0 5% (w/v) ammonium persulfate and 1 mL of 0.5% (w/v) sodium btsullite 13 Maintain the above conditions and allow polymerization to proceed for 1 h (see Note 12) 14 Stop the polymerization reaction by cooling the reaction mixture with ice-cold water. 15. Insolubilize the polymer by adding 2% (w/v) saline solutton of equal volume as the reaction mixture (500 mL). 16 Increase the temperature of the mixture to 40°C for the polymer composed of NIPAM (see Note 13). 17 Collect the resulting precipitate, NAA-AAc, or NAA-GMA copolymer by centrtfugation at 6OOOgand 40°C for 10 min. 18. Redissolve the precipitate by adding cold distilled water 19. Using the dialysis tubing, dialyze the solution against distilled water for 24 h at 4°C. 20. Evaporate down the polymer solution, and the resultant residue is used as the thermo-responsive polymer. 2 1. Store the polymer at 4°C until required for immobihzation. 22. Properties of the thermo-responsive polymer can be evaluated as indicated m Note 14.

3.2. Immobilization of Enzyme (pH Active ~5.0) on Thermo-Responsive Polymer 1. Dissolve 1 g of thermo-responstve polymer, such as NAA-AAc copolymer, m 50 mL of sodium acetate buffer (see Note 15). 2. Add enzyme powder or solution (100-150 mg protein/g of polymer) mto the polymer solution (see Note 16). 3. Mix the solution at 25°C using a magnetic stirrer (see Note 17). 4. Add, intermittently over a period of 30 min, 300 mg of EDC into the mixture (see Note 18). 5. Stir the solution slowly for 3 h (see Note 17). 6 Add saline water to the mixture to a level of 1% (w/v) 7. Increase the :mperature of the mixture to 40°C for the polymer composed of NIPAM. 8. Collect the resultant precipitate (immobilized enzyme) by centrifugation at 6OOOg and 40°C for 10 mm (see Note 19).

106

Hoshino eta/.

9. Wash the unmobilized enzyme twice wrth sodium acetate buffer containing 1% (w/v) salme at 40°C (see Note 20) 10 Dissolve the precipitate m cold sodium acetate buffer. 11, Using the dialysis tubing, dialyze the solution against dtstilled water or sodium acetate buffer for 24 h at 4°C. 12. Store the unrnobil~zed enzyme at 4°C until required for catalysis or for characterization (see Note 2 1).

3.3. Immobilization of Enzyme (pH Active ~5.0) on Thermo-Responsive Polymer 1 Dissolve 1 g of thermo-responsive polymer such as NAA-GMA copolymer, m 50 mL of 0.2M sodium tetraborate buffer (pH between 8 0 and 11 0) (see Notes 7,22) 2 Add enzyme powder or solution (SO-100 mg protein/g of polymer) into the polymer solution (see Note 22) 3. MIX the solution at 25°C using a magnetic stirrer 4. Stir the solution slowly for 48 h (see Note 23). 5 After the munobil~zation, add saline to a concentration of 1% (w/v). 6. Increase the temperature of the mtxture to 40°C. 7. Collect the precipitated immobilized enzyme by centrtfugation at 6000g and 40°C for 10 min (see Note 19). 8 Wash the immobilized enzyme twice with alkaline buffer contammg 1% (w/v) salme at 40°C (see Note 20). 10. Dissolve the precipitate in cold alkaline buffer. 11. Using the dialysis tubing, dtalyze the solution against distilled water or alkaline buffer for 24 h at 4°C 12. Store the irrnnobihzed enzyme at 4°C until requtred for catalysis or for characterization (see Note 2 1).

4. Notes 1 Commercial NAA powder or solution contains p-methoxyphenol as a polymerization inhibitor. Since NAA is water soluble, the inhibitor is extracted into n-hexane or ether. 2. CARE! AAc and MAAc monomers are corrosive and toxic. GMA and NASI monomers are mutagens. Take care in handling these monomers; avoid breathmg vapor during weighing at the balance. 3. These reagents must be prepared fresh before polymerization ts attempted. 4. Smce the dialysis tubing has a porosity of 2 4 run, thermo-reponsive polymer with A4, ~12,000 can be recovered by dialysis If smaller IV, polymer is required, then dialyses tubing with a smaller pore size may be used 5. Many different enzymes can be immobthzed on thermo-responsive polymer Prev~ously, we have nnmobiIized amylase, cellulase, and thermolysin (5,6). In addition, nonenzyme materials, such as glycosides, lectins, and antibodies, can be similarly nnmobilized.

Thermo- Responsive Polymers

107

6. The reagent is deliquescent and care should be taken in weighmgladding to reaction mixture. The reagent should be stored at-20°C to avoid Inactivation at room temperature. 7. Various buffers may be used to suit the enzyme under consideration, e g., Tris-HCl, glycine-NaOH. Phosphate buffer is less satisfactory because it produces an opaque state in the reaction mixture and less enzyme is immobihzed than with other buffers. 8 Experience has revealed that the optimum for immobilization is achieved when the weight ratio between NAA monomer and group-bearing monomer IS P-24.1 Thus, the NAA can vary from 9-24 and the choice will influence the temperature-solubility relationship and the enzyme loading on the polymer 9. The total monomer concentration should be in the range 5-20 g/L. At higher values the polymer Mr will be ~10,000 and cannot be recovered by dialysis or centrifugatlon. At lower values the polymer concentration is too low for efflcient recovery. 10. The temperature for immobilization is 65°C. At lower temperatures, polymerization is less and does not occur at 45’C. At higher temperatures, e.g., 70°C, the yield of polymer is reduced. At 65’C, the yield of polymer is about 0 8-0.9 of the total monomer weight used. 11 Deaeration 1s essential for polymerization, because dissolved oxygen is a polymerization inhibitor. CARE! Nitrogen gas may be bubbled into the mixture for more than 30 min. 12. Polymerization IS complete 60 mm after addltlon of mitlators. 13. Since the temperature for the recovery of polymer is different from that at which NAA is used, the polymerization can be done at a temperature at which the polymer is insoluble. 14 The amount of thermo-responsive polymer produced can be determined by drying the polymer solution at 90% for 24 h. The average molecular weight of the polymer can be measured by intrinsic viscosity (IO), using NIPAM homopolymer as a standard, or by HPLC using a gel permeation column. 15. The optimum pH for unmobilization of enzyme on thermo-responsive polymer prepared by the EDC method is between 4.0 and 6.5. Ideally the pH should be close to the isoelectric point of the enzyme (11,12). The buffer should not contam any surplus compounds with amino, carboxyl, or other groups that may interfere with coupling 16. When 100-150 mg protein is added per 1 g of polymer, the expected level of immobilization should be m the range 50-75 mg protein/g of polymer. 17. For sensitive enzymes, the temperature and time for immobilization may be reduced to 25°C for 3 h, or 4°C for 24 h. 18. Take note that the EDC must be added stepwise m small amounts to the reaction mixture to avoid preclpltatlon of the EDC. 19. Centrifugatlon is a good method for recovery of the munobilized enzyme. Note that centrifugation at higher than 6000g will make it more difficult to redissolve the immobilized enzyme. 20. Washing at least twice 1srecommended to remove unbound enzyme.

108

Hoshino et al.

21 The amount of enzyme immobilized can be determined by bicinichommc acid (13) since the normal Lowry method (14) for protein estimation is not surtable. 22. The best range for pH immobilization is between 8.0 and 11.O and experience has shown that pH 10.5 is the optimum. Note that the amount of enzyme bound usmg this procedure is less, typically 25-40 mg protein/g of polymer than with the other polymer. 23 Immobdizatron reaction using alkaline solutron takes longer than with the EDC method, and the typical range is 48-72 h.

References 1. Matsuno, R. (1975) Koteika koso (m Japanese) Kodansha, Tokyo, 235-249. 2 Epton, R., Marr, G., and Morgan, G. J. (1977) Soluble-polymer-protein conjugates. Polymer 18,319-323. 3. Chen, G. and Hoffman, A. S. (1993) Preparation and properties of thermoreverstble, phase ohgo(N-isopropylacrylamide) conJugates. Bzocon]ugate Chem 4,509-5 14. 4. Rao, M., Seeta, R., and Deshpande, V. (1983) Effect ofpretreatment on the hydrolosls of cellulose by Penicdlum funlculosum cellulase and recovery of enzyme. Blotech. Bzoeng. 25,1863-l 873. 5 Hoshmo, K , Tamguchr, M., Katagni, M., and Fujii, M (1992) Properties of amylase immobilized on a new reversibly soluble-insoluble polymer and its application to repeated hydrolysis of soluble starch J Chem Eng Jpn 25,569--574 6 Hoshino, K., Katagtri, M., Tamguchi, M., Sasakura, T., and Fugu, M. (1994) Hydrolysis of starchy materials by repeated use of an amylase mrmobrhzed on a novel thermo-responsive polymer. J Ferment. Bloeng. 77,407-412. 7. Heskms, M., Gutllet, J. E., and James, E. (1968) Solution properties of poly (N-isopropyl acrylamide). J. Macromol. Ser. Chem A2, 1441-1445 8. Schid, H. G. and Tirrell, D. A. (1990) Microcalorimetric detection of lower critical solution temperature in aqueous polymer solutions J Phys Chem 94,4352-4356 9. Ito, S (1989) Phase transition of aqueous solution of poly (N-alkyl acrylamide) derivatives: effects on side-chain structure (in Japanese) Kobunshz Ronbunshu 46, 437-443. 10. Fujishige, S. (1987) Intrmsic viscosity-molecular weight relationships for poly (N-isopropyl acrylamide) solutions. Polymer, J. 19,297-300. 11 Riehm, J. P. and Scheraga, H. A. (1966) Structural studies of ribonuclease. XXI. The reaction between ribonuclease and a water-soluble carbodumrde Blochemrstry 5,99-l 15. 12. Kondo, A., Imura, K., Nakama, K., and Higashitam, K (1994) Preparation of immobihzed papain using thermo-sensitive latex particles. J Ferment Bioeng 78, 24 l-245 13. Smith, P. K., Krohn, R. I., Hermanson, G. T., Malha, A. K., Gartner, F. H., Provenzano, M. D., FuJimoto, E K., Goeke, N. M., Olson, B J , and Krenk, D. C. (1985) Measurement of protein using bicinchommc acid. Anal. Biochem. 150,76-85 14. Lowry, 0. H , Rosenbrough, N. J., Farr, A. L., and Randall, R. J. (195 1) Protem measurement with the Folm-phenol reagent. J Bzol. Chem. 193,265-275

14 Immobilization of Photosynthetic Membranes in an Albumin-Glutaraldehyde Crosslinked Matrix Robert Carpentier 1. Introduction The photosynthetic membranes of chloroplasts are responsible for capturing light energy and converting it into chemical intermediates. During this process, negatively charged species are formed and water molecules are cleaved. These properties confer to thylakoid membranes a great potential for various biotechnological appltcations. However, the isolated membranes have a relatively short active life time that limits their effective use. To circumvent this limitation, various immobilization techniques have been designed to improve the stability of biological functions. Isolated chloroplasts or thylakoid membranes have been immobilized using various procedures, but immobilization in an albumin-glutaraldehyde crosslinked matrix provides a better preservation of the native thylakoid activity and maintains a greater functional stability when compared to immobilization by several other techniques (1,2). This immobilization method provides good protection against aging and strong illumination (3-5). It can be used not only in whole thylakoid membranes (6) but also in submembrane fractions enriched in photosystem I or photosystem II (7,s). In this chapter, methods are described to obtain membrane preparations of either whole thylakoid membranes (9) or isolated submembrane fractions (10, II), followed by then immobilization in an albumin-glutaraldehyde crosslinked support. The storage properties of the immobilized material are also mentioned since these are essential for the proper use of immobilized photosynthettc membranes. 2. Materials 2.1. Isolation of Whole Thylakoid 1. Fresh spinachleaves. From

Membranes

Methods m Bofechnology, Vol I. Immobrbatfon of Enzymes and Cells Edlled by G F BIckerstaff Humana Press Inc , Totowa, NJ

109

Carpentier

110

2. Homogenization buffer 1. 20 mM TES-NaOH buffer, pH 7.5, containing 5 mM MgC12, 330 mM sorbitol, and 1 mM phenylmethylsulfonyl fluoride (PMSF) (see Notes l-4). 3. Cheesecloth 4. Acetone 80% (v/v) in distilled water. 5. Resuspension buffer 1.60 mMTES-NaOH buffer, pH 7.5, contammg 1 mMNaC1, 5 mM MgC&, 1 mMNH&l, and 330 mM sorbitol (see Notes 1,5)

2.2. Isolation

of Photosystem

II Submembrane

Fractions

1 Fresh spinach leaves. 2 Homogenization buffer 2: 50 mM Tricme-NaOH buffer, pH 7.6, containing 10 mM NaCl, 5 mM MgC12, 400 mM sorbitol, 6 mM ascorbate, and 1 mM phenylmethylsulfonyl fluoride (PMSF) (see Notes 14). 3 Cheesecloth 4 Hypotonic buffer 2. 50 mM Trtcme-NaOH buffer, pH 7.6, contammg 10 n-J4 NaCl, 5 mM MgC12, and 6 mM ascorbate. 5. Acetone 80% (v/v) m distilled water. 6 Incubation buffer 2: 20 mMMES-NaOH buffer, pH 6.5, containing 15 rnA4NaCl and 10 mMMgC12 7 Resuspension buffer 2: 20 mM MES-NaOH buffer, pH 6.5, containing 1 mM NaCl and 5 mM MgCl*. 8. 4% (v/v) Triton X- 100 solution.

2.3. Isolation

of Photosystem

I Submembrane

Fractions

1 Fresh spinach leaves. 2. Homogenization buffer 3: 50 mM Tricme-KOH buffer, pH 7.8, contammg 10 mM KCI, 10 mMNaCl,5 nGt4 MgC12, 700 mM sorbitol, and 1 mM phenylmethylsulfonyl fluoride (PMSF) (see Notes 14). 3. Cheesecloth 4. Hypotonic buffer 3: 20 mM Trtcine-KOH buffer, pH 7.8, containing 20 m&f MgCl*, and 1 mA4phenylmethylsulfonyl fluoride (PMSF) (see Note 4). 5. Isotonic buffer 3. 20 mM Tricine-KOH buffer, pH 7.8, containing 20 mM KCl, 20 mMNaC1,500 mMsorbito1, and 1 mMphenylmethylsulfony1 fluoride (PMSF) (see Notes l-4) 6. Acetone 80% (v/v) m distilled water. 7 Incubation buffer 3: 20 mMTricine-KOH buffer, pH 7.8, containing 10 mMKC1, 10 mM NaCl, 5 mM MgC12, and 250 mM sorbitol (see Notes l-3). 8. Resuspension buffer 3: 20 mA4 Tricine-KOH buffer, pH 7.8, containing 10 mA4 KCI, 10 mMNaC1, and 5 mM MgCl,. 9. 0.2% (w/v) digitomn solution.

2.4. Immobilization

Procedure

1. Immobilizatton buffer: 50 miU phosphate buffer, pH 7.1, for whole thylakoid membranes; 20 mM MES-NaOH, pH 6.5, for photosystem II preparations; or

111

immobilized Photosynthetic Membranes

20 mM Tricine-NaOH, pH 7.8, contaimng 10 mM KCl, 10 mA4 NaCl, and 5 mA4 MgCl*, for photosystem I preparations. 2. 20% (w/v) bovine serum albumm (BSA) solution. 3. 1.5% (v/v) glutaraldehyde solution.

3. Methods 3.1. Isolation

of Whole Thylakoid

Membranes

1. Weigh 100 g of devemed spinach leaves. 2. Clean the spinach leaves in cold distilled water and dry on absorbent paper. 3. Cut the leaves in small pieces and place them with 300 mL of homogenization buffer 1 in a Waring blender with sharp blades (see Note 6) 4. Homogenize for about 30 s using the pulse mode. 5. Filter the slurry through 12 layers of cheese cloth 6. Centrifuge the filtrate for 1 min at 2OOOg. 7. Resuspend the pellets in 40 mL of a 20-fold dilution of homogenization buffer 1. 8 Centrifuge the solution for 1 min at 4000g. 9. Resuspend the pellets (thylakoid membranes) in a small volume of the resuspension buffer 1 and then dilute to 3 3 mg chlorophyll/ml 10. Chlorophyll IS determined in 80% acetone as described in Section 3 5.

3.2. isolation

of Photosystem

II Submembrane

Fractions

1. Weigh 100 g of devemed spinach leaves. 2. Clean the spinach leaves m cold drstilled water and dry on absorbent paper. 3. Cut the leaves in small pieces and place them with 300 mL of homogenization buffer 2 in a Waring blender with sharp blades (see Note 6). 4. Homogenize for about 30 s using the pulse mode 5. Filter the slurry through 12 layers of cheesecloth. 6. Centrifuge the filtrate for 5 min at 2OOOg. 7. Resuspend the pellets in 40 mL of hypotonic buffer 2 8. Centrifuge the solutton for 5 min at 2000g. 9. Resuspend the new pellets in the incubation buffer 2 to obtain a chlorophyll concentration of 1 mg/mL after the further addition of 4% (v/v) Triton X-100 (see Notes 7,8). 10. Incubate the mixture for 20 min in the dark at 4’C with gentle stirring. 11. Add more incubation buffer 2 to obtain a twofold dilution. 12. Centrifuge the solution for 10 min at 36OOg. 13. Pool the supernatants and centrifuge for 30 min at 36,000g. 14. Resuspend the pellets (photosystem II preparation) in a small volume of resuspension buffer 2 and then dilute to a chlorophyll concentration of 3.3 mg/mL. 15. Chlorophyll is determined in 80% acetone as described in Section 3.5.

3.3. Isolation

of Photosystem

I Submembrane

Fractions

1. Weigh 100 g of deveined spinach leaves. 2. Clean the spinach leaves m cold distilled water and dry on absorbent paper.

112

Carpen tier

3. Cut the leaves m small pieces and place them with 300 mL of homogenizatron buffer 3 m a Waring blender with sharp blades (see Note 6) 4. Homogenize for about 30 s using the pulse mode. 5 Filter the slurry through 12 layers of cheesecloth. 6 Centrifuge the filtrate 5 mm at 3000g 7 Resuspend the pellets m 40 mL of hypotonic buffer 3. 8. Incubate the solution for 10 mm on ice in the dark 9. Add 40 mL of isotonic buffer 3 10 Centrifuge for 5 mm at 3000g 11. Resuspend the new pellets m the incubation buffer 3 to obtain a chlorophyll concentration of 2 mg/mL after the further addition of 0.2% digitonm (see Note 9) 12 Incubate the solutton for 30 min in the dark at 4°C with gentle stirring. 13 Add more incubation buffer to obtain a threefold dilution 14 Centrifuge for 30 mm at 36,000g 15. Pool the supernatants and centrifuge for 1 h at 100,OOOg 16 Resuspend the final pellets (photosystem I preparation) in a small volume of the resuspension buffer 3 and then dilute to a chlorophyll concentration of 3.3 mg/mL 17 Chlorophyll is determined in 80% acetone as described in Section 3.5

3.4. lmmobiliza tion Procedure 1 Mix 1 65 mL of immobilization buffer specific for the preparation of photosynthetic membranes used with 1.25 mL of 20% BSA and 1 mL of 1 5% glutaraldehyde (see Note 10) 2. Incubate for 2 min at room temperature. 3 Add 0 6 mL of photosynthetic membranes diluted to 3 3 mg chlorophyll/ml (see Note 11). 4. Mix for 34 s using a vortex mixer 5. Pour the preparation in appropriate vessels, such as Petri dishes or test tubes, to obtain the desired shape and volume of immobilized material (see Note 12) 6. Store for 2 h at -20°C. 7. Thaw the immobilized preparations at 4°C m the dark for at least 2 h before use Best activities are obtained after 12 h. 8. Wash with distilled water using a gentle vacuum before use (see Note 13) 9 The immobilized membranes are less sensitive to strong light and to room temperature than are the free ones (3,4). However, immobilized membranes start to lose their activity immediately at room temperature and light. They are fully stable for 24 h m the dark at room temperature and for 200 h m the dark at 4°C Under the latter conditions, 40% of the initial activity was retained after a storage period of 1000 h (12).

3.5. Determination

of Ch/orophy//

Concentration

1. Add 10 pL of photosynthetic membrane to 5 mL acetone 80% in a conical tube and mix carefully using a vortex mixer (see Note 14). 2. Centrifuge in a bench-top centrifuge for a few minutes to remove precipitated proteins

lmmobllized Photosynthetic Membranes

113

3. Verify the exact volume (5 mL) and adjust if necessary to compensate for evaporation. 4. Measure the absorbance at 663 and at 645 nm. 5. Taking the dilution of the membranes in the acetone solution into account, the chlorophyll concentration (mg/mL) in the membrane preparation is calculated from the following equation: 0.5 [22.22 (A& + 9.05 (A,&], m which A643 and AG6srepresent the absorbancies at the respective wavelengths (see Note 15)

4. Notes 1. This buffer should be prepared just before use. 2. Sorbitol is used to keep the medium isotonic; sugars are also known to help in the protection of btological membranes against denaturation. 3 Sorbitol can be prepared in advance as a concentrated solution (2M) and kept at -2OOC. It is diluted to the required concentration during the preparation of the buffer 4. Phenylmethylsulfonyl fluoride is used as an inhibitor of proteases It should be prepared as a concentrated solution (1M) in dioxane and diluted to the proper concentration during the preparation of the buffer. 5. NH&l is added as an uncoupler that prevents the formation of a pH gradient across the thylakoid membrane. This increases the rates of electron transport, 6. The chamber of the blender and all the solutions used must be ice cold when used. During the preparation, care must be applied to always keep the material m a cold (near 4°C) environment. 7. Triton X-100 should be prepared as a 20% (v/v) solution. Mixing should be done carefully to avoid the formation of foam. 8 The membranes should be first resuspended in a small volume for chlorophyll determination. Then the final volume required to obtain 1 mg chlorophyll/ml is calculated and the volume of Triton X-100 required (to obtain 4% in the final volume) is subtracted from the final volume. The remaining volume of buffer is added before Triton X-l 00. Triton X- 100 is finally added drop by drop with gentle stirrmg on ice. 9. The membranes should be first resuspended in a small volume for chlorophyll determination. Then the final volume required to obtain 2 mg chlorophyll/ml is calculated and the actual volume of the preparation IS subtracted from thts volume. The volume of mcubation buffer 3 to be added to adjust the final concentration is used to prepare a digitonin solution containing the amount of detergent required to obtain 0.2% in the final volume. It may be necessary to heat this solution for about 5 min to improve the solubility of digitonin. Cool down the solution and add it progressively to the membrane suspension with gentle stirring on ice 10. Several immobilizatton buffers were tested for each type of photosynthetic membranes and the buffer used here provides the best retention of native photosynthetic activity and storage stability. 11. The chlorophyll concentration in the immobilization medium is important to obtain the chlorophyll/albumin-glutaraldehyde ratio that provides the best

114

12

13. 14

15.

Carpentier preservation of the photosynthetic membrane integrity together with optimal immobilization. The final immobilized material presents a soft sponge-like green tissue structure It is important to choose the appropriate vessel for immobilizatton since it can be used to mold the immobilized material to any shape or volume required. Small samples containing an exact amount of photosynthetic membranes (m terms of chlorophyll concentration) mnnobihzed m test tubes were routinely used m our studtes. For some applications, the immobilized material may be crushed into small particles usmg a mortar and pestle (12) The method of Arnon (13) was used for many years and is now used with the corrections of Porra et al. (14). The sample of photosynthetic membranes should be added to the acetone while mixing. This is necessary to minimize the amount of chlorophyll that remains bound to the precipitated proteins Several replicates should be done to obtain a better estimation of the chlorophyll concentration If the concentration is above 6 mg/mL, it is better to prepare a first dilution just above the required final concentration and to determine the chlorophyll concentratton m this predilution before final dilution.

References 1. Cocquempot, M. F., Thomasset, B., Barbotin, J. N., Gelif, G., and Thomas, D. (198 1) Comparative stabilization of biological photosystems by several mrmobihzation procedures Eur J. Appl. Mzcroblol Bzotechnol. 11, 193-198. 2 Thomasset, B., Barbotm, J. M., Thomas, D., Thomasset, T., Vejux, A., and Jeanfils, J. (1983) Fluorescence and photoacoustic spectroscopy of munobihzed thylakoids. Blotechnol Btoeng 25,2453-2468. 3. Carpentier, R., Leblanc, R. M., and Mimeault, M. (1987) Photoinhibition and chlorophyll photobleaching m immobilized thylakoid membranes. Enzyme Mzcrob Technol. 9,489-493. 4. Carpentier, R., Leblanc, R. M., and Mimeault, M. (1988) Monitoring electron transfer by photoacoustic spectroscopy in active and munobillzed thylakoid membranes Blotechnol Bloeng 32,64-67. 5. Lemieux, S. and Carpentier, R. (1988) Properties of immobilized thylakoid membranes m a photosynthetic photoelectrochemical cell. Photochem Photoblol 48, 115-121. 6. Thomasset, B., Thomasset, T., Vejux, A., Jeanfils, J., Barbotin, J. N., and Thomas, D (1982) Immobthzed thylakoids in a crosslinked albumin matrix. Plant Physiol 70,714-722. 7 Carpentier, R. and Lemieux, S (1987) Immobilization of a photosystem II submembrane fraction m a glutaraldehyde crosslmked matrix. Appl Brochem Biotechnol 15,107-l 17 8 Bonenfant, D. and Carpenner, R. (1990) Stabilization of the structure and functions of a photosystem I submembrane fraction by mrmobihzation in an albumin glutaraldehyde matrix. Appl. Biochem Biotechnol 26,59-7 1

Immobilized Photosynthetic Membranes

115

9. Purcell, M , Leroux, G. D., and Carpentier, R. (1990) Atrazine action on the donor side of photosystem II in triazme-resistant and -susceptible weed biotypes Pest Blochem Physlol. 37, 83-89.

10 Berthold, D. A., Babcock, G. T., and Yocum, C. F. (1981) A highly resolved, oxygen evolving photosystem II preparation from spinach thylakoid membranes: EPR and electron-transport properties. FEBS Lett 134,23 l-235. 11 Peters, F. A. L. J., Van Wielink, J. E., Wong Fong Sanf, H. W., De Vries, S., and Kraayenhof, R. (1983) Studies on well coupled photosystem I-enriched subchloroplast vesicles. Content and redox properties of electron transfer components. Bzochlm. Blophys. Acta 722,460-470. 12 Loranger, C. and Carpentier, R. (1994) A fast bioassay for phytotoxicity measurements using immobihzed photosynthetic membranes. Biotechnol. Bzoeng 44, 178-183 13. Arnon, D I. (1949) Copper enzymes m isolated chloroplasts. Polyphenoloxydase in B. vulgans. Plant Physzol. 24, 1-15. 14. Porra, R. J., Thompson, W A., and Kriedemann, P. E. (1989) Determination of accurate extinction coeffictents and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Blochim Bzophys Acta 975,384--394.

15 Poly(Ethylene Glycol) Crosslinked to Albumin as a Support for Enzyme Immobilization Guy Fortier, Nicole Demers, Jacques Jean-Franqois, Jean-Charles Gayet, and Edith M. D’Urso 1. Introduction Medical applications of enzymesare found in various areas, such as diagnosis, prognosis, and therapy. They are used as soluble or immobihzed forms m replacement and detoxrfrcation therapies, as scavengers and as antmeoplasic drugs, and also for the prevention of clot formation (I). When immobihzed enzyme systems are needed for in vivo or ex vivo applications, the use of a biocompatible and a nonthrombogenic immobilization support is an important requirement. Based on the findings that poly(ethylene glycol) IS a biocompatible polymer (the work of Abuchowski [2/ demonstrated that a protein modified with PEG has a reduced immunogenicity and a longer plasmatic life), and that the surface modificatron of an enzyme with this polymer increases its structural and catalytic stabilities (3), it was apparent to us that poly(ethylene glycol) is a good polymer for the elaboration of a biocompatible matrix for enzyme immobilization. We have evaluated the feasibility to reticulate enzyme with activated polyethylene glycol in order to obtain a urethane lmk between PEG and ammo groups of the protem. Following observations that multisubunit enzymes,such as catalase and glucose oxidase, redissolved after support matrix polymerization because of the dtssociation of the subumts during the swelling phase of the support matrix, we have introduced bovine serum albumin (BSA) as a coreticulatmg protein for hydrogel preparation. From these preliminary studies, we have developed a family of hydrogels based on crosslinking poly(ethylene glycol) of different molecular masses(actrvated with 4-nitrophenyl chloroformate) with free ammo From

Methods m Botechnology, Vol 1 /mmob//uabon of Enzymes and Cells Edlted by G F Blckerstaff Humana Press Inc , Totowa, NJ

117

Fortier et al.

118

groups (from lysyl residues) of serum albumin (4,5). Poly(ethylene glycol) of molecular mass ranging from 2,00&35,000 inclusively, can be used to prepare hydrogels of varying rrgrdity. The use of low molecular mass of PEG tends to harden the hydrogel, whereas the use of higher molecular mass of PEG (X3000) provides hydrogels with more elastrcity. The BSA-PEG hydrogel family is characterized by a 96% water content, translucidity, and good mechanical properties (6). Also, they can be used as supports for drug-controlled release (7) and for enzyme immobilization (8,9). The diffusion coefficrents for small drugs are only a few orders of magnitude smaller than those observed m bulk solution, and the transport of these drugs throughout

the support matrix follows a Fickian difiusion mechanism (7). Good diffusion properties are tmportant for free movement of substrate and product and a good rate of reactton with immobihzed enzymes. The shelf and operatronal stabthties of various enzymes,immobilized in thesehydrogels, were substanttally increased (8). Recent studies have indicated that these hydrogels have a fairly good bro-

compatibility when implanted subcutaneously or mtraperitonially in mice or rats (8). The residual activity of a crosslmked enzyme,such asasparaginasem a BSAPEG hydrogel

(M,. lO,OOO), was as high as 70% after 3 mo of implantation

into

the peritoneal cavity of rats. In this chapter, we describe the preparation of BSA-PEG

hydrogels in general, and m particular BSA-PEG-enzyme

2. Materials 2.1. Poly(Ethylene

hydrogels.

Glycol) Derivatization

1. Polyethylene glycols (PEG) of molecular masses 3350, 4600, 8000, and 10,000 (Sigma, St. Louis, MO); PEG molecular mass 20,000 (Fluka, Buchs, Switzerland, Terochem, Edmonton, Canada). 2 4-Nitrophenyl chloroformate and triethylamine (Aldrich, St. Louis, MO) 3. Buffer solution for the hydrolysis of activated PEG: 0. 1M sodrum borate buffer, pH 8.5. 4 Acetonitrile, HPLC grade 5 Diethyl ether, reagent grade. 6. Anhydrous sodium sulfate (BDH, Montreal, Canada) 7. A Soxhlet extractor with a cellulose extraction thimble (Whatman [Chfton, NJ], 33 x 94 mm). 8. A reflux apparatus 9. Buchner funnel and filter paper (Whatman #l) 10. A hot plate with magnetic stirrer. 11. A spectrophotometer with a kinetrc module.

2.2. Synthesis

of the Hydrogel

1. Serum albumin (SA) solution for hydrogel synthesis when a PEG of a molecular mass ~5000 IS used. 100 mg of SA dtssolved in 1 mL of 400 mM sodmm borate buffer, pH 9.4. (see Note 1).

PEG-Album/n Support

119

Table 1 Protein and PEG Composition for Hydrogel Formation Using Activated PEGS of Varying Molecular Mass M, of PEG”

Amount of protein,b mg/mL

Amount of PEG,” mg/mL

100

212 154 236 272 252

3350 4600 8000 10,000 20,000

100 100

100 100

OSeeNotes 11,16 hDlssolved m 400 mM sodium borate buffer, pH 9.4 or 8.5 The protein includes the SA and the enzyme CBased on optlmal molar ratio (OH:NH2) Prepare in distilled water (see Notes 2,17)

2. Serum albumin solutton for hydrogel syntheses when a PEG of a molecular mass >5,000 is used: 100 mg of SA dissolved in 1 mL of 400 mM sodium borate buffer, pH 8.5 3 Poly(ethylene)glycol solution: Depending on the molecular mass of PEG, the amount indicated in Table 1 (see Note 2) is weighed and dissolved in 1 mL of cold distilled water This solution should be prepared just before use 4. Washing solution for the hydrogel 100 mA4 sodium borate buffer, pH 9 4 5. Reagent kit for protein assay using BCA protocol (Pierce, Rockford, IL): Solution A contained bicmchonimc acid, sodium carbonate, sodium bicarbonate, and sodium tartrate in 0.2N NaOH; solution B contained 4% copper sulfate solution and BSA solution at 2.0 mg/mL. 6. Solutions for PEG assay: Prepare standard solutions of PEG of various molecular masses up to 20 pg/mL in water, barium chloride 5% (w/v), 0. 1N iodine m water (see Note 3). 7. Solutions for assay of free NH2 groups using fluorescamme procedure: Prepare standard glycme solutions from 20-200 pM m 100 mM potassium phosphate buffer, pH 9.0; fluorescamine 0.3 mg/mL of acetone (see Note 4). 8. A 5-mL syringe with appropriate needle. 9. An electrophoresis gel maker such as Protean II or MiniProtean II (Bio-Rad, Richmond, CA), two glass plates, and two spacers of appropriate thickness 10. An orbital shaker. 11. A cork former or a calibrated mesh sieve of appropriate size.

2.3. Synthesis of the Enzyme Hydrogel 1. Serum albumin/enzyme solution for the synthesis of the enzyme hydrogel: 90 mg of SA and 10 mg of the enzyme (based on the protein content of the enzyme

720

Fortier et al.

preparatton) IS drssolved in 1 mL of 400 mA4 sodmm borate buffer, pH 8 5 or 9 4, depending on the molecular mass of PEG used (see Notes 4-7). 2. Washing solutron for the enzyme hydrogel: Use a suitable buffer solutron for the immobilized enzyme at the appropriate molarity and pH used for the correspondmg soluble enzyme (see Note 8).

3. Methods 3.1. Poiy(Efhy/ene

Glycol) Derivatization

1 PEGS (Mr 3350,4600, 8000, 10,000, and 20,000) are derivatrzed using 4-nitrophenyl chloroformate to obtain a series of poly(ethylene glycol) dmitrophenyl carbonates (10,ll) 2. Before the dertvatrzatron step, all PEGS are dehydrated by dissolvmg 1 mm01 of a PEG (e.g., 10 g of PEG A4, 10,000) in 100 mL of acetonitrrle containmg 1.5 mm01 of trtethylamme (see Note 9). 3 The mixture IS refluxed at 8O’C for 4 h (corresponding to 80 cycles) in a Soxhlet extractor contammg 2 g of anhydrous sodmm sulfate m the cellulose thimble 4. The dehydrated PEG solution is derrvattzed by directly adding 5 mm01 of 4-mtrophenyl chloroformate/mmol of PEG to the dehydrated PEG acetonitrile solutton. 5 The reaction mixture is heated at 60°C for 5 h and left overnight at room temperature. 6. The reaction mixture 1sfiltered on a filter paper (Whatman #l) under vacuum to eliminate the chloride salt. 7. The PEG-dinitrophenyl carbonate IS precipitated by the addition of 5 vol of cold drethyl ether. The solution is stored overnight in a freezer at -20°C 8 Thereafter, the precipitate is filtered on a Buchner funnel and redissolved in acetonitrile and precipitated again with cold diethyl ether 9 The precrprtate is filtrated and dried under vacuum and stored m a freezer at -20°C (see Note 10) 10. Yield and purity are evaluated spectrophotometrrcally at 400 nm by calculatmg the amount of p-mtrophenol released from the hydrolysis solutron containing a determined amount of activated PEG (see Notes 11-14).

3.2. Synthesis of the Hydrogei

or the Enzyme Hydrogel

1. Crosslinkmg of the activated PEG with the protein 1sachieved by adding 1 mL of the SA solution or 1 mL of the SA-enzyme solutron to 1 mL of the activated PEG solution of the selected molecular mass (see Notes 2,10) 2. The mixing of the two solutions is done on ice and kept cold until air bubbles disappear from the solutron (l-5 mm) 3. The polymerrzation solution is rapidly cast with a syringe between the two glass plates mounted m the gel maker device. 4. Polymerizatron takes place at room temperature for at least 4 h at pH 8 5 (see Note 14) or 2.5 h at pH 9.4 (see Note 14). 5 The top of the plates is covered with plastic wrap or equivalent to avoid evaporation during the polymerrzation step.

PEG-Albumin Support

121

6. The hydrogel or the enzyme hydrogel is demolded and cut wtth a cork former of appropriate size or crunched through a calibrated mesh sieve of selected porosity (see Note 15). 7, The hydrogel or the enzyme hydrogel is extensively washed at 4’C using an orbital shaker until all the p-mtrophenol, free PEG, and proteins are released (see Note 16). 8. This step is performed with a suitable buffer for the enzyme, which can contain NaNs at 0.02% (w/v), if necessary. 9. Wash 1 vol of hydrogel with 50 vol of buffer solution. The washmg solution IS frequently changed during a period of 72 h. 10 The washing solutions can be evaluated for their content m p-mtrophenol at basic pH, their PEG content by iodine assay, and their protein content by BCA assay (see Note 16). 11. Iodine assay: Mix 2 mL of sample with 0.5 mL of barium chloride and 0.25 mL of iodine solutton. Vortex the mixture then incubate in dark at room temperature for 15 min. Read the absorbance at 535 nm. 12. p-Nitrophenol assay: Adjust the pH of solution to 9.0 by adding NaOH and read the absorbance of a 1 mL aliquot taken from the washing solution at 400 nm. 13 Protein assay. Add 1 mL of the working reagent to 0.1 mL of the sample. Incubate at 37°C for 30 mm and read the absorbance at 562 nm. 14. The enzyme hydrogel can now be tested for its activity. Apparent K,,, and V,,, can be determined using appropriate techniques and data analysis. 15 Enzyme hydrogel can be stored in their own buffer solution at 4°C or if they are in a bead form, they can be lyophilizated and stored at -20°C.

4. Notes 1. Human, mouse, or rat serum albumin can be used (at the same amounts) instead of BSA. This is especially interesting for in vivo use of the hydrogel because the same source of albumin may be selected to be compatrble with the receiver 2. All amounts in column 2 of Table 1 are calculated for activated PEG of 100% purity. Thus, it is necessary to correct the amount of PEG required by taking mto account the purity of your batch. Never use a PEG of ~85% purity. 3. The standard curve for PEG assay should be prepared with the same molecular mass of PEG used for the synthesis of the hydrogel. 4. The fluorescamine assay can determine the number of free NH2 groups of the enzyme with a standard curve of glycine. If the enzyme has few lysyl residues, it would be necessary to compensate for this shortage by adding more BSA or by maintaining BSA concentration at 100 mg/mL in the polymerization solution. 5. It is possible to use higher amounts of enzyme, up to 60 mg of protein, and a smaller amount of SA, down to 40 mg. The protein in the polymerization solution should be maintained at a final concentration of 50 mg/mL. 6. It is preferable to evaluate the stability of the enzymatic activity at pH 8.5 or 9.4 overnight before trying to make an enzyme hydrogel. Also, to protect the active site during hydrogel synthesis, it is possible to add a competitive inhibitor without free amino group.

122

Fortier et a/.

7 Be sure that a stabilizer, such as glycme, is not present in the enzyme solution because this can react with the activated PEG. 8 Normally, enzyme m-mobilized in this hydrogel has a variation of its opttmal pH of about 0.5 U. 9 Water has to be avoided durmg the PEG activation step if high yield is wanted Dehydration of PEG and trrethylamme in acetomtrile is a crucial step 10 Always allow the flask containing the activated PEG to rest at room temperature before opening it. Water condensation on the flask will hydrolyze the activated PEG. 11. The molar extmctton coefficients (a) for p-nitrophenol at 400 nm are 15,620 and 16,750 M-Vcmfor pHs 8.0 and 8.5, respectively. At pH >8.5 in O.lMborate buffer, the E value is constant at 17,600 IMilcm. The a were obtamed from p-nitrophenol calibration curves at different pHs and were lmear up to 15 umol/mL. 12. The calculation of purity IS based on the ratio between the amount ofp-mtrophenol released and detected spectrophotometrically vs the amount of p-mtrophenol expected to be released 13. The yield of reaction is calculated by multiplying the purity with the amount of product recovered after the second precipitation. 14. The rates of the different activated PEGS are 35.8 x le3/mm at pH 9.5 and 5.66 x 1p3/min in 100 n-&Yborate buffer, pH 8.5, at 25’C 15 If disks or particles of the enzyme hydrogels are requtred, it is posstble to cut or to process the hydrogel before or after the washing procedure step 16 Percentages of the released proteins and PEG vary wtth the molecular mass of the PEG used for synthesis of the hydrogels Normally, no more than 46% of the initial content of protein are released m the washing solution. The percentage of PEG released varies from 20-30% and depends on the molecular mass and the purity of activated PEG used durmg the synthesis. 17. Optimal molar ratios have been calculated taking into account 2 mol of activated group per mol of PEG and 27 lysyl restdues accessible at the surface of BSA

References 1 Fortier, G. (1994) Biomedical apphcattons of enzymes and their poly(ethylene glycol) adducts. Bzotechnol. Gen Engln Rev 12,32!9--356. 2. Abuchowski, A., van Est, T, Palczuk, N. C., and Davis, F. F. (1977) Alteration of mnnunological properties of bovine serum albumm by covalent attachment of polyethylene glycol. J. Blol. Chem. 252,3578-3581. 3. Laliberte, M., Gayet, J.-C., and Fortier, G. (1994) Surface modification of horseradish peroxidase wtth poly(ethylene glycol) of various molecular masses II Relation between the molecular masses of PEG and the stability of horseradish peroxidase-PEG adducts under various denaturing conditions. Blotechnol Appl Bzochem 20,397-413. 4 D’Urso, E. M. and Fortier, G. (1994) New bioartificial polymeric material. poly(ethylene glycol) crosslmked with albumin. I. Synthesis and swellmg properties. J Bioactwe Compat. Polym. 9,367-387.

PEG-Aibumm Support

123

5. D’Urso, E. M. and Fortter, G. (1994) New hydrogel based on poly(ethylene glycol) crosslmked with bovine serum albumin Biotechnol. Techn. 8,7 l-76 6. Gayet, J.-C. and Fortter, G. (1996) New bioarttficial hydrogels characterization and physical properties, in Hydrogels and Biodegradable Polymers /or Bzoapplications, ACS symposium book series # 627 (Ottenbrite, R., Huang, S , and Park, K., eds.), pp. 17-24. 7. Gayet, J.-C. and Fortter, G. (1996) High water content BSA-PEG hydrogel for controlled release device* evaluation of the drug release properties J Control Rel 38,177-184. 8. D’Urso, E. M., Doillon, C , Jean-Francois, J., and Fortier, G. (1995) Poly(ethylene glycol) crosslinked BSA hydrogels as matrix for enzyme irrmrobtlization biomedical applications. Art$ Cells Blood Subst Immob Biotechnol 23,587-597. 9. D’Urso, E M , Jean-Francots, J., and Fortier, G. (1996) New btoartifictal hydrogels. biomedical matrix for enzyme immobilization, in Hydrogels and Biodegradable Polymers for Bioappbcatlons, ACS symposium book series # 627 (Ottenbrtte, R., Huang, S., and Park, K , eds.), pp. 25-41. 10 Fortter, G. and Lahberte, M. (1993) Surface modification of horseradish peroxtdase wtth poly(ethylene glycol)s of various molecular masses. Preparation of reagents and charactertzatton of horseradish peroxidase-poly(ethylene glycol) adducts. Blotechnol. Appl Biochem. 17, 115-130. 11. Veronese, F. M., Largajolli, R., Boccu, E., Benasst, C. A., and Schtavon, 0. (1985) Surface moditicatton of proteins. Activation of monomethoxy-polyethylene glycols by phenylchloroformate and modificatton of rtbonuclease and superoxtde dismutase. Appl. Biochem Blotechnol 11, 141-152

16 Poly(Carbamoyl

Sulfonate)

Andreas Muscat and Klaus-Dieter

Hydrogels Vorlop

1. Introduction Conventional biopolymers for entrapment of cells (e.g., calcium algmate, K-carrageenan) are nontoxic to cells because of the natural raw materials and the gentle immobilization procedure. However, they have disadvantages in their poor mechanical stability (e.g., they are very sensitive to abrasion in stirred reactors) and biodegradability under nonsterile conditions. Polyurethane (PUR) hydrogels show good mechamcal and chemical stability (I), but the raw material (isocyanate-prepolymer) is toxic to living microorganisms (2). Furthermore, the short handling time (seconds) during the immobilization process makes it nearly impossible to prepare a large amount of spherical biocatalysts. Our approach to solve these physical and engineering problems was based on the idea of using chemically blocked isocyanates for the synthesis of polyurethane supports. Isocyanate-prepolymers react with the blocking agent NaHS03 to form the poly(carbamoyl sulfonate) PCS prepolymer at room temperature (3). When mixed with water, conventional PUR prepolymers crosslink at once (seconds) to form a foam or hydrogel. In contrast to that of the PUR prepolymer, PCS prepolymers show an adjustable gelation time that depends mainly on the pH of the solution (see Fig. l), and the temperature (the higher the temperature, the faster the gelation). At pH 8.5 and room temperature, an aqueous solution of PCS prepolymers ~111gel in 10 s to form a PCS hydrogel but at pH c5.0 the solution can be handled up to 10 h. Precise gelation times can be determined by measuring the viscosity characteristics during the conversion of the PCS solution to a PCS hydrogel network using a rotating cylinder viscometer. Because of this typical gelation behavior, PCS prepolymer solutions, (PCS From

Methods in Biotechnology, Vol 1 /mmobr/rIabon of Enzymes and Cells Edlted by G F Bickerstaff Humana Press Inc , Totowa, NJ

125

126

Muscat and Vorlop

0’

4

I 6

I

I

8

10

12

PH Fig. 1. Relationship between gelation time and pH of an aqueous10% (w/w) solution of PCS at room temperature.

content, 34-45% w/w) can be stored at pH 12.0, and at a temperature of-20°C for some months. Pure PCS hydrogel strips (PCS content lO-15% w/w) have good mechanical characteristics that have been estimated by measurmg the modulus of elasticity and the elongation at breaking point using a tensile testing machine. The PCS hydrogels have a higher elongation (80-120%) at breaking point and a lower modulus of elasticity (0.02-0.04 N/mm2) than other biopolymers, such as agar, calcium alginate, and K-carrageenan (4). Additionally, PCS hydrogels have a higher degree of flexibility in contrast to the natural polymers, which show a plastic behavior when compressed. An important factor for consideration when employing entrapment as a method of munobthzation IS that of drffuston (2). Because diffusion properties are an important characteristic for successful application of an tmmobihzed biocatalyst, we tested the diffusion properties of several substancesm both PCS hydrogel and calcium alginate membranes (see Table 1). The evaluation method for diffusion coefficrent IS described by Daynes (5). The diffusion coefficients of calcium alginate and the PCS hydrogel are in the same range. Also, the molecular weight cutoff of the PCS hydrogel membrane ISbetween the molecular weights

127

PCS Hydrogels Table 1 Diffusion Coefficients and Calcium Alginate

(D) for PCS Hydrogel Gel (25OC). Diffusion coefficient (D) lo-* cm2/s

Substance (A4,)

PCS hydrogel

Calcium algmate gel

Nitrite ion (46) Nitrate ion (62) Ethanol (46) Glucose (180)

1.45 1.41 0 77 0 50

1.69 1.56 0.87 051

of myoglobin (M, 17,800) and albumin (M, 67,000), thus substanceswith a A4,> 67,000 dtd not diffuse through the PCS hydrogel (6). These advantages enable PCS hydrogels to be used in a wide range of biotechnology applications, including agitated reactors as well as m fluid-bed reactors. Previously, we immobilized a nitrifying mixed culture and cells of Paracoccus denitrzficans m calctum algmate beads, PCS hydrogel beads (prepared by the dropping method), and conventional hydrogel cubes. The entrapped nitrrfymg

bacteria were found to have almost the same activity in calcium algi-

nate and PCS hydrogel beads (61, whereas cells in PUR hydrogels had no sigmficant activity because of the toxic raw material (isocyanate-prepolymer). Entrapped I? denztrificans in PCS hydrogel beads had lower activity than entrapped cells in calcmm alginate beads (3). However, Wilke et al. (9) found a reversible deactivation ofI? denitrzjkans in PCS hydrogels during the immobilization procedure. Further work with entrapped Saccharomyces cerevisiae showed that PCS beads (prepared by the dropping method) had similar activity as calcium alginate beads directly after the immobilization, whereas no activity was observed in the case of the conventional PUR hydrogels. The PCS hydrogel membranes have been used to immobilize cells (6) and enzymes (8) In thts chapter, we provtde details for production of PCS hydrogel membranes and PCS hydrogel beads. Two procedures are described for production of beads; dropping method and suspension method. The latter method has the advantages that more beads can be produced quickly, it is possible to produce beads of diameter Cl mm, and there are no buffer restrictions (e.g., phosphate) that apply to calcium

alginate

formation.

2. Materials 2. I. Membranes 1. Aqueous solutions of PCS prepolymer (PCS content: 38% [w/w], pH 1.0; see Notes 1,2) (SensLab, Leipzig, Germany).

128 2 3. 4. 5

Muscat and Vorlop 10 &phosphate buffer, pH 7 0 (see Note 2). 20% (w/v) NaOH solution. PTFE (Teflon) plate with a silicone rubber gasket around the plate or a Petri dish. Cells (bacterial or yeast)

2.2. Formation

of Beads by Dropping

1. Aqueous solutions of PCS-prepolymer (PCS content: 38% [w/w], pH 1 0; see Notes 1,2) (SensLab). 2 1% (w/v) CaCl* solution. 3 20% (w/v) NaOH solution. 4. 0.75% sodium algmate (Protanal LF 20/60, Protan, Drammen, Norway) solution adjusted to pH 8 5 with 1% (w/v) NaOH solution. 5. 1% (w/v) phosphate buffer (e.g., sodium tripolyphosphate), pH 8.5. 6. Cells (bacterial or yeast) m appropriate buffer (see Notes 2,3).

2.3. Formation

of Beads by Suspension

1 Aqueous solutions of PCS-prepolymer (PCS content: 38% [w/w], pH 1.0, see Notes 1,2) (SensLab) 2. 20% (w/v) NaOH solution. 3 Vegetable oil with dynamic viscosity of approx 60 (20°C) and 32 mPa/s (37’C) 4 Sieve (0.1 or 0.2 mm). 5. Cells (bacterial or yeast) in appropriate buffer

3. Methods 3.1. Membranes 1 Add 4 g of an aqueous PCS prepolymer solution to 5 g of a suitable buffer (pH 7 0) and mix well. If necessary, add concentrated NaOH dropwlse (syrmge) to bring the pH of the mixture to 7.0. Take care to ensure thorough mixing m-unediately (see Note 4). 2. Add 1 g wet cell mass to this solution quickly and mix well (see Note 5). 3. Pour the mixture onto a glass plate or the Petri dish (see Note 6). 4. Spread the solution uniformly over the surface. 5. Cover the plate or dish to prevent the PCS hydrogel membrane from drying 6. After approx 0.5-l h, submerge the plate or dish m buffer required for the cells (approx pH 8.5). 7. After 1 h, the PCS hydrogel-m-moblhzed cell membrane 1seasily removed from the plate.

3.2. Formation

of Beads by Dropping

1 Add 2 6 g of aqueous PCS prepolymer solution to 6.4 g of CaC12 solution, mix well, and adJust the pH to 6.5 wrth NaOH solution (dropwlse). Take care to ensure thorough mixing immediately (see Note 4). 2. Add 1 g wet cell mass to this solution and mix quickly (see Note 5).

129

PCS Hydrogels Cell mass

PH

PCS-solution ~~

u Solution

l

of CaCl2

Adjustment of pH up to 6.5

I H

p2

’

?a**.*..

..**... *...... .*..... .**.*.. l

*.*...

*..*.** ,

Simultaneous formation of a Ca-alginate layer and gelation of the core

l

.**.*.

,

Na-alginate solutio pH 8.5

Fig. 2. Production of spherical PCS hydrogel beads by the dropping method. PCS-Solution mixed with ~$1 mass

PCS-solution

Adjustment of pH up to 7.5

PCS-solution mixed with cell mass

Vegetable oil T = 37 ‘C

Fig. 3. Production of spherical PCS hydrogel beads by the suspension method. 3. Transfer the suspension to the apparatus for immobilization (see Fig. 2) (7). 3. Drop the resulting suspension from the apparatus into 200 mL of sodium alginate solution, softly stirred by a magnetic stirrer (see Note 7). 4. After a suitable time (90% m each case.

5 Wash the RBC three times with PBS to remove the excess of biotm 6. Resuspend at 10% Ht.

3.3. Deferminafion

of the Number of Biofin Molecules

on RBCs

1 To 600 PL of RBC suspension (Ht lo%), add the BSA stock solution to reach a final concentration of 0 2% (w/v) and ‘251-avidin to provide 1 x lo6 cpm (see Note 6) 2 Incubate 1 h at 4°C 3. Wash three times with PBS and resuspend to 1.O mL 4. Count the radioactivtty and the cell number m each sample. 5 Calculate the number of biotin molecules/RBC (see Table 1, Note 7)

3.4. Determination

of the Number of Biofin-Labeled

RBCs

1. To 5 l.tL of diluted biotinylated RBC suspension (2-5% Ht), add 20 pL of FITCstreptavidm previously diluted. Set a control with unbiotmylated RBC. 2. Incubate m the dark at 4°C for 30 min. 3. Wash three times with PBS. 4. Resuspend to 2.0 mL with PBS. 5. Analyze IO4 cells using a FACScan (Becton Dickrnson). 6. The number of biotin-labeled RBCs is the number of fluorescent positive cells in your sample (see Fig. 2)

3.5. Biofinylafion

of Proteins with NHS-Biofin

1. Add 6 pL/mL of NHS-Biotm 2 Incubate for 1 h at 4°C

(see Note 8)

stock solution to the protein solution (1 mg/mL)

Chiarantini and Magnani

BIOTINYLATED

RBC

FLUORESCENCE INTENSITY

L

1’

Fig. 2. Flow cytometry of biotmylated human RBCs. Human RBCs were blotmylated with NHS-blotin or NHS-LC-blotm or biotin hydrazlde as described under Sections 2. and 3. Biotmylated cells were detected with FITC-streptavldm. As a negative control, unblotmylated RBCs were used. 3. Add the blocking solution to reach a final concentration of 0.M. 4. Incubate for 10 min at 4°C. 5. To remove the excess of blotin, dialyze the solution overnight against PBS using dialysis tubing with a cutoff compatible with the size of your protein (see Note 9). 6. Assay the protein concentration using the Bio-Rad method following mstructlons supplied with the kit. 7. Store at -20°C or better at -80°C m small aliquots Blotinylation 1s stable for several months

3.6. Bio tiny/a tion of Proteins with Bio tin Hydrazide (see Note 70) 1. To the protein solution, add the oxidizing solution to reach a final concentration of 10 m&f. This reaction has to be performed for 30 min on ice in the dark.

Immobilization on Red Blood Cells

149

2. To stop the oxidation, add the glycerol to reach a final concentration of 15 mM Allow it to react for 5 mm at room temperature. 3 Dialyze the sample overnight at 4°C against 0. IA4 sodium acetate buffer, pH 5.5. 4 To the dialyzed sample, add the biotin hydrazide stock solution to reach a final concentration of 5 0 mM. 5. Incubate for 2 h at room temperature. 6. To remove the excess of biotin see Section 3.5., step 5 and Note 9. 7. Assay the protein concentratton using the Bio-Rad method according to the instructions provided with the kit. 8. Store at -20°C or better at -80°C in small aliquots. Biotmylation IS stable for several months.

3.7. Avidin Binding to Biotinylated RBCs 1. Add BSA to biotmylated RBCs to reach the final concentration of 0 2% (w/v) and dilute the biotmylated RBC suspension 1: 1 (v/v) with the stock solution of avidin. 2 Incubate for 1 h at 4°C under constant gentle agitation to minimize the agglutination process. 3. Wash three times with PBS to remove the excess of avidin. 4. Resuspend the RBC to 10% Ht.

3.8. Determination of the Number of Biotin-Avidin-Labeled

RBCs

1. To 5 l.tL of diluted biotm-avidin-labeled RBC suspension (2-5% Ht), add 20 pL of diluted FITC-biotm solution. Set a control with untreated RBC 2. Incubate in the dark at 4°C for 30 mm. 3. Wash three times with PBS. 4. Resuspend to 2.0 mL with PBS 5. Analyze lo4 cells using a FACScan (Becton Dickmson). 6. The number of biotin-avidin-labeled RBCs is the number of fluorescent-positive cells in your sample (see Fig. 3).

3.9. Binding of Biotinylafed Proteins to RBC Membrane 1. Add (under agitation) the stock solution of BSA to your biotin-avidin-labeled RBC to reach a final concentration of 0.2% (w/v). 2. Add the biotinylated protein (from Section 3.6.) under constant gentle agitation (see Note 11). 3. Incubate for 1 h at 4°C under constant gentle agitation. 4. Wash twice with PBS to remove unreacted biotinylated protein. 5. Resuspend to 10% Ht. 6 The RBCs are ready to be used for further experimentation.

4. Notes 1. Adjust the pH and sterilize by filtration using Millipore filter of 0.22 pm. Collect in sterile bottles and store at 4’C for not more than 2 mo.

Chiarantini and Magnani BIOTIN-AUIDIN

LABELLED

RBC

FLUORESCENCE INTENSITY

Fig. 3. Flow cytometry of biotin-avidm-labeled RBCs. The NHS-biotinylated and NHS-LC-biotinylated cells were incubated with avidm. Biotm-avidin-labeled RBCs were detected wtth FITC-biotin as described under Sections 2. and 3. As a negattve control, biotinylated RBCs were used.

2. This molecule is an N-hydroxysuccinimide (NHS) ester of biotin with a M, of 341.38, and it reacts with primary ammes. 3. This molecule is a biotin analog with an extended spacer arm of approx 22.4 nm m length with aM, of 556.58, and it reacts with primary amine as the NHS-Biotm 4. This molecule reacts with the aldehyde groups of carbohydrates generated by a mild oxidation of cis-diol groups with NaI04 (ZO). 5. Avidin is a glycoprotein that contains four identical subunits with a molecular mass of 67 kDa. Avidm is able to bind four distinct molecules of btotm (Ka = lO’U4). 6 We recommend a set of control incubations using unbiotmylated RBCs, and that all the determinattons be done in duplicate 7. Calculate the number of biotin molecules/RBC (see Table 1) according to Suzuki and Dale (II), using the following formulas:

Immobilization on Red Blood Cells A=(Bl-B2)/CxR

151 (1)

A: Avidin concentratton (pg/mL RBC). B 1: Radioactivity of the sample (cpm). B2: Radioactivity of the control sample (cpm). C: Specific radioactivity of 1x51-avidin (cpm/pg) R: Total cells in the assay. N = (A x 6.02 x 1023)/6.8 x 1O’O (2) N: Number of avidm molecules per RBC. il& of avidin 6.8 x 10i”(pg/mol). Avogadro’s number 6 02 x 1O23(molecule/mol). 8 This procedure should be performed in advance respect to RBC biotinylation 9. An alternative method to remove excess biotin: Wash the biotinylated protein three times using the Centricon 10,30, or 100 centrifugation tubes as indicated in the instructions provided by the supplier. This can be done by repeated dilutions and centrifugal concentrations of your sample (i.e., 0.1 mL of biotmylated protem solution diluted to 2.0 mL and reconcentrated to 0.1 mL). 10 Biotmylation of proteins with biotin hydrazide: This procedure shouId be performed in advance respect to RBC biotinylation. This procedure is recommended if you are working with glycoproteins. Il. To evaluate the quantity of the protein that you have to add to the cell suspension you must know the relative number of biotin molecules/RBC This number changes from one species to another (see Table 1) (e.g , for mouse RBCs you can have approx 1000 molecules of biotin/RBC, and consequently a maximum of 1000 molecules of avtdin/RBC. Do not forget that avidin can bmd 4 molecules of biotm at the same time. Making this theoretical calculation you can have a maximum of 3000 molecules of biotinylated protein/RBC)

References 1. Chiarantini, L., Droleskey, R., Magnani, M., Kirch, H., and DeLoach, J. R (1992) Targeting of erythrocytes to cytotoxic T-cells, in The Use of Resealed Erythrocytes as Carrier and Bioreactors (Magnani, M. and DeLoach, J. R., eds.), Plenum, New York, pp. 257-267. 2. Magnam, M , Chiarantmi, L., Vittoria, E., Mancim, U., Rossi, L., and Fazi, A. (1992) Red blood cells as an antigen-delivery system. Bzotechnol. Appl Biochem. 16,18&194.

3. Magnani, M., Mancini, U., Bianchi, M., and Fazi, A. (1992) Comparison of uricase bound and uricase-loaded erythrocytes as bioreactors for uric acid degradation, in The Use of Resealed Erythrocytes as Carriers and Bioreactors (Magnani, M. and DeLoach, J. R , eds.), Plenum, New York, pp. 189-194. 4. Muzykantov, V R , Samokhin, G. P., Smnnov, M. D., and Domogatosky, S. P. (1995) Hemolytic complement activity assay in micro titration plates. J, Appl Blochem. 7,223-227.

5. Gold, E. R. and Fudenberg, H. H. (1967) Chromic chloride: a coupling reagent for passive hemagglutmation reactions. J. Immunol. 99,859-865.

152

Chiarantini and Magnani

6 Jon, Y H and Banker& R. B. (198 1) Couplmg of protein anttgens to erythrocytes through dtsulphide bond formatton: preparatton of stable and sensmve target cells for immune hemolysw. Proc. Nat1 Acad Scr USA 78,2493-2496. 7 Mounetmne, Y., TOSI, P F., Barhoulmi, R., and Ntcolau, C. (199 1) Electromsertion of xeno proteins in red blood cell membranes yield a long lived protem carrier m circulation Bzochzm Bzophys. Acta 1066, 83-89 8 Samokhm, G. P, Smunov, M. D., Muzykantov, V R., Domogatsky, S P, and Smirnov, V N (1983) Red blood cell targeting to collagen-coated surfaces FEBS Lett 154,257-261. 9 Magnam, M., Chiarantmi, L., and Mancini, U (1994) Preparation of characterrzatton of btotmylated red blood cells Blotechnol. Appl Blochem 20,335-345 10. O’Shannessy, D. J., Vaarstad, P. J., and Quarles, R H (1987) Quantitation of glycoprotems on electroblots using the biotin-streptavtdm complex. Anal Bzochem 163,204-209.

11. Suzuki, T. and Dale, G. L. (1987) Biotinytated erythrocytes: zn vzvo survival and zn vztro recovery Blood 70,791-795.

