Immobilized Enzymes and Cells

Contributors to V o l u m e 136 Article numbersare in parenthesesfollowingthe names of contributors. Affiliationslisted ...

Author: Nathan P. Colowick | Nathan P. Kaplan | Klaus Mosbach

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Contributors to V o l u m e 136 Article numbersare in parenthesesfollowingthe names of contributors. Affiliationslisted are current. OBSIDIANA ABRIL (25), Research and De-

PETER S. J. CHEETHAM (40), PPF Internavelopment, Angenics, Inc., Cambridge, tional, Ashford, Kent TN24 OLT, England Massachusetts 02139 ICHmO CHIBATA (41, 43), Research andDeL. ANDERSSON (I 1), Ferring AB, S-200 62 velopment Headquarters, Tanabe SeiyMalm6, Sweden aku Co., Ltd., Yodogawa-ku, Osaka 532, Japan RICHARD ARMENTA (9), Molecular Devices Corporation, Palo Alto, California 94304 DEBBIE C. CRANS (25), Department of Chemistry, Colorado State University, D. AURIOL (23), BioEurope, F-31400 TouFort Collins, Colorado 80523 louse, France PIERO CREMONESl (14), ltalfarmaco, MASAKI AZUMA (36), Technical Research S.p.A., Milan, Italy Laboratories, Kyowa Hakko Kogyo Co., Ltd., Hofu Plant, Hofu-shi, Yamagachi M. J. DANIELS (34), Genencor, Inc., South Prefecture 747, Japan San Francisco, California 94080 JOHANN BADER (28), Deutsches Patentamt, M. DELUCA (8), Department of Chemistry, D-8000 Miinchen 2, Federal Republic of University of California, San Diego, La Germany Jolla, California 92093 J. L. BARET (38), Novo lndustrie Enzymes ROBERT K. DINELLO (9), Chiron CorporaS.A., F-75017 Paris, France tion, Emeryville, California 94608 L. A. BEHIE (30), Department of Chemical G. FLEMINGER (17), Department of Biotechand Petroleum Engineering, The Univernology, The George S. Wise Faculty of sity of Calgary, Calgary, Alberta, Canada Life Sciences, TeI-Aviv University, RaT2N 1N4 mat-Aviv, 69 978 TeI-Aviv, Israel LARRY G. BUTLER (22), Department of BioSABURO FUKUI (27), Department of Induschemistry, Purdue University, West Latrial Chemistry, Faculty of Engineering, fayette, Indiana 47907 Kyoto University, Yoshida, Sakyo-ku, Kyoto 606, Japan G. J. CALTON (45), RhOne.Poulenc Research Center, Savage, Maryland 20763 MURRAY C. FUSEE (42), Research Division, W. R. Grace & Co., Washington ReBERNARD CAMBOU (12), Laboratory of Apsearch Center, Columbia, Maryland plied Biochemistry, Department of Ap21044 plied Biological Sciences, Massachusetts Institute of Technology, Cambridge, G. M. GAUCHER (30), Division of BiochemMassachusetts 02139 istry, Department of Biological Sciences, Faculty of Science, The University of CalM. L. CAMPBELL (45), RhOne.Poulenc Regary, Calgary, Alberta, Canada T2N 1N4 search Center, Savage, Maryland 20763 S. GESTRELIUS (32), Ferring AB, S-200 62 GIACOMO CARREA (14), Istituto di Chimica Malm6, Sweden degli Ormoni, Consiglio Nazionale delle Ricerche (C.N.R.), 20131 Milan, Italy IAN GIBBONS (9), Biotrack, Inc., Sunnyvale, California 94086 Z. M. S. CHANG (7), Artificial Cells and Organs Research Centre, Faculty of Medi- HELMUT G/3NTHER (28), Lehrstuhl fiir Orcine, McGill University, Montreal, Queganische Chemie und Biochemie im Orbec, Canada H3G 1 Y6 ganisch-Chemischen lnstitut der Technisix

X

CONTRIBUTORS TO VOLUME 136

chen Universitgit Miinchen, D-8046 REINHOLD KELLER (47), Hoechst AktiengeGarching, Federal Republic of Germany sellschaft, D-6230 Frankfurt am Main 80, Federal Republic of Germany NORIO HAGI (46), Biotechnology Research Center, Toyo Soda Manufacturing Co., ALEXANDER M. KLISANOV (12), LaboraLtd., Yamaguchi-ken 746, Japan tory of Applied Biochemistry, Department of Applied Biological Sciences, MassaS. HARBRON (13), Department of Biochemchusetts Institute of Technology, Camistry, University College London, London bridge, Massachusetts 02139 WCIE 7JE, England ANDREAS KONNECKE (18), Department of URSULA HAUFLER (26), Fachbereich BioloBiochemistry, Biosciences Division, Karlgic, Universitgit Bremen, D-2800 Bremen Marx-University, DDR-7010 Leipzig, 33, Federal Republic of Germany German Democratic Republic LARS HEDSYS (21), Pure and Applied BioY. KONNO (49), Dowa Engineering Co., chemistry, Chemical Center, University Ltd., Tokyo 100, Japan of Lund, S-221 O0 Lund, Sweden RIET HILHORST (20), Department of Bio- BETTINA Koep (29), Henkel KGaA, D-4000 Diisseldorf 1, Federal Republic of Gerchemistry, Agricultural University Wamany geningen, 6703 BC Wageningen, The FRIEDRICH KRAUSS (28), Lehrstuhlfar OrNetherlands ganische Chemie und Biochemie im OrBERNARD L. HIRSCHBEIN (25), Imaging Reganisch-Chemischen lnstitut der Technissearch, Polaroid Corporation, Camchen Universitdt Miinchen, D-8046 bridge, Massachusetts 02139 Garching, Federal Republic of Germany T. IMAIZUMI (49), The University of Tokyo, L. J. KRICKA (8), Department of Clinical Shibuya-ku, Tokyo 113, Japan Chemistry, Wolfson Research LaboratoKEIICHI INUZUKA (36), Kyowa Hakko ries, University of Birmingham, BirmingKogyo Co., Ltd., Ube Plant, Ube-shi, ham B15 2TH, England Yamaguchi Prefecture 755, Japan MARIA-REGINA KULA (2), lnstitut far EnSHIGEAKI IRINO (46), Chemical Research zymtechnologie der Universitgit DiisselCenter, Toyo Soda Manufacturing Co., dorf in der Kernforschungsanlage Jiilich Ltd., Yamaguchi-ken 746, Japan GmbH, D-5170Jiilich 1, Federal Republic HANs-DIETER JAKUBKE (18), Department of of Germany Biochemistry, Biosciences Division, KarlCOLJA LAANE (20), Unilever Research LaMarx-University, DDR-7010 Leipzig, boratorium, 3130 AC Vlaardingen, The German Democratic Republic Netherlands VIOLETA G. JANOLINO (39), Department of Food Science, North Carolina State Uni- KEITH J. LAIDLER (6), Department of Chemistry, Faculty of Science, University versity at Raleigh, Raleigh, North Caroof Ottawa, Ottawa, Ontario, Canada lina 27695 KIN 6N5 VILLY J. JENSEN (33), Molecular Biology, PER-OLOE LARSSON (21), Pure and Applied Novo Industri A/S, DK-2880 Bagsvaerd, Biochemistry, Chemical Center, UniverDenmark sity of Lund, S-221 O0 Lund, Sweden VOLKER KASCHE (26), Arbeitsbereich M. D. LILLY (13), Department of Chemical Biotechnologie H, Biotransformation and Biochemical Engineering, University und -Sensorik, Technische Universitiit College London, London WC1E 7JE, EnHamburg-Harburg, D-2100 Hamburg 90, gland Federal Republic of Germany A. LOPEZ (23), Departamento de Alimentos ROMAS J. KAZLAUSKAS (25), Research and DEPg, Facultad de Quimica, Mexico Development Center, General Electric D.F., Mexico Company, Schenectady, New York 12301


xi

P. L. LuIsI (19), lnstitut fiir Polymere,

SHOGO NOJIMA (35), Catalysts and Chemi-

Swiss Federal Institute of Technology, EidgenOssische Technische Hochschule (ETH)-Zentrum, CH-8092 Zurich, Switzerland HIDEKATSU MAEDA (3), Fermentation Research Institute, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, Ibaraki-ken 305, Japan MATS-OLLE MXNSSON (1, 10, 11), Pure and Applied Biochemistry, Chemical Center, University of Lund, S-221 O0 Lund, Sweden M. ABDUL MAZID (6), Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2 P. MONSAN (23), BioEurope, F-31400 Toulouse, France KAZUYUKI MORIHAgA (16), Kyoto Research Laboratories, Toho Pharmaceutical Industry Company, Limited, Kyoto Prefecture 617, Japan KLAUS MOSBACH (1, 10, 11, 21, 32), Pure and Applied Biochemistry, Chemical Center, University of Lund, S-221 O0 Lund, Sweden RYONOSUKE MUNEYUKI (16), Research Division, S. T. Company, Limited, Shimoochiai, Shinjuku-ku, Tokyo 161, Japan T. MURAYAMA(49), Oga Operation Office, Akita Oil Storage Co., Ltd., Funagawa, Funagawa-ko, Oga City, Akita Prefecture 010-05, Japan MINORU NAGASHIMA (36), Technical Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Hofu Plant, Hofu-shi, Yamaguchi Prefecture 747, Japan T. J. NAI~NDgANATHAN (13), Wellcome Biotechnology Ltd., South Eden Park, Beckenham, Kent BR3 3BS, England STEFAN NEUMANN (28), Consortium far Elektrochemische, Industrie GmbH, D8000 Miinchen, Federal Republic of Germany SADAO NOGUCHI (36), Technical Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., Hofu Plant, Hofu-shi, Yamaguchi Prefecture 747, Japan

cals, Industry Co., Ltd., Chiyoda-ku, Tokyo I00, Japan TATSUSHI OKA (16), Shionogi Research Laboratories, Shionogi and Company, Limited, Osaka 553, Japan HIROSUKE OKADA (4), Department ofFermentation Technology, Faculty of Engineering, Osaka University, Osaka 565, Japan KIYOTAKAOYAMA(46), Chemical Research Center, Toyo Soda Manufacturing Co., Ltd., Yamaguchi-ken 746, Japan F. PAUL (23), BioEurope, F-31400 Toulouse, France URSULA PFITZNER (31), Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114 LuTz RIECHMANN(26), Fachbereich Biologie, Universitgit Bremen, D-2800 Bremen 33, Federal Republic of Germany G. P. ROYER (15), Biotechnology Division, Amoco Corporation, Amoco Research Center, Naperville, Illinois 60566 J. DAVID ROZZELL (44), Genetics Institute, Cambridge, Massachusetts 02140 SUSANNERUG8 (33), Process Development, Novo Industri A/S, DK-2880 Bagsvaerd, Denmark T. SAKATA (49), Metal Mining Agency of Japan, Minato-ku, Tokyo 105, Japan HmOTOSHX SAMEJIMA (36), Kyowa Medex Co., Ltd., Chiyoda-ku, Tokyo 100, Japan MERTEN SCHLINGMANN (47), Hoechst Aktiengesellschaft, D-6230 Frankfurt am Main 80, Federal Republic of Germany NILS SIEGBAHN(10), Perstorp Biolytica AB, S-223 70 Lund, Sweden HELMVT SIMON (28), Lehrstuhlfiir Organische Chemie und Biochemie im OrganischChemischen Institut der Technischen Universitiit Miinchen, D-8046 Garching, Federal Republic of Germany PAUL J. SKUDDER (39), Process Development Department, APV International Ltd., West Sussex RHIO 2QB, England B. STEINMANN-HOFMANN(19), Institutfiir Polymere, Swiss Federal Institute of

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Technology, EidgenOssische Technische CEES VEEGER (20), Department of BiochemHochschule (ETH)-Zentrum, CH-8092 istry, Agricultural University WaZurich, Switzerland geningen, 6703 BC Wageningen, The Netherlands" HIDEO SUZUKI (5), Fermentation Research Institute, Agency of Industrial Science CHRISTIAN WANDREY (2), Institut fiir and Technology, Ministry of International Biotechnologie 2 in der KernforsTrade and Industry, Ibaraki-ken 305, chungsanlage Jiilich GmbH, D-5170 JiiJapan lich 1, Federal Republic of Germany SIGFRID SVENSSON (21), Carbohydrate ICHIRO WATANABE (48), Central Research Chemistry, Chemical Center, University Laboratory, Nitto Chemical Industry Co., of Lund, S-221 O0 Lund, Sweden Ltd., Tsurumi-ku, Yokohama 230, Japan HAROLD E. SWAISGOOD (39), Department GEORGE M. WmTESIDES (25), Department of Food Science, North Carolina State of Chemistry, Harvard University, CamUniversity at Raleigh, Raleigh, North bridge, Massachusetts 02138 Carolina 27695 G. WIENHAUSEN (8), Department of BiolSATORU TAKAMATSU (43), Research Laboogy, University of California, San Diego, ratory of Applied Biochemistry, Tanabe La JoUa, California 92093 Seiyaku Co., Ltd., Yodogawa-ku, Osaka ROLF WOERNLE (47), Hoechst Aktienge532, Japan seUschaft, D-6230 Frankfurt am Main 80, ISAO TAKATA (41), Research Laboratory of Federal Republic of Germany Applied Biochemistry, Tanabe Seiyaku Cm-HuEY WONG (25), Department of Co., Ltd., Yodogawa-ku, Osaka 532, Chemistry, Texas A&M University, ColJapan lege Station, Texas 77843 ATSUO TANAKA (27), Department oflndusL. L. WOOD (45), RhOne.Poulenc Research trial Chemistry, Faculty of Engineering, Center, Savage, Maryland 20763 Kyoto University, Yoshida, Sakyo-ku, TOMIAKI YAMADA (35), Biotechnology ReKyoto 606, Japan search Department, Research and DevelTAKASHI TANAKA (37), Central Research opment Division, JGC Corporation, Laboratories, Ajinomoto Co., Inc., KaMinami-ku, Yokohama 232, Japan wasaki-ku, Kawasaki 210, Japan IORDANIS THANOS (28), Lehrstuhl fiir Or- K o z o YAMAMOTO (41), Research Laboratory of Applied Biochemistry, Tanabe ganische Chemie und Biochemie im OrSeiyaku Co., Ltd., Yodogawa-ku, Osaka ganisch-Chemischen Institut der Technis532, Japan chen Universitiit Miinchen, D-8046 SHIGERU YAMANAKA (37), Central ReGarching, Federal Republic of Germany search Laboratories, Ajinomoto Co., TETSUYA TOSA (41, 43), Research LaboraInc., Kawasaki-ku, Kawasaki 210, Japan tory of Applied Biochemistry, Tanabe Seiyaku Co., Ltd., Yodogawa-ku, Osaka YOSHIMITSU YAMAZAKI (3, 5), Fermenta532, Japan tion Research Institute, Agency of Industrial Science and Technology, Ministry of JOHANNES TRAMPER (24), Department of International T r a d e and Industry, Food Science, Food and Bioengineering lbaraki-ken 305, Japan Group, Agricultural University Wageningen, 6703 BC Wageningen, The A. YARON (17), Department of Biophysics, Netherlands The Weizmann Institute of Science, 76 100 Rehovot, Israel EDWIN F. ULLMAN (9), Syva Company, Palo Alto, California 94304 MEINHART H. ZENK (31), Lehrstuhl PharITARU URAaE (4), Department of Fermentamazeutische Biologie, Universitgit Mantion Technology, Faculty of Engineering, chen, D-8000 Miinchen 2, Federal RepubOsaka University, Osaka 565, Japan lic of Germany

Preface Volumes 135 through 137 of Methods in Enzymology, Immobilized Enzymes and Cells, Parts B through D, include the following sections: (1) Immobilization Techniques for Enzymes; (2) Immobilization Techniques for Cells/Organelles; (3) Application of Immobilized Enzymes/Cells to Fundamental Studies; (4) Multistep Enzyme Systems and Coenzymes; (5) Immobilized Enzymes/Cells in Organic Synthesis; (6) Enzyme Engineering (Enzyme Technology); (7) Analytical Applications with Emphasis on Biosensors; (8) Medical Applications; and (9) Novel Techniques for and Aspects of Immobilized Enzymes and Cells. The first three sections appear in Volume 135, the next three in Volume 136, and the last three in Volume 137. Immobilization techniques for enzymes, Section (1), has already been treated in Volume XLIV of this series. Immobilization techniques for cells/organelles, Section (2), an area which seems to have great potential, especially for the application of immobilized yeast and plant and animal cells, is covered for the first time in these volumes. Sections (3) and (4) have been dealt with previously. Section (5), the use of immobilized enzymes/cells in organic synthesis, has probably not been covered before. It is my firm opinion that in the not too distant future we will see a number of processes employed which are based, in part, on the examples given in this section. Section (6) on industrial uses updates the material presented in Volume XLIV. The examples given are, to the best of my knowledge, in operational use today or, at least, on a pilot plant level. Section (7), analytical applications with emphasis on biosensors, is the subject of a great deal of research at present, and it may very well be that in the not too distant future we will witness a breakthrough, i.e., many applications of a number of such devices. The medical area, covered in Section (8), seems promising, but certainly more research is required to fully exploit any underlying potential. Finally, in Section (9), I have collected a number of contributions that did not seem to fit in any of the other sections, but do address important and novel developments. I would like to note that although major emphasis in these volumes has been placed on immobilization in its strictest sense, preferentially, covalent attachment of enzymes or entrapment of cells, one should not view immobilizedsystems in too limited a manner. In fact, bioreactors confined by ultrafilter membranes or hollow fiber systems belong in this category, and the various systems appear to overlap. Immobilization techniques as applied to affinity chromatography or immunoassays such as ELISA are not included to any extent in these volumes since they have xiii

xiv

PREFACE

been adequately covered in other volumes of this series (e.g., Volumes XXXIV and 104 on affinity techniques). An area that was originally scheduled for inclusion is synzymes or artificial enzymes. These include attempts to create catalysts mimicking enzymes by coupling of functional groups to, for instance, cyclodextrin [e.g., D'Souza et al. (Biochem. Biophys. Res. Commun. 129, 727-732, 1985) and Breslow et al. (J. Am. Chem. Soc. 108, 1969, 1986)], to crown ethers [Cram et al. (J. Am. Chem. Soc. 107, 3645, 1985)], or to solid matrices [Nilsson and Mosbach (J. Solid-Phase Biochem. 4, 271, 1979) and Leonhardt and Mosbach (Reactive Polymers, in press)]. Related to these studies are attempts to create cavities in polymers with substrate-binding properties ]notably by Wulff et al. (e.g., Reactive Polymers 3, 261, 1985; and previous publications by these authors) and Arshady and Mosbach (Makromol. Chem. 182, 687, 1981)]. This exciting area is presently in a rapid state of development, and the methodology involved should soon be made available in a more comprehensive context. Mention should be made of the developments in the utilization of recombinant DNA technology for the immobilization (and affinity purification) of biomolecules. I refer to the reported fusion of "affinity tails" as polyarginine (Smith et al., Gene 32, 321, 1984), of polycysteine [B01ow and Mosbach, Proceedings of the VIII International Conference on Enzyme Engineering, Annals of the New York Academy of Sciences, in press (presented 1985)], or of protein A (Nilsson et al., EMBO J. 4, 1075, 1985) to enzymes facilitating their purification and immobilization. These preparations can be obtained by fusion of the respective groups as "tail" to the NH2 or COOH termini of the enzyme or by site-directed mutagenesis leading to substitution on the enzyme structure. DNA technology can also be usefully employed to create new multienzyme complexes, fusing enzymes acting in sequence to one another (B01ow et al., Bio/Technology 3, 821, 1985) as an alternative to their co-immobilization on supports; similarly, attachment of "tails" allowing reversible coenzyme binding may be accomplished. The same technology has also been used recently in attempts to prepare esterase mimics from the ground up (Biilow and Mosbach, FEBS Lett. 210, 147, 1987). Since this is such a rapidly moving area, I advise the reader, apart from the usual standard books in this area, to read the proceedings of the Enzyme Engineering Conferences 1-8 (Wiley, first conference; Plenum Press, second-sixth conferences; and Annals of the New York Academy of Sciences, seventh and eighth conferences); Biochemical Engineering, Volumes I-III and subsequent volumes; Annals of the New York Academy of Sciences, 1983; the patent book "Enzyme Technology, Recent Advances" (S. Torrey, ed.), Noyes Data Corporation, Park Ridge, New

PREFACE

XV

Jersey, 1983; and Biotechnology Review no. 2. In addition, in the following journals many articles relating to immobilized enzyme and cell research can be found: Biotechnology and Bioengineering (John Wiley & Sons); Trends in Biotechnology (Elsevier, The Netherlands); Bio/Technology (Nature Publishing Co., U.S.); Applied Biochemistry and Biotechnology (The Humana Press, Inc., U.S.); Applied Biochemistry with Special Emphasis on Biotechnology; Biotechnology Letters (Science and Technology Letters, England); Applied Microbiology and Biotechnology (Springer-Verlag, Germany); Enzyme and Microbial Technology (Butterworth Scientific Limited, England); Biosensors (Elsevier Applied Science Publishing Ltd., England). In studies with immobilized systems, sometimes useful, not immediately obvious "by-products" may be obtained. I refer to the finding that immobilized Escherichia coli cells, when kept in media without selection pressure, show improved plasmid stability (de Taxis du PoEt, P., Dhulster, P., Barbotin, J.-N., and Thomas, D., J. Bact. 165, 871, 1986). An additional example would be the improved regeneration of plants using immobilized protoplasts discussed in Section (2). I would like to express the hope that these volumes present an overview of the various areas in which immobilized enzymes and cells are used, act as a stimulus for further research, and provide methodological "know-how." The proper choice of support and/or immobilization technique for a particular application may not always be easily accomplished, but I hope that guidance to do so is found in these volumes. Putting these volumes together has been a time-consuming and, at times, frustrating undertaking. Without the coeditors, Drs. Lars Andersson, Peter Brodelius, Bengt Danielsson, Stina Gestrelius, and Mats-Olle M~nsson, the volumes would not have materialized. Because of the number of coeditors, some heterogeneity in the editing has resulted. Contributors to the various sections are from substantially different disciplines, and again this has contributed to the heterogeneity that can be found. Part of the editing of the three volumes was carried out in Ziirich, where I held a chair in biotechnology at the Swiss Federal Institute of Technology. Without the enormous efforts and skills of the staff of Academic Press, these volumes would never have reached production. I also owe much gratitude to my secretaries, notably lngrid Nilsson, for their highly qualified help. Finally, I would like to thank the contributors for their efforts. These volumes are dedicated to the memory of the late Professors N. O. Kaplan and S. P. Colowick, with whom I had highly fruitful discussions, especially at the beginning of this undertaking.

K L A U S MOSBACH

METHODS IN ENZYMOLOGY EDITORS-IN-CHIEF

Sidney P. Colowick and Nathan O. Kaplan

VOLUME VIII. Complex Carbohydrates

Edited by ELIZABETH F. NEUFELD AND VICTOR GINSBURG VOLUME IX. Carbohydrate Metabolism

Edited by WILLIS A. WOOD VOLUME X. Oxidation and Phosphorylation

Edited by RONALD W. ESTABROOK AND MAYNARD E. PULLMAN VOLUME XI. Enzyme Structure Edited by C. H. W. HIRS VOLUME XlI. Nucleic Acids (Parts A and B)

Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XlII. Citric Acid Cycle

Edited by J. M. LOWENSTEIN VOLUME XIV. Lipids

Edited by J. M. LOWENSTEIN VOLUME XV. Steroids and Terpenoids

Edited by RAYMOND B. CLAYTON VOLUME XVI. Fast Reactions

Edited by KENNETH KUSTIN VOLUME XVII. Metabolism of Amino Acids and Amines (Parts A and B)

Edited by HERBERT TABOR AND CELIA WHITE TABOR xix

XX

M E T H O D S IN E N Z Y M O L O G Y

VOLUME XVIII. Vitamins and Coenzymes (Parts A, B, and C)

Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME XIX. Proteolytic Enzymes

Edited by GERTRUDE E. PERLMANN AND LASZLO LORAND VOLUME XX. Nucleic Acids and Protein Synthesis (Part C)

Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME XXI. Nucleic Acids (Part D)

Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XXII. Enzyme Purification and Related Techniques

Edited by WILLIAM B. JAKOBY VOLUME XXIII. Photosynthesis (Part A)

Edited by ANTHONY SAN PIETRO VOLUME XXIV. Photosynthesis and Nitrogen Fixation (Part B)

Edited by ANTHONY SAN PIETRO VOLUME XXV. Enzyme Structure (Part B) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVI. Enzyme Structure (Part C)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVII. Enzyme Structure (Part D)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XXVIII. Complex Carbohydrates (Part B)

Edited by VICTOR GINSBURG VOLUME XXIX. Nucleic Acids and Protein Synthesis (Part E)

Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME XXX. Nucleic Acids and Protein Synthesis (Part F)

Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN VOLUME XXXI. Biomembranes (Part A)

Edited by SIDNEY FLEISCHER AND LESTER PACKER

METHODS IN ENZYMOLOGY

xxi

VOLUME XXXII. Biomembranes (Part B)

Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME XXXIII. Cumulative Subject Index Volumes I - X X X

Edited by MARTHA G. DENNIS AND EDWARD A. DENNIS VOLUME XXXIV. Affinity Techniques (Enzyme Purification: Part B)

Edited by WILLIAM B. JAKOBY AND MEIR WILCHEK VOLUME XXXV. Lipids (Part B)

Edited by JOHN M. LOWENSTEIN VOLUME XXXVI. Hormone Action (Part A: Steroid Hormones)

Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN VOLUME XXXVII. Hormone Action (Part B: Peptide Hormones) Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN VOLUME XXXVIII. Hormone Action (Part C: Cyclic Nucleotides)

Edited by JOEL G. HARDMAN AND BERT W. O'MALLEY VOLUME XXXIX. Hormone Action (Part D: Isolated Cells, Tissues, and Organ Systems) Edited by JOEL G. HARDMAN AND BERT W. O'MALLEY VOLUME XL. Hormone Action (Part E: Nuclear Structure and Function)

Edited by BERT W. O'MALLEY AND JOEL G. HARDMAN VOLUME XLI. Carbohydrate Metabolism (Part B)

Edited by W. A. WOOD VOLUME XLII. Carbohydrate Metabolism (Part C)

Edited by W. A. WOOD VOLUME XLIII. Antibiotics Edited by JOHN H. HASH VOLUME XLIV. Immobilized Enzymes

Edited by KLAUS MOSBACH VOLUME XLV. Proteolytic Enzymes (Part B)

Edited by LASZLO LORAND

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VOLUME XLVI. Affinity Labeling

Edited by WILLIAM B. JAKOBY AND MEIR WILCHEK VOLUME XLVII. Enzyme Structure (Part E)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME XLVIII. Enzyme Structure (Part F)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEEF VOLUME XLIX. Enzyme Structure (Part G) Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME L. Complex Carbohydrates (Part C)

Edited by VICTOR GINSBURG VOLUME LI. Purine and Pyrimidine Nucleotide Metabolism

Edited by PATRICIA A. HOFFEE AND MARY ELLEN JONES VOLUME LII. Biomembranes (Part C: Biological Oxidations) Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LIII. Biomembranes (Part D" Biological Oxidations)

Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LIV. Biomembranes (Part E: Biological Oxidations)

Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LV. Biomembranes (Part F: Bioenergetics)

Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LVI. Biomembranes (Part G: Bioenergetics)

Edited by SIDNEY FLEISCHER AND LESTER PACKER VOLUME LVII. Bioluminescence and Chemiluminescence

Edited by MARLENE A. DELUCA VOLUME LVIII. Cell Culture

Edited by WILLIAM B. JAKOBY AND IRA PASTAN VOLUME LIX. Nucleic Acids and Protein Synthesis (Part G)

Edited by KIVIE MOLDAVE AND LAWRENCE GROSSMAN


xxiii

VOLUME LX. Nucleic Acids and Protein Synthesis (Part H)

Edited by KIVlE MOLDAVE AND LAWRENCE GROSSMAN VOLUME 61. Enzyme Structure (Part H)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 62. Vitamins and Coenzymes (Part D) Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME 63. Enzyme Kinetics and Mechanism (Part A: Initial Rate and Inhibitor Methods) Edited by DANIEL L. PURICH VOLUME 64. Enzyme Kinetics and Mechanism (Part B: Isotopic Probes and Complex Enzyme Systems) Edited by DANIEL L. PURICH VOLUME 65. Nucleic Acids (Part I)

Edited by LAWRENCE GROSSMAN AND KIVIE MOLDAVE VOLUME 66. Vitamins and Coenzymes (Part E) Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME 67. Vitamins and Coenzymes (Part F) Edited by DONALD B. MCCORMICK AND LEMUEL D. WRIGHT VOLUME 68. Recombinant DNA

Edited by RAY Wu VOLUME 69. Photosynthesis and Nitrogen Fixation (Part C)

Edited by ANTHONY SAN PIETRO VOLUME 70. Immunochemical Techniques (Part A) Edited by HELEN VAN VUNAKIS AND JOHN J. LANGONE VOLUME 71. Lipids (Part C)

Edited by JOHN M. LOWENSTEIN VOLUME 72. Lipids (Part D)

Edited by JOHN M. LOWENSTEIN

xxiv


VOLUME 73. Immunochemical Techniques (Part B) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 74. Immunochemical Techniques (Part C) Edited by JOHN J. LANGONEAND HELEN VAN VUNAK1S VOLUME 75. Cumulative Subject Index Volumes XXXI, XXXII, XXXIV-LX Edited by EDWARD A. DENNIS AND MARTHAG. DENNIS VOLUME 76. Hemoglobins

Edited by ERALDO ANTONINI, LUIGI ROSSI-BERNARDI, AND EMILIA CHIANCONE

VOLUME 77. Detoxication and Drug Metabolism Edited by WILLIAM B. JAKOBY VOLUME 78. Interferons (Part A)

Edited by SIDNEY PESTKA VOLUME 79. Interferons (Part B)

Edited by SIDNEY PESTKA VOLUME 80. Proteolytic Enzymes (Part C)

Edited by LASZLO LORAND VOLUME 81. Biomembranes (Part H: Visual Pigments and Purple Membranes, I) Edited by LESTER PACKER VOLUME 82. Structural and Contractile Proteins (Part A: Extracellular Matrix) Edited by LEON W. CUNNINGHAM AND DIXIE W. FREDERIKSEN VOLUME 83. Complex Carbohydrates (Part D) Edited by VICTOR GINSBURG VOLUME 84. Immunochemical Techniques (Part D: Selected Immunoassays)

Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS


XXV

VOLUME 85. Structural and Contractile Proteins (Part B: The Contractile Apparatus and the Cytoskeleton) Edited by DIXIE W. FREDERIKSEN AND LEON W. CUNN1NGHAM VOLUME 86. Prostaglandins and Arachidonate Metabolites

Edited by WILLIAM E. M. LANDS AND WILLIAM L. SMITH VOLUME 87. Enzyme Kinetics and Mechanism (Part C: Intermediates, Stereochemistry, and Rate Studies) Edited by DANIEL L. PURICH VOLUME 88. Biomembranes (Part I: Visual Pigments and Purple Membranes, II) Edited by LESTER PACKER VOLUME 89. Carbohydrate Metabolism (Part D)

Edited by WILLIS A. WOOD VOLUME 90. Carbohydrate Metabolism (Part E)

Edited by WILLIS A. W o o d VOLUME 91. Enzyme Structure (Part I)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 92. Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 93. Immunochemical Techniques (Part F: Conventional Antibodies, Fc Receptors, and Cytotoxicity) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 94. Polyamines

Edited by HERBERT TABOR AND CELIA WHITE TABOR VOLUME 95. Cumulative Subject Index Volumes 61-74, 76-80

Edited by EDWARD A. DENNIS AND MARTHA G. DENNIS VOLUME 96. Biomembranes [Part J: Membrane Biogenesis: Assembly and Targeting (General Methods; Eukaryotes)] Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER

xxvi


VOLUME 97. Biomembranes [Part K: Membrane Biogenesis: Assembly and Targeting (Prokaryotes, Mitochondria, and Chloroplasts)]

Edited by

SIDNEY FLEISCHER AND BECCA FLEISCHER

VOLUME 98. Biomembranes (Part L: Membrane Biogenesis: Processing and Recycling) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 99. Hormone Action (Part F: Protein Kinases)

Edited by JACKIE D. CORBIN AND JOEL G. HARDMAN VOLUME 100. Recombinant DNA (Part B)

Edited by RAY Wu, LAWRENCE GROSSMAN, AND KIVIE MOLDAVE VOLUME 101. Recombinant DNA (Part C)

Edited by RAY Wu, LAWRENCE GROSSMAN, AND KIVIE MOLDAVE VOLUME 102. Hormone Action (Part G: Calmodulin and Calcium-Binding Proteins) Edited by ANTHONY R. MEANS AND BERT W. O'MALLEY VOLUME 103. Hormone Action (Part H: Neuroendocrine Peptides)

Edited by P. MICHAEL CONN VOLUME 104. Enzyme Purification and Related Techniques (Part C)

Edited by WILLIAM B. JAKOBY VOLUME 105. Oxygen Radicals in Biological Systems

Edited by LESTER PACKER VOLUME 106. Posttranslational Modifications (Part A)

Edited by FINN WOLD AND KIVIE MOLDAVE VOLUME 107. Posttranslational Modifications (Part B)

Edited by FINN WOLD AND KIVIE MOLDAVE VOLUME 108. Immunochemical Techniques (Part G: Separation and Characterization of Lymphoid Cells) Edited by GIOVANNI DI SABATO, JOHN J. LANGONE, AND HELEN VAN VUNAKIS

METHODSIN ENZYMOLOGY

xxvii

VOLUME 109. Hormone Action (Part I: Peptide Hormones)

Edited by LUTZ BIRNBAUMERAND BERT W. O'MALLEY VOLUME 110. Steroids and Isoprenoids (Part A) Edited by JOHN H. LAW AND HANS C. RILLING VOLUME I 11. Steroids and Isoprenoids (Part B)

Edited by JOHN H. LAW AND HANS C. RILLING VOLUME 112. Drug and Enzyme Targeting (Part A)

Edited by KENNETH J. WIDDER AND RALPH GREEN VOLUME 113. Glutamate, Glutamine, Glutathione, and Related Corn-

pounds

Edited by ALTON MEISTER VOLUME 114. Diffraction Methods for Biological Macromolecules (Part A)

Edited by HAROLD W. WYCKOEE, C. H. W. HIRS, AND SERGE N. TIMASHEFF

VOLUME 115. Diffraction Methods for Biological Macromolecules (Part

B) Edited by HAROLD W. WYCKOEE, C. H. W. HIRS, AND SERGE N. TIMASHEFF

VOLUME 116. Immunochemical Techniques (Part H: Effectors and Mediators of Lymphoid Cell Functions) Edited by GIOVANNI DI SABATO, JOHN J. LANGONE, AND HELEN VAN VUNAKIS

VOLUME 117. Enzyme Structure (Part J)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 118. Plant Molecular Biology

Edited by ARTHUR WEISSBACHAND HERBERT WEISSBACH VOLUME 119. Interferons (Part C)

Edited by SIDNEY PESTKA VOLUME 120. Cumulative Subject Index Volumes 81-94, 96-101

XXVIn


VOLUME 121. Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies) Edited by JOHN J. LANGONE AND HELEN VAN VUNAKIS VOLUME 122. Vitamins and Coenzymes (Part G)

Edited by FRANK CHYTIL AND DONALD B. MCCORMICK VOLUME 123. Vitamins and Coenzymes (Part H)

Edited by FRANK CHYTIL AND DONALD B. MCCORMICK VOLUME 124. Hormone Action (Part J: Neuroendocrine Peptides)

Edited by P. MICHAEL CONN VOLUME 125. Biomembranes (Part M: Transport in Bacteria, Mitochondria, and Chloroplasts: General Approaches and Transport Systems) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 126. Biomembranes (Part N: Transport in Bacteria, Mitochondria, and Chloroplasts: Protonmotive Force) Edited by SIDNEY FLEISCHER AND BECCA FLEISCHER VOLUME 127. Biomembranes (Part O: Protons and Water: Structure and Translocation) Edited by LESTER PACKER VOLUME 128. Plasma Lipoproteins (Part A: Preparation, Structure, and Molecular Biology) Edited by JERE P. SEGREST AND JOHN J. ALBERS VOLUME 129. Plasma Lipoproteins (Part B: Characterization, Cell Biology, and Metabolism) Edited by JOHN J. ALBERS AND JERE P. SEGREST VOLUME 130. Enzyme Structure (Part K)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 131. Enzyme Structure (Part L)

Edited by C. H. W. HIRS AND SERGE N. TIMASHEFF VOLUME 132. Immunochemical Techniques (Part J: Phagocytosis and Cell-Mediated Cytotoxicity) Edited by GIOVANNI DI SABATO AND JOHANNES EVERSE


xxix

VOLUME 133. Bioluminescence and Chemiluminescence (Part B)

Edited by MARLENE DELUCA AND WILLIAM D. MCELROY VOLUME 134. Structural and Contractile Proteins (Part C: The Contractile Apparatus and the Cytoskeleton) Edited by RICHARD B. VALLEE VOLUME 135. Immobilized Enzymes and Cells (Part B)

Edited by KLAUS MOSBACH VOLUME 136. Immobilized Enzymes and Cells (Part C)

Edited by KLAUS MOSBACH VOLUME 137. Immobilized Enzymes and Cells (Part D) (in preparation)

Edited by KLAUS MOSBACH VOLUME 138. Complex Carbohydrates (Part E)

Edited by VICTOR GINSBURG VOLUME 139. Cellular Regulators (Part A: Calcium- and CalmodulinBinding Proteins) Edited by ANTHONY R. MEANS AND P. MICHAEL CONN VOLUME 140. Cumulative Subject Index Volumes I02-119, 121-134 VOLUME 141. Cellular Regulators (Part B: Calcium and Lipids)

Edited by P. MICHAEL CONN AND ANTHONY R. MEANS VOLUME 142. Metabolism of Aromatic Amino Acids and Amines

Edited by SEYMOUR KAUFMAN VOLUME 143. Sulfur and Sulfur Amino Acids

Edited by WILLIAM B. JAKOBY AND OWEN GRIFFITH VOLUME 144. Structural and Contractile Proteins (Part D: Extracellular Matrix) Edited by LEON W. CUNNINGHAM VOLUME 145. Structural and Contractile Proteins (Part E: Extracellular Matrix) Edited by LEON W. CUNNINGHAM

XXX


VOLUME 146. Peptide Growth Factors (Part A) (in preparation)

Edited by DAVID BARNES AND DAVID A. SIRBASKU VOLUME 147. Peptide Growth Factors (Part B) (in preparation)

Edited by DAVID BARNES AND DAVID A. SIRBASKU VOLUME 148. Plant Cell Membranes (in preparation)

Edited by LESTER PACKER AND ROLAND DOUCE VOLUME 149. Drug and Enzyme Targeting (Part B) (in preparation)

Edited by RALPH GREEN AND KENNETH J. WIDDER VOLUME 150. Immunocheraical Techniques (Part K: In Vitro Models of B and T Cell Functions and Lymphoid Cell Receptors) (in preparation) Edited by GIOVANNI DI SABATO VOLUME 151. Molecular Genetics of Mammalian Cells (in preparation)

Edited by MICHAEL M. GOTTESMAN VOLUME 152. Guide to Molecular Cloning Techniques

Edited by SHELBY L. BERGER AND ALAN R. KIMMEL VOLUME 153. Recombinant DNA (Part D) (in preparation)

Edited by RAY Wu AND LAWRENCE GROSSMAN VOLUME 154. Recombinant DNA (Part E) (in preparation)

Edited by RAY Wu AND LAWRENCE GROSSMAN VOLUME 155. Recombinant DNA (Part F) (in preparation)

Edited by RAY Wu

[1]

IMMOBILIZED

ACTIVE COENZYMES

3

[1] I m m o b i l i z e d A c t i v e C o e n z y m e s By MATS-OLLE MANSSON and K t n u s MOSBACH This section includes a number of contributions concerning immobilized multienzyme systems and coenzymes. Other contributions addressing these systems in a different context are also found in Vols. 135 and 136 (articles by Rozzell, Luisi and Steinmann-Hofmann, Scheller et al., Carrea and Cremonesi, Murachi and Tabata, Laane and Veeger, and Chibata et al.). Furthermore, in a previous volume in this series I similar questions and methodologies have been presented. In the following we wish to discuss briefly general aspects especially related to immobilized coenzymes and also to provide a number of additional references. Coenzyme-dependent enzymes are of great potential interest for the synthesis of interesting stereospecific compounds and for analytical applications. Paralleling attempts to use immobilized coenzymes or coenzyme analogs in affinity chromatography 2 the question of their coenzymatic activity in the immobilized state has been addressed. The first immobilization, e.g., of NAD(H), showing coenzymatic activity was reported in 1971.3 Later, synthesis of chemically better defined analogs, several of which can be seen in Table I, was carried out. The main interest has focused on the redox coenzyme NAD, but NADP, ATP, and CoA have also been immobilized as active coenzymes. Preparation of Immobilized Active Coenzymes Coenzymes usually have to be modified to allow proper immobilization and regeneration. This modification of a coenzyme can be accomplished in basically two ways. One is the preassembly approach and the other has been called the solid-phase modular approach. In the preassembly approach the coenzyme is first modified and then assembled with a spacer molecule to increase the steric availability for the coenzyme of the enzyme and subsequently coupled to a support. In the solid-phase modular approach the coenzyme or a modified coenzyme is coupled to a matrix previously substituted with spacer molecules carrying reactive functional groups. 1 K. Mosbach (ed.), this series, Vol. 44. 2 K. Mosbach, in "Advances in Enzymology" (A. Meister, ed.), Vol. 46, p. 205. Wiley, New York, 1978. 3 p. O. Larsson and K. Mosbach, Biotechnol. Bioeng. 13, 393 (1971).

METHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

4

[1]

M U L T I S T E P E N Z Y M E SYSTEMS A N D C O E N Z Y M E S

TABLE I SUBST1TUENTS USED FOR MODIFICATION OF N A D AND A T P IN THE

Compound NAD

Substituent

N-6 POSITION Reference

CH2COOH CH2~NH(CH2)6NH2

a

O CH2~HCH2COOH OH (CH2)2NH2 (CH2)6NH2 (CH2)2~NHCH(CH2)4NH~CH~-~-CH2 O

C~O

c,d e f

O

I

OCH 3 CH2CHCH2OCCH~CH2

I

II

II

II

OH O CH2CNHCH2CNHCH2CHCH2NHCC~CH2

I

III

O O OH CH2CNH(CH2)6NHCC~CH2

II

OCH3

III

O OCHs (CH2)2CNH(CH2)2CNH(CH2)2NHCC~---CH2

II

II

III

O O OCH3 CH2CNH(CH2)2CNH(CH2)2NHCC---~CH2

II

II

O O (CH2)2COOH (CH2)2~NH(CH2)2NH2 ATP

O CH2COOH CH2~NH(CH2)6NH2

III

OCH3

k k

O CNH(CH2)6NH2

II

O CHzCNH(CH2)6NHCC~CH2

II

O (CH2)6NH2

III

OCH3

M. Lindberg, P. O. Larsson, and K. Mosbach, Eur. J. Biochem. 40, 187 (1973). b p. Zappelli, A. Rossodivita, and L. Re, Eur. J. Biochem. 54, 475 (1975). c H. L. Schmidt and G. Grenner, Eur. J. Biochem. 67, 295 (1976). a A. F. Buckmann, M.-R. Kula, R. Wichmann, and C. Wandrey, J. Appl. Biochem. 3, 301 (1981). e D. B. Craven, M. J. Harvey, and P, D. G. Dean, FEBS Lett. 38, 320 (1974). a

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IMMOBILIZED ACTIVE COENZYMES

5

In the authors' opinion the preassembly approach has significant advantages. First, chemically well-defined preparations can be obtained and second, the preassembled coenzyme-spacer molecule can be characterized prior to coupling and studied in homogeneous solution, yielding useful information in advance concerning kinetic constants such as Kdi~s and Km. The different variations of the solid-phase modular approach all suffer from the drawback that heterogeneous preparations containing excess spacers on the matrix may be obtained which can give rise to unwanted nonspecific interactions with the enzyme. The syntheses of NAD(P) and ATP analogs have been described in a previous volume of this series.l The probably most widely applied synthetic route involves alkylation of the N-1 nitrogen of the adenine nucleus [a part of both NAD(P) and ATP] by reagents such as iodoacetic acid, 4 3,4-epoxybutanoic acid, 5 or ethyleneimine. 6,7 After reduction of the alkaline-labile NAD to the stable NADH, a Dimroth rearrangement is carried out to convert the N-1 derivative to the corresponding desired N-6 analog. This N-6 analog is then used as a starting material for the synthesis of analogs with different spacers carrying different reactive groups (Table I). For an immobilized coenzyme to be used as an active coenzyme in an enzyme reactor or as a component of a biosensor, special requirements have to be fulfilled. Water-soluble polymers should be used as they do not cause as much diffusional hindrance as a water-insoluble matrix. In addition the degree of substitution is of importance since it has been reported that a high degree of substitution will lead to inhibition of the enzyme. 8 Widely used water-soluble polymers are dextran and polyethylene glycol 4 M. Lindberg, P. O. Larsson, and K. Mosbach, Eur. J. Biochem. 40, 187 (1973). 5 p. Zappelli, A. Rossodivita, and L. Re, Eur. J. Biochem. 54, 475 (1975). 6 H. L. Schmidt and G. Grenner, Eur. J. Biochem. 67, 295 (1976). 7 A. F. Btickmann, M.-R. Kula, R. Wichmann, and C. Wandrey, J. Appl. Biochem. 3, 301 (1981). s S. Furukawa, I. Urabe, and H. Okada, Eur. J. Biochem. 114, 101 (1981).

I M . Muramatsu, I. Urabe, Y. Yamada, and H. Okada, Eur. J. Biochem. 80, 111 (1977). g F. LeGoffic, S. Sicsic, and C. Vincent, in "Enzyme Engineering" (W. H. Weetall and G. P. Royer, eds.), Vol. 5, p. 127. Plenum, New York, 1980. h y. Yamazaki and H. Maeda, Agric. Biol. Chem. 45, 2277 (1981). i y. Yamazaki, H. Maeda, A. Satoh, and K. Hiromi, Biochem. J. 95, 109 (1984). J S. Adachi, M. Ogata, H. Tobita, and K. Hasimoto, Enzyme Microb. TechnoL 6, 259 (1984). , M. Lindberg and K. Mosbach, Eur. J. Biochem. 53, 481 (1975). I H. Suzuki and Y. Yamazaki, this volume [5]. my. Yamazaki and H. Maeda, Agric. Biol. Chem. 45, 2091 (1981). " W. Berke, M. Morr, C. Wandrey, and M.-R. Kula, Ann. N.Y. Acad. Sci. 437, 257 (1984).

6

MULTISTEP ENZYME SYSTEMS AND COENZYMES

[1]

(PEG), but polymers such as polyethyleneimine (PEI) have also been used. Polymerizable coenzymes such as the acrylic derivatives listed in Table I are immobilized in a polymerization reaction together with other monomers, e.g., acrylamide. The coenzyme content of the final polymer will then depend on the ratio of coenzyme monomer to the other monomers added. The polymeric derivatives obtained in this way are heterogeneous mixtures of various polymer chains. The "coenzymatic activity" of such a coenzyme moiety placed on a polymer chain may depend on its position in the polymer and on the size of the polymer. 9 Properties of Immobilized Active Coenzymes A coenzyme analog synthesized according to the preassembly method has, as mentioned before, the advantage that it can be thoroughly studied, both chemically and enzymatically prior to immobilization. One aspect of great importance is whether the modification has influenced on the interactions between enzyme and coenzyme. Generally, the N-6 position of NAD is the point of choice for substitution since it has been shown that for many dehydrogenases the exocyclic amino group of the parent NAD molecule points out of the active site. 1° Thus, spacers attached at this position also will project out from the active site of the enzyme, and interference with coenzyme binding will be minimized or nonexistent. However in some instances the C-8 position has also proved useful, at least in affinity chromatography (for an overview, see Ref. 11). The easiest way to determine the amount of immobilized coenzyme on a water-soluble polymer is to record the UV spectrum for the coenzyme and, based on the absorption peak, determine its concentration. 1 It is important that the reference cell during recording of the UV spectrum carries the equivalent amount of nonsubstituted polymer. The degree to which an immobilized coenzyme can be enzymatically reduced or oxidized is also of great interest. The kinetic properties of immobilized NAD derivatives vary depending on both what enzyme and what coenzyme derivative are being used. In general the Km values are higher and the Vmaxvalues are lower for the immobilized NAD than for the free NAD. In a comparison of coenzymatic activity between free NAD, N-6-substituted NAD, dextran-NAD,

9 S. Adachi, M. Ogata, H. Tobita, and K. Hashimoto, Enzyme Microb. Technol. 6, 259 (1984). J0 C. I. Br~inden, H. JOrnvall, H. Eklund, and H. Furugren, in "The Enzymes" (P. D. Boyer, ed.), Vol. 11, 3rd Ed., p. 103. Academic Press, New York, 1975. " M. O. M~nsson and K. Mosbach, in "Coenzymes and Cofactors" (D. Dolphin, R. Poulson, and O. Avramovic, eds.). Wiley, New York, in press.

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IMMOBILIZED ACTIVE COENZYMES

7

and agarose-bound NAD, the relative initial rates toward alcohol dehydrogenase were found to be 100, 61, 16, and 0.7, respectively. ~2 Apart from the different number of interactions possible between coenzyme and support that are dependent on the size of the latter, there are two factors which influence these initial reaction rates when compared to the free coenzyme. First, the modification of the coenzyme can alter its binding to the active site. Also, the polymer to which the coenzyme is anchored may influence the binding of the coenzyme to the active site, e.g., because of its hydrophilic/hydrophobic character. In different reports, the effect of polymer molecular weight, 9 NAD density on the polymer, 8 and length of the spacer ~3 on the enzymatic reaction have been discussed. Applications of Immobilized Active Coenzymes Immobilized active coenzymes such as NAD have found use in analytical systems as well as in enzyme reactors. The reactor of choice is the membrane reactor in which a semipermeable membrane retains the high molecular weight immobilized coenzyme together with the enzymes. 14 The design of the reactor will of course be different if the enzyme-coenzyme system is of a totally "self-sustained" form where both the enzyme and the coenzyme are integrated parts of the same polymer. ~5Alternative systems that allow continuous processes to be carried out have also been studied. Following one approach, an NAD analog has been coupled directly onto an enzyme, allowing it to act as a prosthetic group~6,~7; alternatively the enzyme is coimmobilized with an NAD analog to a support in a predetermined configuration. 16,~8 An important issue both in the reactor systems and for analytical systems is the requirement for regeneration of the coenzyme because of its high cost and the fact that it is needed in stoichiometric amounts relative to the product formed. Regeneration can be accomplished chemically, electrochemically, or enzymatically. At present, enzymatic regeneration has the most advantages, especially because of its high specificity in regeneration. However, electrochemical procedures are gaining in importance (in this context see Ref. 18a). Table II summarizes several appli12 p. O. Larsson and K. Mosbach, FEBS Lett. 46, 119 (1974). J3 y . Yamazaki, H. Maeda, A. Satoh, and K. Hiromi, Biochem. J. 95, 109 (1984). ~4 M.-R. Kula and C. Wandrey, this volume [2]. ~5Y. Yamazaki and H. Maeda, this volume [3]. 16 M. O. Mhnsson, P. O. Larsson, and K. Mosbach, this series, Vol. 89, p. 457. x7 C. Woenckhaus, R. Koob, A. Burkhard, and H. G. Schaefer, Bioorg. Chem. 12, 45 (1983). ~s K. J. Laidler and M. A. Mazid, this volume [6]. ~8~I. Thanos, J. Bader, H. Giinther, S. Neumann, F. Krauss, and H. Simon, this volume [28].

8


[1]

TABLE II APPLICATIONS OF IMMOBILIZED N A D ANALOGSa

Product or compound analyzed Enzyme reactors with Alanine Alanine Leucine Benzaldehyde Lactate Lactate Lactate Lactate Lactate Alanine Malate Malate Propanediol Propanediol Lactate Phenylalanine Analytical systems Glutamate Pyruvate Alcohol Alcohol Lactate Alcohol Alcohol Lactate Glutamate Lactate Glutamate

Coenzyme

Enzymesb

coupled enzyme regeneration Dextran-NAD Dextran-NAD PEG-NAD Polyacrylate-NAD PEG-NAD Dextran-NAD PEI-NAD Polylysine-NAD Dextran-NAD PEI-NAD Polyacrylate-NAD Dextran-NAD Coimmobilized NAD Enzyme-bound NAD Enzyme-bound NAD PEG-NAD

GalDH + AlaDH AIaDH + LDH FDH + LeuDH ADH (*) ADH + LDH ADH + LDH ADH + LDH ADH + LDH ADH + LDH AIaDH + LDH FDH + MDH ADH + MDH ADH (*) ADH (*) ADH + LDH FDH + PheDH

Dextran-NAD Dextran-NAD Polyacrylate-NAD Coimmobilized NAD Electrode-bound NAD Electrode-bound NAD Dextran-NAD Dextran-NAD Dextran-NAD Dextran-NAD Dextran-NAD

GDH + LDH GDH + LDH ADH + diaphorase ADH LDH ADH ADH LDH GDH LDH GDH

Reference

c e : e h J k m

n.o n P q q r s s t t ' "

GalDH, Galactose dehydrogenase; AIaDH, alanine dehydrogenase; FDH, formate dehydrogenase; LeuDH, leucine dehydrogenase; ADH, alcohol dehydrogenase, LDH, lactate dehydrogenase; MDH, malate dehydrogenase; GDH, glutamate dehydrogenase; and PheDH, phenylalanine dehydrogenase. b (,) Indicates coupled-substrate enzymatic regeneration. c p. Davies and K. Mosbach, Biochim. Biophys. Acta 370, 329 (1974). a M.-R. Kula and C. Wandrey, this volume [2]. e C. W. Fuller, J. R. Rubin, and H. J. Bright, Eur. J. Biochem. 103, 421 (1980). z H. Okada and I. Urabe, this volume [4]. g P. O. Larsson and K. Mosbach, FEBS Lett. 46, 119 (1974). h j. R. Wykes, P. Dunill, and M. D. Lilly, Biotechnol. Bioeng. 17, 151 (1975). i y . Yamazaki, H. Maeda, and H. Suzuki, Biotechnol. Bioeng. 18, 1761 (1976). J Y. Morikawa, I. Karube, and S. Suzuki, Biochim. Biophys. Acta 523, 263 (1978). k W. Marconi, G. Prosperi, S. Giovenco, and F. Morisi, J. Mol. Catal. 1, 111 (19751976).

[2]

CONTINUOUS ENZYMATIC TRANSFORMATION

9

cations of immobilized active N A D in analysis and as a component in enzyme reactors. In addition a polyethylene glycol-bound ATP ~9 and N A D P 2° have recently been used in enzyme reactor studies. It should be kept in mind that native coenzymes for a number of applications remain an alternative (see, e.g., the contribution on ATP recycling). 2~ In this context a recent publication on "an affinity chromatographic reactor for highly efficient turnover of dissociable cofactors" applying free NAD in an ultrafiltration hollow fiber tube should be mentioned. 22 19 W. Berke, M. Morr, C. Wandrey, and M.-R. Kula, Ann. N. Y. Acad. Sci. 437, 257 (1984). 20 K. Okuda, I. Urabe, and H. Okada, Eur. J. Biochem. 151, 33 (1985). 2~ D. C. Crans, R. J. Kazlauskas, B. L. Hirschbein, C.-H. Wong, O. Abril, and G. M. Whitesides, this volume [25]. 22 O. Miyawaki, N. Osato, and T. Yano, Agric. Biol. Chem. 49, 2063 (1985).

I y . Yamazaki and H. Maeda, this volume [3]. m j. Grunwald and T. M. S. Chang, J. Mol. Catal. 11, 83 (1981). n M. O. MAnsson, P. O. Larsson, and K. Mosbach, this series, Vol. 89, p. 457, o S. Gestrelius, M. O. MAnsson, and K. Mosbach, Eur. J. Biochem. 57, 529 (1975). p E. Schmidt, E. Fiolitakis, and C. Wandrey, Enzyme Eng. 8 (in press). q H. Suzuki and Y. Yamazaki, this volume [5]. • K. J. Laidler and M. A. Mazid, this volume [6]. s T. Yao and S. Musha, Anal. Chim. Acta 110, 203 (1979). t A. Malinauskas and J. Kulys, Anal. Chim. Acta 98, 31 (1978). " Y. Sakaguchi, M. Sugahara, J. Endo, and T. Murachi, J. Appl. Biochem. 3, 32 (1981).

[2] C o n t i n u o u s E n z y m a t i c T r a n s f o r m a t i o n in an Enzyme-Membrane Reactor with Simultaneous NADH Regeneration B y M A R I A - R E G I N A K U L A a n d CHRISTIAN WANDREY

Enzymes as catalysts are especially noted for their high stereo- and regiospecificity. To accomplish the synthesis of chiral compounds often not only an enzyme is needed as catalyst but in addition a low molecular weight coenzyme which participates in the reaction. For a large number of redox processes NAD(H) or NADP(H) serves as coenzymes for dehydrogenases and is utilized in stoichiometric amounts in the course of the reaction. These coenzymes are readily dissociable from the enzyme and require a separate second reaction for regeneration. I S. S. Wang and C. K. King, Adv. Biochem. Eng. 12, 119 (1979). METHODSIN ENZYMOLOGY,VOL. 136

Copyright© 1987by AcademicPress, Inc. All rightsof reproductionin any form reserved.

[2]


9

cations of immobilized active N A D in analysis and as a component in enzyme reactors. In addition a polyethylene glycol-bound ATP ~9 and N A D P 2° have recently been used in enzyme reactor studies. It should be kept in mind that native coenzymes for a number of applications remain an alternative (see, e.g., the contribution on ATP recycling). 2~ In this context a recent publication on "an affinity chromatographic reactor for highly efficient turnover of dissociable cofactors" applying free NAD in an ultrafiltration hollow fiber tube should be mentioned. 22 19 W. Berke, M. Morr, C. Wandrey, and M.-R. Kula, Ann. N. Y. Acad. Sci. 437, 257 (1984). 20 K. Okuda, I. Urabe, and H. Okada, Eur. J. Biochem. 151, 33 (1985). 2~ D. C. Crans, R. J. Kazlauskas, B. L. Hirschbein, C.-H. Wong, O. Abril, and G. M. Whitesides, this volume [25]. 22 O. Miyawaki, N. Osato, and T. Yano, Agric. Biol. Chem. 49, 2063 (1985).

I y . Yamazaki and H. Maeda, this volume [3]. m j. Grunwald and T. M. S. Chang, J. Mol. Catal. 11, 83 (1981). n M. O. MAnsson, P. O. Larsson, and K. Mosbach, this series, Vol. 89, p. 457, o S. Gestrelius, M. O. MAnsson, and K. Mosbach, Eur. J. Biochem. 57, 529 (1975). p E. Schmidt, E. Fiolitakis, and C. Wandrey, Enzyme Eng. 8 (in press). q H. Suzuki and Y. Yamazaki, this volume [5]. • K. J. Laidler and M. A. Mazid, this volume [6]. s T. Yao and S. Musha, Anal. Chim. Acta 110, 203 (1979). t A. Malinauskas and J. Kulys, Anal. Chim. Acta 98, 31 (1978). " Y. Sakaguchi, M. Sugahara, J. Endo, and T. Murachi, J. Appl. Biochem. 3, 32 (1981).

[2] C o n t i n u o u s E n z y m a t i c T r a n s f o r m a t i o n in an Enzyme-Membrane Reactor with Simultaneous NADH Regeneration B y M A R I A - R E G I N A K U L A a n d CHRISTIAN WANDREY

Enzymes as catalysts are especially noted for their high stereo- and regiospecificity. To accomplish the synthesis of chiral compounds often not only an enzyme is needed as catalyst but in addition a low molecular weight coenzyme which participates in the reaction. For a large number of redox processes NAD(H) or NADP(H) serves as coenzymes for dehydrogenases and is utilized in stoichiometric amounts in the course of the reaction. These coenzymes are readily dissociable from the enzyme and require a separate second reaction for regeneration. I S. S. Wang and C. K. King, Adv. Biochem. Eng. 12, 119 (1979). METHODSIN ENZYMOLOGY,VOL. 136

Copyright© 1987by AcademicPress, Inc. All rightsof reproductionin any form reserved.

l0


[2]

Since NADH and NADPH are complex and rather labile organic chemicals they are quite expensive. For the application of dehydrogenases in the enzyme-catalyzed synthesis of chiral compounds an efficient coenzyme regeneration is therefore needed in order to make such a process economically viable. A simple comparison of cost of I mol of NADH (665 g/mol, value -$1000) with 1 mol of chiral a-hydroxy or a-amino acid (-100 g/mol, value -$2) shows that separation, external regeneration, and recycling of the coenzyme would require an overall yield of 99.98% per pass to keep the coenzyme cost at 10% of the product value. Simultaneous regeneration offers a chance to overcome the stoichiometric relation between product and coenzyme by an efficient internal recycling. In principle several experimental approaches are possible. ~-6 Here we concentrate on an enzymatic regeneration of NADH by a second enzyme-catalyzed reaction. Our approach follows the strategy developed in cellular metabolism in which NADH is produced and consumed by a series of balanced reactions. 6-~° Considering ultimately a technical application it should be noted that besides the anticipated conversion, a second substrate is now needed in a stoichiometric amount and two products are formed in equimolar concentration. Therefore the cosubstrate should be cheap enough and neither cosubstrate or coproduct should be obnoxious to the enzymes involved. In the authors' laboratories formate dehydrogenase is preferred as a general NADH-regenerating enzyme for several reasons: the enzyme is readily available in large amounts from Candida boidinii grown on methanolH,~2; the equilibrium of the reaction catalyzed lies far on the side of CO2 and therefore NADH formationS1; and the 2 H. Simon, J. Bader, H. Giinther, S. Neumann, and J. Thanos, Ann. N. Y. Acad. Sci. 434, 171 (1984). 3 Z. Shaked, J. J. Barber, and G. M. Whitesides, J. Org. Chem. 46, 4100 (1981). 4 M. O. M~nsson, P. O. Larsson, and K. Mosbach, this series, Vol. 89, p. 457. M. Aizawa, R. W. Coughlin, and M. Charles, Biotechnol. Bioeng. 18, 209 (1976). 6 R. Wichmann, C. Wandrey, A. F. Biickmann, and M.-R. Kula, Biotechnol. Bioeng. 23, 2789 (1981). 7 C. Wandrey, R. Wichmann, W. Leuchtenberger, A. B/ickmann, and M.-R. Kula, U.S. Patent 4,326,031, European Patent 80,104,346.4; U.S. Patent 4,304,858, European Patent 80,104,345.6. s W. Leuchtenberger, C. Wandrey, and M.-R. Kula, DOS 3,307,094.6, Spanish Patent 530,155. 9 A. F. Bfickmann, U.S. Patent 4,443,594, European Patent 79,102,954.9. ~ C- Wandrey and R. Wichmann, in "Application of Isolated Enzymes and Immobilized Cells to Biotechnology" (A. Laskin, ed.), p. 177. 1985. H H. Schtitte, J. Flossdorf, H. Sahm, and M.-R. Kula, Eur. J. Bioehem. 62, 151 (1976). ~2 K. H. Kroner, H. Schiitte, W. Stach, and M,-R. Kula, J. Chem. Technol. Bioteehnol., Biotechnol. 32B, 130 (1982).

[2]


HCO0-

reduced subsfrate

u.~

PEG2oooo-NAD"

/z~

•

PE52oooo-NAOH k0H

~A

C0z

1l

oxidized subsfrafe

FIG. 1. Reaction scheme. FDH, Formate dehydrogenase; E, enzyme; PEG 20000-NAD + NADH, coenzyme derivatives.

coproduct CO2 is easily separable. The overall reaction scheme is outlined in Fig. 1. It is apparent that for fast NADH recycling, ready access to both enzymes becomes crucial and reactor systems with inherent mass transfer resistances should be avoided. Therefore an enzyme-membrane reactor was chosen. Here the reaction will proceed in homogenous solution. 6-1° The enzymes are retained in the reactor by virtue of their high molecular weight, utilizing an ultrafiltration membrane as a selective barrier. However, the difference in molecular weight between product and coenzyme is not sufficient to achieve satisfactory results by a membrane separation. Therefore we attempted to increase the molecular weight of the coenzyme by attaching it covalently to a water-soluble polymer such as polyethylene glycol (PEG). 9 This way the retention of the coenzyme by an ultrafiltration membrane could be accomplished together with separation of the coenzyme from the product stream. Provided the coenzyme activity is not lost upon modification, the basic design of the reactor should allow continuous enzymatic conversion and regeneration of NADH to proceed. Coenzyme D e r i v a t i v e s The synthesis of the PEG-NADH is outlined in Fig. 2 and has been described in detail by Bfickmann et al. 9,13 The modified coenzyme is readily soluble in water, and its concentration can be determined from absorbance at 340 or 259 nm, respectively. Assuming that the molar absorption coefficients are identical with the native coenzyme the following values have been used for calculation: 6220 M -~ cm -I for NADH at 340 nm, and 18,000 M -~ cm -~ for NAD at 259 nm. A number of dehydrogenases has been tested with regard to their ability to accept the modified coenzyme. u A. F. Biickmann, M.-R. Kula, R. Wichmann, and C, Wandrey, J. Appl. Biochem. 3, 301 (1981).

12


[2]

NH2 ~NH~ "~N/~"~ N

LRib-- p--p

\N /

"k

2H*

--.,bJ

PEG--COO-

L.ib-- p-- p--Rin /

CQrbodiimide NH2

reduction

h R i b - - p - - P--Rib ~

Dimroth

H

PEG--

C - - N --CH~--CH~~'- NH

H

H

in reorrQngement

O

o

N

N

NH2

LRib-- p--p--Rib

-]

FIG. 2. General synthesis route to modified coenzymes. Rib, Ribose; P, phosphate; PEG, polyethylene glycol.

TABLE I KINETIC PARAMETERS OF DEYHYDROGENASES FOR NATIVE NADH AND P E G - N A D H a

Conditions

Native NADH

PEG-NADH

Temperature

Vmax

Km

Vmax

Krn

Enzyme b

Substrate

pH

(°C)

(U/rag)

(mM)

(U/mg)

(raM)

AIaDH LeuDH PheDH L-LDH D-LDH L-HicDH b-HicHD FDH

Pyruvate Ketoleucine Phenylpyruvate Pyruvate Pyruvate Ketoleucine Ketomethionine Formate

9 8 8.5 9 9 8.5 8 8

25 25 25 25 25 25 25 40

20.9 13.2 37.2 308.0 37.0 511.0 259.0 1.2

0.022 0.033 0.047 0.001 0.289 0.044 0.170 0.300

10.3 11.5 17.7 150.0 4.3 214.0 240.0 2.6

1.290 0.029 0.099 0.080 0.060 0.144 0.240 0.330

a Activity per milligram protein in the enzyme preparation. b AlaDH, Alanine dehydrogenase; LeuDH, leucine dehydrogenase; PheDH, phenylalanine dehydrogenase; L-LDH, L-lactate dehydrogenase; D-LDH, D-lactate dehydrogenase; L-HicDH, L-hydroxyisocaproate dehydrogenase; D-HicDH, D-hydroxyisocaproate dehydrogenase; FDH, formate dehydrogenase.

[2]

13


The results are listed in Table I. Often the Vmaxvalue obtained with the derivative is in the range 50-100% in comparison with the native coenzyme, while the Km value may change by an order of magnitude. It should be noted that formate dehydrogenase exhibits an even higher initial reaction velocity with the modified coenzyme than with the native. 6 Besides the examples listed in Table I the following enzymes are known to accept P E G - N A D ( H ) as coenzyme: alcohol dehydrogenase, isopropanol dehydrogenase, malate dehydrogenase, glutamate dehydrogenase. So far, glucose dehydrogenase from Bacillus megaterium is the only dehydrogenase detected that does not accept P E G - N A D H as a substrate. Retention

The retention of the coenzyme in the reactor becomes very important for continuous operation. In Fig. 3 the loss of coenzyme by incomplete retention in the reactor is plotted as a function of the operating time. The data show that retentions better than 99.9% are necessary to avoid excessive loss of coenzyme at a typical mean residence time of 1 hr. The high

\

" " " ........ \

8.8

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

~

O

\ e. 4

\

.........

PEG_20000_NAD(H) R = 9 9 93 kELU = 1 7 X/d

.........

PEG-1000g-NADCH)~""~ R

\\

= gg82

kELU =

\,

\,

4 3

X

X/d

N ""

0.2 R

~'". 2 4 . 0 ~/cl "'"--.....,...

= 99.00

kELU I 8

2

I 4

I I~

I 8

I 18

I 12

I 14

1 10

I 18

I

I

2a

22

24

TIME l D Flo. 3. Elution of P E G - N A D ( H ) across a YM5 (Amicon) membrane in a continuously operated membrane reactor; residence time Z = l hr. R, Retention (R = 99.00% for comparison); ke~u, elution loss (due to incomplete retention); PEG 10000 and PEG 20000, molecular weight 10,000 and 20,000.

14


[2]

retention is a very stringent requirement when choosing the right membrane and depends on the average molecular weight of the coenzyme derivative and also on the pore size distribution determining the actual cutoff behavior of a given membrane. The performance of a membrane selected should be checked by experiments lasting for several days in order to allow assessment of the data with sufficient confidence. Retention R is defined by Eq. (1): R = (Cr-

Cf)/Cr

(1)

where Cr and Cf are the concentration of the compound of interest in the retentate and filtrate, respectively. The washout can be described as a function of time by Eq. (2): Cr/Cro = e -[~1 - R)/~-]t

(2)

Cro is the retentate concentration at time zero, t is the elapsed time, and r the mean residence time. The apparent retention R can be calculated according to Eq. (2) from a plot of l n ( C r / C r o ) versus operating time. The elution loss Kelu is then defined by Eq. (3): Kelu = (1 - R ) / r

(3)

Even for rather high retention, washout becomes critical as residence times go down, since it depends on residence times as well as retention. In Fig. 4 the retention of PEG, carboxylated PEG, and the final preparation of the P E G - N A D H are represented. Experiments were performed within the membrane reactor as described below. Concentration of PEG was measured by refractive index; P E G - N A D H was determined by UV absorbance at 260 nm employing a cuvette with a 1-mm light path for the retentate and a 10-mm light path for the filtrate to compensate partly for the large difference in concentration of the different streams. It should be noted, however, that the apparent retention depends on the hydrodynamics of the system employed. Considering its importance for coenzymedependent processes the apparent retention should be reexamined if other reactor configurations are evaluated or hydrodynamic conditions are drastically changed. The absolute loss of coenzyme is also a function of the coenzyme concentration, which should therefore be as small as possible in the reactor. The lower limits of coenzyme concentration are defined by the necessity to saturate the enzymes involved in order to operate with their maximal catalytic efficiency. Table I shows that P E G - N A D H derivatives exhibit comparatively low Kmvalues, so that in many cases stationary coenzyme concentrations below 0.5 mM are sufficient for operation. Since the enzymes employed in the membrane reactor are much larger

[2]

15

CONTINUOUS ENZYMATIC TRANSFORMATION t

8.6

8.4

A D ¢

8.2 o

8

PE8-28888 PEG-28088-COOH PEG-28888-NADH

-

0

I

I

I

I

I

I

I

2

3

4

5

6

TIME

7

/ D

FIG. 4. Elution of P E G and P E G derivatives across a YM5 (Amicon) m e m b r a n e ; residence time ~" = 1 hr.

than the coenzyme derivative, elution losses of the catalyst itself are negligible. Experimental Setup of an Enzyme-Membrane Reactor The ultimate goal of an experiment determines the degree of sophistication the experimental setup of an enzyme-membrane reactor has to meet. A simple homemade version utilizing commercially available equipment is illustrated in Fig. 5. It has been successfully used for the determination of the stereospecificity of enzymes in crude extracts during a screening procedure.~4 A similar version could be employed if the preparation of small amounts (1-100 mmol) of a certain chiral compound is of prime interest. However, if the enzyme-membrane reactor is operated to evaluate coenzyme utilization and to use the data for feasibility studies or the design of a large-scale process several, safety and control features as well as precise measuring devices have to be installed. 14 W. H u m m e l , H. Schiitte, and M.-R. Kula, Ann. N . Y. Acad. Sci. 434, 194 (1984).

16


[2]

p-

i

i . . . . . . . . . . . . . . .

FIG. 5. Simple version of a laboratory enzyme-membrane reactor. The glass vessel (1) contains the substrate solution in 0.3 M ammonium formate or sodium formate, pH 7.0-7.5. In the thermostated (25 ° water bath) reactor (2), 0.5 ~mol/ml PEG-NADH, 3 U/ml formate dehydrogenase, and 3-5 U/ml of the second dehydrogenase are placed in 10 ml substrate solution containing 0.3 M formate. The reaction mixture is circulated by means of a peristaltic pump (4) passing through an ultrafiltration device (3). A CEC unit from Amicon or a similar device fitted with a YM5 membrane (Amicon) can be conveniently employed. With a CEC unit the circulating rate is set to approximately 30 ml/hr. This will create a small pressure differential and lead to a filtrate flux of approximately 3 ml/hr and a residence time of 3 hr. Filtrate flux is controlled and readjusted manually about twice a day if necessary. Filtrate is collected in bulk or with a fraction collector. The reactor (2) is closed airtight and the liquid level kept constant by the pneumatic overhead arrangement (1). The reactor is stirred using a magnetic stirrer to ensure proper mixing.

Figure 6 gives a flow diagram of a continuous process including all measuring points. Figure 7 shows a laboratory reactor which has been assembled in the mechanics workshop of the authors. The guiding principles in the design were to achieve a high ratio of membrane surface to internal retentate volume, to incorporate temperature control, and to allow easy access to the membrane for setup and changes between experiments. Polarization control is effected by operating the membrane reactor

~

enzyme ~~c ° e no z y mI e ~ ~

~J I product

go substrate I 2 S

METERINfi PUMP STERILE FILTER ENZYME MEMBRANE REACTOR

4 5 6

PHOTOMETER POLARIMETER RECORDER

F

FIG. 6. Flow diagram of a laboratory-scale membrane reactor with magnetic stirrer and thermostating jacket.

[2]

17


lid

se mc se sti

bc )strate sa

FIG. 7. Details of a laboratory membrane reactor (the magnetic bar is inserted in a plastic disk to minimize the retentate volume).

on a magnetic stirrer and inserting a magnetic bar underneath the membrane. In general the speed of the stirrer is set to 200 rpm. For long-time operations special care has to be taken to achieve and maintain sterile conditions in the reactor. After complete assembly a solution containing a suitable disinfectant such as 0.1% peroxyacetic acid is pumped through the reactor, and at the same time all tubing and filters are flushed. The disinfectant is replaced after several hours, at least 5, by sterile water followed by substrate solution. At this point the integrity of all seals is also tested. Finally the selected enzymes and PEG-NADH are added aseptically or pumped into the reactor and concentrated on the retentate side. Reaction starts at zero conversion and the time course until steady state is obtained can be followed and utilized to test the kinetic model based on measurement of initial rates, which has previously been developed to describe the reactor performance. 6,1° Because chiral compounds are produced, polarimetry is the method of choice to follow conversion in the reactor effluent. A port to the retentate side, closed by a septum, serves to draw periodically samples from the reactor using sterile syringes and to measure coenzyme content and enzyme activities. If desired, on-line control of enzyme activity and coenzyme levels is possible, but this requires for each single case a detailed

18


[2]

100

80 g,g 80

z cl H OC bJ Z 0 (J

40

SPACE-TIME-YIEL = D214 G/(LND) 100 MMOL/L KETDISOCAPROATE 400 MMOL/L NH4-FORMATE pH = 8 , 0 , T = 2S * • = 7S MIN

20

0

0

|

I

I

I

}

1

I

I

10

20

3B

48

SO

50

70

80

TIME /

90

D

FIG. 8. L-Leucine production with F D H , L e u O H , and PEG 2 0 0 0 0 - N A D H .

298 U/KG PRODUCT

i~ rl D -I i~r~

O

(3 b.

FDH I

I

I

0

20

30

I

40 TIME

/

ADDITION

1

I

I

i

50

80

70

80

D

FIG. 9. L-Leucine production and deactivation of F D H .

90

[2]

19


261U/KGPRODUCT1 ._1 \

I w 1

t LEUDH

o 0

-

I

ADDITION

I

I

1

I

1

I

I

I

10

20

30

40

SO

60

70

80

90

TIME / D FIG. 10. L-Leucine production and deactivation of LeuDH.

kinetic model in order to differentiate the activity of the enzymes involved. 15 The two enzymes and the coenzymes utilized in the coupled reaction will have different rates of decomposition. The component limiting the performance of the reactor can be identified and added with the substrate stream in order to restore conversion and performance. After sufficiently long operating times the consumption of enzyme activity and coenzyme loss per unit weight of product can be determined with confidence and put into an economic model of the reaction in order to optimize the process studied. For calculation and design it is important to maintain a constant flow. A pulse-free piston pump (Reichelt Chemietechnik, Heidelberg) was found satisfactory for prolonged operation with the low flow rates required for the laboratory reactor. Performance of Reactor Figures 8-11 demonstrate 3 months' performance of a laboratory enzyme-membrane reactor for the production of L-leucine. A cycle number of 80,000 was obtained for the coenzyme. The cycle number is defined 15 R. Wichmann and C. Wandrey,

Enzyme Eng. 6,

311 (1982).

20


[2]

I

100000

O.B

00000

86

60808

0.4

48000 ~ \

0.2

20000

d

0

0 0

10

20

38

40 TZME /

50

60

70

80

90

D

FIG. 11. L-Leucine production, deactivation of PEG 20000-NADH, and development of cycle number.

here as moles of product obtained per moles of coenzyme lost in the process (for whatever reasons). Figure 11 shows that the cycle number increases from the start of the experiment and levels off after - 2 0 days. Figures 9 and 10 demonstrate that the reactor was operated at rather low stationary enzyme concentrations. Increasing the enzyme content in the reactor would give higher space-time yields. Productivities up to 1 kg/ liter-day have been realized, which is much higher than reported values for fixed bed reactors. The optimal ratio of the enzyme activities depends on the kinetic constants, and concentrations are not necessary equal. It is one of the advantages of the enzyme-membrane reactor that the concentration of the enzymes involved in the process can be chosen and maintained at will. The system is amenable for modeling based on kinetic data and can therefore be optimized on rational grounds. Enzymes isolated from different organisms can be mixed and applied together in homogeneous phase. For this reason the enzyme-membrane reactor appears to have advantages compared to the utilization of whole cells, as long as the number of single reactions taking place remains fairly small and the enzymes employed are sufficiently stable in solution during operation. Results shown in Figs. 9 and 10 indicate that these conditions can be fulfilled. The average enzyme

[3]

COIMMOBILIZATION OF

NAD

AND DEHYDROGENASES

21

activity consumption with respect to both enzymes was less than 300 U/ kg product over a period of 3 months of continuous operation. Acknowledgment The coenzyme preparation was from A. F. Biickmann, and the enzymes were isolated by H. Schiitte and K. H. Kroner. The chemical engineering measurements at continuous operation were carried out by Mrs. U. Mackfeld. The engagement of all these co-workers is gratefully acknowledged.

[3] C o i m m o b i l i z e d S y s t e m o f N A D w i t h D e h y d r o g e n a s e s B y YOSHIMITSU YAMAZAKI a n d HIDEKATSU M A E D A

Recycling of NAD is a prerequisite for the industrial application of dehydrogenases. The most effective way of realizing this is to maintain both NAD and the coupled dehydrogenases in one reactor system (bioreactor) and recycle NAD in situ. Several methods have been developed for this purpose: NAD bound to a water-soluble polymer and the dehydrogenases are either placed all together in an ultrafiltration apparatusf1-3 enclosed with a semipermeable membrane, 4,5 microencapsulated, 6 or immobilized in a collagen membrane. 7 Solid-phase coimmobilization of NAD and a dehydrogenase ~,9 and the covalent binding of an NAD derivative in or near the active site of dehydrogenases ~°,~ have also been reported.

l y . Yamazaki and H. Maeda, Agric. Biol. Chem. 50, 3213 (1986). S. Furukawa, N. Katayama, T. Iizuka, I. Urabe, and H. Okada, FEBS Lett. 121, 239 (1980). 3 R. Wichmann, C. Wandrey, A. F. Btickmann, and M.-R. Kula, Biotechnol. Bioeng. 23, 2789 (1981). 4 p. Davies and K. Mosbach, Biochim. Biophys. Acta 370, 329 (1974). 5 A. Malinauskas and J. Kulys, Anal. Chim. Acta 98, 31 (1978). 6 j. Grunwald and T. M. S. Chang, J. Appl, Biochem. 1, 104 (1979). 7 Y. Morikawa, I. Karube, and S. Suzuki, Biochim. Biophys. Acta 523, 263 (1978). 8 S. Gestrelius, M.-O. M~nsson, and K. Mosbach, Fur. J. Biochem. 57, 529 (1975). 9 M. A. Mazid and K. J. Laidler, Biotechnol. Bioeng. 24, 2087 (1982). 10 M.-O. Mhnsson, P.-O. Larsson, and K. Mosbach, Eur. J. Biochem. 86, 455 (1978). I~ M.-O. M~nsson, N. Siegbahn, and K. Mosbach, Proc. Natl. Acad. Sci. U.S.A. 80, 1487 (1983).



[3]

COIMMOBILIZATION OF

NAD

AND DEHYDROGENASES

21

activity consumption with respect to both enzymes was less than 300 U/ kg product over a period of 3 months of continuous operation. Acknowledgment The coenzyme preparation was from A. F. Biickmann, and the enzymes were isolated by H. Schiitte and K. H. Kroner. The chemical engineering measurements at continuous operation were carried out by Mrs. U. Mackfeld. The engagement of all these co-workers is gratefully acknowledged.

[3] C o i m m o b i l i z e d S y s t e m o f N A D w i t h D e h y d r o g e n a s e s B y YOSHIMITSU YAMAZAKI a n d HIDEKATSU M A E D A

Recycling of NAD is a prerequisite for the industrial application of dehydrogenases. The most effective way of realizing this is to maintain both NAD and the coupled dehydrogenases in one reactor system (bioreactor) and recycle NAD in situ. Several methods have been developed for this purpose: NAD bound to a water-soluble polymer and the dehydrogenases are either placed all together in an ultrafiltration apparatusf1-3 enclosed with a semipermeable membrane, 4,5 microencapsulated, 6 or immobilized in a collagen membrane. 7 Solid-phase coimmobilization of NAD and a dehydrogenase ~,9 and the covalent binding of an NAD derivative in or near the active site of dehydrogenases ~°,~ have also been reported.

l y . Yamazaki and H. Maeda, Agric. Biol. Chem. 50, 3213 (1986). S. Furukawa, N. Katayama, T. Iizuka, I. Urabe, and H. Okada, FEBS Lett. 121, 239 (1980). 3 R. Wichmann, C. Wandrey, A. F. Btickmann, and M.-R. Kula, Biotechnol. Bioeng. 23, 2789 (1981). 4 p. Davies and K. Mosbach, Biochim. Biophys. Acta 370, 329 (1974). 5 A. Malinauskas and J. Kulys, Anal. Chim. Acta 98, 31 (1978). 6 j. Grunwald and T. M. S. Chang, J. Appl, Biochem. 1, 104 (1979). 7 Y. Morikawa, I. Karube, and S. Suzuki, Biochim. Biophys. Acta 523, 263 (1978). 8 S. Gestrelius, M.-O. M~nsson, and K. Mosbach, Fur. J. Biochem. 57, 529 (1975). 9 M. A. Mazid and K. J. Laidler, Biotechnol. Bioeng. 24, 2087 (1982). 10 M.-O. Mhnsson, P.-O. Larsson, and K. Mosbach, Eur. J. Biochem. 86, 455 (1978). I~ M.-O. M~nsson, N. Siegbahn, and K. Mosbach, Proc. Natl. Acad. Sci. U.S.A. 80, 1487 (1983).



22


[3]

We report still another method in which the dehydrogenases and a polymerizable NAD derivative are coentrapped in a polyacrylamide gel. The gel is prepared by radical copolymerization, and consequently NAD is bound on the matrix which encloses the enzymes. The purpose of this article is to outline the preparation of the coimmobilized gels of NAD and dehydrogenases and to discuss their application to bioreactors for synthesis and analysis. Synthetic Procedures Preparation o f Polymerizable N A D Derivatives

The principle used is the carbodiimide-mediated coupling of N6-carboxymethyl-NAD (1) with amines containing a methacryloyl group. The amines 2 and 3 (see Fig. 1) were synthesized as described below. Muramatsu et al., ~2 Le Goffic et al., ~3 and Yamazaki et al. 14 also reported methods for synthesis of polymerizable NAD derivatives. 6-(Tritylamino)hexylamine dihydrochloride (4). ~5 To a solution containing 30.6 g of 1,6-diaminohexane and 39.2 g of triethylamine in 200 ml of isopropanol is slowly added a solution of trityl chloride (88 g in 100 ml of isopropanol plus 150 ml of chloroform). The reaction mixture is stirred at room temperature for 3 hr and then the solvents and the remaining triethylamine are removed under reduced pressure. The residual solid is mixed with benzene (1.2 liters) and water (1.2 liters) and vigorously shaken in a separatory funnel. After separation of the two phases, the aqueous layer is discarded and the organic layer is washed successively with water (1 liter) and water containing 2.5% KH2PO4 (0.7 liters x 2). Centrifugation (1800 rpm for 10 min) is necessary to promote the phase separation. As part of the product is contained in the aqueous layers, it is recovered by extraction with benzene (0.5 liters each time). All organic layers are combined and evaporated under reduced pressure. The oily ~2M. Muramatsu, 1. Urabe, Y. Yamada, and H. Okada, Eur. J. Biochem. 80, 111 (1977). 13 F. Le Goffic, S. Sicsic, and C. Vincent, Eur. J. Biochem. 108, 143 (1980). 14 y . Yamazaki, H. Maeda, A. Satoh, and K. Hiromi, J. Biochem. (Tokyo) 95, 109 (1984). In this work, stopped-flow reaction kinetics was investigated on the binding of several species of polymer-bound NAD derivatives to yeast alcohol dehydrogenase in the presence of an inhibitor pyrazole. The result showed that the binding of the polymer derivatives of NAD to the enzyme was not essentially weaker and slower than that of native NAD, but was even faster in some cases in spite of an expected large steric hindrance between the polymer derivatives and the enzyme. ~5This section is reprinted with permission from Y. Yamazaki and H. Maeda, Agric. Biol. Chem. 45, 2091 (1981).

COIMMOBILIZATION OF NAD AND DEHYDROGENASES

[3] H2N 7 ~ f ~ . N H

2 ~ 3 c'CI

.~,~'cooet*

)

~ ) 3 c _ ~ N H

~-4

> ¢, ~C-N~COOEt

CI-

3 DCC

H 3 N ~

~'-7;"°"

>

~'-

5

N

2

__ ,~c-N.'ff"'J'-/""=

23

o

~coo,.occ

6 N

N

~/A¢OH

T,.o,

+ ,c ,.~;o

%~ _"~ TosO-

)

N,~L,,./N

7

3

....--.(R 2or3 NAD-> "> N6-carboxymethyI-NAO ~ I

tk~ ~

o

'%-'%-'

~

: r-o+o-~-o-7 .,o~ I I~Ho~ o- o.