19 Cellulose

Paper Support for Immobilization

Marion Paterson and John F. Kennedy 1. Introduction The use of cellulosic materials as supports for immobihzation of small molecules, proteins, and cells has received considerable attention for many years and possible applications have been pursued extensively (l-24). Chemically, cellulose IS composed of @+glucopyranosyl units linked by (1 + 4) bonds (Fig. 1) and with additional interchain interaction through hydrogen bonds, some of which form the so-called elementary fibrils (15,. Elementary fibrils contain highly ordered crystalline regions and more accessible amorphous regions of a low degree of order. Cellulose IS available in many different physical forms, such as fibers, microgranules, microcrystals, beads, gel particles, capsules, and membranes. Less pure cellulosic materials are used in industrial processesm the form of ropes, pulps, chippings, cloths, and paper. Cellulose has been used most often as a support in ion exchange (26,Z 7) and affinity chromatography (ZS). Its advantages are accessibility, low cost, hydrophilic character, and hydroxyl groups on the surface capable of chemical reaction (see Chapter 1). Its disadvantages as a support are low mechanical resistance, biodegradation, and the heterogeneity m the matrix caused by the crystalline-amorphous regions (the amorphous regions swell more completely and allow deeper penetration of large molecules into the matrrx). Therefore, cellulose is currently employed as a support for immobihzation of proteins to a lesser extent than other polysaccharides, e.g., dextran and agarose. Paper 1sdefined as a thm or layered network of randomly oriented cellulose fibers bound together by hydrogen bonds (19). The relatively low avarlabrlity of potential reactive sites on natural cellulose makes it a medium of low binding capacity, which IS particularly disadvantageous when isolatron and recovery of appreciable quantities of specific biological materrals are desired. However dtsFrom

Methods m Botechnology, Vol 1 /mmob/bat!on of Enzymes and Cells Edlted by G F BIckerstaff Humana Press Inc , Totowa, NJ

753

154

Paterson and Kennedy

Yo”+o-++ HzOH Fig. 1.

Chemical structureof cellulose

advantageous this factor is for the utrhzation of natural cellulose as a general purpose support for chromatography, it is desirable for its apphcation as a support for immobilization of large brological molecules for diagnostic purposes (20). Currently, many proteins are being used for a variety of diagnostic purposes, and there has been a steady increase in the development and use of these protems in the immobilized form (21). The widely spaced substitution sites on the surface of the cellulose microfibril are accessibleto large molecules, such as enzymes, antibodies, antigens, and so on. This minimizes steric hindrance from neighboring macromolecular substituents bound to the support, which m turn permits free diffusion and accessto the active sites on the substituents. Many procedures are available for the immobrlization of biological molecules onto cellulosic materials (3,22-27). Immobihzation of protems onto supports by covalent coupling usually leads to very stable preparations compared with other immobilization procedures, i.e., physical adsorption and romc bindmg. The hydroxyl groups of cellulose are not reactive enough to form covalent bonds between the protein and the support without previous activation. However, cellulose can undergo all reactions associated with polyhydric alcohols to produce a wide range of active materials. Once activated, the support can then react with nucleophihc groups of the protein. Included are the lysyl residues; L-tyrosine, L-histidine, L-arginine, and L-cysteme residues. The permeability, the surface area available for protein attachment, and the reactivity of cellulose largely depend on the degree of crystallinity, the nature, and the size of the compound to be bound, and the swellmg-induced capacity of the activation medium (3). The aim of this chapter is to exemplify two approaches for the covalent immobilization of proteins onto a paper-like cellulosic material by considering the specific example of a-amylase. The cyanogen bromide and carbonyldiimidazole rmmobilization procedures are described. Both procedures follow essentially three steps: a preliminary activation step of the cellulose resulting m the mtroduction of reactive groups, the coupling of the enzyme, and the removal of loosely bound enzyme. The determination of the actlvrty of the immobilized enzyme is also described.

Immobilization on Cellulose Paper

155

Cyanate ester Protein

support

Isourea derivative

Fig. 2. Activation of hydroxyl-containing support using cyanogenbromide In the presenceof triethylamine andcoupling of protein to activated support.

O-TN2 t

0

Activated support

+

& t

Protein

o-fr

Uredane”&ative

Fig. 3. Coupling of protein to carbonyldnmidazole-activatedsupport

Activation of polysaccharlde supports by cyanogen bromide (CNBr) 1s a widely used immobihzation method. The original CNBr activation technique uses a strong base to enhance the nucleophilicity of the support, by transforming some hydroxyl groups into reactive alkoxide ions (28,29). An alternative approach involves the enhancement of the electrophilicity of CNBr by means oi’a suitable cyano-transfer reagent, e.g., triethylamine (TEA) (30,31). Activating the support by means of TEA has several important advantages. It leads to a considerable increase in the overall reaction yield (hydrolysis of CNBr by the reaction medium is slow) and the hydrolysis of active cyanate esters on the support is prevented (uncontaminated by carbamates). Smce imidocarbonates can only be formed by the reaction of cyanate ester with neighboring, suitably oriented alkoxide ions, the use of TEA also precludes the formation of lmidocarbonates on the support (30). Consequently, this procedure yields activated polymers that contam cyanate esters (Fig. 2). The coupling reaction of protein to CNBr-activated support using TEA is also shown in Fig. 2. 1,l ‘Carbonyldiimidazole (CDI), a carbonylating agent, has been proven to be suitable for the activation of hydroxyl-containing matrices for affinity chromatography (32-36). The activation of a support containing hydroxyl groups by CD1 and the subsequent coupling of the protein to the support is shown in Fig. 3.

156

Paterson and Kennedy

Because of the sensitivity to hydrolysis of the carbonylatmg reagent, the acttvation reaction is carried out in anhydrous media, such as dioxane. The mtermediate activated support (an imidazolyl carbonate) is relatively stable to hydrolysis, but reacts smoothly with N-nucleophiles, such as free amino groups in proteins, to give nonbasic uncharged N-alkyl carbonate (urethane derivatives) (32). The choice of method for determination of bound active protein is dictated by the characteristics of the protein in question. In this chapter, the amount of immobilized enzyme activity 1s determmed from measurements of initial reaction rates under defined experimental conditions and compared with the activtty of the free enzyme. a-Amylase (1,4-a-n-glucan glucanohydrolase, EC 3.2.1.1) hydrolyses in an endo-action the (1 + 4)-linkages between a-n-glucopyranosyl residues in starch, but the (1 + 6)-linkages are not hydrolyzed (3 7). The enzymic activity of ol-amylase is measured by the hydrolysis of starch using the dinitrosalicylic acid assay (38,39) to quantify the reducing sugars in the samples.

2. Materials 2.1. Preparation

of CNBr-Activated

Cellulosic

Support

1 Paper-like cellulosic rod (r = 6 mm) consistmg of a tightly rolled fine waved paper with a surface area of ca 2 m*/g (Chembiotech, Birmmgham, UK) 2 Cyanogen bromide (CNBr): Wear appropriate respirator, chemical-resistant gloves, safety goggles, other protective clothing, and use only m a chemical fume hood. Keep tightly closed and store at 4°C (see Note 1). Caution CNBr is highly toxic and irritant. 3. 30% (v/v) acetone solution. Use caution, acetone is flammable. 4. 1.5 M triethylamme solution, in acetone 30% (v/v) Triethylamme is flammable, an irritant, and a mutagen. 5. Hydrochloric acid-acetone solution, 0. lMHCl,30% (v/v) acetone solution (1: 1, v/v). Hydrochloric acid is corrosive. 6 0 1M sodium carbonate&carbonate buffer, pH 9.5 7. Glass syringe with appropriate diameter to fit the cellulosic rod (e.g., 2 mL), polyvinylchloride tubing (I = -6 cm), and screw clamp.

2.2. Immobilization Support 1. a-Amylase

of a-Amylase

(1,4-a-o-glucan

onto CNBr-Activated

glucanohydrolase,

Cellulosic

EC 3.2 1 1, from Badus

subtilu)

2. 3. 4. 5. 6.

0.M sodium carbonattiicarbonate buffer, pH 9.5. 0.05M sodium acetate buffer, pH 5.0. O.lMpotassmm phosphate buffer, pH 6.9. 0.M sodium bicarbonate buffer, pH 8 5. O.lMethanolamine, pH 8.5. Ethanolamine is harmful, an irritant, and a mutagen.

Immobilization on Cellulose Paper 2.3. Preparation

of CD/-Activated

157

Cellulosic Support

1. Paper-like cellulosic rod (Y = 6 mm) consisting of a tightly rolled, fine waved paper with a surface area of ca. 2 m21g (Chembiotech). 2. 1, I’-Carbonyldrimidazole. Care: harmful. 3. Dtoxane. Care: flammable and suspected carcinogen. 4. Dtoxane-water (3:7, v/v). 5. Dioxane-water (7:3, v/v). 6. Glass syringe with appropriate diameter to fit the cellulostc rod (e.g., 2 mL), polyvmylchloride tubing (1 = -6 cm), and screw clamp

2.4. Immobilization onto CD/-Activated 1. 2. 3. 4. 5.

of a-Amylase Cellulosic Support

a-Amylase (1,4-cl-u-glucan glucanohydrolase, EC 3.2.1.1 from Baczllus subtzlzs). O.lM sodium bicarbonate buffer, pH 8.5. 1M sodmm chlorrde solution 0. 1M potassium phosphate buffer, pH 6.9 O.lM ethanolamme, pH 8 5. Ethanolamine is harmful, an irritant, and a mutagen.

2.5. Dinitrosalicylic Acid-Reducing for a-Amylase Activity (39)

Sugar Assay

1. 2. 3. 4

3,5-Dimtrosalicylic acid. Care: harmful and an irritant. Sodium potassium tartrate (Rochelle salt). Care: harmful. Sodium hydroxide Care: corrosive, harmful and an irritant. Dmrtrosalycrlic acid (DNS) reagent: Drssolve 0.25 g of 3,5-dimtrosalycilrc acid and 75 g of sodmm potassium tartrate in 50 mL of 2M sodium hydroxtde solution, and dilute to 250 mL with dtstilled water. The DNS reagent is sensitive to COz. It is stable for several weeks if stored purged wrth helium or nitrogen Otherwise, it should be freshly prepared 5. D-Glucose solution: Prepare 10 mg/mL in 0.1Mpotassiun-r phosphate buffer, pH 6.9. 6. Starch substrate: Prepare 10 mg/mL by suspending soluble starch in O.lMpotassium phosphate buffer, pH 6.9, and heat the suspension until the starch has completely dissolved.

2.5. Lowry Protein Assay (40) 1. 2. 3 4.

Sodium carbonate. Copper sulfate pentahydrate. Care: harmful. Sodium potassium tartrate. Care: harmful. Copper reagent: Dissolve 20 g of sodium carbonate m 260 mL of distilled water; dissolve 0.4 g of copper sulfate pentahydrate in 20 mL of drstilled water and 0.2 g of sodium potassium tartrate m 20 mL distilled water. Mix the solutions. 5. 1% (w/v) sodium dodecyl sulfate (SDS). SDS is harmful and an irritant. 6. 1M sodium hydroxide solution, Sodium hydroxide is corrosive, harmful, and an imtant.

Paterson and Kennedy

158

7. 2X Lowry concentrate: Mix three parts of copper reagent wtth one part of SDS solution and one part of sodium hydroxide solution. Prepare this solution munediately before use. This reagent is stable for 2-3 wk. If a white precipitate forms, warm the solution to 37’C. 8. 0 2N Folin reagent Mix 10 mL of 2N Folin reagent (Care: harmful and an irritant) with 90 mL of distilled water. This solution is stable for several months at ambient temperature if stored in an amber bottle 9. 0.25 mg/mL bovine serum albumin (BSA) solution. 10 0.25 mg/mL a-amylase solution.

3. Methods

3.1. Preparation of CNBr-Activated 1 Connect the polyvmylchlorlde

2

3. 4. 5. 6

7

8.

Cellulosic Support (30)

tube to the end of the glass syrmge with a screw

clamp on the outlet and weigh the system. Cut the paper-like cellulosic rod to the required length (e.g , 1 = 18 mm) and weigh it (dry weight) Introduce the paper-like cellulosic rod mto the glass syringe (see Note 2). Clamp the syringe to a support. Wash the cellulosic support three times with 1 mL of 30% (v/v) acetone solution, using the plunger to flow the solution through the support. Drain the support and weigh the total system. Determine the weight of the drained support. Cool the system to ca. -15°C (see Note 3). Dissolve 20 mg CNBr/g drained support (see Notes 1,4) m 300 uL of 30% (v/v) acetone solution (see Note 5), and add to the support. Use the plunger with an upand-down movement to ensure a good distribution of the liquid through the support. While cooling the system at ca. -15’C, add 179 pL of 1 5M TEA solution (see Note 6) dropwise to the CNBr solution over a period of l-3 min. Use the plunger to ensure a good distribution of the liquid through the support Wash the enzyme support with 2 mL of cold 30% acetone-O.lM hydrochloric acid solution, 2 mL of cold 30% acetone, 2 mL of cold water, and 2 mL of 0 1M carbonate buffer, pH 9.5. Proceed immediately with the coupling step.

3.2. Immobilization of a-Amylase onto CNBr-Activated Cellulosic Support 1. Dissolve 14 mg of a-amylaselg dry support (see Note 7) in 300 pL of O.lM sodium carbonattiicarbonate buffer, pH 9.5 (see Note 8) and add to the support. Use the plunger for a uniform dtstributton of the liquid through the support 2. Leave the enzyme to react with the support for 2 h. Drain the support. 3. Wash the enzyme support with 2 mL of 0.05M acetate buffer, pH 5.0, 2 mL of 0 1Msodium bicarbonate buffer, pH 8.5, and 2 mL of 0. 1Mpotassium phosphate buffer, pH 6.9, to ehminate excess adsorbed enzyme (see Note 9). 4. Treat the enzyme support with 300 pL of 0. 1M buffered ethanolamine, pH 8.5, for 3 h to block excess activated groups.

Immobihzation on Cellulose Paper

159

5. Wash the enzyme support three times with 2 mL of 0. 1M potassium phosphate buffer, pH 6.9. Store the immobilized enzyme in O.lM potassium phosphate buffer, pH 6 9, at 4°C 6. Determine the enzymic activity of the immobilized a-amylase using the DNS assay (see Section 3.5).

3.3. Preparation of CD/-Activated Cellulosic Support (32,33) 1. Connect the polyvinylchloride tube to the end of the glass syringe with a screw clamp on the outlet and weigh the system. 2. Cut the paper-like cellulosic rod to the required length (e.g., I = 18 mm) and weigh it (dry weight). Introduce the paper-like cellulosic rod into the glass syringe (see Note 2). Clamp the syringe to a support. 3. Wash the cellulosic support sequentially with 2 mL of water; three times wtth 2 mL of dtoxane water (3:7), three times with 2 mL of dtoxane-water (7.3); and five times with 2 mL of dtoxane. Use the plunger to flow the solution through the support. 4. Drain the support and weigh the system. Determine the weight of the dramed support. 5. Dissolve 29 mg of CDI/g drained support (see Note 10) in 300 uL of anhydrous dioxane and add to the support. To ensure a good distribution of the solution through the support, use the plunger with an up-and-down movement 6. Leave it to react for 15 min at ambient temperature. Wash the activated support five times with 2 mL of dioxane to elimmate the reaction products and the excess of reagent. Drain the support.

3.4. immobilization of a-Amylase onto CD/-Activated Cellulosic Support 1. Dtssolve 14 mg of a-amylaselg dry support (see Note 7) in 300 pL of 0 IA4 sodmm carbonate-bicarbonate buffer, pH 8.5 (see Note 1l), and add to the support. Use the plunger for a uniform distribution of the liquid through the support. 2. Leave the enzyme and support to react for 21 h (see Note 12). 3. Drain the support. Wash the enzyme support wtth 2 mL of 0 IA4 sodmm btcarbonate buffer, pH 8.5,2 mL of 1Msodium chloride, and 2 mL of 0. Mpotassmm phosphate buffer, pH 6.9, to eliminate excess adsorbed enzyme (see Note 9). 4. Treat the enzyme support with 300 l.tL of 0. 1M buffered ethanolamine, pH 8.5, for 3 h to block excess activated groups. 5. Wash the enzyme support three times with 2 mL of 0.M potassium phosphate buffer, pH 6.9. Store the immobilized enzyme in 0.M potassium phosphate buffer, pH 6.9, at 4°C 6. Determine the enzymic activity of the immobilized a-amylase using the DNS assay (see Section 3.5).

3.5. DNS-Reducing Sugar Assay for a-Amylase Activity (38,39) 3.5.1. Free a-Amylase 1. Warm 2 mL of the starch substrate (10 mg/mL) to 37°C in a water bath. 2. Start the reaction by addition of 200 i.tL of a-amylase solution (see Note 13) to the substrate.

160

Paterson and Kennedy

3. During the period of mcubation, remove samples of 100 pL at various intervals (O-30 mm) and termmate the reaction by adding 1 mL of DNS reagent. Mix the samples well and heat at 100°C for 10 min 4. Cool to ambient temperature and measure the absorbance at 570 nm. 5. To 100 pL aliquots of o-glucose standard solution (O-5 mg/mL in 0. IA4 potassium phosphate buffer, pH 6.9) and controls, add 1 mL of DNS reagent. MIX the samples well and heat at 100°C for 10 mm. 6. Cool to ambient temperature and measure the absorbance at 570 run (see Note 14).

3.5.2. Immobilized a-Amylase 1 Warm 2 mL of the starch substrate (10 mg/mL) to 37°C in a water bath. 2. Start the reaction by addition of 300 PL of the starch solution to the coupled enzyme support. Allow the substate to flow through the enzyme support at various retention times (O-30 min) For each retention time, two 100~pL aliquots of drained solution are transferred to stoppered test tubes 3, The reaction of one sample is terminated immediately by the addition of 1 mL of DNS reagent. The reaction of the second sample is terminated 10 min later by the addition of 1 mL of DNS reagent. This is carried out to verify the presence of desorbed enzyme from the support (see Note 15) 4 Wash the enzyme support three times with 2 mL of distilled water after conversion of each batch of starch (see Note 16)

3.6. Lowry Protein Assay (40) 1. Add 400 pL 2X Lowry concentrate to 400 l.tL a-amylase solution (approx 50 pg protein) and to 400 pL BSA standard solution (O-100 c(gprotein) 2 Incubate at ambient temperature for a minimum of 10 min. 3. Add 200 pL of Folin reagent (0.2iV) and mix immediately after each addition. This mixing is important since the reagent decomposes rapidly. 4. Incubate for an additional 30 min at ambient temperature. 5. Use glass or polystyrene cuvets to read the absorbance at 750 nm. If the absorbances are too high, they may be read at 500 nm 6. A standard curve (absorbance at 750 nm vs amount of BSA) 1sused to determine the protein content of the a-amylase

4. Notes 1 CNBr is a white, crystalline solid that is sufficiently volatile at ambient temperature to generate highly poisonous and irritant vapors. All work should be carried out m a ventilated hood 2. This is a simple system given as an example Other systems may be used, e.g., the reaction may be carried out in a glass vial using gentle agitation 3. Cooling bath: For temperatures down to about -20°C an ice-salt freezing mixture may be used A mixture of sodium chloride with crushed ice at the ratio of 1:3 will theorettcally produce a temperature of about -20°C (41). However, m practrce, the ice-salt mixtures give temperatures of -5 to -18°C.

Immobilization on Cellulose Paper

161

4. There IS a linear relationshtp between the amount of CNBr employed and the activity of the support, m cases in which the optimized CNBr procedure is used (30). Therefore, the concentration of CNBr used wtll depend on the level of activation required. The only limit to the acttvtty of the support will be the amount of hydroxyl groups avatlable on the support for reaction with CNBr. Very high degrees of activation yield multiple-bond formation to the protem, possibly resulting in steric interference problems and loss of protein activity. 5. The volume of acetone solutton shown 1s enough to wet and cover the support (dimensions: r = 6 mm; I = 18 mm). 6 The overall reaction yteld 1s higher at -15°C than at ambient temperature. Also the best results are obtained with a final molar ratio of CNBrTEA of about 1:1.5 (30). 7. The concentratton of the protein used during the couplmg step should be constdered. In general, htgh loadings (e.g., 50-100 mg enzyme/g support) will saturate the support 8. The retention of btological activity of the immobtlized protein depends directly on the characteristtcs of the protein, the buffer conditions, and the pH of immobilization. 9. Protems can be coupled to the support by undesirable nonspecific adsorptton. This can be overcome by extensive washing of the inittal coupled product to yield a support containing covalently coupled protein as exclusive as possible (42,43). 10. The appropriate choice of activation level should be optimized as part of the investigation since it does not necessarily follow that the highest activation levels will necessarily yield the best protein capacity. 11. Many proteins are sensitive to extremes of pH. This lab&y precludes couplings being carried out at pH 9.0-10.0, which would give the most efficient coupling condittons for CDI-acttvated supports. However, coupling at a pH range of 7.5-9.0 can be performed with high couplmg yields provided an activated support of moderately high substitution is used (35). 12. Typical coupling times with CDI-activated supports are 10-l 8 h at pH 10.0 at 4°C (36). Because protein couplmg reactions are carried out in an aqueous milieu, the competing hydrolysis of the activated support can occur. A two-stage hydrolytic process appears to occur under basic pH condittons wtth approx 75-80% of the active groups hydrolysing in 21-24 h with buffers at pH 8.5, whereas the residual activation groups require >30 h for complete hydrolyses (36). 13. For the measurement of the catalytic activity of an enzyme, the reaction must proceed slowly so that only a small proportion of the substrate is converted by the end of the measurement. This is done to be able to monitor the reaction rate and to stay within the linear range of the conversion curve. The enzyme sample should be diluted appropriately. 14. A standard curve, absorbance at 570 nm vs glucose concentration, is plotted and the linear regression equation, which is used to determine the reducing sugars content (expressed as o-glucose) of samples, 1s derived The imttal velocity of productton of reducing sugars by the enzyme is obtained by estimatmg the tan-

Paterson and Kennedy

162

gent of maximum slope, which passes through zero time, of the curve corresponding to the rate of hydrolysis of starch. 15. Nonspecifically bound cl-amylase (if present) may be either active or inactive on the cellulose support, subsequently leaking into solutton when contacted with the macromolecular substrate (starch) This desorbed enzyme can contribute very markedly to initial a-amylase activity 16 The unit of a-amylase activity (U) is defined as the amount of enzyme required to hberate 1 umol of o-glucose/mm from a starch solutton (10 mg/mL m potassium phosphate buffer, 0 IM, pH 6.9), at 37°C. The specific catalytic activity ofa-amylase is expressed as the a-amylase activity per milligram of protein The protein content is determined according to the method of Lowry (‘40) (see Section 3.6 ) The mmal velocity of production of reducing sugars by the mnnobilized enzyme is determined as described in Section 3.5.1. The amount of active protein immobtlized onto the support 1sdetermined from the measurements of initial reaction rates and compared with the catalytic activity of the free a-amylase (see Section 3.5.1 )

References 1. Gemeiner, P., Stefuca, V., and Bales, V (1993) Biochemical engmeermg of biocatalyst immobilized on cellulosic materials. Enzyme Microb. TechnoE 15, 55 l-566 2 Gemeiner, P., Rexova-Benkova, L , Svec, F., and Norrlow, 0. (1994) Natural and synthetic carriers suitable for immobilization of viable cells, active organelles, and molecules, in Immobilized Biosystems-Theory and Practical Applzcatzons (Veliky, I A. and McLean, R J. C., eds.), Blackie, London, pp. 1-128. 3. Cabral, J. M. S. and Kennedy, J. F (199 1) Covalent and coordination immobthzation of protems, m Protein Immobrllzatlon-Fundamentals and Applications (Taylor, R. F., ed.), Marcel Dekker, New York pp. 73-138. 4. Rosenthal, A., Schwertner, S., Volkmar, H., and Hunger, H.-D. (1985) Solid-phase methods for sequencing of nucleic acids. I. Simultaneous sequencing of different ohgodeoxyribonucleotides using a new, mecharncally stable anion-exchange paper. Nuclezc Acid Res. 13, 1173-l 184. 5. Rosenthal, A., Jung, R., and Hunger, H.-D. (1986) Optimized conditions for sohdphase sequencmg: simultaneous chemical cleavage of a series of long DNA fragments immobihzed on CCS anion-exchange paper, Gene 42, 1-9. 6 Lapicque, F. and Dellacherie, E. (1986) Specific-enzyme release of cellulose-bound drugs, experimental and theoretical study. J Appl. Polym Scl 32,285 l-2866 7. Przybyt, M and Sugier, H. (1988) Immobilization of glucoamylase on cellulose Starch/Starke

40,275-279.

8. Ewhler, J , Beyrmann, M , and Biernert, M. (1989) Application of cellulose paper as support material in simultaneous solid phase peptide synthesis. Collect Czech Chem Commun. 54,1746-l 75 1. 9 Eichler, J., Pinilla, C , Chendra, S., Appel, J. R., and Houghten, R. A. (1994) Synthesis of peptide libraries on cotton carriers: methods and applications, in Soled Phase Synthesis-Peptides, Proteins and Nucleic Acids (Epton, R., ed.), Mayflower, Birmingham, pp. 227-232

Immobilization on Cellulose Paper

163

10. Frank, R. (1994) Spot-synthesis: an easy and flexible tool to study molecules recognition, in Solid Phase Synthesis-Pepttdes, Proteins and Nucletc Acids (Epton, R , ed.), Mayflower, Birmingham, UK, pp. 509-5 12. 11. Gil, M. H., Alegret, S., Silva, M. A., Alegria, A. C., and Ptedade, A. P. (1993) The use ofmoditied cellulose for biosensors, m Cellulostc Materrals for Selecttve Separatzons and Other TechnoZogies (Kennedy, J. F , Phillips, G O., and Williams, P A., eds.), Ellis Horwood, Chwhester, UK, pp. 163-170. 12. Ohnishi, M., Iwata, K. Tomita, T., Nishikawa, U., and Hiromi, K. (1990) Kinetic properties of Rhizopus glucoamylase and Bactllus a-amylase, which are immobilized on Cellulofine. StarchKtarke 42,486-489. 13. Manganaro, J. L. and Goldberg, B. S. (1993) Protein purification with novel sheets containmg derivattzed cellulose. Biotechnol. Prog. 9,285-290. 14. Kennedy, J. K. and Paterson, M. (1993) Application of cellulosic fast-flow column filters to protein tmmobihzation and recovery. Polym. Znt 32,7 l-8 1. 15. Krassig, H. A. (1993) The tibre structure, in Cellulose, Structural Accessibiltty and Reactivity, Gordon and Breach, Yverdon, Switzerland, pp, 6-42. 16 Grubhofer, N. (1991) Cellulose ton exchangers, m Ion Exchangers (Dorfner, K., ed.), Walter de Gruyer, Berlin, Germany, pp. 443460. 17. Peterson, E. A. (1980) Cellulose ton exchangers, m Laboratory Techniques tn Biochemistry and Molecular Btology (Wort T. S. and Work E., eds.), Elsevier, Amsterdam, The Netherlands, pp. 233-254. 18 Villems, R. and Toomik, P. I. (1993) Overview, in Handbook of A&& Chromatography, vol. 63 (Kline, T., ed.), Marcel Dekker, New York, pp. 3-60. 19 Kline, J. E. (199 1) Pulping, in Paper and Paperboard-Manufacturrng and Converting Fundamentals, 2nd ed., Miller Freeman, San Francisco, pp. 38-73. 20. Kremer, R. D. and Tabb, D. (1989) The beneficially interactive support medium for diagnostic test development. Int. Lab. Jul./Aug., 40-45. 2 1. Walsh, G. and Headon, D. R. (1994) Proteins for diagnostic purposes, in Protein Biotechnology, Wiley, Chichester, UK, pp 268-301 22. Goldstein, L. and Maneke, G. (1976) The chemistry of enzyme immobilization, in Applied Btochemistry and Bioengineering-Immobilized Enzyme Principles, vol 1 (Wingard, L. B., Katchalski-Katzir, E , and Goldstein, L., eds.), Academic, New York, pp. 23-126 23. Woodward, J. (1985) Immobihzed enzymes: adsorption and covalent coupling, in Immobiltzed Cells and Enzymes (Woodward, J., ed.), IRL, Oxford, UK, pp. 3-17. 24. Kennedy, J. F. and Cabral, J M. S. (1985) Immobilization of biocatalyst by metallink/chelation processes, in Immobiltzed Cells and Enzymes (Woodward, J , ed ), IRL, Oxford, UK, pp. 19-37. 25. Scouten, W H. (1987) A survey of enzyme coupling techniques, in Methods tn Enzymology, vol. 135 (Mosbach, K, ed.), Academic, Orlando, FL, pp. 30-65 26. Kennedy, J. F. and Cabral, J. M. S. (1987) Immobilisation of enzymes on transition metal-activated supports, in Methods in Enzymology, vol. 135 (Mosbach, K., ed.), Academic, Orlando, FL, pp. 117-l 30.

164

Paterson and Kennedy

27. Giovenco, S., Marconi, W, and Pansolli, P (1987) Microbial cells entrapped m cellulose acetate beads, m Methods in Enzymology, vol 135 (Mosbach, K., ed ), Academrc, Orlando, FL, pp. 282-293. 28. March, S. C., Parikh, I., and Cuatrecasas, P. (1974) A simphfied method for cyanogen bromide activation of agarose for affinity chromatography. Anal. Biochem 60, 149-152. 29 Kohn, J. and Wilchek, M. (1981) Procedures for the analysis of cyanogen bromideactivated Sepharose or Sephadex by quantitative determination of cyanate esters and imidocarbonates Anal Bzochem l&375-382 30. Kohn, J. and Wilchek M. (1982) A new approach (cyano-transfer) for cyanogen bromide acttvatlon of Sepharose at neutral pH, which yields activated resins, free of interfering nitrogen denvattves. Biochem Blophys Res Commun 107,878-884. 3 1. Kahn, J and Wilchek, M (1983) New approaches for the use of cyanogen bromide related cyanylatmg agents for the preparation of activated polysacchandes resins, in Affinnlty Chromatography and Biological Recognition (Chaiken, I. M., Wilchek M., and Pa&h, I., eds.), Academic, Orlando, FL, pp. 197-207. 32 Bethell, G S , Ayres, J S., Hancock W S., and Hear-n, M. T. W. (1979) A novel method of activation of crosslinked agarose with 1, I’-carbonylditmidazole whtch gives a matrix for afftnity chromatography devoid of additional charged groups. J Biol. Chem 254,2572-2574. 33. Hearn, M. T. W., Bethell, G. S., Lyres, J. S., and Hancock, W. S. (1979) Applicatton of 1,l’-carbonyldiimidazole-activated agarose for the purification of proteins. II. The use of an activated matrix devoid of additional charged groups for the puriticanon of thyroid protems J Chromatogr 185,463+70 34 Heam, M. T W., Harris, E. L., Bethell, G. S., Hancock, W S , and Ayres, J. A (198 1) Application of 1, I’-carbonyldiimtdazole-activated matrices for the purification of proteins. m. The use of l,l’-carbonyldiimidazole-activated agarose in the biospecitic affinity chromatography isolation of serum antibodies. J. Chromatogr 218,509-518.

35. Bethell, G. S., Ayres, J. S., Heam, M. T. W., and Hancock, W. S. (198 1) Investigation of the activation of crosslinked agarose with carbonylatmg reagents and the preparatton of matrices for affimty chromatography purifications. J Chromatogr, 219,353-359.

36. Heam, M T W. (1987) 1, l’-Carbonyldiimidazole-mediated munobrlizatton of enzymes and affimty hgands, m Methods zn Enzymology, vol. 135 (Mosbach, K., ed.), Academic, Orlando, FL, pp. 102-l 17. 37. Kennedy, J. F, Cabral, J M. S., Sa-Correia, I., and White, C. A. (1987) Starch biomass: a chemical feedstock for enzyme and fermentation processes, m Starch. Propertres and Potential (Galhard, T., ed.), Wiley, Chichester, UK, pp. 115-148. 38. Bemfeld, P. (1955)Amylases, c1and f3,lnkfethods in Enzymology, vol. 1 (Colowtck, S. P and Kaplans, N. O., eds.), Academic, London, UK, pp. 149-158. 39. Chaplins, M. F. (1994) Monosaccharides, in Carbohydrate Analysis-A Practical Approach (Chaplm, M. F. and Kennedy, J. F., eds.), IRL, Oxford, UK, pp. 1-41

Immobilization on Cellulose Paper

165

40 Stoscheck, C. M. (1990) Quantitatton of Protein, in Guide to Protein Purljkatlon Methods rn Enzymology, vol. 182 (Deutscher, M P., ed.), Academic, San Diego, pp. W-68. 4 1 Harwood, L. M. and Moody, G. J. (1989) Organic reactions: from starting materials to pure organic products, in Experimental Organic Chemutry--Principles and Practice. Blackwell, Oxford, UK, pp 102-104.

42. Barker, S. A., Sommers, P. J , and Epton, R. (1969) Preparation and stability of exoamylolytic enzymes chemically coupled to microcrystalline cellulose. Carbohyd Res 9,257263.

43. Barker, S. A., Sommers, P. J., and Epton, R. (1970) Recovery and re-use of watermsoluble amylase derivatives. Carbohyd. Res. 10,323-326

20 Immobilization of Cells Using Electrostatic Droplet Generation Mattheus F. A. Goosen, Eltag S. E. Mahmud, Abdullah S. Al-Ghafri, Hamad A. Al-Hajri, Yousuf S. Al-Sinani, and Branko Bugarski 1. Introduction A major concern m cell and btoactive agent mnnobilization has been the productron of very small microbeads to mmlmize the mass transfer resistance problem associated with large diameter beads (i.e., >lOOO pm) (I). Conventional technology involves production of alginate beads with diameters ranging from 500-2000 pm using compressed air to quickly pass the cell/gel solution through a nozzle (2). Surprisingly, only recently have attempts been made in the application of electric fields to the production of micron-size polymer beads for cell tmmobilization (3-s). The two primary advantages of electrostatrc droplet generation, over, for example, an air jet extruder, are the production of much smaller beads with conventtonal needles and easeof bead size control by simply varying the applied potential, Electrostatics can be configured to produce smaller droplets. When a liquid is subjected to an electric field, a charge is induced on the surface of the liquid. Mutual charge repulsion results in an outwardly directed force. Under surtable conditions, for example extruston of a liquid through a needle, the electrostatic pressure at the surface forces the liquid drop into a cone shape. Surplus charge is ejected by the emission of charged droplets from the tip of the liquid. The emission process depends on such factors as the needle diameter, distance from the collecting solution, applied voltage (i.e., strength of electrostatic field), and electrode geometry. Under most circumstances, the electrical spraying process is random and irregular, resulting in drops of varying size and charge that are emitted from the capillary tip over a wide range of angles. However, when the From. Methods m B/ofechno/ogy, Vd 7 * Immob~l~zatron of fnzymes and Cells Edited by G F Bickerstaff Humana Press Inc , Totowa, NJ

167

Goosen et al.

168 electrostatic generator configuration has been adjusted applied voltage, electrode spacing, and charge polarity, can become quite regular and periodic. In this chapter we describe an electrostatic technique alginate droplets. Experimental parameters that control presented. A technique for encapsulating/immobilizing demonstrated (3-5).

2. Materials 2.1. Electrostatic

for liquid pressure, the spraying process for producmg small microbead size are animal cells is also

Droplet Generation

1. Syrmge pump: Attach the pump to vertical stand. Use a 10 mL plastic syringe and 22- or 26gage stainless steel needles (see Note 1 for alternative set-up). 2. Voltage power supply: A variable high voltage (O-30 kV) power supply with low current (CO 4 mA) is required. We have used a commerctal power supply model 230-30R from Bertan (Hwksville, NY). A homemade power supply can also be built (see Note 2). 3 Collecting solution: Prepare 1.5% (w/v) CaCl, m saline (0.85 g NaCl in 100 mL distilled water). Saline can be replaced wtth distilled water if an algmate solution without cells is being extruded. Place the CaCl, solution in a Petri dish on top of an adjustable stand The stand allows for fine-tuning of the distance between the needle tip and collectmg solutton. 4 Sodium alginate solution* Prepare 1.5% (w/v) low viscostty sodium algmate. Dissolve alginate powder with stnrmg in a warm water bath (see Note 3). Slowly add the 1.5 g sodium alginate to 100 mL warm saline solution (or distilled H,O), stirrmg continuously. It may take several hours to dissolve all of the algmate.

2.2. Cell Immobilization/Encapsulation

Solutions

1. Cell lines: A variety of cells can be immobilized using electrostatics, ranging from hybridomas and insects to islets of Langerhans (5-S). 2. 0.03% sodium algmate solution: Add 2 mL of 1 5% alginate solution to 98 mL saline. 3 CaCl, solution: For 1.1 and 1.5% CaC12 solutions, add 14.6 g or 19.9 g of CaC12*2H20, respectively, to 1000 mL of distilled water. 4. 2-(n-Cyclohexylamino) ethane sulfmic acid (CHES): Prepare a 0.1% CHES solution by adding 5 mL of CHES stock solution to 95 mL of 1.1% CaC12 solutton. CHES stock solution is made by dlssolvmg 2 g of CHES and 0.5 1 g ofNaC1 m 90 mL of distilled water, adjusting the pH to 8.2 with NaOH, and increasing the volume to 100 mL. 5. 0.05M sodium citrate solution: Dissolve 2.58 g of sodium citrate and 0.85 g of NaCl m 200 mL of distilled water. 6. Polylysine (PLL) solution: Dissolve an appropriate amount of PLL hydrobromide in sterile saline. Caution: Allow the tube containing PLL powder to reach room temperature before opening, because PLL readily takes up moisture out of the air.

Electrostatic Droplet Immobilization

169

3. Methods

3.1. Electrostatic Droplet Generation 1. Add about 8 mL of 1.5% algmate solution to a lo-mL plastic syringe, put back the plunger, and attach the syringe to the upright syringe pump. Make sure that the stainless steel needle, 22-gage, is firmly attached and that the syringe plunger is m firm contact with the movable bar on the pump. 2. Position the Petri dish (or beaker) containing CaClz solution so that the needle tip 1sabout 3 cm from the top of the CaC12 hardening solution. This is the primary reason for using an adjustable stand. 3. Attach the postttve electrode wire to the stainless steel needle and the ground wire to the collecting solution. The wires may need some additional support to prevent them from bendmg the needle. 4. Switch on the syringe pump and wait for the first few drops to come out of the end of the needle. This could take a minute or two Doing it this way also ensures that the needle is not plugged (see Note 4). After the first drop or two has been produced, switch on the voltage power supply. 5 Make sure that the voltage is set low, ~5 kV If thts IS the first time that you have tried electrostatic droplet generation, raise the voltage slowly and observe what happens to the droplets. The rate at which they are removed from the needle tip increases unttl only a fine stream of droplets can be seen. The changeover from individual droplets to a fine stream can be quite dramatic. 6. The most effective electrode and charge arrangement for producing small droplets is a posttively charged needle and a grounded plate (Fig. 1C). Two other arrangements are also possible; positively charged plate attached to needle (Fig 1A) and positively charged collecting solution (Fig. 1E). 7. Make sure that the positive charge is always on the needle. This ensures that the smallest microbead size 1s produced at the lowest applied potential (see Fig. 2B,C). With a 22-gage needle and an electrode spacing of 2.5-4.8 cm there will be a sharp drop in microbead size at about 6 kV. This can be noticed visually by observing the droplets coming from the needle tip. 8. Standard commercially available stainless steel needles can be employed However, when going from a 22- to a 26-gage (or higher) needle, needle oscillation may be observed (‘4). This needle vibration will produce a bimodal bead size distribution with one peak around 50 pm diameter beads and another around 200 pm.

3.2. Cell Immobilization/Encapsulation 1. Remove culture medium from a 75-cm2 subconfluent flask of cells (e.g., mammalian or insect cells, ref. 9) and add about 10 mL of fresh medium. Gently shake the flask to dislodge cells from the side. Pellet the cells by centrifugation at 1000 rpm for 10 min and remove medium by vacuum aspiration. Resuspend cell pellet in 5 mL of 1.5% sodium alginate solution (see Note 5). 2. Extrude 5 mL of the algmate/cell suspension mto 200 mL of a 1.5% CaC12 solution using the electrostatic droplet generator. Take a sample of the gelled

Goosen et al. Needle

*

Noodle

. H

C

+ VOLTAGE Needle

: H

Fig. 1. Electrode and charge arrangements, (A) Parallel plate set-up with positively charged plate. (B) Positively charged collectmg plate. (C) Positively charged needle (6). (L = distance that needle extends below plate, H = distance from plate to collecting solution)

Electrostatic Drop/et immobilization

171

SPACING

4.8

cm

2.5

cm

T

2000

i 1000 4.8 cm

C ~?+A.,~ 0

i

2.5 cm

Fig. 2. Effect on microbead size of: (A) applied potential and electrode spacmg; (B) positively charged collecting plate; and (C) positively charged needle (alginate concentration lS%, 22-gage needle for all three configurations, ref 6). microbeads containing the cells and examine them for size and shape consistency using a dissecting microscope. 3. At this point, the calcium alginate gel immobilized cells can be transferred to the appropriate culture medium and used for studies requiring only gel nnmoblllzed

172

4

5

6

7

8.

9

10.

Goosen et al. cells If you need to produce semipermeable microcapsules containing entrapped cells then proceed to the next step. Transfer the gel beads to a sterile 50-mL plastic centrifuge tube with a conical bottom. Wash the beads (-10 s) with 30 mL each of 0.1% CHES and 1 1% CaC12 solutions. After allowing the beads to settle (may take up to 60 s), reduce the volume of the supernatant after each washing with a vacuum aspirator. A semipermeable capsule membrane is then formed by reacting the gel droplets with 30 mL of a 0.05% (w/v) PLL solutton (PLL of M, = 22,000) for 6 mm. During the reactton, cap the tube and gently rock it end-to-end either manually or with the aid of a mtxmg platform Allow capsules to settle, aspirate off the excess PLL solutton, and then wash capsules with 30 mL each of 0 1% CHES and 1 1% CaC12 and wtth two 30-mL aliquots of salme. Resuspend the capsules m 30 mL of 0.03% sodium algmate solution for 4 mm to form an outer layer on the capsules and to neutralize free reactive groups on the PLL membrane. Recent studies have shown that this step is crucial If the capsules are to be employed for cell transplantation (5). The interior of the microcapsules are reliquified by suspendmg them in 30 mL of a 0.05M sodium citrate solution for 6 min. Sodium citrate chelates the divalent calcium ions and allows replacement m the gel with monovalent sodium ions. Wash the capsules several times m saline to remove excess citrate and then rock end-to-end for 30 min to allow lower-mol-wt algmate to diffuse out of capsules and for the capsules to swell toward then equilibrium state (see Note 6). Incubate the encapsulated cells in 100 mL medium m a 75 cm2 culture flask If there is concern about possible contammation, place 1 mL capsule ahquots m 30 mL of medium per culture flask. Capsule membrane molecular weight cutoff can be controlled by varying the PLL molecular weight, concentration, and reaction time. For example, tf a higher membrane molecular weight cutoff is desired, then employ a higher-mol-wt PLL, a lower PLL concentration, and a shorter reactron time in the encapsulation procedure (9, IO).

4. Notes 1. If a syringe pump is not available, remove the syringe plunger and attach an air lme with a regulator to the end of the syringe The algmate extrusion rate can be controlled by varymg the air pressure output on the regulator 2. For logistics and/or financial reasons It may be necessary to build your own DC high-voltage power supply. (We have done thts three times over the past 15 years) Caution: A competent electrician IS required to build the power supply. A step-down transformer (converts 120-240 V AC to 12 V AC) is attached to a power rectifier (converts 12 V AC to 12 V DC). The rectrfier is attached to a high voltage source (converts 12 V DC to > 5 kV DC). Check to make sure that the current associated with the high voltage output is ~1 mA.

Electrostatic Droplet lmmobihza tion

173

3. Lumps of sodium alginate often form if the powder is added all at once to the warm salme. Sprinkle the algmate powder into the saline a small amount at a time with gentle mixing. Once it has all dissolved (up to l-2 h), allow the viscous solution to cool and then transfer it to several plastic test tubes, cap, and store in the refrigerator until required This prevents bacterial growth. If the alginate solution is very viscous, air bubbles will be trapped during stirring. These bubbles will disappear if the viscous solution is left to stand overnight 4. If the needle is plugged, place it m dilute citrate solution for a few minutes. Passing a fine wire through the needle also helps. 5 Resuspendmg the cells in 1 5% (w/v) sodium alginate solution will dilute the alginate solution and could give tear-drop shaped capsules when the solution is extended. To solve this problem, increase the concentration of sodium algmate solution to 2 or 2.5%. 6. The higher the PLL molecular weight employed in the reaction, the greater the expansion of the capsules m the citrate step and the higher the membrane molecular weight cutoff

References 1. Fonsecam, M., Black, G. M , and Webb, C (1986) Reactor configuration for immobilized cells, in Process Engineermg Aspects of Immobilized Cell System Instttutton of Chemtcal Engzneers, Rugby, Warwtckshire, UK, pp. 63-70 2. Klein, J., Stock, J., Vorlop, D. K. (1983) Pore size and properties of spherical calcium algmate biocatalysts Eur. J. Appl. Microbial. Biotechnol. 18, 86-92. 3. Goosen, M. F. A., O’Shea, G. M., Gharapetian, H., and Sun, A M. (1986) Immobilization of living cells in bio-compatible semipermeable microcapsules* biomedrcal and potential biochemical engineering applications, m Polymers tn Medzctne (Chielhm, E , ed.), Plenum, New York, pp. 235-246. 4. Bugarski, B., Li, Q,, Goosen, M. F. A,, Poncelet, D., Neufeld, R. J., and Vunjak, G. (1994) Electrostatic droplet generation: mechanism of polymer droplet formation. Am. Inst. Chem. Engineers J. 40(6), 1026103 1. 5. Sun, A. M. (1994) Microencapsulation as bioartificial organs. allografts and xenografts, in Pancreatic Islet Transplantation, vol. III Immunotsolatton of Pancreatx Islets (Lanza, R. P and Chick, W. L , eds ), R. G. Landes, Austin, TX, pp 45-58. 6. Bugarski, B., Amsden, B., Neufeld, R. J., Poncelet, D., and Goosen, M. F. A. (1994) Effect of electrode geometry and charge on the production of polymer microbeads by electrostatics, Canad .I. Chem Engineer. 72,5 17-52 1. 7. Bugarskr, B., Smith, J., Wu, J., and Goosen, M. F. A. (1993) Methods for animal cell immobilization using electrostatic droplet generation, Bzotechntq. Techniq. 7, 677-682 8. Goosen, M.F.A. (1994) Fundamentals of microencapsulation,

in Pancreatic Islet Transplantation, vol. III, Immunotsolation of Pancreatic Islets (Lanza, R P and Chick, W. L., eds.), R. G. Landes, Austin, TX, pp, 2144.

174

Goosen et al.

9. King, G. A., Dauguhs, A. J., Faulkner, P., and Goosen, M F. A. (1987) Algmate polylysme microcapsules of controlled membrane molecular weight cut-off for mammalian cell culture engineering. Bzotechnol. Prog 3,23 l-240. 10 Okhamafe, A. and Goosen, M. F A (1993) Control of capsule membrane permeabtlity, in Fundamentals ofAnimal Cell Encapsulation and Immobdtzation (Goosen, M F A., ed.), CRC, Boca Raton, FL, pp. 64-74.

21 Hepatocyte Immobilization in Agarose and Functional Integrity Testing Hassan Farghali and Sixtus Hynie 1. Introduction Isolated liver cells are extensively used in various studies and find wide applications in the fields of biochemistry, physiology, molecular biology, pharmacotoxicology, testing chemical pollutants, mutagenicity, carcinogenicity, and in other biomedical studies. The importance of this experimental cellular model stems from the fact that it is an intermediate system between the whole animal model or the isolated perfused liver on one side, and those systems of isolated subcellular organelles or solubilized enzymes on the other side (1). Freshly isolated hepatocytes, primary cultures of hepatocytes, and clonal cell lines are examples of well established cellular models. Certainly, experiments on these hepatocyte models have contributed significantly to our understanding of liver biology and pathophysiology. Nevertheless, the routinely used hepatocyte cellular models have the drawback of being metabolically less active and may be described as static. This is because such systems suffer from hypoxia and waste product buildup that adversely affect cell physiology (2,3). Consequently, the development of a perfusion method for the heptocytes that normalizes the processes of oxygenation and waste removal is of prime importance. Various methods of cellular immobilization, such as microcarrier beads, hollow fiber systems,and the thread technique, are available, but the latter one is employed here because of its popularity, simplicity, and applicability to a wide variety of cells. The method described in this chapter is based on the gel system reported by Foxall et al. for mammalian cells (4) and has been applied to hepatocytes and Sertoli cells (5-8). A system of immobilized and perfused hepatocytes has potential for applications in many biomedical fields with unparalleled advantages over other celFrom

Methods IR Bofechnology, Vol 1 /mmobf/rzafion of Enzymes and Cells Edlted by G F Bickerstaff Humana Press Inc , Totowa, NJ

175

Farghali and Hynie

176

lular systems. One attractive development for such bioreactors is the apphcation of magnetic resonance spectroscopy(NMR) for studies on cells ($6,~12) NMR is a powerful analytical technique that can be used for noninvasive and online analyses of many biochemical events of definitive populations of cells (real time measurements). 31PhosphorusNMR spectroscopy has revealed that a period of 45-60 min of perfusion at the begining of the experiment, after clearing cell debris, is essential to enable a stable and high ATP peak, which indicates maximum functional activity. The perfusion rate used in the methods described below was selected on the basis of our previous studies using this cellular model and NMR (5). Using the system described below, the investigator may continuously monitor several events m the perfusate or inside the cells nondestructively. In this chapter we present a method of rat hepatocyte immobilization for perfusion purposes on a small research scale bioreactor model (2). An outline is given for the isolation of rat hepatocytes, since it is well described elsewhere (2). Hepatocyte integrity and functionality in the gel matrix can be monitored by various physiological, biochemical, or histological approaches, and some of these methods are described below. The small scale laboratory bioreactor can work for 3 d, provided aseptic conditions are available. More laborious methods involving the use of hollow fibers can be used for extended periods of time (3). A similar perfusion system for hepatocytes has been used for estimation of intracellular calcium, intracellular pH, intracellular magnesium, and free radical formation under anoxia/reoxygenation injury (9-12). 2. Materials 2.7. Isolation

of Rat Hepatocytes

(see Note 7)

1 Male Wrstar rats weighing 200-250 g 2 Buffer 1 (modified Hanks A): Dissolve analytical grade ingredients in deronized water. Ingredients in g/L are: NaCl, 8.0; KCl, 0.4; MgS04*7H20, 0.2; Na2HP04*2H20, 0.06; KH2P04, 0.06; NaHCO,, 2.19; 0.5 mM EGTA; adjust to pH 7 4 (see Note 2) 3. Buffer 2 (modified Hanks B): Prepare buffer 1 wrthout EGTA; include 0.07% collagenase and 4.0 rm!4 Ca2+ and adJust to pH 7 4 (see Note 2) 4. Buffer 3 (Krebs-Henseleit): Dissolve analytical grade Ingredients m deionized water Ingredients m g/L are: NaCl, 6 9, KCI, 0 36, MgS04*7H20, 0.295; CaC12*2H20, 0 426; KH2P04, 0 13; NaHCOs, 2.0. Add 2% (w/v) bovine serum albumm (BSA) to the prepared buffer 3 solution (see Note 2). 5. Collagenase for hepatocyte isolation (collagenase H, Boehringer Mannhelm, Mannheim, Germany) 6. Heparin injection (5000 U/mL). 7 Anesthetic ether. 8 BSA, 9899%

177

Hepatocyte Immobihzation in Agarose 9. 10. 11. 12. 13. 14.

Ethylene glycol-bts (2-ammoethylether) N,N,NY’-tetra-acetic 0.4% Trypan blue (Stgma, St. LOUIS, MO). Cell counter (hemocytometer). Stainless steel cannula for rat portal vein cannulation. Liver perfusion system Carbogen.

acid (EGTA).

2.2. Immobilization of Hepatocytes in Agarose Threads and Subsequent Perfusion 1. Prepare the low-gelling agarose solution in warm buffer 3 at 70°C as 1.8% Sea Plaque agarose solution (low-gelling temperature agarose, FMC, Rockland, ME). Cool the agarose solution to 37°C. 2. Thin-wall Chem fluor TFE tubing (I.D. 0.5 mm, Berghof/Amertca, Concord, CA). 3. Crushed ice bath for cooling the hepatocyte-gel slurry during threading. 4. A convenient threading tube wrth the appropriate inlet and outlet for perfusion purpose made of normal glass or plexiglass (see Note 3). 5. Sintered-glass support PE 20 MU filters, 2.5 cm (Kontes, Vineland, NJ) (see Note 3) 6. RPM1 1640 perfusion medium with L-glutamine, without phenol red and sodium bicarbonate (Sigma). 7 Roller pump (Masterflex L/S with multichannel, variable occlusion, cartridge pump head system). 8 Carbogen.

2.3. Assessment of Hepatocyte Functionality and integrity in the Gel Support (see Notes 4,10) 1. Diagnostic kit for lactate dehydrogenase measurement (LD-L, Sigma diagnostic kit). 2. Diagnostic kit for urea biosynthesis measurements (Sigma, BUN kit). 3. Precise pipeting devices. 4. Cuvets with optical properties for UV range and 1 cm hghtpath. 5. UV spectrophotometer (e.g., Uvivon 932, Kontron). 6. A machine for measuring the oxygen consumption by cells, CO1 concentrations, and the pH of the medium simultaneously that is fast and applicable to microvolumes of the bioreactor perfusion medium (ABL 5, Radiometer, Copenhagen, Denmark).

3. Methods

3.7. Isolation of Rat Hepatocytes For the isolation of hepatocytes from the liver of rats, we use the standard two-phase perfusion method according to Moldeus et al. (2) with minor modification (8). 1. Thermostat carbogen.

all perfusion

solutions at 37’C and continuously

bubble with

178

Farghali and Hynie

2. Perfuse the liver with calcmm- and collagenase-free, EGTA-containing Hank solution (buffer 1) for 5 min with a roller pump in a recirculating mode. 3. Perfuse the liver with the calcium- and the collagenase-contammg Hanks solu tion (buffer 2) m a recirculating mode for 15 mm. 4. Disintegrate the whole hver carefully in 50 mL of buffer 3 contammg 2% BSA 5. Filter the mixture of cell suspenston through a 75-pm pore nylon filter. 6 Separate the liver parenchymatous cells (hepatocytes) from other liver cells (Kupfer cells, endothelial cells, disintigrated hepatocytes, Ito cells) in the tiltrate by differential centrifugation of cellular suspension m buffer 3 containing 2% BSA at 50g. 7 Repeat the process of washing with buffer 3 containing 2% BSA and centrifugation one or more times (see Note 5). 8. Count the hepatocytes by using a cell counter and monitor cell viability microscopically by using Trypan blue exclusion method (10 pL diluted hepatocyte suspension + 10 uL 0.4% Trypan blue). Usually hepatocytes exhibit vtabiltty between 90 and 95% and should not be used if the Trypan blue exclusion is ~85%.

3.2. immobilization of Hepatocytes in Agarose Gel Threads and Subsequent Perfusion 3.2.1. Hepatocyte Immobilization in Agarose Threads (see Fig. 1) Mix equal volumes of the thermostatted agarose solution at 37’C with the isolated hepatocytes (e.g., 5 mL each) at a density of 4-5 x 10’ cells/ml Introduce the liquid agarose-hepatocytes mixture into a thermostatted two-necked bottle with magnetic stirrer. The hepatocytes are immobilized in agarose threads by passing the agarose-cell mixture through cooled Chem fluor TFE tubing (0.5~mm internal diameter). This is performed by means of the release of a smooth stream of carbogen into the well-sealed, two-necked glass bottle contammg 10 mL of the cell-agarose mixture. Hepatocyte agarose mixture extrusion can also be achieved manually by using a syringe with an appropriate needle. Use an ice bath to cool the agarose-cell mixture as it passes through the Teflon tube. The tube that collects and houses the extruded hepatocyte gel threads contams precooled RPM1 1640 medium (see Note 3). Pass the agarose-hepatocyte slurry in the two-necked bottle continuously through the cooled TFE tubing where the agarose solidifies and entraps the cells and is completely extruded into the tube containing the medium.

3.2.2. Perfusion of Immobilized Hepatocytes (see Fig. 2, Note 6) 1. Compress the threads gently into the perfusion tube (see Note 3) to form a densely-packed column. 2. Connect the lower end of the tube to the perfusion roller pump by tygon tubing through a three-way stopcock. This stopcock is used: to eliminate any air bubbles by aspiration; to introduce any compound rapidly as a bolus by a syrmge; and to connect another roller pump for perfusion of chemicals or drugs.

179

Hepatocyte Immobilization in Agarose stream

Carbogen ‘t’e f 1 on

‘Tube

+

1iepatocytes z Perfusion

Inlet

Fig. 1. Schematic illustration of the apparatus used to immobihze hepatocytes withm agarose gel threads (reprinted with permission from Physiol. Rex; ref. 6).

Perfuslo Medium

Gas

Mixture

Fig. 2. Schematic illustration of the perfusion system of immobilized hepatocytes m agarose gel threads. On the left side, there is a thermoregulated and oxygenated perfusion fluid flowing at a rate of 10 mL/min by pump 1. Pump 2 1s connected, tf necessary, to the perfusion system by a three-way stopcock. On the right side, there 1s a heat exchanger that enables the inflow permsate to be warmed prtor to flowing mto the threading tube (reprinted with permisston from Bzochzm. Blophys. Acta, ref. 5).

180

Farghali and Hynie

3. Seal the upper end of the cell perfusion tube with a tubber seal cap. The latter contams an air-tight, fixed tygon tube to serve as an oulet for perfusion medium; a means of keepmg the Teflon plug and filter m position and a means of keeping the dead volume of the cell perfusion medium above the plug mimmum 4. Adjust the perfusion flow rate of the oxygenated and thermoregulated (37°C) RPM1 1640 to 10 mL/min per 2 0 x lo8 cells 5. Use a second pump, if necessary, to deliver the compound of interest to the perfusate at a predetermined concentration using the three-way stopcock. Otherwise, add the compound of interest as a bolus through the three-way stopcock located adjacent to the port of the inflowmg perfusate to the immobilized cells or directly to the perfusate reservoir 6 Start the perfusion m a nonrecirculatmg manner for 30 min to remove unattached hepatocytes and cell debris until a clear outlet perfusate is obtained that can be checked both visually and microscopically 8 Perfuse m a recirculating mode for membrane integrity testing using the rate of lactate dehydrogenase leakage and the functionality assessment by ureogenesis (see Notes 78) 9. Adjust the total volume of the perfusion medium in the system to 100 mL m the case of a nonrecirculating mode 10. Stabthze the cellular system by contmuously recnculatmg perfusion with the oxygenated thermoregulated RPM1 medium for 1 h before carrying out the functionality test. 11 Perfuse in a nonrecirculating system for assessing the consumption of hepatocytes O2

3.3. Assessment of Hepatocyte Integrity and Functionality in the Gel Support (see Notes 4,lO) 3.3. I. Measurement of LD Leakage in Perfusate for Membrane integrity Assessment (see Fig. 3) 1 Measure the rate of LD leakage from the immobilized cells into the perfusate by using LD-L Sigma kit after 30 min of stabilization, and then every 30 mm up to several hours of the perfusion (see Note 7). 2. Add 50 pL samples of the perfusate to 1 mL LD-L reagent, mix by inversion, incubate at 30°C for 30 s, and record the absorbance at 340 nm 3. Contmue the incubation and record the absorbance 60 s later Subtract the first lower absorbance from the second higher value to obtain absorbance change per minute. Calculate LD activity (U/L) from the relationship absorbance change per mmute x 3376

3.3.2. Measurement of Urea Synthesis by Hepatocytes in the Perfusion Medium (see Fig. 4) 1 Add 5 pL samples of the perfusate to 1 mL BUN (Endpoint) reagent, mix by mversion, and incubate at ambtent temperature for 5 min (see Note 8)

Hepatocyte Immobihzation in Agarose

181

1001 80 2 2

60

40 2 -I---=-20 0-

Fig. 3. The time-course

0

100

200

Time

(min)

of cumulative

300

lactate dehydrogenase leakage from tm-

mobihzed hepatocytes.

o-

0

100

200

Time

(min)

300

Fig. 4. The time-course of cumulative urea synthesis by immobrlized

hepatocytes

2. Record the absorbance at 340 nm against reagent blank using RPM1 1640 medium instead of the sample. 3. Subtract the sample absorbance from the blank absorbance. 4. Calculate urea concentration (mg%) from the relationship: blank absorbancesample absorbance x 45

3.3.3. Oxygen Consumption by Cells (see Fig, 5) 1. Take a 100 PL sample, usmg a microsyringe, at the inlet of the perfusion medium after 1 h perfusion with RPM1 1640 medium for cellular stabilization. 2. Immediately introduce mto the injection inlet of ABL 5 (see Note 9) and record the O2 tension as the inflowmg O2 tension (pOq’“flow). 3 Repeat the same estimation but using a sample from the perfusion outlet and the 0, tension as the outflowing O2 tension (p020ufflow).

Farghali and Hynie

182

100

Time

260

(mini

-Go

Fig. 5. The time-course of the rate of oxygen consumption by mnnobihzed hepatocytes.

4. Perform the same steps in control expertments using the perfusion arrangement wtth hepatocyte-free agarose threads and calculate the difference in O2 tension between the mflowmg and the outflowing perfusate which IS termed ~02 blank. 5 Calculate the net rate of O2 consumption of the cells from the relationship* ] x flow rate and express results m terms of [(PO2 ~mw - PO2outflow) _ po2blank nmol/min + 1tF cells

4. Notes 1. The isolation and the preparation of hepatocytes m the agarose threads requires 2-3 h, depending on experience. 2. Stock solutions 1, 2, and 3 can be prepared at high concentrations (e.g., 1 x 10 without NaHCOs) and are stable for several months Workmg soluttons can be prepared 1 d before hepatocyte isolation 3. The tube used for perfusion of the gel threads has 2-cm internal diameter and is 10 cm in height. The lower inlet is used for perfusion of the immobtlized cells m the threads by means of a roller pump. The tube has two filters* one IS fixed below the hepatocyte-agarose threads before threading, whereas the second one overlays the hepatocyte-agarose threads after threading to keep the latter m position. The lower filter is made of a sintered-glass support PE 20 MU filter (Kontes), and is meant to allow free passage of water and solutes m the perfusate while trapping the threads, any unattached cells, or cellular debris that had to be washed out during the first nonrectrculating phase of perfuston The upper filter device is made of either nylon sieve with 110~pm pore size or several layers of medical gauze. It is an arrangement that allows loose cells and debris to be washed out until the perfusate is clear, while restraining the cell-gel threads, which are kept tightly in position by a perforated Teflon plug. The perforated Teflon plug, which fits the tube tightly, is mtroduced and removed from the tube by a specially designed plastic rod that can easily fit into the plug. Various forms of threading tube can be devised to fulfill a specific purpose and can be moditield accordmgly.

Hepa tocyte lmmobiliza tion in Agarose

183

4. Various tests are available that can be used for the present system m the same way as for isolated hepatocytes. Trypan blue exclusion, albumin synthesis, ATP content, the response to various hormones, the drug metabohzmg capacity (specifically sulfation pathway), maintenance of transmembrane Na+ and K+ tonic gradients, alanine aminotransferase and aspartate aminotransferase leakage, gluconeogenesis, lactate dehydrogenase leakage, oxygen consumption, and ureogenesis are examples among many others, The last three parameters are selected for demonstration of these methods. 5 Isodensity Percoll centrifugation by passing over 30% Percoll (Pharmacia, Piscataway, NJ) can be used for purification of hepatocytes if time is available and highly purified hepatocytes are needed. 6. Perfusion of the immobilized hepatocytes in a recirculatmg or nonrecirculating mode depends on the specific goal of the study and the design of a particular compartmental model to describe a solute kinetics. 7. The principle of the method depends on the fact that LD catalyzes the conversion of lactate to pyruvate with simultaneous production of the reduced form of mcotinamide adenine dinucleotide (NADH) from nicotinamide adenine dmucleotide (NAD). This reaction results in a gradual increase in UV light absorbance whose rate is proportional to LD actrvrty in the perfusate. Figure 3 depicts the timecourse of LD activrty in the per&ate during several hours of munobihzed hepatocyte perfusion. It is clear that LD leakage rate is practically constant during the observation time, which indicates the relative stability of the cell membrane during perfusion of the hepatocytes immobilized in the gel matrix. 8. Urea synthesis is followed by monitoring the hydrolysis of urea by urease with the production of ammoma, which in turn serves to aminate a-ketoglutarate to glutamate, with the simultaneous oxidation of the reduced form of NADH to NAD m the presence of glutamate dehydrogenase. This reaction results m a gradual decrease in UV light absorbance that is proportional to urea concentration in the perfusate. Ureogenesrs is one of the most sensitive parameters of metabolic competence of the isolated perfused liver cells, and Fig. 4 depicts the time-course of urea synthesis in the permsate during several hours of immobtlized hepatocyte perfusion. It is clear that the rate of urea synthesis increased during the observation time, which indicates good functionality of the hepatocytes in the gel support during perfusion of the immobilized hepatocytes. 9. Immobiltzed liver cell viabihty and function are assessed periodically by measuring O2 consumption usmg an ABL 5 blood gas analyzer that also gives an estimate of the pH of the perfusate. Figure 5 demonstrates that the rate O2 consumption is constant over several hours, and the pH values of the perfusate are practically constant during the same period. 10. Histological evaluation of hepatocytes in the gel matrix indicates that hepatocytes are almost regularly dispersed in the agarose threads. They are of nearly round or oval shape and their surface is furnished by irregular short microvilli at the electron microscopic level, with spherical nuclei containing predominantly euchromatm and large nuclei, indicating a high level of activity and viabrhty, and

Farghali and Hynie

184

well developed formations of granular endoplasmic reticulum, mitochondria, Golgi complex

and

References 1, Moldeus, P , Hogberg, J., and Orrenms, S (1978) Isolation and use of liver cells, in Methods in Enzymology, vol 52 (Fleisher, S. and Packer, L., eds.), Academic, New York, pp 60-7 1 2 G&es, R J., Chresand, T J , Drury, P. D., and Dale, B. E. (1986) Design and application of bioreactors for analyses of mammalian cells by NMR. Rev Magn Res Med 1, 155-179. 3. Gillies, R J., Galons, J. P., McGovern, R. A , Scherer, P. G., Lien, Y. H., Job, C., Ratcliff, R., Chapa, F , Cerdan, S , and Dale, B. E. (1993) Design and application of NMR-compatible bioreactor circuits for extended perfusion of high-density mammalian cell cultures NMR Boomed 8,95-104. 4. Foxall, D. L., Cohen, J S., and Mitchell, J. B. (1984) Continuous perfusion of mammalian cells embedded m agarose gel threads. Exp. CelE Res 154,52 l-529 5. Farghali, H , Rossaro, L., Gavaler, J S , Van Thiel, D H , Dowd, S R , Williams, D. S., and Ho, C. (1992) 3*P-NMR Spectroscopy of perfused rat hepatocytes immobilized m agarose threads: application to chemical-induced hepatotoxicny. Blochim Biophys Actu 1139, 105-114. 6. Farghah, H., Kamenikova, L., and Hynie S. (1994) Preparation of functionally active immobilized and perfused mammalian cells* an example of hepatocyte bloreactor. Phystol. Res. 43, 121-125. 7. Farghah, H., Williams, D. S., Caracem, P., Borle, A. B., Gasbarrini, A., Gavaler, J., Rilo, H. L., Ho, C., and Van Thiel, D H. (1993) Effect of ethanol on energy status and mtracellular calcium of Sertoh cells: a study on immobilized perfused cells Endocrinology

133(6), 2749-2755.

8. Farghali, H , Machkova, Z., Kamenikova, L., Janku, I , and Masek, K (1984) The protection from hepatotoxicity of some compounds by synthetic immunomodulator muramyl dipeptide (MDP) m rat hepatocytes and in VEVO.Meth. Fmd Exptl. Clan Pharmacol

6,449-456.

9 Gasbarrmi, A., Borle, A. B , Farghah, H., Bender, C., Francavilla, A, and Van Thiel, D. H. (1992) Effect of anoxia on intracellular ATP, Na,+, Ca,2+ and cytotoxicity m rat hepatocytes. J Blol Chem 267,6654-6663 10 Gasbarrmi, A., Borle, A. B , Farghah, H., Francavilla, A., and Van Thiel, D H (1992) Fructose protects hepatocytes from anoxic qury. Effect on mtracellular ATP, Ca,2+, Mg,2+, Na,+ and p H, J Blol Chem 267,7545-7552 11. Gasbamm, A., Borle, A. B., Farghah, H., Caracem, P., and Van Thiel, D. H. ( 1993) Fasting enhances the effect of anoxia on ATP, Ca,+ and cell iqury m isolated rat hepatocytes. Blochgm. Biophys. Acta 1150(l), 9-20. 12. Caraceni, P., Gasbarrini, A., Van Thiel, D. H., and Borle, A B. (1994) Oxygen free radical formation by rat hepatocytes durding ostanoxic reoxygenatton: scavenging effect of albumin. Am J Physlol 266, G45 l-G458.

22 Immobilized Hepatocytes in Xenobiotic Biotransformation

Studies

Sixtus Hynie, Ludmila Kamenikova, and Hassan Farghali 1. Introduction In Chapter 2 1, we described a method for hepatocyte immobilization in agarose threads that enables perfusion of the cells and facilitates varied biomedical studies on the biochemtcal properties of hepatocytes. Since the liver 1sthe most important and unique organ m metabolic processmg of both endogenous and exogenous compounds; this chapter provrdes details of an application of immobilized and perfused hepatocytes for xenobiotic biotransformation as an indication of many other potential applications. The liver cells possess high nonspecific enzymatic activity toward individual substrates that, however, may be considered specrfic toward certain chemical groups. These metabolic enzymes, which are genetically determined in the organism, are affected by many exogenous and endogenous factors. The chemical structure of the foreign compound is an important factor for determining its own metabolic fate. It is well known that xenobiotrc biotransformation, which takes place predominantly in liver cells, occurs in two phases (Z-3). In phase I, the major reaction involved is oxidation catalyzed by enzymes, known as mono-oxygenases or cytochrome P450 species, in addition to other reduction and hydrolytic reacttons. In general, phase I reactions reduce the biological activity of the foreign compound or, less frequently, produce highly active metabolites. In phase II, the hydroxylated or other products formed in phase I are converted by specific enzymes to various polar metabolites by conjugation reactions. In this chapter, we demonstrate the application of an immobilized and perfused hepatocyte cellular system (small-scale laboratory hepatocyte bioreactor) as a means to study representative phase I and phase II biotransformation reactions. The de-ethylation of 7-ethoxycoumarin (7-EC) is presented to illustrate a phase I From

Methods II) Bfotechnology, Vol 1 /mmobr/rzat~on of Enzymes and Cells E&ted by G F Buckerstaff Humana Press Inc , Totowa, NJ

185

Hynie, Kamenikova, and Farghali

186

biotransformation. Biotransformation of umbelliferone (UM) and 4-nitrophenol (4-NP) are presented to illustrate phase II reactions (sulfate and glucuromde conjugation). 2. Materials 1. Male Wistar rats (200-250 g body wt) 2. Sodium phenobarbital. 3 Perfusion medmm RPM1 1640 (Sigma, St. Louts, MO). Prepare accordmg to suppher instructions 4. Immobihzed hepatocytes for perfusion: Prepare as described m Chapter 2 1. 5 Pemcillm G, potassmm salt for injection (500,000 IV). 6 Streptomycin sulfate, BP 7 0.5MTris-HCl, pH 7 6 8. 1 mM7-Ethoxycoumarm (7-EC) stock solution m O.SMTris-HCl, pH 7.6: Weigh 4 8 mg of 7-EC and add to 25 mL of 0.5MTris-HCl, pH 7.6; shake contmuously by using a mechanical shaker (e g., Vortex Geme 2) for several hours. Leave the partially dissolved 7-EC solution at room temperature overnight for complete solution. 9. Umbelliferone (UM) stock solution. Dissolve 4 mg of UM m 50 mL of 0 5M Tris-HCI, pH 7.6 10 36 yM4-Nitrophenol(4-NP) stock solution* Dissolve 50 mg of 4-NP in 100 mL of 0.5M Trts-HCl, pH 7.6 11. Sahcylamide solutton: Dissolve 5 mg of sahcylamtde m 50 mL of RPMI-1640 solution. Prepare a fresh solution before the experiment. Add an appropriate volume to produce a concentration of 140 @f to the perfusion medium as an mhibitor of the next conjugation reactton (phase II) 12. UV/visible spectrophotometer (Uvicon 932, Kontron; and fluorescence spectrophotometer, Perkin-Elmer 3000 [Norwalk, CT]). 13. pH Meter. 14. Roller pump (Masterflex L/S with multichannel, variable occlusion, cartridge pump head systems).

3. Methods

3.1. Metabolism of 7-EC and lJM by Immobilized (see Figs. 1,2) 3.1.1. 7-EC

Hepatocytes

1. Inject the rats intraperitonially with 0.5 ml/animal of physiologtcal salme (controls) for four successivedays or with 80 mgikg body wt of sodium phenobarbital dissolved in an equivalent volume of physiological saline for four successive days (see Note 1). 2. After 24 h from the last dose, isolate the hepatocytes from the relevant rat by the

standardtwo-phase perfusion method, asdescribedin Chapter 21. 3 Xmmobihze the hepatocytes prepared from control rats or phenobarbital-treated rats in the agarose gel support (4,s) as described m Chapter 2 1. Immedtately per-

Hepatocyte Biotransformation

Studies

187

--e-- induced -o-

20

40

noninduced

60

80

Time [mrt]

Fig. 1. The mean time course of accummulation (k SEM, n = 5) of the de-ethylated product UM (umbelliferone) during de-ethylation of 7-EC (7-ethoxycoumarm) m the perfusate of induced and nonmduced hepatocytes.

-o- noninduced --•-- Induced

0

0

I 20

I 40

I 60

, 60

Time [mln]

Fig. 2. The mean time course of the disappearance (+ SEM, n = 5) of UM (umbelliferone) m the perfusate of induced and noninduced hepatocytes.

fuse the immobilized hepatocytes with a thermostatted and oxygenated (95% 02, 5% CO*) RPMI- 1640 medium at a rate of 10 mL/min in a nonrecirculating system using a peristaltic pump for about 30 min or until the perfusate becomes clear. 4. Recirculate the perfusate at the same rate (10 mL/min) in a total perfusion volume of 100 mL of RPM1 for 1 h to stabilize the cells. 5. Add to the perfusion reservoir 20 mL of salicylamide solutton and perfuse continuously for an additional 20 min (see Note 2).

188

Hynie, Kamenikova, and Farghali

6 Add 2 mL of 7-EC solution to the perfusion reservoir (20.2 @4 final concentration). Ten minutes later collect a 2 mL sample from the reservoir, which IS replaced immediately by fresh prethermostatted RPM1 1640 medium (37°C). In the same way, continue collecting perfusate samples from the reservoir at 20,30, 45,60, 80, and 90 min after 7-EC addltlon. Use these samples for assessment of the 7-EC de-ethylation product, namely umbelliferone (UM), by fluorimetry. 7. Repeat the experiments using hepatocytes either from phenobarbital-treated rats or physlological saline-treated rats (control) 8. Construct a calibration curve for UM fluorescence, with standards m concentration ranges between 5 and 800 ng/mL, using a fluorlmeter with excltatlon 366 nm and emission 454 nm 9 Extrapolate the production of 7-EC O-de-ethylation m the perfusate sample from the previously constructed cahbratlon curve. 10 Plot a graph to demonstrate the time-course of UM appearance m the perfusate after normalization to amount/mllhon hepatocytes for data obtained from induced and nonrnduced rats (see Notes 3,10)

3.1.2. UM 1. Prepare the immoblhzed hepatocyte system as described m steps 1-4 above, but excluding the addition of salicylamide 2. Add to the perfusion medium 188 pL of UM stock solution (150 ng/mL mitral concentration) 3 Continue as for steps 6-7 above, but replacing 7-EC with UM. 4. Repeat the experiments using hepatocytes from phenobarbital-treated rats and physiological saline-treated rats (control) and UM as a substrate 5. Extrapolate the decline of UM concentration m the perfusate (as a result of conjugation) from the previously constructed calibration curve, which gives an estimate of uncoqugated UM in the perfusate. 6 Plot a graph to demonstrate the time-course representing the decline of UM concentration in the perfusion medium after normalization to an amount in pmol/ million hepatocytes for data obtained from phenobarbital-induced and nomnduced rats (see Notes 4, IO).

3.2. Metabolism (see Fig. 3)

of 7-EC During Three Successive

Days

1 Prepare the immobilized hepatocyte system as described in Section 3.1.) steps l9, but using hepatocytes only from phenobarbital-treated rats 2. At the end of 7-EC sampling, use a nonrecirculating system to perfuse the hepatocytes for at least 1 h to wash out 7-EC and its metabohte 3. Add to the perfusion medium m the reservoir penicillin G and streptomycm sulfate to concentrations of 50 U/mL and 50 pg/mL, respectively. 4. PerfUse the antibiotic-containmg medium for 30 mm in a nonrecn-culatmg system. 5. Stop perfusion and carefully unlock the tube containing the lmmoblhzed hepatocytes with the threads in the antibiotic-containing medium. The tube with its

Hepatocyte Biotransformation 120 u

100

Studies

189

-o- lstday --a-- 2nd day

20 40 so 80 Time [mln]

Fig. 3. The mean time-course of accummulation (& SEM, n = 5) of the de-ethylated product UM (umbelliferone) during de-ethylation of 7-EC (7-ethoxycoumarin) obtamed from the same bioreactor over three successive days.

seal cap and lock from the lower inlet is simply stored in a refrigerator (4-8’C) overnight. 6. On the second day, leave the tube containing the cells at room temperature for 1 h. Then repeat steps l-5. 7 On the third day, repeat step 6, then terminate the experiment (see Note 4) 8. Plot a graph to demonstrate the ttme-course of the de-ethylation product of 7-EC or UM appearance in the perfusate after normalization to amount in pmol/million hepatocytes for data obtained from the induced rats (see Notes 5,lO).

3.3. Metabolism

of 4-NP (see Fig. 4)

1. Prepare the immobilized hepatocyte system as mentioned in steps 14 of Section 3.1.) but using hepatocytes from untreated rats and without the addition of sahcylamide to the perfusate. 2. Add 10 mL of 4-NP stock solution (i.e., 5 mg) to the perfusion reservoir. Ten minutes later, collect a 2-mL sample from the reservoir, which is replaced immediately by a fresh, prethermostatted RPS 1640 medium (37’C). In the same way, continue collecting permsate samples from the reservoir at 20,30,45,60,80, and 90 min after 4-NP addition. 3. Construct a calibration curve for 4-NP light absorbance at pH 11.5 and wavelength 405 nm, with standard concentrations of l-15 pg/mL, using the spectrophotometer. 5. Add 0.4 mL of 2N NaOH to 2 mL sample perfusate to give a final pH of 11.5. Measure the concentration of 4-NP in the perfusate by the method of Burchell and Weatherill (6).

Hynie, Kamenikova, and Farghali

190

3b

i0

i0

Time [mn]

Fig. 4. The mean ttme-course of the disappearance (+ SEM, n = 6) of 4-NP (4-mtrophenol) m the perfusion medmm.

6 The decline of 4-NP concentratton (1 e., conjugation) m the pet&ate sample is extrapolated from the previously constructed calibration curve 7 Plot a graph to demonstrate the time-course of 4-NP UDP-glucuronyltransferase activity in the perfusate (the reduction in color resulting from the formation of the glucuronide product). Normalize the data to amount in pmol/million hepatocytes (see Notes 6-9) 4. Notes 1. This cellular model is very sensitive for detection of low levels of enzyme metabolizing activity. Previously, it was reported that rat liver microsomes had almost no 0-dealkylatmg activity toward 7-EC (7). However, the present work demonstrates that low levels of de-ethylase actrvlty can be detected with high accuracy m a hepatocyte bioreactor, but not m isolated mrcrosomal preparations or even isolated cultured cells. After induction of the microsomal enzymes with phenobarbital the de-alkylase activity 1s increased severalfold The results mdtcate that the activity of 7-EC 0-de-ethylase is linked to an lsoenzyme form of cytochrome P450 that is mductble. In addition, UM accumulated m the perfusate after 7-EC addition, or when used as a substrate per se, 1s almost exclusively changed to the comugated form (sulfatton and glucuromdation). The rate of conjugation of UM obtained from nonmduced and induced rats 1s stmtlar. This demonstrates the nature of transferases (in this case, glucuronyl transferase and sulfotransferase) that are cytosolic and are not inducible by phenobarbital. 2. Sahcylamide 1sadded to the perfusion medium before 7-EC to inhibit the subsequent sulfatton and glucuronidation (conjugation) steps. Thus, the de-ethylatton product UM will accumulate and can be quantitated in the medium.

Hepatocyte Biotransformation

Studies

191

3. Ftgure 1 demonstrates UM appearance in the perfusate as a dealkylation product of 7-EC for hepatocytes obtamed from both induced and noninduced rats. 4. Figure 2 demonstrates the decline of UM concentration in the perfusate as a result of its conjugation for hepatocytes obtained from both phenobarbttalinduced and nonmduced rats. Note that there IS no difference in UM ehminatton kmetics as obtained from induced and noninduced hepatocytes. 5 Figure 3 demonstrates UM appearance in the perfusate as a dealkylation product of 7-EC for hepatocytes obtained from induced rats, using the same cellular system for three successive days. This shows that under appropriate preservation conditions, the mnnobtlized perfused hepatocytes can be used for more than 1 d and the potential use of the same system for relatively longer periods of time has economic and ethical implicatrons. 6. Figure 4 demonstrates the time-course of 4-NP disappearance m the perfusate because of glucuronyl conJugation. 7 It is possible to challenge the mnnobllized cells m one bioreactor model with several substrates successively, provided that the substrate concentrations are nontoxic to the cells. 8. In many cases, direct assay of the metabolites is possible without exhaustive extraction or pretreatment. Obviously, this depends on the analytical method of the relevant compound. Both 7-EC 0-de-ethylation and IJM conjugation are evaluated directly m the permsate, whereas 4-NP conjugation requn-es the modification of the pH of the perfusion medium for the assay. 9 Simultaneous measurements of xenobiotic cellular kinetics and the effects of the added compounds can be evaluated in the perfusion medium 10. The calculations of the metabolite appearance or the substrate disappearance reported herem graphically so far were not corrected because of sampling of the perfusion medium and replacing by fresh medium. Correction methods, however, can be applied if necessary.

References 1. Murray, R. K. (1991) Metabolism of xenobiotics, in Harper’s Bzochemzstry, 22nd ed. (Murray, R. K., Mayes, P.A., Granner, D. K., and Rodwell, V W , eds.), Appleton & Lange, Norwalk, CT, pp. 645-649. 2. Kamenikova, L., Farghali, H., Misekova, D., Lincova, D., and Hynie, S. (1994) Application of the hepatocytes bioreactor to xenobiotic biotransformation. Physiol. Res. 43, 127-130. 3. Hyme, S., Kren, V., Bila, V., Mraz, M., Gaier, N., Kamenikova, L., and Farghah, H. (1994) A preliminary evaluation of drug biotransformation in hepatocytes of genetically defined rat strains. Physiol. Res. 43, 13 1-135. 4. Foxall, D. L., Cohen, J S., and Mitchell, J. B. (1984) Continuous perfuston of mammalian cells embedded in agarose gel threads. Exp. Cell Res 154,52 l-529. 5. Fargahli, H , Kamemkova, L., and Hynie, S (1994) Preparation of functionally active munobilized and perfused mammalian cells: an example of hepatocyte btoreactor Physzol Res. 43, 121-125.

192

Hynie, Kamenikova, and Farghali

6. Burchell, B. and Weathenll, P. (198 1) 4-Nitrophenol UDP-glucuronyltransferase (rat liver), mM&ods in Enzymology, vol. 77. Academic, New York, pp. 169-177. 7 Ullrich, V, Rommer, U., and Weber, P. (1973) Charactertsatton of cytochrome P-450 species in rat liver mtcrosomes, I. Differences m the 0-dealkylation of 7-ethoxycoumarin after pretreatment with phenobarbital and 3-methylcholanthrene. HoppeSeyler 2. Physiol. Chem 3545 14-520.

23 Immobilization in Gel Beads

of Liposomes and Proteoliposomes

Eggert Brekkan, Qing Yang, Gerhard Viel, and Per Lundahl 1. Introduction The lipid bilayers of hposomes are structurally similar to those of biological membranes and are often used as membrane models. Solubilized membrane proteins (transporters, receptors, enzymes,and so on) can be reconstituted into the bilayers (1-3). Interactions between the reconstituted proteins or the lipid bilayers and various substancesof biological interest have been studied by several methods, including chromatographic analysis with immobilized proteoliposomes or liposomes as stationary phase (4-11). Lipid monolayers covalently coupled in silica gel beads have also found chromatographic applications (immobilized artificial membrane chromatography) (12-l 4). Bed reactors with immobilized membrane-bound enzymes in lipid bilayers have been designed (1.516). Methods are described here for immobilization of (proteo)liposomes in gel beads by steric entrapment (17,28) or by use of hydrophobic ligands (29,20). Interactions between the lipid bilayers of immobilized liposomes and drugs or peptides have been studied by chromatographic techniques, as have interactions between the reconstituted and immobilized red cell glucose transporter and glucose or the transport inhibitor, cytochalasin B. Specific capacity factors and dissociation constants were determined. These and other examples of the analytical and preparative use of (proteo)liposomes immobilized in gel beads are briefly presented in Section 3.3. 2. Materials 2.1. Preparation

and Immobilization

of (Proteo)liposomes

1. Egg yolk phosphohprds (MO, 95, and 99% phosphatidylcholine [PC]) (catalog nos. 241601, 14 1601 and 83005 1, respectively; Avant1 Polar Lipids, Alabaster, From. Methods MI Botechnology, Vol I Immobrlmtron of Enzymes and Cells Edlted by G F BIckerstaff Humana Press Inc , Totowa, NJ

193

194

2 3. 4. 5. 6. 7 8 9 10 11 12 13. 14. 15 16 17 18. 19. 20. 2 1. 22. 23

Brekkan et al. AL) Bovine brain extract lipids (type III, 80-85% phosphatidylserine, Sigma, St. LOUIS, MO) (see Note 1). Chloroform. Anhydrous diethylether (reagent grade) N2 gas (~5 ppm 02, ~5 ppm H20, free from oil) Hz0 from Milhpore deiomzer with active carbon cartridge and 0.2~pm filter 750 mA4 stock solution of sodium cholate, pH 8.4. Prepare by addition of 2M NaOH to cholic acid (>99%, Fluka, Buchs, Switzerland) m H20. BufferA: 10 mMTris-HCl, pH 7 4,150 mA4NaC1,l mA4Naz EDTA (see Note 2) Vortex mixer (Lab-Line Instruments, Melrose Park, IL). Sephadex G-50M gel beads, column 2 (id) x 40 cm, peristaltic pump, UV momtor (see Note 3), and fraction collector for hposome preparation. Concentration cells (Mmicon B- 15, Amicon, Beverly, MA). 14-mL polystyrene tubes (e.g., Falcon tube, Falcon, Los Angeles, CA) Dry ice (CO,(s)) 99 5% Ethanol Gel beads for unmobihzation Superdex 200 prep grade, average bead size 34 pm, (Pharmacia) (see Note 4). H 5/5 glass columns (Pharmacia), 5 mm id, gel bed height 4-6 cm (see Note 5) Pump, preferably of high-performance liquid chromatography (HPLC) type, giving constant flow rate for immobilized-liposome chromatography Cellulose dialysis tubing, flat width 32 mm, M, cutoff 12,000-14,000, (Viskasi [Chicago, IL] or Spectra/Par 2 or 4, same cutoff, or Spectra/Par 1, cutoff 60008000, Spectrum [Houston, TX]), and tubing closures from the latter company. Column, 1 (id) x 6 cm, for equilibrating gel beads with lipid solution before dialysis. Smooth-edged, U-shaped plastic frame with lid that fits m the dialysis tubing (see Fig. 1 for dimensions). Dialysis buffer: 20 mA4Tris-HCl, pH 7.0, 150 mMNaC1, 1 mA4Na2EDTA, 1 mA4 2-mercaptoethanol (see Note 6) Dialysis vessel with stirring or with rotation and buffer flow (see Note 7). Na(BH,) (95%, Merck, Darmstadt, Germany) 1,4-Butanedlol diglycldyl ether (70%, Sigma) (see Note 8). 1-Octanethiol (97%, Fluka)

2.2. Amount of Immobilized Phospholipids and Proteins 2.2.1. Phosphorus Analysis by Modified Bartlett Method (21) 1 2 3 4. 5 6. 7. 8 9

1-Amino-2-naphthol-4-sulfomc acid (Merck) 15% (w/v) Na2S205, freshly prepared. Anhydrous Na$!lOs. 15-mL test tubes (see Note 9). 0 65 mM phosphorus standard (Sigma) 5M H2S04. Oven (160°C). H202 (Perhydrol, 30%). 0 22% (w/v) ammonium molybdate, (NH4)6M07024 *4HzO (Merck)

195

Immobilization of Liposomes

t 4I!&

30

3

-30-

Fig. 1. Plastic frame (Perspex), with lid. A flat-shaped dialysis cell is formed by inserting the frame mto tight-fitting dialysis tubing. Dimensions are m mrlhmeters The inside height with inserted lid is 20 mm.

2.2.2. Protein Analysis by Modified Lowry Method (22) 1. 2 3. 4 5 6.

I mg/mL bovine serum albumin (>98% BSA, product no A7030, Sigma). 18% (w/v) trichloroacetic acrd (TCA). 2% (w/v) Na2C03 m O.lM NaOH, freshly prepared. 2% (w/v) CuSO4 * 5H20 (Merck). 4% (w/v) K, Na-tartrate (BDH, Poole, England). Folin and Ciocalteu’s reagent (BDH), diluted 1+3 with Hz0

3. Methods The procedures are performed at room temperature wise stated, and the pH values given refer to 23OC.

(-23°C)

unless other-

3.1. Preparation of (Proteo)liposomes 3.1.1. Phospholipid Solution 1. Dissolve the lipids in chloroform in a 100-mL round-bottomed flask (see Note 10) 2. Remove the solvent by rotary evaporation until a dry lipid film is formed on the walls of the flask 3. Repeat the dissolution and removal of solvent twice with diethylether. 4. Flush the lipid film with N2 until solvent odor has disappeared. 5. Disperse the lipid film with 125 or 250 rniV cholate in buffer A to obtain 100 or 200 mM phospholiptd, respectively, by vigorous mixing (vortex) followed by rotation under mild vacuum (- 100 mbar) for 30 min. The lipid film volume can be neglected (see Note 11) 6. Store the lipid solution at -7O’C (2 mL portions are convenient).

3.1.2. Chromatographic

Detegent-Depletion

Method

1. Apply 4 mL of 100 mMphospholipid solution on a Sephadex G-50 M gel bed, 2 (id) x 40 cm, in buffer A at a flow rate of 2 mL/min at 5-9°C. Or, to prepare

Brekkan et al.

196

proteoliposomes, use 200 mM phosphohpid solution mixed with detergent-solublllzed membrane protems at the ratlo 1: 1 (v/v) or as required (see Note 12) 2 Collect the (proteo)liposomes (-14 mL, 15-20 mM) eluting at the void volume (see Note 13). 3. Transfer the (proteo)liposome suspension gradually to two 5-mL chambers of the Minicon B- 15 concentrator by use of a long-tipped Pasteur plpet. 4. Concentrate the suspension at 44X (5-10 h) to a total final volume of 1.5-2 mL (1 O&l 50 rnI14 lipid) (see Note 14).

3.1.3. Lipid Hydration Method 1. Steps l-4 in Section 3 1 1 2. Add buffer A to a final composition of 100-150 &phosphollpid, 150 mA4NaC1, 1 mA4Na2EDTA, and 10 mMTrls-HCl, pH 7.4, and a final volume of 1.5-2 mL The lipid film volume can be neglected. 3. MIX (vortex) thoroughly until the lipid IS nearly completely dispersed and rotate for 30 min under mild vacuum (- 100 mbar) (see Note 11).

3.1.4. Immobilization of (Proteo)liposomes 3.1 4.1. FREEZE-THAWING

IMMOBILIZATION

OF (PROTEO)LIPOSOMES

PREPARED

BY THE CHROMATOGRAPHIC METHOD (see FIGS. 2,3A)

1. Wash the Superdex gel beads with increasing concentrations of ethanol (20, 50, 99.9%) on a Buchner funnel with water aspiration. 2 Store the partially dried beads under vacuum in a desiccator with anhydrous CaCl* until solvent odor has disappeared 3 Add 1 5 mL concentrated (proteo)hposome suspension to the dry gel beads (corresponding to 1 mL gel bed) in a 14 mL polystyrene tube and mix (vortex) vigorously (see Note 15) 4. Degas the mixture and allow the gel to swell for 3 h at 4’C. 5 Prepare COz(s)/ethanol solution of -70°C (see Note 16) and water bath of 25°C 6. Freeze-thaw the mixture m the tube (-7O”C, 10 min; 25’C, 10 mm or as needed) and mix (vortex) thoroughly. 7 Repeat step 6 8 Transfer the freeze-thawed mixture of gel beads and (proteo)liposomes to a centrlfugatlon tube. 9. Add buffer A to a final volume of about 10 mL and mix. 10. Centrifuge at 350g for 3 min. 11. Remove the supernatant containing nonimmobllrzed (proteo)liposomes and replace with buffer A 12. Repeat steps 9 and 10 several times until a clear supernatant is obtained (see Note 17). 13. Pack the gel with the immobilized (proteo)liposomes in a 5 mm (id) column (HR 515) at suitable flow rate and equilibrate with at least 15 column volumes of buffer A (see Note 18).

Immobilization of Liposomes

197

Fig. 2. Schematic illustration of enlargement of liposomes induced by freeze-thawing. The implied mechanisms are hypothetical. Large liposomes that are formed in gel bead pores on freeze-thawing of mixtures of beads and liposomes of suffkiently high concentration become sterically immobilized (entrapped).

Fig. 3. Schematic illustration of proteoliposomes immobilized in gel beads by (A) freezethawing or dialysis (steric immobilization) or by (B) adsorption to hydrophobic ligands (hydrophobic immobilization).

3.1.4.2.

FREEZE-THAWING IMMOBILIZATION OF LIPOSOMES PREPARED BY THE LIPID HYDRATION METHOD (see FIGS. 2,3A)

1. Prepare COz(s)/ethanol solution of -70°C (see Note 16) and water bath of 25°C. 2. Freeze-thaw the liposome suspension twice (-70°C, 10 mitt; 25°C 10 min or as needed) with vigorous mixing (vortex) after thawing. 3. Steps l-4 in Section 3.1.4.1.

Brekkan et al.

198

4. Freeze-thaw the suspenston (-70°C 5 min; 25’C, 5 mm or as needed) and mrx (vortex) vigorously. 5 Repeat step 4, four times. 6. Steps 8-13 of Section 3.1 4 1. (see Note 19). 3.1.4.3.

DETERGENT DIALYSIS IMMOBILIZATION (SEE FIG. 3A)

1. Thread the dialysis tubing onto the U-formed plastic frame, close the tubing at one end to obtain a dialysis cell, and unmerse the cell m dialysis buffer (see Fig. 1, Note 20). 2 Dilute 0.7 mL of 100 mA4 phosphohpid solutton with 0.7 mL of buffer A. Or, to prepare proteoltposomes, mix the phospholipld solution with an equal volume of detergent-solubrltzed membrane protems (see Note 12). 3. Pack gel beads (corresponding to 1 1 mL gel bed) in a 5 mm (id) column (HR 515) 4. Fill the 1.1 -mL gel bed with the phosphohpid solution and leave to equilibrate for 20 min 5 Fill the dialysis cell with the gel by pushing it out of the column by use of an end piece. 6 Put on the frame lid, close the tubing, and place the cell m the dialysis vessel 7 Dialyze against 4 x 500 mL of dialysis buffer with magnetic stirring for 2 d or with rotating cell and continuous flow overnight (see Note 2 1) 8. Remove nonimmobihzed material by repeated centrtfugal washing, pack the gel beads (corresponding to 1 mL packed gel) mto a column, and equilibrate with buffer A (see Notes 17,18). 3.1.4 4. HYDROPHOBIC IMMOBILIZATION (SEE FIG. 3B) 1 Dry gel beads partially by water aspiration on a Bdchner funnel and mix 1 g of the moist beads with 1 mL 0.6M NaOH (containing 2 mg of Na[BH4]) and 0.5 mL of 1,4-butanedtol diglyctdyl ether in a round-bottomed flask. Stir gently overnight (15 h). 2. Wash the beads thoroughly with water (250 mL) on a Buchner funnel and transfer them into a round-bottomed flask 3 Add 0.8 mL of lMNaOH, 10 mg of Na(BH& and 0.8 mL of freshly prepared solution of 95% ethanol contammg 20 pmol of the octanethiol to be coupled (see Note 22) 4. Stir for 20 h at room temperature. 5 Wash the derivattzed gel on a Bdchner funnel wrth water followed by 95% ethanol (-3 mL of each). Repeat the washing cycle several times until the thiol smell has disappeared. 6. Pack the dertvatized gel beads (corresponding to 1 mL gel bed) suspended in buffer A into a 5-mm (id) column (HR 5/5) and equilibrate with the buffer. 7. Recirculate the (proteo)liposome suspension through the column overnight (see Note 23). 8. Equilibrate the column with at least 30 column volumes of buffer A

Immobilization of Liposomes

799

3.2. Amount of Immobilized Phospholipids and Protein 3.2.1. Phosphorus Analysis by Modified Bartlett Method (21) 1. Dissolve 0.25 g of l-amino-2-naphthol-4-sulfonic acid in 100 mL of freshly prepared 15% Na+$Os. 2. Add 0.5 g of anhydrous Na#Os. 3 Heat the solution gently, filter, and label it Fiske-Subbarow Reagent (see Note 24). 4. Take 0, IO, 30,50,75, 100, 125, and 150 pL of phosphorus standard solution and suitable aliquots (O-2 mL) of the samples (duplicates) (see Notes 25,26) 5. Add 0.25 mL of 5M H2S04 6 Heat in an oven at 160°C for at least 3 h. 7 Cool the tubes to room temperature, add 200 pL of H202, and heat at 160°C for at least 3 h (see Note 27). 8. Cool the tubes to room temperature and add 2.3 mL of ammonium molybdate solution and 0.1 mL of Fiske-Subbarow reagent. Mix thoroughly (vortex) before and after addition of the reagent. 9 Immerse the tubes m boiling water for 7 mm. 10. Cool to room temperature and measure the absorbance at 830 nm within 30 mm. 11 Calculate the phosphorus amount (phospholtptd amount) in the samples by use of the standard curve.

3.2.2. Protein Analysis by Modified Lowry Method (22, see Note 28) 1 Take 0,20,50, 100, and 200 pL of BSA standard solution and suitable aliquots of the samples (duplicates) and dilute to 600 pL with sample buffer. 2 Add 14 mL Hz0 and 1 mL of 18% TCA, mix thoroughly, and incubate for 10 mm on ice. 3. Centrifuge at 600g for 15 min, decant the supernatant, and make sure that the tubes are dry by blotting away any drops on the walls. 4 Solutton 1: Add 255 uL of the 2% CuS04 and 255 pL of the 4% K,Na-tartrate to 50 mL of the 2% Na$Os solution. 5. Add 3 mL of solution 1 to the samples (pellets), mix thoroughly, and incubate for 10 min at room temperature. 6. Add 300 pL of the diluted Folin and Ciocalteu’s reagent; incubate for 30 mm to 2 h. 7. Measure the absorbance at 700 nm. 8. Calculate the protein amount in the samples by use of the standard curve.

3.3. Application Examples 3.3.1. Analysis of the Interaction Between Lipid Bilayers and Drugs or Peptides Partitioning of drugs into lipid bilayers of immobilized phosphatidylcholme liposomes, can be studied and the specific capacity factor can be defined that relates the retention of drugs to the amount of immobilized hposomes in the columns (9,10). Such data can be helpful for prediction of transmembrane

200

Brekkan et al.

uptake of drugs in cells. Other similar chromatographic studies have been reported (14,15,23-25). The immobilization of hposomes m gel beads can be studied (IO), and stable immobilization of liposomes m gel beads can be achieved with high flow rates and reproducible chromatographic results (see Fig. 4). Chromatographlc analyses can also reveal interactions between llposomes and water-soluble peptides or amphiphilic peptides, such asthose corresponding to certain postulated transmembrane segments of the glucose transporter Glutl. The retardation of peptides is related to their water-to-o11 transfer free energies and the distribution of this parameter within the peptides. Also, C-terminal cysteine residues contribute to increased retention (II). 3.3.2. Immobilized Proteoliposome and Membrane-Vesicle Affinity Chromatographic Analysis of Interactions Between the Facilitative Glucose Transporter Glut1 and Glucose or the Inhibitor Cytochalasin B (CB) Proteoliposomes containing purified Glut1 can be nnmobillzed in gel beads and the dissociation constants for Glut 1-CB and Glut 1-o-glucose can be determined by quantitative zonal affimty chromatography (8). The same constants have recently been determined by frontal affinity chromatography (E. Brekkan, in press). Frontal affinity chromatography has the advantage over the zonal method in that the amount of active and available bmdmg sites of the protem can be determined, which gives more accurate dissociation constants than when the total amount of protein immobilized is used. Furthermore, there is no need for purification of the protein prior to reconstitution and immobilization when frontal analysis is used. Even Glut1 in immobilized membrane vesrcles can be subjected to frontal analysis. 3.3.3. Ion-Exchange Chromatography on Charged Llposomes Prepared and Immobilized by Dialysis At low ionic strength, proteins can be bound to the surfaces of immobilized charged liposomes (26,27), and the maximal binding is estimated to correspond to surface charge neutralization. Refined treatments have recently been reported (28,29). The method can be used to separate water-soluble proteins and discriminate between BSA monomers and dlmers (2627). 3.3.4. Transport Retention Chromatography Immobilized on Hydrophobic Ligands

on Proteoliposomes

Proteoliposomes containing purified glucose transporter Glut1 can be immobilized on octylsulfide ligands on hydrophilic spacer arms as described m Section 3.1.4.3. (7,20). n-14C-glucose can be eluted slightly but significantly later than L-3H-glucose, presumably owing to the transport of only the n-enan-

Immobilization of Liposomes

-0.5 IA 0

201

/B

’ a ’ ac’ c aa3* a3 ’ 8’ 0.5 1.0 2 6 10 14 Flow rate (ml/mm) Days from immobilization

Fig. 4. Immobilized liposome-chromatography of three drugs of different hydrophobicities (0, hydrocortisone; 0, metoprolol; a, atenolol) on phosphatidylcholme liposomes immobilized in Superdex 200 prep grade gel beads by freeze-thawing (IO). The specific capacity factor K, of the three drugs is plotted against (A) the flow rate (5 mm td column) and (B) the number of days from immobilization. KS is defined by the expression: K, = [ V, - (Vo - VL)] / [( Vo - V,)B] w here VR is the retention volume on the liposome column, Vo is the retention volume after elution of the liposomes with detergent, VL 1sthe liposome volume, and B is the concentration of immobihzed phospholtpids (mol/L) (reproduced with permission from Elsevier Science Publishers, Amsterdam). tiomer through the liposomes. The difference in elution volume decreasing activity of the protein as the pH is lowered (7).

decreases with

3.3.5. Immobilization of Active GABA-Benzodiazepine Receptor Proteoliposomes in Gel Beads by Freeze-Thawing (30,31)

Solubilized membraneproteins from calf brain can be reconstitutedin lipid vesicles and nnmobilized in gel beads. Whereas flunitrazepam binding activity of the GABA benzodiazepine receptor can be detected using a standard receptor assay (32); binding cannot be detected by use of immobilized proteolipo-

someaffinity chromatographybecauseof the low abundanceof this receptorin brain tissue and therefore in the immobilized

proteoliposomes.

Further progress

in receptorpurification may increasethe number of immobilized binding sites. 4.

Notes 1. Other phospholipids can be used. In our laboratory we prepare egg yolk phospholipids, essentially as described earlier (33). We use a somewhat different solubihzation protocol (29) for this material than that described in Section 2.1 The preparation contains 70% phosphatidylcholme, 21% phosphatidylethanolamine,

202

2.

3 4.

5.

6. 7.

8. 9 10

11. 12.

Brekkan et al. and 9% other phospholipids and lysophospholipids on the basis of TLC analyses with phosphorus determinations, and small amounts of cholesterol and other components, asjudged by TLC analyses (Merck 5628,lO x lo-cm precoated sihca gel 60 Fzs4 plates) with chloroform:methanol:water (65 + 25 + 4) (IS). This TLC system can also be used to check the purity and composition of other phosphohptds. Buffers are filtered (0.22 urn) and degassed before use Other buffer solutions can be used For determination of phosphohptd amounts by phosphorus analysis, phosphate buffers must be avoided. The monitor can be set at 280 nm The signal will depend on light scattering and will be influenced by the detector geometry. Other gel beads can be used, but may require certain adjustments to the nnmobihzatron protocol because of different characteristics, e.g , water uptake on swelling and pore size distrrbutron, (8,lO, 29,20,26) Some types of gel beads may be damaged and even fracture on freeze-thawing for mnnobtllzation of hposomes. No such problem has been encountered with the Superdex 200 prep grade beads Liposomes can also be immobihzed m continuous beds (34) by use of hydrophobic ligands (C.-M. Zeng, Y. Zhang, S. Hjerten, and P. Lundahl, in press) This column is suitable for 1 mL gel beds, but if smaller or larger gel beds are required H 5/2 (0.3-0.6 mL) and H 5/10 (1.8-2.2 mL) columns are available. Other types of columns can be used 2-Mercaptoethanol has an unpleasant odor and can be replaced by dithioerythritol of lower concentration A rotary dialysis apparatus was constructed m our laboratory for the dialysisnnmobiltzation to enhance the mixing and prevent the gel beads from sedimenting (26). One or two dialysis cells were fixed on a plastic rotor that was rotated gently in a 300-mL buffer container by a magnetic driver, whereas the buffer was pumped contmuously (flow rate, 100-l 50 mL/h) through the container overnight at 23“C or at 5-9°C if desired. Harmful by inhalation and contact with skin. Protect eyes. Care must be taken to avoid phosphate-contammg cleanmg agents. To remove phosphohpids, chloroform rmsmg and heating with 5M HzS04 may be needed The amount of lipids to be used to obtain 100 or 200 mM ltptd m the final solution is calculated by use of the (average) A4,of the lipids If the average A4, is not listed, it can be determined by phosphorus analysis The type of phospholiptd and composition can be chosen at will Avoid dehydration of the phospholipid solutton For solubilization of bovine brain extract lipids the molar ratio of cholate to lipid should be 2.1. For different proteins, different solubihzation protocols are used, and we therefore refrain from presenting any general scheme. The prepared membrane is solubilized by addition of a sufficient amount of an appropriate detergent. The excellent treatment of this subject by Ktihlbrandt m his review on three-dimensional crystallization of membrane proteins is recommended (35). Nonsolubihzed material is removed by ultracentrifugation, and chromatographic, electrophoretic,

Immobilization of Liposomes

13. 14.

15

16. 17.

18. 19.

20 2 1. 22.

203

or other separation methods are employed for purification of the membrane protein(s) of interest in the presence of detergent at a concentratron exceeding the detergent’s critical micelle concentration Our studies have centered on the human red cell integral membrane proteins, mainly the glucose transporter (Glutl). Preparation of the red cell membrane, solubilization of the integral membrane proteins, and the purification of the detergent-Glut1 complex have been described in detail (8,36,3 7). The Sephadex G-50M column adsorbs liposomes to a certain extent To avoid this, the adsorption sites may be saturated by recirculation of liposomes. Each Mimcon B-15 concentrator chamber can be reused 3-4 times. The liprd recovery is only 50-60% the first time, but improves on further use of the same chamber The water absorption capacity for each concentrator unit (eight chambers) is at most 80 mL. The amount of gel beads can be varied, but the amount of swelling of the dry gel beads has to be considered. Different gel beads swell to a different extent. The volume of the swelled gel beads should be about two-thirds of the volume of added proteoliposome suspension. The scale of the immobihzation procedures reported here is for preparation of 1 mL gel beds but the procedures can be moditied when larger or smaller columns are required. Wrap the block of dry me in cloth and crush it into small pieces with a hammer. Add the dry ice carefully to 95% ethanol in a thermos. Improved stabilization of immobilized proteoliposomes may be achieved by additional centrifugal washes with hypotonic buffer followed by hypertomc buffer, containing 1M NaCl, and finally buffer A. This may be crucial in certain apphcatrons (8). Percoll (Pharmacla) can be used during the centrifugal washes. Most of the nonimmobrlized liposomes will then be concentrated at the top after the first centrifugation and fewer centrifugal washes are therefore needed (I 0). The flow rate should be higher than the running flow rate to make sure that the gel bed does not compress during the expenment. The liposomes prepared by lipid hydration are large and multilamellar, and the procedure for the immobilization is therefore modified compared to the nnmobllization procedure for liposomes prepared by the chromatographic method, where the liposomes are small and mostly unilamellar. The tedious step of concentrating the liposomes can be omitted, but a disadvantage is that lO-20% of the itutially immobilized hposomes are released during packing, which IS not the case when chromatographically prepared hposomes are immobihzed. Take care not to rupture the dialysis tubing when inserting the frame. Extended dialysis lowers the level of residual detergent in the proteohposomes Alkyl thiols with 4-l 2 carbons can be used and 2-l 00 pmol of the alkyl thiol can be added to obtain a suitable ligand density (19,20,38). Adsorbents with dodecyl chains require longer adsorption times than do octyl-chain adsorbents (19,20) The procedure can be scaled up without affecting the degree of substitution or the coupling yield (38).

Brekkan et al.

204

23 Liposome suspensions obtained by chromotographic preparation, omitting the concentration step The liposomes become concentrated on adsorptron in the gel bed with hydrophobic ligands. 24. Keep the solutton in the dark at room temperature and it can be used for several months. Decant carefully to avoid crystals. 25. To determine the initial amount of immobiltzed phospholipids, resuspend the gel beads m buffer A after the last centrifugal wash and take out aliquots for analysis (see Sections 3.1.4.1. and 3.1.4.2.). For the final amount of unmobthzed phosphohpids, elute them with 100 mMcholate solution (5-10 column volumes) and take out aliquots for analysts 26 Long 15 mL tubes are necessary because of vigorous boiling of the solution when heated at 160°C 27. If the solutions are brownish (incomplete oxtdatton) after 3 h, repeat the addttton of HZOZ. 28. An alternative is to use quantitative amino acid analysis of a hydrolyzed sample or send a freeze-dried sample m suttable buffer with low salt content and contaming 10-100 pg of protein to a laboratory for automated analysrs (David Eaker, Amino Acid Analysis Laboratory, Department of Biochemistry, Biomedical Center, Uppsala Umversity, Box 576, S-751 23 Uppsala, Sweden. Please enquire beforehand for updated cost and further details).

References 1, Racker, E. (1979) Reconstitution of membrane processes. Methods Enzymol 55, 699-7 11 2 Racker, E (1985) Reconsltutlon of Transporters, Receptors and Pathological States, Academic, Orlando, FL. 3 Rigaud, J.-L , Levy, D., and Seigneuret, M. (1990) Membrane protein reconstitution into liposomes: mechanisms of protem-lipid association during detergentmediated reconsitutions. Trends Blomemb Bioenerg 1, 1l-27. 4 Lundahl, P and Yang, Q. (1990) Liposome chromatography: a new mode of separation using liptd bilayer. Protein Nucleic Acid Enzyme (Tokyo) 35, 1983-1998. 5 Lundahl, P and Yang, Q. (199 1) Ltposome chromatography: liposomes immobtlized in gel beads as a stationary phase for aqueous column chromatography. J Chromatogr

544,283-304.

6. Lundahl, P., Yang, Q , Greijer, E., and Sandberg, M. (1993) Immobilizatton of hposomes in gel beads, in Liposome Technology, 2nd ed. vol. 1 (Gregoriadis, G., ed.), CRC, Boca Raton, pp. 343-36 1 7. Lu, L., Brekkan, E., Haneskog, L., Yang, Q., and Lundahl, P. (1993) Effects of pH on the actrvity of the human red cell glucose transporter Glut 1: transport retention chromatography of u-glucose and L-glucose on immobilized Glutl-liposomes. Bzochim. Bzophys Acta 1150, 135-146. 8. Yang, Q, and Lundahl, P. (1995) Immobilized proteoliposomes afftnity chromatography for quantitative analysis of specific interactions between solutes and membrane proteins. Interaction of cytochalasm B and n-glucose with the glucose transporter Glut 1. Blochemwtry 34,7289-7294.

Immobilization of Liposomes

205

9. Beigi, F., Yang, Q., and Lundahl, P. (1995) Immobilized-liposome chromatographic analysis of drug partitioning into lipid bilayers. J Chromatogr A 704,3 15-321. 10. Brekkan, E., Lu, L., and Lundahl, P (1995) Properties of immobilized-hposome chromatographic supports for interaction analysis. J Chromatogr A 711,33-42 11 Zhang, Y, Aimoto, S , Lu, L , Yang, Q., and Lundahl, P (1995) Immobihzed liposome chromatography for analysis of interactions between lipid bilayers and peptides. Anal. Biochem 229,29 l-298. 12. Pidgeon, C. and Venkataram, U. V. (1989) Immobilized artificial membrane chromatography: supports composed of membrane lipids. Anal. Bzochem. 176,36-47. 13. Kaliszan, R , Nasal, A., and Bucinski, A. (1994) Chromatographic hydrophobtcity parameter determined on an immobilized artificial membrane column. relationships to standard measures of hydrophobicity and bioactivity. Eul: J Med Chem 29,163-l 70. 14. Pidgeon, C!., Ong, S , Choi, H , and Lm, H. (1994) Preparation of mtxed hgand immobilized artificial membranes for predicting drug binding to membranes. Anal Chem 66,2701-2709

15. De Cuyper, M. and Jomau, M. (1990) Immobilization of membrane enzymes mto magnetizable, phospholiptd bilayer-coated, inorganic colloids. Prog ColEold Polym SCl. 82,353-359.

16. Gotoh, T., Shidara, M., Iwanaga, T., Kikuchi, K.-I., and Hozawa, M. (1994) Immobilization of y-glutamyl transpepttdase, a membrane enzyme, m gel beads via liposome entrapment. J. Ferm Bioeng. 77,268-273. 17. Wallsten, M., Yang, Q., and Lundahl, P. (1989) Entrapment of liptd vesicles and membrane protein-lipid vesicles in gel bead pores. Biochlm Biophys. Acta 982, 47-52.

18. Yang, Q. and Lundahl, P. (1994) Steric immobilization of hposomes in chromatographic gel beads and mcorporation of integral membrane proteins into their lipid bilayers. Anal. Bzochem. 218,2 1O-22 1. 19. Sandberg, M., Lundahl, P., Greijer, E., and Belew, M (1987) Immobihzation of phospholipid vesicles on alkyl derivatives of agarose gel beads. Blochrm. Bzophys. Acta 924,185-192

20 Yang, Q., Wallsten, M., and Lundahl, P (1988) Immobilization of phosphohpid vesicles and protein-lipid vesicles containing red cell membrane proteins on octyl derivatives of large-pore gels. Blochzm. Biophys. Acta 938,243-256. 21. Bartlett, G. R. (1959) Phosphorous assay in column chromatography. J Biol Chem. 234,466-468. 22. Clark, S. (1984) Receptor purification procedures, in Receptor Biochemutry and Methodology, vol. 2 (Venter, J. C. and Harrison, L. C., eds.), Liss, New York, p. 49.

23. Nasal, A., Sznitowska, M., Bucinski, A., and Kaliszan, R (1995) Hydrophobic parameter from high-performance liquid chromatography on an rmmobilized artificial membrane column and Its relationship to bioactivtty. J. Chromatogr. 692,83-89.

24. Ptdgeon, C., Ong, S., Lm, H., Qui, X., Pidgeon, M., Dantzig, A. H., Munroe, J., Hornback, W. J , Kasher, J. S , Glunz, L., and Szczerba, T. (1995) IAM chromatog-

206 raphy: an In vitro screen for predicting drug membrane permeabthty. J Med. Chem 38,590-594. 25. Ong, S., Liu, H., Qm, X., Bhat, G., and Ptdgeon, C (1995) Membrane partition

coefficients chromatographically measured using immobilized artificial membrane surfaces. Anal Chem 67,755-762. 26 Yang, Q. and Lundahl, P. (1990) Binding of lysozyme on the surface of entrapped phosphatidylserme-phosphattdylcholine vesicles and an example of high performance lipid vesicle surface chromatography. J. Chromatogr 512,377-386. 27 Yang, Q., Wallsten, M , and Lundahl, P. (1990) Lipid-vesicle-surface chromatography. J Chromatogr 506,379-389. 28 Helmburg, T. and Marsh, D (1995) Protein surface-dtstrtbutton and protein-protein interactions in the binding of peripheral proteins to charged lipid membranes. Btophys. J. 68,536-546

29. Arnold, M., Ringler, P., and Brtsson, A. (1995) A quantitative electrophoretic migration shift assay for analyzing the specific binding of proteins to lipid ligands in vesicles or micelles. Bzochzm Blophys. Acta. 1233, 198-204. 30. Schofield, P. R., Darhson, M. G., Fujita, N., Burt, D. R., Stephenson, EA., Rodriguez, H., Rhee, L M., Ramachandran, J., Reale, V, Glencorse, T. A., Seeburg, P H., and Barnard, E. A (1987) Sequence and functional expresston of the GABA* receptor shows a hgand-gated receptor super family Nature 328,22 l-227. 31 Kuriyama, K., Hirouchi, M., and Nakayasu, H. (1993) Structure and function of cerebral GABA, and GABA, receptors. Neuroscl Res. 17,9 l-99. 32 Hulme, E. C (1990) Receptor-binding studies, a brief outline, in Receptor Bzochemutry-A Practical Approach (Hulme, E. C., ed.), IRL, Oxford, UK, pp. 303-3 15 33. Mascher, E. and Lundahl, P. (1988) The human red cell glucose transporter in octyl glucostde. High specific activity of monomers in the presence of membrane lipids Bzochlm Biophys Acta 945,350-359.

34 HJerten, S., Li, Y.-M., Liao, J.-L , Mohammad, J., Nakazato, K., and Pettersson, G. (1992) Contmuous beds: high-resolving, cost-effective chromatographic matrices Nature 356,810,811. 35. Kuhlbrandt, W. (1988) Three-dimensional crystalhzation of membrane proteins Quart. Rev Blophys

21,429--477.

36. Lundahl, P, Mascher, E., Andersson, L., Englund, A.-K., Greijer, E , Kameyama, K., and Takagi, T. (199 1) Active and monomeric human red cell glucose transporter after high performance molecular-sieve chromatography m the presence of octyl glucoside and phosphatidylserine and phosphattdylcholme. Bzochim Btophys Acta 1067, 177-186. 37. Lundahl, P, Greijer, E., Cardell, S., Mascher, E., and Andersson, L. (1986) Improved preparation of the integral membrane proteins of human red ceils, with special reference to the glucose transporter. Biochim Bzophys Acta 855,345-356 38. Maisano, F., Belew, M., and Porath, J. (1985) Synthesis of new hydrophobic adsorbents based on homologous series of uncharged alkyl sulphide agarose derivatives J. Chromatogr: 321,305-3 17.

Cell Immobilization Alcohol (PVA) Gel

with Phosphorylated

Polyvinyl

Kuo-Cheng Chen and Jer-Yiing Houng

1. Introduction Phosphorylated polyvinyl alcohol (PVA) gel is nontoxic and can be economically feasible for industrial scale applications, The use of PVA for cell immobilization has recently attracted much attention (Z-5). In this chapter, we provide details of a novel cell immobilization technique based on use of phosphorylated PVA as a support. PVA is first crosslmked with boric acid for a short time to form a spherical structure. This is followed by solidification of the gel beads by esterification of PVA with phosphate (phosphorylation). The short contact time with boric acid avoids severe damage to microorganisms. The phosphorylated PVA beads satisfy the requirements of supports having strong consistency, durability, and high cell viability as well as being of low cost (see Chapter 1). This method can be applied to immobilize cells of bacteria, yeast, or activated sludge. The characteristics of denitrifying sludge entrapped in phosphorylated PVA gel beads have been examined in our previous work (6). Furthermore, the PVA-immobilized denitrifying sludge has been found to have good stability over a long period of operation in a CSTR (6,7). However, the minuteness of the crosslinked gel structure results in a poor gas permeability of the PVA gel. This property can cause accumulation of gases inside the beads and force them to float up toward the surface of the bulk solution in the reactor. This would occur under circumstances in which an anaerobic fermentation processwas operated and accompanied by gas production, such as carbon dioxide, Modification of the gel structure to increase gel voidage would offer a highly promising alternative to enhance gas permeability. Alginate-modified beads can be produced by addition of a small amount of calcium alginate to PVA gel From

Methods m Botechnology, Vol 7 Immobrkaf/on of Enzymes and Cells Edited by G F BIckerstaff Humana Press Inc , Totowa, NJ

207

Chen and Houng

208

solution during gelation to modify the structure of PVA gel beads. The calcium algmate 1s removed by treating the beads with phosphate solution, and the resultant PVA gel beads have improved gas permeation. In this chapter, we describe the production of PVA gel beads and the algmate modification to produce beads with greater gas permeation. 2. Materials 2.1. Preparation

of PVA-Immobilized

Cell Beads

1, Polyvinyl alcohol (Chang Chun Petrochemical, Taiwan), with a sapomflcation of 78-99 9%, and 1500-2000 degree of polymerization 2 0.2-l .5M monosodium phosphate solutions, pH 5 O-S.0 3 Saturated boric acid solution (e g ,5.5% w/v). 4 Cells can be bacteria, yeast, or activated sludge. The cultivated cells should be filtered or centrifuged. Before use, the harvested bacterial or yeast cells should be adjusted to 20-50 g dry wetght/L or adjust the activated sludge to 20-40 g VSS/L sludge solutton, where VSS is the volatile suspended sohds commonly used to express the biomass in activated sludge.

2.2. Preparation

of Calcium Alginate-Modified

PVA Beads

1 Sodium algmate (Wako Pure Chemical, Osaka, Japan) 2. Saturated boric acid solution containmg 0.5-2% CaC12.

2.3. Measurement of Mechanical of PVA-Immobilized Beads

Strength

The experimental apparatus for the measurement of mechanical strength of PVA-immobilized beads is illustrated in Fig. 1. It includes a rheometer (Model NRN-2002 D.D, Fudoh Kogyo Co., Japan) equipped with a tested needle (l-mm diameter with a flat tip), and a recorder (Model T250, Eyela, Japan). 3. Methods 3.1. Preparation

of PVA-Immobilized

Cell Beads (see Note 7)

1 Prepare a 15-20% (w/v) of PVA solution with water. Heat the solutron to completely dissolve PVA. 2. When the PVA aqueous solution has cooled down to about 30-4O”C, mix one portion of 15-20% PVA aqueous solution thoroughly with an equal volume of concentrated cell solution. 3. InJect the resulting mixture dropwise into the saturated boric acid solution through a syrmge and gently stir for 10 mm to 2 h (see Note 2) to form spherical beads. 4. Screen the PVA beads with a mesh, and then rinse wtth tap water to remove the residual boric acid.

P VA Gel Immobilization

209

Rheometer

Recorder

\ \

‘\

\ \ 1 I /I

/I

Fig. 1. Experimental

apparatus for measurement of gel strength of beads.

5. Transfer the formed gel beads to a 0.2-l .5M sodium phosphate solution, pH 5.0-80 (see Note 3), for 15 mm to 2 h for hardening (see Note 4). 6 Separate the phosphorylated beads by filtration and rmse the beads with tap water

3.2. Preparation

of Calcium Alginate-Modified

PVA Beads

1 Mix amounts of sodium algmate with PVA to produce final sodium algmate concentrations of 0.5-2 0% (w/v) and final PVA concentrations of 15-20% (w/v) (see Note 5) Heat the mixture until dissolved 2. Cool the mixture to about 30-4O”C, and then add the concentrated cell solution in a ratio by a volume of 1: 1. 3. Inject the mixture dropwise mto a saturated boric acid solution containing 0.5-2.0% CaC12 to form a spherical soft structure (see Note 6).

Chen and Houng

210

4. Place these algmate-modified PVA beads into a 0 1-I .OMphosphate solutron for 15 mm to 2 h to srmultaneously carry out phosphorylation of the gel and dtsmtegrate the calcium-alginate polymer (see Note 7). 5. Collect the beads by filtration and rinse them with tap water.

3.3. Measurement of Mechanical of PVA-Immobilized Support

Strength

Measurement of the gel strength of PVA beads ISbased on stress-strain tests (8). Mechanical strength measurement for such gel beads as calcium-algmate has been generally expressed by a critical compressive stresswhen abrasion of gel beads occurs (9). However, PVA gel beads are difficult to rupture by compression. Stress-strain testshave conventionally used mechanical tests,including applrcatron for elastic gel material (8,).Therefore, gel strength expressed by the slope of the mitial linear portion of a stress-strain curve (“Young’s modulus”). The measurement can be made as follows: 1. Place gel beads m a semisphere hole of a metal holder and compress to deformation by a tested needle, at a constant deformation rate of 20 mm/mm, employmg a rheometer. 2. An expenmental curve related to the load applied (kg) and the deformatron gener-

ated(mm) canbe obtainedfrom the recorderwith a paperfed speedof 60 cm/mm. 3. The experimental curve IS further converted by a transformation of scale to obtain the desired stress-stram curve as follows. 6 = FIA,

&=(Lo-L)/L()x = ALILO x 100%

(1)

100%

(2) (3)

where 6 and E are the stress (kg/cm2) and strain (%), respectively; F is the compressive load; A0 is the cross-secttonional area of the fiat needle; L, is the orrginal length of beads (diameter); L IS the compressed length of beads; and ti IS the momentary compressive deformation The larger the Young’s modulus, the higher the mechanical strength of the beads.