8

oH OH u

9

R

=

o

H

I0 R

OH H /

o H/

: N~7-~.~N~

0

Fro. I. Synthetic scheme of polymerizable NAD derivatives. (b3C-, Trityl group; DCC, dicyclohexylcarbodiimide; TosOH, p-toluenesulfonic acid; AcOH, acetic acid; EDC, ethyldimethylaminopropylcarbodiimide hydrochloride.

residue is dissolved in 1 liter of chloroform. This solution is divided into 4 portions and each is extracted 3 times with 0.5 liters of ice-cold 0.1 M HCI. Phase separation is promoted by centrifugation. The aqueous layers are combined, adjusted to a pH of about 13 with 2 M NaOH, and saturated with NaCI. The aqueous solutions (l.5 liters × 4) are respectively extracted with chloroform (0.5 and 0.3 liters). The chloroform layers are combined, dried over Na2SO4, and evaporated under reduced pressure. The residue is dissolved in 400 ml of ether. Into the solution is bubbled dry HCI gas at - 2 0 ° to give a white precipitate. The gas bubbling is stopped when no more precipitate appears. The precipitate is collected by suction filtration, dried in vacuum, and crystallized from ethanol-ether to give 28 g (25% yield) of 4 as prisms, dec. 207 °. 6-Methacrylamidohexylammonium chloride (2).15 Preparation 4, 27.6 g, is dissolved in a mixture of 40 ml of dimethylformamide, 150 ml of chloroform, and 13 ml of triethylamine. To the ice-cold solution is slowly added a chloroform solution (30 ml) containing 22.4 g of methacrylic acid and a small amount of hydroquinone, and then another chloroform solution (40 ml) containing 28.2 g of dicyclohexylcarbodiimide is added under

24


[3]

stirring. The reaction mixture is stirred at 4 ° for 15 min and then at room temperature for 1 hr. The formed dicyclohexylurea is filtered off and the filtrate is concentrated to about 50 ml in an evaporator at 40°. To the concentrate is added 800 ml of benzene. The insoluble materials are filtered off and the filtrate is washed successively with alkaline water (pH 13 with NaOH, 300 ml × 2), water (200 ml), and 2.5% KH2PO4 (200 ml x 2). After drying over Na2SO4, the benzene layer is evaporated under reduced pressure. The oily residue, N-[6-(tritylamino)hexyl]methacrylamide, shows a substantially single spot in TLC [Rf = 0.66 on silica gel F254 developed with benzene/methanol (9 : 1), detected under UV light and by the ninhydrin spray method], but crystallization does not occur. So, the total amount of the residue is dissolved in a mixture of acetic acid (70 ml) and water (30 ml) to remove the trityl group. The solution is maintained at 100° for 10 min and then cooled. To the ice-cold solution is added I00 ml of water. The precipitate of triphenylcarbinol is filtered off. A small amount of hydroquinone is added to the filtrate, which is concentrated to about 10 ml in an evaporator at 30°. The addition of water (100 ml), filtration, and concentration are performed again. Then, 100 ml of toluene is added to the concentrate to remove the remaining acetic acid by azeotropic distillation. After concentration under reduced pressure, 70 ml of 1 M HC1 is added to the oily residue. This mixture is concentrated in an evaporator and then the remaining water and acetic acid are removed under reduced pressure as azeotropic mixtures with ethanol and toluene, respectively. The final residue is crystallized from ethanol-ether to give 10 g (69% yield) of 2 as needles, mp 113-114 °. Ethyl 2-(tritylamino)ethanoate (5). Commercially available glycine ethyl ester hydrochloride, 15 g, is dissolved in 160 ml of chloroform. To this solution was added 24 g of triethylamine and 32 g of trityl chloride. The mixture is stirred at room temperature for 6 hr and then 160 ml of benzene is added. The insoluble salt is filtered off and the filtrate concentrated to give a syrupy residue, which is crystallized from methanol. Thus 36 g (98% yield) of 5 is obtained as prisms, mp 116°. N-(3-Amino-2-hydroxypropyl)-2-(tritylamino)acetamide (6). 15 A solution containing 12.5 g of 5, 70 g of 1,3-diamino-2-propanol (Tokyo Chemical Industries Ltd., Tokyo), and 20 g of ethylene glycol in 40 ml of isopropanol is refluxed for 4 hr. The solvents and the unreacted diamine are removed in vacuum, and the resultant glassy residue is dissolved in 200 ml of chloroform. After washing with water (200 ml), the solution is dried over NaESO4 and concentrated under reduced pressure. The residue is finally precipitated from ethanol-ether-hexane to give 6 as a crystalline solid (13.5 g, 96% yield, dec. 138-141°).

[3]

COIMMOBILIZAT1ON OF NAD AND DEHYDROGENASES

25

N - [ 2 - Hydroxy - 3 - [ 2 - ( tritylamino ) acetamido ] propyl ] methacrylamide (7). 15 Preparation 6, 14.3 g, is treated with methacrylic acid and dicyclohexylcarbodiimide in a similar manner to that of 4, but dichloromethane is used instead of chloroform and triethylamine is not used. The reaction mixture is similarly worked up. Then, the washed, benzene layer is divided into 4 portions. Each portion (200 ml) is extracted three times with 0.05 M HCI (ice-cold solutions of 800,500, and 500 ml). The aqueous layers are combined, adjusted to a pH of about 13 with 2 M NaOH, saturated with NaCl, and extracted three times with chloroform [600,400, and 200 ml for each combined solution (1.8 liters)]. All the chloroform layers (1.2 liters x 4) are combined, dried over Na2SO4, and evaporated under reduced pressure. The viscous residue is crystallized from chloroform-benzene to give 7 as fine needles (11.14 g, 66% yield, dec. 155157°). [N-(2-Hydroxy-3-methacrylamidopropyl)carbamoyl ]methylammonium p-toluene sulfonate (3). 15 The trityl group of 7 (11.1 g) is removed with acetic acid in the same way as described for 2. In the purification stage, p-toluenesulfonic acid (4.62 g, monohydrate, dissolved in 10 ml of ethanol) is used instead of HCI since the corresponding hydrochloride is very hygroscopic. The product is precipitated from ethanol-ether as a crystalline solid (7.7 g, 82% yield, dec. 110-115°). N6-[ N-[ N - ( 2 - H y d r o x y - 3 - m e t h a c r y l a m i d o p r o p y l ) c a r b a m o y l m e t h y l ] c a r b a m o y l m e t h y l ] - N A D (9) and N6-[N-(6-methacrylamidohexyl)carba m o y l m e t h y l ] - N A D (10). 16 N6-Carboxymethyl-NAD (1) is prepared according to the method of Mosbach et al. 17 Then 1.2 g o f l , 2.7 g of 3, and 50 mg of hydroquinone are dissolved in 14 ml of water and the pH is adjusted to 4.8 with 2 M LiOH. To the solution is added 0.53 g of ethyldimethylaminopropylcarbodiimide hydrochloride (8, Fluka) with continuous stirring at room temperature. The pH is maintained at 4.8 with 2 M LiOH and 1 M HC1. After l hr, another portion of 8 (0.28 g) is added and the stirring is continued. After completion of the coupling [within 3 hr from the initial addition of 8; monitored by TLC on PEI-ceUulose F (Merck) developed with 0.1 M LiCI], the reaction mixture is applied to a gel permeation column (BioGel P-2, 2 cm x 1.5 m; elution with water). The nucleotides are eluted in effluent 130-220 ml separately from other compounds (the unreacted amines, etc.). The main fractions are pooled and concentrated to about 10 ml in an evaporator at 30°. The concentrate is applied to a Dowex l-X2 column (1.5 x 50 cm, HCOO- form) and the elution is performed by a linear gradient made of 500 ml of 0.2 M formic i6 y . Yamazaki and H. Maeda, Agric. Biol. Chem. 45, 2277 (1981). 17 K. Mosbach, P.-O. Larsson, and C. Lowe, this series, Vol. 44, p. 859.

26


[3]

acid and 500 ml of 2 M formic acid. Fractions of the main peak (effluent 480-600 ml) are combined and concentrated in an evaporator at 30°. An excess amount of chilled acetone is added to the concentrate. The white precipitate is collected by centrifugation, washed with acetone, and dried in vacuum to give 1.05 g of 9 as white powder; 68% yield from 1, based on UV absorption measurement ('~266 = 23.0 × 103 M 1 c m - 1 ) 16 Similarly, 10 is prepared by the coupling of 1 with 2 in 27% yield. These NAD derivatives show a single spot in various TLC systems with the following Rf values: 0.53 (9), 0.74 (10), and 0.46 (NAD) on cellulose F (Merck) developed with isobutyric acid/1 M aqueous NH3 (5/3, v/v) saturated with EDTA; 0.08 (9), 0.03 (10), and 0.16 (NAD) on cellulose F developed with 0.1 M phosphate buffer (pH 6.8)/ammonium sulfate/n-propanol (100/60/2, v/w/v); and 0.57 (9), 0.57 (10), and 0.28 (NAD) on PEI-cellulose F developed with 20 mM LiC1. The spots are located by viewing under UV light (254 nm).

Preparation of Coimmobilized Gel of NAD and Dehydrogenases Enzymes. Yeast formate dehydrogenase (FDH; lyophilized powder), horse liver alcohol dehydrogenase (LADH; suspension in 20 mM phosphate buffer, pH 7.0, containing 10% ethanol), and pig heart diaphorase (dihydrolipoamide dehydrogenase; suspension in 3.2 M ammonium sulfate) were purchased from Boehringer-Mannheim. Thermus thermophilus malate dehydrogenase (MDH; solution in 50% glycerol) was given to us by Dr. N. Miwa of Mitsubishi Petrochemical Co., Ltd. (Ibaraki), whom we wish to thank for his generosity. Standard Assay Procedures. 18 A Hitachi 124 spectrophotometer with a thermostated cell holder is used to follow the absorbance change at 340 nm in the dehydrogenase assays. The following substrate solutions are used: LADH--0.56 M ethanol, 2 mM NAD, 75 mM semicarbazide-HC1, 9.8 mM glutathione, and 62 mM sodium pyrophosphate-18 mM glycine buffer (pH 8.7) in a total volume of 3.00 ml; FDH--35 mM sodium formate, 1 mM NAD, and 87 mM potassium phosphate buffer (pH 7.5) in a total volume of 3.00 ml; MDH--0.52 mM potassium oxaloacetate, 0.2 mM NADH, 98 mM potassium phosphate buffer (pH 7.5) in a total volume of 2.98 ml; and diaphorase--5 mM sodium olAipoate, 0.2 mM NADH, 0.3 mM NAD, 0.94 mM EDTA, 0.69 mg/ml bovine serum albumin (Miles), and 80 mM potassium phosphate buffer (pH 5.9) in a total volume of 2.88 ml. The reaction is started by addition of the enzyme solution (20 p.l) and is This section is reprinted with permission from Y. Yamazaki and H. Maeda, Agric. Biol. Chem. 46, 1571 (1982).

[3]

COIMMOB|LIZATION OF

NAD

AND DEHYDROGENASES

27

the temperature is maintained at 30° in all cases. Proteins are determined by Lowry's method with bovine serum albumin as standard. ~9 F D H - M D H - N A D Gel. 2° The gel containing FDH, MDH, and NAD is prepared as follows: 15 mg (14.7/xmol) of the polymerizable NAD derivative 9 is dissolved in 0.15 ml of 0.05 M sodium pyrophosphate buffer (pH 8.5) and neutralized with 0.1 M NaOH. To the solution are added 0.69 g of acrylamide (recrystallized from benzene), 36.3 mg of methylenebisacrylamide, and 1.1 ml of the pyrophosphate buffer. Nitrogen gas is bubbled into the solution for 5 min under cooling with ice. Then, the following solutions are added successively to the above solution: 0.25 ml of MDH solution (in 50% glycerol; containing 342 U and 3.08 mg protein), 0.3 ml of FDH solution (in 0.1 M phosphate buffer, pH 7.5; containing 7.02 U and 2.93 mg protein), and two 0.15-ml solutions in the pyrophosphate buffer containing 4.5 mg of K2S208 and 11.25 mg of dimethylaminopropionitrile. The final solution is removed from the ice bath and N2 gas is bubbled into it at room temperature for about 5 min until viscosity increases. Then the solution is kept in the ice bath for 20 min. The formed gel is crushed in a blender with 50 ml of ice-cold 0.1 M phosphate buffer, pH 7.5, and thoroughly washed with the buffer on a Btichner funnel. The total weight of the gel (in a wet state) was 3.51 g. The particle size (average of the major and minor diameters) of the crushed gel was in the range of about 0.01-0.8 mm. The protein content, enzyme activity, and NAD content of the gel and their immobilization yield or recovery were as follows: protein content, 1.38 mg/g (81%); FDH, 0.16 U/g (8%); MDH, 31 U/g (32%); NAD, 3.6 /zmol/g (86%). Assay procedures for the gel are described below. L A D H - d i a p h o r a s e - N A D Gel. ~8 This gel is prepared as described above, but using the following amounts of reagents: 60 mg (56.4/zmol) of the polymerizable NAD derivative 10 in 0.2 ml of the pyrophosphate buffer (pH 8.5), 1.2 g of acrylamide, 66 mg of methylenebisacrylamide, 6 mg of bovine serum albumin, 1.5 ml of the buffer, 0.6 ml of LADH suspension (20.1 U and 6.60 mg protein), 0.6 ml of diaphorase suspension (137 U and 6.42 mg protein), 3 mg K2S208 in 0.15 ml of the buffer, and 7.5 mg dimethylaminopropionitrile in 0.15 ml of the buffer. This gel showed the following properties: protein, 2.43 mg/g (59% recovery); LADH, 0.23 U/g (5%); diaphorase, 3.7 U/g (12%); NAD, 7.5/~mol/g (61%). 19 O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 2o This section is reprinted with permission from Y. Yamazaki, H. Maeda, and K. Kamibayashi, Biotechnol. Bioeng. 24, 1915 (1982).

28


[3]

Estimation o f Immobilized Enzyme Activity, Protein, and N A D 18 The activities of the immobilized dehydrogenases were assayed by the flow method using a substrate solution of the same composition as in the standard assay procedure. The apparatus consisted of a test tube held in a water bath (30°) and a flow system with a flow cuvette in the spectrophotometer and a peristaltic pump. A net was attached at the inlet of the flow system to filter the gels. At first, the flow system was filled with the substrate solution (total volume of 1.3 ml) and then another substrate solution (three times the volume in the standard procedure) was added to the test tube. After equilibration for 3 min at 30°, the reaction was started by addition of the gel to the test tube with a constant circulation of the solution through the whole system at a flow rate of 2.3 ml/min. The reaction mixture in the test tube was also stirred by a magnetic stirrer. The absorbance change at 340 nm was continuously monitored. The experiment was repeated with different amounts of the gel and the free enzyme. The reaction rate (A340/min) was proportional to the number of milligrams of the wet gel as well as the number of units of the free enzyme determined by the standard method. From these data the enzyme activity (U/g wet gel) was calculated. The immobilized proteins were estimated by amino acid analysis (with a Hitachi 835 amino acid analyzer with an autosampler) after acid hydrolysis. A sample of 100-400 mg of the gel was mixed with 15 ml of 6 M HC1. The mixture (in a sealed vial) was kept at 120° for 30 hr and then the insoluble materials were removed by filtration. A 13-ml portion of the filtrate was evaporated to dryness at 60°. The residue was redissolved in 1.5 ml of 0.06 M HC1 and applied to the analyzer. The protein content (mg/g wet gel) was calculated from the combined peak area of 11 species of amino acids (Asp, Thr, Set, Glu, Pro, Ala, Val, Ile, Leu, Phe, and Lys; other amino acids had decomposed or overlapped with unknown compounds on the chromatograms) using the corresponding calibration curves which were prepared with the hydrolyzates of the free proteins (FDH + MDH and LADH + diaphorase + albumin, mixed in the same ratios as in the immobilization). The combined peak area was proportional to the used amount of the gels as well as that of the free proteins. The coexistence of polyacrylamide gels had no effect on this protein analysis as shown by a comparative experiment with albumin plus protein-free gels and albumin only. The NAD content was estimated from phosphate determinationsz~ after exhaustive washing of the gel with 1 M NaCl. 21 G. R. Bartlett, J. Biol. Chem. 234, 466 (1959).

[3]


29

Applications

Continuous Production of L-Malate by the Coimmobilized F D H - M D H - N A D Gel 2° The principle is the coupled reaction: formate

matrix-bound FDH /

CO2

~ matrix-bound ) NADH

t.-malate MDH ~ oxaloacetate

The gel (1.4 g) was packed in a column tube and a solution containing 0.2 M sodium formate and 2 mM sodium oxaloacetate in 0.1 M phosphate buffer (pH 7.5) was passed upward through the column at a flow rate of 2.4 ml/hr. The temperature was kept at 30°. The substrate solution was saturated with chloroform before use for preventing bacterial growth in either the solution or the column. It was further kept in an ice bath and changed to a freshly prepared one, usually every 12 hr. At the points indicated by arrows A and E in Fig. 2, the substrate solution was changed to a solution lacking only formate. It was again changed to the original substrate solution as indicated by arrows B and F. A similar experiment was run for removal and addition of oxaloacetate at the points C and D. The eluent from the column was divided into fractions of 1-hr duration and then held at 100° for 5 min to decompose the unreacted oxaloacetate. The L-malate in the fractions was determined by the method using hydrazine, NAD, and pig heart malate dehydrogenase (Boehringer-Mannheim). 22 The result is shown in Fig. 2. The L-malate production by the F D H - M D H - N A D gel was continued for 3 weeks. When formate was removed from the substrate solution (as indicated by arrow A), the L-malate production ceased. It was, however, restored to the original level by the addition of formate to the solution. This result clearly shows that L-malate was produced with recycling of the matrix-bound NAD. The L-malate production was, of course, stopped by the removal of oxaloacetate from the substrate solution (as indicated by arrow C) and resumed by the addition of the substrate. The concentration of the produced L-malate gradually decreased with time, but even at the final stage of the operation about 60% the initial value was observed and 22O. H. Lowry and J. V. Passonneau, in "A Flexible System of Enzymatic Analysis," p. 203. Academic Press, New York, 1972.

30


3E E

[3]

LO

O

0

J

I I00

~ i

L

I

r

,

3

,

~

5

-~'B , , 7

200 .--

400

300

_ 1 ~ * _ F r o c t l o n NO. h (.,,/U, , , , 9 II 13

,

,

15

L

, 17

500 ,E ~" 19

'~,'F ,

i 21

Days

FI6.2. Continuous production of L-malate in a column reactor packed with the F D H M D H - N A D gel. 2° The arrows indicate the removal of formate (A and E) and oxaloacetate (C) from the substrate solution and the addition of the removed substrate to the solutions (B, D, and F).

the recycling of the matrix-bound NAD was again confirmed by the second experiment concerning the removal and addition of formate at the points E and F. The total amount of L-malate produced during the operation was about 1300/zmol. Therefore the turnover number for each NAD molecule was 260 cycles, assuming that the total number of bound NAD molecules were available to the enzymes. The gel was recovered from the column and the contents of protein, the enzymes, and NAD were determined. The data and the residual percentages were as follows: protein, 0.77 mg/g (56%); FDH, 0.16 U/g (100%); MDH, 1.4 U/g (5%); NAD, 3.6 /xmol/g (100%). The value of NAD was obtained from phosphate analysis and the residual percentage of enzymatically active form could not be determined. In spite of this ambiguity, it can be concluded that long-term production of L-malate was achieved by the stable supply of the matrix-bound NADH. This is because the rate-limiting step of the coupled reaction is considered to be the reaction of FDH since the content of FDH was remarkably less than that of MDH, and moreover, no loss of FDH was found for the recovered gel. Therefore, the gradual decrease in the L-malate production by the F D H M D H - N A D gel must be due to the loss of MDH. This enzyme is very

[3]


31

Sample solution

r~x ,.~,~jlnjector I

~

~

.~-" ~._r~ '~ J

.~

~

Gels of coimmobilized

/

[~I I

~a[~]l I

LADH-Diaphorase-NAD spectrophotometel E x 560nm

i~ ~i;_~ ~'~ Em580nm I~---~ ;--'L~L____~ FlOwcell ~,

(+~-Lipoatesolution j / ~

Resazur n so ut on

FIG. 3. Illustration of the ethanol analyzer using the coimmobilized LADH-diaphoraseNAD gel. ~sThe solutions in the two reservoirs are mixed at an equal flow rate by a peristaltic pump and sent to the column via the injector (Rheodyne, Model 7125). The column is packed with the L A D H - d i a p h o r a s e - N A D gel (5 x 110 mm). The eluent is led to the flow cell (14 p.l) in the fluorescence spectrophotometer (Hitachi 204), which is operated at an excitation wavelength of 560 nm and an emission wavelength of 580 nm. When a solution containing ethanol is added by the injector, resorufin is formed in the column by the coupled reaction of LADH and diaphorase, and the amount thereof (detected as fluorescence intensity) is continuously monitored.

stable as it maintains the whole activity in the incubation at 70° for 3 hr. 23 The reduced protein content of the recovered gel suggests a leakage of MDH from the gel. This was supported by the following experiment. A gel containing only MDH was similarly prepared, packed in a column, and continuously washed with the above substrate solution for 3 weeks in the same way as in the operation of the reactor column. The residual percentages of enzyme activity and protein content of the recovered gel were 4 and 15%, respectively. It was therefore concluded that MDH was removed with time from the reactor column by leakage, but that since its content was large compared with the content of FDH, the loss of MDH was not sharply reflected in the rate of L-malate production.

Ethanol Analyzer Using the Coimmobilized LADH-Diaphorase-NAD Gel 18 The L A D H - d i a p h o r a s e - N A D gel was applied to an ethanol analyzer composed of a column reactor and a fluorescence spectrophotometer (Fig. 3). The system is based on the enzymatic oxidation of NADH with 23 N. Miwa, personal communication (1981).

32

MULTISTEP ENZYMESYSTEMSAND COENZYMES

[3]

"G

i

_=

\

B

1T

[ EtOH] I0 (rnM)

25

50

I0

25

50

I0

25

5O

FIG. 4. Reproducibility of the ethanol analyzer/8 To the system shown in Fig. 3 were applied 0.12-ml solutions of ethanol in 0.05 M phosphate buffer (pH 7.5). The ethanol concentrations are given in the figure. This figure shows the trace of the fluorescence intensity. The operational conditions were as follows: buffer, the same as above; DLlipoate, 5 mM in the reservoir; resazurin, 32 tzM in another reservoir; temperature, 25°; flow rate, 0.95 ml/min; column size, 5 x 110 mm; sensitivity of the photometer, 6 x 10; recorder range, 5 mV. Injection points are indicated by arrows. resazurin, 24 but practically, lipoate (a natural substrate of diaphorase) was

also used to promote the oxidation. The response of this analyzer (the ethanol ~

/matriN-bDUnd ~x~

LADH

/

~N/~"~/"O~o~~

O

~hor~ (fluorescent)

acetaldehyde

0~.~.~

matrix-bound NADH

0 ....~jO

-

N O resazurin sum of fluorescences represented by peak areas) was reproducible with regard to the applied amount of ethanol as shown in Fig. 4. The response increased with increasing ethanol concentration but seemed to saturate at high concentration. The fluorescence intensity reached maximum at 4.5 min after the sample injection at a flow rate of 0.95 ml/min and then gradually returned to its original level. The time required for the return and the response intensity were affected by many factors: pH, temperature, concentrations of lipoate and resazurin, column size, and flow rate. From an investigation on these factors, :8 the optimal conditions were chosen as given in the legend to Fig. 4. The long-term use of the ethanol 24G. G. Guilbault and D. N. Kramer, Anal.

Chem.

37, 1219 (1965).

[3]

COIMMOBILIZATION OF N A D AND DEHYDROGENASES

33

analyzer was tested in the same way as in Fig. 4, but the temperature was maintained at 15° during the operation. Between 9 and 12 samples were applied each day, and then the column was washed with the lipoate solution only and stored in a refrigerator until the next operation. Thus, this analyzer was used 84 times in 1 week for a total operation time of 41 hr. The responses gradually decreased with time, but about 50% of the initial value was still obtained at the final stage. The residual contents (percentage relative to the initial value) of the enzymes and NAD in the recovered gel were as follows: protein, 87%; LADH activity, 41%; diaphorase activity, 21%; and NAD, 100% as phosphate recovery. In a stability experiment it was found that both the free enzymes maintained 100% of the initial activity for 43 hr at 15° in a solution of the same composition as in the analyzer column. The decrease of the analyzer response is due to the loss of the enzymes and it must have been caused by leakage from the gel. Repeated use of NAD as an analytical reagent has also been studied by Davies and Mosbach 4 and Malinauskas and Kulys 5 based on a potentiometric or amperometric procedure. Their systems use a semipermeable membrane to retain a water-soluble polymer-bound NAD along with enzymes. Concluding Remarks As this article shows, the entrapment of dehydrogenases in polyacrylamide gels prepared with the usual monomers plus a radically polymerizable NAD derivative is a useful technique for the immobilization of coupled enzyme systems including the coenzyme. The advantages of the present coimmobilization method are (1) the simplicity, in that NAD and the enzymes are simultaneously immobilized in a single step, and (2) the applicability of the resultant gel to simple column-type reactors, without any need for an additional material such as ultrafiltration membrane. The principal disadvantage is that the reaction mixture cannot be supplemented with the enzyme even when it has been removed from the gel and the bound NAD still remains intact. The ultrafiltration method has an advantage in this connection. Thus, it is essential in the present method to use stable enzymes. The second important problem of this method is the leakage of enzymes from the gels. The molecular weight of Thermus MDH has been found to be relatively low (6 × 104).23 A cross-linking pretreatment to increase its molecular size or preparation of the gel at higher monomer concentrations (to reduce the pore size of the gel 25,z6) z5 C. J. O. R. Morris, in "Protides of the Biological Fluids" (H. Peeters, ed.), p. 543. Elsevier, Amsterdam, 1%6. 26 j. S. Fawcett and C. J. O. R. Morris, Sep. Sci. 1, 9 (•966).

34

MULTISTEP

ENZYME

SYSTEMS AND COENZYMES

[4]

would be effective to prevent the leakage. In contrast, FDH was well retained in the gel even though this enzyme is also not such a large protein (MW = 7.4 x I04, information from the maker). These facts suggest that the degree of leakage of proteins from the gel is dependent not only on the molecular size of the proteins but also on other factors, for instance, their shape and affinity to the matrix. It may be possible to increase the effect on enzyme retention of the affinity factor by selecting more suitable gels and/or chemically modifying the surface of the proteins. Since the present work has shown that long-term recycling of matrix-bound NAD by the simultaneously immobilized enzymes is possible, this method can be expected to find wide use once the above problem of the leakage has been solved.

[4] P o l y m e r i z a b l e N A D D e r i v a t i v e a n d M o d e l E n z y m e Reactor with Recycling of Polyethylene Glycol-Bound NAD By HIROSUKE OKADA and ITARU URABE

Many enzymes require the participation of readily dissociable cofactors such as NAD for their catalytic activities. The continuous utilization of these enzymes requires the retention and regeneration of the coenzymes. For this purpose, several methods of covalently attaching NAD to water-soluble polymers have been reported. 1,2These NAD derivatives are also useful for the study of the interaction between enzymes and NAD. In this chapter, methods for preparing a polymerizable NAD derivative, N A D - N 6-[N-( N-acrylo yl- l -metho x ycarbon yl- 5-aminopentyl )propio amide], and a polyethylene glycol-bound NAD derivative (PEG-NAD) are described (Fig. 1). The polymerizable NAD is a unique derivative, which easily copolymerizes with other vinyl monomers such as acrylamide to give macromolecular NAD derivatives (polymeric NAD). 3 Furthermore, various kinds of polymeric NAD can be obtained by copolymerization with different vinyl monomers in different molar ratios: .~ From investigations into the coenzymatic properties of the polymeric NAD, it has been suggested that NAD derivatives of smaller molecular 1 K. Mosbach, P.-O. Larsson, and C, Lowe, this series, Vol. 44, p. 859. 2 C. R. Lowe, in "Topics in Enzyme and Fermentation Biotechnology" (A. Wiseman, ed.), Vol. 5, p. 13. Horwood, Chichester, England, 1981. 3 M. Muramatsu, I. Urabe, Y. Yamada, and H. Okada, Eur. J. Biochem. 80, 111 (1977). 4 S. Furukawa, Y. Sugimoto, I. Urabe, and H. Okada, Biochimie 62, 629 (1980). 5 S. Furukawa, I. Urabe, and H. Okada, Eur. J. Biochern. 114, 101 (1981).



34

MULTISTEP

ENZYME

SYSTEMS AND COENZYMES

[4]

would be effective to prevent the leakage. In contrast, FDH was well retained in the gel even though this enzyme is also not such a large protein (MW = 7.4 x I04, information from the maker). These facts suggest that the degree of leakage of proteins from the gel is dependent not only on the molecular size of the proteins but also on other factors, for instance, their shape and affinity to the matrix. It may be possible to increase the effect on enzyme retention of the affinity factor by selecting more suitable gels and/or chemically modifying the surface of the proteins. Since the present work has shown that long-term recycling of matrix-bound NAD by the simultaneously immobilized enzymes is possible, this method can be expected to find wide use once the above problem of the leakage has been solved.

[4] P o l y m e r i z a b l e N A D D e r i v a t i v e a n d M o d e l E n z y m e Reactor with Recycling of Polyethylene Glycol-Bound NAD By HIROSUKE OKADA and ITARU URABE

Many enzymes require the participation of readily dissociable cofactors such as NAD for their catalytic activities. The continuous utilization of these enzymes requires the retention and regeneration of the coenzymes. For this purpose, several methods of covalently attaching NAD to water-soluble polymers have been reported. 1,2These NAD derivatives are also useful for the study of the interaction between enzymes and NAD. In this chapter, methods for preparing a polymerizable NAD derivative, N A D - N 6-[N-( N-acrylo yl- l -metho x ycarbon yl- 5-aminopentyl )propio amide], and a polyethylene glycol-bound NAD derivative (PEG-NAD) are described (Fig. 1). The polymerizable NAD is a unique derivative, which easily copolymerizes with other vinyl monomers such as acrylamide to give macromolecular NAD derivatives (polymeric NAD). 3 Furthermore, various kinds of polymeric NAD can be obtained by copolymerization with different vinyl monomers in different molar ratios: .~ From investigations into the coenzymatic properties of the polymeric NAD, it has been suggested that NAD derivatives of smaller molecular 1 K. Mosbach, P.-O. Larsson, and C, Lowe, this series, Vol. 44, p. 859. 2 C. R. Lowe, in "Topics in Enzyme and Fermentation Biotechnology" (A. Wiseman, ed.), Vol. 5, p. 13. Horwood, Chichester, England, 1981. 3 M. Muramatsu, I. Urabe, Y. Yamada, and H. Okada, Eur. J. Biochem. 80, 111 (1977). 4 S. Furukawa, Y. Sugimoto, I. Urabe, and H. Okada, Biochimie 62, 629 (1980). 5 S. Furukawa, I. Urabe, and H. Okada, Eur. J. Biochern. 114, 101 (1981).



['fir]

POLYMERIZABLE N A D DERIVATIVE AND P E G - N A D

L- Lysine 1) CHz=CHCOCI 2) HCIICH3OH C.OOCH3 H2NCH(CH2)4NHCOCH=CH 2 i

F

I

;oocm

35

NAD Polyethylene glycol 1) Propiolactone I SOCt2 ~v 2) Rearrangement CI(CHzCHtO)nCHtCHzCI NHCH2CH2COOH I NH~ J. ~CONH 2 ~' N" j'~I---N ~ ~t" H2N(CH2CH20)nCH2CH2N H2 /

I,"-C2" 40 /

10 mM

20

0

I

I

I

1

2

3

-c(h) FIG. 2. Theoretical relationship between residence time (r) and the yield of L-lactate (Y) at steady state with different ethanol concentrations in the eluent ([ethanol]0). Conditions for calculation are [lactate dehydrogenase] = 0.12/xM, [alcohol dehydrogenase] = 2.88/zM, [C]~ = lmM, [pyruvate]0 = 5 mM.

44


[4]

100

8O

60

> 40

20 I

0

I

I

50 100 150 [ E t h a n o L ] o (raM)

200

FIG. 3. Effect of ethanol concentration in the eluent ([ethanol]0) on the yield of L-lactate (Y) at steady state. (0) Y determined experimentally; error bars indicate fluctuation range; (O) Y calculated for each set of experimental conditions; ( ) Y calculated with the average conditions of these experiments. The average conditions: [lactate dehydrogenase] = 0.11/,~M, [alcohol dehydrogenase] = 2.64/zM, [C]t = 0.749 mM, [pyruvate]0 = 5.0 mM, ~- = 2.61 hr.

ultrafiltration membrane, and the apparatus was placed in a temperaturecontrolled chamber (Tabai LN-110). The continuous enzyme reaction was carried out at 30° in 30 mM sodium phosphate buffer, pH 7.5, containing 0.1% streptomycin and 0.02% NaN3 by continuous ultrafiltration with substrate solution containing pyruvate and ethanol in the buffer as eluent under N2 pressure, and the volume of the reaction mixture was kept at 6.5 ml. The filtrate was collected in fractions at 1-hr intervals, and the Llactate concentration and volume of each fraction were measured. LLactate was assayed with lactate dehydrogenase and NAD.13 The concentration of L-lactate became constant 10-15 hr after the start of the reaction. The steady-state values of L-lactate concentration and residence time 0") were obtained as average values during the steady state over 10 hr (10 measurements). Their fluctuation range was less than 10% of the average value. The concentration of PEG-NAD and the activities of the enzymes in the reactor decreased gradually during the continuous operation. The quasi-steady-state values were estimated from initial and final values by assuming a first-order rate of decrease. The difference between the initial and final values of a steady state was less than 20% of the average value. Figure 3 shows the effects of ethanol concentration in the eluent at a t3 I. Gutman and A. W. Wahlefeld, in "Methods of Enzymatic Analysis" (H. U. Bergmeyer, ed.), 2nd Ed., p. 1464. Academic Press, New York, 1974.

[5]

E N Z Y M E REACTOR BASED ON a q T W O - P H A S E SYSTEMS

45

fixed value of z. The solid and open circles show experimental and theoretical values, respectively. The solid line was calculated with the average conditions of fixed variables in the figure. Deviation of an open circle from the solid line is due to the deviation of the fixed variables of the experiment from their average values. Generally, the steady-state values obtained by continuous operation of the enzyme reactor are lower than those obtained by calculation based on the kinetic model. These differences are thought to be due to the simplifications made for the kinetic model, in which we assumed a simplified ordered Bi-Bi mechanism and neglected the effects of substrate inhibition and so on. However, the differences are not large in the region of Ef -> 0.03 and [C]t -> 0.7 raM, and the steady-state behavior of the enzyme reactor can be explained and predicted semiquantitatively by the simple kinetic model.

[5] A d e n o s i n e 5 ' - T r i p h o s p h a t e R e c y c l i n g in a n E n z y m e Reactor Based on Aqueous Two-Phase Systems B y HIDEO SUZUKI a n d YOSHIMITSU YAMAZAKI

The retention and in situ regeneration of coenzymes (e.g., NAD or ATP) in a reactor are prerequisites for the industrial use of dehydrogenases, kinases, or ligases. One possible approach to this is to bind the coenzymes to water-soluble polymers and use them with the enzymes in an ultrafiltration apparatus. J-3 However, this method has the disadvantage that enzymes as well as the polymers carrying coenzymes tend to be adsorbed on the ultrafiltration membrane and thus lost from the reaction mixtures. J The need for developing a better method for separating polymers from low molecules reminded us of countercurrent partitioning using aqueous two-phase systems. These systems were developed by Albertsson 4 and l y. Yamazaki, H. Maeda, and H. Suzuki, Biotechnol. Bioeng. 18, 1761 (1976). 2 S. Furukawa, N. Katayama, T. Iizuka, I. Urabe, and H. Okada, FEBS Lett. 121, 239 (1980). 3 R. Wichmann, C. Wandrey, A. F. B0ckmann, and M.-R. Kula, Biotechnol. Bioeng. 23, 2789 (1981). 4 P.-A. Albertsson, "Partition of Cell Particles and Macromolecules," 2nd Ed. Wiley, New York, 1971.



[5]

E N Z Y M E REACTOR BASED ON a q T W O - P H A S E SYSTEMS

45

fixed value of z. The solid and open circles show experimental and theoretical values, respectively. The solid line was calculated with the average conditions of fixed variables in the figure. Deviation of an open circle from the solid line is due to the deviation of the fixed variables of the experiment from their average values. Generally, the steady-state values obtained by continuous operation of the enzyme reactor are lower than those obtained by calculation based on the kinetic model. These differences are thought to be due to the simplifications made for the kinetic model, in which we assumed a simplified ordered Bi-Bi mechanism and neglected the effects of substrate inhibition and so on. However, the differences are not large in the region of Ef -> 0.03 and [C]t -> 0.7 raM, and the steady-state behavior of the enzyme reactor can be explained and predicted semiquantitatively by the simple kinetic model.

[5] A d e n o s i n e 5 ' - T r i p h o s p h a t e R e c y c l i n g in a n E n z y m e Reactor Based on Aqueous Two-Phase Systems B y HIDEO SUZUKI a n d YOSHIMITSU YAMAZAKI

The retention and in situ regeneration of coenzymes (e.g., NAD or ATP) in a reactor are prerequisites for the industrial use of dehydrogenases, kinases, or ligases. One possible approach to this is to bind the coenzymes to water-soluble polymers and use them with the enzymes in an ultrafiltration apparatus. J-3 However, this method has the disadvantage that enzymes as well as the polymers carrying coenzymes tend to be adsorbed on the ultrafiltration membrane and thus lost from the reaction mixtures. J The need for developing a better method for separating polymers from low molecules reminded us of countercurrent partitioning using aqueous two-phase systems. These systems were developed by Albertsson 4 and l y. Yamazaki, H. Maeda, and H. Suzuki, Biotechnol. Bioeng. 18, 1761 (1976). 2 S. Furukawa, N. Katayama, T. Iizuka, I. Urabe, and H. Okada, FEBS Lett. 121, 239 (1980). 3 R. Wichmann, C. Wandrey, A. F. B0ckmann, and M.-R. Kula, Biotechnol. Bioeng. 23, 2789 (1981). 4 P.-A. Albertsson, "Partition of Cell Particles and Macromolecules," 2nd Ed. Wiley, New York, 1971.



46


[5]

are useful in collecting cell particles 5 and biopolymers. 6'7 They are prepared by mixing aqueous solutions of different polymers. For instance, when a solution of dextran in water is mixed with a solution of polyethylene glycol (PEG) in water, the mixture becomes turbid and after a while it separates into two phases. If proteins or other polymers are added to the mixture, they are often asymmetrically partitioned to the upper and the lower phases, depending on the experimental conditions. Low molecular weight molecules are usually almost symmetrically partitioned into both phases. Thus one can see the possibility of an enzyme reactor based on the principle that enzymes and the polymer-bound coenzymes are retained in the lower phase while the low molecular weight products are extracted to the upper phase. The purpose of this article 8 is to present an outline of such a reactor in the case of kinases, especially for the coupled system of hexokinase and acetate kinase. Design of the System In constructing a reactor of this type, the first step is to find an aqueous two-phase system in which all catalysts (here, acetate kinase, hexokinase, and polymer-bound ATP) are mostly partitioned to either the upper or the lower phase, and in which phase the enzyme reaction can actually proceed. There are many factors affecting aqueous two-phase systems: (1) the species of polymers and their ratios, (2) the species of buffers, their pH, and concentrations, (3) the species of salts in the media and their concentrations, (4) temperature, and so on. An additional factor in the present case is the structure of the ATP carrier, which must be carefully selected with consideration given to what polymers are used for the aqueous two-phase system. We chose dextran as the ATP carrier and a combination of dextran and PEG for the basic phase system) In this system, the upper phase is rich in PEG and the lower phase is rich in dextran; therefore dextran-bound ATP goes to the lower phase by the tendency of similar materials to meet. In contrast, Johansson ~° reported 5 A.-L. Smeds, A. Veide, and S.-O. Enfors, Enzyme Microb. Technol. 5, 33 (1983). 6 K. H. Kroner, H. Hustedt, and M.-R. Kula, Biotechnol. Bioeng. 24, 1015 (1982). 7 A. Veide, A.-L. Smeds, and S.-O. Enfors, Biotechnol. Bioeng. 25, 1789 (1983). 8 The original publication is Y. Yamazaki and H. Suzuki, Biseibutsu Kogyo Gijutsu Kenkyusho Kenkyu Hokoku 52, 33 (1979). 9 A recent review of the syntheses of polymer-bound ATP is by Y. Yamazaki and H. Maeda, Yuki Gosei Kagaku Kyokaishi 41, 1088 (1983). to G. Johansson, Biochim. Biophys. Acta 222, 381 (1970).

[5]

ENZYME REACTOR BASED ON aq TWO-PHASE SYSTEMS

47

that acidic proteins were mostly partitioned in the lower phase when a charged PEG derivative, PEG sulfonate, was used in preparing the system of PEG and dextran. This can possibly be accounted for by electric repulsion. Dextran-bound ATP is negatively charged in neutral solutions owing to the phosphate groups. Thus, a more favorable partition of dextran-bound ATP to the lower phase can be expected in a phase system supplemented with a negatively charged PEG. Hexokinase (pI 4.5-4.81~) and serum albumin (pI 4.7-4.9, j2 used as enzyme stabilizer) are also expected from their isoelectric point to show a higher affinity to the lower phase in such a system. Johansson used PEG sulfonate ( - O 3 S - C H z C H z O (CH2CHzO)n-CHzCH2-SO3-), but we used a more easily preparable polymer, PEG sulfate (-03S-O-CH2CH20-(CH2CH20),-CH2CHz-O-S03-). Synthetic procedures of dextran-bound ATP and PEG sulfate are described below. Preparation of Dextran-Bound A TP 13

The principle used is the introduction of a spacer chain having a terminal amino group to the N-6 site of adenine nucleus and the succeeding coupling to BrCN-activated dextran using the amino group. In the reaction shown in Fig. 1 ADP is used as the starting material, but the ATP derivative, N6-[N-(6-aminohexyl)carbamoyl]-ATP, which is formed probably from the disproportionation (2ADP = ATP + AMP), is purified by column chromatography. One gram of ADP (free acid, Boehringer-Mannheim) is dissolved in 50 ml of hexamethylphosphoramide with stirring at 70°, and then 20 ml of freshly distilled hexamethylene diisocyanate is added. After incubation at 75 ° for 2 hr, the reaction mixture is cooled to 0 ° and slowly poured into an ice-cold two-phase mixture of chloroform (750 ml) and acidic water (750 ml, pH 1 with HC1), under vigorous stirring. The resultant emulsion is transferred into a separatory funnel and allowed to stand overnight at room temperature. The aqueous layer becomes clear and is separated from the chloroform layer, which is reextracted with 750 ml of acidic water (pH 1). The combined aqueous layer is neutralized with 1 M LiOH and concentrated on a rotary evaporator at 40 ° to approximately 100 ml. To the concentrate is added 1 liter of an ice-cold mixture made of ethanol u M. Kunitz and M. R. McDonald, J. Gen. Physiol. 29, 393 (1946). t2 E. G. Young, in "Comprehensive Biochemistry" (M. Florkin and E. H. Stotz, eds.), Vol. 7, p. 22. Elsevier, New York, 1963. u This section is reprinted with permission from Y. Yamazaki, H. Maeda, and H. Suzuki, Eur. J. Biochem. 77, 511 (1977),

48


H N . , ~ N . " % / " k ~ NC0 ADP

OCNo (CHI) Q- NCO •

t~j ~ - - ~ "~N'~'N /

[5]

HN . - " ~ N ~ / ~ I )

N.~..,,~ N ~'~NI~"N "~

OH OH

H3 CB-

OH OH

Chromatographlc Separation I Dextran-bound ATP

F M N + R C O O H + H20 + hv

(3)

This reaction by itself is of limited analytical usefulness unless it is coupled to the production of FMNH2, a reaction which is catalyzed by an NADH- or NADPH-specific oxidoreductase [Eqs. (4) and (5)]. NADH + H + + FMN NADPH + H + + FMN

oxidoreductase oxidoreductase

~ N A D + + FMNH2

(4)

~ N A D P ÷ + FMNH2

(5)

By coupling the luciferase with the oxidoreductase, any dehydrogenase which produces N A D H or NADPH can be assayed, as shown in reaction (6). XH~ + NAD(P) +

E

, X + NAD(P)H + H +

(6)

Either XH2, the reduced substrate, or the enzyme catalyzing the oxidation of XH2 can be measured. What are the advantages of using coimmobilized enzymes for these assays? In general the enzymes are more stable than the soluble forms. The sensitivity of the assays is greatly increased due to the fact that the immobilized luciferase is present in a microenvironment with locally high concentrations of ATP or FMNH2. For example, coimmobilized N A D H : FMN oxidoreductase and bacterial luciferase produce about 100 times more light per picomole of NADH than comparable amounts of soluble enzymes. Another important aspect is that immobilized enzymes can be incorporated into flow cells where they can be used for multiple assays. Source

of the Enzymes

The experiments reported here have all been performed with enzymes purified in our laboratory. Firefly luciferase is purified as described in Vol. LVII of this series.~ Bacterial luciferase is purified from Vibrio harveyi according to Hastings et al. z The oxidoreductases are purified also from V. harveyiJ There is a diaphorase from microorganisms available 1 M. D e L u c a and W. D. McElroy, this series, Vol. 42, p. 3. 2 j. W. Hastings, T. O. Baldwin, and M. Z. Nicoli, this series, Vol. 42, p. 135. 3 E. Jablonski and M. D e L u c a , Biochemistry 16, 2932 (1977).

84


[8]

from Boehringer-Mannheim which can be used instead of the oxidoreductases. This enzyme will use either NADH or NADPH as a substrate. We do not have any experience in immobilizing commercially available enzymes; however, there is one report in which the commercial bacterial oxidoreductase and luciferase have been coimmobilized on nylon tubing. 4 Other experiments have indicated that the commercial enzymes can be successfully immobilized onto Sepharose using the same procedures described here. 5 A combined luciferase/oxidoreductase preparation partially purified from Photobacterium fischeri can be purchased from the following companies: Boehringer-Mannheim, Lumac/3M, Calbiochem-Behring/ Hoechst, LKB-Wallac, Analytical Luminescent Laboratory, and Packard Instruments. Sigma Chemical Co. Ltd. sells the enzymes either from P. fischeri or as a preparation from V. harveyi. Firefly luciferase and luciferin are commercially available from the following companies: Boehringer-Mannheim, Calbiochem-Behring/ Hoechst, Sigma Chemical Co. Ltd., LKB-WalIac, Lumac/3M, Turner Design, Packard Instruments, and Analytical Luminescent Laboratory. Instrumentation

The instruments used for measuring light emission can be a very simple design with a photomultiplier tube, power supply, and a light-tight reaction chamber. It is also possible to buy more sophisticated automated luminometers with microprocessor-controlled automatic injection systems. The various commercially available instruments were reviewed by Picciolo et al. in 1978. 6 A more recent discussion of the then currently available instruments can be found in an article by Stanley. 7 A different approach to measuring the light emission from luminescent assays is the use of a high-speed (ASA 20000) instant photographic film, as described by Green et al. ~ Assays

Firefly Luciferase. This enzyme is assayed by injecting 100 ~1 of 0.02 M ATP into 500/.d of 0.025 M glycylglycine buffer, pH 7.8, containing 1 × 10 -4 M D-luciferin, 8 x 10 -3 M MgC12, and an appropriate amount of 4 A Roda, S. Girotti, S. Ghini, B. Gricolo, G. Carrea, and R. Bovara, Clin. Chem. 30, 206 (1984). 5 K. Green, L. J. Kricka, G. H. G. Thorpe, and T. P. Whitehead, Talanta 31, 173 (1984). 6 G. L. Picciolo, J. W. Deming, D. A. Nibley, and E. Chappelle, this series, Vol. 42, p. 550. 7 p. E. Stanley, in "Clinical and Biochemical Luminescence" (L. J. Kricka and T. J. N. Carter, eds.), p. 219. Dekker, New York, 1982.

[8]

BIOLUMINESCENT ASSAYS

85

either soluble or immobilized luciferase. The amount of enzyme to be used is dependent on the sensitivity of the luminometer. The peak light intensity is proportional to the concentration of enzyme present and is expressed in relative light units. If the amount of enzyme is kept constant and limiting amounts of ATP are injected, then light intensity is proportional to the ATP concentration. Bacterial Luciferase. Either the soluble or immobilized enzyme is assayed by the injection of 100 Izl 1.5 x 10 -4 M FMNH2 into 500 tzl of 0.1 M potassium phosphate buffer, pH 7.0, containing 0.0005% (v/v) decanal and luciferase. The FMNH2 is prepared by photoreducing FMN in the presence of 5 × 10-3 M EDTA. 8 The buffer-decanal mixture is made from a 0.05% decanal-water suspension which should be prepared freshly every 4 hr. Oxidoreductases. These enzymes are assayed spectrophotometrically by measuring the initial rate of oxidation of NADH or NADPH as the decrease in absorbance at 340 nm. The reaction is initiated by adding I00 /A of 2 mM N A D H or NADPH to 0.9 ml of potassium phosphate buffer, pH 7.0, containing the enzyme and 0.13 mM FMN. The immobilized oxidoreductases are assayed in the same way except the reaction mixture in the cuvette is stirred continuously to avoid settling of the Sepharose. Diaphorase. The conditions for the determination of the activity of the diaphorase are the same as those used in the oxidoreductase assays. The oxidation of N A D H or NADPH in the absence of added FMN is subtracted from that in the presence of FMN.

Coupled Reaction of Coimmobilized Oxidoreductase and Luciferase. Maximum initial light intensity is measured upon the addition of 10-20/~1 of the Sepharose-bound enzyme suspension into 0.5 ml of 100 mM potassium phosphate buffer, pH 7.0, containing 0.04 mM NADH or NADPH, respectively, 0.003 mM FMN, and 0.0005% decanal. Other Enzymes. All of the other enzymes which are coimmobilized with the oxidoreductase and luciferase are assayed spectrophotometrically according to published procedures. 9 Immobilization Procedure The mixture of enzymes to be immobilized contain 5 mg of bacterial luciferase and 2-3 IU of either the NADH or NADPH : FMN oxidoreduc8 j. Lee, Biochemistry 11, 3350 (1972). 9 H. U. Bergmeyer, "Methods of Enzymatic Analysis." Verlag Chemie Weinheim/Academic Press, New York, 1971.

86

MULTISTEP

c.,

P

r~

ENZYME

c,l

t"q

SYSTEMS

AND

COENZYMES

e,l

[8]

i-,,I

~

t'q

?

?

?

e~ i

×

E

?

X

T T T x

~

b

T

x

? ~-

x

T

x

T

x

~

x

-×××~-×~-×-m M d ~

md

md----

,t"q e,~

0 .
. ,
0

[19]

SYNTHESIS

40

{

! I

=~ 11L1

¢o

22!2 33'3 ~Ez s%--%

21o 3'o to

5'o oo

% H20

2t,~

protein powder

2O T ~1~

9A~6 ~,o u n ~,~2

1'

O

~cZ.

/

_J

111

o

2~2 3~3

Wo ~.'o z'o 3.'0

%H~O

J

4M.

555

d.o

s'o

60

FIG. 5. Solubilization by phase transfer from the solid state. (a) Extraction from the solid state. (b) Solubility of lysozyme during extraction from the solid states, expressed in overall concentration (A) and concentration in the water pool (B). To 50 mM AOT/isooctane containing various amounts of water (0,©) or aqueous buffer (A, A) solid lysozyme was added, followed by a short shaking, as described in the text. From Grandi e t a l . 19

It is interesting to observe both from Fig. 5 and from the literature 18 that the maximal solubilization does not take place at the largest w0 values, but instead at rather small ones. In the case of lipophilin discussed above, the French group found the maximal solubility at w0 = 5.6, and the same value was obtained from the myelin basic protein, another component of the myeline structure that is however soluble in water. This sur-

[19]

ENZYMES IN REVERSE MICELLES

197

prising feature is a clear indication that the water inside the water pool is not "normal" water, but acquires novel properties, including a novel solubilization power.

Characterization of Protein-Containing Reverse Micelles The experimental determination of the size and structure of the micellar aggregates which host the biopolymers is more complicated than that of the "unfilled" micelles, mostly because the system under study will generally be heterogeneous, i.e., composed by "filled" (containing the biopolymer) and "unfilled" micelles. Actually, it is rather difficult (with the exception of a few cases) to obtain a high degree of occupancy of the micelles, and most of the experimental methods for the determination of the molecular weight or size (light scattering, osmometry, photon correlation, fluorescence, etc.) will reach an average value. Since also the occupancy degree is generally not known a priori (this depend on the model for the protein uptake), the. knowledge of the molecular weight of the enzyme-containing micelles is indeed not straightforward. One needs an experimental method that is able to discriminate a small amount of filled micelles over a larger background of unfilled ones. Ultracentrifugation with a UV-monitoring device seems to be the best method, since measuring at around 280 nm, where only the protein absorbs, makes the unfilled micelles "invisible." Furthermore, by adding an appropriate chromophoric small molecule to the system, which is present in both filled and unfilled micelles, as suggested by Martinek's group, 2° one may be able to demonstrate both filled and unfilled micelles. One such sedimentation run is shown in Fig. 6A. However, it is not easy to proceed from these data to molecular weight evaluation. Sedimentation and diffusion coefficients must be extrapolated to infinite dilution (as shown in Fig. 6B) of micelles in order to avoid artifacts due to intermolecular association (as is always the case in macromolecular systems having finite virial coefficients). But this is difficult because micelles dissociate at very low surfactant concentration, and there are some conceptual problems in the definition of the partial specific volume of the sedimenting particle. Equilibrium sedimentation runs are probably best suited for determining the molecular weight, but in order to determine the dimensions (Stokes radius) one needs again diffusion coefficients, which have to be measured in independent runs, and extrapolated to c = 0. Finally the molecular weight should be determined as a function of w0 and other micellar parameters. 20 E. Martinek, A. V. Levashov, Y. L. Khmel'nitskii, N. L. Klyachko, V. Y. Chernyak, and, V. I. Berezin, Dokl. Akad. Nauk SSSR 258, 1488 (1981).

198

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS

°i/-

[19]

A

OD

S, S'

18 ¸

16

/

DNP ÷ LYS

LYS

1,

/ ,

2'5

go

w6o

c A O T (mM)

FIG. 6. Ultracentrifugation experiments with AOT reverse micelles. (A) UV scanner traces of the time-dependent sedimentation profile (concentration in terms of optical density at 350 or 296 nm versus radial position) of three different micellar solutions (w0 = 7, 7, 100 m M AOT in isooctane) at 48,000 rpm and 25 °. DNP, 2,4-Dinitrophenol; Lys, lysozyme. (B) Extrapolation of the determined sedimentation coefficients (S) to infinite dilution (AOT in isooctane, w0 = 6.9) for reverse micelles with and without entrapped lysozyme: - - , without lysozyme; - . - , with lysozyme ([lysozyme]/[AOT] = 10 3). From Zampieri et al. 21a

Table I collects some typical experimental data on the molecular weight determination of filled and unfilled micelles. One notices in all cases an enlargement of molecular mass, which is larger than the sum of the two initial components (unfilled micelles and lysozyme, which has a molecular mass of 14,500 Da). The simplest way to interpret the data is shown in Fig. 7. This water shell model, based mainly on geometrical considerations and mass balance, has been elaborated into a method which permits the calculation of the dimensions of the filled micelle as a function of w0 once the molecular weight and dimensions of the protein and the unfilled micelle are known. 2~ However, on the basis of recent measurements, it appears that this first-approximation model may need s o m e r e v i s i o n . 21a 2i F. J. Bonner, R. Wolf, and P. L. Luisi, J. Solid-Phase Biochem. 5, 255 (1980). 21a G. G. Zampieri, H. J~ickle, and P. L. Luisi, J. Phys. Chem. 90, 1849 (1986).

[19]


199

TABLE I MOLECULAR WEIGHTS OF LYSOZYME-CONTAINING REVERSE MICELLES a

W0

AOT (mM)

Molecular weight b

6.9 7.8 10. 11.7

100 100 50 100

60,000-168,000 c 40,000-96,000 d 91,000-122,000 d 81,000-303,000 c

o The aqueous solution is 20 m M borate buffer, pH 8.5. The overall e n z y m e concentration is in the range 50-100 /~M. All measurements are performed at room temperature. From G. Zampieri e t al. 2Ia w0 = [H20]/[AOT]. b The first figure is related to "unfilled," the • second to "filled" micelles. c F r o m equilibrium measurements. d F r o m sedimentation and diffusion measurements.

Conformation and Activity of Enzymes in Reverse Micellar Solutions

Some General Experimental Considerations The concentration of protein in the hydrocarbon micellar solution can be easily checked by spectroscopic methods, for example, by measurement of the optical density at 280 nm. Once the enzyme is solubilized in the hy~irocarbon micellar solution, its activity needs to be assayed. This can be done in the same way as for an aqueous solution of the enzyme, namely by addition of the substrate to the enzyme solution, or vice versa. Several possibilities of this operation protein

® reverse micelle

FIG, 7. E n z y m e s in reverse micelles. The water shell model.

200


[19]

can be used in a micellar solution. (1) The two reagents (enzyme and substrate) can be added to each other as two hydrocarbon miceUar solutions; (2) one of the two reagents can be added to the hydrocarbon micellar solution in small amounts (microliter quantities) as an aqueous buffered solution; (3) finally the substrate, when directly soluble in hydrocarbon, can be added in the pure liquid form (e.g., linoleic acid to lipoxygenase). 22 Which of the assay methods is best depends on the particular system under study. Perhaps a word of warning is necessary concerning the order of mixing. When the enzyme is not very stable in the micellar solution, it is better to add the enzyme as the last component from the aqueous stock solution, to minimize the time that the enzyme stays in the micellar solution. One such case is lysozyme which we will discuss later. The study of the enzyme properties in the micellar solution, from this point on, can be carried out exactly following the procedures used for aqueous solutions. The micellar enzyme solutions are in fact clear, and spectroscopic enzyme assays to measure activity as well as circular dichroism, absorption, fluorescence, or NMR studies can be readily performed. However, there is one conceptual difficulty that sets these systems apart from normal aqueous solutions: the definition of concentration (and therefore of the Michaelis-Menten constant Kin) for water-soluble reagents, such as hydrophilic enzymes or their water-soluble substrates, is not unambiguous. One can refer the concentration to the overall volume (ov) of the solution (hydrocarbon plus water) or to the volume of the water alone. For a micellar solution containing 1% water (v : v), and for the simple case where the guest substance is only present in the water pools, the two numbers differ by a factor of 100. Km,ovis a phenomenological parameter, i.e., it refers to the analytical concentration in the whole system4'23; Km,wpassumes a structural model, i.e., one in which water is segregated in the water pools (wp), which very rapidly exchange materials with each other. There is no reason a priori to prefer to express the concentration as overall or as water pool, but misconceptions may arise. A numerically larger Kr~ value is in fact associated with a lower affinity of the enzyme for the substrate. One should be careful at this point not to confuse a trivial mathematical transformation with physical reality. In our case, enzyme saturation (or half-saturation at Km) is obtained with the same amount of added substrate regardless of whether one considers 22 p. L. Luisi, P. L~lthi, I. Tomka, J. Prenosil, and A. Pande, Proc. 7th Enz. Eng. Conf., Ann. N.Y. Acad. Sci. 434, 549 (1984). 23 p. D. I. Fletcher, N. F. Perrins, B. H. Robinson, and C. Toprakcioglu, in "Reverse Micelles" (P. L. Luisi and B. E. Straub, eds.). Plenum, New York, 1984.

[19]


201

overall or local concentration. Or in other words, if Km is numerically larger when expressed in water pool units, the substrate concentration is larger by the same factor, so that the enzyme fractional saturation is the same. Another controversial conceptual point, which is very important for the experimentalist, is the determination of the pH of the miceUar solutions. The argument has been much debated, z4,25 and no general agreement has been reached. We believe that the situation can be summarized as follows. In principle, there is no simple direct way either to define or to determine the pH in the water pool, mainly because the water of the water pool is a novel solvent, for which no calibration has been as yet offered. However, it can be accepted with fair reliability that the pH of the water pool is close to that of the stock buffered aqueous solution used to prepare the water pool of the reverse micelles, particularly at w0 values larger than 10-15. A useful experimental check is the determination of the apparent acidity by using the 3tp NMR of phosphate buffers in the water pool. 25,1° After this discussion of the main operational problems encountered in the field, we can now present an analysis of the conformation and activity of enzymes solubilized in apolar solvent via reverse micelles. We will analyze the various classes of enzymes, trying to emphasize in each case the major changes in activity and conformation brought about by the micellar environment with respect to bulk water. Most of the data presently available in the literature about proteins in reverse micellar systems are presented in Tables II-V. In particular, Table II reports data about proteases, Table III about hydrolases other than proteases, Table IV about oxidoreductases and related enzymes, and Table V about nonenzymatic proteins. A collection of kinetic parameters (Km and kcat) in reverse miceUes, compared with aqueous solutions, is offered in Table VI. Proteases

As apparent from Table 1I, 1'9'16'27-33 the most widely studied enzyme in hydrocarbon micellar solutions is a-chymotrypsin. The first report de24 O. A. El Seoud, in "Reverse Micelles" (P. L. Luisi and B. E. Straub, eds,), p. 81. Plenum, New York, 1984. 25 R. E. Smith and P. L. Luisi, Heir. Chim. Acta 63, 2302 (1980). z6 p. L. Luisi and C, Laane, Trends Biotech. 4, 153 (1986). 27 p. L. Luisi, F. Henninger, M. Joppich, A. Dossena, and G. Casmati, Biochem. Biophys. Res. Commun. 74, 1384 (1977). 18 K. Martinek, A. V. Levashov, N. L. Klyachko, V. I. Pantin, and I. V, Berezin, Biochim. Biophys. Acta 657, 277 (1981). 29 F. M. Menger and K. Yamada, J. Am. Chem. Soc. 101, 6731 (1979).

202

IMMOBILIZED

ENZYMES/CELLS

o

t-

IN ORGANIC

•- ¢ , , ~

~

•. ~

~

,~

'~.~

:~

.~

~

:~

~

o

.~ ,-.,

[19]

SYNTHESIS

•

~ .'~

~ . ~ . ~ :~

:~

~

'~ ~,

~

~

z

g~ o

o

~-

¢h

~,

~a

z ua 0

E 0

o

¢11

.~.

4~

• t " ,.~ ~'~ P"-. O

106 fl/cm). Entrapment of Enzymes in Reversed Micelles According to Luisi et al., 6 Martinek et al., 8 and Menger and Yamada, 9 there are essentially three techniques that can be used to solubilize enzymes into reversed micellar media (Fig. 2). Procedure.

1. A small amount (few % v/v) of a concentrated aqueous stock solution of enzyme is added by a microsyringe under vigorous stirring to the reversed micellar medium (Fig. 2A). In general the final overall enzyme concentration is within the range 1-20/zM, which is well below the actual micelle concentration in most reversed micellar media studied so far. For a 0.1 M AOT in heptane solution the micelle concentration is approximately 0.5 mM at w0 = 20, l° and for a 0.2 M CTAB in octane medium the concentration is within the range 1-4 mM (Laane, unpublished results). Hence most reversed micelles are empty. This technique is the most frequently used since it is rapid and extremely simple. Instead of mechanical agitation the medium can also be sonicated) ~ We prefer, however, mechanical agitation with a Vortex mixer. 8 K. Martinek, A. V. Levashov, N. L. Klyachko, and I. V. Berezin, Dokl. Acad. Nauk. USSR 236, 920 (1977); Dokl. Biochem. (Engl. Transl.) 236, 951 (1977). 9 F. M, Menger and K. Yamada, J. Am. Chem. Soc. 101, 6731 (1979). 10 p. D. I. Fletcher and B. H. Robinson, Ber. Bunsenges. Phys. Chem. 85, 863 (1981). H p. Douzou, E. Keh, and C. Balny, Proc. Natl. Acad. Sci. U.S.A. 76, 681 (1979).

220


A

B

[20]

c

Flo. 2. The three techniques to solubilize a protein in a reversed micellar medium. (A) Injection technique, by which a few microliters of the buffered stock solution of the protein are added to the surfactant solution in hydrocarbon or to a solution that already contains water. (B) Extraction from the solid state, in which the protein powder is added to a solution of hydrocarbon containing the surfactant and water. (C) Phase transfer technique in which the hydrocarbon solution of the surfactant is in contact with an aqueous solution containing the protein. From Lnisi and Wolf. 6

A few minor disadvantages of this technique are that it is difficult to encapsulate high concentrations of enzyme in the reversed micellar solution and that the enzyme may be inactivated slightly due to a "concentration shock" during the injection. Furthermore, one has to be aware of the possibility that even a few hours after injection the solution may be thermodynamically unstable due to microscopic structural rearrangements. These problems are generally overcome by the second technique. 2. The enzyme is added in the solid state as a fine powder to a complete reversed micellar solution (Fig. 2B). Subsequently the protein is dissolved by vigorous shaking or sonication. 9 This procedure takes much more time than the first one, and is reserved mainly for relatively stable and hydrophobic proteins. When it is necessary to obtain highly concentrated solutions of protein, the dry protein is added in excess and the portion remaining undissolved after a few hours is removed by centrifugation or filtration. As was shown by Martinek and co-workers ~2the solubility of a-chymotrypsin in 0. l M AOT micelles reached a maximum of - 1 mM which is probably more than the micellar concentration in this medium. Hence all reversed micelles will contain at least one protein. 3. The enzyme is solubilized in the water pools of the micellar medium by a phase transfer from a bulk aqueous solution to the reversed ~2A. V. Levashov, Yu. Khmelnitski, N. L. Klyachko, and K. Martinek, in "Surfactants in Solution" (K. L. Mittal and B. Lindman, eds.), Vol. 2, p. 1069 (1984).

[20]

ENZYMATIC REACTIONS IN REVERSED M1CELLES

221

micellar solution (Fig. 2C). This technique may be useful for studying the kinetics and mechanism of solubilization. 6 At present it is not possible to give general guidelines for the solubilization of enzymes in reversed micelles. Neither is it possible to predict whether an enzyme will be active in such media. Too many unpredictable factors such as the local pH around the enzyme and electrostatic and hydrophobic interaction between the enzyme and its microenvironment determine the solubility and the activity of a given protein. Likewise, the temperature is a factor that should be considered. For example the solubility of liver alcohol dehydrogenase in AOT-isooctane reversed micelles increases substantially when the temperature is raised from 22 to 300. 6 Furthermore, one should be aware of the fact that some proteins containing a noncovalently bound cofactor can loose this cofactor upon solubilization. For example, flavodoxin from Megasphaera elsdenii loses its FMN upon solubilization in CTAB-hexanol-octane reversed micelles, but not in an AOT-isooctane medium (A. J. W. G. Visser, unpublished results). To date, the solubilization of proteins in reversed micellar media is in the "trial and error" stage. Yet, the general consensus seems to be that for every enzyme it is possible to design a reversed micellar medium in which the enzyme is active.

Procedure for 20fl-Hydroxysteroid Dehydrogenase Encapsulation. Prior to use 20fl-hydroxysteroid dehydrogenase from Boehringer-Mannheim is diluted to a concentration of - 2 mg protein/ml and dialyzed against 7 mM HEPES (pH 7.6) to remove ammonium sulfate. According to technique 1 the enzyme is then added by microsyringe to the standard CTAB reversed micellar medium.

Determination of Enzyme Activity in Reversed Micelles

Principle. Since reversed micellar solutions are optically transparent, enzymatic reactions can be followed using conventional spectroscopic techniques. Alternatively, enzyme activities can be followed under these conditions with a common 02 electrode, standardized by adding known amounts of Oa to the micellar system, in the case of oxidases, monooxygenases, and dioxygenases (Laane, unpublished results). Another powerful technique is HPLC, in particular for apolar compounds such as steroids. 1-3 Procedures for 20fl-Hydroxysteroid Dehydrogenase. 20fl-Hydroxysteroid dehydrogenase requires both NADH and a 20-ketosteroid for catalysis. Hence, its activity can be followed spectroscopically by measur-

222


[20]

ing the rate of NADH consumption at 340 nm or by following the rate of steroid conversion by HPLC. Procedure 1. A dialyzed stock solution of 20fl-hydroxysteroid dehydrogenase is prepared as described above. Freshly prepared 50 mM solution of steroid in chloroform, 30/zl, is pipetted into a Bausch and Lomb 0.5-in. test tube. After evaporation of the chloroform 1.5 ml of a H E P E S hexanol-CTAB-octane micellar solution is added together with 5 ttl 5 mM N A D H in 50 mM HEPES (pH 7.6). While mixing the reaction is started by the addition of 5/zl stock solution of 20fl-hydroxysteroid dehydrogenase. Subsequently the reaction is monitored at 340 vs 380 nm using any kind of spectrophotometer. Procedure 2. At time intervals 25-/xl aliquots from a complete reaction mixture (see procedure 1) is added to 100/zl acetonitrile in a 1-ml Eppendorf centrifuge tube. This step is required to stop the reaction and to precipitate CTAB specifically, because CTAB interferes with the detection procedure. After freezing for about 20 min at - 2 0 ° the samples are centrifugated for 2 min at 12,000 g in an Eppendorf centrifuge. Under these circumstances most steroids remain in solution and can be analyzed by HPLC equipped with a Phase Sep Spherisorb 10/~ ODS column. The steroids can be detected in most cases between 200 and 280 nm and can be eluted from the column using a mixture of acetonitrile and water. For progesterone this ratio is 80 : 20 (v/v) and for prednisone 25 : 75 (v/v). For the determination of initial enzyme activity procedure I is preferred, since it is the most rapid and the simplest. In Fig. 1 the area is shown in which initial 20fl-hydroxysteroid dehydrogenase activities could be measured. Procedure 2 is preferred when long-term experiments are performed (see Fig. 6). Determination of the Intrinsic Rate Parameters of Enzymes in Reversed Micellar Media

Principle. The methods employed to determine the intrinsic rate parameters kcat and Km in aqueous media are also applicable in reversed micellar media. The only difference with an aqueous system is that in a reversed micellar medium one can relate the Km of a water-soluble substrate either to the overall (K °v) or to the water pool concentration (KWp). This in contrast to the Km for apolar compounds, which of course is always related to the overall concentration. In most studies so far the observation is that the K wp is much larger than in water, whereas the K °v is about the same as in water. 6,j3 We disagree with these authors that the 13 S. Barbaric and P. L. Luisi, J. A m . Chem. Soc. 103, 4239 (1981).

[20]

E N Z Y M A T I C REACTIONS IN REVERSED M1CELLES

223

close resemblance between the K~nv and the K m in water is accidental. In our opinion the only physically meaningful expression for Km in reversed micellar media is K~v . Theory. When an enzymatic reaction is considered that obeys Michaelis-Menten kinetics the following reactions take place: kl E + S .

k2 " ES

, E + P

k-i

(1)

where E is the enzyme, S the substrate, and P the product, kl, k_ ~, and kz are rate constants. It is clear that the velocity (v) of such a reaction does not depend on the way the concentrations are expressed. Hence Vmax

Vmax

v = 1 + (K°V/S °v) = 1 + (KWp/S wp)

(2)

When 0 is the volume fraction of the water phase, the water pool and overall concentrations are related as follows S °v = OS wp

(3)

As a result Eqs. (4) and (5) can be written K ° v / o s wp = K w p / S wp

K °v = OK wp

(4) (5)

Since Km is defined as K m = (k-1 + kz)/kl

(6)

and k_~ and k2 are first-order rate constants, k~ v=

(1/O)k~ p

(7)

the correction factor (1/0) indicates that the substrate is dispersed over the whole volume of the solution and in fact reflects the frequency factor of collision and exchange between micelles. Hence K °v is relevant and not Kwp.

Deviations of K°mv from Km values found in aqueous media may originate either from microenvironmental effects on the enzyme itself or from interface-substrate interactions. Km a n d keat V a l u e s f o r 20fl-Hydroxysteroid D e h y d r o g e n a s e . For catalysis 20fl-hydroxysteroid dehydrogenase requires the water-soluble substrate NADH and in case of a reversed micellar system an apolar steroid, for example progesterone. The K °v values for NADH and for progester-

224


[20]

TABLE I MICHAELIS-MENTEN PARAMETERSFOR 20/~-HYDROXYSTEROID DEHYDROGENASE IN AQUEOUS AND REVERSED MICELEAR MEDIAa K~nv (~M) Medium Aqueous Reversed micellar

NaC1 (mM)

NADH

Progesterone

kcat (sec -~)

0 500 0 500

3.6 3.4 11.0 3.7

10 n.d. 400 n.d.

10.9 6.2 14.4 7.6

a Experiments were performed in 50 mM Bistris, pH 6.3, or in reversed micellar solutions of 0.2 M CTAB in octane, 10% hexanol, w0 = 10, containing 50 mM Bistris, pH 6.3, in the water pool. Similar solutions were prepared containing 500 mM NaCI. For the determination of K °v for NADH the progesterone concentration was kept constant at 5 mM and for the determination of K °v for progesterone the overall NADH concentration was fixed at 50 ~M. n.d., Not determined. (Taken from ref. 3.)

one as well as the kcat values are listed in Table I both for an aqueous and for a CTAB reversed micellar medium. Compared to an aqueous solution, kcat increases in CTAB reversed micelles. This increase is not caused by a shift in pH of the water pool, since in aqueous solutions of pH 7.0 and 6.3 similar values for kcat a r e obtained. Upon enclosure in reversed micelles, K °v for NADH increases. Considering that NADH is negatively charged and CTAB positively, it can be envisaged that NADH is associated with the interface. Hence, the effective concentration available to 20/3-hydroxysteroid dehydrogenase could be lower, resulting in an increase in K°mv. In a reversed micellar medium the differences in K °v for NADH and kcat decrease at high ionic strength, indicating that the changes in k~atand K °v for NADH are caused mainly by electrostatic effects imposed by the charge of the head groups of the surfactant. In contrast to the K °v for NADH, that for progesterone increases markedly upon enclosure in reversed micelles, which could be due to a relatively low concentration of progesterone in the microenvironment of the enzyme compared to the bulk concentration. In conclusion it can be said that the enzyme experiences an essentially aqueous environment in reversed micelles, since kcat and K °v for NADH hardly change upon enclosure in the presence of salt, whereas the change in K°mv for progesterone is not caused by an effect on the enzyme itself, but by changes in the microenvironment.

[20]

ENZYMATIC REACTIONS IN REVERSED MICELLES

225

Determination of the Parameters That Regulate the Enzymatic Conversion of Apolar Substrates in Reversed MiceUes

Principle. When an enzyme is encapsulated in a reversed micelle and the substrate is present in the oil phase, the substrate has to cross barriers such as the interphase and, if present, the aqueous layer surrounding the enzyme before catalysis takes place. It can therefore be expected that for apolar substrates the enzyme activity will be influenced by (1) the thickness of the water layer around the protein, and (2) the concentration of substrate in the interphase. Consequently, a reversed micellar medium should be designed in such a way that the water content is as low as possible and that the solubility of the substrate in the interphase is high with respect to the continuous phase. The water layer around the enzyme can easily be varied by adjusting w0. The radius of the water pool increases when w0 increases. TM As a measure for the solubility of a substrate in the interphase the hydrophobicity value of the interphase, log P i , c a n be used. 2,3 Since the interphase contains only cosurfactant and CTAB the semiempirical formula log Pi -

ao

a0+ 1

log Pcosurfactant +

1

log PCTAB

(8)

can be defined, where a0 is the molar ratio of the cosurfactant to CTAB in the interphase. Similarly, a log P for the continuous phase, log Pcph, can be defined according to Eq. (9), log Pcph = Co log ecosurfactant -1- (1 - c0) log Porganic solvent

(9)

where Co is the mole fraction of the cosurfactant in the continuous phase and thus equal to E q . (10). In this formula Ccph, p, M °, and My are the amount of cosurfactant in the continuous phase (/zl/ml), the density (kg/liter), and the molecular weight of the organic solvent (o) and the cosurfactant (c), respectively. CcphpC/MCr CO = CcphpC/MCr "k (1000 -- Ccph)p°/M °

(10)

By determining the log P values of the different components of the system, Ccphand a0 (see below), log Pcph and log Pi c a n be calculated for any given composition of the reversed micellar medium. Hence a quantitative measure for the hydrophobicity of the different phases in a reversed mi14 R. A. Day, B. H. Robinson, J. H. R. Clarke, and J. V. Doherty, J. Chem. Soc., Faraday Trans. 1 75, 132 (1979).

226

IMMOBILIZED

ENZYMES/CELLS

IN ORGANIC

SYNTHESIS

[20]

S-

F 'T

E % E

>, >,

ru -I-

C3 tf~

0

01 02 03 04 }logPi- [ogPs I

\\0%

FIG. 3. The influence of ling P~ - log Psi and Ilog Pc~h -- log Psi on the 20/3-hydroxysteroid dehydrogenase activity with prednisone as substrate. For explanation, see text; for details, see Hilhorst e t al. 2.3

cellar medium can be obtained, which can be compared to the hydrophobicity of the substrate (log Ps) and related to the enzyme activity. Since small differences between log P values indicate good mutual solubilities, high substrate concentrations in the interphase and thus high enzyme activities are expected when Ilog P i - log Psi is minimal and when [log Pcph - log Psi is as large as possible. With these rules for any given apolar compound a medium can be composed that assures optimal enzymatic conversion. Figure 3 shows the results obtained with 20fl-hydroxysteroid dehydrogenase in H E P E S - C T A B - h e x a n o l - o c t a n e reversed micelles with prednisone as substrate. The results clearly show the effects of Ilog P~ - log Psi and ]log Pcph -- log Psi on the enzyme activity and that these effects are additive and thus amplify each other. Determination of log P. Log P is defined as the logarithm of the partition coefficient in an octanol water two-phase system. J5 For simple molecules log P values are known or can be calculated from hydrophobic fragmental constants according to Rekker. 15 For more complicated structures such as steroids log P can be determined easily as follows. One micromole of apolar compound is shaken overnight in sealed vials containing 1 ml octanol and 1 ml of an aqueous solution. The composition of the aqueous solution and the temperature should be the same as those used in the enzymatic assay. The concentration of the apolar com~5 R. F. Rekker, " T h e Hydrophobic Fragmental Constant." Elsevier, Amsterdam, 1977.

[20]

ENZYMATIC REACTIONS IN REVERSED MICELLES I

I

I

227

I

300

3 -5

200

x

~oo-~vl I

I

I

I

1

2

3

4

V ocfane (m[)

Fi6. 4. Typical example of a titration curve for the HEPES-hexanol-CTAB-octane medium. For explanation see text. Ccphis the amount of hexanol in the continuous phase (in ~l/ml); V~ is the amount of hexanol (in p.1) in the interphase, and V denotes total volume.

pound in both phases can be determined afterward using the appropriate detection technique. Determination ofccph and ao. The mole fraction of cosurfactant in the interphase (a0) and its amount in the continuous phase (Ccph) are determined by phase boundary titrations according to Bowcott and Schulman. 16 For the HEPES-hexanol-CTAB-octane system the procedure is as follows. A 2 ml solution containing 0,2 M CTAB in octane and 50 mM HEPES (pH 7.6) at the desired water content is prepared in a Bausch and Lomb 0.5-in. test tube. This solution is titrated using a microsyringe with hexanol to clarity under vigorous shaking. Upon dilution with octane the solution becomes turbid again because of extraction of the hexanol from the interphase into the continuous phase. This process of dilution and titration is repeated 5 times in the same tube. Each titration is carried out in duplicate or triplicate at the temperature used in the enzyme assay. A plot of the minimal amount of hexanol required for the formation of a clear solution vs the total volume of octane added should yield a straight line (Fig. 4). The slope represents the amount of hexanol in the continuous phase in microliters hexanol per milliliter continuous phase (ccph), whereas the intercept on the Yaxis (V~) represents the amount of hexanol in microliters present in both the interphase and the water pool. Since 16 j. E. Bowcott and J. H. Schulman, Z. Elektrochem. 59, 283 (1955).

228

IMMOBILIZED ENZYMES/CELLS IN ORGANIC SYNTHESIS (c0)surfattant~.~

2~'' '~÷

N'~'+

2 - ~ / f - MV ~

i

~H2ase

,

.

.

.

'+

[20]

.

~/~

APOLAR

AUM t - I ~ K E T O S T E R O I O

.~L,pOH

",..,2My++2H+J ~ N A D

..........

j% HSOH~

+'

J~APOLAR

A~

~ofl-HYDROXYSTER0ll]

FIG. 5. Scheme for the Hz-driven regeneration of NADH and the subsequent reduction of an apolar steroid in a reversed micellar medium. For clarity all the water-soluble components of the system are drawn in one micelle. A more realistic view is that all components are distributed among all micelles that are in rapid exchange with each other. H2ase, Hydrogenase; LipDH, dihydrolipoamide dehydrogenase; HSDH, 20/3-hydroxysteroid dehydrogenase; and MV, methyl viologen. From Hilhorst et al. ]

hexanol is very poorly soluble in water, the latter amount is negligible. V~ times the density of the cosurfactant and divided by its molecular weight yields the amount of hexanol in the interphase (ci) in micromoles. Since all surfactant molecules are localized in the interphase, a0 can be calculated according to Eq. (11), ao

= ciMS/S

(11)

where S is the amount of surfactant in the assay in milligrams and M s the molecular weight of the surfactant. Multienzymatic Reactions in Reversed Micellar Media Principle. An important property of reversed micelles is that they are in rapid exchange with each other. Hence, enzymes distributed among these microdroplets can communicate with each other either directly or through low molecular weight mediators. Figure 5 shows an example of a carrier-mediated multienzymatic system in a reversed micellar medium. ~ Cytochrome-c3 hydrogenase (EC 1.12.2.1) and dihydrolipoamide dehydrogenase (EC 1.8.1.4) are used for the in situ regeneration of NADH which is consumed by 20fl-hydroxysteroid dehydrogenase during the site- and stereospecific reduction of an apolar ketosteroid to its corresponding 20fl-hydroxyform. Procedure. An aqueous solution (70 tzl) is injected into 1.5 ml of a Vortex-stirred 0.2 M solution of CTAB in hexanol-octane 1 : 4 (v/v) containing 1.0 mM steroid unless stated otherwise. Stirring is continued until the solution becomes clear. Any assay vial can be used, provided it contains a gastight rubber septum that allows the withdrawal of aliquots from the reaction mixture.

[20]

ENZYMATIC REACTIONS IN REVERSED MICELLES I

i

229

i

~E E

o

i

co i

~i lC

x o

o

=~ 0 ~.

0

~l

I

6

I

9

Time (hrs)

FIG. 6. Time course of 20fl-hydroxypregn-4-en-3-one formation at different concentrations of progesterone in a HEPES-hexanol-CTAB-octane reversed micellar medium. See text for procedure. Progesterone concentrations: 0.2 mM (IlL 1.0 mM (A), and 5.0 mM (N). From Hilhorst et al. 1

The aqueous solution contains pig heart dihydrolipoamide dehydrogenase (2.5 /.tM), 20fl-hydroxysteroid dehydrogenase (3.0/zM), methyl viologen (2.5 mM), and NAD ÷ (1.0 mM) in 50 mM HEPES (pH 7.6). After six cycles of 30 sec evacuation, 15 sec flushing with O2-free argon, and 5 min of bubbling with O2-free H2, 10/xl hydrogenase isolated from Desulfovibrio vulgaris 17 (final concentration of 0.6/xM in the water pool) is added to initiate the reaction. During the reaction the incubation is shaken mildly at 25 ° . Figure 6 shows the time course of progesterone reduction. The results clearly demonstrate that the reaction rate declines when the conversion is almost complete. Furthermore, at higher concentrations the reaction rate increases and the reaction proceeds steadily for >10 hr, indicating that the multienzyme system is stable for a considerable length of time. Acknowledgments We wish to thank Mrs. J. C. Toppenberg-Fang for typing the manuscript, Mr. M. M. Bouwmans for drawing the figures, and the Pharmaceutical Industry Duphar BV, Weesp, and the Netherlands Organization for the Advancement of Pure Research (Z.W.O.) for financial support. 17 H. M. Van der Westen, S. G. Mayhew, and C. Veeger, F E B S L e t t . 86, 122 (1978).

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[21] D i s a c c h a r i d e S y n t h e s i s w i t h Immobilized fl-Galactosidase B y P E R - O L O F L A R S S O N , L A R S H E D B Y S , S I G F R I D S V E N S S O N , and

KLAUS MOSBACH Many carbohydrates are known to interfere in cellular recognition mechanisms in a very efficient way. j This fact forms the basis for the design of new drugs that would, for example, prevent the adhesion of bacteria on mucosal surfaces. The carbohydrate drug could be envisaged to block the bacterial receptors, making them unable to recognize or bind to the host cell, a process known to be a crucial initial step of the pathogenesis. The interest in such biologically active carbohydrates has prompted the development of new methods for carbohydrate synthesis. Normal organic chemical methods for di- and oligosaccharide synthesis are usually laborious and time consuming, due to a number of protection and deprotection steps. The use of enzymes for carbohydrate synthesis is conceptually very attractive, as the procedure often is a single-step reaction. However, several isomers may be formed. When only a few isomers are formed, as is the case here, the workup is also straightforward, making the overall procedure very convenient. Under the proper conditions, the enzyme fl-galactosidase will catalyze a transgalactosylation reaction (Fig. 1). 2 The fl-galactoside, lactose, combines initially with the enzyme to form a galactosylated enzyme with concomitant release of glucose. The galactosylated enzyme may experience two fates. Either it is hydrolyzed with water to give free enzyme and galactose, or it may transfer its galactosyl moiety to a second sugar, in this case N-acetylgalactosamine. This transgalactosylation reaction leads to the formation of galactosyl-N-acetylgalactosamine. Under normal aqueous conditions the final equilibrium is shifted far toward hydrolysis. However, the transfer reaction is comparatively fast, and during an intermediate phase a substantial yield from the transfer reaction is obtained. A third route is also evident from the scheme, namely the reversal of the hydrolysis reaction. For any substantial synthesis to occur, the water activity must be kept low. The procedures given below describe the synthesis of the disaccharide galactosyl-N-acetylgalactosamine [fl-D-Gaip-(I-6)-D-GalNAc] in good 1 N. Sharon and H. Lis, Chem. Eng. News March 30, p. 21 (1981). 2 K. Wallenfells and R. Well, in " T h e E n z y m e s " (P. Boyer, ed.), Vol. 7, 3rd Ed., p. 617. A c a d e m i c Press, N e w York, 1972.


Copyright © 1987by AcademicPress, Inc. All rights of reproduction in any form reserved.

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DISACCHARIDESYNTHESIS

231

Galactosyl transfer

"GIc E + GaI-GIc

+ GalNAc E-Gal

ll

+H20

GaI-GalNAc + E

Hydrolysis

E+Gal

FIG. 1. Simplified scheme of proposed mechanism of/3-galactosidase, showing the transfer and the hydrolysis reactions. E,/3-Galactosidase; Gal-Glc, lactose; GalNAc, N-acetylgalactosamine; Gal-GalNAc, galactosyl-N-acetylgalactosamine, Reproduced from Hedbys et al. 4 with permission.

yield from lactose and N-acetylgalactosamine and in low yield from galactose and N-acetylgalactosamine via reversal of the hydrolysis reaction. The enzyme was used in the immobilized form to facilitate its reuse and its easy removal from the reaction mixture when the yield was at its maximum. In addition, and of relevance for future studies, immobilization may lead to more stable enzyme preparations of importance when carrying out enzymatic synthesis at elevated temperatures and in organic solvents and/or solvents with low water activity. Methods

Immobilization of fl-Galactosidase to Sepharose CL-4B The immobilization is carried out by the tresyl chloride m e t h o d ) : Sepharose CL-4B (Pharmacia Fine Chemicals, Uppsala, Sweden), 5 g of moist gel, is washed with water and then transferred stepwise to organic solvent by washing with a series of solvents: 30 : 70, 50 : 50, and 70 : 30 acetone:water, followed by acetone, and finally acetone dried with a molecular sieve) The gel is filtered off, carefully avoiding any moist air to be sucked through the gel bed, and then transferred to a 10-ml beaker containing 1.13 ml dry acetone and 95/xl pyridine. Tresyl chloride, 6 125 3 K. Nilsson and K. Mosbach, this series, Vol. 104, p. 56. 4 L. Hedbys, P.-O. Larsson, K. Mosbach, and S. Svensson, Bi oc he m . Biophys. Res. Commun. 123, 8 (1984). 5 One hundred grams of Linde molecular sieve type 4 A is added to 1 liter of acetone. Drying time is 24 hr. 6 Tresyl chloride (2,2,2-trimethylsulfonyl chloride) was obtained from Fluka, Buchs, Switzerland. Tresyl chloride-activated Sepharose gel is available from Pharmacia Fine Chemicals, Uppsala, Sweden.