3.4. Determination

of Gas Permeability

1. Put 10 g of immobilized cell beads into a bottle containing 80 mL of cultrvatlon medium. Flush and fill the head space of the bottle with helium gas. Seal the bottle with a rubber stopper.

2. Placethe bottle m a rotary shakerandcultrvate for a certain duration (e.g., 24 h). 3 At the end of the cultivatton, count the number of immobihzed particles floating up to the solution surface. The ratio of floating particles to the total number is defined as the particle floating ratio

PVA Gel Immob~hza tion

211

4. The gas permeabihty of the mnnobihzed sludge is determined indirectly by estimation of particle floating ratio. The higher the ratio value, the worse the gas permeability of the immobilized beads.

3.5. Immobilization of Denitrifying Sludge 3.5.1. Preparation of PVA-Immobilized Cell Beads 1 Prepare a PVA solution at a suitable concentration. Heat the solution to completely dissolve PVA. 2. When the PVA aqueous solution has cooled down to about 30-4O”C, mtx one portion of PVA aqueous solution thoroughly with an equal volume of ca 30 g VSS/L of denitrifymg sludge solution (see Note 8). 3. Inject the resulting mixture dropwise into the saturated boric acid solution through a syringe and gently stir for 30 mm to form spherical beads. 4. Screen the PVA beads with a mesh and then rinse with tap water to remove the residual boric acid. 5. Transfer the formed gel beads to a sodium phosphate solution for 30-50 min for hardening of the beads (see Note 9). 6. Separate the phosphorylated beads by filtration and rinse the beads with tap water (see Note 10)

3.5.2. Preparation of Calcium Alginate- Modified PVA Beads 1. Certain amounts of sodium alginate are mixed with PVA solutton to produce a final sodium alginate concentration of 1.O% (w/v) and a final PVA concentration of ca. 10% (w/v) (see Note 11). Heat the mixture until dissolved. 2. Cool down the mixture to about 30-40°C, and then add the denitrifying sludge solution (30 g VSS/L) in a ratio by volume of 1: 1. 3. Inject the mixture dropwise into a saturated boric acid solution containing 1 0% CaC& to form a spherical soft structure (see Note 12). 4. These alginate-modified PVA beads are then placed into a phosphate solution for 15-I 20 min to simultaneously carry out phosphorylation of the gel and disintegration of the calcium-alginate polymer (see Note 13). 5. Collect the beads by filtration and wash with water.

3.5.3. Determination

of Bioactivity (see Note 14)

1. Suspend 6 mL of tmmobilized sludge beads in a 115 mL serum bottle with 80 mL of synthetic wastewater containing 10 mMphosphate, pH 7.9, as buffer, methanol as a carbon source, and KN03 (100 mg N/L). The methanol-to-nitrogen ratio is 5: 1 (w/w). 2. Purge the solution with helmm gas and seal the bottle. 3. Incubate the sludge beads for 3 h in a rotary shaker at 30°C. Withdraw the samples under anaerobic conditions and centrifuge at 2000g for 10 mm to remove the solid particles. 4. Analyze the nitrate concentration by ion chromatograph (Model 2OOOi, Dtonex, Sunnyvale, CA) equipped with a conductivity detector. Use an anion analytical

212

Chen and Houng

column (Ionpac AS4A, Dronex) with 1.7 mMNaHCOJ1.8 mA4 Na$Os as eluent, and a flow rate of 2 mL/min. 5 The denitrifymg activity of the gel beads is determined as the consumption of mg of nitrate tons/h/g of immobtlized sludge.

3.5.4. Repeated Batchwise Operation 1 Place 10 g on immobilized sludge beads into bottles containing 80 mL of SW medium (see Note 8). 2. Flush and till the head space of each bottle with helium gas Seal the bottles with a rubber stopper and then cultivate at 30°C and 130 rpm in a rotary shaker 3. At the end of each run (24 h), separate the beads, wash with water, and replace with a fresh medium for the subsequent incubation.

4. Notes 1. The immobihzation conditions should be optimized depending on the charactertstics of the cells used, the requirements of gel strength, bioacttvity, and the limitation of the mass transfer resistance of the beads 2. The contact time should not be too long to prevent cell damage in such an aseptic and strong acidic environment. 3 The esterification reaction of PVA with phosphate is quite sensitive to pH changes The pH range of 4 O-6.0 IS suitable for bead formation and gel strength. Between pH 6.0-8.0, the higher the pH means the lower the gel strength. The PVA beads are not stable at a pH value greater than 8.0, whereas gel strength is drastically reduced at a pH value lower than 4.0. 4. An increase in phosphate concentration or contact time in the esterification of PVA may result in an increase in gel strength, but also increased diffusion resistance, whtch results from a more compact structure of the gel matrix. 5 The mixture viscosity of sodium alginate and 20% (w/v) PVA would be too high to be injected through a needle Therefore, a lower PVA concentration is suggested here 6 The mechanical strength of the gel generally increases with an increase in the calcium ion concentration (from 0.5-2%) during the solidification process. 7. As indicated in Note 4, phosphate concentration might affect the gel strength m the esterification of PVA. In the preparation of algmate-modified PVA beads, phosphate hardens the PVA gel structure through the esterification reaction and simultaneously disintegrates the polymer of calcium-algmate mside the PVA beads. Therefore, the phosphate concentration may affect the gel structure and subsequently the gel strength, gas permeabihty, dtffusion, and bioactivity of the beads 8 Demtrifymg sludge was obtained from a mixed liquor from an aerobic sewage treatment plant, acclimated with SW medium m an anoxic 10-L continuous stirred tank reactor for over 1 yr. The SW medium is composed of 0 722 g/L KN03,

213

PVA Gel Immobilization

Strain

(%I

Fig. 2. Typical compressive stress-strain relationships of phosphorylated PVA beads prepared in various concentrations (M) of sodium phosphate solution with a constant contact time of 1 h. The dashed lme indicates the curve of PVA-boric acid gel beads that were prepared according to Hashimoto and Furukawa (1) (reprinted with permlssion from Enzyme and Microb. Technol., ref. 6).

0.5 g/L CH30H, 0.418 g/L Na2HP04*2H20, 0 202 g/L KH2P04, 0.02 g/L MgS04.7H20, 0.01 g/L CaC12.2H20, 0.005 g/L FeS04.7Hz0, 0.0025 g/L MnS04*H20, and 0.0025 g/L NazMo04*2Hz0, and the pH is adjusted to 7.0. 9. Figures 2 and 3 show the relationships of PVA-immobilized denitrifymg sludge beads prepared under various phosphate concentrations (0.35-l .3&I) and contact times (15-120 min). An increase in phosphate concentration or contact time may result in an increase in gel strength, but is accompanied by a decrease in bloactivity of the beads because of the higher diffusion resistance that results from a more compact structure of the gel support. 10. The optimum pH values for the bloactivlty of the free and immobilized demtrifying sludge are observed to be almost the same. The optimum temperature of gel beads is 10°C higher than that of free sludge. The discrepancy indicates that the PVA-immobihzed sludge may be more stable than the free form. 11. The influence of sodium algmate concentrations at 0.5, 1.0, and 2.0% (w/v), hardened in 2% CaClz solution, followed by the immersion of the beads in 1M phos-

Chen and Houng

214

0

25

50 Stratn

75

1

3

1%)

Fig. 3. Typical compressrve stress-strain relationships of phosphorylated PVA beads prepared with various contact times in sodium phosphate solutron (reprinted with permission from Enzyme and Mcrob. Technol., ref. 6).

phate solution IS gtven in Table 1. After one day of cultivation, the PVA beads wtth 0.5 or 1.O% algmate addition display similar bioactrvtty as that of the control, i.e., which IS without any modification Meanwhile, the beads modified by 2.0% alginate display a 20% lower btoactivrty than the control. Moreover, in the case of 1.0% algmate addition, the particle-floatmg ratio after seven repeated batches of cultrvation decreases to roughly 30%. 12. The effect of 0 5, 1.0, and 2.0% (w/v) CaC12 m saturated boric acid solution toward the alginate-modified PVA bead properties is shown in Table 1. The braactivity of the beads hardened m 0.5% CaC12 solutton is close to that of the control. The beads gelated in a higher calcmm ion concentration exhtbtt lower bioactlvrty than that at lower concentratron. The beads hardened in 2% CaC12 solution decrease broacttvlty by 20% On the other hand, It can be seen from Table 1 that a significant improvement in gas permeability is obtained with the alginate-modified beads gelated in 1% CaCl, solution. 13 The influences of phosphate under various concentrations ranging from 0. l-l .OM are also presented in Table 1. Regardless of whether the beads are modified by algmate or not, the highest activities are obtained by treatment with 0.3M phos-

215

PVA Gel immobilization Table 1 Effects of Alginate, CaCll and Phosphate and Gas Permeability of Alginate-Modified Condttton Controla 0.5% Alginateb 1.O% Alginate 2.0% Alginate 0.5% CaCl.$ 1.O% CaClz 2 0% CaC12 0. 1M Phosphated 0.3M Phosphate 0.5M Phosphate 1.OM Phosphate

on the Bioactivity PVA Gel Beads

Relative btoactivity, %

Particle floating ratio, %

100 97.4 100.5 80 7 102.7 94.0 80.7 99.2 1192 63.9 73.2

41.8 42.2 29.9 41.0 23.4 15.6 410 16.1 21.2 21.6 16.4

aPVA gel beads, without any modtticatton bEffect of algmate when 2% CaC& and 1Mphosphate IS used in the immobihzation process CEffectof CaClz when 2% algmate and IMphosphate is used in the unrnobthzation process. dEffect of phosphate when 1% algmate and 1% CaC121sused m the tmmobihzation process

phate. Moreover, when the PVA beads are modified with 1% alginate and hardened m 1% CaClz solutton, the effect of phosphate concentration on gas permeability of the gel 1s insignificant. 14. Btoactivity is expressed by nitrate reduction rate.

References 1 Hashimoto, S. and Furukawa, K. (1987) Immobilization of activated sludge by PVA-boric acid method. Biotech Bioeng. 30,52-59. 2. Ariga, O., Takagi, H., Nishizawa, H., and Sano,Y. (1987) Immobilization of microorganisms with PVA hardened by iterative freezing and thawing. J, Ferment Technol. 65,65 l-658. 3. Shindo, S. and Kamimura, M. (1990) Immobilization of yeast with hollow PVA gel beads. J. Ferment. Bioeng. 70,232-234. 4. Myoga, H., Asano, H., Nomura, Y., and Yoshida, H. (1991) Effects of immobillzation conditions on the nitrtfication treatabihty of entrapped cell reactors usmg the PVA freezing method. Wut. Scz. Tech. 23, 1117-l 124. 5 Wu, K. Y. A. and Wtsecarver, K. D. (1992) Cell immobilization using PVA cross-linked with boric acid. Biotechnol. Bioeng 39,447-449.

216

Chen and Houng

6. Chen, K. C. and Lm, Y. F. (1994) Immobilization of microorganisms with phosphorylated polyvinyl alcohol (PVA) gel. Enzyme Mcrob. Technol 16,79-83 7. Lm, Y. F. and Chen, K. C. (1995) Denitrification and methanogenesis in co-immobilized mixed culture system. Wat Res 29,35-43. 8. Nielson, L. E. (1974) m Mechanical Properties of Polymers and Composites, vol. l., Marcel Dekker, New York, pp. S-9. 9 Chen, K. C and Huang, C. T. (1988) Effects of the growth of Trzchosporon cutaneum on alginate beads upon bead structure and oxygen transfer rates. Enzyme Microb Technol. 19,284-292

25 Covalent Immobilization to Graphitic Particles

of Enzymes

Marco F. Cardosi 1. Introduction

The use of carbon as a support material for enzymes is receiving increasing attention, parttcularly in the field of enzyme electrodes, in which use is made both of the conducting nature of carbon and the degree of surface chemical reactivity associated with the material. This final conslderatton makes it possible to attach enzymes directly to the surface of carbon by using suitable bidentate linking reagents. Although there are a large number of carbon materials available that differ in their chemical and physical properties, the basis of almost all carbonaceous material is the layer plane of carbon atoms that forms the hexagonal graphite crystal where each carbon atom in the basal plane is cr trigonally bonded to three adjacent coplanar atoms m sp2 hybridization (see Fig. 1). Generally speaking, the surface of a carbonaceous solid (for example a carbon particle) represents the borderline caseof a lattice defect. At this interface, the coordination of each carbon atom must be different from that of the interior atoms. Consequently, the surface carbon atoms will have some unsaturated valence able to react with other elements or compounds. Under normal conditions, these dangling valences become satisfied by reaction with dioxygen and water (by the process of chemisorption) present in the atmosphere, resulting in the formation of chemical functionalities like carboxylic, hydroxyl, phenolic, quinone, and lactone, which are bound as a monolayer to the substratum of the material (Fig. 1). In crystalline graphite, the amount of edge area is small compared to that of the basal plane. Consequently, graphite does not exhibit significant oxygen chemisorptton. Mtcrocrystalline carbons, such as activated carbons, blacks, and charcoals, on the other hand, have more disordered strucFrom

Methods m &otechnology, Vol 1 lmmobrlrzafron of Enzymes and Cells Edlted by G F hckerstaff Humana Press Inc , Totowa, NJ

217

218

Cardosi

Fig. 1. Schematicrepresentationof the layer plane of carbon atoms in graphite showing various surface functional groups.

tures and thus more reactive edge area, which results in a larger propensity of chemisorption. Since tt is by reaction with these surface groups that enzymes can be covalently bound to carbon, correct choice of starting material is particularly important. In addition, the presence of surface hydroxyl and carboxylit acid moiettes is parttcularly useful for the immobilization of proteins. Depending on the nature and mode of preparation of the material, different carbons will have their own particular surface properties and hence chemical reactivities. It must be noted, however, that carbon-oxygen complexes are not formed exclusively by reaction with 02, but can also be formed by reactions with oxidizing solutions. The chemical formation of reactive groups on the surface of carbon has been and continues to be an active area of research (1-3). Treatment of carbon with concentrated nitric acid at room temperature, for example, can lead to the formation of carboxylic acid and hydroxyl groups, whereas treatment with sodium hypochlorite results in the formation of both carboxylic acid and aromatic hydroxyl groups (phenols). For certain applications, carbon is an ideal immobilization matrix, although the material does have certain disadvantages. The most serious of these is the lack of knowledge regarding the specific surface chemistry of the material, which is crucial to the immobilization strategy employed. Consequently, without the aid of complex surface analysis equipment, immobilization to carbon is at best empirical. Suitable surface analysis techniques include Auger spectroscopy, X-ray photoelectron spectroscopy (XPS) and energy dispersive X-ray

Carbon Support for Immobilization

219

analysis (EDX). Although beyond the scope of this chapter, analytical applications of both Auger spectroscopy and EDX analysts in this context are presented elsewhere (46). Another problem results from the interfacial binding energies that occur at the surface of carbon. These are large, and can in some casesresult in the denaturation of a protein. Indeed, the need to chemically treat carbon, thereby Iowering the interfacial energiesand thus preventing protein denaturation, has recently been discussed in the literature (7). A final problem relating to the use of carbon is that because of the n-electron density of the basal carbon plane, the material is conducive to strong chemisorption interactions, particularly where unsaturated compounds and aromatic rings are involved. Consequently, much of the covalent coupling of enzymes to carbon may be caused by nreversible adsorption. Careful design of control experiments and judicious chorce of wash conditions should, however, help to minimize this uncertainty.

In this chapter, two methods for immobilizing enzymes to oxidized carbon particles will be outlined. The first uses carbodiimide linking chemistry. The second uses 2,4,6,trichloro-1,3,5-triazine as the linking agent. Both methods assume the presence of hydroxyl groups on the surface of the carbon (see Note 1).

2. Materials 2.1. Immobilization

of Glucose Oxidase to Carbon Particles (4)

1. Vulcan XC-72, a carbon black with a volatile content of 2%, a surface area of 254 m*/g, and a mean particle size of 30 nm (E-Tek, Framingham, MA). 2. Nitric acid (oxidizing solution): Prepare a 20% (v/v) solution of nitric acid in ultrapure water. Store in an amber bottle until required. Care: Nitric acid is a powerful oxidizing agent and corrosive solution. 3. N-hydroxysuccinamide (NHS) solution: Prepare a 0.25M stock solution in redistilled dimethyl formamide immediately before use (alternatively, use HPLC grade dimethyl formamide). Store this at 4°C m an amber bottle until required 4. N-(dimethyl ammopropyl)-N’ethylcarbodiimtde (DAEC) solution: Prepare a OSM solution of DAEC in colhdine-HCl buffer, pH 5.5. Take care when handling DAEC Wear gloves and avoid breathing in DAEC dust when weighing out. 5. Whatman No. 1 filter disks, filter funnels, 25-mL conical flasks, orbital shaker, Soxhlet extraction apparatus, and a Bdchner flask. 6. Glucose oxidase (Btozyme, Gwent, UK).

2.2. immobilization of Horseradish to Carbon Particles (6)

Peroxidase

1. Vulcan XC-72, a carbon black with a volatile content of 2% and a surface area of 254 m*/g, and a mean particle size of 30 nm (E-Tek Inc.)

Cardosi

220

2. Nitric acid (oxidizmg solution): Prepare a 20% (v/v) solution of nitric acid m ultrapure water. Store m an amber bottle until required. Care: Nitric acid is a powerful oxidizing agent and corrosive solution. 3 Cyanuric chloride (CC) solution. Prepare a 2% (w/v) solution of cyanuric chloride in a 1: 1 binary mixture of 1,4-dioxane and xylene. Prepare this immediately before use. Care: Cyanuric chloride is a corrosive substance and caution should be exercized not to inhale any dust. 4 Whatman No 1 filter disks, filter funnels, 25mL conical flasks, orbital shaker, Soxhlet extraction apparatus, petroleum ether (60-80°C) fraction, and a Buchner flask 5. Horseradish peroxidase (HRP) (Biozyme).

3. Methods 3.1. Immobilization

of Glucose Oxidase to Carbon Particles

1. Prior to use, extract (Soxhlet) the carbon black material in AnalaR petroleum ether (60-80°C) fraction for 24 h. Collect the carbon, dry at room temperature and store desiccated over silica gel until needed. 2. Weigh out 0.1 g of extracted Vulcan XC-72 (because of the small particle diameter, avotd creating dust). Add this to a 25-mL conical flask. 3 Add 10 mL of the 20% nitric acid solution. Loosely stopper the flask with a cotton wool plug. (At this stage, the carbon should form a suspension m the OXIdizmg solution.) Place the flask on an orbital shaker and leave the carbon m contact with the solution for 48 h at room temperature 4. After this time, collect the oxidized carbon particles by vacuum filtration through Whatman No 1 filter paper. Thoroughly wash the carbon with ultrapure water until the pH of the washings is no longer acidic. (It is useful during this procedure to resuspend the carbon to maximize the contact area.) When the carbon particles have been sufficiently washed, dry the material at room temperature and store over silica gel until required. 5 Take 0.1 g of the oxidized carbon particles and place in a 25-mL conical flask. Add 5 mL of 0 25MNHS m dimethyl formamide. To this, add 3 mL of the 0 5M DAEC-collidine-HCl solution Place the flask on an orbital shaker and mix the contents overnight at room temperature. 6 After the activation treatment, collect the carbon particles by filtration under vacuum. Wash with ultrapure water until the washings contain no UV (260 nm) absorbing material. Finally, wash the carbon with 100 mL of anhydrous acetone and store under vacuum over sihca gel (see Notes 2,3). 7. Take 0.05 g of the activated carbon and add to a 25-mL comcal flask. Suspend the carbon in 10 mL of O.lMphosphate buffer, pH 7.6, contaming 14 mg of glucose oxidase. Gently mix for 24 h at room temperature. 8. After this time, collect the carbon by vacuum filtration through Whatman No 1 filter paper. Wash with ice-cold phosphate buffer containing OSMKCl until

Carbon Support for Immobilization

221

no more enzyme is detected in the washings (OD2s0 nm and enzyme assay). Finally, wash the carbon with 100 mL of me-cold ultrapure water and dry over silica gel. 9. The immobilized enzyme should be stored at -4’C untrl required. Under these conditions, the preparation should be stable for at least 2 mo (see Note 4)

3.2. lmmobiliza tion of Horseradish Peroxidase to Carbon Particles (see Notes 5,6) Because this method also relies on the generation of surface hydroxyl groups, the procedure above.

for the oxidation

of the carbon is exactly as outlined

1. Place 1 0 g of oxtdized carbon into a 100 mL round bottomed flask Add 50 mL of CC solution. Allow the carbon to react for 10 h at room temperature. (A magnetic sttrrer and star bar should be used to ensure that the carbon remains in suspension.) 2. After this time, remove the carbon by filtration and allow to dry at room temperature. Put the solid into a cellulose extraction thimble and extract the carbon (Soxhlet) with petroleum ether for 24 h to remove any adsorbed CC. When this procedure is complete, remove the carbon and dry under vacuum. 3. Hydrate 0.5 g of the activated carbon in 10 mL of 0. 1Msodmm acetate buffer, pH 5.0, containing 11.4 mg of HRP. Leave the carbon in contact with this solution for 12 h at room temperature. 4 Collect the carbon by vacuum filtration through a Whatman No. 1 filter paper. Wash the carbon with 50 mL of O.lM sodium acetate buffer, 50 mL of O.lM NaCl, and finally 80 mL of ultrapure water (check the washings to ensure that they contam no HRP activity). 5. Dry the material over sihca gel and store at 4’C until required. Under these conditions, the preparation should be stable for up to 4 mo, showing little loss of immobtlized activity (see Note 7).

3.3. Assay of Immobilized

Glucose Oxidase Activity (see Note 8)

1. Set up an oxygen sensor (e.g., a Rank Brothers polarographic oxygen electrode) according to the manufacturer’s instructions. Thermostat the reaction cell to 25°C. 2. Take 10 mg of carbon and wash with 10 mL of distilled water (this serves to wet the carbon). 3. Resuspend the wetted carbon in 1 mL of 0.2M sodium acetate buffer, pH 5.6 (at this stage it is important to keep the carbon suspended in the buffer by using a micro stir-bar and magnetic stirrer). Collect the carbon by centrtfugation. Keep the supernatant because this serves as a control. Resuspend the carbon m 1 mL of fresh 0.2M sodium acetate buffer, pH 5.6. 4. Inject a suitable sample volume (e.g., 20-100 pL) dtrectly into the working chamber of the oxygen sensor that has been primed with 0.2M sodium acetate buffer,

222

Cardosi pH 5.6, contammg 50 miV P-o-glucose. Monitor the rate of oxygen consumption over a period of 5 mm (the exact time will depend on the activity of the unmobrhzed enzyme). For the procedure described here, typical values of dOZ/dt are on the order of -30% O2 tension/min (see Note 9).

3.4. Assay for Immobilized

Horseradish

Peroxidase

1 Prepare the following stock solutions in 0 1M sodium acetate buffer, pH 5.0, 2 mA4hydrogen peroxide solution, and4.7 mM2,2’-azino-bis-(3-ethyl-benzthiazolinesulfonic acid) (ABTS) solution 2 Take 10 mg of carbon and wash with 10 mL of distilled water (this serves to wet the carbon). 3. Resuspend the wetted carbon m 1 mL of 0. 1M sodium acetate buffer, pH 5.0 (at this stage it is important to keep the carbon suspended in the buffer by using a micro stir-bar and magnetic stirrer.) Collect the carbon by centrifugation. Keep the supernatant because this serves as a control Resuspend the carbon m 1 mL of fresh 0 2M sodmm acetate buffer, pH 5.6 4. Add 1 mL of hydrogen peroxide solution and 1 mL of ABTS solution to a plastic cuvet. Use the resultant solutron to zero a spectrophotometer set at a recording wavelength of 420 nm. Add an ahquot of the carbon suspension to the cuvet (20-100 PL depending on the immobilized enzyme activity). Incubate the reaction mixture at room temperature for 5 mm (see Note 10) 5. After the allocated reaction trme, transfer the solution to an Eppendorf tube and pellet the carbon particles by centrifugation. Carefully decant the supernatant into a clean plastic cuvet and measure the absorbance at 420 nm. Control carbon samples should result in no colored product being formed

4. Notes 1. It must be stressed that actual knowledge of the absolute population and chemical reactrvities of the various groupings on the surface of carbon is still limited and an area of intense research activity. Although one of the treatments mentioned above may appear to enhance the efficacy of a chemical coupling procedure, this rn itself 1sindirect evidence that presupposes that the couplmg reaction proceeds as planned The reader IS referred to refs. 1-3, which deal with the surface chemistry of carbons in greater detail. 2. The basis of this activation procedure is the activatton of a carboxyhc acid group by a water-soluble carbodnmrde (DAEC) to form an o-acyl urea intermediate (Fig. 2) This then reacts with an amine to form a peptrde bond. This tmmobrhzation method for carbon was first described by Cass et al. (5) 3. The o-acyl urea is, however, prone to hydrolysis, thus leading to reduced coupling efficiencies (this IS particularly serious in the case in which the density of surface hydroxyls may be low). Carbodiimides used in conJunction wrth NHS, however, result in active esters that are more resistant to hydrolysis than the o-acyl urea, thereby increasing the couplmg efficiency of the reaction (4).

223

Carbon Support for Immobilization

II 0 Carbodilmide

surface

carboxyllc

cl-acy1

acid

(activated

surface

attached

protein

urea carbon)

urea

Fig. 2. Schematic representation of the activation of a carboxyhc acid by a water soluble carbodiimide to give the o-acyl urea derivative. This then reacts with a primary amine to form a peptide bond.

4. A flow chart for the covalent attachment of glucose oxidase to Vulcan XC-72 is shown m Fig. 3 5. As an alternative to the carbodiimtde tmmobilization chemistry, tt is also possible to use 2,4,6,-trichloro-1,3,5triazine (cyanurtc chloride) as the lmkmg agent. The use of this reagent has recently been described by Cardost for the immobilization of horseradish peroxidase (UP) to nitric acid oxidized Vulcan XC-72 (6) 6 The basis of the attachment using cyanuric chloride (CC) is the formatton

of a covalent bond between the chloro-s-triazine nucleus and hydroxyl groups present on the surface of the carbon. Substitution of one chlorine atom on the s-trtazine nucleus with an ester-type bond does not greatly reduce the activity of the ring to further substitution, thus allowing the molecule to act as a bidentate ligand. One of the advantages of usmg CC as a linkmg agent IS that it can undergo a variety of reactions. For example, it can react with hydroxyl and amino groups, with alkyl and aryl Grignard reagents, and wtth organic hydrazine reagents In addition, it produces an ether-type linkage to the carbon that is stable and, because of the aromatic framework of the molecule, provides a short (1 nm) spacer. 7. A flow diagram for the activation of carbon using CC is shown in Fig. 4. The chemistry for the covalent attachment of horseradish peroxidase to the activated carbon is summarized m Fig. 5

Card&

224 Vulcan XC-72

Soxhlet extraction for 24 hours m petroleum ether

Oven drymg for 30 mmutes

Oxldabon m 20% mtnc acid for 24 hours at room

temperature

1

Activation m NHUDAEC

solutton oven&t

1

Soxhlet extrachon for 24 hours

in petroleum ether

Collect the carbon by filtration and wash

1

Immobilize glucose oxldase to carbon

Fig. 3. Flow chart showing the covalent attachment of glucose oxidase to particles of Vulcan XC-72.

8. The overall biochemtcal reaction catalyzed by glucose oxrdase is shown below Glucose oxrdase Glucose + O2 --------------------------> Gluconlc acid + H202 (1) Because the enzyme uses dioxygen, glucose oxidase acttvtty can be followed using a conventional Clark-type polarographtc oxygen sensor. The immobrhzed enzyme can be measured by followmg the decrease m disolved oxygen tension at saturating concentrations of glucose. A typrcal protocol for measuring rmrnobtlized glucose oxidase activity 1sshown above.

carbon

particle

Fig. 4. Flow chart showing

CC activated

24

hours

acetate

pn

5.0

covalantly

attached

proten

the covalent attachment of horseradish peroxidase to particles of Vulcan XC-72

0.m

Cardosi

226 Vulcan XC-72

Soxhlet extraction for 24 hours in petroleum ether 1 Oven drying for 30 minutes

Oxidation in 20% nitric acid for 24 hours at room temperature 1 Activation in CC solution 1 Soxhlet extraction for 24 hours in petroleum ether Fig. 5. Synthetrc scheme for the attachment of horseradrsh peroxldase to the surface of a CC-activated carbon particle (4)

Horseradish peroxidase catalyzes the two-electron reduction of hydrogen peroxide. A large number of organic compounds can provide the reducing power for this reaction. Hence, a number of assay methods exist for horseradish peroxldase. A convenient assay method that was found suitable for measuring peroxidase activity on carbon is descrrbed in Section 3 4. Although these experiments verify the presence of unmobllized enzyme, care should be used m any kinetic interpretation This is because any kinetic analysis is complicated by such factors as partitionmg at the interface, matrix diffusion, possible steric complications, and subpopulations of active, partially active, and inactive enzyme. Thus, any KM and V,,, values that may be obtained should be interpreted with caution, particularly if they are to be used m estimating the amount of enzyme that is bound to the support matrix. 9. A suitable control for this method 1sto lmmoblhze either a neutral protein, such as bovine serum albumin, or apo-glucose oxidase (glucose oxldase that has been inactivated through the loss of the FAD cofactor). In both cases, the endogenous (blank) rate of oxygen consumption should be zero It is also prudent to filter an ahquot of carbon from the resuspension buffer and assay the filtrate. This will confirm that the observed activity is indeed caused by immobihzed glucose OXIdase. A typical experimental trace is shown in Ftg. 6.

227

Carbon Support for Immobilization Addition of unmobthzed glUc0~~ 1 Blank response

1.... .. .. ...-.. , 100% oxygen

”

--I-

+-

._...._. ._. . .. ,

Zero % oxygen

.

c

lOOseconds

Fig. 6. Expertmental trace for the assay of immobihzed glucose oxidase activity using an oxygen sensor. At the point indicated on the trace (arrow), 0.6 mg of modified carbon was added to the cell. A blank result is also shown. Here, BSA was mmobthzed to the carbon particles instead of glucose oxidase.

10. Because the carbon particles will interfere with the passage of the light beam through the solution, it is best to perform this as an end-point assay.

References 1. Donnet, J. B. (1968) The chemical reactivity of carbons. Carbon 6, 16 l-l 76. 2. Walker, P. L. (1962) Carbon-an old but new material. Am Scl June, 259-293. 3. Wang, J. (1981) Reticulated vitreous carbon-a new versattle electrode material. Electrochim. Acta 26, 172 l-1726. 4. Cardosi, M. F. and Birch, S W. (1993) Screen printed glucose electrodes based on platmised carbon parttcles and glucose oxidase. Anal. Chim Acta 276,69-74. 5. Cass, A. E. G., Davis, G , Francis, G D., Hill, H. A. O., Higgins, I. J., Plotkin, E. V, Scott, L D., and Turner, A. P. F. (1984) Ferrocene-mediated enzyme electrode for the amperometrrc determmatron of glucose. Anal. Chem 56, 667-67 1. 6. Cardosi, M. F. (1994) Hydrogen peroxide-sensttrve electrode based on horseradish peroxidase-modified platinized carbon, Eiectroanalysls 6, 89-96. 7. Alvarez-Icaza, M. and Schmid, R. D. (1994) Observatton of direct electron transfer from the active centre of glucose oxidase to a graphite electrode achieved through the use of a mild mnnobilization. Bzoelectrochem Broenerg 33, 191-l 99.

Enzyme Immobilization Complexes Severian

Dumitriu,

Using Chitosan-Xanthan

Pierre Vidal, and Esteban Chornet

1. Introduction The immobilization of enzymes is a techmque extensively studied since the late 1960s (I), and the knowledge accumulated on enzyme immobilization studies has grown steadily since then (24). Hydrogels are a particular category of support material that can be used for convenient immobilization. The preparation of hydrogels can be achieved by a variety of methods: reticulation of linear polymers, grafting of synthetic polymers onto naturally occuring macromolecules, chelation of polycations, and complexation between polyanions and polycations (7-11). In this chapter, we describe novel hydrogels prepared from naturally occurring polymers, namely chltosan and xanthan. Chitosan and chitin are both produced industrially from crustacean shell wastes. Like cellulose, chitin is a p-(1-4)-linked glycan, but is composed of 2-acetamido-2-deoxyd-glucose (N-acetylglucosamine) (Fig. 1). Chitosan is the deacetylated form of chitin (Fig. 1). Xanthan is an extracellular heteropolysaccharide produced by Xanthomonas, and xanthan gum has a p-D-( 1-4)-linked glucan backbone with short trisaccharide sidechains consisting of a-D-mannose, P-D-glucuronic acid, and P-D-mannoseon alternating glucose residues (12). The terminal P-o-mannopyranosyl unit is glycosidically linked to the O-4 position of the P-D-glucopyranosyluronic acid unit, which in turn is glycosidically linked to the O-2 position of an a-o-mannopyranosyl unit. The mannose contains pyruvate groups located at approximately every other sidechain (23) (Fig. 2). Polymer complexes are formed by the association of two or more complementary polymers, and may arise from electrostatic forces, hydrophobic interactions, hydrogen bonding, van der Waals forces, or combinations of these interactions (14,15). Many polymer complexes form as a result of electrostatic From

Methods m Botechnology, Vol. I * lmmobrlrzatron of Enzymes and Cells Edlted by G F Blckerstaff Humana Press Inc , Totowa, NJ

229

Dumitriu, Vidal, and Chornet

230

Fig. 1. Structure of chitin (A) and chitosan (B).

forces and polymeric acids may form complexes by proton transfer to complementary polymeric bases, resultmg m polycation:polyanion pairs. Finally, simple polyelectrolytes in solution may be linked together by multivalent ions to form gels or coacervates (16).

2. Materials 2.1. Preparation 1. 2. 3. 4 5 6. 7. 8.

of Chitosan-Xanthan

Spheres

Xanthan sohd (Aldrich, Milwaukee, WI). Isopropyl alcohol. 50:50 (v/v) Ethanol. Xanthan solution (6 5 g/1000 mL). Chitosan sohd (Sigma, St. Louis, MO). O.lN HCl solution. 0 1NNaOH solution. O.lM acetate buffer, pH 5.6.

2.2. Immobilization

and Assay of Xykmase

1. Chitosan solution: Dissolve 5.45 g of chitosan in 300 mL of O.lNHCl The solution is neutralized with O.lN NaOH to pH 6.3. Then distilled water is added to a final volume of 1000 mL 2. Xanthan solution. Dissolve 6.5 g in 1000 mL of distilled water. 3. Endo- 1,4-P-xylanase (E.C. 3 -2.18) from Trlchoderma virzde (Fluka, Buchs, Switzerland).

Chitosan-Xanthan

Complexes

231

Fig. 2. Structure of xanthan. 4. 0.5Macetate buffer, pH 5.3. 5 0.05Macetate buffer, pH 5.3. 6. Remazol Brilliant Blue R-Xylan (RBB-xylan) 0.05M acetate buffer, pH 5.4.

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

(Sigma) Prepare 5.75 mg/mL in

and Assay of Protease

Protease type XIX (E.C. 3.4.2.1.19) from Aspergillus sojae, 0.4 U/mg (Sigma). Hemoglobin powder (Sigma). 0.25M phosphate buffer, pH 7.2. 0.05M acetate buffer, pH 5 3. 0.5Macetate buffer, pH 5.3. Trichloroacettc acid, 5 wt% m water. Substrate: Prepare 2% hemoglobin by dissolvmg (wtth gentle stirring) 5 g of hemoglobm in a solution of urea (80 g urea/80 mL water). Heat the mixture at 37°C for 60 mm, then add 50 mL of 0.25Mphosphate buffer, pH 7.2. Adjust the pH of solution to 7.2, then dilute the solution to 250 mL with water.

2.4. Immobilization

and Assay of Lipase

1. Lipase type II (EC. 3.1.1.3) from porcine pancreas, 0.4 U/mg (Sigma) 2. 0.55Mphosphate buffer, pH 7 5.

Dumitriu, Vidal, and Chornet

232

3, 6N HCl solutton 4. Iso-octane. 5. Olive oil-water emulsion (Sigma): Prepare 50% (v/v) stabilized olive oil emulsion, containing 0 1% sodium azide as preservative. 6. Olive 011. 7. Copper reagent: Prepare according to Lowry and Tinsley (17) Prepare a 5% (w/v) aqueous solutton of cupric acetate, filter, and adjust the pH to 6 1 using pyridine

3. Methods

3.1. Preparation of Chitosan-Xanthan

Spheres

1, Purify xanthan by dissolvmg it m water (1 wt% of xanthan) then precipitate with isopropyl alcohol. Use four dissolution-precipitation cycles (see Note 1). 2 Dissolve the purified xanthan in water (1 wt%), then precipitate with 50.50 (v/v) ethanol, filter, and rinse with ethanol/water mixtures (varying from 2: 1 to 1.2 by volume). Dtssolve the resultant xanthan in water (1 wt%) and lyophihze 3. Place 20 mL of 0.545 wt% chitosan solution m a reaction flask, and to this add 20 mL of 0.65% xanthan solution. Add the xanthan solution slowly at a rate of 0.5 mL/mm using a 0.2-0.5 mm syringe and agitate the vessel slowly to avoid aggregation of the spheres 4. After addition of the xanthan, agitate the mixture for a further 30 min. 5 Dilute the reaction mixture with 200 mL of O.lMacetate buffer, pH 5.6 6. Decant the spheres and wash with 500 mL of the same acetate buffer until no free xanthan or chitosan 1s found in the washings (see Note 2). 7 Store the spheres in the same buffer until required.

3.2. Immobilization

and Assay of Xylanase

1 Dissolve xylanase to give a final concentration of between 0 5 and 1.5 wt% in 0 65 wt% xanthan solution. 2. Mix the solution for 15 min at 15°C before introducing it dropwise, using a syringe, mto an agitated reaction vessel containing 0.545 wt*/ chnosan solution (the pH of the chitosan solution had been adjusted to 4 3). 3. After formation of spheres, the mixture is agitated for 15 min at room temperature (20-22V) 4. Decant the immobiltzed enzyme spheres, wash with 0.5Macetate buffer, pH 5.3, and then store m 0 05M acetate buffer, pH 5.3, at 5°C. 5. Mix 0.02 g of immobilized enyme spheres with 0.5 mL of preheated (30°C) 5 75 mg/mL RBB-xylan solution, pH 5.4. 6. Incubate the reaction mixture at 30°C for 60 mm Then terminate the reaction by addition of 1 mL of ethanol. 7. Allow the reaction mixture to equilibrate at room temperature for 30 min. Then remove the remaining precipitate by centrifugation at 2000g for 1.5 min. 8 Measure the absorbance of the supernatant at 595 nm with reference to a blank (see Notes 3,4)

Chltosan-Xanthan

Complexes

3.3. Immobilization

233

and Assay of Protease

1. Dissolve the protease to give a final concentratton of between 0.5 and 1.8 wt% in 0.65 wt% xanthan solution. 2. Mix the solution for 15 mm at 15°C before introducmg it dropwise, using a syringe, into an agitated reaction vessel containing 0.545 wt% chitosan solution (the pH of the chitosan solution had been adjusted to 6 3) 3. After formatton of spheres, agitate the mixture for 15 min at room temperature

(20-22°C). 4. Decant the immobilized 5. 6.

7. 8. 9.

enzyme spheres and wash with 0.5M acetate buffer, pH 5.3, and then store m 0 05Macetate buffer, pH 5.3, at 5°C. To 0.02 g of immobilized protease, add 5 mL of hemoglobin substrate solutton (pre-equilibrated at 25”(Z), and mix thoroughly but gently to prevent frothing Allow the reactton to proceed for a set time, not greater than 10 min, then terminate the reaction by additron of 10 mL of 5% trichloroacetic acid (TCA) to the mixture. Prepare a blank by adding the TCA to the enzyme before addition of substrate Remove the precipitated material by centrifugation as in Section 3 2., step 7 Measure the absorbance at 280 nm with reference to the blank.

3.4. Coimmobilization

of Xylanase and Protease (see Note 5)

1. Dissolve protease and xylanase to give individual final concentrations of between 0.5-l .8 wt% in 0.65 wt% xanthan solution. 2. Prepare the immobtlized enzymes as indicated in Section 3 3. from steps 2-4, inclusive

3.5. Immobilization

and Assay of Lipase

1. Dissolve lipase to give a final concentration of between 0.3-2.2 wt% in 0.65 wt% xanthan solution 2. Mix the solution for 15 mm at 15°C before introducing it dropwise, using a syringe, into an agitated reaction vessel containing 0.545 wt% chitosan solution (the pH of the chitosan solution had been adjusted to 6.3). 3 After formation of spheres, agrtate the mixture for 15 mm at room temperature (ZO-22°C). 4. Decant the immobilized enzyme spheres and wash with 0.5M acetate buffer, pH 5 3, and then store in 0 05M acetate buffer, pH 5.3, at 5°C 5. Prepare standard samples of free fatty acids (18) containing 2.0-50 pmol oletc acid by dissolving them m test tubes with 5 mL of iso-octane. 6. Add 1.0 mL of cupric acetate-pyridme reagent and mix the two phases thus formed vigorously for 90 s using a vortex mixer. Allow the mixture to stand still for about 10-20 s until the aqueous phase is separated clearly from the solution of iso-octane and fatty acid. Plot standard curves of oleic acid vs absorbance of iso-octane solutton at 715 nm against the control, which contains no free oleic acid.

Dumitriu, Vidal, and Chornet

234 350

300 250

E $

200

ii 2 %

150

!3 100

50

0 o.i2

0;s

1:o

[XYl==l

1:2

1.3

I 56

(I)

Fig. 3. Relative protease activity as a function of varying concentrations of cormmobrlized xylanase. Protease 1 wt%, mcubatron time 50 min.

3.51. Hydrolysis of Olive Oil- Water Emulsion 1. Prepare a reaction medium containing 0.2 g dry weight of immobilized hpase, 2.5 mL olive oil-water emulsion (Sigma 50% v/v, stabilized olive oil emulsion), and 2.0 mL OSMphosphate buffer, pH 7.5. 2 Incubate this mixture at 37°C using a shaker. 3. Withdraw samples from the reaction mixture at various time intervals. 4. Stop the reaction after 10 mm by addition of 2 mL of 6N HCl solution, mix vigorously, and boil for 5 mm. 5. To this mixture, add 5 mL of iso-octane, mix for 2 mm, then centnmge the mtxture 6. The tso-octane layer is separated and analyzed for oleic acid as for the determmanon of lipase activtty.

3.5.2. Hydrolysis of Olive Oil in /so-Octane 1 Prepare 0.2 g dry weight of immobilized lipase and 5 mL total volume containing various amounts (1 O-60 wt%) of olive oil. 2. Incubate the mixtures at 34’C for 24 h. 3. At the end of the incubation period, withdraw 3 mL of the iso-octane layer and analyze for oleic acid content as for the determination of lipase activity.

3.6. Coimmobilization

of Lipase and Xylanase

(see Note 6)

1. Dissolve lipase and xylanase to give individual final concentrations of between 0 3 and 2.2 wt% m 0 65 wt% xanthan solutron.

Chitosan-Xanthan Complexes

235

s 160-l

0

0.2

0.4

0.6

0.8 ~xylmse1

1

1.2

1.4

1.6

(%I

Fig. 4. Activity of the xylanase coimmobilized at different concentrations with lipase m a xanthan-chttosan matrix. Substrate RBB-xylan; incubation temperature 3O’C; incubation time 30 mm. 2. Prepare the immobilized inclusive.

enzymes as indicated m Section 3.3. from steps 2-4,

4. Notes 1. Four dissolution-precipitation cycles will lower the nitrogen content from 0.7 (initial xanthan) to 0.15 wt% in purified xanthan 2. Verified by tsopropyl alcohol precipitation for xanthan, and 1ONNaOH solution precipitation for chitosan. 3. Enzyme activity is expressed in terms of enzyme units per unit weight of dry microsphere support. 4. The immobilizatton reaction yield varies between 74 and 98%, this being a function of the concentration of enzymes dissolved in the xanthan solution. 5. The coimmobilization of protease and xylanase induces a synergistic effect, the proteolyttc activity is increased because of the presence of the xylanase (Fig. 3). 6 The xylanase activity, in a system coimmobilized with ltpase, is increased because of the synergistic effect of the lipase (Fig. 4).

References 1. Silman, I. H. and Katchalski, E. (1966) Water-insoluble derivatives of enzymes, antigens, and antibodies. Ann. Rev. Biochem. 35, 873-877.

236

Dumitriu, Vidal, and Chornet

2. Crumbhss, A L , Stonehuerner, J G , and Henkens, R W. (1993) Carrageenan hydrogel stabilized colloidal gold multi-enzyme btosensor electrode utilizing unrnobiltzed horseradish peroxidase and cholesterol oxidase/cholesterol esterase to detect cholesterol m serum and whole blood. Bzosensors and Bzoelectron 8,33 l-335 3, Khbanov, A M (1983) Immobihzed enzymes and cells as practical catalysts Sczence 219,722-725

4 Dumitrm,

S. (1991) Processes with immobilized enzymes and cells, m Bzoconverszon of Waste Materzals to Industrzal Products (Martin, A. M , ed ), Elsevter, Lon-

don, pp. 64-l 16. 5. Wehtje, E., Adlercreutz, P , and Mattiasson, B (1993) Improved activity retention of enzymes deposited on solid supports. Bzotechnol Bioeng 41, 171-179 6. Dua, R. D., Vasudevan, P , and Kumar, S. (1984) Carboxypeptidase unmobiltzation on a cellulostc matrix. J Macromol. Ski. Chem. 21,43-5 1. 7 Crescenzt, V., Dentmi, M , and Rizzo, R. (199 1) Polyelectrolytic behavior of ionic polysacchartdes, m ACSSymposzum Series 150. Amencan Chemtcal Society, Washington, DC, pp. 33 l-346. 8. Yamagiwa, K., Shimtzu, Y., Kozawa, T., Onodera, M , and Ohkawa, A (1992) Formation of calcium-algmate gel coating on biocatalyst immobilization carrier J Chem Eng Jpn 25,723-730.

9. Fukuda, H (1980) Polyelectrolyte complexes of chitosan with sodmm carboxymethylcellulose. Bull Chem Sot. Jpn. 53, 837-842. 10. Tsuchida, E. and Abe, K. (1986) Polyelectrolyte complexes, in Developments zn Ionzc Polymers (Wilson, A. D and Prosser, H J , eds.), Elsevier, London, pp 191-199 11. Williams, P. A., Clegg, S M., Day, D. H., Phillips, G. O., and Ntshmari, K. (199 1) Mixed gels formed with komjac mannan and xanthan gum, in Food Polymers, Gels, and ColZozds (Dickinson, E , ed.), Royal Society of Chemistry, Cambridge, UK, pp. 339-347 12. Jansson, P. E , Keene, L , and Lmdberg, B (1975) Structure of the extracellular polysaccharide from Xanthomonas campestris Carbohydr Res 45,275-28 1. 13. Stankowski, J D., Mueller, B. E , and Zeller, S G (1983) Location of a second Oacetyl group m xanthan gum by the reductive-cleavage method. Carbohydr Res 241,321-326. 14. Bekturov, E. and Bimendina, L A. (1980) Interpolymer complexes. Adv PoZym sci 41,10@-109

15. Scranton, A. B., Klier, J , and Aronson, C. L (1992) Complexation of polymeric acids with polymeric bases, m Polyelectrolyte Gels, Propertzes, Preparatzon, and Applzcatzons (Harland, R. S. and Prud’homme, R. K., eds ), ACS Symposium Series 480, American Chemical Society, Washmgton, DC, pp. 17 1-188. 16 Good, W R. and Mueller, K. F. (198 1) Hydrogels and controlled delivery AZChE Symp. Ser 77,42-50.

17 Lowry, R. R. and Tmsley, I. J. ( 1976) Rapid colorimetrtc determination of free fatty acids. J Am Ozl Chem Sot. 53,47@-472. 18. Kwan, D. Y. and Rhee, J S. (1986) A simple and rapid calortmetric method for determmation of free fatty acids for llpase assay J Am. OzE Chem Sot 63, 89-92

27 Calcium Alginate Film Formed on a Stainless Steel Mesh Harold E. Swaisgood

and Flavia M. L. Passos

1. Introduction

Viable cells can be immobilized in calcium alginate to improve the volumetric productivrty of the fermentation process by increasing the cell numbers per unit of bioreactor volume and using the cells in their most productive growth phase. Also, in a continuous process, nnmobilizatron prevents cells from washing out of the bioreactor with the fermentation product. Hence, the feed rate of fresh substrate medmm per unit of bioreactor volume (i.e., the dilution rate) is uncoupled from the growth rate of the cells. This is not the case for a continuous, freely suspended cell bioreactor in which the product withdrawal rate must be carefully balanced with the cell growth rate to avoid washout. To obtain highly active cells, the nnmobilization should not be harmful to the cells and should have a minimal effect on the cell phystology (unless the desired product is not growth-assocrated, in which casethe immobrlization and operation condrtions can be manipulated to induce the physrological changes of interest). Procedures using entrapment in alginate gels are very effective m maintaining a high degree of cell viability. Cell growth in the gel after immobilization yields high cell concentrations per gram of support. A disadvantage of entrapment procedures is the mass transfer limitations because there is no appreciable convectrve mixing (I). Limitation of both substrate and product mass transfer affects cell productivity. Such effects are especially significant rf the desired compound is growth-associated. Thus, reduction of mass transfer limitations is necessary to achieve desirable btotransformation rates with gelimmobilized cell systems. Cells grow in large numbers in a thin layer near the surface of the gel and, m the steady state, they continue to grow and are released into the medium at a From

Methods m Biotechnology, Vol 7 lmmobrlrzatron ol Enzymes and Cells Edlted by G F BIckerstaff Humana Press Inc , Totowa, NJ

237

Swaisgood and Passos

238

constant rate. Klem and Wagner (2) have noted that justification for the use of immobilized cell systems requires that cells released into the medium do not dominate the fermentation. Consequently, the fluid residence time should be much shorter than the duplication time of the free cells. This can be achieved by using a biocatalyst with a high loading of very active cells. To minimize mass transfer limitations and assure high volumetric productivity, the ratio of gel surface area to bioreactor volume should be maximized. The choice of bioreactor type should take into account productivity and mass transfer limitations as well as the characteristics of the substrate medium. Although gel beads are the most commonly used configuration for alginateimmobilized cells, other techniques that increase the surface area of the support have been suggested. As an alternative bloreactor design, we have proposed a configuration, in which cells are unmobihzed in a thm film of alglnate on the surface of a spiral mesh. As an example, we describe here an immobilized Lactococcus bloreactor for milk acidification and inoculation. In this case, the immobilized cell bioreactor not only preacidifies the milk, but also inoculates it with a highly viable cell population. Such maculation 1s very desirable for productlon of cultured milk products. 2. Materials 2.1. Construction of a Spiral Stainless-Steel Mesh A rectangular stainless-steel mesh with 0.89~mm open-width and 14 x 29 cm dimensions giving an area of 406 cm2. 2.2. Preparation 1. Lactococcus

2. 3 4. 5. 6.

of Cells for Immobilization lactis species lactls C2.

M 17 broth with 0.5% lactoseand 20% glycerol (3) Elllker broth (Difco, Detroit, MI). 200 mMpotassium phosphate,pH 7.0. 0.1% peptone water. 4% solution of sodium algmate (ProtanalLF 10/60,Protan,Drammen,Norway).

2.3. Cell Entrapment in Calcium AIginate 1. 2% CaCl, solution. 2. Autoclaved skim milk. 2.4. Construction and Operation of the Spiral Mesh Bioreactor 1. A 250-mL, Jacketed,stirred reservoir (Wheaton,Mlllville, NJ). 2. Three peristaltic pumps, one of which is capable of pumping at a rate of 1 L/mm, (Masterflex, Cole-Parmer, Nlles, IL). 3 Nonfat dry milk powder, reconstituted to 11% concentration in water. 4. A dual channel pH controller (Model 4000-120-60, New Brunswick Scientific, Edison, NJ) equipped with an Ingold pH electrode

239

Stainless Steel Bioreactor

3. Methods 3.1. Construction of a Spiral Stainless Steel Mesh Roll the rectangular stainless-steel mesh into a spiral with a 0.3-cm space between each round. Fix the spiral into place by fastening with small lengths of stainless steel wire at the top and bottom. 3.2, Preparation

of Cells for immobilization

1. Culture the lac+ Lactococcus lactis species lactic C2 at 30°C in Ml7 broth contaming 0.5% lactose (3) and store as a stock culture at -85°C in Ml7 broth wtth 0.5% lactose and 20% glycerol. 2. Propagate the cells at 30°C in 500 mL of Elliker broth. 3. Harvest the cells in the late exponential growth phase by centrifugation at SOOOg for 15 min at 4Y!. 4. Wash the pelleted cells twice with 200 mMphosphate buffer and resuspend them in 20 mL of 0.1% peptone water 5. Steam 20 g of sodmm alginate at 100°C for 30 min, then dissolve rt m 500 mL of sterile water and cool to room temperature. Mix the cell suspension with the alginate solution

3.3. Cell Entrapment

in Calcium Alginate (4,5)

1. Dip the stainless steel mesh into the 2% CaC12 solution, remove, and drain on a paper towel. This is most effectively accomplished by placing the CaC12 solutron in a graduated cylinder with a diameter slightly larger than that of the spiral mesh 2 An alginate-cell film is formed by dipping the mesh into the sodium alginate-cell mixture for 3 min. This results m a thin film covering the mesh, including the open spaces in the mesh. 3. The film is transformed into a strong gel by dipping the spiral mesh mto the 2% CaC& solution (ionotropic gelation). Allow the spiral mesh to stand in the CaC12 solution for 30 mm to form a firm gel. Typically, this results in a gel film with a thicknessof 300-400 pm. 4. The spiral mesh biocatalyst may be activated by overnight incubation in reconstituted skim milk at 30°C to allow the cells to grow and occupy most of the gel pore volume.

3.4. Construction (4,5, see Note I)

and Operation

of the Spiral Mesh Bioreactor

1. Place the spiral mesh with the immobilized alginate Lactococcus film in a glass column (drmensrons: 48 x 170 mm). The spiral mesh IS held in the center of the column by two perforated stainless steel rings so it does not touch the column surface. 2. Connect the column to the jacketed, stirred reservoir with tubing through the Masterflex pump (bioreactor pump) to allow recirculation of the skim milk at 1 L/min (see Note 2).

Swaisgood and Passos

240

3 Steam reconstituted skim mtlk (11%) for 45 min, cool by refrrgeration and place m a 4-L reservoir 4 Connect the the 4-L reservoir by tubing to the 250-mL,Jacketed, stirred reservotr through a Masterflex pump (feedstock pump) that 1scontrolled by the dual channel pH controller 5. The pH of the milk in the bioreactor (the column wtth the spiral mesh and the 250-mL stirred reservorr) is controlled at pH 5 7 using the pH controller with the electrode inserted mto the Jacketed, stirred reservoir (see Note 3). 6 Maintain the temperature of the bioreactor at 30°C by circulatmg thermostated water through the Jacket. 7 Maintain the workmg volume of the broreactor constant by inserting a tube (outflow tube) into the bioreactor reservoir at the desired volume level, and connect the tube to a continuously operatmg peristaltic pump (outflow pump). A schematic illustration of the bioreactor is shown m Fig. 1 8 After 8-10 generations, steady-state conditrons are reached as characterrzed by a constant dtlution rate, a constant rate of release of biomass (free cells released mto the product outflow stream), and a constant rate of lactic actd productton (measured in the product outflow stream, see Notes 4,5)

3.5. Enumeration

of Immobilized

Cells

1 Turn off the btoreactor and drain the milk from tt 2 Connect the feedstock pump that was disconnected from the 4-L reservoir to dram the bioreactor to a reservoir containing 0. IM EDTA and recirculate thus solution through the spiral mesh m the column at 1 L/mm 3. The alginate gel should dissolve m 15-20 min, and the cells can be collected by centrifugation at SOOOgfor 10 min at 4°C. 4. Determine the viable cells by plating on Elliker agar using standard conditions

4. Notes 1. The productivtty of the bioreactor depends on the ratio of irnmobtlized biocatalyst surface area to the bioreactor volume. Increasing this ratio increases the productivity, which increases the dilution rate for the bioreactor operation The procedure given represents an example and should not be confused with the potential optimal productivity such a design could yield The example given typically provides about 1OO-fold more cells in the broreactor than can be maintained in a similar free-cell broreactor (4,5). The resulting dilution rate is about 2 h-1 and a productivity around 3 g lactate/h/L of bioreactor volume. These numbers could be increased by decreasing the bioreactor reservoir volume and by decreasmg the spacing between rounds of the spiral and the volume of the column. 2. The total bioreactor volume is represented by the sum of the volume of the column containing the spiral mesh, the volume of the bioreactor reservoir, and the recirculation tubing volume. 3 The pH controller mamtains the pH by turning on the feedstock pump when the pH falls below the set point (PH 5.7, for example) resulting from lactic acid productton.

Stainless Steel Bioreactor

1. 2. 3. 4.

Bioreactor Reservoir pH Electrode pH Controller Bioreactor Column with Immobilized Cells 5. Milk Feedstock

241

6. Oufflow Pump 7. Feedstock Pump 6. Bloreactor Pump 9. Acidified Milk Product 10. Thermostated Water Bath

Fig. 1. Schematrc dtagram of the immobihzed

cell broreactor.

4. Because a dual channel pH controller 1sused, the other channel may be used to operate a free-cell bioreactor at the same time under the same conditions. 5. Use of this particular strain of Lactococcus (protease-positive) results in clottedmilk protein film formation on the gel surface after 3 d of continuous operation. Such a film limits mass transfer and lowers productivity. However, when the protease-negative strain is used, film formation is not observed (4,.5). Also, lactococcr are inhibited by the product lactic actd; thus, immobihzed cells exhibit lower activity because of product mass transfer limitation. Cells that do not have thus characteristic are less susceptible to the influence of product mass transfer limitations.

References 1. Karel, S. F., Salmon, P. M., Stewart, P. S., and Robertson, C. R. (1990) Reaction and diffision in immobthzed cells fact and fantasy, in Physiology of Immobilized Cells (de Bont, J. A. M., Hisser, J., Mattrason, B., and Tramper, J., eds.), Elsevter, Amsterdam, The Netherlands 2. Klein, J. and Wagner, F. (1987) Different strategies to optimize the production phase of munobtlized cells. Ann NYAcad. Sci. 501,306-3 16.

242

Swaisgood and Passos

3. Terzaghi, B. E. and Sandine, W. E. (1975) Improved medium for lactic streptococci and their bacteriophage Appl Mtcrobiol. 29,807-813. 4. Passos, F. M. L. and Swaisgood, H. E. (1993) Development of a spiral mesh bioreactor with immobilized lactococci for continuous inoculation and acidification of milk. J Duq Scr 76,2856-2867. 5 Passos, F M. L , Klaenhammer, T R., and Swaisgood, H E (1994) Response to phage infection of immobihzed lactococct during continuous acidification and maculation of skim milk. J Davy Res. 61, 537-544

Preparation of Immobilized Subunits of a Multisubunit Enzyme Gordon

F. Bickerstaff

1. Introduction It is well established that many mtracellular enzymes are oligomeric with drmers and tetramers being the most common of the possible subunit structures (I, 2). Evaluation of the functional significance of the quaternary structure is an important part of the fundamental studies on the catalytic properties of an enzyme that are necessary to obtain a complete understanding of enzyme structure, function, mechanism, and applications (3-5). For some enzymes the correlation between allosteric properties, subunit mteractions, and enzyme activity is generally well understood. However, in many other enzymes, considerably less is known about the occurrence of subunit interactions or the significance of the oligomeric structure (6-9). An experimental drawback that has hindered progress in this field is the tenacity of the noncovalent bonding between subunits. In most enzymes, the native oligomeric structure does not dissociate easily to enable comparative studies to be made on the native monomeric form, which would thereby reveal the importance of the oligomeric structure. The use of urea and guanidine-HCl to effect artificial dissociation is usually unsatisfactory because it leads to subunit unfolding and consequent loss of catalytic activity. A more readily applicable technique is the use of enzyme immobilization to restrict the freedom of movement of an enzyme. In this approach, covalent attachment is made via a single subunit of the enzyme, which allows experimental separation of the subunits to provide a means of obtaining isolated subunits of an oligomeric enzyme. The basic strategy 1s outlined in Fig. 1. The experimental conditions concerning the activation of the support material are specifically designed to favor covalent attachment of each enzyme molecule to the support via one subunit From

Methods m Botechnology, Vol 1’ ImmobNzafron of Enzymes and Cells E&ted by G F BIckerstaff Humana Press Inc , Totowa, NJ

243

Bickerstaff

244

Immobilized

enzyme

I Denaturation

Renaturation

Immobilized

subunit Denaturation -a Unfolded

Soluble enzyme

Immobilized re-associated

Fig. 1. Scheme for preparation of immobilized ated enzyme.

subunit and immobilized

reassoci-

only. In practice, this is achieved by using low concentrations of CNBr to activate the support, thereby ensuring that the coupling points on the support are sufficiently spread out so that not more than one coupling point on the support is available to react with a given enzyme molecule (48). The preparation of immobilized subunits involves two stepsthat are designed to remove the noncovalently bound subunits of the oligomeric enzyme (see Fig. 1). In the first step, the immobilized enzyme is washed with a denaturing buffer containing guanidine-HCI to facilitate dissociation and removal of the noncovalently bound subunits. In the second step, the denaturing buffer is replaced with a renaturing buffer to allow the immobilized unfolded subunits to refold and renature. Since denaturation and renaturation are essential stepsin this technique, it is essential to establish for a given soluble enzyme that renaturation is possible and discover any special requirements (e.g., presence of reducing agent)

Immobilized Subunits

245

Immobilized

subunit (inactive)

Denaturation

-03

Soluble enzyme (inactive) Immobilized re-associated enzyme (50% activity) Fig. 2. Scheme for evaluation of enzyme activity in immobilized

inactive subunits.

for renaturation of the enzyme. In this respect, it may be necessary first to examine the reversible denaturation of the soluble enzyme with a view to optimizing the experimental conditions for maximum renaturation of the subunits. Confirmation of the presence of immobilized subunits is essential and can be obtained by observing the highly specific ability of the immobilized subunit to reassociate with added soluble subunits that are formed transiently during renaturation from a denatured state (see Fig. 1). Reassociation between immobilized and soluble subunits to reform the original oligomeric structure can occur with high yields of immobilized reassociated enzyme. A novel feature of this technique is that added subunits from isoenzyme species can be used to attempt to discover if intermediate/mixed oligomeric structures are possible. The use of immobilization to study the properties of enzyme subunits is increasing and is contributing valuable information to fundamental enzymology that may have significant importance in genetic engineering of enzymes for use in industry and medicine. The technique is generally capable of deciding whether or not the subunits of an enzyme are catalytically active. If the immobilized subunit is active, then a comparison of its enzymic properties with those of the corresponding immobilized oligomer can yield useful information on the role of the oligomeric structure in the function, properties, and control of the enzyme (1 O-l 5). If the immobilized subunits are found to be catalytically inactive, then it is important to demonstrate that the loss of activity is not caused by an incorrectly immobilized subunit or by some extraneous influence exerted by the support material. A useful test, illustrated in Fig. 2, is effectively a hybridiza-

Bickers ta ff

246

tion experiment m which the inactive immobilized subumt is reassociated with a soluble subunit that has been irreversibly inactivated by chemical means. The resultant hybrid is an immobihzed reassociated enzyme consistmg of one potentially active immobilized subunit and one completely mactive subunit. If the reconstituted subunit--subunit mteractions generate catalytic activity m the immobilized subunit, this is evidence that subunit interactions are essential for catalytic activity. Studies can then be undertaken on the mnnobilized subumt. In this chapter, details are provided for the preparation of immobilized subunits and immobilized reassoctated enzyme using a model enzyme, creatine kmase, from rabbit muscle. The transitions between immobilized subunit and oligomer enzymes can be followed by monitoring changes in protein content and enzyme activity. Detailed mformatlon on the appropriate methods for esttmating protem content and enzyme activity are given m Chapter 29. 2. Materials 2.1. Preparation of the CNBr Reagent 1. Acetonitrile (BDH, London) Care: highly toxic. 2. Cyanogen bromide (CNBr) (100 g bottle, Sigma, St. Louis, MO). Care: highly toxic

3 Anhydrous magnesium sulfate (BDH). 4. Fume cupboard, disposable gloves, face protection.

2.2. lmmobi/ization

of the Enlyme

1. 2. 3. 4. 5. 6. 7. 8. 9 10. 11.

Sepharose 4B gel (Pharmacla, Uppsala, Sweden). 2M sodium carbonate solution. CNBr-acetomtrile solutron, prepared as described m Sectton 3. I. 1 .OMsodium bicarbonate solution pH 9.0 (cold). Double-distilled water (cold). 10 mM sodium phosphate buffer, pH 8.0, containing 1 mM EDTA (see Note 1). Creatme kinase from rabbit muscle (Boehrmger, Mannhelm, Germany). 10 mMsodmm phosphate buffer, pH 8.0, containing 1 mMEDTA and 1.OMNaCl. O.lM glycme solution End-over-end mixer. Sintered-glass filter funnels, 25 and 100 mL, Buchner flask, and connectton to suction pump. 12. Bench-top centrifuge and rotor suitable for 15-mL centrifuge tubes. 13 Graduated glass 15-mL centrifuge tubes with graduations @-lo mL. 14. Pasteur pipets and bulb.

2.3. Preparation

of GeLBuffer

Suspensions

1 Sintered-glass filter funnels, 25 and 100 mL, Buchner flask, and connectton to suction pump.

Immobilized Subunits 2 3. 4. 5.

247

Bench-top centrifuge and rotor suitable for 15-mL centrrfuge tubes Graduated glass 15-mL centrifuge tubes with graduatrons O-10 mL. Pasteur prpets and bulb 10 mJ4 sodium phosphate buffer, pH 8.0, containing 1 mM EDTA (see Note 1).

2.4. Preparation of immobilized

Subunits

1 2. 3. 4.

Immobilized enzyme from Section 3.1. Parafilm, Clingfilm, or similar. Guanidme-HCl (AnstaR grade, BDH, see Note 2). Denaturing buffer. Prepare O.lM Tris-HCl buffer, pH guanidine-HCl and 5 mM dnhioerythritol (DTE) (see Note 5. Renaturing buffer: Prepare O.lM Tris-HCl buffer, pH 7 dithioerythrtol (DTE). 6. 10 mM sodium phosphate buffer, pH 8.0, containing 1 mM

2.5. Preparation of Immobilized

7.5, containing 6M 3). 5, containing 5 mM EDTA (see Note 1).

Reassociated Enzyme

1. Immobilized subunits from Section 3.3. 2. Small magnetic stir-bar 8-10 mm long, suitable for operating in a test tube 3. Denatured soluble enzyme. Dissolve 3 mg of creatine kinase m 1 mL of denaturing buffer (see Note 4) 4. 10 mM sodium phosphate buffer, pH 8.0, containing 1 mM EDTA (see Note 1). 3. Methods

3.1. Preparation of the CNBr Reagent 1. Use a standard drstillation apparatus, set up in the fume cupboard to redistill the acetonitrrle (see Note 5 for safety advice). 2. Add 2 g of anhydrous MgS04/50 mL of redistilled acetonitrile and allow the mixture to stand at room temperature for 24 h. 3 Use a standard filter funnel and Whatman No. 1 filter paper to filter off the MgSO+ 4. Collect the acetonitrile and immediately add 50 mL directly to the manufacturersupplied bottle containing 100 g of CNBr to provide a stock solution of CNBr acetonitrile. Note. This procedure must be undertaken m a fume cupboard set at maximum extraction (see Note 5). Ensure that the stock solution is securely stoppered and store inside a sealed container m a fume cupboard 5. Prepare a working solution of approx 50 mg/mL CNBr-acetomtrile. Add, using an automatic prpet, 0.5 mL of stock CNBr-acetonitrile to 9.5 mL of redistilled and dried acetonitrile solution Mix and store in a securely stoppered bottle in the refrigerator.

3.2. Preparation of Immobilized

Enzyme

1. Wash 15 mL packed volume of Sepharose in a sintered-glass funnel connected to a Buchner flask to provide mild suction. Wash with 5 aliquots of 50 mL distilled water to remove preservatives from the gel (see Note 6).

Bickerstaff

248

2. Transfer 10 mL packed volume of washed Sepharose to a 25-mL beaker and add 20 mL of 2A4 sodium carbonate solutton. MIX the suspension gently using magnetic stirrmg. 3. Transfer the beaker contaimng the suspension to a magnetm stirrer in a fume cupboard set to maximum extraction. 4 To the stirred suspension add, using an automatic pipet, 1 0 mL of acetomtrlle containing 50 mg of CNBr and increase the stirring to ensure rapid mixing 5 After 2 mm rapid stnrmg, transfer the gel onto a smtered-glass funnel and wash the remainder of the gel from the beaker onto the glass funnel wrth dlsttlled water. 6 Wash the gel under gentle suction successrvely with 100 mL each of cold 1 OM sodium bicarbonate solution, pH 9.0, 100 mL of cold distilled water, and 100 mL of cold 10 mA4 phosphate buffer, pH 8.0, containing 1 mA4 EDTA. 7. Transfer the 10 mL of activated gel to a 25-mL screw-top bottle and add 8 mL of phosphate/EDTA buffer containmg 10 mg of creatme kinase. Retain 1 mL of enzyme solution to wash the residual gel from the funnel into the beaker (see Note 7). 8. Cap the bottle and mix the suspenston on an end-over-end mtxer overnight at 4°C 9 Transfer the gel from the bottle to the smtered-glass funnel using phosphate/ EDTA buffer to wash the remamder of the gel from the bottle to the funnel. 10 Wash the gel alternately with 50 mL aliquots of phosphate/EDTA buffer contaming or not containing NaCl using 4 aliquots of each buffer. Start with buffer contaming NaCl and finish with buffer contammg no NaCl (see Note 8). 11. Transfer the gel to a clean 25-mL screw-top bottle using 0 1Mglycme solutron to wash the gel from the smtered-glass funnel into the bottle. Add 10 mL of 0 1M glycme solution to the bottle and mix by end-over-end rotation for 2 h at room temperature. 12. Transfer the gel to the smtered-glass funnel and use phosphate/EDTA buffer to wash the residual gel from the bottle to the funnel (see Note 7). 13 Wash the gel with 200 mL of cold phosphate/EDTA buffer and prepare suspensions as indicated in Section 3.3.

3.3. Preparation

of GekBuffer

Suspensions

1. Transfer and distribute the 10 mL of gel from the sintered-glass funnel into three 15-mL glass graduated centrrfuge tubes and use phosphate/EDTA buffer to wash the gel from the funnel to the centrifuge tubes (see Note 7). 2. Use phosphate/EDTA buffer to wash the gel beads down the walls of the centrtfuge tubes and add further phosphate/EDTA buffer to the lo-mL mark. 3. Centrifuge the test tubes at 200-4OOg for 2-3 mm to pack the gel beads. 4. Prepare a 1:2 (v/v) suspensron of gehbuffer in each centrifuge tube. Read the packed gel volume on the graduated centrifuge tube and withdraw phosphate/ EDTA buffer from the tube using a Pasteur pipet to provide the correct volume of buffer requned to give the suspenston (see Fig. 3)

Immobilized Subunits

249

Volume of +

Graduated centrifuge tube Fig. 3. Graduated centrifuge tube for determination of packed gel volume of immobilized enzyme. 5. Store the suspensions at 4’C until required.

3.4. Preparation

of Immobilized

Subunits

1. To 4 mL of packed immobilized enzyme gel from Section 3.1. in a graduated centrifuge tube add 10 mL of denaturing buffer. 2. Cover the centrifuge tube with parafilm, clingfilm, or similar and mix the suspension by end-over-end rotation for 1 h at room temperature (see Note 9). 3. Transfer the gel to a sintered-glass funnel and wash the gel with 5 aliquots of 10 mL of denaturing buffer. Drain the last aliquot of denaturing buffer. 4. Wash the gel with 5 aliquots of 25 mL of renaturation buffer. 5. Suspend the gel in 20 mL of renaturation buffer, and using a Pasteur pipet, transfer the gel suspension to a 25-mL screw-top bottle. Use a few milliliters of renaturation buffer to transfer the residual gel from the funnel to the bottle. 6. Allow renaturation to continue for 3 h at room temperature followed by 18 h at 4’C (see Note 10). Stirring or mixing is not required. 7. Transfer the gel suspension from the bottle to a sintered-glass filter funnel using a Pasteur pipet and use phosphate/EDTA buffer to wash the residual gel from the bottle to the funnel. 8. Wash the gel with 8 aliquots of 40 mL of phosphate/EDTA buffer.

250

Bickerstaff

9. Transfer the immobilized subunit gel to a glass graduated centrifuge tube using a Pasteur pipet and use phosphate/EDTA buffer to wash the residual gel from the funnel to the centrifuge tube. 10. Prepare a gehbuffer (v/v) suspension as indicated m Section 3 3.

3.5. Preparation

of Immobilized

Reassociated

Enzyme

1. Prepare a 1.5 (v/v) suspension of immobilized subunit m renaturation buffer using 1 mL packed volume of gel and 4 mL of renaturation buffer 2. Using a Pasteur ptpet, place the suspension m a lo-mL standard test tube containmg a small magnetic stir-bar, typically 8-10 mm long 3. Stir the suspension gently and add 5 separate ahquots of 0 10 mL of denatured soluble enzyme with mtervals of 5 min between each ahquot addition (see Note 11) 4. At 5 min after the last addition, stop stirrmg and allow reassociation to occur at room temperature for 3 h followed by 18 h at 4°C. 5 Using a Pasteur pipet, transfer the gel from the test tube to a smtered-glass funnel Wash the gel with 5 ahquots of 25 mL of phosphate/EDTA buffer and finally prepare a gel.buffer (v/v) suspension as indicated in Section 3 3.

4. Notes 1 This buffer is the most suitable for the enzyme creatme kmase and can be substituted with a buffer appropriate for the enzyme to be mnnobihzed. 2. Guamdine-HCl must be of the highest purity because impure grades have various cyanate derivatives that are potent enzyme mhibitors AristaR grade is expensive, and if necessary, reagent grade guanidine-HCl can be purified by recrystallization from 98% (v/v) ethanol at least three times or until the absorbance at 280 nm of an aqueous 6M solution is CO.1. 3 Note that preparation of a 6M solution involves addition of a large quantity of solid guamdine-HCl, which will take up a good proportion of the volume. Thus, if preparing 50 mL, dissolve the solid in only 10-l 5 mL of distilled water and brmg the pH of the solution to 7.5 using concentrated HCl solution dropwtse. Inclusion of DTE IS optional and IS added to the buffers for creature kinase because this enzyme has essential thiol groups that must be reduced during denaturation. 4 Use the denaturing buffer from Section 2.4., step 4, and allow the denaturing to occur for at least 1 h at room temperature. 5. Acetonitrile and CNBr are highly toxic and can be fatal at low levels by mhalation and skm contact. All steps involving these two agents must be undertaken m an efficient fume cupboard with adequate protection for skm and inhalation as well as contingency precautions for dealing with spillage handling m the event of an accident 6. Packed gel volume refers to Sepharose gel that has been centrifuged in a bench-top centrifuge at low centrtfugal force of 200-4OOg for 2-3 mm in a graduated glass centrifuge tube. Note that some graduated centrifuge tubes have approximate graduations as a rough guide Use centrifuge tubes with accurate graduations and check the accuracy using a pipet and water

Immobilized Subunits

251

7. Transfer the gel from tubes using a spatula to move most of the solid gel, then use a Pasteur pipet and a small volume of buffer to wash the residual gel from the walls of the tube. Then transfer it to the next container. 8. At all times when the gel is on the smtered-glass filter funnel do not allow the suction to dry the gel Use the suction to remove liquid until the moment when the gel forms a firm cake, then stop the suction If suction is continued, the gel will change in color from gray (moist) to white (dry) and if enzyme is unmobilized it may become inactivated. 9. Invert the tube a number of times to ensure that the suspension is completely mixed before placing it on the end-over-end mixer. 10 The renaturation conditions indicated here are appropriate for the enzyme creatme kinase. Other conditions and chemical requirements may be required for other enzymes 11. Typical protein content for immobilized subunits of creatine kmase using the procedures described here is 200 pg/mL of packed gel For reassoctation, it IS necessary to add a fivefold excess of enzyme protein. Therefore, at least 1000 ug of denatured/unfolded soluble enzyme is added to 1 mL of immobilized subunits.

References 1. Bickerstaff, G. F. (1980) Immobihzed enzymes and their use in evaluating subunit interactions Int J Bzochem. 11,201-206. 2. Bickerstaff, G. F. (1984) Applications of mnnobtlized enzymes to fundamental studies on enzyme structure and functton, in Topics zn Enzyme and Fermentation Biotechnology, vol. 9 (Wiseman, A, ed), Ellis Horwood, Chichester, UK, pp 162-201 3. Martinek, K. and Mozhaev, V. V. (1985) Immobilization of enzymes. an approach to fundamental studies in biochemistry. Adv. Enzymol. 57, 179-247. 4. Chan, W. W.-C. (1976) Some experimental approaches for studying subunit mteractions in enzymes. Can. J Biochem 54,521-528. 5. Chang, G.-G., Huang, T.-M., and Chang, T.-C. (1993) Immobilization of the tetramenc and monomeric forms of pigeon liver malic enzyme on Sepharose beads. Eur. J. Blochem. 213,1159-l 165. 6. Aguire, R. and Kasche, E. (1983) Catalytically active monomer forms of immobtlized arginase. Eul: J Blochem. 130,373-38 1. 7. Porter, D. H. and Cardenas, J. M. (1981) Single subunits of Sepharose-bound pyruvate kinase are active Biochemistry 20,2532-2537. 8. Bickerstaff, G. F. and Price, N C. (1978) Properties of matrix-bound dimer and monomer derivatives of unmobilized creatine kinase from rabbit skeletal muscle. Blochem J. 173,85-93 9 Iborra, J. L., Ferragut, J. A., and Lozano, J. A. (1981) Subunit interactions in tyrosmase from frog epidermis in immobilized enzyme systems. Bzochem. J 197,58 l-589 10. Carrea, G. and Pasta, P. (1987) Properties of immobtlized subunits of 20/3hydroxysteroid dehydrogenase, in M&z&r in Enzymology, vol. 135 (Mosbach, K., ed.), Academic, London, pp. 475-483.

11 Douzhenkova, I. V., Asryants, R. A., andNagradova, N. K. (1988) Glyceraldehyde-3phosphate dehydrogenase subunit cooperativity studied using immoblhzed enzyme forms. &ochzm Brophys Acta 957, o-70. 12. McCracken, S. and Meighen, E. (1987) Immobrhzed subumts of alkaline phosphatase, in Methods in EnzymoEogy, vol. 135 (Mosbach, K., ed.), Academic, London, pp. 492-501. 13. Rudge, J. and Bickerstaff, G. F. (1984) Thermal stability properties of immobtlrzed creatine kinase Blochem Sot Trans. 12,3 11,3 12. 14. Toher, J., Kelly, A. M., and Bickerstaff, G. F. (1990) Stabtltty properties of two supports for mrmoblhzation of enzymes. Bzochem Sot Trans 18,3 13,3 14 15. Dtckson, S. R. and Bickerstaff, G. F. (1992) Properties of munobilized bromelam Biochem Sot. Trans. 20,23,24.

29 Characterization of Enzyme Activity, Protein Content, and Phiol Groups in Immobilized Enzymes Gordon

F. Bickerstaff

1. Introduction Immobilization is designed to restrict the freedom of movement of an enzyme and, in doing so, places limitations on the enzyme and the biotransformation catalyzed by the enzyme (see Chapter 1). In practical terms, this often means that the normal procedures used for assay of the soluble enzyme activity, protein content, and so on, must be redesigned to accomodate the presence of the support material. In general, problems are likely to be experienced when the active agent in an assay procedure binds dnectly to the enzyme and measurement is based on the enzyme-reagent complex. Support materials are likely to cause interference in such procedures. Determination of protein content in immobilized enzymes can be estimated by direct and indirect methods (Z-3). The indirect approach involves protein determination before and after immobilization, and is less convenient and less accurate when extensive washing of support material is required to remove unbound protein. The direct method involves reaction of the immobilized protein with protein assay reagent, and it is essential that the products of the reaction are released into solution so they can be separated from the support material and assayed without interference (45). For example, protein assay based on Coomassie Blue dye binding is not suitable for immobilized enzyme because the dye binds to the immobilized protein (6) and in effect becomes immobilized to the support material. The Lowry protein assay is more appropriate for immobilized enzymes because the colored product is released into solution and can be separated from the mnnobihzed enzyme by filtration or centrifugation. An alternative method involves use of polyethylene glycol to diminish light scattering of agarose beads and subsequent direct spectrophotometric measurement of bound protein at 280 nm (3). From

Methods in Botechnology, Vol 1 fmmobihzatron of Enzymes and Cells EdIted by G F BIckerstaff Humana Press Inc , Totowa, NJ

253

Determination of enzyme activity is generally less of a problem for most enzymes because the unmobilized enzyme can be readily separated from the product either during or after the assay. On some occasions it 1s desirable to have a continuous assay. In these circumstances It is essential to ensure that the lmmoblhzed enzyme ts effectively stn-red in the substrate mixture (4,7). Efficient stirring will mmimlze the limitations imposed by diffusion A particularly useful assay for some immobilized proteins is detemmatlon of available thiol groups (if present and accessible) using Ellman’s reagent (8) 5,5’-dithiobis(2-mtobenzoic acid) (DTNB). The product of the reaction between DTNB and thlol groups is 2-mtro-S-thiobenzolc acid, which is released into solution and IS conveniently measured at 4 12 run (9, IO). In this chapter, details are provided for protein assay, continuous enzyme assay and thiol group assay of irnmoblhzed creatine kmase to illustrate the type of approach required to overcome the problems presented by the presence of the support material.

2. Materials Use double-distilled,

2.1. Preparation 1 2. 3. 4. 5

deionized

water in preparation

and Pipeting of GekBuffer

of all solutions.

Suspensions

Automatic pipet 20-200 pL and disposable tips. Scalpel and new (sharp) blade. Long thin microspatula. 10 mM sodium phosphate buffer, pH 8.0, containing 1 mM EDTA (see Note 1). Immobilized enzyme suspension m 10 mM sodium phosphate buffer, pH 8 0, containing 1 mM EDTA, prepared as described in Chapter 28 (see Note 2).

2.2. Protein Assay of ImmoMized

Enzyme

1. Lowry reagent A: Prepare a 2% solution of NaC03 solution containing O.lM NaOH. 2 Lowry reagent B* Prepare a 1% solution of CuS04 3. Lowry reagent C* Prepare a 2% solution of sodium potassium tartrate. 4. Folin and Ciocalteu’s phenol reagent (Merck, Poole, UK): Prepare a I :2 dilution with distilled water 5. Bovine serum albumin (BSA): Prepare a solution of 1 mg/mL in distilled water. 6. Magnetic stirrer and SIX magnetic stir-bars of S-10 mm m length, suitable for stirring in a standard 15-mL test tube (Merck). 7. Immoblllzed enzyme suspension in 10 mM sodium phosphate buffer, pH 8.0, containing 1 mM EDTA, prepared as described in Chapter 28 (see Note 2). 8 Whatman No. 1,42-mm diameter filter paper.

2.3. Enzyme Activity Assay of Immobilized

Enzyme

1. Themostatted cell holder (Merck). 2. Water bath with circulator for external temperature control.

Immobilized Enzyme Assays

255

3. Micro stir-bar, typically 3-4 mm in length. Cut several 3-4 mm pieces of metal from the end of a straightened-out standard metal paper clip using wire cutters. Take a Pasteur plpet and seal the thin end m the flame of a Bunsen burner. Drop one of the small pieces of paper clip down the pipet so that it is located at the sealed end. Use glass cutters to cut, then break the glass about 1 mm beyond the piece of paper clip. Use forceps to pick up the cut end with the piece of paper clip Inside, and seal the open end in the flame of a Bunsen burner. Allow the micro stir-bar to cool before use 4. O.lM glycme-NaOH buffer, pH 9.0, containing 40 mM creatme, 4 mM ATP, 5 mA4 magnesium acetate, 1 mM phopho(enol)pyruvate, and 133 pJ4 NADH. 5. Pyruvate kinase/lactate dehydrogenase enzyme mixture (Boehringer Mannheim, Dorval, UK) for coupled enzyme assays (see Note 3). 6 10 mA4 sodium phosphate buffer, pH 8.0, containing 1 mM EDTA. 7. Immobihzed enzyme suspension in 10 mJ4 sodium phosphate buffer, pH 8.0, containing 1 mA4 EDTA, prepared as described in Chapter 28 (see Note 2). 8. Spectrophotometer with recorder/printer attachment.

2.4. Thiol Group Assay of Immobilized

Enzyme

1 Small stir-bar, typically 8-10 mm m length (Merck). 2. 5 25 mA45,5’-dlthio-bis(2-mtrobenzoic acid) (DTNB) (Sigma, St. Louis, MO) in distilled water. 3. Bench-top centrifuge and rotor for test tubes, centrifuge test tubes (Merck). 4 Spectrophotometer. 5 A 1 mL semlmicro spectrophotometric cuvet (Merck).

3. Methods 3.1. Preparation

and Pipeting of GekBuffer

Suspensions

1. Use the scalpel to carefully cut approx 2 mm off the thm end of the disposable tips. Make sure the cut is made cleanly and does not squash the end of the tip. 2. Adjust the gel:buffer suspension by adding or withdrawing phosphate/EDTA buffer to provide the buffer suspension required for the particular assay (see Chapter 28, Fig. 3). 3. Before taking measured ahquots of suspension, use the mlcrospatula as a central stirring paddle to make the suspension homogeneous (see Note 4). 4. Use the automatic pipet with the cut tips to withdraw measured aliquots of suspension (specified later) from the homogeneous suspension. 5 Wipe the outslde of the pipet tip with paper tissue to remove any gel attached to the tip to avoid carry over of undefined amount of gel mto the next solution.

3.2. Protein Assay of immobilized

Enzyme

1 Prepare a test tube rack containing 12 test tubes (SIX plus six duplicates). 2. Into the six tubes add 0.00,0.02,0.04,0.06, 0.08, and 0.1 mL of BSA solution. Repeat the additions to the corresponding duplicate tubes.

256

Bickerstaff

3 Adjust the volume m each tube to 0.6 mL with addttron of distilled water. 4. Prepare a separate test tube rack (see Note 5) with SIX test tubes (three plus three duplicates), and mto each test tube place a magnetic stir-bar. 5 Prepare a I.1 (v/v) gel buffer suspension and stir the suspension to make it homogeneous as described in Section 3.1.) step 3 (see Note 6) 6. Use the automatic prpet with cut tips to transfer 0.025 mL of suspension mto the first test tube and then into the duphcate tube Add 0 05 mL of suspension mto the second test tube and duplicate, and 0.075 mL of suspension mto the thud test tube and duplicate 7. Adjust the volume in each tube to 0.6 mL with addition of distilled water. 8 Place the test tube rack contaunng the tmmobtlized enzyme on the magetic stirrer and stir the suspensions gently. 9. Prepare Lowry reagent mixture by mixing 50 mL of reagent A with 0 5 mL of reagent B and 0 5 mL of reagent C Prepare fresh and ensure the components are well mixed. 10 To each test tube add 2.5 mL of Lowry reagent mixture and allow the mixtures to react at room temperature for exactly 10 min If necessary, increase the stirrmg of the tmmobihzed enzyme tubes to ensure efficient mixing. 11 To each tube add 0.3 mL of diluted Folin reagent. Vortex mix the BSA tubes to ensure rapid mixing, and allow the reaction color to develop for 30 min at room temperature 12. Filter the immobilized enzyme suspensions through Whatman No. 1 filter paper to remove the gel Collect the gel-free supematants and note the absorbances at 625 nm. 13. Prepare a calibration graph of standard protein vs absorbance using the BSA absorbances. Determine the protein content of the immobrlized enzymes and express the protein content in terms of pg/mL of packed gel (see Note 7).

3.3. Enzyme Activity Assay of /mmobilized Enzyme 1. Place the themostatted cell holder on the magnetic sttrrer and connect the water bath circulator. Place a cuvet in the holder containing 3 mL of water. Adjust the temperature until the water in the cell stabihzes at 30°C (see Fig. 1). 2 Place a micro-stirrer bar in a cuvet and place the cuvet m the cell holder. Add 2.90 mL of 0. Mglycine-NaOH buffer, pH 9.0, containing 40 Mcreatine, 4 rnM ATP, 1 mA4 phospho(enol)pyruvate, 5 mM magnesium acetate, 133 uM NADH Add 24 U each of pyruvate kinase and lactate dehydrogenase (see Note 3). Stir the mixture for 2-3 mm to allow the solution to equilibrate at 3O’C. 3. Dilute 0.1 mL of innnobihzed enzyme 1: 1 (v/v) suspension to 2 mL with phosphate/EDTA buffer (see Note 6). 4. Initiate the reaction by adding 0.05 mL of diluted immobilized enzyme suspension to the stirred cuvet. 5. At 1 min intervals, transfer the cuvet from the thermostatted cell holder into a recording spectrophotometer to record the absorbance change for a few seconds, then return the cuvet to the thermostatted cell holder for further temperature control and stnrmg (see Note 8).

Immobilized

Enzyme

257

Assays Themostatted cell

Water

Magnetic stirrer Fig. 1. Stirred, thermostatted cell block for continuous stirring and temperature control of immobilized enzyme during enzyme assay. 6. Express immobilized enzyme activity in terms of U/n& of packed gel and specific activity in terms of U/mg of immobilized protein.

3.4. Thiol Group Assay of Immobilized

Enzyme

1. Transfer 2 mL of a 1: 1 (v/v) gel:buffer suspension of immobilized enzyme to a standard test tube containing a small, 8-10 mm magnetic stir-bar (see Note 9). 2. Stir the suspension on a magnetic stirrer for l-2 mm, then add 0.1 mL of 5.25 mA4 DTNB solution. 3. Continue the stirring gently for 15 min at room temperature. 4. Centrifuge the suspension for 5 min at 2500g and collect the supernatant. 5. Determine the absorbance of the supernatant at 412 nm using a 1 mL semimicro cuvet. 6. Calculate the thiol group content of the immobilized enzyme (see Note 10).

4. Notes 1. This buffer is appropriate for the enzyme creatine kinase and may be replaced by any appropriate buffer for the enzyme under evaluation. 2. Typical protein contents for creatine kinase immobilized under the conditions indicated in Chapter 28 are immobilized enzyme, 400 pg/mL of packed gel; immobilized subunit, 210 pg/mL of packed gel; and immobilized reassociated enzyme, 390 pg/mL of packed gel. 3. The coupling enzymes are supplied as an ammonium sulfate suspension. Calculate the volume of suspension required to provide 24 U of each enzyme for each assay. For example, the suspension contains 450 U of each enzyme/ml, therefore

Bickers ta ff

258

4

5.

6 7.

8

10 assays will require 240 U and that will be provided by 0.53 mL of suspension Transfer the measured suspension to a centrifuge tube and centrifuge the suspension at 2500g for 5 min. Carefully remove the supernatant with a Pasteur pipet and bulb, and dissolve the ammonmm sulfate pellet in 0.6 mL of cold 0. 1M glytine solution, pH 9.0. Store on tee and add 0.05 mL to the enzyme assay cuvet. Use the microspatula to agitate/stir the suspension by hand Keep the gel in the suspension and avoid allowing the gel to attach to the wall of the centrifuge tube above the suspension level because this will disturb the 1: 1 (v/v) ratio. When a uniform suspension has been prepared, take samples without delay or restn the suspension. A uniform suspensron is essential for accuracy in pipeting samples. A wooden or all plastic test tube rack may be required if a metal or wire rack interferes with the stirring This will be dependent on the performance of the magnetic stirrer. Prepare a a suitable control by performmg the immobilization procedures exactly as detailed m Chapter 28, but replacing the addition of enzyme with phosphate/ EDTA buffer Use the cahbratton graph to convert the absorbances obtained for the immoblhzed enzyme to pg of protein, then calculate the protein content m terms of pg of protein/ml of packed gel For example, if 0.05 mL of 1.1 (v/v) suspenston is found to contam 10 pg of protein, then the protein content of the gel IS 400 ug/mL of packed gel. From the chart recording, determine the change m absorbance/min and use the formulae below to calculate volume activity in U/mL and specific activity m terms of U/mg for immobilized enzyme. A unit is defined as the conversion of 1 pmol of substrate/mm under the assay condition described. Assay volume, 3 mL: extinction coefficient for NADH, 6.22 (tit/cm); change in absorbance/ min, 0.026; protein content of gel, 0.4 mg/mL of packed gel; protein concentration of diluted suspension, 0.01 mg/mL suspenston, sample volume, 0.05 mL of suspension Volume activity = (3 mL x 0.026)/(6.22 x 0 05 mL) = 0 25 U/mL Specific activity = (0.25 U/mL)/(O.Ol

mg/mL) = 25 U/mg

(1) (2)

9 It is important to prepare an appropriate control as described in Note 6, especially for the immobdlzed subunit and immobilized reassociated forms of the enzyme. The presence of DTE in the renaturmg buffer can generate high blank values unless it is exhaustively washed from the gels. Effective washing will reduce the background because of residual DTE to around lo%, which will not interfere with the results. 10 The &extinction coefficent for the product of the reaction between DTNB and throl groups is 13.6 mM-l/cm. A typical absorbance of 0.16 at 4 12 nm is obtained wtth 2 mL of munobtlized enzyme 1: 1 (v/v) suspension containing 0.5 mg/mL of packed gel. Thus, the thiol content would be 0.16/13.6 = 1 17 x 1O-2 mmol/L = 1 17 x 1t5 mmol/mL. The molecular weight of rabbit muscle creatme kmase is 82,000, so the protein concentration of the immobilized enzyme would be

Immobilized Enzyme Assays

259

0.5 mg/82,000 = 6.1 x lOA mmol/mL. Therefore, the number of thiol groups per enzyme molecule is (1.17 x 1t5)/(6.1 x 1V) = 1.9

References 1. Koelsch, R., Lasch, J , Marquardt, I , and Hanson, H. (1975) Application of spectrophotometric methods to the determmation of protein bound to agarose beads Anal Biochem. 66,556-567. 2. Lowry, 0. H., Roseburgh, N. J., Farr, A. L., and Randall, R. J. (195 1) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193,265-275 3. Golovina, T. O., Cheredmkova, T. V., Mevkh, A. T., and Nagradova, N. K. (1977) A convenient method for estimation of Sepharose-bound protein. Anal. Btochem 83,778-78 1. 4. Bickerstaff, G. F. and Price, N. C. (1978) Properties of matrix-bound dimer and monomer derivatives of immobilized creatine kinase from rabbit skeletal muscle. Blochem J. 173,85-93. 5. Rudge, J. and Bickerstaff, G. F. (1984) Thermal stability properties of immobtltzed creatine kinase Biochem. Sot Trans 12,3 11,3 12 6. Bickerstaff, G. F. and Zhou, H (1993) Protease activity and autodigestion (autolySIS) assays usmg Coomassie Blue dye bmdmg. Anal Bzochem. 210, 155-158 7. Mort, J S , Chong, D. K. K., and Chan, W. W.-C. (1973) Continuous spectrophotometric assay of Sepharose-bound enzyme and its use to study kinetics of coupling of the enzyme to Sepharose. Anal Biochem 52,162-l 68. 8. Ellman, G. L. (1959) Tissue sulphydryl groups. Arch Biochem. Btophys 82,70-77. 9. Pratt, M. L., Gold, J. C , and Roche, T. E. (1982) Sepharose-msolubihzatton of the dihydrolipoyl transacetylase core component of the pyruvate dehydrogenase complex* preparation and characterization J Biochem. Bzophys Meth 6, 119-l 3 1 10. Bickerstaff, G. F., Paterson, C., and Price, N. C. (1980) The refolding of denatured rabbit muscle creatine kinase. Biochtm. Bzophys. Acta 621,305-3 14.

30 Immobilization of Enzymes Acting on Macromolecular Substrates Reduction of Steric Problems Jose M. GuisBn, Guadalupe Penzol, Pilar Armisen, Agatha Bastida, Rosa M. Blanco, Roberto Fernandez-Lafuente, and Eduardo Garcia-Junceda 1. Introduction Substrates of small molecular size can reach the active site of unmobilized enzymes without suffermg significant steric hindrances generated by the sup-

port. The situation is very different when macromolecular substrates are used (Fig. 1). Some of these reactions are of great interest; for example, hydrolysis of starch by amylases, limited

proteolysis

catalyzed by proteases, and hydroly-

sis of RNA or DNA by nucleases. Immobilized proteins for affinity chromatography (protein A, antibodies, and so on) are a further example m which the interaction may involve a macromolecule (I). In this chapter, we present three different strategies to reduce the steric problems associated with immobilized enzymes: 1. Use of supports with different internal morphology; 2. Preparation of enzyme derivatives with different orientations molecule on the support; 3. Use of adequate spacer arms.

of the enzyme

The porous supports used for mnnobilization can be grouped into two mam classesaccording to their internal morphology. First are supports with internal morphology as void spaces surrounded by surfaces (e.g., cylindrical pores, fibrous solids, and so on). Examples of this type of support are porous glass, ceramics, agarose gels, cellulose, and so on. When used for immobilization of From

Methods in Botechnology, Vol I lmmobilrzabon of Enzymes end Cells EdIted by G F Bickerstaff Humana Press Inc., Totowa, NJ

261

Guish

262

et al.

SUBSTRATE

SUPPORT-ENZYME

Fig. 1. Steric problems in processescatalyzedby immobilized enzymes.When immobilized enzymesact on macromolecular substrates,the presenceof the support surface can strongly restrict the accessof thosesubstratesto the enzymeactive center.

enzyme, an orientation is produced in which the active site is faced to the support surface. This surface would promote a high steric hindrance for subsequent interaction of the immobilized enzyme with macromolecular substrates (Fig. 1). Agarose gels are used here to exemplify this type of steric hindrance (2,3). Second are supports with internal structure formed by networks of isolated linear macromolecular chains. In these supports, structure is maintained by very stable chemical crosslinking of different macromolecular chains. Examples of these supports are found in some of the new polymeric supports that are now commercially available, such as polyvinyl, polyacrylic, and polymethacrylate resins (Toyopearls [Tosohaas, Stuttgart, Germany], Eupergit [Riihn Pharma, Darmstadt, Germany], Biosynth [Riedel-de-Haen, Seelze,Germany], and so on). At first glance, steric hindrance arising from the presence of these supports should be minimal (Fig. 2). An example of this type of support is the polymer carrier VA-Epoxy (Biosynth). The method of activation can direct the orientation of an enzyme on a support through selective use of different areas of the enzyme surface (4,5). Thus, in some cases the enzyme-active site may be oriented opposite to the support surface, and be fairly accessible to macromolecular substrates (Fig. 3). In this chapter we provide strategies of immobilization that allow protein to be immobilized via three different types of residues: amino-terminal, lysine residues,

Reduction

of Steric

Problems

263

SUBSTRATE

SUPPORT-ENZYME

Fig. 2. Reduction of steric problems by using supports with adequate internal morphology composed by networks of isolated macromolecular chains.

and carboxylic groups (Fig. 4). These are the most frequent residues found on the surfaces of proteins and form the basis for the generality of the strategies outlined below. For immobilization of enzymes through their amino-terminal residues (6,,, it can be assumed that any s-amine of lysine residues of a protein exposed to the medium should have a pK value next to that of the free amino acid (10.5-I 0.7). However, the amino terminus of a protein has a lower pK value (around pH 7.0-8.0). Thus, the amino terminus at a mild pH (i.e., pH 8.0) will be more than 300-fold more reactive than the average lysine residue. Therefore, any immobilization method able to irreversibly immobilize a protein via a single amino group-support bond should direct the immobilization toward the surface area of the protein containing the residue with a lower pK value. In this way, most of the methods used for immobilization of enzymes via amino groups should produce this type of immobilization when the N-terminal is exposed to the medium. As an example of this methodology, we use the glutaraldehyde activation of supports as a method for irreversible immobilization of enzymes through a single amino-support link. Enzymes can be immobilized by utilizing surface areas containing a high density of amino groups (3). Glyoxyl supports that are weakly activated give poor immobilization yields, however, when using moderately or highly activated supports, and moderately high pH value (e.g., pH 10.0) enzymes are

264

Guisrin et al.

Fig. 3. Reduction of the steric problems by modulation of the final orientation of the immobilized enzyme. Use of differently activated supports involves different enzyme residues in the immobilization process, and by careful choice, enzymes are immobilized using residues that are located away from the active center.

immobilized quickly and apparently irreversibly on these supports. Initial binding occurs via formation of at least two point enzyme-support attachments. Hence, the driving force of immobilization is not the reactivity of a single amino group on the enzyme surface, but the high density of amino residues in a given area. Clearly, these areas have multiple possibilities to form more than one attachment. Immobilization of enzymes can be readily achieved through surface carboxy groups (7-9). Although carboxylic moieties are the most abundant groups on the surface of proteins, they have hardly been used for immobilization of enzymes, even though activation by carbodiimide to amino supports is a reliable method. However, the high pK value of most amino groups makes it very difficult to perform a mild immobilization protocol. We have developed a new amino-support, namely monoaminoethyl-N-aminoethyl (MANA) gels, with a layer of primary amine groups having low pK (around 7.0) that allows the use

-CH2-CH.$W

-CH2XH2f‘H

-6Uamldehyde

-Gktaddehyde

ENZYME

+

ENZYME

ENZYME

4

SUPPORT(aldehyde)-ENZYhllE(amine(t))

SUPPORT(amine)-ENZYME(carboxilic)

SUPPORT(aldehyde)-ENZYME(amine)

Fig. 4. Chemtstry of three different immobilizatton protocols involving different residues on the enzyme surface On glyoxyl-agarose gels, the enzyme will be immobtlized via an area rich in lys residues On MANA- agarose gels, the enzyme will be unmobtlized via an area rich in asp and glu residues. On the glutaraldehyde-agarose gels, the enzyme will be immobtlized by the N-terminal amme

GLUTARALDEHYDE-AGAROSE

-CH2CH2+H

-CH2CH24H

MANA-AGAROSE

GLYOXYL-AGAROSE

C~XHO

-CH2CH0

266

GuisAn et al.

SUBSTRATE

ARM

Fig. 5. Reduction of steric problems by using hydrophihc and inert spacer arms

of carbodlimide under optimal conditions (pH values between 5.0 and 6.0) using moderate concentrations of carbodiimide (around 10-100 mM). These three methods allow direct enzyme immobilization through different areas on protein surfaces. Another way to reduce steric hindrance is the use of a spacer arm to distance the enzyme from the support surface (Fig. 5). In our opinion, the effectiveness of this strategy depends on a number of key charactenstlcs. 1 The spacer arms must be long enough to promote effective separation of the enzyme from the support 2. The spacer arm should be very flexible to provide high mobility to the resulting immobilized enzyme and enable maximum interaction between the enzyme and the macromolecular substrate 3 A long spacer arm should be completely hydrophlhc and inert to prevent addltional interactions (hydrophobic, electrostatic, and so on) between the spacer arm and the enzyme. 4. The amount of spacer arm on the support should be very low (e.g., less than one spacer arm for each enzyme molecule to be unmobihzed). High levels of spacer arm on a support produce a spacer wall and create new sterlc hindrance

267

Reduction of Steric Problems

Dextrans partially oxidized by periodate seem to fulfill these requirements. They are commercially available in many different sizes, are easily activated, unstructured, and very flexible polymers (see Chapter 32). Reduction of these polyaldehyde polymers with sodtum borohydride, after reaction with the enzyme, produces very stable enzyme-dextran-support bonds, and converts the remaining aldehyde groups to inert and hydrophilic hydroxy groups (1Z).

2. Materials 2.1. Preparation

of Amine Agaruse

1. Prepare glyoxyl-agarose beads as described in Chapter 3 1, Section 3.1 2 Ethylenediamine (EDA) in water Place the preparation vessel (e.g., beaker) m an ice bath and adJust the solution pH to 10.0 by adding concentrated HCI very slowly to mamtain the temperature at 25°C. Correct the volume with water to get a final EDA concentration of 2M (see Note 1). 3. Sodium borohydrrde. 4. O.lM acetate buffer, pH 4.0, containing 1M NaCl. 5. O.lM borate buffer, pH 9 0, containing IM NaCl

2.2. Preparation

of Glutaraldehyde

Agarose

1 Prepare amine agarose beads as described in Sectton 3.1. 2. 25% Glutaraldehyde, commercial solution, 3. 200 mMphosphate buffer, pH 7.0.

2.3. Preparation

of Aldehyde

Biosynth

1. Btosynth (Polymer Carrier VA-Epoxy, Rtedel-de Ha&n, Germany): Swell 1 g of the commercial product m 100 mL water. Keep the suspension under mtld stirring overnight at room temperature. Avoid magnetic stirring 2. 1N sulfuric acid solution. 3 O.lM sodium periodate solution.

2.4. Preparation of Agarose with a Spacer Arm 2.4.7. Preparation of Low-Activated Amine Agarose (3 pmol/mL) 1. 2. 3. 4. 5. 6.

Agarose 4B CL O.lM sodium periodate solution. 2M ethylene diamine solution, 50 mM bicarbonate buffer, pH 10.0. Sodium borohydride. 50 m&I phosphate buffer, pH 7.0.

2.4.2. 50% Oxidation of Dextran M, 20,000 1, Dextran Mr 20,000, commercially 2. Sohd sodium periodate.

available.

Guish

268

et al.

2.4.3. Attachment of Dextran to Amine Agarose 1 2. 3 4. 5.

0. 1M phosphate buffer, pH 7 0 Trimethyl aminoborane (TMAB) Amme agarose (3 ymol/mL) prepared as described in Section 3 4 1 Partially oxidized dextran prepared as described in Section 3.4.2. Solid sodium borohydride.

2.4.4. Activation of Immobilized Dextran 1. O.lM sodium pertodate solution. 2 Dextran immoblhzed on amine agarose (3 umol/mL) Section 3.4.3 3. 10% potassium iodide solutton 4. Saturated

sodium

2.5. Immobilization 1. 2. 3. 4. 5. 6

solutton.

of Enzymes on Amino Supports

Amme agarose beads prepared as described in Section 3.1. 4 mg/mL enzyme solution in appropriate buffer for the enzyme. Solid 1-ethyl-3-(3-drmethylammopropyl)carbodiimtde (EDC). O.lM bicarbonate buffer, pH 9.0, containing 1M NaCI. O.lM acetate buffer, pH 5.0, containing 1M NaCl. OSM hydroxylamine, pH 7 0.

2.6. Immobilization 1. 2. 3. 4 5

bicarbonate

prepared as described in

of Enzymes on Glutaraldehyde

Supports

4 mg/mL of enzyme solution m 50 Wphosphate buffer, pH 8.6. Glutaraldehyde agarose prepared as described m Section 3.2 50 mM bicarbonate buffer, pH 10.0. Solid sodium borohydride. 50 mA4phosphate buffer, pH 7.0.

3. Methods 3.7. Preparation

of Amine Agarose Amine agarose IS available commercially (Hispanagar S.A., Burgos, Spain) or it can be prepared as described below. 1. 2 3. 4. 5.

Suspend 10 mL of glyoxyl-agarose in 40 mL of 2M ethylenedramme, pH 10.0. Stir this suspension for 2 h at room temperature. Add 400 mg of sohd sodium borohydride. Keep the suspension under mild sturing for 2 h at room temperature. Filter and wash the gel with the acetate/NaCl buffer The procedure for filtermg the gel IS always vaccum filtration using a Buchner flask with smtered-glass filter funnel 6 Filter and wash the gel with the borate/NaCl buffer. 7. Filter and wash the gel with distilled water.

Reduction of Steric Problems

269

8. Filter the support to dryness. This involves removal of water between the agarose beads so they do not stick together, but not removal of water from the internal porous structure of the gel.

3.2. Preparation of Glutaraldehyde Agarose 1 Suspend 10 mL of the amine agarose in 20 mL of 15% glutaraldehyde-200 phosphate buffer, pH 7.0, and adjust the pH to 7.0. 2. Keep the suspension under mild stirring for 15 h at 25°C. 3. Filter and wash the gel exhaustively with the phosphate buffer. 4. Wash the gel thoroughly with distilled water and then filter it to dryness.

mM

3.3. Preparation of AIdehyde Biosynth 3.3.1. Acid Hydrolysis of the Epoxide Groups 1. Wash the Biosynth support extensively with distilled water. 2. Suspend 100 mL of wet support in 900 mL of water and adjust the pH to 2.0 with sulfuric acid. 3. Incubate this suspension at 40°C for 14 h. 4. Wash the glycol support thoroughly with distilled water.

3.3.2. Oxidation of the Glycol Groups 1. Suspend 100 mL of the glycol-Biosynth m 900 mL of distilled water. 2. Add to this suspension 130 mL of 0. Mperiodate solution (this support contains 130 pmol epoxide/mL of support). 3. Keep the suspension under mild stu-rmg for 90 mm. 4. Wash the support thoroughly with distilled water and filter to dryness.

3.4. Preparation of Agarose with Spacer Arm 3.4.1. Preparation of Low-Activated Amine Agarose (3 pmol/mL) 1 2. 3. 4. 5. 6. 7. 8. 9 10. 11. 12. 13.

Suspend 10 mL of the agarose 4B CL in 88.3 mL of distilled water (see Note 2) Add 1.7 mL of 0.M sodium periodate solution. Stir this suspension gently for 90 min at room temperature. Wash the gel thoroughly with distilled water and filter it to dryness. Suspend 10 mL of the gel in 90 mL of bicarbonate buffer, pH 10.0 Add 100 mg of solid sodium borohydride and stir the suspension for 30 mm at room temperature. Wash the gel with phosphate buffer, pH 7.0. Wash the gel thoroughly with distilled water and then filter it to dryness. Suspend 10 mL of this 3 nmol/mL glyoxyl-agarose (obtained in step 8) in 89.7 mL of distilled water. Add 0.3 mL of 0.M sodium periodate solution. Stir the mixture for 90 mm at room temperature. Wash the gel thoroughly with distilled water and filter it to dryness. Activate this glyoxyl-agarose to amme agarose as described m Section 3.1.

GuisAn et al.

270 3.4.2. 50% Oxidation of the Dextran 20,000 (see Note 3) 1. 2. 3. 4

Dissolve 2 5 g of dextran m 35 5 mL of distilled water Add 3 g of sodmm periodate (see Note 4) Strr the mixture at room temperature for 3 h. Transfer the mixture to dialysis tubing and dialyze this solutton against water at room temperature. Dialyze against 5 L of water with four changes Change the water every 3 h, and rf one of the changes 1sovernight, then carry out that step m the cold room at 4°C.

3 4.3. Attachment of Dextran to Amine Agarose 1 Dissolve 984 mg of trimethyl aminoborane m 30 mL of phosphate buffer, pH 7.0 (to produce 150 nnI4 TMAB) 2 To this solutron add 30 mL of the partially oxidized dextran from Section 3 5 2. 3 Suspend 30 mL of the amme agarose gel (3 pmol/mL) m this solutron 4. Stir the mixture for 2 h at 25°C. 5 Adjust the pH to 10 0. 6 Add 450 mg of sodmm borohydride (5 mg/mL of suspension) 7. Wash the support with phosphate buffer, pH 7.0. 8. Wash the support thoroughly wtth water and filter tt to dryness

3.4.4. Activation of Immobilized Dextran 3.4.4.1.

TITRATION OF THE REMAINING GLYCOLS IN THE AGAROSE-DEXTRAN (SEE NOTE 5)

1 Take a 1 mL ahquot of agarose-dextran and suspend it in 9 mL of dtstilled water Add 1 mL 0 1M sodium perrodate solution to this suspension (correspondmg to 100 pmol perrodate/mL support). 2 Take a 1 mL ahquot of amine agarose (3 pmol/mL), suspend it in 9 mL of drstilled water and add 1 mL of O.lM sodium periodate solution to this suspension (control) 3. At time zero and then at periodic times, withdraw 0.1 mL aliquots of the supernatant of both suspensions (for example, every 15 mm). A typical reaction period would be 60-90 mm 4. Prepare a blank solution of sodium pertodate in water in the same proportion as the suspensions. 5. Spectrophotometrrc assays: a Fill a cuvet with 3 mL of a solution of 1: 1 (v/v) potassium iodide and saturated sodmm bicarbonate (prepare fresh) Add 0.2 mL blank and measure the absorbance at 4 19 nm. The reading corresponds to 0% perrodate consumed b Add 0.2 mL of the supernatant of the control suspension to the cuvet contammg 3 mL solution 3 and measure the absorbance at 4 19 mn Repeat the assay wtth ahquots withdrawn at different times. Compare the readings with the ones obtained m step a. to calculate the perrodate consumed by the amme agarose support (X).

Reduction of Steric Problems

271

c. Add 0.2 mL supernatant of the suspension of agarose-dextran on the cuvet containing 3 mL solutron 3 and measure the absorbance at 419 nm Repeat the assay with ahquots withdrawn at different times. Compare the readings with the ones obtained in step a to calculate the perrodate consumed by the amine agarose plus the dextran (Y). d. When the value of Y remains constant, then (Y - X) = periodate consumed only by the dextran This value corresponds to the glycol groups remaining m the partially oxrdrzedlreduced dextran attached to the agarose. 3.4.4.2. OXIDATION OF THE AGAROSE-DEXTRAN (GLYCOL) 1. Assuming that the glycol concentration (Y-X) is 20 umol/mL support, suspend 10 mL of the support in 88 mL of distilled water. 2. Add 2 mL of 0.M sodium periodate (correspondmg to 20 umol/mL). 3 Stir for 90 min at room temperature. 4. Wash the support thoroughly with distilled water and filter it to dryness (see Note 6).

3.5. Immobilization

on Amino Supports

1. Assay the catalytic activity of the enzyme solution in acetate buffer, pH 5 0 2. Suspend 8 mL of amme agarose in 72 mL of the 4 mg/mL enzyme solution and adjust the pH to 5.0 (see Note 6). 3. Keep the suspension under mild stirring at room temperature 4. Withdraw aliquots from suspension and supematant and assay their catalytic activities at different times. 5. Confirm that all the enzyme has been immobilized ionically when the activity remains in the suspension and there is no activity left m the supernatant. 6. Add 153 mg of solid EDC (10 rnM, see Note 7). Add the same to the control suspension. 7. Check that the pH is 5.0. 8. Keep the suspension under mild stirring for 90 mm at room temperature 9. Filter the gel without washing it. 10. Suspend the gel in bicarbonate-NaCl buffer, pH 9.0, and measure catalytic activity of the supematant (see Note 8). 11. Once the catalytic activity of the supematant is constant, filter and wash the derivative thoroughly with bicarbonate-NaCl, buffer pH 9.0. 12. Filter and wash the derivative with acetate-NaCl buffer, pH 5.0. 13. Filter and wash the derivative thoroughly with distilled water and then filter it to dryness. 14. Suspend 8 mL of the derivative in 72 mL of hydroxylamme solutron and stir at room temperature for 4 h (see Note 9). 15. Wash the gel thoroughly with distilled water and then filter to dryness.

3.6. Immobilization

on Glutaraldehyde Supports

1. Prepare 20 mL of 4 mg/mL of enzymesolution in phosphatebuffer, pH 8.6, and assayits catalytic actrvrty using the appropriate assay procedure.

272

Guisin et al.

2. 3. 4 5.

Suspend 1 mL of glutaraldehyde agarose in the 20 mL of enzyme solution. Adjust the pH to 8.6. Stir the suspension at 4°C for 2 h. At different times (e.g., every 15 mm) withdraw 0.1 mL aliquots from the suspenston and the supematant and assay for catalytic activities. The frequency of samplmg must be established for each immobtlizatton. Vacuum filter the derivative and resuspend it in 9 mL of bicarbonate buffer, pH 10.0, containing 10 mg of sodium borohydrtde. Stir the mixture for 30 mm at room temperature. Filter and wash the derivative with phosphate buffer, pH 7 0. Wash the derivative thoroughly with distilled water and then filter tt to dryness.

6. 7 8 9

3.7. Activity of Different Rennin Derivatives and Macromolecular Substrates

on Small

Prepare derivatives of the enzyme rennm from A4ucor mzehei according the strategies described m this chapter.

to

1 Agarose support: Use glyoxyl, amino, and glutaraldehyde agarose to unmobthze rennm with different orientations 2 Agarose dextran support: Prepare 75% oxidized dextran A4, 10,000 attached to amine agarose (20 pmol/mL). Reduce the support and oxidize the remaining 25% glycols of the dextran to aldehyde prior to enzyme mrmobilization. Use this derivative to test the effect of the spacer arm 3. Immobiltze rennin on Btosynth aldehyde to test the influence of the internal morphology of the support. 4 Assay the activities of all these derivatives using a small synthetic substrate, such as the hexapeptide leu-ser-phe(N02)-ile-ala-1euOMe (assayed spectrophotomettcally at 3 10 run), and K-casem, the natural macromolecular substrate of rennin. Table 1 shows the activittes of the derivatives of rennin fomM miehez (1 l-l 3). 5. There is hardly any effect of the immobilization method when the synthetic substrate is assayed. However, the activities with macromolecular substrate are clearly dependent on the procedure used for immobilization. The effect of different orientations of the enzyme on the three different agarose supports is seen by the poor activity on glyoxyl-agarose, slightly improved actlvtty on amine agarose, and significantly improved activity on glutaraldehyde agarose. 6. Orientation of the enzyme through amines from lys residues causes the largest stertc hindrances to the accestbtlrty of substrate. However, with this same ortentation, stertc hindrance was reduced by the use of spacer arms, and also by the use of supports having a network structure. In these cases, the activmes were close to that obtained with the optimal orientation of the enzyme on agarose (glutaraldehyde agarose as support).

4. Notes 1. Aliphattc amines are oxidizable at high temperatures, yielding yellow mtrocompounds. Hence, adJustment of pH of this solution should be made very care-

Reduction of Steric Problems

273

Table 1 Activity of Different Immobilized Derivatives of Rennin from Mucor miehei Assayed Using a Small Synthetic Substrate and Macromolecular Substrate Casein Activity support Aldehyde-agarose Amine-agarose Glutaraldehyde-agarose Agarose-dextran Biosynth aldehyde

Hexapeptidea

Casemb

100 92 100 100 100

2.5 4.75 33.3 25 25

aThe actiwties are referred to by the actwity of soluble enzyme (100%) hThe activities are referred to by the time of clotting measured wtth soluble enzyme (rate of clottmg 100%)

2.

3.

4.

5.

fully by stepwise addition of aliquots of concentrated acid in an ice bath m order to maintain the temperature of the solution below 25°C. Crosslinkmg of 4% agarose (4B CL) produces 20 pmol of glycols/mL of gel. To obtain a gel with 3 pmol/mL rt IS necessary to block 17 of them by oxtdation to aldehyde and to reduce them to hydroxyl. Otherwise, these remaining glycols could give rise to undesirable reactions in further oxidation steps In a first step, the spacer arm must be linked to the support. To achieve this, the dextran must be partially oxidized and the resulting aldehyde groups reacted with the amines from the support The reduction yields irreversible agarose-spacer arm bonds and it also reduces all the remaining glyoxyls of the polymer to inert hydroxyl groups. The glycol groups remaining in the dextran can now be oxidized and transformed to new glyoxyl groups on which the enzyme can react through its amines. The reaction with periodate is stoichiometric. The concentration of periodate given in the methods is that required for 50% oxidation of dextran, and corresponds to one periodate molecule per glucose molecule, i.e., per two glycol groups The process of attachment of partially oxidized dextran to amine agarose is not quantitative because not all the molecules of the polymer offered to the gel are linked. These molecules remam in the supernatant and are eliminated during the succesive washing and filtration steps. We had previously oxidized 50% of the total glycols of the dextran to lmk the polymer to agarose and reduced all the remaining aldehydes. There are another 50% of the glycol groups available to be oxidtzed again to enable linkage of the enzyme. The most accurate way to calculate the total amount of glycols is to titrate the consumption of periodate of this dextran agarose support. Amine agarose supports can also consume periodate, so it is also neccesary to perform a control titration of periodate consumption of the amine agarose gel.

274

Guisdn et al.

6. The nnmobillzation takes place ionically and has to be performed at a pH value at which both the ammo groups from the support and the carboxyl groups from the enzyme are ionized. Most enzymes can be linked to these low pK amine supports at pH 5.0 and 6.0, although the optimal value must be studied for each particular enzyme 7. EDC is a strong modifying agent of carboxyl groups. Although 10 mM and 90 mm are typical conditions, the concentration and reaction time must be studied carefully for each particular enzyme. Thus, the conditions must be established to prevent extensive reaction that could produce modifications leading to enzyme inactivation 8 After the reaction with EDC, all the molecules of the enzyme might not be attached covalently to the support, By suspenston of the derivative m alkaline pH value and high ionic strength, molecules adsorbed only ionically will desorb from the support The suspension in these conditions will ensure that the activity of the derivative is derived only from covalently attached enzyme molecules. 9. EDC may produce side reactions with the hydroxyl group of tyrosmes and thiol groups from cysteines. The reaction with hydroxylamine may be necessary to eliminate these possible undesirable modifications of the enzyme.

References 1. Hermanson, G. T., Malha, A. K., and Smith, P. (1992) Zmmobillzed Af$mty Llgund Techniques. Academic, San Diego, CA. 2. Carlson, A , Hill, G. C , and Olson, N F (1986) The coagulation of milk with munobilized enzymes. a critical review. Enzyme Mxrob. Technol 8,642-650. 3. Guisan, J. M. (1988) Aldehyde-agarose gels as activated supports for immobilization-stabilization of enzymes. Enzyme Microb. Technol. 10, 375-382. 4. Rodriguez, V. (1995) Estabilizacion de enzimas de estructura compleja: biotransformation enzimattca de antibioticos p-lactamicos. Doctoral Thesis, Umversidad Complutense de Madrid. 5. Penzol, G. (1992) Inmovilizacion de proteinas industriales que actuan sobre sustratos macromoleculares. Hidrolisis de caseina por derivados de renina Doctoral Thesis, Universidad Complutense de Madrid. 6. Monsan, P. (1977) Optimization of glutaraldehyde activation of a support for enzyme immobihzation. J. Mol Cut 3,371-384 7. Femindez-Lafuente, R., Rosell, C., Rodriguez,V., Santana, M. C., Soler, G., Bastida, A., and Guisan, J M. (1993) Preparation of activated supports contammg low pK amino groups. A new tool for protein immobilization via the carboxyl coupling method. Enzyme Microb. Technol l&546-550. 8. Timkovich, R. (1977) Polymerization side reactions during protein modifications with carbodiimide Blochem Biophys Res Commun 74,1463-1468. 9. Carraway, K L. and Koshland, D. E. (1972) Carbodiimide modification of proteins. Meth. Enzymol 25,616-650 10. Schacht, E.H (1987) Modification of dextran and application m prodrug design, m Industrial Polysaccharfdes Engineering. Genetic Engineering, Structure/Property Relations and Applications (Yalpam, M., ed.), Elsevier, Amsterdam.

Reduction of Steric Problems

275

11. Marlin, P., Raymond, M. N., Bricas, E., and Ribadeau, D. B. (1980) Kinetic studies on the action ofMucorpusillus, Mucor mihei acid proteases and chymosins A and B on a synthetic chromophoric hexapepttde. Bzochim. Biophys. Acta 612,41&420. 12. Raymond, M. N., Gamier, J., and Bricas, E. (1972) Studies on the specificity of chymosin (rennin) Biochlmle 54, 145-154. 13. Mashaly, R. I , Saad, M. H., El-Abassy, F., and Wahban, A. A (1988) Coagulation of milk by calf rennet, pepsin and Mucor miehez rennet immobihzed on large agarose beads. Milchwzssenschaft 43,79-82.