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/zl, is added under vigorous magnetic stirring to rapidly disperse the activating agent. The mixture is then stirred at a slow rate for 15 min at room temperature. The activated gel is washed with acetone and then transferred to an aqueous solvent by washing with 70 : 30, 50 : 50, and 30 : 70 acetone : 5 mM HC1 in water, followed by 5 mM HCI. The activated gel is stored at 4° until used. The activated gel, about 5 g wet weight, is washed with 0.20 M sodium phosphate buffer, pH 8.0, and mixed with 6 mg of Escherichia coli [3galactosidase 7 in 6 ml of the same buffer. The suspension is shaken gently for 2 hr at room temperature. The [3-galactosidase-carrying gel is washed with the above buffer, followed by 0.2 M Tris-HCl, pH 8.0, in which it is kept for 30 min to block any remaining tresyl groups. The gel is then washed with 0.5 M NaC1, water, and finally 0.10 M sodium phosphate buffer, pH 7.0, containing 2 mM MgCIz. The activity of the gel is routinely determined from its ability to hydrolyze o-nitrophenyi-[3-o-galactoside. The gel typically has an activity of 48 U/g of gel, which corresponded to about 50% of added activity.

Enzyme Assays [3-Galactosidase activity is determined from initial rate measurements of o-nitrophenyl-[3-D-galactoside hydrolysis at 24°. The assay mixture contains 1.0 mM o-nitrophenyl-[3-D-galactoside, 2 mM MgC12, and 0.10 M sodium phosphate buffer, pH 7.0. The hydrolysis reaction is followed at 420 nm, where the product has a molar absorption coefficient of 2050 Mcm -~ (pH 7). 8 When immobilized enzyme is assayed, a spectrophotometric cell equipped with a magnetic stirring device is used to keep the particles suspended. Normally 2-5 mg of gel is used per assay (2 ml assay volume).

Preparation of Galactosyl-N-acetylgalactosamine [[3-D-Galp-(1-6)-D-GalNAc] via Transglycosylation Immobilized enzyme, 1.4 g (70 U), is suspended in 17 ml 0.10 M potassium phosphate buffer, pH 6.5, containing 0.22 M N-acetylgalactosamine and 0.45 M lactose. The mixture is shaken gently at room temperature, and samples taken intermittently for HPLC analysis to determine the correct time for breaking the reaction, i.e., when the yield was at its 7 Grade VI was obtained from Sigma Chemical Co., St. Louis, MO. The preparation used had a specific activity of 80 U/mg with the assay conditions given under "Enzyme Assays." 8 The molar absorption coefficient is very pH sensitive around pH 7 (pK, for o-nitrophenol = 7.2). To avoid this problem, the assay may be run at pH 8.

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DISACCHARIDESYNTHESIS

233

maximum. Samples are diluted with 9 volumes acetonitrile : water 3 : 1, and injected on a LiChrosorb-NH2 column (Merck; 5-/~m particles, 25 cm length). The mobile phase consists of acetonitrile : water 3 : I. The effluent is monitored at 235 nm, at which wavelength acetamido sugars are detected. The k' value of N-acetylgalactosamine was determined to be 1.6 and the k' value for the product galactosyl-N-acetylgalactosamine was 3.6. After 30 hr incubation, the reaction is stopped by spinning down the gel. As an extra measure of precaution, the supernatant is boiled for 3 min to denature any traces of enzyme, and freeze-dried. The yield, according to HPLC measurements, is at this stage about 25%. The product /3-DGalp-(1-6)-D-GalNAc is isolated in pure form by using a combination of chromatographic techniques. 4

Preparation of Galactosyl-N-acetylgalactosamine [/3-D-Galp-(1-6)-D-GalNAc] via Reversal of the Hydrolysis Reaction Immobilized enzyme, 5 U, is suspended in 0.50 ml of 0.10 M potassium phosphate buffer, pH 6.5, containing 0.31 M galactose and 0.31 M 2acetamido-2-deoxy-D-galactose. The mixture is gently shaken at room temperature for 24 hr before the reaction is stopped and the mixture treated as above. Comme~s The described transferase-type synthesis gave a very good yield of galactosyl-N-acetylgalactosamine [fl-D-Galp-(1-6)-D-GalNAc]. Analysis of the reaction mixture showed that small amounts of the disaccharides/3D-Galp-(1-6)-D-Gal and/3-D-Galp-(1-6)-D-GIc also had been formed. Remarkably, only (1-6)-linked GalGalNAc could be f o u n d . 4 Preliminary results with enzymes from other sources indicate that other linkages are possible to obtain. The product galactosyl-N-acetyl-galactosamine had not been described earlier and was therefore identified/characterized by sugar and methylation analyses using gas chromatography-mass spectrometry as analytical tools. Proton NMR data and the observed optical r o t a t i o n (o~D = 26°) were also in agreement with the proposed structure? The reversed hydrolysis reaction, on the other hand, resulted in only low yield under the conditions given here. To make the latter reaction suitable for preparative applications, the conditions must be altered substantially. However, recent preliminary studies with several glycosidases, including/3-galactosidase, suggest that satisfactory yields (10-50%) are possible, provided that a combination of measures are taken such as very high substrate concentration and high temperature (to increase substrate solubility and decrease water activity).

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[22] S y n t h e s i s o f U r e a w i t h U r e a s e in W a t e r - O r g a n i c Solvent Mixtures B y LARRY G . B U T L E R

The synthesis of urea in animals involves several enzymes and is driven by hydrolysis of 3 mol of ATP per mole of urea synthesized. 1 Urea can be readily synthesized nonenzymatically from the inorganic salt ammonium cyanate; this original synthesis by W6hler in 1828 was a landmark in the history of chemistry. 2 It has now become possible to demonstrate the synthesis of urea from an inorganic salt in a reaction catalyzed by a single enzyme, independent of hydrolysis of ATP or any other high energy compound. The inorganic salt is ammonium carbonate; the enzyme is urease, which catalyzes urea synthesis by reversal of urea hydrolysis2 Reversal of hydrolytic reactions was originally considered as a biosynthetic route; the first claim of an enzyme-catalyzed synthesis reaction was by reversal of the hydrolytic reaction catalyzed by maltase (a-glucosidase). 4 Because water is not only the solvent but is a reactant at high concentration, the equilibrium for hydrolytic reactions lies far toward hydrolysis when carried out in aqueous media. Methods for overcoming this thermodynamic constraint have been developed so that reversal of peptide bond hydrolysis has become a useful method for semisynthesis of proteins 5 and is being extended to large-scale synthesis of specialized materials such as aspartame. 6 Hydrolysis of urea is equivalent to hydrolysis of two peptide bonds, so the equilibrium lies even farther toward hydrolysis than for comparable reactions such as peptide bond hydrolysis. 7 Nevertheless, synthesis of urea from ammonium carbonate can readily be detected and quantitated. Replacement of part of the solvent water by water-miscible organic solvents dramatically increases the yield of urea. 3 This increase was presumed to be due to the corresponding decrease in water activity, shifting the equilibrium toward urea synthesis, 3 but an effect of the organic solJ H. 2 F. 3 L. 4 C. 5 R. 6 K. 7 A.

A. Krebs and K. Henseleit, Hoppe-Seyler's Z. Physiol. Chem. 210, 33 (1932). W#hler, Poggendorff's Annalen 12, 253 (1828). G. Butler and F. J. Reithel, Arch. Biochem. Biophys. 178, 43 (1977). Hill, J. Chem. Soc. 73, 634 (1898). E. Offord, "Semisynthetic Proteins." Wiley, New York, 1980. Oyama and K. Kihara, CHEMTECH 14, 100 (1984). Kapune and V. Kasche, Biochem. Biophys. Res. Commun, 80, 955 (1978).


Copyright © 1987 by Academic Press, Inc. AI! rights of reproduction in any form reserved.

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UREASE-CATALYZED UREASVNTHEStS

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vent on ionization a may also be involved. Several problems associated with synthesis by reversal of enzymatic hydrolysis reactions are illustrated by investigation of the effects of reaction conditions on the rate and extent of urea synthesis as catalyzed by urease. Source of Enzyme. Jack bean urease extracted and purified by the standard acetone crystallization procedure 9 with minor modifications ~° was used in demonstrating urease-catalyzed urea synthesis) Utilization of urease from other sources should be possible, although conditions for good reaction rates would have to be established for the particular enzyme used. Conditions which determine the position of equilibrium of the urea hydrolysis reaction (see below) are of course independent of the source of the enzyme. Assays

Urea Synthesis. In order to obtain readily measurable amounts of urea it is necessary to use relatively high concentrations of substrate. Typical conditions are 0.75 M (NH4)zCO3, 5 mM EDTA, and 5 mM 2-mercaptoethanol (no additional buffer is necessary). The urease concentration should be about I Sumner Unit 11 per milliliter. Under these conditions Jack bean urease catalyzes urea synthesis at about 2% of the rate at which it hydrolyzes urea (conditions for urea hydrolysis specified below). Urea concentration is determined spectrophotometrically by a modification ~2of the Fearon reaction.13 Incubated samples, zero time blanks, or urea standards containing up to 1/zmol urea in a total volume of 0.2-1.0 ml in screw-capped tubes are quenched by addition of 5 ml of 52% H3PO4 (600 ml of 85% H3PO4 diluted with water to 1 liter). Samples can be kept at room temperature at this stage for up to an hour without significant loss of urea, so that several samples can be run together through the remainder of the procedure. To each quenched sample is added 1.0 ml of an aqueous solution containing 6 g of 2,3-butanedione monoxime and 0.3 g thiosemicarbazide (both from Aldrich Chem. Co.) per liter. After heating for 30 min on a boiling water bath, samples are cooled to room temperature and their absorbance at 530 nm is determined immediately. The assay is not affected by high concentrations of ( N H 4 ) 2 C O 3 . At smaller sample volumes (0.2-0.5 ml) the apparent extinction coefficient for urea (approxi8 G. A. Homandberg, J. A. Mattis, and M. Laskowski, Jr., Biochemistry 17, 5220 (1978). 9 G. Mamiya and G. Gorin, Biochim. Biophys. Acta 105, 382 (1965). ~0C. M. Kaneshiro, Ph.D. Dissertation, University of Oregon, Eugene (1975). N j. B. Sumner, this series, Vol. 2, p. 378. ~2 M. K. Schwarz, this series, Vol. 17, p. 857. 13 W. R. Fearon, Biochem. J. 33, 902 (1939).

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mately 2 × l06 M -~ c m - 0 is relatively insensitive to the presence of organic solvents in the sample. For samples in which the equilibrium concentration of urea is being determined, it is advisable, after measuring the urea concentration, to add fresh urease and incubate again until the urea concentration is constant. Urea Hydrolysis. For comparison to urea synthesis, urea hydrolysis can be measured by an adaptation of the same assay using 2 mM urea as substrate, in the presence of 0.2 mM EDTA, 5 mM 2-mercaptoethanol, and 20 mM imidazole acetate, pH 7.0. This urea concentration is below the Km for urea 14 so the rate decreases continually throughout the incubation. Data points at four different reaction times are plotted semilogarithmically; the first-order rate constant is determined from the slope. Effect of Organic Solvents on Enzyme Stability The use of organic solvents to enhance the yield of urea is possible only if urease activity can be maintained in the presence of the solvents. Fortunately, Jack bean urease is known to be rather stable in organic solvents such as glycerol.~5 Moreover, it is extracted and crystallized in 32% acetone. 9 In connection with these experiments it was observed that urease in 50% aqueous dimethylformamide, ethanol, or pyridine irreversibly lost most of its activity in a period of a few hours at room temperature. However, activity was recovered in high yield from urease dissolved in 50% aqueous solutions of acetone or polyhydroxylic alcohols such as glycerol and 1,2-propanediol. In an attempt to stabilize Jack bean urease against denaturation and loss of catalytic activity by high concentrations of organic solvents, the enzyme was immobilized and cross-linked by two independent methods. In neither case, however, did immobilized cross-linked urease have improved stability in the presence of organic solvents) Subsequent work was done only with soluble urease. Effect of Organic Solvents on Reaction Rate Organic solvents affect urea hydrolysis in much the same way that they affect urea synthesis .3 The effect of organic solvents on reaction rate can thus be conveniently determined by measuring urea hydrolysis, which requires less enzyme than urea synthesis. The effects are strongly dependent on the nature of the organic solvent, as well as its concentra~4 K. R. L y n n , Biochim. Biophys. Acta 146, 205 (1967). ~5 C. C. Contaxis and F. J. Reithel, J. Biol. Chem. 246, 677 (1971).

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U R E A S E - C A T A L Y Z E D UREA SYNTHESIS

237

tion. The solvents were reagent grade but not redistilled; further purification of the solvents might be warranted. When compared at the same concentration (50%), the rate of urea hydrolysis ranged from undetectable (in ethanolamine) up to equal activity in acetone to that observed in strictly aqueous solution. 3 Rates in 50% aqueous glycerol, dimethylformamide, ethanol, and pyridine were 15, 49, 56, and 65% of the rate in water. In acetone and glycerol, in which urease is stable indefinitely, the activity as a function of organic solvent concentration shows rather different patterns, with most of the activity retained up to 75% acetone and much less activity observed at relatively low glycerol concentrations) Solvent selection for proposed urea synthesis processes wouldrequire optimization of the particular solvent for enzyme stability, reaction rate, and yield of urea (see later section). Effect of Organic Solvent, Substrate, and Hydrogen Ion Concentration on Yield of Urea The equilibrium concentration of urea, determined in solvents in which urease is stable so that achievement of equilibrium can be assured, increases as the water activity (related to concentration) diminishes. The yield of urea at equilibrium is approximately a second-order function of water concentration (activity) as predicted by the equilibrium constant. 3 The yield of urea as a function of (NH4)2CO3 concentration, at constant solvent composition, strongly increases as the substrate concentration increases. 3 In 50% glycerol, the observed urea yield was greater than the theoretical yield (which assumes concentration and activity are equivalent) at low substrate concentrations and lower than the theoretical yield at high substrate concentrations. The yield of urea is greatly enhanced in alkaline conditions) H + is a by-product of urea synthesis from HCO3-, according to Eq. (1). 2 NH4 + + H C O 3 - --0 u r e a + 2 H 2 0 + H +

(1)

Effect of the Nature of the Organic Solvent on Yield of Urea The equilibrium concentration of urea differs greatly depending upon which organic solvents are utilized to replace part of the water. Compared at a concentration of 50%, the equilibrium concentration of urea in aqueous acetone is over 12-fold higher than in aqueous glycerol. 3 Other organic solvents tested gave yields between these values, with hydroxylic solvents generally showing the lowest yields) As determined by measurement of the solubility of urea and ammonium carbonate, the primary effect of organic solvents is on the activity of the salt, rather than urea.

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Use of Organic Solvents to Study the Reaction Mechanism It has been reported that the products of urease-catalyzed urea hydrolysis are ammonium and carbamate ions. 16 Measuring urea synthesis rather than hydrolysis, it is possible to show that maximum initial rates are obtained only if the substrate ammonium bicarbonate has been dissolved for an hour before urease is added; freshly dissolved substrate gives much slower initial rates of urea synthesis s This difference is likely due to a requirement for synthesis from carbamate, which forms nonenzymatically at a slow rate after ammonium bicarbonate is dissolved. In order to prove this point, carbamate concentration should be measured in both fresh and aged substrate solutions, and the rate of synthesis from ammonium carbamate should be directly compared with the rate of urea synthesis from ammonium carbonate. Significance The small amounts of urea synthesized (approximately O. 1% yield) by reversal of the urease reaction are not likely to warrant consideration of this process for large-scale urea production. Urease does, however, provide an interesting model for investigation of the reversal of hydrolytic enzyme reactions, both because of the historical significance of urea synthesis, and because urease illuminates several factors which limit utilization of the technology of reversal of enzymatic hydrolysis. Perhaps most important is the realization that no single set of conditions is optimal for all the parameters affecting urea synthesis. For example, conditions which give high urea yields also give slow rates of synthesis, and conditions in which synthesis is rapid give poor yields. Enzyme efficiency and stability and substrate solubility likewise may be affected differently by organic solvents. Some of these problems may be partially overcome by using mixtures of different organic solvents.

~6R. L. Blakely, J. A. Hinds, H. E. Kunze, E. C. Webb, and B. Zerner, 1991 (1969).

Biochemistry 8,

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IMMOBILIZEDDEXTRANSUCRASE

239

[23] D e x t r a n S y n t h e s i s U s i n g I m m o b i l i z e d Leuconostoc rnesenteroides D e x t r a n s u c r a s e

By P. MONSAN, F. PAUL, D. AURIOL, and A. LOPEZ Dextransucrase [ a ( 1 ~ 6)-glucan:o-fructose-2-glucosyltransferase E.C.2.4.1.5] catalyzes the synthesis of a glucose polymer, dextran, according to the reaction: n Sucrose ~ (glucose)n + n fructose dextran

Dextran is a branched polymer in which the linear parts result from linear condensation of o-glucopyranosyl units by a ( 1 --~ 6)-osidic bonds, whereas branching points result from a(1 ~ 3), a(1 ~ 2), or a(1 ~ 4) bonds. The degree, nature, and length of branching are related to the strain of bacteria which produced the enzyme and the conditions of polymer synthesis.~ Dextransucrase from Leuconostoc mesenteroides NRRL B512 F, a nonpathogenic lactic bacterium, produces a low-branched dextran polymer containing 95% a ( 1 ~ 6) bonds and 5% o~(1~ 3) bonds, and which is highly soluble in water. This explains the exclusive use of this strain for industrial production of dextran. Produced in the same manner since 1950, dextran is mainly used as a blood plasma substitute (clinical dextran) and chromatography support. 2 Dextran is also involved in the process of dental caries: pathogenic bacteria of the genus Streptococcus produce insoluble dextran, which favors the adhesion of bacteria on the teeth and causes the formation of dental plaque. 3 Leuconostoc mesenteroides dextransucrase is an exocellular enzyme induced by its substrate, sucrose, the only inductor known at present. 4 Dextran synthesis is thus performed outside the cell, without the intervention of activated precursors or cofactors, as is the case for the synthesis of the majority of polysaccharides.5 In vitro dextran synthesis is thus A. Jeanes, W. C. Haynes, C. A. Wilham, J. C. Ranki, E. H. Melvin, M. J. Austin, J. E. Cluskey, B. E. Fisher, H. M. Tsuchiya, and C. E. Rist, J. Am. Chem. Soc. 76, 5041 (1954). 2 A. Jeanes, in "Extracellular Microbial Polysaccharides" (P. A. Sandford and A. Laskin, eds.), Symp. Ser. Vol. 45, p. 284. American Cancer Society, Washington, D.C., 1977. 3 R. L. Sidebotham, in "Advances in Carbohydrate Chemistry and Biochemistry" (R. S. Tipson and D. Horton, eds.), Vol. 30, p. 371. Academic Press, New York, 1974. 4 W. B. Neely, Arch. Biochem. Biophys. 79, 154 (1959). 5 R. C. W. Berkeley, G. W. Gooday, and D. C. EUwood, "Microbial Polysaccharides and Polysaccharases." Academic Press, New York, 1979.



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possible using a cell-free purified enzyme preparation. The energy required for the condensation of o-glucopyranosyl residues is provided by hydrolysis of the glucose-fructose osidic bond of the sucrose molecule. This energy is conserved by the formation of o-glucosyl-enzyme covalent complexes. The action mechanism of dextransucrase described by Robyt e t al. 6"7 involves two identical fixation sites of D-glucosyl residues, from which the latter are transferred to the reducing end of the growing dextran chain. This chain remains covalently bonded to the enzyme. During polymer synthesis, the enzyme forms an enzymatically active covalent complex with both glucose and dextran. Dextran release occurs when the fructose produced during the reaction is sufficiently concentrated to act as acceptor of the dextran chain. As fructose is a weak acceptor, the molecular weight of the polymer is extremely high, greater than 106.3 Fructose may also act as acceptor for D-glucosyl residues, resulting in the formation of leucrose, o~-o-glucopyranosido-(1 ~ 5)-fructopyranose. 8 However, other molecules, not involved in the reaction, may also act as acceptors for D-glucosyl and dextranosyl residues. Maltose is a particularly efficient acceptor causing the release of small oligodextrans containing a maltose residue at the reducing end. 9,1° These oligodextrans are themselves excellent acceptors.11,12 Control of dextran molecular weight during synthesis with the resulting direct production of polymers of sizes required for pharmaceutical and analytical applications may thus be envisaged. Along with a deeper understanding of the transfer mechanism of oglucopyranosyl residues and the possibilities of molecular weight control of dextran, dextransucrase production and purification have also been improved, in particular using fed-batch culture and ultrafiltration.13.14 We have recently developed an original purification process involving liquidliquid extraction of dextransucrase directly from culture broth. 15The enzymatic preparation thus obtained has a very high specific activity, 6 j. F. Robyt, B. K. Kimble, and T. F. Walseth, Arch. Biochem. Biophys. 165, 634 (1974). 7 j. F. Robyt and H. Taniguchi, Arch. Biochem. Biophys. 174, 129 (1976). F. H. Stodda, E. S. Sharpe, and H. J. Koepsell, J. Am. Chem. Soc. 78, 2514 (1956). 9 H. M. Tsuchiya, N. N. Hellman, H. J. Koepsell, J. Corman, C. S. Stringer, S. P. Rogovin, M. O. Bogard, G. Bryant, V. H. Feger, C. A. Hoffman, F. R. Senti, and R. W. Jackson, J. Am. Chem. Soc. 77, 2412 (1954). ~0j. F. Robyt and T. F. Walseth, Carbohydr. Res. 61, 433 (1978). n F. Paul, E. Oriol, D. Auriol, R. M. Willemot, and P. Monsan, Eur. Congr. Biotechnol. 3, (1984). 12 j. F. Robyt and S. H. Eklund, Carbohydr. Res. 121, 279 (1983). 13 p. Monsan and A. Lopez, Biotechnol. Bioeng. 23, 2027 (1981). 14 j. F. Robyt and T. F. Walseth, Carbohydr. Res. 68, 95 (1979). 15 F. Paul, P. Monsan, and D. Auriol, French Patent 8,307,650 (1983).

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IMMOBILIZEDDEXTRANSUCRASE

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greater than 170 U/mg protein and is free from contaminating saccharolytic activity. ~5,~6 Little research has been made into immobilization of L. mesenteroides dextransucrase. Robyt et al. 6,7 immobilized dextransucrase on BioGel P2 activated by glutaraldehyde in order to show the synthesis mechanism of dextran, and, in particular, the synthesis of a(1 ---* 3) branching points on the dextran linear chain. Recently, Reilly's group immobilized the enzyme by covalent bonding on various supports. ~7 But the most active derivatives were obtained by dextransucrase adsorption on phenoxyacetyl cellulose powder. 18 In our case, dextransucrase was immobilized on porous silica activated by glutaraldehyde in order to compare the kinetic behavior of the enzyme in free and immobilized form, and the nature of the dextran thus obtained. 13,19,2° Assay Methods Dextransucrase A s s a y . Enzyme activity is determined by measuring the initial production rate of reducing sugars in a solution containing 2 ml 3.3 M sucrose solution (Merck, Germany) in water, 0.24 ml 1 M sodium acetate buffer (pH 5.2) containing 2.5 g/liter calcium chloride, a volume of enzymatic solution with an activity between 2 and 20 U, and a volume of distilled water to obtain a final volume of 12 ml. Reaction takes place at a controlled temperature of 30° with stirring by magnetic rod. The amount of reducing sugars is determined using the reagent 3,5-dinitrosalicylic acid, 2~ with aliquots taken at 5-rain intervals over a period of 20 min, diluted if necessary to maintain reducing sugar concentration between 0.2 and 2 g/liter. Evolution of optical density as a function of time allows the initial production rate of reducing sugars to be determined. Dextransucrase activity (U) is defined as the amount of enzyme releasing 1/~mol of reducing sugar per minute. Protein Determination. Protein concentration is determined by the method of Lowry et al. 22 using bovine serum albumin as reference. 16 F. Paul, D. Auriol, E. Oriol, and P. Monsan, Enzyme Eng. 7, in press (1986). 17 n . Kaboli and P. J. Reilly, Biotechnol. Bioeng. 22, 1055 (1980). ~8 H. N. Chang, Y. S. Ghim, Y. R. Cho, D. A. Landis, and P. J. Reilly, Biotechnol. Bioeng. 23, 2647 (1981). ~9A. Lopez and P. Monsan, Biochimie 62, 323 (1980). 2o p. Monsan and A. Lopez, in "Advances in Biotechnology" (M. Moo-Young, C. W. Robinson, and C. Vezina, eds.), Vol. 1, p. 679. Pergamon, New York, 1981. 2~ j. B. Sumner and S. F. Howell, J. Biol. Chem. 108, 51 (1935). 52 O. H. Lowry, N. H. Rosebrough, A. L. Farr, and R. H. Randall, J. Biol. Chem. 193, 265 (1951).

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[23]

Dextran Determination. Dextran concentration is determined by the anthrone method using glucose as reference. 23

Production of Dextransucrase by Fed-Batch Culture of Leuconostoc mesenteroides Dextransucrase is produced by cultivation of the strain L. mesenteroides N R R L B512 F in a nutritive medium at 27° containing 2,4 yeast extract (Biokar, France), 20 g/liter; dipotassium hydrogenophosphate, 20 g/liter; sucrose, 20 g/liter; sodium chloride, 0.01 g/liter; magnesium sulfate (MgSO4"7H20), 0.2 g/liter; manganese sulfate (MnSO4. H20), calcium chloride (CaCI2" 2H20), and iron sulfate (FeSO4" 7H20), 0.01 g/liter. The pH of the medium is adjusted to 6.9 by pure orthophosphoric acid addition before sterilization. After the addition of a volume of preculture corresponding to 10% of total culture volume, cell growth is measured by following optical density at 650 nm of the culture medium diluted 10 times with distilled water. At the beginning of the exponential growth phase, a concentrated sucrose solution (800 g/liter) is added at a rate of 20 g/hrliter of culture medium. Sucrose must remain present in the medium to fulfill its role as growth carbon source, inducer, and substrate for dextransucrase, pH is maintained at 6.7, corresponding to optimum enzyme production. 24Cell growth and dextransucrase production are shown in Fig. 1. The addition of sucrose allows both the cell growth phase and dextransucrase production phase to be prolonged. Under these conditions, final culture activity was 8.8 U/ml, corresponding to a culture productivity of 1.2 U/ml-hr. 19 On completion of the growth phase, the culture medium is recuperated and centrifuged (20 rain, 4°, 10,000 g). The supernatant is immediately adjusted to pH 5.2 by the addition of pure orthophosphoric acid, thus constituting crude enzymatic extract. Purification of Dextransucrase Ultrafiltration and Gel Permeation. Crude enzymatic extract, 250 ml, is subjected to ultrafiltration in a hollow fiber cartridge (HIP 100-20, Amicon) with a nominal molecular weight cutoff of 100,000. The solution is diafiltered with an amount of 20 mM acetate buffer (pH 5.2) containing 0.05 g calcium chloride/liter and corresponding to 10 times the volume of the solution to be purified. The experiment is carried out at 4 °. Because of 23 T. A, Scott and E. H. Helvin, Anal. Chem. 25, 1956 (1953). 24 H. M. Tsuchiya, H. J. Koepsell, G. Bryant, M. O. Bogard, V. H. Feger, and R. W. Jackson, J. Bacteriol. 64, 521 (1952).

[23]

IMMOBILIZEDDEXTRANSUCRASE I

I

I

243 I

s

2

2

a

6

g

TIME (hr)

FIG. 1. Production of dextransucrase during fed-batch culture of L. mesenteroides NRRL B512(F). Absorbance (El); dextransucrase activity (A).

its very high apparent molecular weight the dextransucrase remains inside the fibers (Table I). This retentate is directly subjected to gel permeation: 40 ml of enzymatic preparation is placed on a 4 x 100 cm column containing 1 liter of Ultrogel AcA 34 (IBF, France), of separation range between 20,000 and 350,000, and eluted with a 20 mM sodium acetate buffer (pH 5.2) containing 0.05 g calcium chloride/liter. Chromatography is performed at 4°. Figure 2 shows the separation obtained19: it may be seen that dextransucrase elutes at the void volume and clearly separates from the greater part of the proteins present in the extract. Specific activity for a typical experiment as described in Table I attained 103 U/mg protein after the two successive steps of ultrafiltration and gel permeation. Final yield was 51%. Phase Partition. We have developed an original process for the purification of dextransucrase which is based on the fact that the latter is of dextranosyl-enzyme form and which takes into account the incompatibility of dextran with polyethers, e.g., polyethylene glycol (PEG). 15 The addition of a PEG aqueous solution to a dextran aqueous solution, when both are at suitable concentrations in the medium, leads to the appearance of two phases, each containing between 80 and 95% water. The top phase is rich in PEG and the bottom phase is rich in dextran. This phenomenon

244

[23]


TABLE I DEXTRANSUCRASE PURIFICATION FROM L. mesenteroides BY ULTRAFILTRATION AND GEL CHROMATOGRAPHY

Property Initial volume (ml) Final volume (ml) Protein concentration (mg/ml) Dextransucrase activity (U/ml) Dextransucrase total yield (%) Specific activity (U/mg protein)

Diafiltration'~

Gel permeation chromatography b

--5.12

250 291 0.075

40 100 0.013

7.63

4.25

1.34

Crude extract

100 1.5

65 56.7

51 103

a Experimental conditions: crude enzymatic extract was diafiltered with 10 times its volume of 20 mM sodium acetate buffer (pH 5.2) containing 0.05 g calcium chloride/ liter, using a HIP 100-20 hollow fiber cartridge (Amicon) with a nominal molecular weight cutoff of 100,000. b Experimental conditions: diafiltered enzyme solution was chromatographied on a 4 x 100 cm column containing 1 liter of Ultrogel AcA 34 (IBF, France) and eluted with a 20 mM sodium acetate buffer (pH 5.2) containing 0.05 g calcium chloride/liter at 4°.

is called phase partition. 25 Such two-phase systems are used for the partition of media containing cells or organelles, 25 cell debris, and enzymes. 26 In our case, the presence of dextran in the solution to be purified only requires the addition of PEG. The covalent association of enzyme and dextran, in particular, results in the presence of dextransucrase in the dextran-rich bottom phase. A 50% (w/v) PEG solution (Merck, molecular weight 1500) in water is slowly added to the solution to be purified (150 ml) in a glass beaker cooled in a 4 ° water bath and continually stirred. Phase partition occurs when the solution, at first translucid, becomes turbid. The system thus obtained is centrifuged at 8000 g for l0 min at 4 °. After centrifugation, the top phase is discarded. The volume of the bottom phase is then adjusted to the initial volume of the solution to be purified (150 ml) using 20 mM sodium acetate buffer (pH 5.2) containing 0.05 g calcium chloride/liter. This enzymatic preparation may be sub25 p. A. Albertsson, "Partition of Cell Particles and Macromolecules." Wiley, New York, 1971. 26 M. R. Kula, in "Applied Biochemistry and Bioengineering" (L. B. Wingard, E. Katchalski-Katzir, and L. Goldstein, eds.), Vol. 2, p. 71. Academic Press, New York, 1979.

[23]

IMMOBILIZEDDEXTRANSUCRASE I

I

I

I

245 I

E r-

--

4

v

C) o0

>-

c~

t

-3

LU !

\

(I) cr

~ t.d (/3

_2~

0 u') Cn

X ttt

20 40 60 B0 FRACTION NUMBER (1.5mr/tube)

100

FIG. 2. Gel filtration of dextransucrase on an Ultrogel AcA 34 column. Experimental conditions: column, 4 x 100 cm; eluent, 20 mM sodium acetate buffer (pH 5.2) containing 0.05 g calcium chloride/liter, 4 °. Absorbance (---); dextransucrase activity (A).

jected to further phase partition as described above. Thus, in a typical experiment, five successive phase partition steps were carried out on crude extract, resulting in a final specific activity of 171 U/mg protein and 1.35 U/mg dextran with a final yield of 95%. The amount of protein represented 0.8% of the total amount of the purified preparation. Comparison of the two methods described shows that repeated phase partition leads to a degree of purity much greater than that obtained by ultrafiltration and chromatography. Phase partition is quick and easy and allows the rapid concentration of crude enzymatic extract which has a positive effect on enzyme stability. Dextransucrase Immobilization Immobilization Procedure Porous Silica. A porous silica (Spherosil, Rh6ne-Poulenc, France) available as beads of various diameters and porosities is used. The support is aminated by y-aminopropyltrimethoxysilane coupling in boiling

246


[23]

T A B L E II CHARACTERISTICS OF SPHEROSIL a POROUS SILICA SUPPORTS Specific area (m2/g)

Pore size (nm)

6 37 120 180 445

550 80 25 15 7

a The diameter o f Spherosil (Rh6ne-Poulenc, France) beads ranges from 100 to 200 ~ m .

xylene for 24 hr. The diameter of the Spherosil particles ranges from 100 to 200/xm and the specific area from 6 to 445 m2/g. The characteristics of these supports are given in Table II. Glutaraldehyde Activation. A 100 mg amino-silica sample is activated with 20 ml of 2% glutaraldehyde solution in 0,05 M pyrophosphate buffer, pH 8.6. The glutaraldehyde solution is prepared from a commercial 25% glutaraldehyde solution in water (Merck) and its pH is adjusted before use. The samples are agitated by roller-mixing (Denley Mixer A 257) for 3 hr at 20 °. The supernatant is sucked up after the silica beads settled. Excess glutaraldehyde is washed off by repeated contact of the support with 20 ml of the above pyrophosphate buffer over a period of 1 hr at 20°. The activated support is washed until the supernatant no longer shows any reaction with Schiff's reagent for aldehyde detection. The support is then washed three times with 20 ml of the buffer solution in which immobilization is then to take place (if not pyrophosphate buffer). This method constitutes the optimization of the parameters influencing support activation and enzyme immobilization on porous silica as previously described.27, 28 Enzyme Immobilization. Dextransucrase immobilization is performed in 10 mM acetate buffer (pH 5.2). Dextransucrase solution, 15 ml, with a specific activity of 65.3 U/mg protein is brought into contact with 100 mg activated silica for 12 hr at 4 ° in 20-ml tubes and roller-mixed as during activation. After removal of the supernatant, the support is first thoroughly washed with 10 mM sodium acetate buffer (pH 5.2), then washed with 1 M sodium chloride solution to desorb the noncovalently grafted enzyme fraction (2 hr at 4°), and finally washed with acetate buffer. A blank is carried out by bringing a nonactivated support into contact with :7 p. M o n s a n , J. Mol. Catal. 3, 371 (1977-1978). 28 p. M o n s a n , Eur. J. Appl. Microbiol. Biotechnol. 5, 1 (1978).

[23]

IMMOBILIZEDDEXTRANSUCRASE l >.k-.-

t

247 I

30

I.(_.)

-X--H

(1) (2)

Two enzymes capable of catalyzing reaction (2) are 2-enoate reductase (EC 1.3.1.31) from a Clostridium tyrobutyricum strain (Clostriclium La 1) and 2-oxocarboxylate reductase (so far no EC number has been given) from Proteus vulgaris or Proteus mirabilis. The latter enzyme is now called (2R)-hydroxycarboxylate-viologen oxidoreductase since the later

C. H. Wong and G. M. Whitesides, J. Am. Chem. Soc. 105, 5012 (1983) and literature cited therein. 2 G. M. Whitesides and C. H. Wong, Angew. Chem. 97, 617 (1985). 3 H. Simon, J. Bader, H. Giinther, S. Neumann, and J. Thanos, Angew. Chem. 97, 541 (1985) [Angew. Chem. Int. Ed. Engl. 24, 539 (1985)]. 4 H. L. Schmidt and H. Giinther, Phil. Trans. R. Sac. Lond. (1987), Discussion Meeting of Biosensors, London, May 1986, in press and literature cited therein. 5 H. Simon, J. Bader, H. Giinther, S. Neumann, and I. Thanos, Ann. N. Y. Acad. Sci. 434, 171 (1984), in "Enzyme Engineering 7" (A. I. Laskin, G. T. Tsao, L. B. Wingard, Jr., eds.).

304


[28]

described reaction (7) is reversible if carbamoylmethylviologen is used. 6 Both enzymes show an astonishingly broad substrate specificity, 3,5,7-13 high stereoselectivity, 3,5,J4 and satisfactory kinetic parameters, 3,~4 and they do not 3,5,7-9require reduced pyridine nucleotides as cosubstrates. An important group of enzymes which can be used for electroenzymatic NAD(P)H regeneration are certain methyl viologen-dependent NAD(P) ÷ reductases3&7-9: 2 MV +. + NAD(P) + + H + --* 2 MV 2÷ + NAD(P)H

(3)

Many organisms produce enzymes that catalyze this reaction, and the enzymes show specific activities of the order of 0.01-40 units/mg protein. 8 Some of the enzymes have been studied, while others have not been characterized in detail s o f a r . 3,8,9 On the basis of reaction (3) and the fact that many pyridine nucleotidedependent reductases are present in cells, electromicrobial or electroenzymatic reductions of the following type are possible: H+

(4) H+

The electrons delivered from the cathode of an electrochemical cell reduce the mediator, i.e., methyl viologen which in turn is enzymatically oxidized by NAD ÷ or NADP + with the aid of enzyme El. The reduced pyridine nucleotides can be used as cosubstrates of reductase E2 for stereospecific reduction of unsaturated substrates. Often E1 and E2 are present in the same cell species. Another possibility is the combination of E1 and E2 from two different cell sources, for example the combination of 6 H. Skopan, H. GOnther, and H. Simon, Angew. Chem., Int. Ed. Engl. 26, 128 (1987). 7 M. Biihler and H. Simon, Hoppe-Seyler's Z. Physiol. Chem. 363, 609 (1982). s H. Simon, H. Giinther, and J. Thanos, in "Enzymes as Catalysts in Organic Synthesis" (M. P. Schneider, ed.), Vol. 35. Reidel Publishing, Dordrecht, 1986. 9 j. Bader, H. Gtinther, S. Nagata, H. J. Schuetz, M. L. Link, and H. Simon, J. Biotechnol. 1, 95 (1984). ~0 H. G0nther, C. Frank, H. J. Schuetz, J. Bader, and H. Simon, Angew. Chem. Int. Ed. Engl. 229 322 (1983). u W. Tischer, W. Tiemeyer, and H. Simon, Biochimie 62, 331 (1980). ~2H. Sedimaier, W. Tischer, P. Rauschenbach, and H. Simon, FEBS Lett. 100, 129 (1979). 13 S. Neumann and H. Simon, FEBS Lett. 167, 29 (1984). ~ H. Gfinther, S. Neumann, and H. Simon, J. Biotechnol, 7, 53 (1987).

[28]

PREPARATIVE ELECTROENZYMATIC REDUCTIONS

305

two different cell species or their crude extracts or the two purified enzymes from different sources. Reactions (4) and (2) can be performed by using cells made partially permeable, for instance by freezing and thawing, or by using pure or partially purified e n z y m e s . 3'5'8-1°'14 If methyl viologen is used in combination with whole cells of CIostridium La 1 or P. vulgar& hydrogen may be formed in a side reaction according to reaction (5): 2 MV +. + 2 H +

hydrogenase~ 2 MV 2+ + H2

(5)

In such cases benzyl viologen substituting for methyl viologen as a mediator can be used instead. However, benzyl viologen is less stable if experiments are carried out for long periods of time. The different possibilities are discussed in the examples given below.

Substrate Specificity and Some Kinetic Data of Enoate Reductase Enoate reductase from Clostridium La 1 (DSM 1460) catalyzes the following reactionS,8: 3R

2My +.+2Ia++

'-R ~

X

~ 2R/-~tR

,

H

+2~v~*

(6)

H

X = COOR I = H, CH3, C2H5, F, CI, Br, NHCHO, OCH3 R 2 = many alkyl groups, COOCH3, substituted phenyl or pyridine tings, but also furyl and thioenyl groups R 3 = residues not too bulky and rigid

The rates of different substrates have been published. 3,5,7,8In the reduced form the enzyme is especially oxygen sensitive. The specific activity of purified enoate reductase determined for the reduction of (E)-2-methyl-2butenoate is about 20 units/mg protein (1 unit protein converts 1/zmol of enoate per minute). This value is about 1.5-fold the reduction rate observed using N A D H as the electron donor. Therefore enoate reductase can also be used in combination with an NADH regenerating system such as formate dehydrogenase from Candida boidinii.11 The following rules have emerged regarding the enzyme's substrate specificity: ~R should not be too bulky; NHCOCH3 and OC2H5 cannot replace N H C H O or OCH3 if 2R is a substituted phenyl ring. The halogens F, C1, or Br are tolerated only in the a position; in contrast, halogens in the/3 position are eliminated. 12The least restrictions apply to functionalities in position 2R. Branching in the 13 position leads to diminished activi-

306


[28]

ties of the enzyme (2-4 units/mg). If 2R and 3R are interchanged, i.e., if E and Z isomers jaa of substrates 3R2RC=CHX are used, the products found are the corresponding enantiomers. This was shown for E- and Z-gerania t e ) The former led to the R and the latter to the S enantiomer of citronellate, respectively• Therefore only pure E and Z isomers should be used if an enantiomeric product containing a chiral center in the/3 position is desired. In substrates such a s / C = C = C R X (i.e., allene carboxylates) ~. or jC---~C--C---~CRX only the a,/3-double bond will be reduced. It can be assumed from reductions of substrates similar to 1 R H C = C R X that the E and the Z isomers will yield identical enantiomeric products• So far, all the products analyzed show an enantiomeric excess greater than 96%. 3,5 If the reductions are carried out in buffered 2H20, stereospecifically deuterated compounds can be obtained. 3,5,9 Using MV.+, enoate reductase shows a pH maximum at 5.2. 5 At pH 4.5 and 6.2 the rate is two-thirds that at pH 5.2. A pH value less than 5.2 should not be used and pH values up to 8.0 do not harm the enzyme• A pH value of 6.0-6.2 is recommended as a compromise considering the reaction rate and the stability of both reduced methyl viologen and enoate reductase. In addition, the reverse direction of reaction (6) has not been observed so far. The kinetic data of enoate reductase are favorable as can be seen in Table I. Substrate Specificity and Some Kinetic Data of (2R)-Hydroxycarboxylate-Viologen Oxidoreductase This oxidoreductase from P. mirabilis (DSM 30115) or P. vulgaris (DSM 30118) catalyzes the following reaction3,5,8,J3,4: O )~ R CO0

OH H +2H++2BV'*°r2MV'*'---+

>~C +2BV2÷or2MV2+ R OO-

(7)

R = CH3, CH2F, CTHls, C6HsCH2, C6H5, (CH3)2CHCH2, C2HsCH(CH3), HOCH2C(CH3)2, -OOC(CH2)~ (n = 1, 2, 3), -OIPI(Me)CH2CH2, 3-indolyl, 5-benzyloxy-3-indolyl II

O

The rates of reduction of different substrates have been published. 5,8,13,14 Except glyoxylate, all tested derivatives of 2-oxocarboxylates were found to be good substrates. The specific activity of purified (2R)-hydroxy_carboxylate-viologen oxidoreductase is about 1000 units/mg protein, using phenylpyruvate as the substrate (100%). Beside 2-oxomonocarboxylates, J4~According to the rules of Cahn, Ingold, and Prelog the notation is Z if the substituents of higher priority are on the same side of the C = C double bond; otherwise, it is E.