31 Immobilization

of Enzymes on Glyoxyl Agarose

Strategies for Enzyme Stabilization by Multipoint Attachment Jose M. Guistin, Agatha Bastida, Rosa M. Blanco, Roberto FernBndez-Lafuente, and Eduardo Garcia-Junceda 1. Introduction Immobilization of enzymeson pre-existing supports containing small aliphatic aldehyde groups (e.g., glyoxyl-agarose) presents a set of interesting features from both a basic and a more practical point of view. The chemistry of this tmmobihzation method is quite different from more popular methods involving glutaraldehyde and the three principal steps (activation of the support, enzyme immobilization, and end-point of immobilization including blocking remaining active groups) are outlined in Fig. 1 (I). Based on this chemistry and on the reactivity/stability properties of the groups involved there are several special and interesting features regarding the nature and stability of the enzyme support linkages, the absence or presence of additronal noncovalent enzyme support interactrons after immobilization, the opportunites for multipoint enzyme support attachments, and the potential for very high loading of enzyme biocatalyst. Glyoxyl groups present an almost unique feature in being very stable and at the same time highly reactive for enzyme immobilization. These properties allow immobilization of a great amount of enzyme per unit volume of support, e.g., it is easy to perform very long immobilizations (without support deacttvation) in order to obtain a monolayer of enzyme that completely covers the inner support surface. Thus, it is possible to immobilize up to 40 mg of enzyme on 6% agarose gels and 100 mg on 10% agarose gels. The stability of the glyoxyl groups strongly enhances the range of practical applications possible for such supports. The concentration of glyoxyl groups on the activated support remains constant after storage for 1 yr at 4°C. From

Methods m Botechnology, Vol 1 Immobi~zahon of Enzymes and Cells Edlted by G F Buckerstaff Humana Press Inc , Totowa, NJ

277

d

MO,

-CH2-C-H

9

THE

-CH2-CH2*

OF

SUPPORT

2 2-H

9

sWPORT($yorybENZfME(amme)

-CH,-CH,-CH

GLYOXYL-*a4RosE

-CH

-CH*X-b

-CH2--C-!-l

-Cn,-CH=NH

9

Fig. 1. Support activation with glyoxyl groups and chemistry of the m-unobillzation process

lMMOBlLRATlON

lMMOBlLlZATlON

END POINT

ENZYME

GLYCERYLdGAROSE

Cl-$-CHCHC~OH

-CH2 -CHOH-cH$)H

ACTNATION

Enzyme Stabilization on Glyoxyl Agarose

279

The acttvation of supports with glyoxyl groups is not only restrrcted to agarose. These groups can also be generated on most of the commercial supports, including both organic (actrvatmg hydroxyl or ammo groups) and inorganic supports (by using glycidoxy propyl trimethoxylsilane reagents). After immobilization, chemical modification of the enzyme is mimmal. On one hand, activation of the enzyme for coupling IS unnecessary and on the other hand, the primary amino groups of the enzyme involved in the immobihzation are only transformed into secondary ammo groups with very similar properties (chemical structure, hydrophilicity, pK value, and so on). The immobilization of enzyme can be performed in different ways, each of which will yield a drfferent enzyme orientation on the support. At pH values close to neutral, immobilization in the presence of mild reducing agents (aminoboranes, cyanoborohydrrde) promotes, in very early stages, irreversible one-point immobrlization involving the surface ammo groups having the lowest pK value (e.g., the amino terminal residues). At alkaline pH values, rltltnobilization can be performed m the absence of reducmg agents (J-3). As has been described in Chapter 30, reversible immobilization produced by this method only becomes an irreversible one when at least a two-point immobilization is performed.

In this way the enzyme becomes immobilized

through its

surface area having the highest density of lysme groups. In both cases,mild borohydride reduction of the immobilized derivatives as a final step provides some useful advantages (3). The enzyme-support linkages become extremely stable (secondary amine groups), and remaining active groups on the support are converted into inert and hydrophilic hydroxy groups with no promotion

of additional

noncovalent

interactions

between the enzyme

and the support surface. Thus, the properties of the derivatives are related only to the intrinsic

properties

of the immobilized

enzyme.

This immobilization method also presents excellent prospects for development of additional strategies for enzyme stabilization through multipoint covalent mnnobihzation (Fig. 2) that can be summartzed as follows: 1. Selection of an adequatesupport (agaroseand some morganrc commercral resins) may allow a good geometrical congruence between the enzyme and the support surface (1,4,.5) 2. The method enables production of very highly activated supports. 3. Immobilization at alkaline pH values orientates the enzyme so that immobihzation takes place via an area of the protein containing a high density in amino groups.

4. The absenceof steric hindrancesstrongly facilitates additional multipomt attachment between the already immobilized enzyme and the activated support, 5. The gross structural modification of the enzyme is still very small, even when a great number of support-enzyme bonds have been established (Fig. 3)

H2 \H2N HP HzN c Gu&in et al.

280

MINIMAL

CHEMICAL

A NUMBER HYDROPHILIC

MODlFlCATlON

OF VERYSTABLE AND COMPLETELY

ff LINKAGES INERT

THE

ENZYME

ATASHORT SUPPORT

DISTANCE

FROM

THE SUPPORT

SURFACE

Fig. 2. Two-step process of enzyme stabilization through multtpomt attachment.

6 The method enables very long multt-interactions to occur between enzymes and highly activated supports because of the high stabthty of glyoxyl groups

These useful characteristics have been utilized in the preparation of highly stable derivatives of a number of industrial enzymes with almost full retentton of catalytic activity (Z-3,6-12, see Table 1). A more complete discussion of these strategies for stabihzatron of enzymes via multtpoint covalent attachment on glyoxyl-agarose is given elsewhere (12). 2. Materials 2.1. Activation of Agarose 2.7.7. Activation of Agarose Gel to Glyceryl-Agarose 1 Agarose 6BCL: Wash commercial agarose thoroughly with distilled water to remove any preservative and filter it to dryness, weight 70 g. (see Note 1) Avoid magnetic stirring of agarose, especially during long reaction times.

Enzyme Stabihzation on Glyoxyl Agarose

281

1

RAPID

CH

=N-

IMMOBILIZATION

-.

Fig. 3. Stablhzation support.

. -slnw

of enzyme by multipoint

covalent attachment on an inert

2. Sodmm borohydrtde. 3. 1.7N NaOH solution. 4. Glycidol(2,3 epoxy propanol): Store between 0 and 5°C (toxic).

2. I .2. Oxidation of Glyceryl-Agarose

to Glyoxyl-Agarose

2.1.2.1. To PREPARE5 ~MOL ALDEHYDEIMLOF GEL (SEE NOTE 2) 1. Glyceryl-agarose prepared as decnbed in Sectron 3.1.1. 2. 0.M sodium pertodate solution. 3. Distilled water. 2.1.2.2. To PREPARE75 ~MOL ALDEHYDE/MLOF GEL (SEE NOTE 3) 1, Glyceryl-agarose prepared as decribed in Section 3.1.1. 2. 0 1M sodium periodate solution 3, Distilled water 2.1.2.3. DEGREES OF ACTIVATION BETWEEN 5 AND 75 ~MOL/ML 1, O.lM sodmm pertodate solution.

GuisAn et al.

282 Table 1 Immobilization-Stabilization Multipoint Attachment

of Enzymes by Covalent on Glyoxyl-Agarose

Enzyme

Activity, %

Stabilization

65 65 65 80 65 90 60 60

8000 1000 100,000 1000 1000 20,000 100 12,000

Pemcillm G acylase FNRa Chymotrypsm P-Galactostdase Esterase n-ammo acid oxidase Thermolysm Trypsinb

“FNR, Ferredoxm NADP reductase bPrepared m the absence of mhlbltor. Activity IS wrth reference to soluble enzyme activity. Stablhzahon IS with reference to soluble enzyme under similar condltlons

2 Calculation N pmol/mL

of volume of 0 1M sodium periodate for a degree of acttvatton of

100 mL agarose x N pmol/mL = X total umol X/l 00 mM sodmm periodate = Y mL 0. 1M sodium periodate

(1) (2)

3 Ratio gel:suspension (v:v) = 1 10 1000 mL = total volume of the suspension 1000 - (100 mL agarose + Y mL sodmm periodate) = Z mL distilled water

(3) (4)

4. Glyoxyl-agarose gels are very stable when stored at 4°C as wet gels (filtered to dryness). The support is fully preserved m these conditions for more than 1 yr. The gels are filtered to dryness, which requires vaccum filtration to remove the water between the agarose beads so that they do not stick to each other, but not removal of the water inside the porous structure of the gel.

2.1.2.4. MONITORINGANDCONTROLOF THEOXIDATION 1. Solution 1: Prepare a solutton of sodium periodate m water m the same proportion as the suspension to be tested. This solution will be used as a blank. 2. Solution 2: Prepare a solution of 1: 1 (v:v) 10% potassium iodide and saturated sodium bicarbonate This must be freshly prepared.

2.2. lmmobiliza tion of Enzyme 1. 50 mM bicarbonate buffer, pH 10.0. 2. 90 mL enzyme solution m bicarbonate buffer (see Section 3.3.). 3. If the presence of competitive mhrbitor of the enzyme is reqmred, dissolve the mhtbttor m the enzyme solutton of step 2 (see Note 4 and Section 3.3.).

Enzyme Stabilization on Glyoxyl Agarose

283

4. 10 mL glyoxyl-agarose (wetgh 7 g of gel previously filtered to dryness). Either glyoxyl-agarose 5 pmol/mL or glyoxyl-agarose 75 pmol/mL. 5. Solid sodium borohydride 100 mg (1 mg/mL suspension). 6. 25 mM sodium phosphate buffer, pH 7.0.

2.3. Preparation of a Multipoint Derivative of Trypsin-Agarose Containing 40 mg of Trypsin/mL of Support 1. 2. 3. 4.

Trypsin (Sigma, St. LOUIS, MO). Benzamrdine (Sigma). Sodium borohydride. 25 mM sodium phosphate buffer, pH 7.0.

3. Methods 3.1. Activation of Agarose 3.7.7. Activation of Agarose Gel to Glyceryl-Agarose 1. Suspend 100 mL of agarose 6BCL m distilled water up to a total volume of 120 mL at room temperature. 2. Add to this suspension 34 mL of 1.7N NaOH solution containing 0.95 g of solid sodium borohydride.

3. Take the vessel to an ice bath, keep the suspension gently stirred, and add 6.7 mL of glycidol, dropwise, very slowly to prevent the temperature rising over 25% 4. Stir the suspension overnight (18 h) at 25°C. 5. After this time, wash the gel thoroughly by adding several vol of distilled water and vacuum filter after each addition. Use a Btichner flask with glass-sintered funnel connected to a vaccum line for filtration. 6 After the last addition of water, exhaustrvely vacuum filter the gel to remove water between the particles (see Section 2.1.1.3., step 4).

3.1.2. Oxidation of Glyceryl-Agarose

to Glyoxyl-Agarose

3.1.2.1. To PREPARE 5 ~MOL ALDEHYDE/ML GEL (SEE NOTE 2) 1. Suspend 100 mL (70 g) of glyceryl-agarose, obtained as described in Sectron 3.1,1., in 895 mL of distilled water. 2. Add 5 mL of O.lMsodium pertodate slowly to this suspension while stirring. 3. Keep the suspension under mild stirring for 1 h. 4. Wash the gel thoroughly with distrlled water and filter it to dryness as described above (Section 3 1.1.) steps 5,6). 3.1.2.2. To PREPARE 75 ~MOL ALDEHYDE/ML GEL 1. Suspend 100 mL (70 g) of glyceryl-agarose, obtained as described m Sectton 3.1.1,) in 825 mL of distilled water. 2. Add 175 mL of 0. 1M sodium periodate slowly to this suspension while stirring. 3. Keep the suspension under mild stirring for 90 min. 4. Wash the gel thoroughly with drstilled water and filter it to dryness.

Guistin et al.

284

3.1.2.3. DEGREES OF ACTIVATION BETWEEN 5 AND 75 ~MOL/ML (SEE NOTE 3) 1. Suspend 100 mL of glyceryl-agarose m Z mL of distilled water. 2. Add Y mL of sodmm periodate slowly while stn-rmg. 3. Follow steps 3 and 4 in Section 3.1.2.1. 3.1.2.4. MONITORING AND CONTROL OF THE OXIDATION 1. Prepare the blank solution, solution 1. 2 Prepare solution 2 3. Spectrophotometrrc assay. Fill the reference cuvet with 3 mL of solutton 2. Fill the sample cuvet with 3 mL of solution 2 and add 0.2 mL of blank (solution 1) Measure the absorbance at 419 nm. 4 Prepare a reference cuvet containmg 3 mL of solutton 2. In the sample cuvet, place 3 mL of solutton 2 Then add 0.2 mL of supernatant of the suspension to be tested. Compare the absorbance at 4 19 nm with the one obtained m step 3. 5 Repeat the assay with ahquots of supernatant withdrawn at different times until the reading is zero (all periodate has been consumed) or constant (no more periodate can be consumed because all groups have already been oxidized).

3.2. Immobilization

of Enzyme

1. Prepare the enzyme solutton (with or without competitive inhibitor) and test the catalytic activity (see Section 3.3.). 2. Control: Add 1 mL of dlsttlled water to 9 mL enzyme solution (see Section 3.3.) with or without mhlbrtor (as m the mnnoblhzatton mixture). 3. Add 10 mL of activated gel (glyoxyl-agarose 5 or 75 pmol/mL) to 90 mL of enzyme solution. Check the pH value again and correct it if necessary by adding diluted NaOH or HCl solution. 4 Keep the suspension under mild stirring at 25°C; check and maintain the pH at 10.0. 5. At different time intervals (e.g., 15 mm) withdraw 0 1-mL aliquots of supernatant and suspension for assay of enzyme activity. The time intervals need to be determined experimentally. 6. Assay the activity of the control solution using the same time intervals and ahquot volumes as m step 5 (see Note 5). For glyoxyl-agarose 5 pmol/mL the munobilization process terminates when the acttvtty of the supernatant remains constant. For glyoxyl-agarose 75 pmol/mL the immobrhzation process terminates when the activity of the supernatant is zero (see Note 6).

3.2.7. Multi-Interaction Ignore this multi-interaction stepif the degreeof agaroseactwation is 5 pmol/mL (see Note 6). 1. Prepare a suspension of immobthzed enzyme on agarose with a degree of acttvanon of 15 pmol/mL or more. Keep the suspension under mild strirrmg (avoid magnetic stming) for several hours (see Note 7).

285

Enzyme Stabilization on Glyoxyl Agarose

2. If a competitive inhibitor of the enzyme has been added to the enzyme solution, then filter the derivative to remove the inhibitor and resuspend it in 90 mL of 50 mM bicarbonate solution, pH 10.0.

3.2.2. Reduction 1. To a 100 mL suspension of the immobtlized enzyme at pH 10 0, add 100 mg of solid sodium borohydride 2 Stir the suspension for 30 min at room temperature. 3. Wash the suspension with 25 mM sodium phosphate, pH 7.0, while vacuum liltering to eliminate the borohydride. 4. Wash the suspension thoroughly with distilled water and filter to dryness

3.3. Preparation of a Multipoint Derivative Containing 40 mg Trypsin/mL of Support

of Trypsin-Agarose

1 Dissolve 400 mg of solid trypsm in 90 mL of 50 rnMsodmm bicarbonate, pH 10.0. 2. Add to this solutton a competitive inhibitor of trypsm, such as benzamidine, to a final concentration of 3 n&f. 3. Assay the catalytic activity of this solution. Add 10 mL of glyoxyl-agarose activated to 75 pmol aldehyde/mL gel and assay the activity of both suspension and supernatant after 5 mm whtle sttrring. If any activity remains in the supernatant, stir the suspension for 5 more minutes, then repeat the assays. 4. Stir this suspension very gently for 72 h. Avoid magnetic sturing to reduce abrasion of the agarose particles. 5. Add 100 mg of sodium borohydride and allow it react for 30 mm under stnrmg Then wash and filter the suspension with 25 mM phosphate buffer, pH 7.0, and then water as described above. Filter it to dryness (see Note 8).

4. Notes 1. Glyoxyl-agarose is commercially available at different degrees of activation (Hispanagar S. A., Burgos, Spain). The activation method described here was performed with agarose 6BCL kindly donated by Hispanagar. The density of wet gel is 0.7 g/mL. 2. 6BCL agarose gels already possess 5 pmol/mL of glyceryl groups remaimng from the crosslmking reaction. Thus, to obtain this density of active groups, the reaction with glycidol is not necessary if these crosslinked gels are used. 3. Oxidation of glycols with sodium periodate is a stoichiometric reaction. Thus, the degree of activation of agarose can be easily controlled through the concentration of periodate used. Typically, two densities of aldehyde groups/ml of support are used, 5 or 15 pmol/mL to obtain one-point attachment enzyme derivatives and 75 pmol/mL to obtam multipomt derivatives. The procedure to obtam any other activation degree is described in Sections 2.1.2.3. and 3.1.2.3. 4. Competitive inhibitor of the enzyme may be required to avoid enzyme inactivation (for instance in the case of proteases, the inhibitor prevents autolysis), or to preserve the structure of the enzyme

Guisdn et al

286 loom-*

.-e-e-

-

_----

Suspension

80

20

0

0.1

0,3

0.5

9

Time

Fig. 4. Immobilization-stabilizatton tive mhibitor

50

100

(hours)

course of trypsm in the presence of a competi-

5. If the enzyme acttvity decreases resulting from mactivation of enzyme this effect can be distmguished from loss in activity of the supernatant resulting from immobilization. 6 For any activation of agarose over 5-15 pmol/mL the supernatant should show no activity indicating that there is more than one bond linkage between the enzyme and the support. If the attachment is made through one bond only, then all of the enzyme will not be linked to support. The immobilizatton step fimshes when the enzyme activity in the supernatant is constant. In low-activated agarose (typically 5 pmol/mL) the distance between two active groups of the support is larger than the diameter of the protein molecule. Thus, only one bond can be formed between the enzyme and the support. Therefore, it is pomtless to undertake the multi-interaction step in this case, and one point derivatives can be reduced at this point. 7 Multipomt derivatives can be allowed to interact with the support after the nnmobilization has concluded Additional bonds can be formed by keeping the suspension at pH 10.0 (noniomc amme groups are available to react) for a fairly long interaction time at 25°C. This temperature is enough to allow vibrational movement of the protein and correct alignment of reacting groups on the enzyme and support. The longer the time allowed, the more reactions will occur. The optimum multi-interaction time required for each enzyme must be estab-

Enzyme Stabilization on Glyoxyl Agarose

287

lished m every case Prepare enzyme derivatives with different multi-mteractton times and check the thermal stability of each of them The time of chotce is the shortest one that provides the maximal stability (see Frg 4). 8. Figure 4 shows the tmmobiltzation and multi-interaction time course for a multipoint derivative of trypsin (m the presence of inhibitor) A fast drop in the activity of the supernatant indicates a fast immobrlization rate. Nevertheless, the activity of the suspension is fully preserved during the tmmobihzatton and multi-interaction process. Longer contact times of multi-interaction provide higher stabilization of the derivatives without any lost of catalytic activity.

References 1. Gutsan, J M. (1988) Agarose-aldehyde gels as supports for immobrlizatton stabtlization of enzymes. Enzyme Mlcrob. Technol. 10,375-382. 2 Blanco, R M., Calvete, J J., and Guisan, J. M. (1989) Immobihzation-stabtlizatton of enzymes. Variables that control the mtenstty of the trypsm (amine)-agarose(aldehyde) multipoint attachment. Enzyme Mxrob. Technol. 11, 353-359. 3 Blanco, R. M. and Guisan, J. M (1989) Stabihzatton of enzymes by multipomt covalent attachment to agarose-aldehyde gels. Borohydrlde reduction of trypsm agarose derivatives. Enzyme Mlcrob Technol 11,360-366. 4. Mozhaev, V. V., Siksnis, V. A., Torchilin, V. P., and Martinek, K. (1983) Operational stability of copolymerized enzymes at elevated temperatures. Bzotechnol Broeng 25, 1937-1945. 5. Klibanov, A. M. (1983) Stabilization of enzymes against thermal inactivation. Adv Appl. Mlcrobiol. 29, 1-28. 6. Guisan, J. M., Bastida, A., Cuesta, C., Fernandez-LaEuente, R., and Rosell, C (I 99 1) Immobtlization-stabilization of chymotrypsm by covalent attachment to agarose-aldehyde gels. Biotechnol Bloeng. 38, 1144-l 152. 7. Guisan, J M. and Blanco, R. M. (1987) Stabihzatton of trypsin by multiple-point attachment to aldehyde-agarose gels. Ann. NYAcad. SCL 501,67-72. 8. Alvaro, G., Fernandez-Lafuente, R., Blanco, R M., and Gutsan, J M. (1990) Immobilization-stabilization of penicillin G acylase from E. co11 Appl Biochem Biotechnol. 26, 18 1-195. 9. Gutsan, J. M., Fernandez-Lafuente, R., Bastida, A., Blanco, R M., Soler, G., and Garcia-Junceda, E. (1996) Modulation of activity/stabtltty properties of lipase from Pseudomonasjluorescens by multipomt covalent mnnobtlizatlon on glyoxyl-supports, in Engineering Of/lWh Lipases (Malcata, F. X., ed.), Kluwer, Amsterdam, pp. 243-256. 10. Otero, C., Ballesteros, A , and Guisan, J. M. (199 1) Immobilization/stabtlizatton of lipase from Candida rugosa. Appl. Biochem. Biotechnol. 19, 163-175. 11. Guisan, J. M., Alvaro, G., and Femandez-Lafuente, R. (1990) Immobilizatton stabilization of pentcillm G acylase. An integrated approach. Ann NYAcad Scr 613, 552-559 12. Guisan, J. M , Blanco, R. M., Fernandez-Lafuente, R., Resell, C.M., Alvaro, G., and Bastida, A. (1993) Enzyme stabilization by multipoint covalent attachment on activated preexisting supports, in Stab&y and Stabilization of Enzymes (van der Tweel, V J .I., Harder, A., and Buittelaar, R M., eds.), Elsevier, Amsterdam, pp 55-62.

32 Stabilization Modification

of Immobilized Enzymes by Chemical with Polyfunctional Macromolecules

Jo& M. GuisBn, Ver6nica Rodriguez, Cristina M. Resell, Gloria Soler, Agatha Bastida, Rosa M. Blanco, Roberto Fernindez-Lafuente, and Eduardo Garcia-Junceda 1. Introduction For many years, chemical modification of enzymeshas been a key technique to elucidate the residues involved in their catalytic activity. More recently, chemical modification has also been a useful tool in applied biochemistry (e.g., enzyme stabilization by modrfication of key residues, by intramolecular crosslinking, and so on) (14). From this latter point of view, chemical modification continues to be an excellent approach for improvement of remobilized enzyme engineering (enzyme stabilization, modulation of enzyme catalytic propertres, and so on), and an additional complementary technique to improve the properties of genetically engineered enzymes. In this chapter, we propose two approaches for chemical modification of enzymes. First, modification is performed on immobilized enzymes (taking all the advantages of solid-phase protein chemistry), and second, macromolecular polyfunctional reagents are used instead of small mono- or bifunctional reagents. Macromolecular polyfunctional reagents that are large and hydrophilic can be used to chemically modify the surface of enzymes to obtain the followmg advantages (I): 1. Production of generalcrosslinking acrossa number of reactive groups (at different distances)on the enzymesurface. 2. Enzymestabilization by restriction of enzymesurface mobility and, therefore, increasedresistanceto protein unfolding and subunit dissociation. 3. Enzymestabihzationby masking hydrophobic groups of the enzymesurface. From

Methods II) Biotechnology, Vol I Immob/l,ratlon of Enzymes and Cells Edlted by G F BIckerstaff Humana Press Inc , Totowa, NJ

289

290

Guisdn et al.

Fig. 1. Intermolecular crosslinking of enzyme subunits by chemical modification of immobilized derivatives with polyfunctional macromolecules. The use of long and flexible polymers favors the formation of intermolecular crosslinking in polymeric proteins.

4. Enzyme stabilization by creation of a barrier of resistance to attack by chemical reagents (e.g., peroxides), and chemical solvents or other apolar reagents.

Intersubunit crosslinking and generation of artificial environments around the protein surface are two strong features of this approach. Enzymes with quaternary structure may undergo inactivation by dissociation of subunits and stabilization of these enzymes can be achieved by crosslinking the subunits (Fig. 1, refs. 7-9). As this involves crosslinking two different residues on the enzymethat belong to different subunits,the advantagesof using a polyfunctional reagent are apparent, and their large molecular size allows them to cover areas in two or more subunits (Fig. 1). Modification of the enzyme surface properties (charge, hydrophobicity, interactions with different media, and so on) can have a dramatic effect on enzyme properties. Thus, this method can be very useful for alteration of the environment surrounding an enzyme (Fig. 2).

Stabilization

of Immobilized

Enzymes

291

', I

, ’

P2°*1 ORGANIC [ SOLVENT

1

[OXYGEN]

Fig. 2. Modification of the microenvironment of immobilized enzymesby chemical modification with polyfunctional hydrophilic macromolecules.

Dextrans are polymers of (1 + 6) glucose, which are commercially available in a broad range of molecular weights by different companies (see Table 1). Every monomer of glucose contains two glycols that can be easily oxidized by periodate to yield two aldehyde groups per molecule of glucose (IO). That enables production of polyaldehyde polymers that can react with accesible amino groups that are frequently located on the surface of proteins (Fig. 3). Further reduction with sodium borohydride yields irreversible secondary amine bonds between the protein and the polymer, and at the same time it reduces the remaining aldehyde groups present on the polymer. Thus, after the chemical modification, the enzyme remains attached to a highly hydrophilic and inert polymer (polyhydroxyl). The polyaldehydes can be further transformed to polyamines by reaction with ethylenediamine (Fig. 3) and used for interaction with carboxy1 groups on the protein. The primary amino groups introduced into the dextran have a low pH (around 7.0) because of the vicinity to the secondary amino groups (11,12). The resulting polyamine enables modification of proteins through their carboxyl groups using carbodiimide coupling (13,14) under optimal conditions (pH 5.0-6.0), in which the concentration of this reagent is low enough to avoid undesirable chemical modification of the enzyme.

Guisan et al.

292 Table 1 Dextran Available from Fluka (Buchs, Switzerland) and Sigma (St. Louis, MO) Dextran (MJ

Fluka

Sigma

1000-1600 50004000 10,00cM2,000 15,000-20,000

+ + i+ + + + -

+ + + + + + +

40,00&56,000 60,000-90,000 1oo,ooO-200,000

400,000-670,000 2,000,000 5,000,000

Modified dextrans have good potential as polyfunctional macromolecules for protein chemical modrfkation and the avatlabrhty of a vartety of dextran sizes, together with the possibility of different methods and varying levels of actrvation, provides opportunities to tailor polyfunctional reagents to suit particular applications. 2. Materials

2.1. Preparation

of Po/ya/dehydes

1 Commerctal dextran, M, 10,000 mol wt (see Note 1) 2. Solid sodium periodate.

2.2. Modification 1. Immobilized

of immobilized

Enzymes with Polyaldehydes

enzyme derivative (prepared as described in detail m Chapter 30).

2. 25 n-Nphosphate, pH 7.0. 3. 4. 5. 6.

Solid trimethylamine borane. 3.33 mM polyaldehyde solutton prepared as described m Section 3.1. 50 mM bicarbonate buffer, pH 10.0. Sodium borohydride

2.3. Preparation of Polyamines 2.3.1. Activation of Polyaldehyde with EDA 1. 3.33 rnA4 polyaldehyde solution, A4, 10,000 (see Note 2) 2. Trimethyalmme borane. 3. Prepare a solution of ethylenediamine (EDA) in water and take the vessel to an me bath. Add concentrated HCl dropwise very slowly while stirring to avoid the temperature increasing over 25°C (see Note 3), until the pH is 5.0. Adjust

293

Stabilization of Immobilized Enzymes

POLYALDEHYDE

DEXlRAN

OH

OH

POLYAMINES

=

DEXlRAN

-%%A+-

OH n

Fig. 3. The chemistry of the preparation of polyfunctional dextrans.

macromolecules

from

the volume with water to obtain a final concentration of OSMEDA. This must be preformed cautiously m a fume cupboard. EDA IS very toxic and must be handled wearing gloves.

294

GuisCin et al.

4. 10NNaOH solution. 5. Solid sodium borohydride

2.3.2. Titration of Amine Groups in the Poiyamine 1 Prepare a 5 mA4 solution of picryl sulfomc acid (trmttrobenzene sulfomc acid, TNBS free acid) in 50 Wphosphate buffer, pH 7.0 2. 28.5 Wethylenediamine solution in water, pH 7 0 (see Section 2 3.). 3. The polyamine solution must be stored at 4°C or frozen.

2.4. Modification 1 2 3 4 5

of Immobilized

Enzymes with Polyamines

Immobilized enzyme derivative 5 mMphosphate buffer, pH 5.5 3.33 mh4 solution of polyamine A4, 10,000 prepared as described m Section 3 3. Solid l-ethyl-3-(3-dimethylammopropyl) carbodnmide (EDC) 20 Wphosphate buffer, pH 7.5.

3. Methods 3.1, Preparation

of Polyaldehydes

1 Dissolve 1.25 g of dextran Mr 10,000 m 37 5 mL of distilled water (3.33 mM dextran containing 184.8 mM glucose). 2. Add 3 g solid sodium periodate (0 37A4). This corresponds to two pertodate molecules per molecule of glucose (see Note 4) 3. Stir at room temperature for 3 h 4. Dialyze this solution against 5 L of distilled water (five changes) at room temperature. Change the water every 3 h. If necessary one of the changes can be overnight tf performed in the cold room at 4°C. 5 The polyaldehyde solution must be freshly prepared or stored frozen.

3.2. Modification

of Immobilized

Enzymes with Polyaldehydes

1. Dissolve 262.6 mg of trimethylammo borane in 16 mL of 25 mM phosphate buffer, pH 7.0, using strong stirring to provide a TMAB concentration m the suspension of 150 mM. 2. Suspend 8 mL of immobilized enzyme derivative in the phosphate buffer-TMBA solution. 3. Adjust the pH to 7.0. 4 Add 0.8 mL of polyaldehyde solution and keep the suspension under mild stirring (avoid magnetic stirrmg) for 48 h at room temperature (see Note 5). 5. Add 56 mL of bicarbonate buffer, pH 10.0, and adJust the pH to 10.0 6 Add 80 mg of solid sodium borohydride 7. Stir at room temperature for 30 min. 8 Filter and wash the munobtlized enzyme derivative with 50 vol 25 mM phosphate buffer, pH 7.0. 9 Filter and wash the immobilized enzyme derivative with 50 vol distilled water and filter the derivative to dryness

295

Stabilization of lmmobllized Enzymes 3.3. Preparation of Polyamines 3.3.1. Activation of Polyaldehyde with EDA

1. Add 54.71 mg of trimethylamine borane (125 rnA4) to 3 mL of polyaldehyde solution. 2 Take the vessel to an ice bath. 3 To this solution, add a total volume of 3 mL of EDA in four additions of 0 75 mL every 15 min. Keep the solution stirring and cooled at 4°C durmg the reaction. 4. Add 1ONNaOH solution and adjust the pH to 10.0, also at 4°C. 5. Add 120 mg of solid sodmm borohydrtde (20 mg/mL of suspension) 6. Stir the mixture for 1 h at 4’C. 7. Dialyze this solution against distrlled water as before.

3.3.2. Titration of Amine Groups in the Polymer 1. Spectrophotometric method (see Note 6): Add 0.2 mL of polyamine solutron to a cuvet containing 2 mL of TNBS solution. Measure the increase m absorbance at 420 nm. 2. Compare thus slope with the one produced by adding 0 2 mL of a 28 5 mM solution of EDA to the cuvet containing 2 mL of TNBS.

3.4. Modification

of Immobilized

Enzymes with Polyamines

1. Suspend 4 mL of tmmobilized enzyme derivative in 8 mL of 5 mM phosphate buffer, pH 5.5, at room temperature. 2. Add 0.27 mL of polyamine solution and adjust the pH to 5 5. 3. Star the suspension at room temperature for 6 h (see Note 7). 4. Add 230 mg of solid EDC to give 0. 1M concentration m the total volume of the suspension and adjust pH again to 5.5. 5 Stir the suspension for 90 min at room temperature. 6. Filter and wash the suspension several ttmes with 20 mM phosphate buffer, pH 7.5. 7. Filter and wash the suspension thoroughly with distilled water and filter the derivative to dryness.

3.5. Examples 3.5.7. Thermal Stabilization of Penicillin G Acylase-Agarose Modified via Polyaldehyde (see Table 2)

Derivative

1. According to the procedure described, add enough sodium pertodate to dextran M, 10,000 to oxidize only 10% of the polymer. The derivative of PGA-agarose had been previously enriched m amino groups. Modification of thus derivative with the polyaldehyde is performed as described above (15). 2. The half-life of the modified derivative is 8 h when incubated at 56°C and pH 8.0, whereas the half-life of unmodified derivative is 15 min under the same mcubatron condrtions.

Guisan et al.

296 Table 2 Stabilization by Different

of Immobilized Derivatives of Enzymes Strategies Using Polyfunctional Reagents

Enzyme Inactivation

cause

Stabilization vs unmodified enzymeC Stabihzatton vs soluble enzymed

DAO“

PGAb

Chymotrypsin

Dissociation 20

Thermal inactivation 30

Solvent inactivation 6

ND

30 x 8000

6 x 100,000

aD-ammo acid oxldase bPenicillin G acylase CStablllzatlon IS given as the ratio between the half-lives of modified and unmodified enzyme derivatives m the condltlons described in Sections 3.5.1.. 3.5.2., and 3 5 3 dUnmodified derivatives of PGA and chymotrypsin are already stabilized with regard to soluble enzyme m the condltlons indicated ND* not determined

3.5.2. Stabilization of Quaternary Structure of o-Amino Acid Oxidase (see Table 2) 1. The immobilized derivative of D-A0 on glyoxyl-agarose is modified with polyaldehyde in a ratio of 10 molecules of polymer/protein molecule (8). 2. Modification with three polymers of different M, (10,000, 18,300, and 40,000) is performed for 24 h 3. A level of 92% intermolecular crosslinking IS achieved with polymer ofA4, 10,000 and 100% intermolecular crosslmking with the other two polymers. Check the crosslinking by electrophoresis 4. Determine the stability of the derivative modified with polymer of M, 40,000 vs inactivation caused by subunit dissociation by ddutmg 1:30 (v:v) of this derivative and the correspondmg unmodified one and mcubatmg them at pH 9.0 and 42°C. 5. The half-life of unmodified derivative is 30 min in these conditions and the half-life of the crosslinked one is 11 h.

3.5.3. Solvent Stability of Modified Chymotrypsin (see Table 2) 1. Multipoint chymotrypsin-glyoxyl agarose derivative (containing 40 mg enzyme/ mL support) is modified with polyamine IV, 6,000 in a ratio of 12 polymer molecules/enzyme molecule (16) 2 After 6 h of gently stirring of the suspension at pH 5.5, 5 mM EDC is added to obtain formation of covalent polymer-enzyme bonds. The reaction will take 90 min. 3. Incubate this derivative and an unmodified one m 50% iV,N-dimethylformamide at pH 9.0 and 30°C. The residual activity 1s65% in the modified derivative after

Stabilization of Immobilized Enzymes

297

5 d mcubatron, and the residual activrty of the unmodified decreases to the same value in only 20 h.

4. Notes 1. Dextran M, 10,000 is given here as an example, but thts procedure can be applied to any different A4r dextrans without any change in the protocol. Dextrans are commercially available m a broad range of &I, (see Table 1). 2. Polyamines prepared from oxidized dextrans of different M, can be prepared identically. 3. Aliphatic amines are oxidable at high temperature, yielding yellow nitrocompounds. Hence, adjustment of pH of these solutions should be made very carefully by stepwise addttton of ahquot of concentrated acid in an me bath m order to maintain the temperature of the solution below 25°C. 4. Oxidatron with sodmm periodate is stoichlometric. The concentration mdtcated corresponds to total oxidation of the dextran. To achieve partial oxidation of the dextran, calculate the required concentration of periodate. 5. The amount of aldehyde and the contact time between the derivative and the polymer must be established in each case. Different conditions should be tested for each enzyme and each purpose (modification of the surface of the protein, subunit crosslinkmg, and so on) The amount of polyaldehyde gtven here corresponds to 16 polyaldehyde molecules (M, 10,000) per glutaryl acylase molecule and the time required to crosslmk both subunits in 90% of the enzyme molecules. The same crosslinking yteld can be obtained usmg 0.4 mL polymer (8 molecules per enzyme molecule) and a incubation time of 96 h. 6. This procedure enables transformation of 40% of the aldehyde groups on the polymer into amines. Titration of the amine groups can also be determined using a pH stat. 7. The amounts used and reaction times given here correspond to subunit crosslinkmg of 75% of molecules of a derivative of glutaryl acylase-agarose. The parameters must be established for each enzyme and type of modification desired, as well as the polymer size

References 1. Turkova, J., Vohnik S. Helusova, S., Benes, M. J., and Tichl, M. (1992) Galactosylation as a tool for the stabilization and immobilization of proteins. J. Chromatogr 597,19-27 2. Wong, S. S. and Wong, L. J. C. (1992) Chemical crosslinking and the stabilization of proteins and enzymes. Enzyme Microb. Technol. 14,866-874 3. Fernandez-LaFuente, R., Rodriguez,V., Bastida, A., Blanco, R M.,Alvaro, G., and Gursan, J J. (1993) Stabilization of soluble proteins by intramolecular crosslinking with polyfunctronal macromolecules. Poly-(glutaraldehyde-like) structure, in Stabilzty and Stabilizatzon of Enzymes (van de1Tweel, W. J. J., Harder, A , and Buitelaar, R. M., eds ), Elsevter, Amsterdam, pp. 3 15-322.

298

Gush

et al.

4 Torchilm, V. P., Makwmenko, A. V., Smirnov,V. N , Berezin, I V, and Martmek, K. (1979) The principles of enzyme stabilization. V. The possibility of enzyme self stabilization under the action of potentially reversible mtramolecular cross-lmkages of different length. Bzochim. Blophys. Actu 568, l-10. 5. Fernandez-Lafuente, R., Rosell, C M , Alvaro, G , and Guisan, J. M (1992) Additional stabilization ofpemcilhn G acylase derivatives by controlled chemical modification with formaldehyde. Enzyme Mwob Technol 14,489-495 6 Royer, G P , Ikeda, S., and Aso, K. (1977) Cross-lmkmg of reversibly tmmobihzed enzymes. FEBS Letts 80,8!%94 7. Torchilm,V. P., Trubetskoy,V S , Yanenko, V G., and Martmek, K. (1983) Stabihzation of subunit enzyme by mtersubunit crosslinking with bifunctional reagents: studies with glyceraldehyde-3-phosphate dehydrogenase J Moi Catalyszs 19, 291-301 8 Rodriguez, V (1995) Estabilizacion de enzimas de estructura compleJa biotransformation enzimatica de antibioticos /3-lactamtcos. PhD Thesis. Universidad Complutense de Madrid. 9. Klemes, T. and Citri, N (1979) Catalytic and conformational properties of cross-linked derivatives of pemcillmase Biochzm. Bzophys Acta 567,401409. 10. Yamagata, Y., Arakawa, K., Yamaguchi, M., Kobayashi, M., and Ichishima, E. (1994) Functional changes of dextran-modified alkaline protemase from alkalophilic Bacdlus sp Enzyme Mlcrob Technol 16,99-l 03 11. Sakharov, I. Y , Larinova, N. I., Kazanskaya, N. F., and Berezm, I. V. (1984) Stabilization of proteins by modification with water-soluble polysacchandes. Enzyme Microb

Technol 6,27-30

12 Fernandez-Lamente, R , Rosell, C M , Rodriguez, V, Santana, C , Soler, G , Bastida, A , and Gutsan, J M. (1993) Preparation of activated supports containing low pK ammo groups A new tool for protein numobillzation via the carboxyl coupling method. Enzyme Microb Technol 15,546-550. 13. Carraway, K. L. and Koshland, D. E., Jr., (1972) Carbodiimide modification of proteins. Methods Enzymol. 25,616-623. 14. Torchtlm, V. P., Maksrmenko, A. V., Smnnov, V. N., Berezm, I. V., Khbanov, A M , and Martinek, K (1978) The principles of enzyme stabilization III. The effect of the length of intra-molecular cross-linkages on thermostability of enzymes. Bzochzm Blophys Acta 522,277-283.

15. Molma-Rosell, C. (1993) Reacciones de quimica tina catahzadas por dertvados estabihzados de Pemcilma G acilasa. PhD Thesis. Umversidad Complutense de Madrid 16 Soler, G (1995) Disefio de derivados de qulmotripsma coma catahzadores en reacciones de quimica fina. PhD Thesis. Umversidad Complutense de Madrid

33 Covalent Immobilization of Enzymes Using Commercially Available CDI-Activated Agarose George J. Piazza and Marjorie B. Medina 1. Introduction Carbonyldiimidazole (CDI) activated supports for enzyme immobihzatlon are commercially avallable. The urethane linkage that is formed when a protein 1s bound to these supports IS about 20-fold more stable than the N-substituted lsourea linkage formed during protein lmmobllization on cyanogen bromide-activated matrices (1). Complete characterization of the munobllized preparation requires the development of new methodology or the modification of existing methodology to measure the amount of protein bound to, and the enzymatic activity residing in, the immobilized preparation. In this chapter we present detailed descriptions of the immobilization of two enzymes, hpoxygenase (2,3) and lactamase, on CDI-activated agarose, and procedures for characterlzatlon of the immobilized enzymes.

2. Materials Water was purified to a resistance of 18 n&&cm using a Barnstead (Dubuque, IA) NAN0 pure system.

2.7. Lipoxygenase ImmotWzation 1 Soybeanlipoxygenase (LOX EC 1.13 11.12) (lipoxidase, type 1-B, Sigma, St. Louis, MO). 2. CDI-activated support.Reacti-Gel, a 6% crosslinkedagaroseactivated with 1,l’carbonyldilmidazole (CDI) (Pierce,Rockford, IL) 3. Couplmg buffer: 0.2M borate buffer, pH 9.0 (see Note 1). 4. Quenching reagent: 2M 2-amino-2-hydroxymethyl-1,3-propanediol (Tns)-HCl buffer, pH 8.0. From

Methods tn Blofechnology, Vol I lmmobrfrzatron of Enzymes and Cells Edlted by G F BIckerstaff Humana Press Inc , Totowa. NJ

299

300

Piazza and Medina

5. Storage buffer. O.lM sodium phosphate buffer, pH 7.0, containmg 0.9% NaCI, 0 05% bovme serum albumin (BSA), and 0.02% NaN3 6. LabQuake rotary shaker (Lab Industries, Berkeley, CA) 7 IO-mL polypropylene column fitted with a porous polyethylene disk 8. Buchner flask and filter funnel for vacuum filtration of gel.

2.2. Lactamase immobilization 1. P-Lactamase (EC 3 5.2.6) derived from Baczhs cereus 569/H9, lactamase I*lactamase II activity, 10: 1 (Calbiochem, La Jolla, CA). 2 CDI-activated support. Reacti-Gel, a 6% crosslmked agarose activated with 1, l’carbonyidnmidazole (Pierce, Rockford, IL). 3 Coupling buffer 0. 1M borate buffer, pH 8 5, containing 0 9% NaCI. 4. Quenching reagent. 2M Trts-HCl buffer, pH 8.0. 5. Storage buffer: 0.05M sodium phosphate buffer, pH 7.2, containing 0.9% NaCl, 1% gelatm, 0.02% NaN3 6 Dialysis buffer. 0. 1M borate buffer, pH 8.5. 7 Dilution buffer: 0. 1M phosphate buffer, pH 7.2 8. Dialysis tubing with A4, cutoff of 1000. 9 Poly-Prep column with a two-way stopcock (Bio-Rad, Richmond, CA)

2.3. Protein Measurement and Lipoxygenase Activity Assay 2.3.1. Efficiency of Immobilization of Lipoxygenase Protein assay reagent (Bra-Rad). Protein standard containmg min and 30 mg/mL globulin (Sigma).

50 mg/mL

albu-

2.3.2. LOX Assay: HPOD Generation in Aqueous Media The assay is based on the generation (HPOD) from linoletc acid (LA).

of hydroperoxyoctadecadrenom

acid

1 0.2M borate buffer, pH 9.0. 2 Linoleic acid. 3. Sonicator.

2.3.3. LOX Assay: HPOD Generation in Organ/c Media 1. 2 3. 4

Linoleic acid. Water-saturated octane 0.2M borate buffer, pH 9 0 Dtethyl ether

2.3.4. Spectrophotometric

Assay

1 Xylenol orange reagent: 100 @4xylenol orange, sodium salt (Aldrich, Milkwaukee, WI), 250 cul/lammomum ferrous sulfate, 25 mMH2S04, and4 mA42,6-dt-t-butyl4-methylphenol in methanol/water (9: 1, v/v) (4; see Notes 2,4).

301

immobilization on CD/-Agarose 2. Ethanol. 3. Cumene hydroperoxide (Sigma).

2.3.5. Analysis by Thin-Layer Chromatography

(TLC)

1 TLC plates: silica gel HPTLC, with preadsorbent zone, 10 x 10 cm (Analtech, Newark, DE). Plates are developed in a twin-trough TLC tank (Camag, Wrightsvllle, NC). 2. 5% boric acid in methanol 3. Solvent I: diethyl ether, benzene, ethanol, acetic acid (40:50:2*0.2, v/v/v/v) Solvent II: isooctane, diethyl ether, acetic acid (50:50:2, v/v/v). 4. 60% sulfuric acid solution,

2.3.6. Analysis by High Performance Liquid Chromatography (see Note 3)

(HPLC)

1 Methylene chlortde; linoleic acid; 1,3 dilinolein; monolinolein; diolein (Sigma or Nu Chek Prep, Elysian, MN). 2. Dry nitrogen gas. 3 0.3MN-tns(hydroxymethyl)-methylglycine (Tricine), pH 8 0. 4 100 mM deoxycholate in water. 5 15°C water bath (Lab-Line, Orbit Shaker Bath, Melrose Park, IL). 6 IMcitric acid solution. 7 Methanol/chloroform, 2: 1 (v:v) solution.

2.4. Protein Measurement and Lactamase Activity 2.4.1. Efficiency of Immobilization of Lactamase 1. 2. 3. 4.

BCA protein reagent. Dilute as described by suppher (Pierce) Microtiter wells (Immunolon strips frum Dynatech Laboratories, Chantilly, VA). Biotek ELISA Reader (Burlington, VT). DeltaSoft program (Biometallics, Princeton, NJ)

2.4.2. Evaluation of Lactamase Activity: Preparation of P-Lactam Standard 1. 2. 3. 4.

3 mg/mL penicillin G in dilution buffer. 1 mg/mL lactamase m dilution buffer. Dilution buffer: 0. 1M phosphate buffer, pH 7.0. Microfiltration centrifugation tube with 10 kDa filter (Amicon).

2.4.3. Analysis by TLC 1. 35-urn porous polypropylene disk, 2.5 x 1.7 pm (BelArtPmducts,Pequannock,NJ). 2. TLC plates: silica gel HPTLC-GLC, 10 x 20 cm, score at 2.5 cm (Analtech) and high performance LHP-KD, 10 x 10 cm, with preadsorbent zone (Whatman, Clifton, NJ). For developing use a wide mouth 118-mL glass jar for the 2.5 x 10 cm plates and a twin-trough TLC tank (Camag) for the 10 x 10 cm plates.

Piazza and Medina

302 3. 4 5. 6 7.

Methanol Penicilloic acid prepared as described in Section 3 4 2. Developing solvent: chloroform, acetone, acetic acid, 10:9*1 (v*v:v). Iodine crystals 1% aqueous gelatinized potato starch solutton

3. Methods 3.7. /mmobi/~zsfion

of Lipoxygensse

1 Add a 1 mL settled volume of CDI-activated agarose to a 10 mL polypropylene column fitted with a porous polyethylene disk. Remove the acetone, m which the CDI-activated gel IS suspended, from the gel with vacuum filtration without allowing the gel to dry completely. If a polypropylene disk is not available, a Buchner flask and funnel may be used.

2. Wash the gel with 3 x 5 mL of chilled distilled water. Remove the water with vacuum filtration as before. 3. Covalently link the activated gel to the LOX within 30 min of washing. Dissolve 2.0 mg/mL of LOX in chilled coupling buffer and transfer 3 mL to a covered polypropylene container holding the CDI-activated gel. Set aside 0 2 mL for total protein and activity measurements 4. Incubate the gel with the LOX for 42 h at 4°C with gentle mixing using a rotary shaker. Adjust the pH of the gel/enzyme to the original pH (this may require three adjustments) 5. After the mcubation, collect the effluent to determine the concentration of unbound protein and LOX activity. Rinse the gel with 5 mL of the borate coupling buffer and pool with the effluent. 6 Block the remaining active sites on the gel with 4 x 5 mL washes with quenching reagent (2M Tris-HCl, pH 8.0) and allow the last 5 mL to remain m the column for 30 mm at room temperature 7. Rinse the gel containing the unmoblhzed enzyme with 3 x 10 mL washes of cold water and store at 4°C in storage buffer.

3.2. Immobilization

of Lactamase

1. Dissolve 30 mg of p-lactamase m 3 mL of chilled O.lM borate couplmg buffer, pH 8.5, and transfer to a dialysis tubing for dialysis overnight against 1 L of coupling buffer to remove the buffer salts and to equilibrate the protein m the borate couplmg buffer. Change the buffer three times during the dialysis. 2. Transfer the dialysate to a calibrated conical tube to measure the volume of the dialysate 3. Fit a Poly-Prep column with a two-way stopcock and close the bottom outlet. Transfer the CDI-activated agarose gel to the column and allow it to settle to a 2 mL volume. Drain the acetone from the gel using vacuum filtration. 4. Wash the gel with 2 x 1 mL of chilled distilled water, using vacuum filtration between the washes

Immobilization on CD/-Agarose

303

5. Close the bottom of the column. Couple the enzyme to the activated gel within 30 min. 6. Set astde 0.2 mL of the lactamase dialysate from step 2 for total protein measurement. Add 4 mL of the chilled lactamase dialysate m coupling buffer to the CDI-activated column. Rinse the sample tube with 0.5 mL coupling buffer and add to the gel. 7. Cap the top of the column and Incubate the gel mixture for 40-42 h at 4°C with gentle mixing on a rotary shaker Check and maintain the pH at 8.5 8. After the innnobilizatron period, collect the effluent and determine the concentration of the unbound lactamase. 9. Remove excess imidazole from the unbound lactamase by dialysis or ultratiltratton in borate coupling buffer prior to quantification of the protein concentratron Rinse the gel with 2 x 1 mL washes of borate coupling buffer and pool with the effluent. 10. Rinse the column further with 2 x 1 mL washes of borate coupling buffer and quench with 4 x 5 mL washes of 2MTris-HCl buffer, pH 8.0. Allow the last 5 mL to remain in the column for 30 min at room temperature. This step blocks the remaining active sites on the gel. 11. Rinse the gel containmg the lmmobthzed enzyme with 3 x 5 mL washes of degassed storage buffer and store at 4“C.

3.3. Protein Measurement and Lipoxygenase Activity Assay 3.3.1. Efficiency of immobilization of Lipoxygenase 1. Determine the percent of protein load on the React+Gel by measurmg the total protein before and after coupling. The difference between the amount added and recovered protein is divided by the initial amounts and multtplied by 100. The amounts of protein m the LOX/borate buffer mixture are estimated using the Bio-Rad Protein Reagent and Sigma protein standards for calibration (see Note 4). 2. Dilute the protein standards to provide concentrations of 0.0125-l .Omg protein/ mL of 0.2M borate buffer, pH 9.0. 3. Transfer 2O-ltL aliquots of standards and samples (protein solution prior to immobilization and unbound protein) to test tubes in duplicate. 4. Add 0.2 mL of Bio-Rad reagent protein sample and 0.2M borate buffer, pH 9.0 (0.8 mL), to each tube, mix gently, and incubate the mixture for 30 mm at room temperature. 5. Measure the absorbances at 565 nm using a spectrophotometer.

3.3.2. LOX Assay: HPOD Generation in Aqueous Media 1. Remove the storage buffer from the immobilized LOX (IMM-LOX) by vacuum filtration. 2. Wash the IMM-LOX with two 3.0-mL aliquots of 0.2M borate buffer, pH 9.0. 3. Add 0 59 g of IMM-LOX (containing 3 mg protein) to 40 mg of linoletc acid (LA) that had previously been suspended by sonication for 30 min m 20 mL of 0.2M borate buffer, pH 9.0.

Piazza and Medina

304

4. Allow the reaction to proceed at 15°C wtth agitation at 250 rpm. After 15 mm, filter the IMM-LOX from the reaction mixture, wash with two lo-mL aliquots of borate buffer, and place in storage buffer at 5°C or return to a fresh suspension of LA. 5. Assay an ahquot from the reaction solutton for hydroperoxide content as described in Sections 3 3 4 ,3 3 5., and 3 3 6.

3.3.3. HPOD Generation in Organic Media 1. For assays in organic solvent, dissolve 40 mg of LA m 15.0 mL of water-saturated octane m a 125-mL, glass-stoppered Erlenmeyer flask. 2. Wash the IMM-LOX with 0.2Mborate buffer, pH 9.0, then add 1 5 g IMM-LOX containing 1.5 mg protein and 187 nL of 0 2M borate buffer, pH 9.0, to the LA solution. 3. Allow the reaction to proceed at 15°C with agitation at 250 rpm. After 15 mm, filter the gel from the reaction mtxture and wash the IMM-LOX with two lo-mL aliquots of borate buffer that has been similarly filtered. 4 Lower the pH of the water layer to 3.0 using 0. lMHC1 and separate the aqueous and organic layers. 5 Extract the aqueous layer with 3 x 5 mL diethyl ether. Combme the ether and the organic fractions and take an ahquot for hydroperoxtde assay as described m Sections 3 3 4,3 3 5 , and 3.3.6.

3.3.4. Spectrophotometric HPOD concentration orange reagent.

Assay

is deternnned spectrophotometrrcally

using the xylenol

1 Add 2 0 mL of the xylenol orange reagent to IO-50 uL of sample and raise the volume of the mixture to 2.1 mL by addttton of ethanol. 2. Incubate the assays at room temperature for 45 min and then measure the absorbance at 560 nm vs a blank of 2.0 mL xylenol orange reagent and 100 pL of ethanol (see Note 5) 3. Use a commercial preparation of cumene hydroperoxide to prepare a caltbratton curve of the dye reagent. Prepare the calibration curves on the same day as the reaction assay using freshly diluted (m 95% ethanol) cumene hydroperoxide.

3.3.5. Analysis by TLC TLC analysis is performed on each reaction mixture as a check on the hydroperoxtde levels given by the xylenol orange method and to determine if any anaerobic byproduct formation and/or decomposition of HPOD has occurred (5). 1. Dip 10 x 10 cm sthca gel-HL TLC plates m 5% boric acid in methanol and allow to air dry prior to spotting with samples and standards 2. Develop the TLC plates sequentially m developing solvents I and II with air drying between developments

Immobilization on CD/-Agarose

305

3 Visualize the hydroperoxides by charring after spraymg the TLC plates with 60% sulfuric acid (see Note 6).

3.3.6. Analysis by HPLC (see Note 3) Analysis by HPLC is partrcularly convenient for determining the activity of IMM-LOX on several substrates simultaneously. Prepare an equal molar mtx-

ture of substrates containing an internal standard. Measure the loss of the substrate with time caused by oxidation by IMM-LOX and compute the relative oxidation rates (6). 1 Prepare in methylene chloride a substrate mrxture consisting of 30 ymollml linoleic acid, 15 pmol/mL 1,3-dtlinolein, and 30 pmol/mL 1-monolinolein, with 30 pmol/mL diolein as the internal standard. 2. Subject a 20-pL ahquot of the substrate mixture to analysis by HPLC and adjust the concentrations of the substrates accordingly (see Note 3) 3. Add a 0 2-mL ahquot of the substrate mixture to nine lo-mL Erlenmeyer flasks equipped with stoppers. 4. Evaporate the methylene chloride from the flasks under a stream of dry nitrogen 5 Add 1.8 mL of 0.3MN-tris(hydroxymethyl)-methylglycine (Trtcme), pH 8 0, and 0.2 mL 100 mM deoxycholate in water to each flask. 6. Stopper the flasks and place them in a 15°C water bath. Agitate the flasks at 250 rpm. 7 At time zero add IMM-LOX to each flask except for one. The IMM-LOX should contain approx 100 pg LOX. 8. The reaction mixture in the flask: receiving no IMM-LOX should be quenched immediately with 0.4 mL of 1Mcitric acid. Thereafter, each oxidation should be quenched at 5-min intervals. 9 Immediately after quenching, each reaction mixture is extracted with 2 x 1 5 mL methanol/chloroform (2: 1). 10. Evaporate the methanol/chloroform from the flasks under a stream of dry nitrogen. 11. Redissolve the substrates and products m methylene chloride. Filter the solutions and subject them to analysts by HPLC.

3.4. Protein Measurement and Lactamase Activity 3.4.7. Efficiency of Immobilization of Lactamase Determine the coupling efficiency by measuring protein concentration before and after coupling. Use lactamase as a specific protein standard (see Note 7). 1 Prepare lactamase standards in concentrations of 0.0125-l .O mg protein per mL of 0 1M borate buffer, pH 8 5, containing 0.9% NaCl. 2. Transfer 50-PL aliquots of standards and samples (protein solution prior to munobiltzation and unbound dialysate) to the test tubes m duplicate.

Piazza and Medina

306

3. Add I mL of the BCA reagent to each tube, mix gently, and incubate the mtxture for 30 mm at 37°C. 4. Transfer 200~pL ahquots to mtcrottter wells. 5. Measure the absorbances at 562 nm using a Btotek ELISA Reader equtpped with the DeltaSoft program that can analyze and determine the protein concentratton in the samples (see Notes 78)

3.42. Evaluation of Lactamase Activity: Preparation of P-Lactam Standard Use penicillorc

acid as a standard for enzyme actrvrty and prepare in the

laboratory from penicillin G. 1, Transfer 100 pL of 3 mg/mL pemcillin G and 50 pL of 1 mg/mL lactamase to a microfuge tube 2. Adjust the volume to 1 mL by addition of 850 PL of 0 1M phosphate buffer, pH 7.0, (dilutton buffer) and mix m a rotary mixer for 10 mm. 3 Transfer the mixture into a mtcrotiltratton centrifuge tube (10 kDa filter) and centrifuge for 20 min at 10,OOOg.Dilute the pemctlloic acid m the filtrate 100X with dilution buffer and use as a standard (see Notes +l 1)

3.4.3. Analysis by TLC 1 Make a microcolumn by inserting a 35-pm porous polypropylene disk, 2.5 x 1.7 pm into the bottom of a l-mL disposable pipet ttp. 2 Transfer a 100-JJL portion of the immobilized enzyme preparation to a microfuge tube and wash with 1 mL of 0. 1Mphosphate buffer, pH 7.0. Recover the unmobtlized enzyme by centrifugation at 10,OOOgfor 10 mm. 3. Add 100 pL of 3 mg/mL pemcrllm G to the lactamase-agarose and place the mixture in a rotary mixer for 10 min. 4. After centrifugatton at 10,OOOgfor 10 mm, analyze the supernatant by TLC using a modification of a procedure reported by Moats (7) as follows, 5. Clean the TLC plates by Immersion in methanol for 10 mm and subsequent drymg at 85°C for 20 min. 6. Apply l-2 pL samples to the 2.5-cm TLC plates and an dry using a hair dryer. For the detection of low concentrations of penictllm G and penicilloic acid, apply 25pL samples to 10 x 10 cm Whatman TLC plates. 7. Place the TLC plates in developing tanks that have been equilibrated with 10 mL of the developing solvent, chloroform:acetone:acettc acid, 10:9 1 (v:v.v), and develop for 7 min. The Whatman TLC plate is developed for 10 mm with the same solvent mixture in a twin-trough TLC tank in which both chambers are filled with 10 mL of the developing solvent 8. Au dry the developed plate and visualize spots for penicillin G and pemctlloic acid by exposing the plate to iodine vapor for 3-5 mm (see Note 12). 9. Allow the background iodine on the TLC plate to vaporize for 30 s and then spray the spots with a 1% aqueous gelatinized potato starch (8). The starch suspension is previously heated to >7O”C and cooled prior to use as a spray.

Immobilization on CDI-Agarose

307

4. Notes 1. If the influence of coupling pH is to be investigated, use a coupling buffer that contains a mixture of buffers: borate, ethylenediamine-tetraacetic acid (EDTA), and N-tris(hydroxymethyl)methylglycine (Tricine) all at 0 2M. 2. The I&S04 must be added unmediately after the solvent is added. The reagent should be brtght yellow. 3. HPLC analysts is conducted on an Alltech (Deerfield, IL) Cl8 hydroxyethylmethacrylate (HEMA) column (250 x 4.6 mm) installed on a Waters (Milford, MA) LCMl HPLC instrument (9,10). The detector is a Varex evaporative lightscattering detector MK III (Alltech) operated at a temperature of 48”C, and with N2 as the nebuhzing gas at a flow rate of 1.3 mL/mm. The mobile phase has the following composition and gradient: methanol: 10 mh4 aqueous trtfluoroacetlc acid (TFA) (86:14), O-4 min; methanol:10 mM TFA (90:20), 4-8 mm; acetonitrile:methanol:lOmMTFA(30:65:5), S-10mm; acetonltrile:methanol*lO mA4 TFA (5 1:48: l), 10-25 min. The flow rate is 1 mL/min Calibration curves are prepared that allow the conversion of peak area to micromoles for each substrate. 4 Absorbance at 280 nm should not be used to measure protem, since the hydrolysis product of the CDI-activated agarose is imidazole, which absorbs at this wavelength. 5. Old samples of LA exposed to air may be partially oxidized. If the degree of oxidation of LA is not known, the blank should be a substrate mixture that was not exposed to LOX. 6. A 6-mm thick sheet of alummum is placed on top of a laboratory hot plate to give even heat distribution during charring. The Rr values of LA, HPOD, and hydroxyoctadecenoic (ricinoleic) acid were 0.77, 0.62, and 0.53, respectively, Since this TLC developmental system cannot differentiate between fatty acids having differing degrees of unsaturation, the Rfvalue of hydroxyoctadecadienoic acid, the reduced product of HPOD, is also 0.53. 7. Although the absorbance at 562 nm of an equal weight of BSA 1s 10 times higher than that of lactamase I, BSA may still be used as a protein standard for couplmg efficiency, since this parameter is the ratto of the amount of bound lactamase I to the amount of lactamase I mitially added times 100. 8. The coupling efficiency as measured by the BCA protem assay is typically 55%, with approx 3.8 pmol lactamase (M, 3 1,SOO)/mL of agarose gel. 9. p-lactamase I catalyzes the hydrolysis of the lactone ring of pemclllins and p-lactams forming penicllloic acid (11,12). 10. The immobilized enzyme retained its activity and was more stable than the enzyme in solution. 11. Penicilloic acid was used as a standard to determine the activity of the immobllized enzyme. The yield of penicilloic acid as described in Section 3.4.2 was quantitative as shown by a single band on a TLC plate 12. A pinch of iodine crystals is placed in the bottom of the 118-mL glass jar that is capped to allow the iodine vapor to saturate the jar.

308

Piazza and Medina

References 1 Hearn, M. T W, Harris, E L , Bethell, G. S , Hancock, W S , and Ayers, J. A (198 1) Application of 1,l ‘-carbonyldnmidazole-activated matrtces for the purification of proteins. III. The use of l,l’-carbonyldiimidazole-activated agaroses in the biospeciflc affimty chromatographic isolation of serum antibodies. J Chromatogr 218,509-5

18.

2 Parra-Diaz, D , Brower, D. P., Medma, M. B., and Piazza, G. J. (1993) A method for the tmmobthzation of soybean hpoxygenase. Bzotechnol Appl Blochem. 18, 359-367. 3 Piazza, G J., Brower, D P., and Parra-Diaz, D. (1994) Synthesis of fatty acid hydroperoxtde in the presence of organic solvent usmg mnnobtllzed hpoxygenase Bzotechnol Appl Blochem. 19,243-252 4. Jtang, Z.-Y, Woollard, A. C. S., and Wolf, S P (1991) Lipid hydroperoxide measurement by oxidatton of Fe2+ m the presence of xylenol orange Comparison with the TBA assay and an iodometrtc method. Lipids 26, 853-856 5. Gardner, H. W. (1991) Recent mvestigattons into the hpoxygenase pathway of plants. Biochim Biophys Acta 1084,22 l-239 6 Schellenberger, V , Siegel, R. A., and Rutter, W. J. (1993) Analysis of enzyme spectticity by multiple substrate kinetics Biochemzstry 32,4344-4348 7 Moats, W. A. (1983) Detection and semiquantitative estimation of pemcilhn G and cloxacillm in milk by thin-layer chromatography. J Agrlc Food Chem. 31, 1348-1350. 8. Medina, M. B. and Schwartz, D P (1987) A multi-residue TLC screening procedure for anabohc oestrogens and detection of oestradiol, DES or zeranol in chicken muscle tissue extracts. Food Add. Contam. 4,4 15427. 9. Nufiez, A. and Piazza, G. J. (1995) Analysis of hpoxygenase kinetics by high performance liquid chromatography with a polymer column. Lipids 30, 129-133 10. Piazza, G. J. and Nufiez, A. ( 1995) Oxidation of acylglycerols and phosphoglycendes by soybean lipoxygenase. J. Am. 011 Chem. Sot. 72,463-466. 11 Laws, A. P and Page, M. I. (1989) The effect of the carboxyl group on the chemical and p-lactamase reactivity of p-lactam anttbiottcs. J Chem Sot Perkm Trans 2, 1577-1581. 12 Waley, S G. (1992) j3-Lactamase mechanism of action, m The Chemistry of j?-Lactams (Page, M. I., ed.), Blackte, New York, pp. 198-228.

Immobilization of Cells in Polyelectrolyte Complexes Johanna

Mansfeld and Horst Dautzenberg

1. Introduction Polyelectrolyte complexes are rapidly formed when oppositely charged polyions are mixed. There are two basic principles making use of polyelectrolyte complexes for nnmobilization of cells and enzymes-entrapment and microencapsulatron. Because the entrapment in polyelectrolyte complexes (1) ytelds only amorphous polymeric structures complicating handling of these immobilizates, this chapter concentrates on microencapsulation within polyelectrolyte complexes. Microencapsulation is based on membrane confinement. For encapsulatron, the biocatalysts are enclosed in semipermeable membranes retaining the catalyst without limiting the exchange of substrates and products. Generally, the semipermeable membranes can be obtained by boundary layer polymerization, coacervation, liquid drying procedures, and polyelectrolyte complex formation. The latter method, presented in this chapter, is the only one ensurmg mild reaction conditions, avoiding the use of solvents and toxic monomers. A wide variety of materials has been used for this purpose; for instance, combinations of alginate and chitosan (2) or poly+lysine (3), poly(dimethyldiallylammomum chloride)/sodium cellulose sulfate, chitosan/poly-L-glutamic acid (5), chitosan/tc-carrageenan (61, or chitosaticarboxy-methylcellulose (7). In comparison to gel entrapment in which gel beads with a solid core are formed becauseof interaction of polyelectrolytes with small counterions, such as alginate with Ca*+ ions or chrtosan with phosphates, the interaction of high-mol wt polyelectrolytes of opposite charges yields polyelectrolyte complex capsules with a liquid core. In contrast to polyanion salts (e.g., alginate), which are relatively unstable in the presence of phosphates, chelating compounds and at hrgh pH; and polycation salts (e.g., chltosan), which lose their stability at lower pH From

Methods Edlted

In Bofechnology, by

G F BIckerstaff

Vol 1 Immob!l/zat/on Humana

309

Press

of Enzymes Inc , Totowa.

and Cells NJ

Mansfeld and Dautzenberg

310

and require phosphate ions to mamtam the gel structure; polyelectrolyte complex capsules are much less affected by most of these compounds. Therefore, these capsules usually have higher chemical and mechanical stability under various conditions, enlarging the field of possible applications. Another advantage of microencapsulation is that the cells can grow to some extent without damaging the capsules, whereas cell growth in gels leads to destruction of gel beads and leakage of the cells into the culture medium. The immobilization of enzymes in polyelectrolyte complex capsules is only recommended under certain precautions because the pore size of the capsule membrane is usually too large. Another drawback is that the microcapsules are easily distorted when they are packed mto a column reactor. This results in high pressure drop. Binding to insoluble carriers ts generally preferred in mmrobihzation of enzymes. However, with respect to encapsulation of small protein molecules, microcapsules could be of Interest in the field of controlled release of proteins, drugs, or other therapeutic agents (3,10). The poly-L-lysine/alginate system has found commercial application at Damon Biotech (Needham Heights, MA) for production of MAbs. In this chapter, the most frequently used materials and methods in microencapsulation are described.

2. Materials 2.1. Encapsulation

in Sodium Cellulose

Sulfate/PDMDAAC

1. Sodium cellulose sulfate: Prepare from Linters cellulose vra heterogeneous sulfation. Average degree of substitution, 0.3-O 5; average degree of polymerrzation, 300-500; viscosrty of 1% (w/v) solution 25-35 mPa * s (University of Potsdam, Department of Chemistry, Teltow, Germany): Prepare a 4.0-4.2% (w/v) solution of sodium cellulose sulfate m distilled water. 2. Poly(dimethyldrallylammonmm chloride) (PDMDAAC): Prepare via radtcal

polymerization of dimethyldiallylammonium chloride, mean 30004000 M, (University of Potsdam, Department of Chemistry, Teltow, Germany): Prepare a 1.2% (w/v) solution of PDMDAAC in dtstilled water. 3 Cell suspension. 4. Syringe with 0.2-1.0-n-m inner diameter needle or suitable syrmge pump extrusion system. For preparation of capsules with small drameter, an airjet droplet generator system IS recommended

2.2. Encapsulation of Cells in Sodium Cellulose PDMDAAC After Preimmobilization of Cells 2.2.1. Preimmobilization of Cells

Sulfate/

1. 0.25% (w/v) solution of sodium cellulose sulfate in distilled water 2 0.25% (w/v) solution of PDMDAAC in distilled water. 3. Cell suspension.

311

Cells in Polyelectrolyte Complexes 2.2.2. Encapsulation of Preimmobilized Cells in Sodium Cellulose Sulfate/PDMOAAC

1, 4.0-4.2% (w/v) solutron of sodium cellulose sulfate m distilled water. 2. 1.2% (w/v) solutron of PDMDAAC in distilled water. 3. Syringe with 0.2-l .O-mm inner diameter needle or suitable syringe pump extrusion system For preparation of capsules with small diameter an airjet droplet generator system is recommended.

2.3. Encapsulation

in AlginateIPoly-r-Lysine

1. 1.5-l .6% (w/v) sodium alginate (Kelco, Hamburg, Germany) solution in water or a suitable buffer for production of spherical beads; or 2. O&0.8% (w/v) sodium alginate solution in water or a suitable buffer for production of nonspherical, softer beads. 3 0. 1M CaClz solution in water or a suitable buffer. 4. Cell suspension in the desired growth state and suitable medium. 5. Syrmge with 0.2-I .O-mm inner diameter needle or smtable syringe pump extrusion system. For preparation of capsules with small diameter, an airjet droplet generator system IS recommended 6. 0.05-0.075MCaC12 solution in water or a buffer appropriate for the type of cells. 7. 0.02-O. 1% (w/v) poly+lysine (Sigma, St. Louis, MO) solution, 15,000-20,000 mol wt 8. 0.05M sodium citrate buffer, pH 7.4. 9. 0.03-O. 15% (w/v) algmate solution in water or suitable buffer. 10. 0 2% (w/v) poly(ethylene rmine) (Sigma) solutton.

2.4. Encapsulation

in ChitosatVPolyanion

Complexes

1. 2. 3. 4.

Chitosan (Protan, Redmond, CA), final concentration 3% (w/v) Concentrated citric acid solutron. Calcium chloride. Alginate (Kelco)/glucose solution: Prepare solutions consisting of 0 75% and 2.25%, respectively. 5. Cell suspension in the desired growth state and suitable medium. 6. Syringe with 0.2-l .O-mm inner diameter needle or suitable syringe pump extrusion system. For preparation of capsules with small diameter an atrjet droplet generator system is recommended.

3. Methods 3.1. Encapsulation

in Sodium Cellulose

Sulfate/PDMDAA

C

1. Dissolve the sodium cellulose sulfate under shaking for 24 h m distilled water and autoclave the resulting viscous solution for 10 min at 12 1“C (see Notes 1,2). 2. Dissolve the PDMDAAC in distilled water and sterilize the resulting solution by sterile filtration (see Notes 1,3) 3. Grow the cells up to the desired growth state or dissolve the enzyme m a small volume of approprtate buffer for the particular enzyme.

Mansfeld and Dautzenberg

312

4 Suspend 1.0-2 5 g of cells (dry weight) in 100 mL of sodium cellulose sulfate solutton and mix the suspension homogeneously by vtgorous shaking. The final sodium cellulose sulfate concentration should be 4% (see Notes 2,4). 5 Drop the cell/sodmm cellulose sulfate suspension through a syringe needle or using syringe pump extrusion systems into a stirred PDMDAAC solutron. The cell/sodmm cellulose sulfate volume should not exceed one-fifth the volume of PDMDAAC solution used for precipitation. 6 Stir the capsules for 40 mm until the hardening process reaches optimum (see Note 5). The residence time in the PDMDAAC solution has to be optimized and adapted to the particular cell types and capsule-forming polyanions (see Note 6) 7 After finishing the hardening process, remove the PDMDAAC solution and replace with the desired nutrient or production medium. Care should be taken at this step because problems with shrinkage or stickiness of the capsules could arise. A stepwtse exchange IS recommended. 8. The rmmobthzed biocatalyst is now ready for use and can be transferred to the reactor or can be stored at 4°C until rt is needed

3.2. Encapsulation of Cells in Sodium Cellulose PDMDAAC After Preimmobilization of Cells 3.2.1. Preimmobilization of Cells

Sulfate/

1 Dissolve the sodium cellulose sulfate at a concentratron of 0.25% (w/v) by vigorous shaking m distilled water and autoclave the solutton 20 min at 121°C. 2 Dissolve the PDMDAAC at a concentration of 0.25% in distilled water and sterilize by sterile tiltratron. 3. Grow the cells up to the desired growth state. 4 Suspend the 40 mL of cells (typically 1J-2 5 g dry weight) in 60 mL of 0.25% (w/v) sodium cellulose sulfate solution and mix homogeneously by vtgorous shaking. 5. Add the 0.25% (w/v) PDMDAAC solution under stirring until the isoromc point 1sreached and a precipitate is formed. 6. Take the suspension up in a safety pipet and let the PEC (polyelectrolyte complex) precipitate settle m the safety prpet for 5 mm.

3.2.2. Encapsulation of Preimmobilized Cells in Sodium Cellulose Sulfate/PDMDAAC 1 Transfer the precipitate (from Section 3.2-l., step 6) to 200 mL of 4.1% (w/v) sodium cellulose sulfate solutton. 2. Suspend the coarse PEWcell network uniformly in the sodmm cellulose sulfate solution by stirring or shaking to ensure a homogeneous mixture. 3 Perform the next steps of the encapsulation as descrrbed m Sectron 3 l., steps 5-8 (see Notes 6,7).

3.3. Encapsulation

in Alginate/Poly-r-Lysine

1. Dissolve the alginate m water or a suitable buffer (for instance, physrological saline for animal cells) and autoclave at 100°C for 15 min (see Note 8).

Cells in Polyelectrolyte Complexes

313

2 Dissolve the CaCl, m water or a smtable buffer and stenhze by membrane tiltratron. 3. Grow the cells up to the desired growth state or dissolve the enzyme m a suitable buffer (see Note 9). 4. Suspend the cells homogeneously m the alginate solution. Depending on size, type, and vrability of the used cells, suspend OS-5 0 x lo3 cells/ml of alginate. 5. Drop the cell/alginate suspension into the O.lMCaCl, solution through a syringe needle or by the help of a syringe pump extrusion system. The CaCl* solutron should be stirred contmuously, but not too vigorously. The volume of the algmate beads should not exceed one-fifth the calcmm chloride solutron. 6 Leave the gel beads for 20 min to 1 h in the stirred CaCl, solution. 7. Decant the supernatant. 8 Wash the gel beads under stirring with the 0.05-0.075MCaC1, solution m water or the appropriate buffer. Use a fivefold excess of washing soluttons. 9. Remove the supernatant and drain as much away as possrble. 10. Treat 5 mL of gel beads for 6-8 min in 15 mL of poly+lysine solution (see Note 10). 11. Decant the supernatant. 12. Wash the beads with a suitable buffer containing 0.05-0.075M CaClz solution. 13. Remove the supernatant and treat the beads with a fivefold excess of 0.05M cttrate buffer, pH 7.4, for 5-6 mm to rehquefy the inner algmate core and prepare the microcapsules. 14. Wash the microcapsules twice with a suitable buffer. 15. The immobilized biocatalyst is now ready for use and can be transferred mto a suitable nutrtent or into the reactor or can be stored at 4°C until it IS needed 16. For special applications (implantation) the prepared microcapsules are covered with a third layer (see Notes 11-14). 17 For covering with algmate, remove the last wash buffer and wash the mrcrocapsules with the 0 05-0.075M CaClz solution. 18. Remove the calcium chloride solutron and treat the microcapsules for 5 min with a 0.03-O. 15% sodium alginate solution for 4 min. 19 Perform steps 10-15 (see Note 13).

3.4. Encapsulation

in ChitosadPolyanion

Complexes

1. Prepare a chitosan solution of a final concentration of 3% (w/v) by autoclavmg the corresponding amount of chitosan under water and dissolvmg by addition of concentrated citric acid (to give final concentration 0.26M) (see Notes 16-18). 2 Add calcium chloride to a concentratton of 0.45-l .5M (see Note 19). 3. Store the chitosan solution under nitrogen, 4. Dissolve alginate and glucose in distilled water and autoclave at 100°C for 15 min (see Note 17). 5. Grow the cells up to the desired growth state. 6. Suspend 1.5-3.0 g (dry weight) of cells homogeneously m 100 mL of chitosan/ CaC12 solution. 7. Drop the cellkhitosan suspension from a height of approx 15 cm into the 0.75% (w/v) algmate/glucose solution through a syringe needle or by the help of a

314

8 9 10.

11

Mansfeld and Dautzenberg syringe pump extrusion system. The alginate solution should be stirred contmuously, but not too vigorously (see Note 19). The volume of the chitosadalginate capsules should not exceed one-fifth of the algmate solution. Stir the mixture for a further 15 mm (see Note 20). Wash the capsules with distilled water for 5-l 0 mm The immobilized biocatalyst is now ready for use (see Note 2 1) and can be transferred into a suitable nutrient or mto the reactor or can be stored at 4°C until it is needed. For preparation of polyelectrolyte complex capsules a variety of other materials can be used (see Notes 22,23).

4. Notes The toxicity of the used polyions on enzymes or cells has to be proven if sensitive enzymes or cells are used. In some applications the VISCOUSliquid core has been shown to mfluence nutrient or oxygen transport to the cells, resulting in poor growth and productivity of the cells Low polyanion concentrations lead to low viscosities of the polyamoticell suspension, causmg difficulties m microcapsule preparation Weak or deformed capsules are formed that collapse because of breaking of the membranes, allowmg cells to penetrate mto the medium. If desired, stabrhzmg substances or nutrients may be added to the PDMDAAC solution without influencmg capsule formation; for instance, up to 10% glucose. However, it has to be taken into account that high concentration differences between capsule interior and outer medium cause shrinkage of the capsules If enzymes are to be encapsulated, PDMDAAC with higher mol wt, higher concentrations of sodium cellulose sulfate and PDMDAAC solutions and shorter residence times of the capsules m the PDMDAAC solution are recommended to diminish the degree of leakage and mactivation of the enzyme (23). The bmdmg of the enzyme to an msoluble carrier prior to encapsulation reduced the leakage rate of the enzyme remarkably. The capsule properties are dependent on substrtution and polymertzation degree of sodium cellulose sulfate, mol wt distribution of PDMDAAC, residence time m the polycation precipitation solution, added nutrients, and cell species used. Asci or pseudo-asci formmg species create more problems than single cells. With these species, a prennmobilizatron m a coarse network of polyelectrolyte complexes proves to be advantageous (see Section 3.2.). The preunrnobiltzation leads to sigmficantly increased capsule strength because the mcorporation of cells into the capsule membrane IS drastically diminished (12). In some cases, it proves useful to kill the membrane-standing cells by a prolonged incubation of the capsules in the cytotoxic PDMDAAC solution. The residence time m the PDMDAAC solutron determines the capsule membrane thickness. The substitution degree of sodium cellulose sulfate must be high enough to ensure a good water solubility, but not so high as to cause damage of cells. The mmimum degree of polymerization sodium cellulose sulfate is 200.

Cells in Polyelectrolyte Complexes

315

6. Instead of sodmm cellulose sulfate, potassmm poly(viny1 alcohol) sulfate can be used as polyanion (I). 7. As a concluding remark, it is recommended to adapt the described encapsulation process at a small scale to the applied cell species and the used capsule materials to obtain optimal results with respect to capsule properties, stabtlity, and performance. 8. Low concentrations of alginate yield solutions with low viscosities resulting m formation of irregularly shaped gel beads. Spherical beads can be obtained only tf alginate solutrons with viscosities >0.03Nslm2 are used. The upper vtscosity limit IS set by the need to extrude the solution through the apparatus mto the gelling solution. The choice of algmate concentration also depends on the type of cells to be encapsulated. 9. The nutrient medium should contain no divalent cations (I.e., Ca2+ or Mg2+), because gelling of the sodium alginate would occur 10. Wrth the alginate/poly-L-lysine system, capsule properties (mol wt cut off of the capsule membrane) are dependent on molecular weight and concentration of polyL-lysine, and reaction time of alginate with poly-L-lysme. Revtews describing this technology have been published (8,9). The optimum A4, of the poly-L-lysme is in the range between 15,000 and 25,000. At lower A4, no mtcrocapsules can be obtained in the following hquefication process, whereas at M, >35,000, mtcrocapsule walls with very high porostty are formed. The reaction time of the alginate wtth the poly+lysine should not be too short to allow formation of a suffictently thick and stable microcapsule membrane. Times between 6 and 10 mm are recommended. 11. For special applications (implantation), it is useful to cover these microcapsules with a third layer of, for instance, alginate (8), poly(ethylene imine) (3), or poly(ethylene oxide) (14) to overcome the problem of overgrowth with fibroblastor macrophage-like cells on the microcapsules because of interaction of cells with the positively charged surface in the case of the bilayered microcapsules. 12 This treatment creates an outer alginate membrane on the the poly+-lysme membrane because of ionic interaction between the negatively charged alginate and the positively charged poly+lysine 13. If producing trilayered microcapsules, it is preferable to perform the reliqueftcation of the inner alginate core after formation of the third layer. 14. If poly(ethylene imine) is used, a 0.2% solution should be applied according to ref. 3. The A4,of the poly(ethylene imine) should be in the range between 40,000 and 60,000. 15 The alginate/poly-L-lysme system proved to be very useful in encapsulation of animal and hybridoma cells. However, for encapsulation of faster growmg cells, like microbial cells, it is less suited. 16. Chitosan [(1,4)-2-acetamido-2-deoxy-S-D-glucan] is produced by deacetylation of the polysaccharide chitin. 17. Factors influencjng capsule performance and strength in the case of the chttosan/ alginate system are (2) concentration and molecular weight of chitosan, degree

Mansfeld and Dautzenberg

316

18 19

20

21

22. 23.

of acetylation of chitosans, degree of ester&anon of algmates, mannuronic to guluronic acid ratio of the used algmates, and reaction time. The concentration of chitosan should be high enough to generate regularly shaped capsules. Viscoseties of the final chitosan solution should be m the range of 0.75Ns/m2. Addition of a three- to fivefold excess of glucose over chnosan concentration is recommended. A deacetylation degree of 86% proved to be advantageous (2) Instead of citric acid, acetic acid can be used. Addition of calcium chloride to the core material improves the capsule stability significantly. By addition of calcium chloride, presumably an algmate layer on the polyelectrolyte complex membrane was formed, mcreasmg the strength of the capsules. The residence time of the capsules m the alginate solution has to be chosen depending on desired thtckness of the capsule membrane. Very thick layers of alginate around the polyelectrolyte complex capsules lead to hindered diffusion and reduce biocatalyst performance if microbial cells are to be encapsulated As described by Polk et al. and Tomida et al. (10,1.5), polyelectrolyte complex capsules with chitosan can also be prepared by droppmg a solution of a polyamon mto a chitosan solution. K-Carrageenan (IS) or alginate (10) can be used as polyamons. In this case the chitosan concentration should not exceed a concentration of 0 5% (w/v), whereas the core-forming algmate should be used at a concentration between 2 and 3% (w/v). Instead of algmate, tc-carrageenan (15), carboxymethylcellulose (7), or potassium poly(vmy1 alcohol) sulfate can be used to prepare polyelectrolyte complexes (1) Copolymers of acrylic acid, 2-hydroxyethylmethacrylate, methylmethacrylate as polyanion, and copolymers of N,N-dimethylammoethylmethacrylate, ethylmethacrylate, 2-hydroxyethylmethacrylate, and acrylic acid as polycations have also been applied to preparation of polyelectrolyte complex microcapsules (11)

References 1. Kokufuta, E., Himohashi, M., and Nakamura, I. (1987) Continuous column denitritication using polyelectrolyte complex-entrapped Paracoccus denztrificans cells. J Ferment Technol 65,359-361 2. Daly, M. M. and Knorr, D. (1988) Chitosan-alginate complex coacervate capsules. effects of calcmm chloride, plasttctzers, and polyelectrolytes on mechanical stability. Blotechnol. Progc 4,76-81. 3. Lim, F. and Sun, A M. (1980) Microencapsulated islets as bioartificial endocrine pancreas. Sczence 210,908-910. 4 Dautzenberg, H , Loth, F., Pommerening, K., Lmow, K.-J., and Bartsch, D. (1980) Microcapsules and process for the production thereof. Patent No DD 160 393 (Cl BOl J 13/02), GB 2 135 954 (Cl BOl J 13/02). 5. Jarvis, A. P. and Lim, F. (1984) Method for culturmg anchorage-dependent cells. Patent No. US 4,495,288 (Cl C12N 5/00). 6. Beaumont, M. D. and Knorr, D. (1987) Effects of immobihung agents and procedures on vtability of cultured cellery (Apium graveolens) cells. Biotechnol Lett 9,377.

Cells in Polyelectrolyte Complexes

317

7. Yoshtoka, T., Hirano, R , Shtoya, T., and Kako, M. (1990) Encapsulation of mammalian cell with chitosan-CMC capsule. Biotechnol. Bioeng 35,66-72. 8. Goosen, M. F. A. (1987) Insulin delivery systems and the encapsulation of cell for medtcal and industrial use. CRC Cnt. Rev. Blocompatibdity 3, l-24 9. Rupp, R. G. (1985), in Large-Scale Mammalian Cell Culture (Feder, J. and Tolbert, W. R. eds.), Academic, London, pp. 19-38. 10. Polk, A., Amsden, B., DeYao, K., Peng, T., and Goosen, M. F. A. (1994) Controlled release of albumin from chitosan-alginate microcapsules. J Pharm. Scz 83, 178-185. 11. Gharapetain, H., Malekt, M , O’Shea, G. M., Carpenter, R. C., and Sun, A M. (1987) Polyacrylate mtcrocapsules for cell encapsulation: effects of copolymer structure on membrane properties. Biotechnol Bzoeng. 30,775-779. 12. Fdrster, M., Mansfeld, J., Schellenberger, A., and Dautzenberg, H. (1994) Immobtltzatton of citrate-producing Yarrowia lzpolytica cells in polyelectrolyte complex capsules. Enzyme Microb Technol l&777-784 13. Mansfeld, J., Forster, M., Schellenberger, A., and Dautzenberg, H. (199 1) Immobtlization of invertase by encapsulation in polyelectrolyte complexes. Enzyme Microb Technof. 31,240-244

14. Sawhney, A. and Hubbell, J. A. (1992) Poly(ethylene oxtd)-graft-poly(L-lysme) copolymers to enhance the biocompatibility of poly(L-lysine)-alginate microcapsule membranes. Biomaterlals 13,863-870. 15 Tomida, H., Nakamura, C., and Kiryu, S. (1994) A novel method for the preparation of controlled-release theophylline capsules coated wtth a polyelectrolyte complex of k-carrageenan and chitosan. Chem Pharm. Bull. 42,979-98 1.

35 Coimmobilization Johanna

of Enzymes and Cells

Mansfeld and Horst Dautzenberg

1. Introduction Comnnobilization of cells and/or enzymes is performed for three main reasons: first, to enable cells to use other, nonmetabolizable substrates than the natural ones of the correspondmg strain (1,2); second, to enlarge the product spectrum by utilization of the catalytic capabilities of the coimmobilized enzyme or cell (3); and third, to simplify conventional two- or more step processes.A simplification of multiple-step processescan also be obtained by using recombinant strams containing the genes of the needed additional enzymes (4). For enzymes acting m sequence, coimmobilization creates a favorable microenvironment for the second enzyme by reducing time of diffusion of the substrate to the enzyme, thus increasing efficiency of the overall system. Since metabolic pathways include series of reactions, coin-mobilized multienzyme systems can be regarded as models of compartmentalized intracellular enzymes and in vitro models for cellular processes. In the case of coimmobilized cells, undefined or defined mixtures of cells have been used. Therefore, similarities with the use of mixed cultures in conventional fermentation processesexist. A broad variety of methods and materials have been described in the literature with respect to coimmobihzation of enzymes, enzymes and cells, or cells coimmobilized with other cells, reviewed in detail elsewhere (5,. Theoretically, an unlimited number of enzymes can be immobilized in the same microcapsule or gel bead or fixed onto the same surface and used as biocatalyst if suitable conditions can be found for coimmobilization and application of the biocatalyst. In practice, however, problems often arise because of strong deviations of pH and temperature optima, requirements of cofactors, stabilizing substances,growth factors, nutrients, and so on. All of these aspects have to be taken into account when designing a coimmobilized biocatalyst. From

Methods /n B/otechno/ogy, Vol 1 lmmobrlrzatron of Enzymes and Cek Edited by G F BIckerstaff Humana Press Inc , Totowa, NJ

319

320

Mansfeld and Dautzenberg

Furthermore, the optimum ratio between the connmobihzed species has to be determined. Another possibility for using two or more enzymes in one reactor is the separate immobilization of each enzyme on a suitable carrier material and the simultaneous use m the process. One of the methods presented in this chapter is microencapsulation. In comparison with gel entrapment as the most frequently used immobilization method, microcapsules have the advantage of much higher mechamcal stability and stability toward complexmg anions. Gel entrapment owes its popularity to its extreme simphcity and the low cost of the materials (see Chapter 1). Connmobilized enzymes and cells have been used in applications ranging from wine and beer production (6), ethanol production from whey permeate (Z) or cellobiose (21, to vz sztu oxygen supply (7) and citric acid production (8).

2. Materials 2.1. Coimmobilization

of Enzymes and Cells by Gel Entrapment

1. Sodium alginate (Kelco, Hamburg, Germany). 2. N-(3-dimethylaminopropyl)-N’-ethyl-carbod~~m~de~y~ochlo~de (EDC) (Merck, Rahway, NJ) (see Note 1). 3. N-hydroxysuccuumide (Merck). 4. Enzyme solution: Prepare 50-100 mg/mL in water or an appropriate buffer for the particular enzyme (see Note 2). 5. 0. IM CaC& solutron m water or a suitable buffer (see Note 3). 6. Cell suspension at the desired growth state and in appropriate medmm for the particular cells (see Note 4). 7. Syringe supplied with a 0.2-l -mm inner diameter needle or suitable syringe pump extrusion system. For preparation of capsules with small diameter, an airJet droplet generator system is recommended. 5. 0.05M CaCl, solution in water or a suitable buffer,

2.2. Coimmobilization of Enzymes and Cells via Microencapsulation in Polyelectrolyte Complex Capsules 2.2.1. Immobilization of the Enzyme on an insoluble Support 1. Macroporous poly(aminomethy1 styrene) (Wofatrt UF 93; Chemie AG, Bitterfeld, Germany, see Notes 5,6), crosslinked with 10% divinyl benzene, content of amino groups 3-4 mmol/g carrier, bead diameter 10-20 urn. 2. Glutaraldehyde solution 2 5% (v/v) in a suitable buffer (see Note 7) 3. Enzyme solution: Prepare 2-50 mg/mL m an appropriate buffer for the enzyme. In relation to the support, use 1O-200 mg/g (dry weight) of support material (see Note 2). 4. Buffer containing l.OM NaCl. Use the appropriate buffer for the particular enzyme.

Coimmob//ization of Enzymes and Cells

321

2.2.2. Preimmobilization of Cells and Carrier-Bound Enzymes Prior to Encapsulation in Sodium Cellulose Sulfate/PDMDAAC 1. 0.25% (w/v) Solution of sodium cellulose sulfate m distilled water (see Chapter 34, Section 2.1.). 2. 0.25% (w/v) Solution of poly(dimethyldiallylammonium chloride) (PDMDAAC) in distrlled water (see Chapter 34, Section 2.1.). 3. Cell suspension at the desired growth state and surtable medium and carrier-bound enzyme.

2.2.3. Encapsulation of Preimmobilized Cells/Carrier-Bound in Polyelectrolyte Complex Capsules

Enzyme

1. 4.0-4.2% (w/v) Solution of sodium cellulose sulfate in distilled water (see Chapter 34, Section 2.1.). 2. 1.2% (w/v) Solution of PDMDAAC in distilled water (see Chapter 34, Section 2.1.). 3. Syringe with 0.2-l .Omm inner diameter needle or suitable syringe pump extrusion system. For preparation of capsules with small diameter, an aqet droplet generator system is recommended.

2.3. Coimmobilization of Enzymes and Cells by Binding of Enzymes to the Cell Surface 1 2 3 4. 5

Compressed living or dead cells depending on the particular apphcation. Sorbitol powder, Enzyme solution: Prepare 50-100 mg/mL in a small amount of water (see Note 2). 25% (v/v) Glutaraldehyde solution 2% (w/v) Tannin solution.

3. Methods 3.1. Coimmobililation (see Note 8, ref. 9)

of Enzymes and Cells by Gel Entrapment

1. Dissolve 500 mg sodium algmate m 10 mL of water (see Note 2). 2. Dissolve N-hydroxysuccinimide to give a final concentration of O.lM. Dissolve EDC to give a final concentratron of 0. 1M. Dissolve them separately in a small volume of water (see Note 9). 3. Combine the 10 mL of alginate solution from step 1 with 5 mL of the EDC solution from step 2 and 5 mL ofN-hydroxysuccinimide solution from step 2. Stir the mixture gently for 15 mm-1 h at room temperature. The pH should be kept at 4.75 during that time (see Note 10). 4. Dissolve the enzyme in a small volume of water. 5. Add 5 mL (50-100 mg/mL) of the enzyme to the mixture and allow the coupling to proceed overnight in the cold room. Use gentle stirring with a magnetic sturer or end-over-end mixer. With sensitive enzymes, the activated alginate should be dialyzed against water prior to the coupling procedure to remove excess of EDC, tsourea, and IV-hydroxysuccimmrde.

322

Mansfeld and Dautzenberg

6. Grow the cells to be coimmobthzed up to the desired growth state m suitable, compatible media. 7. Add a further 500 mg of sodium algmate m a small volume of water or a surtable buffer to the enzyme-alginate solution. The volume of water will depend on the volume of cells to be added and should be calculated to ensure that alginate concentration after addition of cells is 2%. 8. Suspend 1.5-2.5 g (dry weight) of cells homogeneously in the enzyme-algmate solution. The final alginate concentration should preferably be 2% (see Notes 4,11). 9. Dissolve the CaC12 m water or a suitable buffer and sterilize by membrane filtratron. 10 Transfer the cell-enzyme-algmate suspensron to a syringe or the syrmge pump extrusion system. Drop the cell-alginate suspension slowly from a height of approx 15 cm into the 0. 1M calcium chloride solution through the syringe needle or by the help of a syringe pump extrusion system The CaCI, solution should be stirred continuously but not too vigorously The volume of the alginate beads should not exceed one-fifth of the calcium chloride solution Il. Leave the gel beads for 20 mm-1 h in the stirred CaCl* solution. 12. Decant the supernatant. 13. If necessary, the gel beads can be washed with a 0.05M CaCl, solution. 14 The coimmobilized biocatalyst is now ready for use and can be transferred to the reactor or can be stored in a 0.05M CaCl, solution at 4°C until it is needed.

3.2. Coimmobilization of Enzymes and Cells via Microencapsulation in Polyelectrolyte Complex Capsules 3.2.1. immobilization of the Enzyme on an Insoluble Support (see Notes 5,6) 1. Suspend the polystyrene beads m 15 mL of 2.5% glutaraldehyde solution per 1 g of dry polystyrene carrier matenal. 2. Stir the mixture for l-l .5 h at room temperature (see Note 12). 3 Remove the glutaraldehyde solutron after finishing the activation step. 4. Wash the support material 5-6 times with water until the washing soluttons are free of glutaraldehyde. Use 2,4-dmitrophenylhydrazine or any other reagent specific for aldehydes as an mdicator 5 Dissolve 240 mg/mL of enzyme in buffer (see Note 13). The use of an excess of enzyme 1srecommended and typically 10-200 mg of enzyme is added/l g (dry weight) of support material. 6 Add the enzyme and stir the mixture for at least 3 h up to overnight (see Note 14). 7. Decant the supernatant containing unbound enzyme solutron. 8. Wash the enzyme-carrier complex several times with an appropriate buffer supplemented with 1M NaCl to remove any unbound enzyme, and on the last washmg steps (3-4 times) only with buffer. 9 The enzyme-carrier complex can now be resuspended m an appropriate buffer and stored at 4°C until tt is needed.

Colmmobilization

of Enzymes and Cells

323

3.22. Preimmobilization of Cells and Carrier-Bound Enzyme Prior to Encapsulation in Sodium Cellulose Sulfate/PDMDAAC (see Notes 15,16) 1. Dissolve sodium cellulose sulfate and PDMDAAC as described in Chapter 34, Section 3.2 1. 2. Suspend the cells (10-20 g dry weight/l L of capsule material) and a sufficiently high amount of enzyme-carrier complex homogeneously in the 0.25% sodium cellulose sulfate solution by vigorous shaking. 3. Perform the steps of preimmobilization and encapsulation of preimmobilized cells and enzyme as described in Chapter 34, Secttons 3.2.1. and 3 3 2.

3.3. Coimmobilization of Enzymes and Cells by Binding of Enzymes to the Cell Surface (12) 1. Dehydrate the compressed cells by addition of sorbitol powder. Use an amount that is 10% the weight of the cells. 2. Stir the cell/sorbitol mixture for 10-20 min. 3. Remove excess water with a filter press. 4. Resuspend the dehydrated cells in the enzyme solution (see Note 17). Typically, the enzyme concentration is 50-100 mg/mL and 100 g cells are resuspended m 35-50 mL of enzyme solution. 5 Add the 2% tannin solution. Use a four- to fivefold amount in comparison to the amount of enzyme solution and add glutaraldehyde (to give a final concentratton of 0.1%) to the resulting cell suspension. This will crosslink the enzyme molecules and bind the enzyme layer to the cell wall. 6. Allow the resulting mixture to react for at least 34 h under shaking. 7. Wash the comunobihzed biocatalyst with an appropriate buffer. 8. The coimmobilized biocatalyst is now ready for use and can be transferred to the reactor or can be stored at 4’C until it is needed,

4. Notes 1. For binding of enzymes, water-soluble carbodiimides are preferred Instead of EDC, N-cyclohexyl-N’-2[-(N-methylmorpholino)-ethyl]carbodiimide p-toluenesulfonate salt (CMC) can be used. However, EDC usually gives better results. The purity of the carbodiimide is extremely important. Old samples should not be used. 2 Usually the activation is performed in water. If necessary, buffers can be used, provided no amino, phosphate, or carboxyl groups are present since they compete with the reaction. Note that the enzyme concentration and the total amount of enzyme applied must be determined for each case because enzyme solublhty and other factors will have a great influence. 3. CaC12 conce rations between 0.05 and 1Mcan be used. Using higher concentrations can speed up the hardening process of the gel beads. 4. The nutrient media or buffers used should not contain divalent cations (i.e., Ca*+ or Mg2+), because gelling of the sodium algmate will occur.

324

Mansfeld and Dautzenberg

5 In princtple, the encapsulation of enzymes in sodium cellulose sulfate/ PDMDAAC polyelectrolyte complex membranes is possible (IO, II). Because cell encapsulation requires different conditions in terms of capsule materials, concentration of polyton solutions, and capsule formation process, leakage of enzyme cannot be avoided in this case. The leakage of enzyme from the capsules can be overcome by covalent bmdmg of the enzyme to the free hydroxyl groups of sodmm cellulose sulfate and subsequent capsule formation with the modified sodium cellulose sulfate (8). 6. For the support material, a wide range of materials are available and it is possible to select one well suited for immobilization of the desired enzyme It is also possible to chose a particular method of activation to suit the enzyme used. For nnrnobilization of carbohydrate-sphtting enzymes, the procedure described in this chapter is well suited. The type of support activation can also be chosen to suit the enzyme used. 7. A slightly alkaline buffer is recommended to allow for a sufficient activatton of the carrier material, preferably 0. 1M phosphate buffer, pH 7 5 8 The immobilization of enzymes by gel entrapment has been shown to be less favorable because of severe enzyme leakage from the gel. This can be overcome by covering the gel bead with an acrylatelmethacrylate copolymer, for instance Eudragit, Rohm Pharma, or by covalently binding the enzyme to the gel-forming material (2) However, by crosslmkmg the enzyme, for instance with glutaraldehyde, enzyme leakage can be minimized. 9 The amount of carbodnmide is dependent on the concentration of carboxyl groups present at the gel-forming material. It should be greater than stotchiometric amounts An excess between 10 and 100X is recommended. Usually a concentration of O.lM is sufficient. 10. The pH optimum of the acttvation and coupling process of the alginate is m the range between 4.5 and 6.0. 11. Algmate concentrattons between 1.5 and 4% are used. Low concentrations of alginate yield soft, irregularly shaped gel beads Spherical beads can only be obtained when alginate solutions with viscosities >0.03Nslm2 are used. The upper viscosity limit is set by the need to extrude the solution through the needle or apparatus into the gelling solution The choice of algmate concentration depends, furthermore, on the type of cells to be entrapped. 12. Do not use a magnetic stirrer, because the carrier material may be subject to grinding 13. Since the amino groups of the enzyme are reacting with the glutaraldehyde-acttvated carrier, the enzyme should be dissolved m a buffer with a pH around 7 0 or slightly alkaline depending on the stability of the used enzyme. 14. The temperature of coupling the enzyme to the carrier can be selected depending on the stability of the enzyme. A period of 3 h is usually sufficient. 15. The capsule properties are dependent on substitution and polymerization degree of sodium cellulose sulfate, molecular weight distribution of PDMDAAC, residence time in the polycation precipitation solution, added nutrients, and cell spe-

Coimmobilization

of Enzymes and Cells

325

ties. Asci or pseudo-asci forming species create more problems than single cells With these species, a preimmobilization in a coarse network of polyelectrolyte complexes proved to be advantageous (see Section 3.3.2.). The preimmobilization leads to significantly increased capsule strength since the incorporation of cells into the capsule membrane 1sdrastically diminished (s). In some cases, it proved useful to kill the membrane-standing cells by a prolonged incubation of the capsules m the cytotoxic PDMDAAC solution The residence time in the PDMDAAC solution determines the capsule membrane thickness. The degree of substitution of the sodium cellulose sulfate must be high enough to ensure a good water-solubility, but not too high to cause damage of cells. The minimum degree of polymerization of sodium cellulose sulfate is 200. 16. The toxicity of the polyions used on enzyme or cells has to be proven if sensitive enzymes or cells are used. In some applications, the viscous liquid core has been shown to influence nutrient or oxygen transport to the cells, resulting in poor growth and productivity of the cells. 17. The amount of water m the enzyme solution should be carefully chosen so that nearly all of the water is taken up by the dried cells.

References 1. Hahn-Hagerdal, B. (1985) Comparison between immobilized Klyuveromycesfragilts and Saccharomyces cerevisiae co-immobilized with P-galactostdase, with respect to continuous ethanol production from concentrated whey permeate. Biotechnol. Bioeng 27,914-916.

2. Hahn-Hagerdal, B. (1984) An enzyme co-immobilized with a microorganism: the conversion of cellobiose to ethanol using P-galactosidase and Saccharomyces cerevisiae in calcmm alginate gels. Brotechnol. Btoeng. 26, 77 l-774. 3. Martin, C. K. A. and Perlman, D. (1975) Conversion of L-sorbose to 2-keto-gulonic acid by mixtures of immobilized cells of Gluconobacter melanogenus IF0 3293 and Pseudomonas species. Eur J. Appl. Microbial. 3,91-95. 4. Janse, B. J. H. and Pretorius, I. S. (1995) One-step enzymatic hydrolysis of starch using a recombinant strain of Saccharomyces cerevisiae producing a-amylase, glucoamylase and pullulanase. Appl. Mtcrobiol. Biotechnol. 42, 878-883. 5. Hahn-Hagerdal, B. (1983) Co-immobilization involving cells, organelles, and enzymes, in Immobiltzed Cells and Organelles, vol. 2 (Mattiasson, B., ed.), CRC, Boca Raton, FL, pp. 79-94. 6. Hartmeier, W. (1983) Preparation, properties and possible applications of co-immobilized biocatalysts, in Enzyme Technology (Lafferty, R. M., ed.), Springer Verlag, Berlin, pp 207-2 17 7. Adlercreutz, P., Hoist, O., and Mattiasson, B. (1982) Oxygen supply to immobilized cells: 2. studies on a coimmobilized algae-bacteria preparation with in sttu oxygen generation. Enzyme Mtcrob. Technol. $395400. 8. Mansfeld, J., Fdrster, M., Hoffmann., T., Schellenberger, A., and Dautzenberg, H. (1995) Coimmobilization of Yarrowia lipolytica cells and invertase in polyelectrolyte complex microcapsules. Enzyme Mtcrob. Technol 17, 11-17.

326

Mansfeld and Dautzenberg

9. Cuatrecasas, ?. and Par&h, I. (1972) Adsorbents for affinity chromatography. Use of N-hydroxysuccmimide esters of agarose. Bzochemzstry 11,229 l-2305. 10. Mansfeld, J., Fdrster, M., Schellenberger, A., and Dautzenberg, H. (1991) Immobihzation of invertase by encaplsulation in polyelectrolyte complexes. Enzyme i&rob Technol. 13,240-244.

11. Ristau, O., Pommerenmg, K , Jung, C., Rem, H., and Scheler, W (1985) Aktivitatseigenschaften von Urease m Mikrokapseln. Blamed Biochzm Acta 44, 1105-1111 12 Hartmeier, W. (1990) Co-immobilization Blotechnol 4,399-407.

of enzymes and whole cells Food

13. Hartmeier, W. and Hemnchs, A (1986) Membrane enclosed algmate beads containing Gluconobacter cells and molecular dispersed catalase. Biotechnol Lett 8, 567-572 14 Lee, J M and Woodward,

J. (1983) Properties and apphcation of imrnobdlzed mobilis in calcium alginate. Bzotechnol Bzoeng 25,2441-245 1

P-D-glucosidase

coentrapped with Zymomonas

36 Adsorption

of Lipase on Inorganic Supports

Jose V. Sinisterra 1. Introduction Adsorption is a very economical procedure for immobilization of enzymes. The stability of the adsorbed enzyme derivative will depend on the strength of the noncovalent bonds formed between the support and the amino acid residues on the surface of the protein, Two principal types of bonds can be formed between the enzyme and the inorganic support and they are electrostatic bonds (charge-charge interaction) and hydrogen bonds. The number and nature of these bonds is controlled by the isoelectric point of the protein, and by pH at which the immobilization occurs as these two factors determine the respective surface charges. The main source of surface charged groups on the protein are lys, arg, asp, and glu residues, which are charged at neutral pH. In addition hydrogen bond interactions may involve the hydroxyl groups of ser and tyr residues and the thiol group from cys residues. An important consideration when undertaking immobilization of enzymes is the physical properties of the support (see Chapter 1). In particular, the average pore diameter and surface area can have a significant effect on the final properties of the immobilized enzyme, and therefore it is essential to determine the optimum characteristics for the enzyme and application under investigation. For example, it is known that the pore diameter of the support should be at least 1.5-fold greater than the mam axis of the protein in order to ensure adequate accessof the enzyme molecules into the support. The principal disadvantage associated with adsorption as a method of immobilization is that the interactions between support and enzyme are much weaker than in the case of the covalent binding. Therefore, adsorbed enzymes are easily desorbed from the support surface. However, adsorbed enzymes have been found to be very useful m several areas of biotechnology (see Chapter 1). In From

Methods m Biotechnology, Vol. 1 Immobrlrzahon of Enzymes and Cells Edlted by G F BIckerstaff Humana Press Inc , Totowa, NJ

327

Sinisterra

328 Table 1 Half-Life of Immobilized

Lipase

Enzyme

Derivative9 Support material SiOZ

Soluble enzyme Immobthzed by adsorption

Half-life (h)

A1203

CPGl CPG2 Immobihzed

covalently using TCT

s102 A1203

CPG 1 CPG2 Immobtlized

covalently using GLY

s102 A1203

CPGl CPG2 aDetalls of covalent immobdlzatlon

05 2.5 2.5 5 5 16 38 28 20 17 20 12 20

are grven m Chapter 37

Table 1, half-life studies are shown for lipase from Can&a cylindracea mmobilized on several inorganic supports both covalently and by adsorptton. It can be seen that covalent immobilization provided greater stability than imtnobihzation by adsorptron, but the adsorbed enzyme IS significantly more stable that the soluble enzyme. 2. Materials (see Notes 1,2) 2.1. lmmobiliza tion 1. Si02 Kieselgel60 (Merck, Rahway,NJ). 2. 3 4. 5

Al203 Aluminum 60 (Merck). Controlled-pore glass CPGl (BDH, London, UK) CPG2 (Sigma, St. Louis, MO) Sepiohte (Tolsa, Madrid, Spain). Lipase (E.C.3.1 1 3) solution from C cylzndrucea, Type VII, containing 900 U/mg solid using olive oil as substrate (Sigma): Prepare a solution of 5 mg/mL m 0 lMTris-HCl buffer, pH 7.0.

2.2. Determination

of Half-Life

of immobilized

Enzyme

1 Immobilized enzyme prepared as detailed in Section 3 1 and Chapter 37, Section 3.1. 2. O.lM Tris-HCl buffer, pH 7.0.

3. Waterbath at 50°C, and a water bath at 37’C. 4. Olive oil substrate (Sigma). 5. 0.2M NaOH solution.

Adsorption of Lipase

329

Table 2 Physical Properties of the Inorganic Supports support s102 A1203

CPGl CPG2 Sepiolite

Particle diameter, pm

Pore diameter, nm

Surface area, m2/g

Pore volume, cm3/g

15-40 63-200 40-200 80-120 63-l 80

3-20 4-10 127 300 6-10

316 59 24 7 258

0 79 0241 1 19 0 76 0.44

3. Methods 3.1. lmmobiliza tion 1 2 3. 4.

Add 5 mL of bpase solution to 2 g of solid support m a glass-bottomed flask Stir the mixture for 2 h at 4°C. Decant the supernatant containing unbound enzyme. Rinse the immobilized enzyme with several washes of Tris-HCl buffer to remove loosely-bound enzyme 5. Lyophihze the mixture for 48 h. 6 Store the immobilized enzyme derivative at 4°C until required (see Notes 3,4)

3.2. Determination

of Half-Life

of Immobilized

Enzymes

1. Suspend 0.5 g of the immobilized enzyme in 50 mL of Tris-HCl buffer and mcubate the mixture at 50°C. 2. At suitable time intervals (e.g., every 30 min) withdraw 10 mg of immobilized enzyme. Cool the aliquot to 37°C. 3. Assay the residual enzyme activity by adding a volume of immobilized enzyme to 10 mL of substrate emulsion of olive oil at 37’C (see Note 5). 4. Incubate the immobilized enzyme and olive oil for 15 mm at 37°C and maintain the pH using a pH titrator with NaOH solution to determine the release of fatty acids.

4. Notes 1. Inorganic supports: This methodology can be carried out with different inorganic carriers. Four different supports are selected as the most interesting carriers for biotechnology applications 2. The physical properties of the supports are described in Table 2. All the supports are mesoporus (pore diameter >8 nm) to retain enzyme molecules. 3. In these conditions, all the enzyme molecules remain on the solid surface, but the stability of the enzymatic derivative m hydrolysis conditions is similar to the stability obtained for the native enzyme (Table 1, ref 1).

330

Sinis terra

4. The enzymatic derivative is useful m the esterification and transesteritication reactions m slightly hydrated organic solvents, such as l,l, 1-trichloroethane, isooctane, toluene, and so on. 5 The volume of enzyme taken should be determmed experimentally by first undertaking a preliminary assay to establish the level of immobilized enzyme activity Then ahquots for experiments may be scaled up or down as required.

References 1 Moreno, J M and Sinisterra, J. V. (1994) Immobtlization of hpase from Candzda cylzndracea in inorganic supports. J Mol. Catal 93,357-369. 2. Moreno, J M (1994) Utilizacton de nuevos derivados mmovihzados de hpasa de Candzda cylzndracea en la obtencion de S(+) antuntiamatortos no esteroidicos PhD Umversidad Complutense, Madrid, Spain

37 Immobilization of Enzymes on Inorganic Supports by Covalent Methods Jose V. Sinisterra 1. Introduction Covalent binding of an enzyme to a support is probably the most interestmg method of immobilization from an industrial point of view. In this methodology, the activated groups of the support react with some external functional groups of the protein (see Chapter 1). Generally, reaction between the protein and activated groups of the support takes place by means of surface amino and carboxyl groups because they are accessible and are not normally involved m essential structural or catalytic activity. Immobilization by covalent methods must be carried out in chemical condittons that are compatible with the stability properties of the proteins. Generally, this involves two steps, first, acttvation of the support and second, coupling of the enzyme to the activated support. Actrvated supports have unstable and reactive functional groups, and therefore, unless stepsare taken to preserve the activated support, immobilization of the enzyme should follow immediately after activation of the support. Several covalent immobilization methodologies have been described m the literature (l-3), but the selection of methodology is dependent on the nature of the support and of the enzyme (see Chapter 1). In this chapter, immobilization of lipase from Candida cyhndracea (a glycoprotein) and penicillin G acylase from Kluyvera citrophyla (a dimeric enzyme in which the active site 1sformed between both subunits) is described to illustrate the methods. Two methods are provided in this chapter, immobilization using trichlorotriazine (TCT) (see Fig. l), and immobtlization using glycidol methodology (see Fig. 2). Immobilization of enzymes using TCT chemistry gives excellent results m the tmmobilization of monomeric proteins and glycoproteins. Immobilization of enzymes From

Methods m Btotechnology, Vol 1 Immobr/rzat/on of Enzymes and Cells Edtted by G F BIckerstaff Humana Press Inc , Totowa, NJ

331

Sinis terra

332 I-

on

N

Cl

Cl

LHa

r’o“c “v”

I

Cl

a

NH*-ENZYME

kTa N

0

Fig. 1. Immoblhzatlon

NH-ENZYME

of an enzyme using trichlorotriazine.

using glycidol methodology is recommended if the enzyme is very sensitive to HCl liberated during immobilization using TCT, but It IS not recommended if the enzyme has catalytic functional groups that are sensitive to the redox agents used in this methodology (NaI04 and NaBH4). 2. Materials

2.7. Activation of the Support Material 1, Alumina-60 or Silica-60 (Merck). 2. 2,4,6-trichloro-1,3-5-triazine (TCT)(cyanuric acid, Merck, Rahway, NJ) Caution: This compound is toxic. It must be used in dry conditions (see Note 1)

333

Covalent Methods of Immobilization

G C si-oG G i -OH

+

CHZ-CH-CHZOH

CH2-CH(OH)-CH20H

i-o-

i -0

CH2-HC=O

-CH2-HC=N-ENZYME

Fig, 2. Immobihzatton

Na104 + CO2

+ Hz0 NH2-ENZYME

I

of an enzyme using glyctdol.

3 Toluene (see Note 2). 4. Triethylamine.

2.2. Immobilization

of Lipase

1 Lipase from C cyhndrucea, type VII, containing 900 U/mg solid using olive oil as substrate (Sigma, St. Louis, MO). 2 O.lMTris-HCl buffer, pH 8.0.

2.3. Immobilization

of Penicillin

G Acyiase (4)

1. Pemcillin G acylase from K cytrophilu (Antibiotico, Leon, Spain): Prepare a solution of 80 mg/mL in O.lM phosphate buffer, pH 7.0. 2. O.lMphosphate buffer, pH 7.0. 3. Penicillin G sulfoxide (Antibiotico): Prepare a solution of 240 mg in 20 mL water.

2.4. Activation

of the Support

Material Using Giycidoi

1. Alumina-60 or Silica-60 (Merck). 2. Glycidol (2,3-epoxipropanol, Aldrich): Prepare 2M glycidol in 0 16M NaOH solution. 3. Sodium borohydride (Merck). 4. 0.16M sodium hydroxide solution.

2.5. immobilization

of Lipase on Giycidoi-Activated

Support

1. Lipase of C. cylindracea, type VII, containing 900 U/mg solid using olive oil as substrate (Sigma): Prepare 20 mg/mL m O.lM Tris-HCl buffer, pH 8.0. 2. O.lMTris-HCl buffer, pH 8.0.

334 3. Methods 3.1. Activation

Sin&terra of the Support

Material (see Fig. 7)

1. Dry the inorganic support by heatmg the solid m an oven at 150°C for 4 h (see Note 3) 2 Set up a dry 1-L, glass, round-bottomed flask with a reflux condenser (see Note 4) on a magnetic stirrer with heating facility. 3 In the nozzle of the refluxer, place a glass tube with anhydrous CaC12 to avoid hydration of the reaction mixture by water in the atmosphere 4. Place 50 g of the dry morgamc support m the flask and suspend m 100 mL of dry toluene (see Note 5). Stir the suspension. 5 Add 15 g of TCT (see Note 6) and 30 mL of triethylamme (see Note 7) to the suspension. The amine is added to react with the HCl formed during the activation of the support 6. Connect the flask to the reflux condenser with the calcium chloride glass tube and heat the mixture until a gentle reflux is obtained. Continue to heat the suspension and stir magnetically for 4 h. 7. Allow the mixture to cool to room temperature, then filter by vacuum filtration using a Buchner flask and filter. 8. Wash the solid with 2 x 10 mL of dry toluene (see Note 8) to remove unreacted TCT and trrethylamme Filter the solid by vacuum filtration as before 9. Wash the solid with 3 x 100 mL of dry acetone (see Note 9) to remove any residual organic compounds m the solid. Filter the solid by vacuum filtration as before. This step is very important to avoid possible mactivation of enzymes by residual organic compounds 10. Store the solid at room temperature m dry conditions (see Note 10).

3.2. immobilization The immobrlization

of Lipase to TCT-Activated reaction

involves

Support (see Table 7)

the amino groups from lysine resi-

dues (see Fig. 1). 1. Mix 1 g of TCT-activated inorganic support with 10 mL of 80 mg/mL enzyme m O.lM Trts-HCl buffer, pH 8.0, m a 50-mL glass flask. 2 Stir the mixture for 3 h at 4°C 3. Filter the imrnobtllzed enzyme by vacuum filtration as before and wash with 20 mL of the same Tris buffer and then with 2 x 20 mL of distilled water. 4. Store the munobtllzed enzyme derivative at 4°C.

3.3. Immobilization of Penicillin on TCT-Activated Support (4)

G Acylase

1. In a glass-bottomed flask, mix 1 .OmL pemcillin G acylase with 4.3 mL distilled water and 5 mL penicillin G sulfoxtde (see Note 1 1), and stir for 10 mm at room temperature. Penicillin G sulfoxide serves to protect the active site of the acylase enzyme.

335

Covalent Methods of Immobilization Table 1 Catalytic Inorganic

Efficiency Supports

of Lipase immobilized Activated with TCT

Support material A1203

Si02 CPGl CPG3

on Different

Enzyme bound, mg/g solid

Catalytic efficiency, %”

78 113 121 97

26 44 16 25

OThe catalytic efficiency is obtained comparing the enzymatic activity of 1 g of immobilized derivative and an equivalent amount of grams of the native enzyme in the hydrolysis of olive oil (3) 2. Add 0.5 g of the TCT-activated support and mix with the solution Stir the mixture for 1 h at 4°C. 3. Filter the immobilized enzyme derivative by vacuum filtration as before and wash five times with 5 mL of 0. 1M phosphate buffer, pH 7.0, to remove the suboxtde inhibitor. 4. Store the immobilized enzyme derivative at 4°C. The immobilization yteld is typically 20-40% of the enzyme added,

3.4. Activation

of the Support

Material Using Glycidol

1. Place 1 g of support material solid (see Note 12) mto a lOO-mL glass flask and add 25 mL of 2M glycidol in 0 16A4 NaOH solution to the flask. 2. Add 150 mg of sodium borohydride to the solution. This compound serves as an antioxidant. 3. Stir the mixture magnetically at around 900 rpm for 24 h at 25°C. 4. Filter the mixture as before and wash with distilled water until the pH of the filtrate returns to pH 7.0 (see Fig. 2). 5. Suspend the solid in 10 mL of distilled water. Add 2.5 mL of 0 1 1MNaI04 solution to generate aldehyde groups on the support. Stir the mixture for 4 h at 25°C. 6. Wash the support solid with 2 x 10 mL of distilled water and store the solid at 4°C.

3.5. Immobilization

of the Lipase on Glycidol-Activated

Support

1. Mix 1 g of glycidol-activated solid with 20 mL of 20 mg/mL lipase solution. 2. Stir the mixture for 3 h at 25°C (see Fig. 2). 3. Filter the immobihzed enzyme derivative by vacuum filtration as before and wash with 2 x 20 mL of 0. 1M Trts-HCl buffer, pH 7.0. 4. Store the immobilized enzyme at 4°C. The immobilization yield is typically 20-40% depending on the degree of support activation (1).

Sinis terra

336 4. Notes 1. The hydrolysis of TCT produces HCl.

2. Use toluene from a new, unopened bottle. Toluene from old and open bottle must

first be dried over molecular sieve overnight 3. The adsorbed water must be removed to avoid hydrolysis of TCT, which would

4. 5.

6 7

8

9.

10

11.

12.

produce 2,4,6-trihydroxy-1,3,5triazine because this 1s inactive for support activation. All the glass material must be completely dry to avoid hydrolyses of TCT. Benzene, dtmethylsulfoxide, or dry 1,Cdtoxane can be used, but toluene 1s the most useful because it has a relative high boiling point (110 6”C), whtch determines the reaction temperature under reflux conditions. Also, it can easily be removed by filtratton and tt has relative low toxtctty. TCT should be qutckly weighed to avoid the hydrolysis of the product by water m the atmosphere Use safety glasses and a protective mask against acids Other tertiary aliphatic amines or pyridine can be used instead of triethylamme. Nevertheless, thts amme gives the best results. Secondary and primary ammes cannot be used because they react with TCT to produce unreactive compounds. The sohd ts suspended in 100 mL of dry toluene and gently stirred to put the solid m contact with the liquid The suspension is allowed to stand for 5 min, then IS filtered by vacuum, and the same procedure is repeated one more time Washing of the solid with dry acetone 1s carried out m the same expertmental condtttons as that described above for toluene The degree of acttvatton (mol of TCT bonded/g sohd) can be determined by tttratton of chloride liberated m the hydrolysis of the activated support, using the following procedure: Mix 0.1 g of the activated support with 10 mL of 0 1N NaOH solution and star the mtxture for 2 h at room temperature. Mix 1 mL of the solutton with 10 mL of O.lM phosphate buffer, pH 7 0, containing 0 05 g of K2Cr207. Tttrate with O.OlMAgNOs solution until a red precipitate ofAg2Cr207 forms m the presence of fluorescent as indicator (1). This 1s the endpoint of the tttration The number of moles of chloride obtained are equivalent to the moles of TCT bound to the morgamc support. Pemcillm G acylase is a dimertc enzyme (subumts a and p), whose active site IS located between both subunits. Therefore, immobilization must be carried out m the presence of an inhibitor, penicillin G suboxide, to block the active site and avotd deactivation of the enzyme during nnmobthzatton process by separation of the subumts c1and p. The solid support can be washed with 20 mL of distilled water/gram of solid and then with 2 x 20 mL of dry acetone tf the solid was obtained from an old bottle. Thus treatment cleans the solid

References 1 Moreno, J. M (1994) Utilization de nuevos dertvados inmovthzados de hpasa de Candida cyhdracea en la obtencion de S(+) antnnflamatorios no esterotdtcos PhD Thests. Umversidad Complutense, Madnd, Spain.

Covalent Methods of Immobilization

337

2 Mosbach, K (1978) in Methods in Enzymology, vol. 135 (Mosbach, K., ed ), Academic, London. 3 Moreno, J. M. and Sinisterra, J V. (1994) Immobilization of lipase from Candzda cylindruceu in inorganic supports. J Mol. Catal. 93, 357-369. 4. Aparicio, J. and Smisterra, J. V. (1993) Influence of the chemical and textural properties of the support in the immobilization of penicillin G-acylase from Kluyvera citrophila, on inorganic supports. J. Mol. Catal. 80,269-276.