[28]

307

PREPARATIVE ELECTROENZYMATIC REDUCTIONS TABLE I KINETIC PARAMETERS DETERMINED FOR REACTIONS CATALYZED BY ENOATE REDUCTASE Substrate

Km (raM) °

Inhibitor

Ki (mM) °

Reduced methyl viologen NADH b Cinnamate (Z)-2-Bromocinnamate (E)-2-Methyl-2-butenoate (E)-Geraniate (Z)-Geraniate

0.4 0.012 0.013 0.50 1.5 0.014 0.10

Reduced methyl viologen Methyl viologen NAD ÷ Phenyl propionate Aliphatic carboxylates

10 20 0.84 30 500

Michaelis-Menten constants have been determined by analysis of initial rate studies using variable substrate concentrations and saturating concentration of NADH or reduced methyl viologen, respectively. For details of inhibitor studies, see Biihler and Simon. 7 b Instead of NADH reduced methyl viologen can be used as an electron donor.

2-oxodicarboxylates are also reduced at rates amounting to 50-75% of the rate observed for phenylpyruvate reduction. Branching in the 3-position of a substrate results in diminished reaction rates. For a quaternary carbon atom in the 3-position only about 5-7% of the reduction rate has been observed. The maximal reduction rate is observed at pH 6.75, and at pH 6.0 and 7.5 half of the maximum rate is seen. Neither NADH nor NADPH are electron donors. Typical Km and K~ values are shown in Table II. The use of this enzyme activity for the preparation of different (2S)-hydroxyTABLE II KINETIC PARAMETERS DETERMINED FOR REACTIONS CATALYZED BY 2-HYDROXYCARBOXYLATE-VIOLOGEN OXIDOREDUCTASE Substrate

Km (mM)"

Inhibitor

Reduced methyl viologen Reduced benzyl viologen Phenylpyruvate 2-Oxo-4-methylpentanoate 2-Oxo-3,3-dimethylbutanoate 2-Oxoglutarate

I

"~ 60

I I | I

I

o

i

E

I I I tI

N ¢._~ Q~

! kI

i

>,,

t |I

, 2O

!

,x

/I

4/

Soluble

enzyme

/

k \'o 5

?

9

pH FIG. 2. The effect of pH on the activity of immobilized (closed symbols) and soluble (open symbols) strictosidine synthase. The assay was conducted in the presence of 0.1 M citrate phosphate (A, A), potassium phosphate ( 0 , O), and borate (ll, F-I) buffer, respectively, under standard conditions.

In contrast, the immobilized enzyme shows still about 23% of its maximal activity at pH 4.0 and 52% activity at pH 8.0. This demonstrates that the immobilized enzyme tolerates pH shifts much better than the soluble enzyme. The influence of the secologanin concentration on the activity of the immobilized strictosidine synthase was determined by double-reciprocal plots of velocity against substrate concentration. A linear relationship was observed over a range of 1.2 to 48 mM. The apparent Km value was 2.1 mM and Vmaxwas 9.1 pkat/mg. An apparent Kmof 3.4 mM and Vmaxof 55 pkat/mg for the soluble enzyme has been found previously. 6 With tryptamine as a substrate an apparent Km value of 0.9 mM and Vmax of 18.9 pkat/mg were observed, while the soluble enzyme showed an apparent Km of 2.3 mM and Vmaxof 130 pkat/mg. The comparison of the Km values indicates an equal or even slightly increased affinity for either substrate. Vmaxvalues are obviously lower with the immobilized enzyme, and this may indicate a conformational change of the protein molecule attached to the matrix. A substrate inhibition at tryptamine concentra-

348

[31]


tions exceeding 1 m M had previously been observed with the soluble enzyme 6 and was also found with the immobilized synthase, although this inhibition was less pronounced. While 2.5 mM tryptamine produces 46% inhibition of the soluble synthase, the inhibition of the immobilized enzyme is only 17%. As compared with the soluble enzyme, the immobilized strictosidine synthase is remarkably thermostabile. As depicted in Fig. 3 the immobilized enzyme has a half-life of about 68 days at 37 °. About 6% of its activity can still be observed after more than 1 year at that temperature. The half-lives of the partially purified soluble enzyme and protein extract (0-70% ammonium sulfate precipitation) under identical conditions are only 5 hr approximately. When the immobilized enzyme is stored at 4 ° in the presence of 0.02% NAN3, the preparation retains 100% of its initial activity after a period of more than 3 years. In all cases discussed above, strictosidine was shown by chromatographic means to be the sole reaction product formed by the immobilized enzyme from secologanin and tryptamine as substrates. The stability of the immobilized enzyme is therefore increased about 325-fold. As described below, this property

,oo

"~

I0

Time ( hours )

Time ( days )

!

b

'

1000 Time ( hours )

'

2000

FIO. 3. Stability of immobilized ( 0 ) and soluble (©) strictosidine synthase. The enzyme was kept at 37° in the presence of 0.g2% sodium s i d e . An aliquot was removed from each sample at times indicated, washed free from azide, and assayed for activity under standard conditions. The half-life of immobilized strictosidine synthase was found to be about 68 days, whereas the half-life of the soluble enzyme was 5 hr.

[31]

STRICTOSIDINESYNTHASE

349

makes the enzyme an excellent tool for the preparative synthesis of the common intermediate in the biosynthesis and biomimetic synthesis of indole alkaloids, i.e., strictosidine. Preparative Synthesis of Strictosidine A water-jacketed glass column (1 x 6 cm) was filled with 2 g of Sepharose containing about 40 nkat of bound strictosidine synthase, and the gel was washed thoroughly with water. Aqueous unbuffered 15 mM solutions of tryptamine- HCI and of secologanin were prepared in separate flasks. The substrates were adjusted with acid or alkali to pH 6.5. Both solutions were pumped separately onto the column each at a speed of 3 ml/hr and a column temperature of 37°. The solutions were mixed in the column head and subsequently passed through the catalyst. This precaution was taken in order to prevent any chemical condensation of the reactants, which would yield both stereoisomers, i.e., 3a(S)-strictosidine and 3B(R)-vincoside. The column eluate was collected in fractions of 72 ml and checked chromatographically for product formation. In addition, the condensation reaction was monitored by pulsing with 14C-labeled tryptamine. Under these conditions substrates were transformed to more than 95%. The column was continuously run at 37° for a period of 12 days without any changes in the reaction rate. The freeze-dried column eluate yielded a total of 6 g of strictosidine. The chemical identity and optical purity of strictosidine formed by the immobilized enzyme were confirmed by acetylation of the product and subsequent UV, NMR, MS, IR, and CD spectroscopy. H The compound is stereochemically pure and all physical data are in absolute agreement with the authentic compound) General Discussion Strictosidine synthase is the first enzyme involved in secondary metabolism of plants that has been immobilized successfully. The method described for the synthesis of strictosidine has several distinct advantages. Since the immobilized enzyme is quite stable, one can accumulate relatively large amounts of the glucoalkaloid strictosidine by operating the column continuously; on the other hand, one can put the column in a refrigerator after one run and use it subsequently for another preparation of either labeled or unlabeled strictosidine. This procedure has been performed in this laboratory for a period of up to 3 years with the same n U. Pfitzner and M. H. Zenk, Planta Med. 46, 10 (1982).

350


[31]

column and without loss of catalytic activity of the immobilized enzyme. This column offers an additional advantage in that it can be utilized for the synthesis of the versatile biogenetic precursor molecule strictosidine labeled in particular atoms with either a radioactive or a stable isotope. Furthermore, the material labeled with isotopes can be converted to the alkaloid precursor with very high concentrations of 15N or ~3C, which make these compounds amenable to modern spectroscopic studies such as nuclear magnetic resonance. Strictosidine synthesized from tryptamine and secologanin in this way can therefore be used as a synthon for known J2 or yet unknown chemical and biological transformations to give products of academic and pharmacological interest.

12 R. T. Brown, in "lndole and Biogenetically Related Alkaloids" (J. D. Phillipson and M. H. Zenk, eds.). Academic Press, London, 1980.

[32]

OVERVIEW

353

[32] O v e r v i e w

By S. GESTRELIUS and K. MOSBACH This section will provide the reader with information on immobilized enzyme/cell systems used in industry. Some of the processes now used worldwide have already been presented in Volume 44 of Methods in Enzymology. In Table I we have listed all processes, including those presently being tested on a pilot scale, that to the best of our knowledge are in use today. Relevant references are also provided as are names of some of the companies producing the catalysts:or applying these processes. The reader is also referred to Section II on the use of enzymes/cells in organic synthesis as a number of the examples given in this section will probably be implemented on an industrial level in the near future. In addition, in Volume 137, Section III, some related examples can be found, notably on water purification using immobilized microorganisms. Worth mentioning in this general context may also be the removal of raffinose and stachyose in sugar beet processing using "immobilized" pellets of Mortierella vinacae I and the traditional vinegar process in which cells immobilize naturally to birch twigs. This "industrial" section is heterogeneous in nature. In some cases, complete detail of the methodology is not given, probably because of "proprietary" considerations. Whenever possible the aim has been to describe not only immobilization methods but also methods (and problems) of integrating immobilized enzyme/cell-catalyzed reactions into process flow schemes. Comments on the importance of sterility are found in some of the contributions. The increasingly interesting area of mammalian cell culture technology using microcarriers or other immobilized systems, including entrapment, hollow fiber systems, and ceramics, is not included in this section and only briefly treated in Section II, Volume 135. Because of its rapid development it deserves to become the subject of a special volume in this series. In spite of problems with industrial plants reluctant to reveal their methodologies (understandably though), we hope that this section will provide the reader with an overview and will act as a stimulus for other workers in the field. We would like, in this context, to refer the reader to a recent review article by R. D. Schmid on biotechnology in Japan, which appeared in Appl. Microbiol. Biotechnol. 24, 355365, 1986. This review lists immobilized enzyme/cell systems used in Japan. J. O b e r a , S. H a s h i m o t o , a n d H. S u z u k i , Sugar Technol. Rev. 4, 209 (1976/1977).



354

[32]

E N Z Y M E E N G I N E E R I N G ( E N Z Y M E TECHNOLOGY)

e¢-

z ~r

b..~

..~

~Z

.~.~

~ ~

~

oo ~

......

r~

.4 [-..

o

o

e I-

u

Q e~

.~

oooo

-~

~.~-~

,._7 e~

[32]

OVERVIEW

355

e~

e~

~z

~

~ -

. . . . . .

~o~t~¢.~

"~

~

e e

e

m

m

m

m

m

~

~

~

~z •~,o~ ~ •.

~

~ .

--

t-

~ ¢~'~

"~

~.~

~

~

~

~ ~

~

o

-~ o°

.~

~-z

o

~

r~ .~

~

.=

=

o

~

~,z ~

-

~.~ ~

~ ' ~

~

~o

~ ~

~

~,~

~ ~ ~

.~o

Q

~ ,~ ~.

,~

..~

..

.~

~r~

~

gi~. l~ ~

~

~ ~~

356

ENZYME ENGINEERING (ENZYME TECHNOLOGY)

[33]

The first paper presented in this section is one by V. Jensen and S. Rugh, which summarizes 10 years of experience with industrial-scale production and the application of immobilized glucose isomerase. A more general discussion on the industrial application of immobilized enzymes by M. J. Daniels follows. Subsequently a number of contributions are presented.

[33] I n d u s t r i a l - S c a l e P r o d u c t i o n a n d A p p l i c a t i o n o f Immobilized Glucose Isomerase By VILLY J. JENSEN a n d SUSANNE R U G H

The development of the high-fructose corn syrup process is the story of how process engineering bridged the gap between the enzyme manufacturers and the corn syrup industry in the years around 1974. Cheap soluble enzymes had been known and used by corn syrup producers for decades. However, the industry was not familiar with the application of expensive enzymes which had to be reused and protected from deactivating components in the syrup to achieve an economic process. The enzyme producers, on the other hand, were equally unfamiliar with the production of reusable enzymes for use in such large-scale manufacturing processes. The actual development of today's immobilized glucose isomerase products and application process has therefore been a complex feedback process between enzyme producers and high-fructose corn syrup (HFCS) producers. Better immobilized glucose isomerase (IGI) products allowed improvements in the isomerization process, leading to further development of more sophisticated IGI products. An outline of the different immobilization methods used for glucose isomerase products together with an evaluation of the product characteristics obtained by the various methods is given here. Also the development of the methods used in the isomerization process and the mutual influence of product and process development are described. The glucose isomerase characteristics and the kinetics of the glucose isomerization reaction are well described elsewhere 1,2 and are not dealt with in this paper. The description of products and methods is based on literature, information from the manufacturers, and the authors' experience. It is, however, not necessarily a description of the exact methods used by manufacturers of industrial products, as such information is not available from all manufacturers. W.-P. Chen, Process Biochem. June/July 30 and 36 (1980). 2 j. A. Roels and R. van Tilburg, Starch/Staerke 31, 17 (1979).



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[33]

The first paper presented in this section is one by V. Jensen and S. Rugh, which summarizes 10 years of experience with industrial-scale production and the application of immobilized glucose isomerase. A more general discussion on the industrial application of immobilized enzymes by M. J. Daniels follows. Subsequently a number of contributions are presented.

[33] I n d u s t r i a l - S c a l e P r o d u c t i o n a n d A p p l i c a t i o n o f Immobilized Glucose Isomerase By VILLY J. JENSEN a n d SUSANNE R U G H

The development of the high-fructose corn syrup process is the story of how process engineering bridged the gap between the enzyme manufacturers and the corn syrup industry in the years around 1974. Cheap soluble enzymes had been known and used by corn syrup producers for decades. However, the industry was not familiar with the application of expensive enzymes which had to be reused and protected from deactivating components in the syrup to achieve an economic process. The enzyme producers, on the other hand, were equally unfamiliar with the production of reusable enzymes for use in such large-scale manufacturing processes. The actual development of today's immobilized glucose isomerase products and application process has therefore been a complex feedback process between enzyme producers and high-fructose corn syrup (HFCS) producers. Better immobilized glucose isomerase (IGI) products allowed improvements in the isomerization process, leading to further development of more sophisticated IGI products. An outline of the different immobilization methods used for glucose isomerase products together with an evaluation of the product characteristics obtained by the various methods is given here. Also the development of the methods used in the isomerization process and the mutual influence of product and process development are described. The glucose isomerase characteristics and the kinetics of the glucose isomerization reaction are well described elsewhere 1,2 and are not dealt with in this paper. The description of products and methods is based on literature, information from the manufacturers, and the authors' experience. It is, however, not necessarily a description of the exact methods used by manufacturers of industrial products, as such information is not available from all manufacturers. W.-P. Chen, Process Biochem. June/July 30 and 36 (1980). 2 j. A. Roels and R. van Tilburg, Starch/Staerke 31, 17 (1979).



[33]

IGI INDUSTRIAL-SCALEPRODUCTION-APPLICATION

357

The immobilization methods described are selected so that the sequence of products obtained by these methods illustrates the development of industrial IGI products during the past l0 years. It is possible to reproduce most of the products in a well-equipped biochemical laboratory. In many cases, alternative glucose isomerase-producing microorganisms can replace those mentioned in the descriptions. It is the hope of the authors that many readers will actually perform the trials. Development of Products and Processes Initial P h a s e

The first patent, "On Production of Fructose from Glucose through the Action of Xylose Isomerase," was issued to Marshall 3 in 1960, but 14 years were to elapse before a really large industrial exploitation of the idea was developed. The basic research was carried out in Japan in the 1960s by Takasaki et al. 4 who also patented the first partially reusable glucose isomerase, 5 which was produced according to the following method. A S t r e p t o m y c e s strain (e.g., S t r e p t o m y c e s w e d m o r e n s i s ATCC 21230) is inoculated in 500 ml of a medium containing 3% wheat bran, 2% corn steep liquor, and 0.024% COC12, pH 7. After shaking for 24 hr at 30°, the broth is heat treated at 65 ° for 15 min, and the "immobilized" glucose isomerase is recovered by filtration as the enzyme has become fixed within the cells by the heat treatment. The total activity is sufficient to isomerize 150 ml of 50% glucose solution (70°, pH 5.0-7.5) to a conversion of 48% fructose in 24 hr. After isomerization the cells are recovered by centrifugation and reused in flesh glucose solutions. The activity drops by one-third in about 100 hr, corresponding to an activity half-life of approximately 170 hr. This includes both physical loss of enzyme activity and enzyme inactivation. The product was used in small industrial scale by Clinton Corn Processing Company, a division of Standard Brands, United States, from 1967 until about 1970. The isomerization was initially performed in a batch reactor. Other cell paste products, for instance the flocculated A r t h r o b a c t e r cells patented by the R. J. Reynolds Tobacco Company,6 were also industrially utilized in the initial phase of the development of glucose isomerase. The batch isomerization process with heat-fixed cells was carried out 3R. O. Marshall, U.S. Patent 2,950,288 (1960). 4 y. Takasaki, Y. Kosogi, and A. Kanbayashi, Ferment. Adv. Pap. Int. Ferment. Syrup., 3rd, 561 (1969). 5Agencyof Industrial Scienceand Technology,Japanese Patent Application 27,525 (1965); British Patent 1,103,394(1968). 6R. J. ReynoldsTobacco Company, U.S. Patent 3,645,848 (1972).

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at 60-70 °. The syrup concentration was 40-45% (w/w), and the normal isomerization time was about 20 hr. Because of the long residence time, the pH had to be kept at 6.5-7.0 in order to decrease by-product formation. Approximately 10 mM of Mg2+ was added as enzyme activator. Unfortunately, the low pH necessitated the presence of about 1 mM of Co 2+ in the syrup in order to stabilize the enzyme. This was undesirable from a food approval point of view, and the Co 2+ had to be removed from the syrup after isomerization. The physical appearance of this first industrial glucose isomerase was a soft cell paste. The heat fixation stabilized the cells to some extent because the proteases in the cells were destroyed, and because the solubility of the cell components was decreased. However, the loss of cells, solubilized enzyme, and solubilized cell components to the syrups was inevitable in a batch process with this product. The instability of the product and the long residence time in the isomerization process gave rise to a significant by-product formation in the syrup. These impurities had to be removed from the syrup together with the added Mgz+ and Co 2+ by tedious filtration, ion exchange, and carbon treatment. In addition, the recovery of the cells from the batch isomerization process was difficult in industrial scale.

Industrial Development The First Continuous Isomerization Process. Clinton's work with the labor-demanding and difficult batch isomerization process with heat-fixed cells was soon substituted by a continuous process. In 1970, Lloyd et al. 7 developed a continuous process with the enzyme product in a pressurized leaf filter. A slurry of the glucose isomerase cell paste was pumped into a leaf filter, and each leaf was coated with a 2.5- to 3.5-cm layer. After coating, glucose solution was pumped continuously through the filter at a flow rate which allowed a 45% conversion of glucose to fructose. The other process parameters in the leaf filter isomerization were essentially the same as in the batch process, and Co 2+ in the syrup was still necessary. The loss of active, insoluble enzyme was avoided by the leaf filter process with the heat-fixed cells, but the leakage of soluble components from the heat-fixed cell product to the syrup was still a problem. Purified Reusable Enzyme. The success of fully launching fructose syrup and the problems associated with producing it by means of the heatfixed cells soon resulted in the development of pure reusable enzyme products. In 19708 the Clinton Corn Company patented an improved prod° 7 N. E. Lloyd, L. T. Lewis, R. M. Logan, and D. N. Patel, U.S. Patent 3,694,314 (1972). 8 K. N. Thompson, R. A. Johnson, and N. E. Lloyd, U.S. Patent 3,788,945 (1974).

[33]

I G I INDUSTRIAL-SCALE PRODUCTION--APPLICATION

359

uct, using the following methodology. Streptomyces rubiginosus ATCC 21175 is grown in submerged, aerobic culture. The cells are harvested by filtration. One kilogram of filter cake is slurried in 5 liters of deionized water with 5 mM of Co 2+ and 8 g of a cationic detergent. After stirring at 60 °, pH 6.7-6.8 for approximately 4 hr the cells are disrupted, and the cell debris is removed by filtration. The glucose isomerase containing supernatant is concentrated under vacuum. The enzyme concentrate contains some inactive material which can bind to DEAE-cellulose. This inactive material would decrease the activity of the reusable enzyme complex. Therefore, the impurities are removed by adsorption to a small amount of DEAE-cellulose at conditions which will not bind the enzyme to the DEAE-cellulose. The enzyme in the purified concentrate is then adsorbed onto DEAE-cellulose by mixing further DEAE-cellulose and enzyme at proper conditions. The DEAE-cellulose is thoroughly washed with water in order to remove fine particles before using it for enzyme adsorption. An amount of 6.25 g of moist filter cake can initially isomerize 6.5 g of glucose per hour to 49.6% conversion in a small column at 45% (w/w) substrate concentration, pH 6.5, temperature 60°. The activity halflife is around 200 hr. This purified glucose isomerase replaced the heat-fixed cells in Clinton's filter press reactor. The process parameters were the same, and Co z÷ was still needed. The adsorbed, purified glucose isomerase product represented a major improvement. The enzyme loading was high, and the syrup contamination with inactive, solubilized cell material was avoided. However, the glucose isomerase product was still a very soft gel. An absorbed enzyme system is extremely susceptible to changes in process conditions, even though the enzyme in the leaf filter press can be utilized for several hundred hours. Therefore, a truly fixed enzyme was needed. Cross-Linked Glucose Isomerase for Batch Isomerization. Glutaraldehyde cross-linked glucose isomerase has been available in industrial scale since 1974, and is still widely used by HFCS manufacturers. Novo Industri A/S produces the product Sweetzyme in accordance with the method developed by Amotz et al.9: Cells of Bacillus coagulans NRRL B 5636 are cultivated in a standard fermentation medium containing xylose. A cell concentrate is produced by centrifuging the fermentation broth at 10° to give a sludge containing approximately 12% dry matter. The concentrate is left at pH 7.9, 20 ° for 3 hr with mild stirring. To 1 kg of sludge is then added 40 ml of a 50% glutaraldehyde solution. After 1 hr the mixture has gelled into a coherent mass. The gel is broken up mechanically, diluted with one volume of water, and flocculated with a cationic flocculant to give a clear water phase. The mixture is filtered, and the filter cake is 9 S. Amotz, T. K. Nielsen, and N. O. Thiesen, Belgian Patent 809,546 (1974).

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ventilated with air to remove some of the free water. The filter cake is granulated on a 1-mm screen and dried in a fluid bed with an inlet air temperature of 50 ° (drying at room temperature can be used instead of fluid bed drying). The dried product is ground and sieved to 100-350/zm particles. The activity recovery from the sludge is 50-60%. An amount of 10-15 g of the obtained product can isomerize 1 kg of glucose solids to 45% conversion in 20 hr when the substrate concentration is 40% (w/w), pH 6.5-7.0, temperature 60-65 °. The physical properties of the cross-linked, dried glucose isomerase were significantly better than the physical properties of the cell paste and DEAE-cellulose products, but the pressure drop stability was still insufficient for fixed bed operation, The cross-linked enzyme particles could be recovered from a batch process simply by allowing the particles to settle and decanting the isomerized syrup. The sedimentation was carried out in the batch reactor when the desired degree of conversion had been achieved. The isomerized syrup was then drawn off. The enzyme particles were kept covered by a protective layer of syrup, as the enzyme was susceptible to oxygen at the isomerization temperature. Fresh glucose syrup was added after drawing off the isosyrup. About 5% of the initial activity was lost per isomerization. Therefore, in order to have a constant isomerization time, the activity loss was compensated for by the addition of fresh enzyme after each isomerization. After about 40 batches, the enzyme had to be discarded due to a too high proportion of particles to liquid. The addition of Co 2+ and Mg 2+ to the syrup was still necessary. Typical process cycle durations are given in Table I. The product could also have been used in the filter press continuous process, but this process never became widespread in the syrup industry. In the years of 1974-1976, most isosyrup was produced by a batch process. The standard HFCS syrup has - 7 0 % syrup dry substance (DS), 42% fructose, 54% glucose, and 4% dextrins. The global HFCS production was 285,000 tons DS in 1974 increasing to 890,000 tons in 1976. Although the process had been significantly improved by the use of cross-linked products, it was still tedious to run a batch process, and it TABLE I CYCLE DURATION FOR BATCH ISOMERIZATION Cycle step

Duration (hr)

Isomerization Settling Settling and pump out Filling with fresh syrup

20 1 8 8

[33]


361

was evident that products for very-large-scale continuous processes had to be developed. Immobilized Glucose lsomerase for Fixed Bed Operation. Several glucose isomerase products for fixed bed operations were developed by enzyme manufacturers in the period 1974-1976. Not all of these parallel developments are well documented in the literature, but can be exemplified by the development of Sweetzyme, which is produced by Novo Industry A/S. The development process was described by Hemmingsen 1° and Carasik and Carroll.11 In the early phase of this development, a product for continuous fluid bed operation was developed by McMullen and Carasik ~2in an attempt to overcome the lack of long-range physical stability of the earliest Sweetzyme product for batch isomerization. The industrial-scale experiments, however, turned out negatively. It was impossible to achieve a stable, even flow rate distribution over the reactor cross section, and the fluid bed concept was abandoned. Sweetzyme for fixed bed operations was principally produced in accordance with the method of Sweetzyme for batch operations with a few essential modifications. Before cross-linking the cells are disrupted by pumping the cell sludge through a Manton Gaulin homogenizer with a single-stage homogenizing valve assembly. The pressure drop over the valve is 300-350 kg/cm 2. After the cross-linking and filtration process, the moist, cross-linked aggregate is extruded by means of an axial extruder with a 0.8-mm screen and dried in a fluid bed dryer. The particle fractions 400-1000/xm are used for fixed bed operations. The recovery of apparent activity is approximately 50%. The activity level and physical properties are summarized in Appendix I. Recently, an improved Sweetzyme has been developed. The immobilization method is essentially the same, but the glucose isomerase is produced by a selected strain of Streptomyces murinus. The immobilized glucose isomerase products developed and marketed by Miles (Takasweet) and Godo Shusei are principally produced in accordance with the method of Sweetzyme, i.e., the particle shaping is performed after immobilization. However, the microorganisms used are different, and there are also differences in some of the unit operations used during immobilization. The product developed and marketed by Gist Brocades (Maxazyme) is produced by a different process as patented by van Velzen 13 and de10 S. H. Hemmingsen, "Applied Biochemistry and Bioengineering," Vol. 2, p. 157. Academic Press, New York, 1979. 11 W. Carasik and J. O. Carroll, Food Technol. Oct., 85 (1983). 12 W. H. McMullen and W. Carasik, U.S. Patent 4,138,290 (1976). J3 A. G. van Velzen, U.S. Patent 3,838,007 (1972).

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scribed by van Tilburg14: Actinoplanes missouriensis (NRRL B-3342) cells containing glucose isomerase are produced by submerged fermentation under aerobic conditions. After achieving the desired activity, the pH is adjusted to 8.6, and the temperature is raised to 72° in order to kill the microorganisms. The cells are recovered by centrifugation. Gelatin (in solution?) is added to the cell sludge at a temperature slightly above 40 °. The final gelatin concentration is 8% (w/v). After deaeration in a vacuum vessel, the mixture at 40 ° is sprayed into butylacetate at 6°. By this procedure, small spherical gelatin/cell particles are formed. The spheres solidify because of the low temperature, but in order to obtain particles that are sufficiently rigid in the isomerization process, the particles are crosslinked with glutaraldehyde in a concentration of 2.5% (w/v) at 10-12 °. Excess glutaraldehyde and soluble impurities are removed by several water washings. The immobilized glucose isomerase particles are then transferred into a 25% propylene glycol/water solution (preservative), drained, packed, and stored. Also, the product developed by Nagase/Denki Kagaku Kogyo, (Sweetase) differs from the traditional cross-linked products in that the enzyme-containing cells are bound to an ion-exchange resin without glutaraldehyde cross-linkinglS: Streptomyces phaechromogenes is cultivated by aerobic fermentation at 30° in a medium containing xylan extract, corn steep liquor, and salts. The culture broth is then heat treated in order to avoid autolysis and enzyme leakage from the mycelium. The mycelia are collected by filtration. The enzyme-containing mycelia are then immobilized with a water-insoluble anion-exchange resin. The anion-exchange resin contains quaternized nitrogen in pyridine rings. After immobilization, dehydration, and granulation, the granulated enzyme is dried at 60 ° for 3 hr. The main characteristics of the most important glucose isomerase products for fixed bed operation are summarized in Appendix I of this chapter. The data are based on literature, brochures, and personal communications. A strictly scientific comparison of the products is impossible as data for different products refer to slightly different application conditions, and as "optimal conditions" for a given product depend on the factor optimized (see for instance Table III for different optimizations of Sweetzyme). Comparison of Batch and Fixed Bed Processes. Table II summarizes the main differences in the isomerization parameters between the batch process and the plug flow process at the time when the Sweetzyme product for fixed bed operation was introduced into the market. The advan14 R. van Tilburg, Ph.D. Thesis, Delft University of Technology, The Netherlands (1983). J5 Japanese Patent Application 49-19082, corresponding to U.S. Patent 3,915,797.

[33]


363

TABLE II COMPARISON BETWEEN BATCH AND PLUG FLOW PROCESSES

Parameter

Batch process (1974-1976)

Plug flow process (1976)

Syrup concentration pH Temperature Co 2+ Mg 2+ R e s i d e n c e time Half-life of e n z y m e Productivity

40-45% w / w 6.5-7.0 65 ° 3.5 × 10 -4 M 20x (Ca 2+) 20 hr 300 hr - 9 0 0 kg a

40-45% w / w 8.4 (column inlet) 65 ° -20x (Ca 2+) 0 . 5 - 4 hr 500 hr - 1 5 0 0 kg a

Calculated as kilogram of syrup DS per kilogram of enzyme. tages of the plug flow process in comparison with the batch process are obvious. The immediate productivity increase was substantial, so the enzyme cost of conversion was nearly halved even before the strict optimization of the plug flow process. Co 2+ was no longer needed for enzyme stability, and by-product formation decreased considerably because of the short residence time in the column. A detailed description of the process design for the plug flow process and an analysis of its merits are given by Poulsen and Zittan in Vol. 44 of this series.16

Process Optimization 1976-1979 was a period of increase in IGI and isosyrup production capacity. Much effort was given to the optimization of both the IGI manufacturing process and the isomerization process. Optimization of isomerization process concentrated on feed purity as well as on isomerization parameters. The purity of the glucose substrate is a critical factor for the enzyme activity and stability. A saccharified corn starch glucose syrup contains both particulate matter and soluble impurities such as amino acids, peptides, lipids, and ions. Many of these naturally occurring impurities are very damaging to the IGI. An insufficiently purified glucose syrup can reduce the IGI productivity to 50% or less of that obtained with a purified syrup. Particulate matter in the feed syrup may cause clogging of the columns and should therefore be removed by centrifugation and filtration. Soluble impurities, such as amino acids, peptides, and certain metal ions, are enzyme poisons and inhibitors, and should be removed by carbon treat~6p. B. R. Poulsen and L. E. Zittan, this series, Vol. 44, p. 809.

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ment and ion exchange. Ca 2+ is a naturally occurring component in starch syrups and a powerful glucose isomerase inhibitor, but many other metal ions also have a negative influence on the activity. The normal purification process comprises carbon treatment with powdered or granular carbon, as described by Herves and Bailley, ~7followed by an ion-exchange process and Mg 2÷ addition. The ion-exchange process consists of a strong cation exchanger and a weak anion exchanger as described by the suppliers of ion-exchange resins. The efficiency of the carbon treatment and the ion-exchange processes is checked by measuring the UV absorption and conductivity. A small amount of Mg z- is added before isomerization because it is a powerful activator, and because it can prevent the inhibitory effect of Ca 2÷ if the Mg 2÷ is in 10- to 20-fold surplus. The work with optimization of the isomerization parameters has been concentrated on adjusting the process to capacity, economy, and syrup quality. The production capacity is usually adapted by adjusting isomerization temperature and age of the enzyme. When a high syrup production capacity is needed, the isomerization temperature is increased to about 63 °, and old low-activity enzyme is replaced by new high-activity enzyme. The lowest cost of conversion is obtained by running at a low temperature, i.e., down to 56-57 °, and by utilization of the enzyme until only 10% residual activity remains. The quality of the syrup has been improved by lowering the isomerization pH as a low pH reduces the formation of color and by-products. Today the isomerization process is run according to the "bottleneck" model, and Table III gives a comparison between Sweetzyme used in the original and the present plug flow processes. The process optimizations have allowed a productivity increase from about 1500 kg of syrup dry substance (DS) per kilogram of enzyme up to about 5000 kg DS/kg of enzyme. The IGI products supplied by the various manufacturers have different activities per kilogram of IGI product, but the IGI prices are adjusted so that the cost of conversion follows the market price for isomerization. The cost of conversion is roughly 10-20 cents per 100 lb of isosyrup dry substance. The global isosyrup production in 1977 was about 1.2 million tons dry substance and has been steadily increased to about 6.7 million tons in 1984. Second Generation Isomerization Processes and Products

55% Fructose Corn Syrup In 1978 a new development took place in the corn syrup market when 55% high-fructose corn syrup became available. This product has a sweeti7 D. V. Herves and C. Bailley, Starch 12, 422 (1977).

[33]

IGI INDUSTRIAL-SCALE PRODUCTION-APPLICATION

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Pilot plant operation has been carried out since April 1982. Figure 6 shows the course of operation beginning in September 1982. In this case, 8.5-9.0% (v/v) alcohol was constantly produced from diluted cane molasses for over 4000 hr (~6 months). The productivity of alcohol is calculated as ~20 g/liter total volume-hr (33 g/liter gel-hr). This means that 600 liters pure alcohol is produced each day using a 1-kl column reactor. Also, this operation has been controlled by a computerized system, with automatic analysis of ethanol and residual available sugar in the fermented broth. Figure 7 shows another run of pilot plant operation. In this case, an alcohol-tolerant strain was employed, and more than 10% (v/v) ethanol was steadily produced for more than 2000 hr. Furthermore, a semicommercial plant was constructed at the Hofu Plant of Kyowa Hakko in April 1983. The plant consists of two 10-kl reactors in series (Fig. 8); operation was started in May 1983. Even in this large-scale plant, more than 8% (v/v) ethanol has been steadily produced for more than 1000 hr, as shown in Fig. 9. Conclusions Compared with the conventional batchwise process, the present process can be evaluated as follows. The productivity of alcohol is more than 20 times higher than with conventional batchwise fermentation. This process does not need any special equipment for the preparation of immobilized yeast cells. This means a significant decrease in the initial investment cost. This process can be operated continuously for more than 6 months without medium-sterilization and seed-fermentation steps. This

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[36]

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[37]

INTERESTERIFICATION OF TRIGLYCERIDE

405

translates into a significant savings of energy and labor costs. This process can be operated automatically when a computer control system is applied. Also, using minimal instrumentation, fixed-rate control of the fermentation is available. Higher alcohol concentration (more than 10% v/v) can be attained if a suitable strain is used. In this case, still more than 10 times higher productivity, compared with conventional batchwise fermentation, can be obtained. Conversion yield is 90-95% versus the theoretical maximum yield. In total, this process should economize the alcohol fermentation process to a great extent. At present, a combined process using this immobilized yeast cell process and a vacuum fermentation technique is under investigation at the pilot plant in Hofu Plant as a project of RAPAD. Accordingly, we expect to obtain much higher alcohol fermentation efficiency through higher productivity of alcohol production and lesser amounts of waste liquor. Acknowledgment This work has been done as a part of the Biomass Utilization Project of the Research Association for Petroleum Alternatives in Japan.

[37] R e g i o s p e c i f i c I n t e r e s t e r i f i c a t i o n o f T r i g l y c e r i d e w i t h Celite-Adsorbed Lipase By SHIGERU YAMANAKA and TAKASHI TANAKA

Chemical interesterification has been used to modify the physical properties of triglyceride mixtures in the oils and fats industry. In the process, a chemical catalyst is used to promote acyl migration among glyceride molecules. The resulting products consist of glyceride mixtures in which the fatty acid moieties are randomly distributed among the glyceride molecules. It is also well known that during hydrolysis or synthesis of triglycerides lipases (EC 3.1.1.3) show specifities toward the carbon position of the glycerol molecule and the acyl residue with the use of such lipases products not obtainable by chemical interesterification methods may be produced. Interesterification with pancreatic lipase in an aqueous system has been reported for obtaining palmitate-enriched glyceride from glyceryl-l-palmitate 2,3-dioleate and palmitic acid. ~ In this reaction sysI R. W. Stevenson, F. E. Luddy, and H. L. Rothbart, J. Am. Oil Chem. Soc. 56, 659 (1979),

METHODS IN ENZYMOLOGY,VOL. 136

Copyright © 1987by Academic Press, Inc. All rightsof reproductionin any form reserved.

[37]


405

translates into a significant savings of energy and labor costs. This process can be operated automatically when a computer control system is applied. Also, using minimal instrumentation, fixed-rate control of the fermentation is available. Higher alcohol concentration (more than 10% v/v) can be attained if a suitable strain is used. In this case, still more than 10 times higher productivity, compared with conventional batchwise fermentation, can be obtained. Conversion yield is 90-95% versus the theoretical maximum yield. In total, this process should economize the alcohol fermentation process to a great extent. At present, a combined process using this immobilized yeast cell process and a vacuum fermentation technique is under investigation at the pilot plant in Hofu Plant as a project of RAPAD. Accordingly, we expect to obtain much higher alcohol fermentation efficiency through higher productivity of alcohol production and lesser amounts of waste liquor. Acknowledgment This work has been done as a part of the Biomass Utilization Project of the Research Association for Petroleum Alternatives in Japan.

[37] R e g i o s p e c i f i c I n t e r e s t e r i f i c a t i o n o f T r i g l y c e r i d e w i t h Celite-Adsorbed Lipase By SHIGERU YAMANAKA and TAKASHI TANAKA

Chemical interesterification has been used to modify the physical properties of triglyceride mixtures in the oils and fats industry. In the process, a chemical catalyst is used to promote acyl migration among glyceride molecules. The resulting products consist of glyceride mixtures in which the fatty acid moieties are randomly distributed among the glyceride molecules. It is also well known that during hydrolysis or synthesis of triglycerides lipases (EC 3.1.1.3) show specifities toward the carbon position of the glycerol molecule and the acyl residue with the use of such lipases products not obtainable by chemical interesterification methods may be produced. Interesterification with pancreatic lipase in an aqueous system has been reported for obtaining palmitate-enriched glyceride from glyceryl-l-palmitate 2,3-dioleate and palmitic acid. ~ In this reaction sysI R. W. Stevenson, F. E. Luddy, and H. L. Rothbart, J. Am. Oil Chem. Soc. 56, 659 (1979),

METHODS IN ENZYMOLOGY,VOL. 136

Copyright © 1987by Academic Press, Inc. All rightsof reproductionin any form reserved.

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[37]

tem the yield of triglyceride is low, probably due to the large amount of buffer solution used. Recently, interesterification with carrier-adsorbed lipase was successfully attempted in organic solvent systems for the production of cacao butterlike fat from fats and oils containing triglycerides with oleic acid residues at the 2-position, fatty acids, aliphatic alcohol esters of fatty acids, or other fats and oils. 2~ In such a reaction system in the presence of a small amount of a trihydric alcohol such as glycerol (more than 0.1% of the raw material), water, or a buffer solution (less than 2 % of the total weight of the reaction mixture), lipase catalyzes the interesterification reaction when a carrier is used as a disperser of the enzyme. This paper describes a regiospecific interesterification reaction method for triglyceride in n-hexane using Celite-adsorbed lipase in the presence of a small amount of glycerol or buffer solution. A simplified reaction scheme is shown below (see also Table I). H2C--O--CO--Ra

H2C--O--CO--Rx

I

H C - - O - - C O - - R b + 2 R~--COOH --~ H C - - O - - C O - - R b + RaCOOH + RcCOOH

H2C--O--CO--Rc

H2C--O--CO--Rx

We will also describe the isolation method for reformed triglyceride from the reaction mixture, and analytical methods for total fatty acid composition and fatty acid in the 2-position of the triglycerides. (Analysis of fatty acid in the 2-position of triglyceride discloses the position of individual fatty acids within a triglyceride.) Experiment A: Preparation and Application of Lipase Adsorbed to Celite Coated with Glycerol

Reagents Olive oil (commercial product of Yoshida Pharmaceuticals Ltd., Japan) T. Tanaka, E. Ono, M. Ishihara, S. Yamanaka, and K. Takinami, Agric. Biol. Chem. 45, 2387 (1981). 3 T. Tanaka, M. Ishihara, and E. Ono, Hakko to Kogyo 41, 375 (1983). 4 T. Tanaka, E. Ono, and K. Takinami, British Patent 2,042,579 (1982). 5 M. H. Coleman and A. R. Macrae, British Patent 1,577,933 (1980). 6 T. Matsuo, N. Sawamura, Y. Hashimoto, and W. Hoshida, British Patent Application 2,035,359A (1980).

[37]

INTERESTERIFICAT1ON OF TRIGLYCERIDE

407

Stearic acid Glycerol [water content in glycerol was less than 1.5% (Karl Fischer titration)] Lipase (Seikagaku-kogyo Co., Japan, Rhizopus delemar, fine grade 200 units/mg solid) (one unit is defined as that liberating 1 /zmol equivalent of fatty acid from oil or fat per minute at 25°) Celite (Johns-Manville Sales Co., United States, No. 535) n-Hexane Procedure. Two grams of Celite is washed three times with about 20 ml of deionized water and dried in vacuo for 2 days at 50°. About 10 ml of methanol containing less than 0.1% water and 0.2 ml of glycerol is added to the dried Celite. After thoroughly mixing, methanol is evaporated at 50° in vacuo. The Celite coated with glycerol is transferred into 40 ml of n-hexane in a 100-ml flask and mixed thoroughly to obtain a suspension. Ten grams of olive oil and 10 g of stearic acid are added to the above suspension. Then finally 40 mg of lipase is added to the suspension. In this way lipase is adsorbed to the glycerol-coated Celite. This reaction mixture is shaken in a rubber-stoppered flask on a reciprocal shaker at 37° for 24 hr (120 strokes/min). Isolation of Triglyceride. When the enzyme reaction is completed, Celite is eliminated by decanting. The reaction mixture containing 0.2-0.5 g of triglyceride is loaded on a Florisil column. (Preparation of column: 30 g of 60-80 mesh Florisil containing 7% water is dispersed in 100 ml of n-hexane, and the suspension is packed in a 2 × 25 cm column. Then the column is washed with 5 column-volumes of n-hexane.) The column is then eluted with the solvent system composed of diethyl ether and n-hexane (1:4, v/v). The eluted fractions (40-80 ml) are collected, and concentrated to about 1 ml. After checking that this concentrate is free from diglycerides, monoglycerides, and fatty acids by the conventional analytical TLC method, it is subjected to the following analyses. Analysis of the Total Fatty Acid Composition. One hundred and fifty milligrams of triglyceride is hydrolyzed with 3 ml of 0.5 N NaOH in methanol at 80° for 10 min in a test tube equipped with a Liebig condenser. Then the fatty acid in the reaction mixture is subjected to methyl ester preparation for further analyses by gas chromatography. Preparation of fatty acid methyl esters is basically carried out using the American Oil Chemists Society method (Ce 2-66). To the hydrolyzate is added 3.5 ml of 7% BF3-methanol reagent and the reaction mixture is boiled for 2 min. After the addition of 2.5 ml of n-hexane the reaction mixture is boiled for an additional minute. After cooling to room temperature, 15-20 ml of saturated NaCI aqueous solution is added to the mix-

408


[37]

ture. One milliliter of the n-hexane layer containing methyl esters of fatty acid is removed with a pipette and dehydrated on anhydrous sodium sulfate. This dehydrated n-hexane layer is used as the sample for analyses. The sample (0.5-5/.d) is injected directly in a gas chromatograph. Analysis is carried out by the conventional method. Analysis of the Fatty Acid in the 2-Position of a Triglyceride. The method is modified from that described by Usui et al. 7 One-tenth gram of triglyceride is added to 7.5 ml of 1% poly(vinyl alcohol) (average degree of polymerization 200), and the suspension is homogenized twice for 3 min using a homogenizer. To 7 ml of the homogenizate, 2 ml of 0.5 M phosphate buffer (pH 8.0) and 0.5 mg of lipase (Sigma Type IV porcine pancreas) are added. The reaction mixture is then kept at 40° for 1 hr. To stop the reaction, 2.5 ml of an acetone-ethanol mixture (1 : I, v/v) is added to the reaction mixture, which is then acidified by the addition of 2.5 ml of 1 N HCI. The acidified reaction mixture is subjected to extraction with diethyl ether 3 times. After the ether layer is washed with water twice, the washed ether layer is dehydrated with sodium sulfate. Monoglyceride isolated by conventional preparative TLC from the dehydrated ether layer is used as a sample for further analyses. Finally, the fatty acid composition of monoglyceride is analyzed by the same method used for total fatty acid composition analyses.

Experiment B: Preparation and Application of Lipase Adsorbed to Celite Coated with Buffer Solution

Reagents Oleic safflower oil (product of the United States, extracted from safflower species having oil rich in oleic acid) Palmitic acid 0.3 M TES [N-tris(hydroxymethyl)methyl-2-amino-ethanesulfonic acid] buffer (pH 6.5) Lipase I (Seikagaku-kogyo Co., Japan, Rhizopus delemar, fine grade 200 units/mg solid) Lipase II (Meito-sangyo Co., Japan, Candida cylindracea, Lipase MY, 42 units/mg solid) Celite (Johns-Manville Sales Co., United States, No. 535) n-Hexane Procedure. TES buffer (0.1 ml) is mixed with 1 g of Celite in a test tube and the mixture is shaken until Celite is coated with buffer solution as 7 H. Usui, H. Kuwayama, and M. Nagakura, Yukagaku 20, 284 (1971).

[37]


409

uniformly as possible. Celite coated with buffer solution is transferred to a 100-ml flask containing 40 ml of n-hexane. The suspension is mixed thoroughly. Then 10 g of oleic safflower oil and I0 g of palmitic acid are added to this suspension. Finally 20 mg of lipase I or 100 mg of lipase II is added to this suspension, respectively. (In this way the enzyme is adsorbed onto the buffer-coated Celite.) The reaction mixture is shaken at 30° for 3 days on a reciprocal shaker (120 strokes/min). Isolation of Triglyceride. Same as in the case of Experiment A. General Considerations Hydrolysis and resynthesis of glycerides occur because lipase reactions are reversible. When the water content in the reaction system is restricted, the hydrolysis of the oil or fat is minimized, permitting lipasecatalyzed interesterification to occur. Free fatty acid exchanges with the fatty acid moieties of the triglycerides to produce novel triglycerides, incorporating the added fatty acid. When 1,3-specific lipase is used, the reaction occurs only at the 1- and 3-positions of the glycerides, whereas if nonspecific lipase is used, the reactions occur at all three positions. 2,s An interesterification reaction using olive oil and stearic acid is described as the first experiment (A). In this experiment, lipase is considered to be activated by a small amount of glycerol which coats the Celite. Activation in this case means that the lipase is put into an active form. In this way it is possible to convert olive oil to a new reformed fat where the oleic acid moieties at the 1- and 3-positions are replaced by stearic acid (25-30%). In the second experiment (B) an interesterification reaction is described in which a small amount of TES buffer (pH 6.5) is used as an activator. Such Celite-adsorbed lipase from Rhizopus delemar converted safflower oil to a reformed fat, where oleic acid moieties at the 1- and 3positions were replaced by palmitic acid moieties. The analytical data on the total fatty acid composition and the 2-position fatty acid composition of the fat are shown in Table I. The oleic safflower oil, composed of about 75% oleic acid and 20% linoleic acid, was converted to an interesterified fat composed of about 50% oleic acid and 40% palmitic acid. When the lipase from Candida cylindracea was used, palmitic acid exchanged with oleic acid at random on all three positions of the glyceride (Table I). s A. R. Macrae, J. Am. Oil Chem. Soc. 611, 291 (1983).

410


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TABLE I FATTY ACID COMPOSITION OF OLEIC SAFFLOWER OIL [NTERESTERIFIED TRIGLYCERIDEa

Interesterified triglyceride Oleic safflower oil

Lipase I

Lipase II

Composition and reaction

Total (%)

2-Position (%)

Total (%)

2-Position (%)

Total (%)

2-Position (%)

Palmitic acid Stearic acid Oleic acid Linoleic acid

6.2 2.2 75.5 17.0

0.2 0.5 77.2 23.2

40.1 0.8 48.4 11.1

2. I 0.1 75.0 23.0

47.0 2.0 42.2 9.1

34.0 0.1 54.1 13.2

Main reaction

I O,L

(+Pal)

I

O,L

t_- ,O,L

P,O,L

~,O,L

LP,O,L P,O,L

I

+ I~,O,L Lipase I, lipase of R. delemar; lipase II, lipase of C. cylindracea. O, oleoyl; L, linoleoyl; P, palmitoyl; and Pal, palmitic acid.

~F-Hexane l Mixing

r ~

Methanol evaporation I

Reaction vessel

(at 3 0 - 4 0 °)

FIG. 1. The lipase-catalyzed interesterification setup; A flowchart of a semiscale enzyme process is given. Addition of carrier, reactants, and catalyst is carried out according to the numbers designated.

[38]

PRODUCTION--APPLICATION OF IMMOBILIZED LACTASE

411

Production of a cacao butterlike fat using 1,3-specific lipases is very interesting to the oils and fats industry. An illustration of an enzymecatalyzed reaction in a semiscale reactor is presented in Fig. 1. The enzyme reaction is carried out in a stirred tank fermenter, equipped with temperature recording and control at 30-40 °, under agitation for 24-72 hr. The reaction mixture is agitated at a speed giving a uniform dispersion. After the enzyme reaction, the Celite particles are separated from the reaction mixture, and the solvent is removed by evaporation. From this oils and fats fraction, the interesterified triglycerides (or cacao butterlike fat fraction) is concentrated and purified, using ethanol in an ordinary fractionation procedure, accompanied by differential scanning calorimetric analyses. In order to make this process practical from an industrial point of view, repeated use of adsorbed lipase as well as utilization of cheap raw material (like the midfraction of palm oil) and efficient fractionation of the products are indispensable.

[38] L a r g e - S c a l e P r o d u c t i o n a n d A p p l i c a t i o n o f Immobilized Lactase By J. L.

BARET

Whey is a major by-product of the dairy industry. Most of it is spraydried or processed and used in a variety of applications as a food, a feed, or a fermentation substrate. Ultrafiltration techniques have also been developing for the past decade and are now becoming a well-established technology to recover whey proteins and also to process milk in cheese making. Secondary by-products from the ultrafiltration, known as whey or milk permeates, are now produced in a significant amount. A continuous effort for better utilization of wheys and permeates is being made by the dairy industry. The hydrolysis of lactose into glucose and galactose appears to be an interesting approach to widen the profitable uses of wheys and permeates. Hydrolyzed lactose is sweeter and more soluble than lactose; it also presents several additional advantages which allow producers to obtain new attractive products. This report presents some aspects of the immoMETHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987by Academic Press, Inc. All rights of reproduction in any form reserved.

[38]


411

Production of a cacao butterlike fat using 1,3-specific lipases is very interesting to the oils and fats industry. An illustration of an enzymecatalyzed reaction in a semiscale reactor is presented in Fig. 1. The enzyme reaction is carried out in a stirred tank fermenter, equipped with temperature recording and control at 30-40 °, under agitation for 24-72 hr. The reaction mixture is agitated at a speed giving a uniform dispersion. After the enzyme reaction, the Celite particles are separated from the reaction mixture, and the solvent is removed by evaporation. From this oils and fats fraction, the interesterified triglycerides (or cacao butterlike fat fraction) is concentrated and purified, using ethanol in an ordinary fractionation procedure, accompanied by differential scanning calorimetric analyses. In order to make this process practical from an industrial point of view, repeated use of adsorbed lipase as well as utilization of cheap raw material (like the midfraction of palm oil) and efficient fractionation of the products are indispensable.

[38] L a r g e - S c a l e P r o d u c t i o n a n d A p p l i c a t i o n o f Immobilized Lactase By J. L.

BARET

Whey is a major by-product of the dairy industry. Most of it is spraydried or processed and used in a variety of applications as a food, a feed, or a fermentation substrate. Ultrafiltration techniques have also been developing for the past decade and are now becoming a well-established technology to recover whey proteins and also to process milk in cheese making. Secondary by-products from the ultrafiltration, known as whey or milk permeates, are now produced in a significant amount. A continuous effort for better utilization of wheys and permeates is being made by the dairy industry. The hydrolysis of lactose into glucose and galactose appears to be an interesting approach to widen the profitable uses of wheys and permeates. Hydrolyzed lactose is sweeter and more soluble than lactose; it also presents several additional advantages which allow producers to obtain new attractive products. This report presents some aspects of the immoMETHODS IN ENZYMOLOGY, VOL. 136

Copyright © 1987by Academic Press, Inc. All rights of reproduction in any form reserved.

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[38]

bilized lactase processes developed by Corning for the hydrolysis of lactose in permeates and wheys. Lactose Hydrolysis Reaction and Applications Lactose in solution can be hydrolyzed. The reaction is slightly exothermic and can be catalyzed with acids, cationic resins in H ÷ form, or enzymes. It is not strictly quantitative because side products may form depending on the nature of the catalyst and on the pH and temperature of the reaction. These aspects were studied with demineralized whey permeates which are fairly purified lactose solutions.l The studies showed that the lactose hydrolyzed products which were obtained by acid or H ÷ resin hydrolysis were less pure than those obtained by enzymatic hydrolysis. With more complex lactose-containing feedstocks available on a large scale such as milk, wheys, and their permeates, the acid and H ÷ resin hydrolysis routes are not industrially attractive because of the complex side reactions occurring during such processes. Lactose hydrolysis by enzymatic routes presents a high selectivity. It can be carried out batchwise using soluble fl-galactosidase, also called lactase. This approach is justified in applications marginally sensitive to the cost of enzyme such as for dietetics purposes or for lactose intolerants. Immobilization of lactases was considered as a means to decrease the cost of enzymatic hydrolysis. The production of hydrolyzed lactose products at low costs is essential for applications as sweeteners or as intermediate food products such as protein-sweetener mixes. Immobilized lactase composites were developed using either Aspergillus niger or Aspergillus oryzae acid lactases. Immobilized lactase systems were designed and operated under industrial conditions to carry out the lactose hydrolysis reaction in lactic acid wheys, acidified sweet wheys, permeates, and demineralized permeates. Lactose hydrolysis can be considered to be a new unit operation which can be integrated in the transformation of a wide range of lactose-containing feedstocks. Starting from wheys, "lactolyzed" whey syrups are obtained which can be used as food ingredients or feed specialities. Depending on the level of demineralization, hydrolyzed permeates can be utilized as fermentation substrates or feed ingredients or elaborated into sweeteners or high DE syrup. (DE stands for "dextrose equivalent," defined as total reducing sugars in the syrup calculated as dextrose and expressed as a percentage of the total dry substance.) G. Coton, Address to the International Dairy Federation, Geneva, September (1979).

[38]

PRODUCTION-APPLICATION OF IMMOBILIZED LACTASE

413

Immobilized Lactase Composites

Preparation of Immobilized Lactase Composites fl-Galactosidases from A. niger and A. oryzae (also called acid fungal lactases) are immobilized on a porous silica carrier based on the procedures defined by Messing and Weetall. 2 This carrier is a controlled-pore SiO2 ceramic of 30/45 US mesh size (or about 0.50 mm mean particle size) and 350/k average pore diameter. The specific area is 45 m2/g, for a total pore volume of 0.6 cm3/g. This carrier is morphologically and chemically stable in a wide range of temperatures, it has good mechanical properties, and it is not biodegradable. Particle size and pore dimension are optimized to reduce external diffusion limitations to mass transfer, to limit pressure drops, and to maximize the enzymatic activity. The lactase is covalently bound to the controlled-pore silica cartier using the silane-glutaraldehyde immobilization procedure already described in the literature. 2 This technique can be briefly summarized as follows. The silanol groups on the silica surface react with 7-aminopropyltriethoxysilane to give an alkylamine-silica derivative. The free available amino groups are then activated by glutaraldehyde. The resulting activated carrier contacts the enzyme in order to obtain the immobilized lactase composite. The coupling efficiency decreases when higher lactase loading is used. This immobilization process was optimized and scaled up from the laboratory procedure to an industrial operation.

Properties of Immobilized Lactases Regarding kinetic behavior, the pH profiles of immobilized lactase composites are fairly similar to the soluble lactase with a shift to the acidic side in the range of 0.5-1 pH units. Immobilized A. niger lactase exhibits an activity of about 500 U/g at 50° at optimal pH in the range 3.5-3.8 (Table I). The Michaelis constant (Km) and the inhibition constant (Ki) were determined as Km= 0.053 M and Ki = 0.005 M. The activation energy calculated from the Arrhenius relationship of reaction rate to temperature is 12 kcal/mol. The deactivation energy determined from the variation of half-life (ti/2) as a function of temperature is 40 kcal/mol. 3 Immobilized A. oryzae lactase has an activity of 400 U/g at 40° at optimal pH in the range 4-4.5. Values of gm = 0.05 M and Ki in the range 0.020.05 M were reported 4 as well as an activation energy of 6 kcal/mol and a 2 R. A. Messing and H. H. Weetall, U.S. Patent 3,519,538. 3 j. R. Ford and W. H. Pitcher, Conf. Whey Prod. Chicago, Sept, (1974). 4 H. Hirohara, H. Yamamoto, E. Kawano, and T. Nagase, Int. Enzyme Eng. Conf., 6th (1981).

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TABLE I MAIN CHARACTERISTICS OF LACTASES AND IMMOBILIZED LACTASES Soluble lactases

Immobilized lactases

Source

Status a

pH optimum

pH stability

Temperature optimum

pH optimum

Activity b

A. niger A. oryzae

GRAS GRAS

3.5-4 4.5-5

3-8 3.5-8

55-60 50-55

3-3.5 4-4.5

500 U/g (50°) 400 U/g (40°)

GRAS, Generally recognized as safe. b Units are micromoles of lactose hydrolyzed per minute at optimum pH and defined temperature. thermal deactivation energy of 70 kcal/mol. The operational characteristics of A. oryzae lactase appeared to be more favorable to processing whey and permeates because of its optimum pH (4.5) and less inhibition by galactose. Engineering Considerations The development of immobilized lactase processes is basically dependent on several parameters: the operational characteristics of the immobilized lactase composite, the nature of the substrate feedstocks, the design of the reactor system, and the operating strategy. The overall performance of the system is the result of the interaction between these different parameters and determines the economy of the process. The key objective is to maximize the amount of hydrolyzed lactose which is processed per unit weight of catalyst. A long operational life is necessary to reach this objective. The deactivation of the immobilized lactase is mainly influenced by the operating temperature and pH, the nature of the feed, and the development of microbial contaminations. In industrial practice, several processes based on immobilized enzyme reactors are currently being used. The reactors have two points in common. First, a substrate feedstock of controlled purity is processed, and second, the development of microbial contaminations can be controlled because the substrate media are deficient and/or a selective environment can be used (temperature, pH, substrate concentration, microbial inhibitots). The situation using wheys and permeates is very different. These media are nutritionally rich and thus are excellent growth media for microorganisms. The possibility of controlling the development of microbial contaminations at low pH (below pH 3.5) and at temperatures above 35 °6 5 H. H. Weetall, N. B. Havewala, W. H. Pitcher, C. C. Detar, W. P. Vann, and S. Yaverbaum, Biotechnol. Biogen. 16, 295 (1974).

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415

is restricted for several reasons. Generally the addition of microbial inhibitors in the feedstock is industrially impossible because the presence of those substances must be avoided in the end product. The control of microbial contaminations in the reactor appeared as a critical technical problem to be solved in order for this technology to be developed.

Feed Substrate Different lactose-containing feedstocks have been evaluated. The main characteristics of these feeds are reported in Table II. The complexity of the feed increases as a function of the presence and concentrations of salts (defined as ashes), nitrogenous matter (total nitrogen x 6.38), true proteins (proteinaceous nitrogen × 6.38), and suspended solids. The highly demineralized permeates are for practical purpose very close to a pure lactose solution. However, their use may be limited because they are costly to produce by current ion-exchange techniques. In order to widen the applications of the technology it was important to develop processes that were adapted to the other different substrate feeds. Processing of permeates and electrodialyzed permeates showed that the presence of salts and different concentrations of cations and anions normally found in these feeds had no adverse effect on the performance and stability of the enzyme. The need for demineralization is therefore related to the application of the end product. The level of suspended solids in wheys and the presence of the colloids in sweet whey were found to be of particular importance during operations in a fixed bed reactor. Microbial contaminations in the feed also has a critical impact as the main source of contamination.

Reactor Design Reactors were designed in order to test the long-term stability performance of the immobilized lactase and the economics of the processes under industrial conditions. The design of the reactor aimed at maximizing the performances of the immobilized lactase reactor while minimizing or controlling at the same time operational problems such as microbial contaminations, pressure drops, and plugging. Three main types of reactors were considered: fixed bed, fluidized bed, and stirred tank reactors (Table III). A perfect fixed bed reactor behaves ideally as a plug flow reactor. An ideal continuous stirred tank reactor would behave as a perfect backmix reactor. Some backmixing is observed in fluidized bed reactors and the extent of bed expansion affects the performance. From a kinetic stand6 M. Harju, Nord. Med. Tidskr. 6, 155 (1977).

416


Z

z z

O

b, Z 0 O ,.=

k~

o~ O

m H

z

I

e~

o~

.r., ©

[38]

[38]


417

TABLE III COMPARATIVE PERFORMANCES OF CONTINUOUS STIRRED TANK AND PLUG FLOW REACTORSa

Normalized residence timeb (units/ml-hr) Degree of conversion (%)

Continuous stirred tank

50 60 70 80 90

14 24.2 42.6 81.6 202.8

Plugf l o w

Relative amount: continuous stirred tank/plug flow reactor

6.4 9.8 14.8 22.6 37.6

2.2 2.5 2.9 3.6 5.4

Aspergillus niger lactase, Ki = 0.0054 M; K m = 0.0528 M; lactose

5% (w/w). b Normalized residence time (E/F). Plug flow reactor (E/F)pF = ~ I_

Ki

SoX + \ Ki

+

Continuous stirred tank reactor 1 [SoX + XKm(1 + XSo/Ki) ] (E/F)csTR = ~ 1 -- X J

where So is the initial lactose concentration (mol/liter); E, amount of enzyme (units); F, volumetric flow rate (ml/hr).

point a plug flow reactor appears to be the most efficient at minimizing the immobilized lactase requirement and the volume of the reactor. H o w e v e r , fixed bed reactors are k n o w n to be sensitive to the presence of suspended solids which m a y be present in feeds. This aspect was found to be of i m p o r t a n c e with w h e y feeds, so p r e t r e a t m e n t of w h e y feeds b e c a m e necessary to run satisfactory operations in a fixed bed mode. Microbial Contaminations

Sources of Contamination T h e r e are three main sources of microbial contamination that m a y affect the operations at industrial scale: microorganisms normally present in the feed substrate, microorganisms present in the reactor or adsorbed on the carrier, and accidental contamination occurring during the handling of the lactase c o m p o s i t e or other operations.

418

ENZYME ENGINEERING(ENZYME TECHNOLOGY)

[38]

It is not realistic to try to operate sterile reactors. However, the control of microbial contaminations to safe levels is an absolute necessity to monitor the performances of the reactor and also to guarantee the quality of the hydrolyzed products. In practice, the level of microbial contaminants in the feed substrate can be controlled by conventional heat treatment processes such as pasteurization which are well known in the dairy industry. The level of microbial contaminants in the product stream leaving the reactor is directly related to the level of contaminants in the feed, and it is also a function of the level of microbial contaminants which are present in the reactor. Because of the short residence time in the reactor, it was found that the increase of contaminants in the products is marginally related to the contaminants in the feed. The relative output/input ratios of microbial contaminants in the product and feed streams were good indicators in establishing the nature and trends of the microbial contaminations in the reactor. Approach to Immobilized Lactase Sanitation The development in the reactor of microbial contaminants, mainly yeasts and bacteria, can be critical. It was apparent that any commercially viable immobilized lactase process must incorporate a sanitizing or disinfecting procedure. This procedure must efficiently destroy the contaminating microorganisms without any appreciable effect on the immobilized lactase. Furthermore, when the product is intended for use in the food industry, the disinfecting agent often must meet governmental regulatory requirements. Methods for disinfecting immobilized enzyme reactors were evaluated. The different sanitizers or bacteriostatic agents were tested. Of them, acetic acid, which is commonly used as a dilute aqueous solution in laboratory studies, gives only limited results in industrial conditions. 7 Other known disinfectants, such as halogen derivatives, quaternary ammonium, and biguanidine polymers, were unsuitable because of partial or complete inactivation of the enzyme. Substituted diethylenetriamines of the following general formula were Ri

x

/

R4

/NCH2CH2NCHzCH2N \ R2 7 j. L. Baret, Brevet Franfais 2,471,192.

R3

R5

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419

found useful at laboratory scale to disinfect the immobilized lactase. Synergistic mixtures of dioctyldiethylenetriamine and trioctyldiethylenetriamine are commercially available from Th. Goldschmidt AG under the name Tego-Diocto BS. Disinfection processes based on the use of this product were scaled to industrial operations. 7 Operating Strategy

Industrial operations with immobilized enzyme reactors are dependent on two critical factors, activity and stability. The activity has to be maintained above some minimum value for an adequate period of time in order to guarantee satisfactory operations. When no particular care is taken, a rapid decrease in the activity is observed as a result of various phenomena such as the deposition of material within the bed, the formation of a coating around the particles, the development of microbial contaminations, channeling, or other problems. The thermal deactivation of the enzyme is not the controlling factor, as the apparent loss in activity was found to be reversible when cleaning-sanitation operations were operated on "dirty" or contaminated immobilized lactase (Table IV). Depending also on the nature and the quality of the feed, some pretreatment may be required to keep the activity constant during a period of time adequate for continuous production (Table V).

T A B L E IV HYDROLYSIS OF RAW WHEY AT p H 3.5 AND 50 °

Parameters

Test A"

Test B b

Flow rate (ml/hr) Immobilized lactase (g) Lactose (%) t = 2hr Glucose (g/liter) Degree of conversion (%) t = 6hr Glucose (g/liter) Degree of conversion (%)

102 4.68 4.26

103 4.68 4.26

17 78

16.5 76

11.8 54

11.4 53

" Test A was performed with freshly prepared immobi-

lized lactase. b Test B was performed subsequently to test A, after cleaning the immobilized lactase for 20 min with 1% aqueous acetic acid solution in a fluidized bed mode.

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TABLE V APPARENT DEACTIVATION DURING HYDROLYSIS OF CLARIFIED AND DEMINERALIZED WHEY WITH IMMOBILIZED LACTASEa

Whey

Operating temperature (°C)

Apparent half-life (hr)

Without heat treatment With heat treatment Without heat treatment With heat treatment

35 35 50 50

7 1980 4 124

a Whey was demineralized to 50% by electrodialysis (Ionics stack-pack) and acidified to pH 3.5 with HC1. It was then clarified by centrifugation on a Alfa Laval LAPX 202. Heat treatment was 1 hr up to 80°.

Hydrolysis and Cleaning-Sanitation Cycles Reactors were operated for long periods of time under semiindustrial conditions on a cyclic mode including a continuous production phase at constant temperature and cleaning-sanitation operations. 8 A first plant was operated at the Milk Marketing Board (MMB) technical division at Crudgington, United Kingdom, on a continuous basis 5 days a week with permeates demineralized by ion exchange. It processed about 350 liters/ hr, achieving 80% hydrolysis for 15-20 hr per day. The immobilized lactase was fluidized every day, when necessary with a dilute acetic acid solution (1%, volume basis). More than 100 operating cycles were carried out, during which the microbial contaminants in the hydrolyzed products were controlled in the range of 100-2000 total counts per milliliter. The conversion was maintained practically constant at the specified value during the operations at constant flow rate.

Impact of Cleaning-Sanitation Procedures on Stability Cleaning-sanitation procedures were developed to improve the operational stability for processing whey f e e d s . 9 In laboratory experiments, crude whey was received from a cheese factory and clarified with an Alfa Laval separator LAPX 202. The clarified whey was demineralized to 50% in an electrodialysis module (Ionics). It was then acidified to pH 3.5 with concentrated HC1 and stored at 2-4 °. The whey was heat-treated up to s L. A. Dohan, J. L. Baret, S. Pain, and P. Delalande, Int. Enzyme Eng. Conf. 5th (1979). 9 j. L. Baret and L. A. Dohan, Brevet Franfais 2,483,748.

[38]


421

80 ° , centrifuged on the same separator, and cooled down to 4 ° . The whey was subsequently used as the feed for three columns, each column containing about 5 g of immobilized lactase, with the whey flow rate at 100110 ml/hr. Hydrolysis was conducted continuously for about 18 hr per day at 35 ° . At the end of the hydrolysis phase, columns were rinsed with water. In column 1, a solution of Alcalase 0.6L, at pH 7.5, 6 Anson units/ liter, from Novo was used to clean the immobilized lactase. It was then disinfected with a 0.1% solution of Tego-Diocto BS (Th. Goldschmidt AG). In column 2, only acetic acid was used at pH 3 and in column 3, only Tego-Diocto BS 0.1% in solution was used. These cleaning-sanitation procedures have a significant impact on the performance as seen from Table VI. This was confirmed during two semiindustrial projects using cottage cheese whey and acidified sweet whey, cleaning-sanitation procedures were optimized to maximize the productivity of the immobilized lactase.

Temperature Program The knowledge of both the activation energy and the thermal deactivation energy allows an estimation of the theoretical life of the enzyme as a

TABLE VI ACTIVITY AND STABILITY OF THE IMMOBILIZED LACTASE DURING HYDROLYSIS OF WHEY IN RELATION TO DIFFERENT CLEANING AND/OR DISINFECTING PROCEDURES

Column 1: protease and Day

Tego-Diocto BS

Column 2: acetic acid

Column 3: Tego-Diocto BS

258 202 163 117 36 (stop)

174 143 125 122 107 104 98 35 90

Activity (units/g) 1 5 10 15 17 20 26 Half-life(days) Stability (%)0

235 296 233 220 223 213 203 55 96

9 60

Stability (%) is the average statistical ratio of the activity after 17 hr to the activity after 2 hr hydrolysis over all the days of operations.

422


[38]

function of the initial operating temperature. The theoretical life will increase when the initial operating temperature decreases, so initial low temperature operation is necessary to maximize the productivity. However, in practice, a pure thermal deactivation of the enzyme may not be the only controlling phenomenon at low temperature. The initial productivities of the reactors were maintained constant at specified values by raising the temperature when it was necessary. Actual gains in activities were about 5-10% per degree centigrade in the range of operating temperatures which were considered (20-45 °) with various feeds. This operating temperature approach in conjunction with an adequate cleaning-sanitation process made it possible to keep the performances of the reactors constant over periods of time sufficient to demonstrate the economic feasibility of the process. Industrial Developments These semiindustrial operations increased our confidence in the technology for hydrolyzing wheys and permeates. The technology was transferred to full-scale operations. Corning established joint ventures with major partners in the food industry: the Specialist Dairy Ingredient company (SDI) with the Milk Marketing Board in England, the Nutrisearch Company with the Kroger Company in the United States, and Corvire with Union Laiti~re Normande (ULN) in France. A plant processing 20,000 liters/day of sweet whey is operated by SDI at Aston (Cheshire). The hydrolysis reactor can process 1000 liters/hr of nondemineralized acidified sweet whey with a load of about 40 kg of lactase composite. Lactolyzed whey products are obtained, and are formulated as "sweet-protein" syrups which can be used as ingredients in different sectors of the food industries. The SDI production was used to develop the market for this new product in confectionary, ice cream, and baked products. Capacity expansion is under way. The most advanced operation is the Nutrisearch plant in Winchester, Kentucky, which combines the immobilized lactase technology of Corning and continuous fermentation technology of Kroger Co. Investments for that plant were 15 million dollars for a nominal capacity of 100,000 gallons of raw cottage cheese whey per day. Whey is processed by ultrafiltration to obtain a protein-rich retentate. This stream is then formulated as a dried whey protein concentrate. The permeate stream is pumped into two hydrolysis columns. These columns are 3-ft-diameter and 15-ft-high vessels filled with about 1500 kg of lactase composite. The lactolyzed permeate stream can then be fermented by a selected Saccharomyces cerevisae strain in a continuous fermenter to produce baker's yeast.

[39]

UHT

STERILIZED MILK TREATMENT USING SOX

423

A semiindustrial unit (10,000 1/day) is operated by Corvire in the U L N plant at Conde-sur-Vire. H o w e v e r , the industrial development was slower there because of the specific regulatory constraints in France. L a c t o s e hydrolysis with immobilized lactase is now an industrial reality. A subsequent expansion in industry is expected in the coming years.

[39] Continuous Treatment of Ultrahigh-Temperature Sterilized Milk Using Immobilized Sulfhydryl Oxidase 1 B y HAROLD E. SWAISGOOD, VIOLETA G. JANOLINO, and

PAUL J. SKUDDER Thiols are often responsible for undesirable flavors in foods because of their extremely low organoleptic threshold concentrations. The " c o o k e d " flavor of ultrahigh-temperature (UHT) 2 sterilized milk is a familiar example of such " o f f flavors." Aseptic packaging of U H T milk allows the product to be merchandized and stored at ambient temperature. If the flavor could be acceptable by a larger fraction o f the population, a potential energy saving could be realized. Consequently, discovery that an e n z y m e indigenous to unheated milk, and thus aesthetically acceptable as a processing aid for milk, could be used to eliminate the c o o k e d flavor 3,4 has important practical value. Isolation of Protein Fractions Having Increased Sulfhydryl Oxidase Activity Sulfhydryl oxidase (SOX) is an iron-containing, glycomembrane enzyme existing primarily in the membrane vesicle fraction of skim m i l k ) -~ This work is Paper No. 9349 of the Journal Series of the North Carolina Agricultural Research Service. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products. 2 Abbreviations used: UHT, ultrahigh-temperature; CPG, controlled-pore glass; SOX, sulfhydryl oxidase; DTNB, 5,5'-dithio-bis(2-nitrobenzoicacid); GSH, reduced glutathione. 3 H. E. Swaisgood, U.S. Patent 4,053,644 (1977). 4 H. E. Swaisgood, U.S. Patent 4,086,328 0978). 5 V. G. Janolino and H. E. Swaisgood, J. Biol. Chem. 250, 2532 (1975). 6 M. B. Sliwkowski, M. X. Sliwkowski, H. E. Swaisgood, and H. R. Horton, Arch. Biochem. Biophys. 211, 731 (1981). 7 M. B. Sliwkowski, H. E. Swaisgood, and H. R. Horton, J. Dairy Sci. 65, 1681 (1982). 8 M. X. Sliwkowski, M. B. Sliwkowski, H. R. Horton, and H. E. Swaisgood, Biochem. J. 209, 731 (1983). METHODS IN ENZYMOLOGY, VOL. 136


[39]

UHT

STERILIZED MILK TREATMENT USING SOX

423

A semiindustrial unit (10,000 1/day) is operated by Corvire in the U L N plant at Conde-sur-Vire. H o w e v e r , the industrial development was slower there because of the specific regulatory constraints in France. L a c t o s e hydrolysis with immobilized lactase is now an industrial reality. A subsequent expansion in industry is expected in the coming years.

[39] Continuous Treatment of Ultrahigh-Temperature Sterilized Milk Using Immobilized Sulfhydryl Oxidase 1 B y HAROLD E. SWAISGOOD, VIOLETA G. JANOLINO, and

PAUL J. SKUDDER Thiols are often responsible for undesirable flavors in foods because of their extremely low organoleptic threshold concentrations. The " c o o k e d " flavor of ultrahigh-temperature (UHT) 2 sterilized milk is a familiar example of such " o f f flavors." Aseptic packaging of U H T milk allows the product to be merchandized and stored at ambient temperature. If the flavor could be acceptable by a larger fraction o f the population, a potential energy saving could be realized. Consequently, discovery that an e n z y m e indigenous to unheated milk, and thus aesthetically acceptable as a processing aid for milk, could be used to eliminate the c o o k e d flavor 3,4 has important practical value. Isolation of Protein Fractions Having Increased Sulfhydryl Oxidase Activity Sulfhydryl oxidase (SOX) is an iron-containing, glycomembrane enzyme existing primarily in the membrane vesicle fraction of skim m i l k ) -~ This work is Paper No. 9349 of the Journal Series of the North Carolina Agricultural Research Service. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products. 2 Abbreviations used: UHT, ultrahigh-temperature; CPG, controlled-pore glass; SOX, sulfhydryl oxidase; DTNB, 5,5'-dithio-bis(2-nitrobenzoicacid); GSH, reduced glutathione. 3 H. E. Swaisgood, U.S. Patent 4,053,644 (1977). 4 H. E. Swaisgood, U.S. Patent 4,086,328 0978). 5 V. G. Janolino and H. E. Swaisgood, J. Biol. Chem. 250, 2532 (1975). 6 M. B. Sliwkowski, M. X. Sliwkowski, H. E. Swaisgood, and H. R. Horton, Arch. Biochem. Biophys. 211, 731 (1981). 7 M. B. Sliwkowski, H. E. Swaisgood, and H. R. Horton, J. Dairy Sci. 65, 1681 (1982). 8 M. X. Sliwkowski, M. B. Sliwkowski, H. R. Horton, and H. E. Swaisgood, Biochem. J. 209, 731 (1983). METHODS IN ENZYMOLOGY, VOL. 136


424


[39]

MILK .Centrifuge (4080 g, 50 min, 30") SKIM MILK

iChymosin (2 ml of Img/ml solution per liter of skim milk) 90 rain, 50 ° Centrifuge (16,300 g, 45 rain, 5 °)

WHEY • Add (NH4)2SO4 to 50*/.

saturation at 4* • Allow to stoodovernight,4* • Centrifuge (16,500 g, 60 rain, 4*)

• Filter (0.4S ~um filter] • Diafilter (100,000 Mr

membrane)against 47 ma sodiumphosphate, pH 7.0, 20-23*

• Filter (0.45 pm filter

,Molecular sieve chrorr~togmphy on 500 nm pore diameter glycerolpropyI-CPG equilibrated with 47 mM sodium phosphatepH 7.0

Pellet

Membrane retentate

Void volume fraction

(Crude skim milk

(Crude skim milk membrane

(Skim milk membrane

membrane fraction)

vesicles)

vesicles)

FlG. 1. Schematic illustration of methods for preparation of various skim m i l k membrane

vesicle fractions.

Several methods have been developed for preparation of active isolates directly from whey which are therefore commercially attractive 5,9-1~ (Fig. 1). Whey is obtained from the skim milk upon removal of the caseins by clotting with chymosin (rennin). This reaction occurs at the pH of milk (6.6-6.8) and results from the hydrolysis of a specific peptide bond in Kcasein; thus other proteins are not proteolyzed in this treatment. Because the preparation of SOX requires that the pH is maintained above 6, commercial whey which meets this criterion may also be suitable for enzyme preparation. Of the three methods listed in Fig. 1 for obtaining preparations of skim milk membrane vesicles, ultrafiltration and molecular sieve chromatography are the most attractive because of their simplicity. The chromatographic method has yielded the most active preparations, typically purified 300-fold from whey. 9 However, larger volumes are more easily processed by diafiltration than by chromatography on CPG-3000. Availability of membranes with a larger molecular weight cutoff would improve the purification obtained during diafiltration of whey. The size of 9 V. G. Janolino, D. A. Clare, and H. E. Swaisgood, Biochim. Biophys. Acta 658, 406 (1981). l0 H. E. Swaisgood, M. X. Sliwkowski, P. J. Skudder, and V. G. Janolino, in "Utilisation des Enzymes en Technologic Alimentaire" (P. Dupuy, ed.), p. 229. Technique et Documentation Lavoisier, Paris, 1982. 11 V. G. Janolino and H. E. Swaisgood, J. Dairy Sci. 67, 1161 (1984).

[39]

U H T STERILIZED MILK TREATMENT USING S O N

425

most of the vesicles appears to be between 200 and 300 nm in diameter, 12 so that methods which select for thissize range should yield the most homogeneous preparations of these membranes. An increase in SOX activity relative to y-glutamyltransferase activity can be obtained by a combination of diafiltration followed by chromatography on CPG-3000. Sulfhydryl oxidase may be further purified by solubilization of the membrane vesicles with nonionic detergent and isolation of this enzyme by transient covalent affinity chromatography on cysteinylsuccinamidopropyl-glass. 8 At present, however, the quantities obtainable by this procedure prevent its commercial application. Immobilization of the Enzyme Several methods involving both covalent attachment and adsorption on inorganic matrices have been investigated as means for immobilization. Continuous processing and aseptic packaging of U H T milk require extremely high flow rates, and the enzyme support must be compatible with this requirement. Porous and nonporous glass or ceramics were chosen because of their mechanical strength, excellent flow characteristics, and the ease with which they can be cleaned. Unlike other enzymes which have been developed for use in immobilized forms, SOX is a membrane enzyme, and consequently, certain considerations are important to the choice of immobilization conditions. The pore size of the support matrix and the degree of enzyme solubilization are major factors in determination of the specific activity of the immobilized catalyst. 1°,11,~3 If the enzyme is not solubilized it is substantially excluded from the pore volume of matrices having pore diameters as large as 300 nmH; accordingly, the specific activity (U/g) of such catalysts is similar to that prepared on a nonporous support of equivalent particle dimensions. 13 However, the enzyme can be solubilized in the nonionic detergent polyoxyethylene 9-1auryl ether without loss of activity. 7 In I% solutions of this detergent substantial penetration of the pore volume of matrices with pore diameters as small as 100 nm has been observed. ~3 The above considerations are substantiated by the activities obtained for various immobilized forms of the enzyme prepared by covalent attachment to controUed-pore glass (CPG) beads. These results, listed in Table I, show that solubilization of the enzyme improves the specific activity of the immobilized catalyst. It is also clear from the data that chromatographically purified membrane vesicles, which had the highest specific t2 V. G. Janolino, C. S. Barnes, and H. E. Swaisgood, J. Dairy Sci. 63, 1969 (1980). 13 V. G. Janolino and H. E. Swaisgood, J. Dairy Sci. 61, 393 (1978).

426


[39]

TABLE I ACTIVITIES OF IMMOBILIZED ENZYME PREPARATIONS OF VARIOUS ENZYME ISOLATES

Activity Preparation

Support

(U/g)

50% saturated (NH4)2SO4

SuccinamidopropylCPG-2000 Aminopropyl-CPG-2000

0.9"

Succinamidopropyl-

1.9 c

precipitate Solubilized diafiltered whey Chromatographically purified skim milk membrane vesicles Solubilized chromatographically purified skim milk membrane vesicles a

1.5 b

CPG-1400

Succinamidopropyl-

6.4 c

CPG-1400

From Janolino and Swaisgood.t4

b F r o m V. G. Janolino and H. E. Swaisgood (unpublished observa-

tions). c From Janolino and Swaisgood. H

activity for SOX, yielded the most active enzyme particles. In each case the succinamidopropyl-CPG was activated with water-soluble carbodiimide using either a simultaneous or a sequential activation-immobilization procedure.14 Solubilized enzyme was immobilized on the aminopropyl-CPG by preactivation of the matrix with 2.5% glutaraldehyde. Independent studies have shown that each of these three covalent immobilization methods yield preparations having comparable activity. Although covalent immobilization of purified membrane vesicles has resulted in more active preparations, our recent studies have resulted in a method of immobilization of SOX which may be more attractive for preparation of a commercial reactor. These results (Fig. 2) show that SOX is adsorbed directly from whey onto the cationic, silica-based matrix Spherosil QMA (P. J. Skudder and H. E. Swaisgood, unpublished results). Furthermore, it appears that SOX is adsorbed in preference to some of the other whey proteins. Thus, a 20-g reactor was loaded with enzyme simply by passing 600 ml of solubilized diafiltered whey through the column of beads. The resulting immobilized catalyst typically displayed activities similar to those obtained by covalently immobilizing the 50% (NH4)2SO4 precipitate. Furthermore, the activity could be stabilized t4 V. G. Janolino and H. E. Swaisgood, Biotechnol.

Bioeng. 24~ 1069

(1982).

[39]

427

U H T STERILIZED MILK TREATMENT USING S O X

i i ~ 4'0 protein coaceatratio~ in retentote

"10 50 t*.

0.03 activity

8

in

dll~lP reteotate

" •0.02

2.0 ~

.>_

3 1,0 ~

0.01

0

0

I

_ _ 1

200

400

0 600

Volume of the SOX isolate passed through matrix (ml) FIG. 2. Adsorption of SOX activity on Spherosil QMA upon passage of solubilized, diafiltered whey retentate through a 20-g column of the beads. Whey was diafiltered using a 100,000 Mr membrane against 47 mM sodium phosphate, pH 7.0, and solubilized with 1% polyoxyethylene 9-1auryl ether. The flow rate was 25 ml/hr at 5°. (©) SOX activity; ( 1 )

protein concentration in the emerging stream.

by cross-linking with a 0.1% (w/v) glutaraldehyde solution, yielding a reactor with an operational stability which appeared to be similar to that observed for covalently immobilized forms. Characteristics of Sulfhydryl Oxidase Reactors Laboratory-scale reactors of immobilized SOX have been operated continuously with UHT milk using both the fixed-bed and the fluidizedbed configurations. Most of the data have been obtained with a 2-liter reactor 1°,15containing SOX covalently immobilized on 170 g of succinamidopropyl-porous silica (200 nm pore diameter, 80/120 mesh) which was operated in both reactor configurations continuously at 30 °, which is near the temperature optimum for enzyme activity. The catalytic efficiency was comparable in either configuration; however, reactor plugging should not be a problem in the fluidized-bed mode. Characteristics of a number of is H. E. Swaisgood, V. G. Janolino, and M. X. Sliwkowski, "Proceedings: International Conference on UHT Processing and Aseptic Packaging of Milk and Milk Products," p. 67. Department of Food Science, North Carolina State University, Raleigh, North Carolina, 1980.

428


[39]

PREPARATION

1

POTENTIAL FOR LONG-TERMSTORAGE BEFORE USE TOTAL POTENTIAL USEFULLIFE > 3 MO

1 IN-LINE PLACEMENT ~ 1 f l 3-6 DAY OPERATION

l REGENERATION WITH 4M UREA ~ REGENERATION ULTRAFILTRATION

RECOVER& REGENERATE SUPPORTBY HEAT TREATMENT FIG. 3. Schematic illustration of the proposed operational protocol for use of an immobilized SOX reactor.

smaller reactors containing various immobilized forms of the enzyme also have been determined. Conclusions drawn from these results suggest that typically a reactor has a half-life of about 1 week during continuous operation with U H T milk, and that the activity may be regenerated by washing the reactor with a sterilized 4 M urea solution. ~5,~6For example, the 2-liter reactor was operated in the fluidized-bed configuration over a 3-month period using six regeneration cycles with 4 M urea, and retained 80% of its original activity. 16 The immobilized enzyme is extremely stable during storage under conditions which are sterile or limiting to microbial growth, e.g., 10% ethanol. 17A suggested protocol for operation of a SOX reactor is outlined in Fig. 3. It should be noted that even for covalently immobilized forms, the support matrix can be regenerated after the enzyme becomes inactive, thus allowing fresh enzyme to be immobilized. Also, the 4 M urea solution can be maintained sterile and can be regenerated by ultrafiltration. The activity of SOX reactors was routinely assayed with 0.8 mM GSH 5 either in 47 mM sodium phosphate, pH 7.0, or in milk ultrafiltrate. The rate of thiol oxidation was measured from the decrease in concentration determined by reaction with DTNB ~ using e4~2 = 13,600 M -I cm-L Typically, 85-/.d aliquots were removed and added to 1.0 ml of 100 # M DTNB in 0.1 M sodium phosphate, pH 8.0, containing 10 mM EDTA, and 16 M. X. Sliwkowski and H. E. Swaisgood, J. Dairy Sci. 63 (Suppl. 1), 60 (Abstr, DR 56) (1980). 17 H. E. Swaisgood, V. G. Janolino, and H. R. Horton, AIChE-I. Chem. E. Syrnp. Set. 74, (No. 172) 25 (1978).