Use of Divalent Metal Ions Chelated to Agarose Derivatives for Reversible Immobilization of Proteins Robert R. Beitle, Jr. and Mohammad

M. Ataai

1. Introduction Immobilization of a protein through coordinate bonds formed with divalent metal ions (e.g., Me(II), Cu(I1)) is becoming an attractive alternative to covalent coupling chemistries. This is primarily a result of the reversible nature of the immobilization, because the protein may be easily removed from the support matrix through interruption of the protein-metal bond. The primary requirement for immobilization via Me(I1) interaction is surface histidine residues (L-4). When such residues are absent, genetic engineering may be used to enhance metal affinity by incorporation of histidine containing metal affinity tails (5-S). Thus proteins of varying sources and enzymatic activity may be immobilized using this technique (9,ZO). Sepharose and agarose derivatives containing metal chelating groups, such as iminodiacetic acid (IDA) or nitrilotriacetic acid (NTA), are suitable supports. Various supports of high porosity and potentially high enzyme loading are available commercially from various sources and sold primarily as chromatography packing, such as immobilized metal affinity chromatography (IMAC) media. Another novel, nonporous agarose derivitive contains a ferromagnetic core with IDA as a chelate group (If, 12). In this chapter, we describe preparation of both conventional and magnetic materials for protein immobilization through Me(I1) interaction. 2. Materials 2.1. Preparation

of Magnetic Agarose (Nonporous

Matrix, see Note I)

1. Magnetic agarose: 10 mL (Perceptive Diagnostics, Cambridge, MA). 2. Sodium borohydride. From: M&hods in Biotechnology, Vol. 1: immobilization of Enzymes and Cells Edited by: G. F. Bickerstaff Humana Press Inc., Totowa, NJ

339

Beitle and A taai

340

3 1,4-Butanedrol diglycidal ether. 4. Iminodiacetic acid, disodium salt. 5 Stock metal solution: Prepare 500 mL of 5 mg/mL metal salt CuS04, ZnCl*, NiCl,, or CoC12 Dissolve the salt in distilled, deiomzed water (ddHzO, see Notes 2,3). 6. Stock 1M NaCl solution: Dissolve 58.5 g NaCl in 1 L of ddHzO and filter if required. 7 Stock 1MNaOH solution. Dissolve 40 g of NaOH in 1 L of ddHzO 8 Stock 2MNa2C03 solution: Dissolve 212 g in 1 L of ddH20. 9 Smtered-glass funnel and gentle vacuum source. 10. Shaking table, rocking platform mixer, or suitable device 11. Enzyme solution (see Note 4)

2.2. Preparation

of Commercial

Supports

IDA or NTA

(Porous Matrix) 1. IMAC media. 50 mL (Pharmacla, Piscataway, NJ, or Qtagen, Chatsworth, CA, see Note 5). 2. Peristaltic pump and chromatography column (see Section 3 2 , step 5) 3 Sintered-glass funnel and gentle vacuum source. 4 Shaking table, rocking platform mixer, or suitable device. 5. Stock metal solution: Prepare 500 mL of 5 mg/mL metal salt CuSO,, ZnCl,, NiC&, or CoC12. Dissolve the salt in ddH20 (see Notes 1,4) 6. Stock 1M NaCl solution: Dissolve 58.5g NaCl in 1 L of ddH20 and filter if required (see Note 6). 7 Enzyme solution (see Note 2)

2.3. Matrix Regeneration 1. Stock 0.05M EDTA (ethylenediamme Prepare 1 L. 2 5% (v/v) Ethanol

3. Methods 3.1. Preparation

tetraacetic acid, disodmm salt) solution:

of Magnetic Agarose

1 Wash by suction 1 mL of magnetic agarose usmg a smtered-glass funnel and approx 75-100 mL of ddHzO Transfer the washed material to a lo-mL screwcapped vial. 2 Prepare a solution containing 1 mL of 0.5Msodmm hydroxide solution and 1 mL of 1,6butanediol diglycidal containing 2 mg sodium borohydride Add this solution to the suction-dried magnetic agarose. 3 Mix the slurry for 8 h at 25°C. 4. Wash the product with approx 50 mL of ddHz0. This may be performed by usmg a magnet placed against the side of the vessel to concentrate the product and decant the solution

341

Metal-Ion Based immobilization

5. To the product of step 4, add 10 mL of 2M sodium carbonate containing 0.2 g of immodiacetic acid and 2.4 mg sodium borohydride and mix the suspension for 8 h. 6. Wash the product of step 5 wrth approx 50 mL of ddH20 m a srmilar fashion to step 4. magnetic agarose in 10 mL of stock metal 7 Suspend the IDA-functionalized solution to charge it with metal ions. A l-h incubation is sufficient with gentle shaking. 8. Wash the product of step 7 with approx 50 mL of ddH*O. Use a magnet to aid m concentrating the media and decanting the solution. The media is now ready for enzyme loading. 9. Enzyme may be loaded by suspending the enzyme (typically l-5 mg/mL) m a solutron of buffer containing O.l-1M sodium chloride; add this suspension to the metal charged media. The beads can accommodate up to 40 mg of protein/ml of functionalized beads. 10. Wash the beads as before with 50 mL of buffer contammg NaCl to remove unbound enzyme.

3.2. Preparation of Commercial Matrix, see Note 5)

Supports

IDA or NTA (Porous

1 Wash by suction 1 mL of commercial IMAC media using a sintered-glass funnel and approx 75-100 mL of ddH,O. Transfer the washed material to a lo-mL screwcapped vial. 2. Suspend the media in 10 mL of stock metal solution to charge rt with metal ions. 3 Wash the product of step 2 with approx 50 mL of ddHzO by placing the media into the sintered-glass funnel again. The media is now ready for enzyme loading. 4. Immobthze the enzyme by placing it m a buffer solution containing O.l-1M sodium chloride. Mix the enzyme solution (l-5 mg/mL) with the metal-charged media, place in the sintered-glass funnel, and wash with buffer to remove unbound enzyme. 5. As an alternate in steps 2-4, place washed media in a small chromatography column (1 cm id). Pass appropriate solutions from each step 2-4 at a flow rate of 0.05 mL/min using a peristaltic pump.

3.3. Matrix Regeneration 1. Mix the solution of EDTA with the support or pass through the EDTA solutron through the support if on a short column. The EDTA will strip both the protein and the metal ion from the supports listed above. 2 Wash the stripped support material with 50 mL of ddHzO to remove any EDTA, Me(II), and protein. 3. After washing, place the media m a solution of 5% ethanol for storage at 4°C.

4. Notes 1. The primary advantages of the magnetic media include the reduction of intraparticle difmsional resistances and the ability to utilize magnetism to either

Beitle and A taai

342

2

3 4

5. 6.

concentrate the medta after protein attachment or contam the media m a magnetic stabilized flmdized bed (I3). The metal ion chosen will greatly influence stability and the possibility of nonspectfic adsorption of contaminant proteins present m the enzyme preparation. In general, the strength of Cu(II)-protein interaction 1s highest when compared to Nt(II), Co(II), and Zn(I1). However, use of the latter metal tons will reduce the possibility of nonspecific adsorption, because they require hrstidme residues m multiplicity and certam geometries (2,14). If necessary, filter through a 0.2~pm filter to remove particulate matter If the enzyme or protein of interest does not exhibit strong afftmty for dtvalent metal ions, consider the use of an expression system that explotts metal affinity as the basis for separation. Several references cite recovery via metal affimty and are referred to m the introduction This approach will facilitate both the puriflcation of the enzyme and subsequent immobilization Pharmacta provides IDA-functionalized media, whereas Qtagen provides NTAcontaining material NaCl is typically required to supress ionic interactions and atd m protein adsorption (15,16) Buffered solutions that come m contact with the immobilized enzyme (e.g., substrate) should contain this component.

References 1. Porath, J., Carlsson, J., Olsson, I., and Belfrage, G (1975) Metal chelate affimty chromatography, a new approach to protein fractionation. Nature 258,598,599. 2. Hemdan, E., Zhao, Y , Sulkowski, E , and Porath, J (1989) Surface topography of histidine residues: a facile probe by immobthzed metal ion affinity chromatography. Proc. Natl. Acad. Scl USA 86,18 1l-l 8 15. 3. Arnold, F (1991) Metal-affinity separations: a new dimension in protein processmg. Bzotechnology 9, 15 l-l 56. 4. Beitle, R. and Ataai, M. (1992) Immobilized metal affinity chromatography and related techniques, m New Developments m Bioseparanon (Ataai, M. and Sikdar, S., eds.), American Institute of Chemical Engineers, New York, pp. 34-44. 5. Beitle, R. and Ataai, M. (1993) One-step purification of a model periplasmtc protein from inclusion bodies by its fusion to an effective metal-bmding peptide Biotechnol. Progr 9,64-69.

6 Hochuh, E., Bannwarth, W., Dobell, H., Gentz, R., and Stuber, D. (1988) Genettc approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbant Biotechnology 6, 1321-1325. 7. Lilius, G., Persson, M., Bulow, L., and Mosbach, K. (1991) Metal affinity precipitation of proteins carrying genettcally attached polyhistidine affiruty tatls. Eur J Biochem

198,499-504.

8 Ljungquist, C., Breitholtz,A., Brink-Nilsson, H , Moks, T, Uhlen, M., and Nilsson, B. (1989) Immobrhzation and affinity purification of recombinant proteins using histidine-pepttde fusions. Eur. J. Biochem. 186,563-569.

Metal-Ion Based Immobilization

343

9 Coulet, P, Carlsson, J., and Porath, J. (1981) Immobtllzatton of enzymes on metal-chelate regenerable carriers. Bzotechnol. Bioeng 23,663-668. 10 Ptesecki, S., Teng, WY., and Hochuh, E. (1993) Immobtlization of P-galactosidase for application m organic chemistry using a chelating pepttde. Bzotechnol Bioeng 42,178-l 84. 11 Abudiab, T. and Beitle, R (1995) Preparation of magnetic immobilized metal affinity separation media and its use in the isolation of proteins. Biophyszcal J., in press. 12. Lee, Y. and Vacqmer, V (1992) Reusable cDNA libraries coupled to magnetic beads, Anal. Biochem. 206,206,207.

13 Sikavitsas, V., Yang, R., and Burns, M. (1995) Magnetically stabilized flutdtzed beds. Indus Eng. Res 34,2873-2880 14. Zhao, Y., Sulkowski, E., and Porath, J. (199 1) Surface topography of histtdme residues in lysozymes Eur J Biochem 202,1115-l 119. 15. Yip, T., Nakagawa, Y., and Porath, J. (1989) Evaluation of the interaction of peptides with Cu(II), Ni(II), and Zn(TI) by high-performance immobilized metal ton affinity chromatography. Anal Bzochem. 183, 159-17 1. 16. Porath, J. (1987) Metal ton-hydrophobic, thiophilic, and p-electron governed interactions and their apphcatton to salt-promoted protein adsorption chromatography. Bzotechnol Prog. 3, 14-21

39 Transition Metal Methods for Immobilization of Enzymes and Cells John F. Kennedy, Joaquim and Marion Paterson

M. Cabral, Maria R. Kosseva,

1. Introduction The use of transition metal chemistry for the immobihzation of biological molecules is long established and well documented (l-17). This method is based on the chelation properties of transition metals, namely titanium(IV) and zirconmm(IV), which seem particularly attractive on account of the nontoxicity of their oxides (2). Investigation of a number of gelatinous hydrous metal oxides has shown that hydrous titanium(IV), zircomum(IV), non(III), vanadium(III), and tin(IV) oxides may be usedper se as matrices for immobilization of biocatalyst (3-5). Such hydrous metal oxide materials have proved to be suitable for the immobilization of amino acids (3), peptides (3), enzymes (l-3), antibiotics (181, polysaccharides (19), and whole living cells (20-28). The transition metal chelation method has also been used for the immobilization of enzymes and cells onto a variety of inorganic (I-@ and organic (2) supports by treatment with a solution of a transition metal salt. Titanium(IV) is the most stable and common oxidation state. The two most common coordination numbers of titanium(IV) are 4 and 6, giving rise to tetrahedral and octahedral geometries, respectively. Titanium(IV) chloride is an example of the tetrahedral configuration; this colorless liquid behaves as a Lewis acid and is readily hydrolyzed by water to give hydrous titanium(IV) oxide. On addition of ammonia to a solution of titanium(IV) chloride, gelatinous, untilterable precipitates are obtained at ammonia-titanium ratios between 2-3.75. On increasing this ratio to 4, titanium is precipitated quantitatively as a white, amorphous precipitate. The reactions are as follows (2): From

Methods III Botechnology, Vol I /mmob/katron of Enzymes and Cells Edlted by G F BIckerstaff Humana Press Inc , Totowa, NJ

345

346

Kennedy et al. TiCl,, + Hz0 + TiOCl, + 2 HCl TiOCl~ + 2HCl+ 4NH40H + TiO(OH)* + 4 NH&I + 2H20

(1) (2)

Heating can play a very important role in precipitating hydrous metal oxides from soluttons. After neutralization by addition of base to a solution of metal compound, or after dilutton of such a solutton, the thermodynamic conditions for precipitation of the respective oxide may be present, but the rate of such precipitation may be very slow. It is frequently practical and convenient to bring about the required precipitation by heating the mixture. Zirconium occurs almost exclusively in its compounds in the oxidatton state 4+. The behavior of zirconium complexes in aqueous solution is characterized by hydrolysis and polymerization. When znconmm(IV) chloride ts dissolved in water, a solution is obtained that behaves identically to the solution obtained from ZrOC12*8Hz0. The reactions that occur can be formulated as shown in the following equations (2): ZrCl, + 8H20 +- [Zr(H20)8]4+

+ 4Cl-

(3) [Zr(H,0),14+ + Hz0 + [Zr(H,0),(OH)]3++ H30+ (4) [Zr(H,0),(OH)]3+ + Hz0 + [ZI(H,O)~(OH),]~+ + H30+ (5) The final complex cation is able to polymerize to give a tetrametric complex: 4[Zr(H,0),(OH),J2+ + [Zr4(H20),6(OH)8]8++ 8H2O+ (6) This tetrametric complex is present m both aqueous solution and the solid znconyl chloride octahydrate. The aqueous solution of zn-conium(IV) chloride has acidic properties, the H+ activity of a 1 mA4solution being almost equal to that of 1 mM HCI. This is because of the hydrolysis of the tetrametric complex (2), Pki(OHh(H2%18+ + 4H20 + [Zr4(OH)8(OHk(H20)1214’+ 4H30+ (7) In the titanium(IV) chloride solution, a proportion of the titanium ions is octahedrally coordinated with molecules or ionic species that are essentially the ligands of the complex ion (ref. 2, see Fig. 1 for examples of complex ions). According to the type of ligand occupying each of the six sites, a whole series of six-coordinated complexed titanium species may be considered to exist in solunon. Nucleophilic groups (e.g., hydroxy, amino, and thiol groups) are effective ligands for the transition metal ions, and therefore it is to be expected that transition metal ions may complex both supports and enzymes.Transition metal ions may complex with polysaccharides (e.g., cellulose) in which the hydroxy groups act as new ligands, replacing others (Fig. 2). Proteins have groups that can act as ligands, such as the free carboxy groups from the C-terminus of acidic amino acids, the phenolic hydroxy groups of tyrosyl residues, the alcoholic hydroxy groups of seryl and threonyl residues, free sulfydryl groups from any

Transition Metal-Based Immobilization

347

L ,*’ 8’ -L

,-Ti L ‘I

L

L = neutral 0

ligand

Fig. 1. Titamum ions m tltanium(IV) chloride-HCl solution. The species depicted are (2) the pure aquo complex, (3) and (4) chloroaquo complexes of various charges, and (5) the chloro complex

I

CARBOHYDRATE

Fig. 2. Representative structures of hydrous titanium(IV) chelate.

oxide-polysaccharide

cysteinyl residues, and ammo groups from the N-terminus of s-amino groups of lysyl residues (3). The techniques of inorganic support activation, e.g., silica, magnetic iron oxrde, and so on (6412) are based on the deposition and interaction of high surface area hydrous metal oxides on the base carrier particles during the precipitation process of the hydrous metal oxides. With inorganic supports, a layer of hydrous metal oxide on the support surface is thought to be responsible for the immobilization of the enzymes more than a chemical interaction between the transition metal and the support (2). This layer is favored to be obtained by drying the support m the presence of an excessof salt solutton. During heating,

Kennedy et al.

348

-Si4H

I +

MC!@H)b

I drying I

-Si4

--WOW

&la-~

+

HCI

I

Fig. 3. Activation of inorganic support with transition metal salts to produce an oxychloride derivative.

the evaporation of water and hydrochloric acid occurs and the formation of hydrous metal oxide and/or oxychloride is induced with concomitant precipitation on the support. In most cases, the immobilization of enzymes onto organic crosslmked polysaccharides or inorganic supports occurs via transitional metal chelating using tttanmm(IV) chloride, pure or in an acidic solution. In such cases, the activation is carried out at low pH values without disruption of the support. In the case of more sensitive supports, namely of the proteinaceous nature (e.g., gelatin), the activation conditions should be as mild as possible with minimum deterioration of the support. Several mild techniques in which the transition metal remains m a soluble and reactive form at high pH values, which gives less disruption of the proteins, have been developed (32,33). Since the gel formation is pH-dependent, the choice of titamum or zirconium reagents is governed by the pH range required for application. Titanium(IV) hydrous oxide 1s more effective in acidic conditions and zuconium(IV) hydrous oxide is more effective at neutral or higher pH (34). Kennedy (1,2,3.5) developed a method of protem immobilization based on the chemistry of the hydrous transmon metals oxides per se. From the practical and economic viewpomt, hydrous titanium(IV) and zirconium(IV) oxides have proved to be satisfactory as matrices for immobilization of enzymes since they are insoluble over the normal physiological pH range, give good retention of enzyme activity, and the immobilization procedure IS accomphshed in one step. Transition metal-activated supports can also be used for immobilization of cells (20,21,24,25,36). This method is based on the activation of the support with transition metal salts, producing in the first step an oxychloride derivative (20,21) (Fig. 3). This derivative can then react with several agents (e.g., water,

Transition Metal-Based Immobilization

-Si-O-

I I

349

M--j=1 I (OH) n-1

Fig. 4. Coupling of whole cells to transition metal activated inorganic support

tannic acid, and 1,6-diaminohexane m carbon tetrachloride) to yield supports with different functional groups (20,21). An example of interaction of cells with a hydrous metal oxide derivative is shown in Fig. 4. Cells of Succharomyces cerevisiae and Escherichia colt have been immoblltzed on hydrous metal oxides and the retention of the living processesof the bound cells were demonstrated by oxygen-uptake experiments (26) and invertase acttvity (27). Cells of speciesAcetobacter have been immobilized on hydrous titanium(IV) oxide and these biocatalysts were used for the continuous production of malt vinegar from wort in a tower fermenter (22). 2. Materials All reagents and solvents are comercially available. 2.1. Immobilization of Enzymes on Titanium-Activated Supports 2.7.7. Heating Procedure (1,2,29)

Inorganic

1 Lead glass ballotini with two sizes Nl: dP = 1.903 mm, ps= 2 905 g/mL or NlO* dp = 0.258 IllIll, ,,8= 2.905 g/mL (Jencons Glass, Leighton Buzzard, UK) 2. 4% (w/v) Solution of ethylenediaminetetra-acetic acid (EDTA) disodium salt (caution: harmful). 3. 1: 1 (v/v) Solution of water:sulfuric acid (caution: corrosive, irritant). 4. 15% (w/v) Titanium(IV) chloride solution in a 15% (w/v) solution of HCl (caution: corrosive). 5. Glucoamylase (1,4-a-n-glucan glucohydrolase, EC 3.2.1.3). 6. 0.02M sodium acetate buftier, pH 4.5. 7 0.02M sodium acetate buffer, pH 4 5, contaming 1M sodium chloride 8. 6M solution of urea (caution: irritant, harmful, possible mutagen).

2.1.1. Neutralizing Procedure (1,2,30,31) 1. 2. 3. 4. 5. 6

Magnetic non oxide [non(II) diiron(II1) oxide Fes04] (Fisons, Loughborough, UK). Titanium tetrachlortde (caution: corrosive) 1M ammonium hydroxide (caution: irritant) solution. 0.2M sodium acetate buffer, pH 5.8, containing O.OlM calcium chlortde. a-Amylase (1,4-a-n-glucan glucanohydrolase, EC 3.2.1.1) from Bacillus subtllu. 0.2M sodium acetate buffer, pH 5.8, containing O.OlM calcium chloride.

Kennedy et al.

350 2.2. Determination

of the Activity of Immobilized

Enzymes

1 3,5-Dmitrosalycilx acid (DNS) reagent (38,39). Follow the same procedure described in Chapter 19, Section 2.5. 2 10 mg/mL D-Glucose solution m 0.02M sodium acetate buffer, pH 4 5 3. 1% ( w/v) Starch solution: Suspend soluble starch in 0 02M sodium acetate buffer, pH 4.5, and heat the suspension until the starch has completely dissolved 4 1% (w/v) Starch solution: Suspend soluble starch m 0.2M sodium acetate buffer, pH 5.8, and heat the suspension until the starch has completely dissolved

2.3. Immobilization of Enzymes on Titanium-Activated Organic Support (32,33) 2.3.1. Preparation of Titanium(W) Chloride-Acrylamide Complex and its Use for Activation of Gelatin Support 1 Acrylamide. Caution: Acrylamide is highly toxic, an irritant, a carcinogen and may cause possible teratogenic and neurological hazards. Wear appropriate respirator, chemical-resistant gloves, safety goggles, and other protective clothing and use only in a chemical fume hood (see Note 1) 2. Dry dichloromethane. Caution: Dichloromethane is an irritant and may cause mutagenic or teratogenic effects (see Notes 2,3) 3. Titanmm(IV) chloride (caution: corrosive). 4 Glutaraldehyde crosslinked gelatin m bead form, 0.25-O 50 mm diameter (Gist-Brocades, Holland). 5 0.5M sodium acetate solution. 6 O.lM sodium acetate buffer, pH 4 5

2.3.2. Preparation of Titanium(lV) Chloride-Lactose and its Use for Activation of Gelatin Support

Complex

1. 15% (w/v) Titanium(IV) chloride solution m a 15% (w/v) solutton of HCl (caution: corrosive). 2. 0 5M lactose solution. 3. 5M ammonia solution (caution: irritant). 4. Glutaraldehyde crosslmked gelatin m bead form, 0.25-0.50-mm diameter (GrstBrocades). 5. 0.M sodium acetate buffer, pH 4.5

2.3.3. Activation of Glutaraldehyde-Gelatin Titanium(lV) Chloride

Support with Uncomplexed

1. Titanmm(IV) chloride (caution: corrosive). 2. Glutaraldehyde crosslinked gelatin in bead form, 0.25-O 50 mm diameter (GtstBrocades). 3. 0. IM sodium acetate buffer, pH 4.5.

2.3.4. Immobilization of Glucoamylase onto Titanium(lV)-Activated Gelatin Beads 1. Glucoamylase (1,4-a-n-glucan glucohydrolase, EC 3.2.1.3) 2. O.lM sodium acetate buffer, pH 4.5.

351

Transition Metal-Based Immobilization 2.4. Determination of the Activity on Organic Supports

of Glucoamylase

immobilized

1. Gelatin-immobilized glucoamyiase. 2 O.lM sodium acetate buffer, pH 4.5. 3. 1% (w/v) Starch solution: Suspend soluble starch in 0. 1M sodium acetate buffer, pH 4.5, and heat the suspension unttl the starch has completely dissolved.

2.5. Immobilization of Glucoamylase on Hydrous Transition Metal Oxides 1. 2. 3. 4. 5

Titanium chloride (caution: corrosive). Znconium(IV) chloride (caution: corrosive). IM ammonium hydroxide solution (caution: irritant). Glucoamylase (1,4-a-n-glucan glucohydrolase, EC 3.2.1.3) O.lM sodium phosphate buffer, pH 7.0, containing O.OlML-cysteine ride and 0 4 mM EDTA.

2.6. Immobilization of Yeast Cells on Titanium-Activated Inorganic Supports

hydrochlo-

(1,26,36)

1. 15% (w/v) Titanium(IV) chloride solution in 15% (w/v) HCl (caution: corrosive). 2. Pumice stone, vulcanic porous material, mainly 67% SiO, and 15% A1203 with a 5-m2/g surface area and particle size 200-300 pm. 3. Baker’s yeast 5. cerevzsze: Use as a source of S-n-fructofuranosidase (EC 3.2.1.26). 4. 0 02M sodium acetate buffer, pH 4.5.

2.7. Immobilization of Yeast Cells on Hydrous Transition Metal Oxides (24) 1. 2. 3. 4.

15% (w/v) Titanium(IV) chloride solution in 15% (w/v) HCl (caution: corrosive). 0.65M zirconium(IV) chloride solution m 1M HCl (caution: corrosive). 2M ammonia solution (caution: irritant). 0.9% (w/v) Saline solution. 5. Baker’s yeast S. cerewsiae: Use as a source of 8-n-fiuctofuranosidase (EC 3.2.1 26).

3. Methods 3.1. lmmobiliza tion of Enzymes on Titanium-Activated Inorganic

Supports

3.7.7. Heating Procedure (1,2,29) 1. Weigh 10 g of lead glass into a flask and heat at 100°C with 40 mL of 4% EDTA disodium salt solution at pH 10.0 for 48 h (see Note 4). 2. Wash the etched glass thoroughly with distilled water. 3. Add 40 mL of a 1: 1 (v/v) solution of sulfuric acid:distilled water and leave to soak at ambient temperature for 5 d.

Kennedy et al.

352

4 Wash thoroughly with distilled water. 5 Add 40 mL of 15% tttamum(IV) chloride solution and reflux the mixture for 3-5 h (see Notes 5,6). 6 Remove the titanmm(IV) chloride expent solution and wash thoroughly with dlstilled water. 7 Add 1 g of the glucoamylase solution in 0.02M sodium acetate buffer, pH 4 5 The enzyme solution to support ratlo (by volume) IS 2: 1 (see Note 7). 8 Leave the enzyme to react with the support at 4°C for 8 h 9 Wash the lmmobillzed enzyme with 0.02M sodium acetate buffer, pH 4.5, with stirring for 30 mm, then 0.02M sodium acetate buffer, pH 4 5, contaimng 1M sodium chloride with stirring for 30 min, then 6Murea solution with stirrmg for 60 mm, and finally 0.02Msodmm acetate buffer, pH 4.5, with stirring for 30 mm 10. Determine the enzymlc activity of the immobilized glucoamylase as indicated m Section 3 2.

3.1.2. Neutralizing Procedure (1,2,30,31) 1 Weigh 100 mg of magnetic iron oxide (see Note 5) 2. Add 50 pL of pure (liquid) tltanium(IV) chloride to the Iron oxide and add manedlately 1 mL of distilled water. Neutralize the mixture to pH 5 8 using lMammonium hydroxide solution with constant stirring. 3 Remove the supernatant by centrtfugation. 4. Wash the activated support with 0.2M sodium acetate buffer, pH 5.8, containing 1 mL of O.OlMcalcmm chloride and add 200 pL (5 mg) of a-amylase solution to the activated support (see Note 7) 5. After 1 h at 4°C with frequent shaking remove the unbound enzyme solution and wash the Immobilized enzyme five times with the 0.2M sodium acetate buffer, pH 5.8, contammg 5 mL of O.OlMcalcmm chloride. Suspend the sohds m 5 mL of the same buffer and store at 4°C. 6. Determine the enzymlc activity of the immoblhzed a-amylase as indicated m Section 3.2

3.2. Determination of the Activity on Inorganic Supports

of hmobilized

Enzymes

Glucoamylase activity 1s measured by the hydrolysis of a 1% (w/v) starch solution in 0.02M sodium acetate buffer, pH 4.5, at 45OC (see Notes 8,9,15). For a-amylase use a 1% (w/v) starch solution in 0.2M sodium acetate buffer, pH 5.8, and 37OC. For yeast cell invertase, use a 2% (w/v) solution of sucrose m 0.02M sodium acetate buffer, pH 4.5, at 30°C. 1. Add 100~pL aliquots of the suspension (immobilized

enzyme) or native enzyme solution to 5 mL of 1% (w/v) starch solution m 0.02M sodium acetate buffer, pH 4.5, at 45’C. 2. At zero time and at various time intervals over a 30-min period of incubation at 45”C, withdraw 100~VL ahquots and terminate the reaction by adding 1 mL of DNS reagent solution.

Transition Metal-Based Immobilization

353

3. Mix the samples well and heat in a boiling water bath for 10 mm. Cool to ambient temperature and measure the absorbances of the soluttons at 570 nm. 4 To 100~pL aliquots of n-glucose standard solution, O-5 mg/mL in 0.02M sodium acetate buffer, pH 4.5, and controls, add 1 mL of DNS reagent. Mix the samples well and heat in a boiling water bath for 10 min. Cool to ambient temperature and measure the absorbances of samples at 570 nm. 5. Measurement of the amount of active enzyme bound to the support is obtained by a comparison of the rate of production of reducing sugars for the samples of immobilized enzyme with that of native enzyme solution (see Chapter 19, Section 3.5.). The unit of glucoamylase activity (U) is defined as the amount of enzyme capable of producing 1 umol of n-glucose/min from a 1% (w/v) starch solution in 0.02M sodium acetate buffer, pH 4.5, at 45°C. The unit of a-amylase activity (U) is defined as the amount of enzyme required to liberate 1 l.tmol of o-glucose/min from a 1% (w/v) starch solution in 0 2M sodium acetate buffer, pH 5.8, at 37°C. The unit of invertase activity (U) is defined as the amount of enzyme required to liberate 1 pmol of o-glucose/min from a 2% (w/v) sucrose solution in 0.02M sodium acetate buffer, pH 4.5, at 30°C.

3.3. Immobilization of Enzymes on Titanium-Activated Organic Supports (32,33) 3.3.1. Preparation of Titanium(l V) Chloride-Acrylamide Complex and Its Use for Activation of Gelatin Support 1, Add a solutton of 7.1 g acrylamide m 400 mL of dry dichloromethane, with stirring over a period of 10 mm, to a solution of 19.0 g titanium(IV) chloride m 40 mL of dry dtchloromethane (see Notes l-3). 2. The solution immedtately turns yellow and a dense, orange liqmd ~111 separate out. On continued stnrmg, thts VISCOUSliquid wtll sohdtfy to a yellow sohd within a few hours. 3. Collect this solid by filtration, grind, and wash wtth dichloromethane, then dry. 4. Wash 2-mL bed volume of gelatin beads with 5 x 10 mL of distilled water. 5. Add a 2-mL bed volume sample of the washed glutaraldehyde crosslmked gelatin particles to a 2-mL (100 mg/mL) solution of titanium chloride-acrylamide complex in dtstilled water at pH 3.0, adjusted with 0.5M sodium acetate solution, 6. Stir the mixture for 30 min at 18°C remove the supematant, and wash the titanium(IV) acrylamide-activated gelatin particles with 5 x 10 mL of 0. 1M sodium acetate buffer, pH 4.5. Use munediately for coupling to the enzyme (see Section 3.3.4., Note 10).

3.3.2. Preparation of Titanium(lV) Chloride-Lactose and Its Use for Activation of Gelatin Support

Complex

1. Add 2 mL of a solution of 15% titanium(IV) chloride HCl to 18 mL of an aqueous solution of 0.5M lactose and adjust the pH to 3.1 with 5M ammoma solution. 2. Wash 2 mL of the glutaraldehyde crosslinked gelatin particles wtth 5 x 10 mL of distilled water.

Kennedy et al.

354

3. Add 5 mL of the titanium(IV) chloride in lactose solution, prepared as described above, to the water-washed, glutaraldehyde crosslinked gelatin particles (see Note 6). 4. Stir the mixture for 60-120 min at 18°C remove the supernatant, and wash the titanium(IV)-lactose activated gelatin particles with 5 x 10 mL of O.lM sodium acetate buffer, pH 4.5. Use immediately for coupling to the enzyme (see Section 3.3.4., Note 10).

3.3.3. Activation of Glutaraldehyde-Gelatin Titanium(lV) Chloride

Support with Uncomplexed

1. Add 1 mL of pure titanium(V) chloride to 5 g of moist, water-washed or 2 g of freeze-dried glutaraldehyde crosslinked gelatin beads. 2. Stir the mixtures occasionally for O.M.0 min. 3. Remove the excess titanium(IV) chloride and wash the activated beads with 0.M sodium acetate buffer, pH 4.5, until the pH of the liquids is 4.5. Use immediately for coupling to the enzyme (see Section 3.3.4. and Notes 11,12).

3.3.4. Immobilization of Glucoamylase onto Titanium(lV)-Activated Gelatin Beads (33) 1. Add 25 mL of a 5-mg/mL glucoamylase solution in 0.M sodium acetate buffer, pH 4.5, to a 5-g sample of titanium(IV)-activated gelatin beads, obtained in Section 3.3.1, 3.3.2., or 3.3.3, and stir for 18 hat 4°C. 2. Remove the enzyme solution and wash the gelatin-enzyme conjugates with 0. 1M sodium acetate buffer, pH 4.5. Determine the enzymatic activity of the immobilized glucoamylase as shown in Section 3.4.

3.4. Determination of the Activity of Glucoamylase on Organic Supports

Immobilized

1. Add a moist sample, containing 5-50 mg of gelatin-immobilized glucoamylase, that has been washed in O.lMsodium acetate buffer, pH 4.5, or an lOO-pL aliquot of a solution of native glucoamylase containing 5-10 ug/mL, in O.lM sodium acetate buffer, pH 4.5, to 2.5 mL of a prewarmed 1% (w/v) starch solution in O.lM sodium acetate buffer, pH 4.5, at 55°C with continuous stirring. 2. Proceed according to Section 3.2., from step 2.

3.5. Immobilization of Glucoamylase on Hydrous Transition Metal Oxide (1,2) 1. Add 20 mL of distilled water to 1 mL containing 1.73 g of pure titanium(IV) chloride (see Notes 12,13) and immediately neutralize the mixture to pH 7.0 with 1M ammonium hydroxide solution. 2. Centrifuge the resultant suspension and discard the supematant liquid. 4. Wash the precipitate thoroughly with 5 x 25 mL distilled water. 5. Centrifuge and discard the washing supematant. 6. Add a 5-mg/mL solution of glucoamylase at pH 7.0 to the washed solid and react at 4’C for 1 h.

355

Transition Metal-Based fmmobilization

7. Remove the unbound enzyme by centrifugation and wash the precipitate (enzyme-bound hydrous titanium(IV) oxide) with 10 x 25 mL of 0.M sodium phosphate buffer, pH 7.0, containing O.OlML-cysteine hydrochloride and 0.4 mA4 EDTA. Store the immobilized enzyme preparation in 25 mL of the same buffer at 4°C. Determine the enzymatic activity of the immobilized glucoamylase using the DNS assay (see Section 3.2).

3.6. Immobilization of Yeast Cells on Titanium-Activated Inorganic Supports (25,26) 1. Add 3 mL of 15% titanium(IV) chloride solution HCl to a 1 g sample of pumice stone. 2. Dry the mixture in an oven for 48 h at 45°C. An oxychloride derivative is obtained (see Note 15). 3. Wash the titanium(IV)-activated support with 3 x 10 mL of distilled water. A hydrous oxide derivative is obtained. 4. Add to the hydrous oxide derivative 10 mL of a 2% (w/v) suspension of baker’s yeast S. cerevisiue in 0.02M sodium acetate buffer, pH 4.5, and mix for 2 h at 4°C. 5. Remove the supernatant of the immobilized cell preparation, 6. Wash the immobilized cell preparation with 3 x 10 mL of distilled water and 10 mL of 0.02M sodium acetate buffer, pH 4.0. Determine the invertase enzymatic activity of the immobilized cells as indicated in Section 3.2. (see Note 16).

3.7. Immobilization Oxides (1,26,36)

of Yeast Cells on Hydrous Transition

Metal

1. To a 15% titanium(IV) chloride solution in HCl solution or a 0.65M zirconium(IV) chloride solution in 1M HCl, slowly add a 2A4 ammonia solution until neutrality (pH 7.0). 2. Wash the precipitate with 3 x 5 mL of 0.9% saline solution. 3. To the hydrous metal oxide prepared above, add 10 mL of a 2% wet (w/v) suspension of 5’. cerevisiae cells in 0.9% saline solution. Agitate gently for 5 min at ambient temperature. 4. Allow the mixture to stand at ambient temperature and the suspension to settle out, leaving a clear supernatant (practically devoid of microorganisms). 5. Consolidate the immobilized cell preparation by centrifugation at low speed and remove the supematant. Determine the invertase enzymatic activity of the immobilized cells as indicated in Section 3.2.

4. Notes 1. Acrylamide is a white crystalline solid, which is toxic, a carcinogen, an irritant, is harmful, and may cause possible teratogen and neurological hazards. Use only in a chemical iume cupboard. 2. Dichloromethane is an irritant liquid and is sufficiently volatile at ambient temperature to generate vapors that may be mutagenic or teratogenic. Use only in a chemical fume cupboard.

356

Kennedy et al.

3. Preparation of dry dichloromethane (40). Anhydrous sodium sulfate can be used as a drying agent for this solvent (at temperatures below 32.4”C) Shake the solvent with a small amount of the drying agent. If sufficient water is present to cause the separation of a small aqueous phase, this must be removed and the liquid treated with a fresh portion of the desiccant. If time permtts, the liquid, when apparently dry, should be filtered and left overnight in contact with fresh drying agent The dessicant should, in general, be separated by filtration (best through a fluted paper). Other common drying agents for organic compounds are anhydrous calcmm chloride, metalhc sodium, and phosphorus pentoxide 4 This treatment ~111 remove part of the lead-etched glass so that the structure would be opened up to facilitate deposition of the metal oxide layer withm the bead surface (2,6,36). 5. An optional step can be included to dry the activated support at 600°C for 8 h before the enzyme coupling (1) 6. The ratros of transition metal salt solution/support weight and acttvation time/ support weight (or volume) are important parameters. For instance, with porous supports these ratios must be optimized in order to give the maximum active immobilized enzyme preparation (2,36). 7 The concentration of the enzyme during the immobilization step IS also an important parameter to be constdered. High protein loadings may saturate the support 8 The biological actrvrty of the immobilized enzyme will depend directly on the characterrstics of the enzyme, the buffer conditions, the temperature, and the pH of immobilization Enzymes from the same EC classification obtained from different sources may have different optimal working conditions (e.g., pH, temperature). The amount of activity remaining in solution or desorbed by washing should also be considered. 9 The observed operational stabilization of immobilized enzymes is generally the sum of the effects of stabilization at the molecular level and the loading of an excess amount of the enzyme. The latter causes the process to be diffusion limited rather than to be kinetically controlled; it leads to a relatively constant activity (37). 10. In general, the methods of immobilization of enzymes on crosslinked gelatin particles activated with complexes (e.g., titanium-acrylamide, titanium-lactose) show low levels of bound enzyme activity. This is probably because of the low reactivity of the activated gelatin particles since the hgands (complexes) cannot be displaced easily by the primary amino groups of the gelatin (2,32,33). 11. Liquid titamum(IV) chloride in its uncomplexed form is much more reactive and is used for the activation of gelatin particles. When the activation is carried out by adding the moist water-washed gelatin particles directly to an excess of titanium(IV) chloride, an exothermic and vigorous reaction takes place. This is mainly because a significant amount of water is still present m the particles. Only short activation times can be employed with this method. Contact times in excess of 5 min result m degradation of the gelatin matrix This is caused by the highly reactive nature of pure titanium(IV) chloride and the evolution of heat during the reaction, resulting m the hydrolysis of the peptide bonds (2,32,33).

Transition Metal-Eased immobilization

357

12. To overcome the problems found with the titanium(IV) chloride activation using moist water-washed gelatin particles, a freeze-drying step is included to remove the water from the gelatm particles. Higher surface-immobilized enzyme activity is obtained by this method (32,33). 13. Instead of using pure titanium(IV) chloride, an aqueous acidic solution of this salt, such as the commercial solution of 15% (w/v) titanium chloride in 15% (w/v) HCl or a 50% (w/v) solution of titanium(IV) chloride in in 6MHC1, can be used (2). 14. Instead of titanium(IV) chloride, other metal chlorides can be used. In the standard method for the preparation of hydrous zirconium oxide, the required amount of ztrconium chloride is dtssolved in 1M HCl to give a 0.65M solution of the metal (2). 15. Oxychloride derivatives are unstable and must be immediately reacted with suitable agents for enzyme/cell coupling (21). 16. Other methods are available for the determination of the activity of immobihzed S. cerevisiue cells, e.g., measurement of the viability of the immobilized cells by their oxygen uptake using an oxygen electrode (26).

References 1. Kennedy, J. F. and Cabral, J. M. S. (1985) Immobihzation of biocatalysts by metallmlclchelatron processes, m Immobilized Ceils and Enzymes (Woodward, J., ed.), IRL, Oxford, UK, pp. 19-37. 2. Kennedy, J. F. and Cabral, J. M. S. (1986) Use of titanium species for the immobilization of bioactive compounds Transition Met. Chem. 11,41-66. 3 Kennedy, J. F., Barker, S. A., and Humphreys, J. D. (1976) Insoluble complexes of the amino acids, peptides and enzymes with metal hydroxides. J. Chem Sot. Perkin IFans. 1,962-967,

4. Kennedy, J. F. and Pike, V. M. (1978) Water-insoluble papam conJugates of hydrous titanium(IV) oxide and of surface-coating materials modified to contain hydrous titanium(IV) oxide. J. Chem. Sot. Perkin Trans. 1, 1058-1066 5. Kennedy, J. F,, Humphrey, J. D., and Barker, S. A. (I 981) Further facile immobilization of enzymes of hydrous metal oxides and use of their immobilization reversibility phenomena for the recovery of peptide antibiotics. Enzyme Microb. Technol. 3, 129-136. 6. Novais, J. M. (1971) Studies on the insolubilization and use of amylolytic enzymes. Ph.D. Thesis, University of Birmingham, U.K. 7. Charles, M., Coughlin, R. W., Paruchuri, E. K., Allen, B. R., and Hasselberger, F X. (1975) Enzymes immobilized on alumina and stainless steel supports. Biotechnol. Bioeng. 17,203-2 10. 8. Flynn, A. and Johnson, D. B. (1978) Some factors affecting the stability of glucoamylase immobilized on hornblende and on other inorganic supports. Biotechnol. Bioeng 20, 1445-1454. 9. Bhatt, R. R., Joshi, S., and Kothari, R. M. (1979)A simple method for the rapid and economical immobilization of glucose isomerase. Enzyme Microb. Technol. 1, 113-116.

358

Kennedy et al.

10. Rokugawa, K., Fujtshtma, T., Kuminaka, A., and Yoshmo, H. (1980) Immobtlization of nuclease Pi on ion exchange resms by titanmm complex method. J Ferment. Technol. 58,423-429. 11. Rokugawa, K , FuJtshima, T , Kummaka, A , and Yoshmo, H. (1980) Immobil~zanon of adenosine (phosphate) deammase on ion exchange resins by titanium complex method. J Ferment Technol 58,583-585. 12. Cabral, J. M. S., Cardoso, P., and Novars, J. M. (1981) Influence of coupling conditions on activity and operational stability of glucoamylase immobilized on titanmm(IV) activated controlled pore glass. Enzyme Microb Technol 3,41-45. 13 Bisse, E. and Vonderschmitt, D J. (1978) Immobtlization of glucose dehydrogenase by titanium tetrachloride. FEBS Lett 93, 102-104 14 Cabral, J. M. S., Kennedy, J F., and Novais, J. M (1982) Investigation on bmdmg mechanism of glucoamylase to alkylamine derivatives of titanmm(IV)-activated porous inorganic support Enzyme Microb Technol 4,337-342 15. Cabral, J. M. S., Kennedy, J F , Novais, J. M., and Cardoso, J P (1984) Composition and compostttonal-behavioural relationships of enzymes immobilized on porous morgamc supports via trtanmm(IV) specres. Enzyme Mzcrob Technol. 6,228-232 16. Cabral, J. M S., Novais, J, M., Kennedy, J. F., and Cardoso, J. P. (1983) Immobihzation of biocatalysts on new route transition metal-activated inorganic support. Enzyme Mrcrob Technol. 5,30-32. 17 Cabral, J. M. S., Novats, J. M., and Cardoso, J. P. (1984) Coupling of glucoamylase on alkylamme derrvative of titanmm(IV)-activated controlled pore glass with tanmc acid. Blotechnol. Bloeng. 26,386-388. 18. Kennedy, J. F. and Humphrey, H D (1976) Active nmnobihzed antibtottcs based on metal hydroxides. Antlmlcrob. Agents Chemotherap. 9,766-770. 19. Kennedy, J. F. and Fox, J. E. (1977) The fully automatic ion-exchange and gel-permeatron chromatography of neutral monosaccharides and ohgosaccharides with Jeoico JLC-6AH analyser. Carbohydr. Res. 57,205-214. 20. Cabral, J. M. S., Novais, J. M., and Kennedy, J F. (1986) Immobilization studies of whole mtcrobial cells on transitton metal activated inorganic supports Appl Mtcroblol. Blotechnol 23, 157-l 62 2 1. Kennedy, J. E and Cabral, J. M. S (1990) Use of titanium species for the immobihzatton of cells. Transztton Met. Chem. 15, 197-207. 22 Kennedy, J. F., Humphrey, J. D., and Barker, S A (1980) Application of hvmg nnmobihzed cells to the acceleration of the continuous conversions of ethanol (wort) to acetrc acid (vmegar)-hydrous titanium(W) oxide immobilized Acetobacter species. Enzyme Microb. Technol 2,209-2 16. 23. Cabral, 3. M S., Fevereiro, P., Novais, J. M., and Pais, S S (1983) Comparison of immobilized methods for plant cells andprotoplasts.EnzymeEng. 7,50 I-503. 24. Cabral, J. M. S. and Kennedy, J. F. (1987) Immobrhzatron of microbial cells on transition metal-activated supports, in Methods in Enzymology, vol 135 (Mosbach, K., ed.), Academic, Orlando, pp. 357-372. 25. Cabral, J M S., Cadete, M. M., Novais, J M., and Cardoso, J. I? (1984) Immobilization of yeasts on transition metal-activated pumice stone. Enzyme Eng 7,483-486.

Tfansition Metal-Based /mmobi/ization

359

26. Kennedy, J. F , Barker, S A., and Humphrey, J. D. (1976) Mrcrobtal cells livmg immobihzed on metal hydroxides. Nature 261,242-244 27. Dias, S. M., Novais, J. M , and Cabral, J. M. S. (1982) Immobihzation of yeasts on titamum activated inorganic support. Biotechnol Lett 4,203-208. 28. Cabral, J. M. S. and Kennedy, J. F. (1991) Covalent and coordination immobilization, in Protein Immobilization, vol 14 (Taylor, R , ed.), Marcel Dekker, New York, pp. 73-138. 29. Cardoso, J. P , Chaplin, M F., Emery, A N., Kennedy, J. F., and Revel-Chton, L. P (1979) The unmobtlizatton of enzymes on titanium-activated inorganic support. J Appl. Chem Biotechnol

28,775-785.

30. Kennedy, J. F., Barker, S. A., and White, C. A. (1977) Immobilization of a-amylase on polyaromatic and titanium compounds mcorporating a magnetic material Starch/ Starke 29,240-243.

3 1. Kennedy, J F. and Whtte, C. A. (1979) Stability and kinettc properties of magnettc nnmobilized a-amylase. Starch/Starke 31,375-38 1, 32. Kennedy, J. F., Kalogarakis, B., and Cabral, J. M. S. (1984) Immobtlizatton of enzymes on crosslinked gelatine particles activated with various forms and complexes of titanium(IV) species. Enzyme Microb. Technol. 6,68-72. 33. Kennedy, J. F and Kalogaraku, B. (1980) Immobthzatton of glucoamylase on gelatine by transition-metal chelatton. Blochzmie 62, 549-56 1 34. Kennedy, J F. and Cabral, J. M. S (1983) Immobilized livmg cells and their applicanon, in Immobdized Microbial Cells, vol. 4 (Chibata, I. and Wingard, L., eds.), Academic, New York, pp 189-280. 35. Kennedy, J. F. and Kay, I. M. (1976) Hydrous titanmm oxides-new support for the simple immobrlization of enzymes J. Chem. Sot. Perkm Trans 1,329-335. 36. Cabral, J. M. S. (1982) Studres on immobilization of enzymes by the method of transition metals. PhD. Thesis, Technical University of Lisbon, Portugal 37. Mattiasson, B. and Kaul, R. (1991) Determination of coupling yields and handling of labile proteins in immobrhzation technology, in Protean Immobdzzation, vol. 14 (Taylor, R., ed.), Marcel Dekker, New York, pp. 16 l-l 79. 38. Bemfeld, P. (1955)Amylases, a and p, m Methods m Enzymology, vol. 1 (Colowtck, S. P and Kaplan, N. 0, eds.), Academic, London, pp 149-158. 39. Chaplin, M. F. (1994) Monosaccharides, in Carbohydrate Analysis-A Practtcal Approach (Chaplin, M. F. and Kennedy, J. F., eds.), IRL, Oxford, UK, pp. lltl 40. Vogel, A. I. (1954) A Text-Book of Practical Organic Chemutry, Longmans, Green and Co., London, pp. 139-144.

Index A Acid phosphatase, assay, 7 I entrapment in poly-HEMA gel, 69 Acttvated nylon chemical modification of, 32 Adsorption, advantages of, 4, 327 bioselective, 13 of biotinylated P-galactosidase, 17 of biotinylated transglutammase, I7 characteristics of, 3 desorptton, 5 dtsadvantages of, 5 morgamc supports for, 328 nonspecific binding, 5 Agarose, activation of, 283 amine derivative, 268 characteristics of, 262 containing metal chelating groups, 339 derivatives, 266 dextran complex, 270 glutaraldehyde derivative, 269 glyoxyl, 277 microarchitecture, 7 multipoint attachment of enzymes, 277-279 spacer arm addition of, 269 Algmate gel, destabtlizatton of, 62 Alginate-poly-L-lysine entrapment of cells, 3 12 Alkylated nylon, chemical modification of, 29

Aluminum activation, with glyctdol, 335 wtth TCT, 334 Aminoaryl derivattve of, Eupergtt C, 47 glass, 46 glycophase-coated CGP, 46 nylon, 46 weak ion-exchangers, 47 Ammoaryl derrvattves, activation of, 47 Ammopropyl glass, removal of bound protein, 24 succmylation of, 23 synthesis of, 23 thionyl chloride activation of, 23 a-Amylase, assay, 159 immobtlizatton to cellulose, 158 Antibodies immobilization of, 24 Avidm-btotin interaction, 13 B Bioactivity of munobilized denitrtfymg sludge, 2 11 Biotin assay, on red blood cells, 147 binding sites of, 16 Biotin-avtdin red blood cells, determination of, 149 flow cytometry of, 150 Biotinylated proteins binding to red blood cells, 149 Biotinylation of, enzyme, 17 proteins, 146

367

362 red blood cells, 146 supports, 15 Biotransformation of, 7-ethoxycoumarin, 186 4-nitrophenol, 189 sucrose, 5 umbelhferone, 188 Bovine serum albumin-polyethylene glycol, hydrogel, characteristrcs of, 118 enzyme rmmobtlizatron, 120 BSA-PEG, see Bovine serum albummpolyethylene glycol C Calcium alginate, characteristics of, 61, 62 entrapment of enzymes in, 63, 321 Calcmm alginate-modified PVA beads, 209 Carbon parttcles, characteristics of, 2 17-2 19 immobihzatton of, glucose oxidase, 220 horesradish peroxidase to, 22 1 Carbonyldiimidazole, activated support commercially available, 299 activatron of cellulose, 159 characteristics of, 155, 156 tc-Carrageenan, properties, 53, 58 structure, 57 CDI, see Carbonyldrimidazole Cells, coimmobihzation wtth enzymes, 319 Cells, nnmobilization in, PCS hydrogels, by dropping, 128 by suspension, 129 poly-HEMA gel, 7 1, 72 Cellulose, activatton with cyanogen bromide, 158 aminoaryl derivative, 46 support, 153-l 56

index Chemically modified nylon, 37, 38 Chitosan-polyamon complex entrapment of cells, 3 13 Chitosan-xanthan, characteristics of, 229 immobilization of, lipase to, 233 protease to, 233 xylanase to, 232 spheres, preparation of, 232 Chlorophyll determmation, 112 Chymotrypsin mnnobihzed on glyoxyl agarose, 296 CM-cellulose, ammoaryl derivative, 47 CM-Sephadex, ammoaryl derivative, 47 CPG, see Controlled pore glass Coimmobrhzatton of, enzymes and cells, 3 19 lipase and xylanase, 234 xylanase and protease, 233 Controlled pore glass, ammoaryl derivative of, 46 cleaning of, 23 microarchitechture of, 7 regeneration of surface of, 18 Covalent binding, 5 Creatme kmase, enzyme assay of, 256 munobibzatlon of, 247 rmmobrhzed subunits, preparatton of, 249 reassociated subunits preparation of, 250 thlol group assay of, 257 Crosslinkmg, characteristics of, 9 electrochemically aided, 95 enzymes onto microelectrodes, 83 Cyanogen bromide, activation of, cellulose, 158 hydroxyl supports, 7, 155,247 Sepharose, 247

Index D o-amino acid oxidase immobilization to glyoxyl agarose, 296 Denitrtfying sludge, characteristics of, 2 11 tmmobtlrzation of, 2 11 Dextran, complex with amme agarose, 270 modification of, 293 Dimtrosaiicylic acid assay for reducing sugars, 159 DNS, see Dinitrosalicylic acid

E Electrochemical-based immobihzation, electrochemically atded crosslmking, 96 entrapment m electrochemically grown polymers, 95 Eletrochemically grown polymers, entrapment of enzymes, 95 Electrostatrc droplet, encapsulation, 169-l 72 generation, 169 Encapsulation, characteristics of, 9 Encapsulation in, alginate/poly-r.-lysine, 3 12 chitosan/polyanion complex, 3 13 photopolymertzed gel, 9 1 polypyrrole, 95 sodium cellulose sulfate, 3 12 Entrapment characteristics of, 8 Entrapment in, calcium alginate, 32 1 eletrochemrcally grown polymers, 95 polyelectrolyte complexes, 309, 3 10 Enzyme tmmobrhzation, on activated nylon, 34-36 in tc-carrageenan, 56 on chemically modified nylon, 37, 38 multipoint attachment, 277-279 in poly-HEMA gel, 7 1, 72

363 stabilization strategy, 277-28 1 on thermo-responsive polymer, 105 Enzymes, coimmobilization with cells, 319 Erwnia cells, immobtltzation of, 3 7-Ethoxycoumarin, btotransformatton of, 188

F Fines, removal of, 18 P-Fructofuranosrdase, assay of, 71 entrapment m poly-HEMA

gel, 69

G P-Galactosrdase, assay of, 18 biotinylation, 17 Gel-buffer suspensron, 248 Gelatin, complex, wrth lactose and titanium, 353 with titanium and acrylamtde, 353 immobtltzation of glucoamylase, 354 Glucoamylase assay of, 352 Glucose oxidase, assay of, 7 1, 22 1 electrochemical crosslinking of, 95 entrapment in, electrochemically grown polymer, 95 photopolymertzed gels, 91 poly-HEMA gel, 69 immobilizatton to carbon parttcles, 220 mnnobilizatron on a microelectrode, 84 P-Glucosidase, assay of, 71 entrapment in poly-HEMA gel, 69 Glutamate dehydrogenase, immobilization of, 24 Glutaraldehyde-agarose, 269

Index

364 Glutaraldehyde, storage problems, 84 Glycidol activation of inorganic support, 3 3 5 Glycoprotein nonspecific binding of, 80 Glyoxyl agarose, 277-280 Graphitic particles, 2 17-2 19 H Hepatocytes, mnnobiltzatton m agarose threads, 178 tmmobilized, assessment of integrity and functionality, 180 biotransformation reactions of, 188, 189 perfusion of, 179 isolation of, 177 Horseradish peroxidase, assay of, 222 immobilrzation to carbon particles, 221 I Immobtlization, to activated nylon, via amide bonds, 34 via amidme groups, 35 via Schiff’s bases, 35 via Ugi’s condensation, 36 of antibodies, 24 of cells in PVA beads, 208 to chemically modified nylon, via amidine groups, 38 via azo groups, 38 via disultide bridges, 38 via Schiff s bases, 37 of creatine kmase subunits, 244 of demtrifying sludge, 211 of electrostatic droplets, 169 of enzymes onto cell surface, 323 of Erwma cells, 3 of lipoxygenase, efficiency, 303

method, selection of, 2 on PEI-coated magnetite with a spacer, 136 on PEI-coated magnetite without a spacer, 135 on red blood cells, 143, 144 visual assessment of, 43-44 Immobilized, biotm measurement of, 16 enzymes, reduction of stertc problems, 261-266 enzyme suburnts, 243-245 hepatocytes, integrity of, 180 metabohsm of, 186 oxygen consumption of, 18 1 perfusion of, 178 phosphohpids, determination of, 199 protein, determmatron of, 199 proteoliposomes, affinity separations, 200 drug partitioning, 199 ligand binding studies, 200 receptor binding, 20 1 reassociated enzyme, 250 Inorganic supports, 328 L Lactamase, assay of, 305, 306 immobilization to CDI-activated agarose, 302 Lactococcus

lactls,

bioreactor, 239-24 1 nnmobllization, 239 Lewatn CNP-80, ammoaryl derivative, 47 Ltpase counmobiltzation with xylanase, 234 Lipase tmmobilizatton, m chitosan-xanthan, 233 hydrolysis of olive oil, 234 to inorganic support, 328,334

index Ltpoxygenase, assay of, by, HPLC, 305 spectrophotometry, 304 thin layer chromatography, 304 tmmobiltzation to CDI-activated agarose, 302 Lowry protein assay, 160, 199 M Magnetic agarose support, 340 Magnetite support material, 133 Microelectrodes, crosslinking enzymes to, 84 electrochemical-based rmmobihzation of, 93, 94 photochemical immobihzation of enzymes to, 87-89 Microencapsulation in polyelectrolyte capsules, 322 Mtcroorganisms, tmmobtlrzation m carrageenan, 56 N 4-Nitrophenol, biotransformation by unmobrltzed cells, 189 Nylon, ammoaryl derrvative, 46 chemical modification, 29, 30 form selection, 38, 39 general properties, 27,28 pretreatment, 33 Nylon activation by, N-alkylation, 34 0-alkylation, 34 partial hydrolysis, 33 0 Olive oil hydrolysis by immobilized lipase, 234 P PCS, see Poly(carbamoy1 sulfonate) PEG, see Poly(ethylene glycol)

365 PEI, see Polyethylenetmme Pencillin G acylase, immobrhzation to inorganic support, 334 Phospholipid, assay of, 199 Phosphorylated polyvinyl alcohol beads, characterrsttcs of, 207 determination of gas permeability, 2 10 mechanical strength, 2 10 modification with calcmrn alginate, 209 immobilizatron of, cells, 208 denitrtfymg sludge, 2 11 Photolithographic patterning of enzymes on microelectrodes, characteristrcs of, 87-89 lift-off technique, 90 photopolymerization, 91 Photosynthetic membranes, tmmobilization of, 112 Photosystem II submembrane fractions, isolation of, 111 Photosystem I submembrane fractions, isolation of, 111 Plant cells, immobilization m carrageenan, 56 Polyaldehyde modification of immobilized enzymes, 294 Polyaldehydes, characteristics of, 290,29 1 formation of polyammes from, 295 immobilization of enzymes with, 294 preparation of, 294 Polyamine modification of immobrllzed enzyme, 295 Poly(2-hydroxyethyl methacrylate), characteristics of, 67,68 entrapment in, 7 1 optimization of, 69 Poly(carbamoy1 sulfonate) hydrogels, beads, dropping of, 128 suspension of, 129

366 characterlstlcs of, 125-127 membranes of, 128 Polyethylenelmme, coated magnetite particles, 133, 134 immobtllzation of glycoenzyme, 136 Poly(ethylene glycol), crosslinked to BSA, 118 derivatization of, 120 Polyfunctional macromolecule from dextran, 293 Poly-HEMA, see Poly (2-hydroxyethyl methacrylate) Protease, coimmobillzatlon with xylanase, 233 immobihzatlon in chitosan-xanthan, 233 Protein, assay of lmmoblllzed enzyme, 255 blotmylation of, with blotm hydrazide, 148 with blotin-NHS, 147 blotmylated, binding to membrane surface, 149 immobilized on rayon/polyester, 79 Proteoliposomes, immoblllzatlon by, detergent dtalysls, 198 freeze-thawing, 196 hydrophobicity, 198 preparation, by detergent depletion, 195 by lipid hydration, 196 from phospholipids, 195 PVA, see Phosphorylated polyvinyl alcohol R Rat hepatocytes, lmmobilizatlon of, 178 isolation of, 177 Rayon/polyester characteristics of, 77, 78 lmmoblllzatlon on, 79

Index Red blood cells, blotinylatlon of, with blotm hydrazide, 146 with NHS-biotm, 146 blotinylated, avidm binding to, 149 determmation of biotm-labeled of, 146 determmatlon of biotm molecules on, 147 Rennin, action on small and large substrates,273 Immoblllzatlon of, 272 Reversible immobilization, 339 S Safety general note, v Sepharose, activation with cyanogen bromide, 247 Silica with, activation, glycldol, 335 TCT, 334 immobilization of, lipase to, 334 Pencillin G acylase to, 334 microachitecture, 7 Sodium cellulose sulfate entrapment of cells, 3 12 Stabilization of, o-amino acid oxidase, 296 chymotrypsin, 296 enzymes, 289-292 Stainless steel, bioreactor, 239 mesh, 239 Steric problems reduction of, 26 l-266 Succinarmdopropyl glass, activation of, 23 Sucrose biotransformation of, 3 Support material, activated nylon, 34-36 agarose, 178 alginate gel, 6 1 alginate-polylysine complex, 3 12 aluminum, 3 34

Index ammoaryl-based, 47 aminopropyl glass, 23 biotinylation of, 15 BSA-PEG hydrogel, 117, 118 calcmm alginate, 6 1, 239 calcmm alginate-modified PVA, 209 carbon particles, 2 17-2 19 K-carrageenan, 56 celite, 13 cellulose, 153-l 56 cellulose-aminoaryl derivative, 46 chemically modified nyon, 37, 38 chitosan-polyanion complex, 3 13 chitosan-xanthan, 229 CM-cellulose, aminoaryl derivative, 47 CM-Sephadex, aminoaryl derivative, 47 CPG, 22 CPG-ammoaryl derivative, 46 dextran-agarose, 270 Eupergit C, 42 gelatin, 353 glyoxyl-agarose, 277 hposomes, 193 magnetic agarose, 340 modified-agarose, 265 PCS hydrogels, 125-l 27 PEI-coated magnetite, 135 polyaldehydes, 290,291 poly-HEMA hydrogel, 67,68 polypyrrole, 95 PVA beads, 208 rayon-polyester, 77, 78 red blood cells, 146 Sepharose, 247 silica, 333 sodium cellulose sulfate, 3 12 thermo-responsive polymer, 105

T TCT, see Trichlorotriazine Thermo-responsive polymer, characteristics of, 10 l-l 03 immobilization on, 105

367 Thiol groups assay, 257 Thionyl chloride, activation of glass, 23 Thylakoid membranes, isolation, 111 Titanium-activated, inorgamc support, 35 1 organic support, 353 Tosyl chloride activation of rayon/ polyester, 79 Transglutaminase, assay of, 17 biotinylation of, 17 Transitton metal immobilization, 345349 Trichlorotriazine, activation of sthca, 334 reagent, 33 1,332 Trypsin immobilized to agarose, 285

U UM, see Umbelliferone Umbelliferone, biotransformation

of, 186

V Vulcan XC-72,224 X Xylanase, coimmobilization with, lipase, 234 protease, 233 immobilization in chitosan-xanthan, 232 Y Yeast cells, entrapment in, calcium algmate, 63 poly-HEMA gel, 69 immobilization on, hydrous transition metal oxides, 355 titanium-activated inorganic support, 355