[39]

U H T STERILIZED MILK TREATMENT USING S O N

I

I

I

I

I

429

t

Z Z O

111 ~e ttl

Z

0 U Z Ill U n, uJ a.

/8'1

100 300 500 N O R M A L I Z E D RESIDENCE T I M E

I

I

700 900 E/F ( U N I T S - M I N / L )

FIG. 4. Conversion of substrate to products as a function of normalized residence time. (1) Column assays; (Is) recirculation assays; (A) batch assays. Reproduced with permission from Swaisgood e t al. 15

the absorbance at 412 nm read after 2 min. 8 One unit of enzyme activity corresponds to 1/zmol SH oxidized per minute at 35°. To facilitate scaling up of the reactors, the percent oxidation of 0.8 mM GSH solutions was determined as a function of a "normalized residence time" (Fig. 4)) °,]5 Using this curve, for example, if the desired

430


100

I

[

L

[39]

[ ...... -

E 80--

Q

ul

~

6 0 ~

--

_z soFU

uJ F-

LU Q

I,,-

~

3 0 - -

--

U E

L

2o

I 320 400 480 560 N O R M A L I Z E D R E S I D E N C E T I M E (GSH U N I T S - M I N / L )

FIG. 5. Relationship between the degree of cooked flavor and the extent of enzyme treatment of UHT milk.

degree of oxidation and the flow rate through the reactor are known, the amount of enzyme required to yield the desired result can be calculated. Flavor Modification of UHT Milk Both trained and untrained consumer panels have been used to organoleptically evaluate enzyme-treated UHT milk and compare it to untreated UHT milk and commercial pasteurized milk. 3'1°']5'18 UHT milk is H. E. Swaisgood, Enzyme Microb. Technol. 2, 265 (1980).

[39]

U H T STERILIZED MILK TREATMENT USING S O X

431

was treated using various immobilized SOX reactors, including the 2-liter reactor, and by injection of filter-sterilized enzyme into aseptic packages. The flavor was evaluated immediately after treatment with immobilized enzyme. Similar results have been observed using both methods of treatment; however, injection of soluble enzyme requires preparation of covalent affinity chromatographically purified enzyme to avoid the occurrence of proteolytic activity in the injected solution. In a triangular test, the untrained panel of judges could not distinguish enzyme-treated U H T milk from commercial pasteurized milk, but all of the panelists could distinguish between UHT milk and enzyme-treated U H T milk by headspace odor alone. To relate the degree of cooked flavor to the extent of enzyme treatment, a panel of experienced milk judges was used to evaluate the flavor of U H T milk which had received varying degrees of enzyme treatment, as measured by the normalized residence time. 10.15,18For this purpose, the units of enzyme activity were determined by assay with GSH; thus the normalized residence time is the same as that shown in Fig. 4. The degree of cooked flavor is expressed as the percentage of the panel which could detect the flavor. The relationship between these two parameters is shown in semilogarithmic form in Fig. 5. The linearity of this relationship suggests that the disappearance of the flavor compound(s) follows first-order kinetics, i.e., the concentration is much less than the Km, as would be expected for such flavor compound(s). The above results suggest that cooked flavor is reduced below the threshold level by a treatment corresponding to 60% oxidation of 0.8 mM GSH. Combination of the results in Figs. 4 and 5, using a normalized residence time of 510 units-min/liter,10 indicates that 8500 units of enzyme activity would be required per 1000 liter/hr processing rate. Typically, the specific activity of immobilized SOX varies between 0.1 and 1.0 units per milliliter of catalyst, which means that the reactor size for a 1000 liter/hr processing rate should be 8.5 to 85 liters. To maintain a constant production rate, one would predict that the most effective system would use multiple reactors staggered with respect to their age so that the oldest could be replaced on a schedule derived from knowledge of the immobilized enzyme's half-life.

432


[40]

[40] P r o d u c t i o n o f I s o m a l t u l o s e U s i n g I m m o b i l i z e d M i c r o b i a l Cells B y PETER S . J .

CHEETHAM

In the last 25 years enzymes have acquired many actual and potential applications in the production of sugars and sweeteners. Undoubtedly the most significant so far is high-fructose corn syrup produced using immobilized glucose isomerase. Others include maltose syrups, Aspartame, and cyclodextrins. In this article I would like to give some reasons why we were interested in isomaltulose as a product and the attractions of using an immobilized cell process for its production. In particular I shall describe some of the methodological advances made and the experimental approaches we found useful, particularly when considering the requirements of a large-scale production process. The objective was to produce a pure, cheap product in as great a yield as is possible and compatible with easy operation of the process.

Properties of Isomaltulose Isomaltulose (alias palatinose, lylose; 6-O-a-D-glucopyranosyl-D-fructofuranose) is a natural component of honey. ~It has a number of interesting properties. First, isomaltulose has a sweetness typical of disaccharides, being only about a third as sweet as sucrose but with a sweetness profile very similar to that of sucrose. Isomaltulose is a reducing sugar and so causes browning reactions to occur in foods to a greater extent than does sucrose, and has potential applications in intermediate-moisture foods where the use of sucrose is restricted by its high sweetness. This low sweetness intensity is also useful in that the flavors of many isomaltulose-containing foods are masked less than in similar products containing sucrose. It can be readily crystallized and is metabolized in a very similar way to sucrose 2,3 and so can be used as a calorific bulking agent, for instance to completely replace the sucrose in foods and drinks. 4,5 Isomaltulose has a calorific value of 4 kcal/g but shows a slower i I. R. Siddiqui and B. Furgala, J. Apic. Res. 6, 139 (1%7). 2 I. Macdonald and J. W. Daniel, Nutr. Rep. Int. 28, 1083 (1983). 3 p. S. J. Cheetham, in "Developments in Sweeteners" (C. K. Lee and M. G. Lindley, eds.), Vol. 3, p. 105. Applied Science, Barking, Essex, U.K., 1982. 4 C. Bucke and P. S. J. Cheetham, United Kingdom Patent 2,066,539B [Tate & Lyle, PLC]

(1983). 5 K. Suzuki, New Food Ind. 26, 1 (1984).



[40]

ISOMALTULOSE PRODUCTION USING MICROBIAL CELLS

433

rate of release of monosaccharides into the blood. Therefore insulin release is correspondingly reduced compared with other simple sugars, creating the possibility of applications in diabetic and sports foods and drinks. Second, it appears to be noncariogenic; that is, it is not used by Streptornyces mutans to form the acid and/3-glucan polymers that are important factors in causing tooth decay.6-8 This property of isomaltulose has been exploited in a caries-preventing sugar composition containing Aspartame and isomaltulose. 9 Third, it is very much more resistant to acidic hydrolysis than. sucrose, making it very much less likely to invert when incorporated, into carbonated beverages, 4 for instance. Fourth, it has the property of being an excellent excipient in tablets, l° Finally whereas most enteric bacteria can metabolize most sugars, isomaltulose can only be utilized by bifidobacteria. Thus isomaltulose encourages the growth of the bifidobacteria and discourages the growth of potentially putrifactive microorganisms that have a tendency to cause diarrhea. It has therefore been tested in food, drink, and medicine formulations, especially those containing dairy products. 11 The crystalline structure of isomaltulose has been established: there is one intramolecular hydrogen bond between the 2 and 2' carbon atoms, and several intermolecular hydrogen bonds are present. IH NMR and 13C N M R spectroscopy has been carried out showing that the a and 13isomers are present in a ratio of 1 : 4 at 34°, very similar to the values measured for fructose.~2.~3 Several chemical derivatives of isomaltulose have been synthesized. 14 Production of Isomaltulose by Fermentation The chemical synthesis formation of isomaltulose time: as an intermediate in the South German Sugar

of isomaltulose is very difficult. The microbial has attracted commercial interest for some Palatinit (or Isomalt) manufacture, initially by Co. who produced it by fermentation using

6 K. R. Roberts and M. C. Hayes, Scand. J. Dent. Res. 88, 201 (1980). 7 y . j. Kadomura, Tokyo Dental College Soc. 82, 407 (1982). s T. Ooshima, A. Izumitani, S. Sobue, N. Okahashi, and S. Hamada, Infect. lmmun. 39, 43 (1983). 9 Mitsui Sugar Co., Japanese Kokai Tokyo Koho Application 8,218,925 (1983). l0 M. G. Lindley and S. Hathaway, United Kingdom Patent Application 2,066,640A (1981). H Calpis Shokuhin Kog (rika), Japanese Patent Application JS 7091-193 (1980). 12 W. von Dreissig and P. Luger, Acta Crystallogr., Sect. B B29, 514 (1973). 13 A. de Bruyn, J. van Beeumen, M. Anteunis, and G. Verhegge, Bull. Soc. Chim. Belg. 84, 799 (1975). 14 yon F. Loss, Z. Zucherind. 19, 323 (1969).

434


[40]

Serratia plymuthica (NCIB 8285) or a strain described as Protaminobacter rubrum (CBS 574.77) 15, and more recently by Bayer AG 16 using the same strains. The cells were isolated from sugar beet refineries in the 1950s and grown on an undefined medium containing 5% sucrose and other nutrients until a high cell concentration was reached after - 2 4 hr and then used to convert a diluted refinery sucrose stream (20% w/w) which is aerated and maintained at pH 7.0 until all the sucrose is completely converted into isomaltulose (-12 hr). The cells are recovered using a jet separator and can be reused approximately six times, provided sterile conditions can be maintained. The isomaltulose solution is then deionized using a strong acidic cation exchanger in the H ÷ form, such as Amberlite 200, and a weak basic anion exchange in the OH- form, such as Amberlite IRA 93. The deionized solution is concentrated under vacuum to 65% (w/w) and cooled and the isomaltulose crystallized in two stages before separation in a basket centrifuge. In a continuous process cells are grown continuously and the cell suspension removed from the fermenter, mixed with a concentrated sucrose solution, and processed in a cascade of three to five stirred vessels so as to achieve complete conversion of the sucrose. The cells are separated and recycled, and the isomaltulose solution purified and crystallized as above. Crueger et al. 16have described the production of isomaltulose by continuous fermentations. Sucrose solutions (25% w/w) were converted at a dilution rate o f - 0 . 1 hr -I. Thus once a steady state had been reached the productivity of their fermenters was - 2 5 g isomaltulose. This value is higher than achieved in batch fermentations where productivities of - 1 7 g/liter-hr were reported in UK Pat. spec. 1,429,334. Since these cells can be reused up to six times total productivity may approach 120 g/liter per 84 hr. The comparable value for our immobilized cell system was 40 g/liter-hr, but of course because the immobilized cells are so stable their productivity aggregated over a long time period such as a year (200 kg/ liter-year) is very much higher than that of the continuous fermentation. The isomaltulose so formed is then hydrogenated at alkaline pH at 100-125 ° using Raney nickel Type B l l 3 Z to form Palatinit which comprises an equimolar mixture of 6-O-a-o-glucopyranosyl-o-sorbitol and 1-O-o~-D-glucopyranosyl-o-mannitol.15.16The uncrystallized product is not directly hydrogenated, presumably because the hydrogenated sugars derived from the trehalulose would make the product less pure and therefore 15 South Germane Sugar Co., United Kingdom Patent Spec. 1,429,334 (1976). ~6W. Crueger, L. Drath, and M. Munir, European Patent Application 78,100,803.2 (1979). t7 H. Schiweck, Alimenta 19, 5 (1980).

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435

less acceptable to regulatory authorities. Isomalt is a white crystalline material of about half the sweetness and calorific value of sucrose and which also exhibits low cariogenicity and good stability toward enzymatic and microbial hydrolysis over a wide pH range. It is manufactured and sold by Palatinit AG, a subsidiary of the South German Sugar Co. and Bayer AG, as a low-calorie bulking agent/sweetener. ~7They now use immobilized cells to produce the isomaltulose (see later) rather than fermentation methods. We have also found that isomaltulose could be formed by a solid-state fermentation of sugar cane. The cane was diced and crushed and inoculated with one of the organisms described in this paper. Following incubation at 30° for several days, isomaltulose could be recovered in low yield by hot-water extraction of the cane. This method depends on the isomaltulose-producing organisms also secreting pectinases to break down the cane, thus liberating sucrose for conversion to isomaltulose. Potential Advantages of the Use of Immobilized Cells Efforts have been made to produce isomaltulose using immobilized cells as they offer several potential advantages over the fermentation process. 18,19 Isomaltulose formation takes place most efficiently when nongrowing cells are used. Therefore, the cell production and product formation steps can be individually optimized since they are performed separately. Very high densities of cells can be achieved, much greater than could be achieved in conventional fermentations without washout occurring. Also high concentrations of substrate that are inimical to cell growth can be used. The high energy inputs required by the need for agitation, aeration, and the maintenance of sterility are reduced, very little substrate is diverted into cell growth, and less wastes are often produced. Last, continuous operation is possible such that the lengthy nonproductive periods that occur between batch fermentations are avoided or greatly reduced. Choice of Microorganism A number of strains of microorganism including several Enterobaceriacae strains were tested for isomaltulose production, z°-22 Since the mi~8p. 19 C. 20 C. :~ P. 22 p.

S. J. Cheetham, Top. Enzyme Ferment. Biotechnol. 4, 189 (1980). Bucke, Philos. Trans. R. Soc. London, Ser. B 300, 369 (1983). Bucke and P. S. J. Cheetham, U.S. Patent 4,359,531 (1982). S. J. Cheetharn, C. E. lmber, and J. Isherwood, Nature (London) 299, 628 (1982). S. J. Cheetharn, C. Garrett, and J. Clark, Biotechnol. Bioeng. 27, 471 (1985).

436


[40]

croorganisms screened were intended for use in a nongrowing immobilized form it proved advantageous to screen them in this state so as to obtain an organism with a high activity, but also with a high stability when used in an immobilized form. Samples of each cell strain were grown and immobilized separately. Growth was aerobic at 30 ° in 500-ml shake flasks, containing 200-ml aliquots of medium consisting of sucrose (40 g/liter), which acts as a carbon and energy source and also induces the enzyme, peptone (10 g/liter), and beef extract (4 g/liter). Several strains with a high activity were obtained, three of which, all Erwinia rhapontici strains, had high operational stabilities when tested in columns. Evidence for a periplasmic location for the isomaltulose-synthesizing activity was provided by the observation that the addition of a nonlethal dose of the antibiotic lincomycin (10 rag/liter) to the complex medium enhanced the cells activity by 60%. Lincomycin is known to limit the synthesis of, or otherwise inhibit proteases that degrade other periplasmic proteins. 23 On a large scale, cells were grown in a defined medium developed as part of our process improvement program, and flocculated with a chemical flocculant prior to concentration by centrifugation. Use of a nonsucrose-limited medium was advantageous so that once growth had finished some sucrose remained to stabilize the enzyme activity. Due to the difficulties in recovering small bacterial cells using a Westphalia disk-stack centrifuge, flocculant was added to aggregate the cells and thus increase their rate of sedimentation. The cationic polyquaternary flocculants Cyanamid C573 and C577 proved to be most effective. The precise dose of flocculant was optimized for each fermentation in laboratory experiments immediately prior to addition to 800 liters of fermenter broth. The flocculant was added diluted 10-fold in water and mixed gently in the fermenter for 10 rain to ensure even distribution prior to recovery of the cells by centrifugation. Addition of excess flocculant was deleterious since the strength of the alginate pellets was weakened. During growth capsular polysaccharide was produced, which interfered with the recovery of the cells in a concentrated form by centrifugation. The extent of polysaccharide production was proportional not only to the amount of sucrose present, but also to the degree of agitation of the culture medium. Since Erwinia is a facultative anaerobe the polysaccharide is probably produced in response to high pO2 values, the capsular polysaccharide imposing a diffusional restriction on the rate of oxygen transfer from the bulk medium to the cells. Polysaccharide production appeared to be at the expense of isomaltulose production such that high 23 M. Levner, F. P. Wiener, and B. A. Rubin, Infect. Immun. 15, 132 (1977).

[40]


437

activity cells were obtained from poorly agitated fermentations and vice versa. Once immobilized, the cells are in a relatively anaerobic environment due to the difficulty oxygen has in diffusing into the pellet, and no further polysaccharide is produced and more isomaltulose is formed. Immobilization The cells are collected by centrifuging at 23,000 g for 20 min at 30° 21 (see Fig. 1). The cells are evenly dispersed as a 20% (wet weight/v) slurry in freshly prepared 5% (dry weight/v) sodium alginate using an overhead turbine impeller. Dissolution of the alginate powder is much easier if warm deionized water is used. Alginate is supplied in a sterile form and can be dissolved and immobilized in autoclaved solutions if required. 24 Immobilization on a small scale is by extruding dropwise from a syringe. On a larger scale the cell slurry could be pumped through narrow-bore tubing. Gelation takes about 2-3 hr to complete at room temperature using a large excess of 0.1 M CaC12 solution (pH 6.5). Even-sized, smooth, spherical pellets with a diameter of 3-5 mm and consisting of a thin, highly polymerized skin and a core of highly porous gel are formed. Sodium alginate (Protanal LF10/60, BDH) extracted from Laminaria hyperborea, which has a high ratio of guluronic to mannuronic acid blocks and thus a high mechanical strength, is preferred in all the experiments. The gel is formed by calcium ions cross-linking guluronic acid residues in adjacent polysaccharide c h a i n s . 24 Many of the alginates tested were relatively unsuitable because of their low gel strength. On a large scale cells were immobilized by extrusion through a stainless-steel plate containing - 3 6 holes of very small diameter. The plate was coated with PTFE to facilitate extrusion. Several liters of pellets could be produced per hour using this method. The size of the pellets is governed by the diameter of the orofice used and the viscosity of the cell slurry. Thus the viscosity of the alginate is very important in order to achieve good results, 2.5% (w/v) Protanal LF10/60 having a viscosity of - 5 0 0 centipoise (cP) at 20°, for example. Spherical pellets were obtained when the droplets were allowed to fall from a height of about 30 cm into the CaCI2. Alginate gel pellets could be solubilized by a variety of substances such as EDTA or sodium hexametaphosphate. 22 The formation of isomaltulose from a pure sucrose solution was then monitored, so that no significant growth of the cells could take place. Several organisms produced isomaltulose; others either did not produce 24 C. Bucke, this series, Vol. 135 [15].

438


[40]

Growth Media preparation & sterilization

l Growth of cells

Harvesting of cells ~ wastes (spent media) (centrifugation; flocculatlon)

Dissolution of alglnate ---gSlurrying of cells in alginate

l Immobilization by extruding into CaCl 2

t Draining off of CaCl 2 and packing into columns

Exhausted ( immobilized biocatalyst (waste)

L

Operation of immobilized cell ( reactors

Substrate preparation

Product (column eluate)

L

Crystallization Wastes (side products etc)

Centrlfugatlon (including washing)

l

Drying and Sieving

1

Product packaging

FIG. 1. A flow diagram showing the m~or unit operations in the immobilized cell process for producing isomaltulose. From Cheetham e t al. 22

[40]


439

isomaltulose at a sufficient rate, were difficult to grow, produced pigment, or were potentially unsafe. 22 Although the Serratia and Protaminobacter strains had higher initial activities, three strains of Erwinia rhapontici, classified as Enterobacteria agglomerans by API tests, proved to be most suitable because their activity was very much more stable when the cells were used continuously in an immobilized nongrowing form. 22 All of the organisms tested produced trehalulose as well as isomaltulose. About 85% of the activity is initially retained following immobilization, comparison of the freshly immobilized pellets being made with free cells shaken in substrate with alginate pellets. The preferred strain of cells NCPPB 1578 was isolated as a secondary pathogen of rhubarb and was obtained in a yield of about 7 g wet packed cells per liter of medium ( - 2 × 109 viable cells per gram wet weight2~). No synthesis or degradation of enzyme appeared to take place following harvesting of the cells and prior to use of the immobilized cells. This is because the activity of both logarithmic and stationary phase cells, and cells stored for 5 hr prior to immobilization, was unaffected by the addition of chloramphenicol either to the growth medium, the alginate, or CaCI2 used for immobilization, or to the sucrose substrate. Characterization of the Cell-Free Isomaltulose-Forming Enzyme Isomaltulose formation is mediated by a single enzyme that is located in the cell's periplasmic space and that is solubilized most easily by osmotic shocking, typically by rapidly resuspending 1.5 g wet weight cells in 150 ml of ice-cold deionized water. 2~,25The enzyme is a previously undescribed hexosyltransferase with no ion or cofactor requirements and with four novel features. Unlike most other glycosyltransferases the enzyme is sucrose specific. Second, it has an intramolecular mechanism using only the glucose and fructose derived from sucrose. Labeled glucose and fructose were not incorporated in experiments in which unlabeled glucose and fructose (both 0.5 M) and ~4C-labeled glucose and fructose were added to 1.6 M sucrose to give a working radioactivity of 10/xCi/ml. After assay at 30° and pH 7.0, 2-tzl samples diluted to 2% (w/v) were chromatographed and the plates incubated with X-ray film (Kodak, Hemel Hempstead, Herts, United Kingdom) for several days before developing. Results were quantified by scraping off each spot and counting in a toluene-based scintillant containing 2,5-diphenyloxazole (4 g/liter), 1,4-bis(5-phenyloxazol2-/xl)benzene (0.1 g/liter), and Fisons Mix No. 1 emulsifier with a 104 cpm external standard. 25 Thus both the glucose and fructose moieties of 25 p. S. J. C h e e t h a m , Biochem. J. 220, 213 (1984).

440


[40]

sucrose appear to be enzyme bound. Third, the enzyme is very unusual in that it resembles chemical catalysts rather than biocatalysts, since it displays reaction nonselectivity forming simultaneously isomaltulose and much smaller quantities of a second hitherto uncharacterized disaccharide which has been given the neologism trehalulose (1-O-a-D-glucopyranosyl-t~-D-fructopyranose). The trehalulose could be purified by chromatography on a 115 × 2.2 cm column of Dowex AGW X40 (200-400 mesh, K + form) or by preparative high-performance liquid chromatography with a Waters Prep LC/system 500A, with four Prep Pak 500/C1s columns (30 × 5.7 cm chain each) used in series by means of an auxiliary radial compression unit, both with degassed distilled water as the eluate. 25 13C NMR showed that the fructose moiety of the trehalulose exists predominantly in the pyranose rather than furanose. Trehalulose appears to be the first such oligosaccharide; previously combined fructopyranose has only been observed in oligosaccharides substituted in the C-5 position of the fructose. 26 An additional characteristic of the enzyme is that upon extended incubation a recycling mechanism causes the concentration of isomaltulose, the kinetically preferred product, to reach a transient maximum and then fall, and the concentration of trehalulose, the thermodynamically preferred product, to rise slowly. Thus after 10 days an isomaltulose to trehalulose ratio as low as 1.15 : 1 is reached. This is because although the rate of formation of trehalulose is only about 15% of that of isomaltulose, trehalulose is broken down much less rapidly than isomaltulose, such that a net accumulation of trehalulose takes place. Isomaltulose, but not trehalulose, could be converted to sucrose by the enzyme working in reverse. Therefore trehalulose appears to be the lower energy species. For a more detailed explanation of this phenomenon see Kashe et al. 27 The enzyme has been named isomaltulose synthease and should be classed in the EC 5.4.99.--group. 25 By contrast the enzyme from P. rubrum is not sucrose specific and can transfer glucose from sucrose to arabinose or xylose,28 which indicate that it does not have an exclusively intramolecular mechanism as with the E. rhapontici enzyme. Only - 5 % yields of products were obtained, but recently Hashimoto et al. 29 have shown that much higher yields are obtained using unnatural substrates, e.g., when 6-chloro-6-deoxysucrose as donor and methyl/3-D-arabinofuranoside as acceptor were used, a 72% yield of 5-O-o~-D-glucosylpyranosyl-/3-D-arabinofuranoside was obtained. z6 R. S. Shallenberger, "Advanced Sugar Chemistry," pp. 223-231. Horwood, Chichester, England, 1982. 27 V. Kashe, U. Haufler, and L. Riechmann, this volume [26]. 2s W. Mauch and S. Schmidt-Berg-Lorenz, Z. Zuckerind. 14, 375 (1984). 29 H. Hashimoto, M. Sekiguichi, and J. Yoshimura, Carbohydr. Res. 144, (1985).

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441


Interestingly neither P. rubrum or E. rhapontici enzymes acted on galactosucrose, the C-4 epimer of sucrose. Choice of Immobilization Method Whole cells were immobilized by various methods and the activity and stability of the freshly immobilized cell preparations tested (Table I). Several methods gave good yields of permanently immobilized enzyme activity and in one case, adsorption to DE-52 cellulose (B.D.H. Ltd) in which 2 g of wet cells was mixed with l0 ml of a thick slurry adjusted to pH 7.0 with Tris-HCl buffer, the activity was slightly higher than that obtained using alginate. However all the preparations were very significantly less stable than the cells entrapped in alginate (Table I). 22 Alginate immobilization also has the advantage of being a cheap and simple method using food-grade materials and uses a material with a relatively low solution viscosity but with a good strength when a gel is formed. On a large scale pellets were formed by extrusion under pressure through multiple holes of 0.5 mm diameter drilled in a 200 x 2 mm stainless-steel plate coated with PTFE. Alginate gels could be formed using calcium or several other periodic group 2 cations such as aluminium. Calcium was preferred because of the higher activities obtained and the greater mechanical strengths of the pellets. The concentration of CaCl2 (0. l M) used and the period allowed for immobilization (2 hr) did not significantly affect the activity of the

TABLE I COMPARISON OF THE ACTIVITY AND STABILITY OF Erwinia rhapontici CELLS IMMOBILIZED BY VARIOUSMETHODSa

Immobilization technique

Activity (g product/ g wet cells-hr)

Half-life (hr)

Conversion achieved (%)

Calcium alginate DEAE-cellulose Polyacrylamide Glutaraldehyde-aggregated ceils r-Carrageenan-locust bean gum Bone char Agar Xanthan-iocust bean gum

0.325 0.583 0.13 0.153 0.263 0.01 0.34 0.10

8,500 400 570 40 37.5 25 27 8

99 87 50 23.5 38 25 27 39.5

From Cheetham et al. 22 Note that no activity was retained following immobilization in collagen or cellulose acetate. Because of the interdependence of stability and the degree of conversion2x both parameters are quoted above.

442


[40]

cells. The mechanical strength of the pellets was proportional to the concentration of alginate used. Pellets were of even size such that handling was easy and sieving prior to use was not required. The gel could also be extruded in the shape of ropes which were then wound or cut into sections, or formed as blocks which were granulated. These preparations had similar activities to those of the pellets, as did cells which had been immobilized in fibrous rather than gelatinous calcium alginate by injecting the cell-alginate slurry into a highly sheared CaC12 solution such as is formed in a domestic food blender. 22 When the gel was formed slowly by the inclusion of dicalcium phosphate and 1,5,-gluconolactone as a calcium-chelating agent, the activity was only a third of that achieved by the standard method. Mechanical Strength of Pellets Columns of alginate-immobilized cell pellets had excellent physical properties. Microbial degradation was never observed although microorganisms possessing alginate lyase did attack sodium alginate solutions stored for long periods. Substrate transfer into the pellets was rapid, no leakage of cells took place, and low-pressure drops were generated even when 55% (w/v) sucrose was pumped through columns of pellets at high flow rate (pressure measured by a pressure gauge between the pump and the column inlet). 3° Columns were relatively incompressible. The compressibility of the column is defined as the reciprocal of the bulk compressive modulus, that is, the change in column volume per unit pressure applied under defined conditions. Constant strain rate and stress relaxation tests in which high pressures were applied to test columns of pellets were used to simulate the behavior of much larger columns. Use of an Instron gel tester Model 1140 (High Wycombe, Bucks, UK) fitted with a 0.019-m-diameter probe moving at 0.83-6.7 × 10-3 m/sec and exerting pressures of up to 13158 kg/m 2 demonstrated that the columns had nonlinear viscoelastic behavior. 3] Compressibility decreased with the concentration of alginate used to form the pellets but increased as the concentration of entrapped cells was increased. Pellets were not fractured unless very high pressures were used and deformation was only partially reversible. Over long periods large creep effects were observed which decreased exponentially with time 3j (creep is the slow and continuous deformation commonly observed in ductile materials which in this case results in an exponential reduction in the void volume of the column). Compres3o p. S. J. Cheetham, K. W. Blunt, and C. Bucke, Biotechnol. Bioeng. 21, 2155 (1979). 31 p. S. J. Cheetham, Enzyme Microb. Technol. 1, 183 (1979).

[40]


443

sion was markedly reduced when the pellets were partially dried in a fluidized bed drier prior to use. Use of Immobilized Cell Reactors A column of regularly packed alginate pellets proved to be the obvious choice of reactor configuration, due to the higher density of pellets that could be achieved than is possible in a stirred or fluidized reactor, and because attrition of pellets occurred during prolonged use in agitated reactors. Moreover high degrees of conversion of substrate into products were achieved easily because of the plug-flow kinetics characteristic of columns. Up to 93% conversion was achieved in batch or continuous stirred reactors whereas greater than 99% conversion was regularly maintained in packed beds of up to 1 m 3 in size with the result that much higher yields of pure crystalline product were obtained. Isomaltulose could be detected by thin-layer chromatography for 16 hr in butanol/ethanol/water, ( 5 : 3 : 2 , v/v/v) using Merck silica gel plates, and then staining with carbazole for free and combined fructose. 32 The concentration of isomaltulose in the column eluates could be measured with - 9 5 % accuracy by assaying the reducing sugar concentration." Separation of sucrose, isomaltulose, and trehalulose by HPLC was difficult but good resolution was obtained using a Spherisorb 5-/zm silica column (Waters Assoc., Northwich, Cheshire, UK). zl Subsequently even better resolution was obtained using a Zorbax amino column (25 x 0.46 cm I.D.) (Du Pont Ltd., Hitchin, Herts., UK) which had been treated with acetonitrile/water (82 : 18, v/v) and maintained at 22°. This solvent was also used as the eluting solvent at 2 ml/min. Sugars in the eluate were detected using a Waters R401 refractive index detector. The assumption is made in calculating results that all the sugars have the same refractive index. Typical retention times obtained using a new column were for fructose 7.1 min, glucose 8.7 min, sucrose 15.1 min, and isomaltulose 17.3 min. Substrate was pumped through the immobilized cell columns using Watson-Marlow pumps (Falmouth, Cornwall). The relationship between the degree of conversion achieved and the residence time is shown in Fig. 2. The relationship was independent of the size of the reactor, provided that the hydrodynamic properties of the column remained constant. This curve is similar to the WF curve used extensively in chemical engineering. The flow rate was adjusted at frequent intervals such that the desired degree of conversion of sucrose into isomaltulose was maintained con32 S. Adachi, J. Chromatogr. 17, 225 (1965). 33 A. Astoor and E. J. King, Biochem J. 56, 44 (1954).

444


[40] II)

100

O

80

1.6 . ~

c~

O

._o

~.---

c~ cO

0

"-~ g 4~

0"8

'0

"~

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0.9

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Flow-rate (column volumes / h) FIG. 2. The effect of the degree of conversion of sucrose into products on the activity of columns of immobilized Erwinia cells. 2~ (11) Activity (g isomaltulose/g wet weight cells per hour); (A) percentage of the immobilized cells. (©) Molar concentration of isomaltulose in the column eluate. The initial flow rate used was about 0.1 empty column volumes of substrate/hr, giving a residence time of just over 3 hr (assuming the column has a void volume of 33%).

stantly high (Fig. 2). Thus the half-life of the column represents the time taken for the flow rate through the column to be reduced to one-half of its original value. The half-life value obtained has direct industrial relevance in that the quantity and quality of the eluate usually need to be maintained constant, for instance to facilitate subsequent processing of the product. A lag phase of about 24-36 hr occurred before a steady state was established, during which concentration gradients of reactants were formed in the column and a very limited amount of cell growth took place. The activity of the immobilized cells was optimal at 30° and pH 7.0, although it was most stable at the acidic pH values (pH 4.0-4.5) maintained by the small quantities of acid formed by the cells. Little color was generated during reaction and no microbial or chemical degradation of the pellets was observed. Activity was maximal with an initial productivity of about 40 g isomaltulose/liter reactor volume per hour when the cells contained 20% (wet wt/v) cells; higher concentrations resulted in excessive internal diffusional restrictions. The activity of the immobilized cells was independent of their aspect ratio (height to diameter ratios 3 to 48 : 1). 22 A contact time of about 3 hr was required to achieve 99% conversion of the substrate. No fouling problems were encountered.

[40]


445

Stabilization

Immobilization in alginate stabilized the activity of both the intact cells and cell-free extracts. However, the stability of the immobilized whole cells was much greater than when a cell-free enzyme extract or mechanically disrupted cells were used (Table II), although the initial activities of these preparations were not appreciably different. Cells were mechanically disrupted by mixing 2 g wet wt cells with 2 g of dry sand and 2 ml of deionized water and shaking at maximum amplitude for 20 min at room temperature in a Mickle shaker (Laboratory Engineering Co., Gomshall, Surrey, UK) or by sonication as a 10% (wet wt/v) suspension in deionized water at 2.8 A for 340 min using a probe with a tip of diameter 2.5 cm and cooling the sample at 5-min intervals. 21 Viable cells were not essential for isomaltulose formation, because the activity of the cells decayed slowly and linearly with time, with a half-life of 1 year, whereas the viable cell count declined rapidly with a half-life of 300 hr, probably due to a lack of nitrogenous and trace nutrients. Viable cells counts were made by grindTABLE II OPERATIONAL STABILITIES OF THE ISOMALTULOSE-FORMING ACTIVITIES OF VARIOUS E. rhapontici

PREPARATIONS a

Type of enzyme preparation used and conditions of use Free cells maintained in exhausted growth medium Free cells used batchwise in 0.12 M sucrose Free cells used batchwise in 1.60 M sucrose (28% conversion) Immobilized cells used with 0.12 M sucrose (70% conversion) Immobilized cells supplied with 0.365 M sucrose in 100 mM HEPES buffer, pH 7.0 (95% conversion) Immobilized cells used with 1.60 M sucrose (70% conversion) Immobilized cells used with 1,60 M sucrose (99% conversion) Immobilized cell debris (45% conversion) A cell-free extract obtained by osmotically shocking cells used batchwise An immobilized cell-free extract (81.5% conversion) Immobilized cells supplied with affination syrup diluted to 1.6 M sucrose and adjusted to pH 7.0 (76% conversion) Immobilized cells supplied continuously with growth medium (90% conversion) Immobilized cells used in a continuous stirred reactor maintained at pH 7.0 Immobilized cells supplied with 1.02 M isomaltulose

Operational stability (half-life, hr) 335 25 41 55 295 2,265 8,625 190 26 620 386 76 341 300

From Cheetham et al. 2~ Unless otherwise stated preparations were tested in packed columns supplied with 1.60 M sucrose substrate. 2~m

446


[40]

ing the pellets in phosphate-buffered saline (PBS), plating serial dilutions of the PBS extracts onto nutrient agar, incubating at 30° for 24 hr, and then counting the numbers of colonies formed. Total suspended cell measurements were calculated from A540 readings of the PBS extracts made versus a suitable blank solution. 21 A small amount of metabolic activity was retained by the nonviable but structurally intact immobilized cells as they continued to produce aketo acids throughout the useful life the columns. 21 The acids were measured as pyruvate equivalent by the method of Slonekar and Orentos. 34 No partitioning of sucrose between the bulk substrate and the immobilized cells was observed. A crucial factor in the success of this process was that the immobilized cells were both most active and maximally stable when high concentrations of sucrose were used, irrespective of the extent of conversion achieved, reaching a maximum at 46% w/v (55% w/w or 1.6 M). 2~ Only the immobilized and not the free cells were markedly stabilized by the concentrated sucrose solutions (Table II). The use of concentrated sucrose was additionally advantageous because the equilibrium between isomaltulose and sucrose was favored. Furthermore the use of concentrated sucrose reduced the formation of gaseous and acidic side products, the growth of contaminant microorganisms, and the activity of endogenous proteases, while greatly reducing the size of equipment required, the volumes of syrup that had to be manipulated, and the volume of water that had to be removed by evaporation prior to crystallization. 2~ The maximum concentration of sucrose that could be used was about 1.6 M. When high degrees of conversion of substrates containing higher concentrations of sucrose were achieved the solubility of isomaltulose at 30 ° was exceeded such that crystals of isomaltulose formed in the upper part of the column. The presence of crystals impeded the flow of substrate, increasing the pressure drop through the column and eventually causing complete blockage. The extent of conversion of sucrose into isomaltulose could be maximized ( - 1 % w/v sucrose remaining) and the generation of trehalulose minimized by controlling the flow rate of substrate through the column and thus optimizing its contact time with the cells (Fig. 2). Increases in the extent of conversion were accompanied by decreases in activity and by a marked increase in the stability of the immobilized cells (Table II). 2L22 Stabilization may be due to stabilization by the product or by both the product and substrate, or to the imposition of diffusional restrictions on the supply of substrate to the immobilized cells, since stability could be increased by raising the concentration of cells entrapped in the pellets. 34j. H. Slonekar and D. G. Orentos, Nature (London) 194, 478 (1962).

[40]


447

Stability may also be connected with the low rate of endogenous metabolism of the cells or with their plasmolysis by the high sugar concentrations. The purity of the sucrose used was also important, since both activity and stability were greatly reduced when affination syrup, an impure sucrose stream produced during refining, was used. Little is known about the control of enzyme turnover in microbial cells 35but it was shown that loss of activity was not due to the action of contaminant microorganisms. No enzyme induction or turnover could have occurred in the immobilized Erwinia cells, first, because of the high purity of the substrate used and second because addition of chloramphenicol or benzylpenicillin (both 500/xg/ml) to the substrate, which inhibit protein and cell wall synthesis, respectively, did not affect stability. Stability was, however, enhanced by the use of buffered substrate (100 mM HEPES) and the use of packed columns rather than a 500-ml continuous stirred reactor containing 200 ml of pellets, maintained at pH 7.0 and supplied with fresh substrate at a rate of 0.016 liter/hr (Table II). Similarly, a fluidized bed reactor, in which the reactants were recycled through the reactor at very high flow rates and neutralized with NaOH after each cycle, was not advantageous. Detailed analysis indicated that the major factor governing the loss of activity is the presence of a very low concentration of cumulative inhibitor in the substrate. This is because the stability of the immobilized cells is best correlated with the volume of substrate passed through the columns. Thus irrespective of the stability achieved, the volume of substrate processed per half-life was virtually constant (Fig. 3). No loss of material from the beads was apparent even after long periods of continuous operation, and the beads retained full mechanical strength as measured with the Instron gel tester. Thus in order to achieve high productivities it is advantageous to allow complete conversion of substrate. Under these conditions the volume of substrate processed by the immobilized cells, the amount of inhibitor the enzyme is exposed to, and consequently the loss in enzyme activity are minimized. 22 The gene for the isomaltulose synthase is in effect "altruistic" since it results in the formation of a product that stabilizes the whole cells and so is beneficial to all the cells genes. Regeneration of the Immobilized Cell Activity After considerable decay of activity had taken place while using sucrose feedstock, regeneration could be achieved simply by pumping growth medium up the column at intervals, provided the medium did not contain an excess of calcium-chelating agents which would cause the 35 R. L. Switzer, Annu. Rev. Microbiol. 31, 135 (1977).

448

ENZYME ENGINEERING

[40]

(ENZYME TECHNOLOGY)

lO00

Volume of substrate processed / unit

I

f

column volume / half-life (llllt12) SO0

0

i $

i lO

Stability (hx 10 3) FIG. 3. The effect of the volume of 1.6 M sucrose substrate passed through a column of immobilized cells (liter of substrate processed/liter of column volume per half-lifeof use) and the operational stability of the isomaltulose-formingactivity of the cells (half-life).From Cheetham et al. 22 alginate to be solubilized. The extent of regeneration was slow, the cells having a doubling time of 64 hr, and was approximately proportional to the viable cell count prior to the administration of nutrients. Regeneration was limited by the space available for fresh cells inside the gel, although some cells did grow out of the alginate gel. Dissolved benzylpenicillin or chloramphenical prevented regeneration, indicating that the d e n o v o activity is entirely accounted for by the growth of fresh cells as enzyme induction, renewal, or derepression could have occurred in the presence of benzylpenicillin. 22 Crystallization of Isomaltulose Syrup eluted from the immobilized cell columns routinely contained less than 0.02 M sucrose and about 1.2 M isomaltulose plus side products: some glucose, fructose and sucrose, but chiefly trehalulose. The crystallization of isomaltulose is exothermic (A G = + 20 kcal/mol). The preferred method of crystallizing isomaltulose is to concentrate the eluate to about 2.0 M isomaltulose by rotary evaporation at no higher than 60 ° to minimize color formation, and then allow the syrup to cool. Seed crystals are next added to the agitated solution. Nucleation and crystallization then take place. The crystals are recovered by basket centrifugation and

[40]


449

washed with water spray, dried in a fluidized bed drier, and sieved to give a purity of greater than 99% and an overall yield of about 65%. Thus the isomaltulose has been considerably purified and concentrated. Crystals contained 1 tool of water of crystallization per mole of isomaltulose 2Lz2 and have a melting point of 118-122 °, a solubility at 30° of - 7 0 g/100 ml water, a bulk density of 0.65-70 g/ml, an absolute density of 1.45 g/ml, and an E R H (equilibrium relative humidity) of 25-32% water at 80% and 22°. As the extent to which the column eluate was concentrated prior to crystallization was increased the yield of crystals increased, but their purity fell. Product was in the form of white, free-flowing crystals with chemical and microbiological purities similar to those of granulated sucrose; that is within twice the American Bottlers Standard (less than 200 mesophiles, 10 yeast, and 10 molds per l0 g). Following crystallization a mother liquor remains that contains sucrose, glucose, fructose, trehalulose, and uncrystallized isomaltulose. Applications for this syrup would be very useful to improve the profitability of the process. Storage Stability It is often important to be able to store immobilized cells without incurring excessive losses in enzyme activity. Such a facility would be useful if the cells need to be stored prior to use or when they are being transported from their site of production to the factory. Common methods of preservation used in the food industry include irradiation, dehydration by drying or freezing, chilling, or use of extremes of pH or osmotic pressures. The centrifuged cell pellet and cells slurried in alginate have moderate stabilities consistent with easy immobilization (Table III), but are insufficiently high to allow for storage, etc. Several procedures were found useful and practical in prolonging storage stability. 36 These included partially drying the immobilized cell pellets, typically to about 4050% of their original weight with a stream of warm air (30-40°), for instance in a fluidized bed dryer, storing the pellets soaked in glycerol (1 : 1-3, v/v) and under an inert atmosphere of nitrogen and including bacteriostatic agents such as benzoic acid esters (Nipa Laboratories, Ltd., Pontypridd, Glamorgan, United Kingdom) at concentrations of 0.10.6%, e.g., Nipasept Na ÷ at 0.06% (w/v). When these methods were employed no losses in activity could be measured after 500 hr storage in sealed containers at room temperature, whereas in the absence of these treatment substantial losses in activity and often considerable microbial 36 p. S. J. C h e e t h a m , U.S. Patent Application 4,443,538 (1984).

450


TABLE III STORAGE STABILITY OF THE ISOMALTULOSE-SYNTHESIZING ACTIVITY OF VARIOUS ORGANISMS AND UNDER DIFFERENT CONDITIONS a

Organisms/conditions E. rhapontici NCPPB 1578' 1739 139 ATCC 29284 pellets plus a 2-fold P. rubrum excess of glycerol

NCIB 2878 S. marcesens NCIB 8285 Freeze-dried Centrifuged cell paste Alginate slurry NaCI (saturated) Ammonium sulfate (saturated) Glycerol : pellets, ratio 1:1 1:2 1:3 1:5 Glycerol + Nipasept Na ÷ (0.06%) + Nipacombin A (0.01%) + Nipaheptyl (0.0125%) Penicillin G (1.76 mg/ml) Chloramphenicol (1.76 mg/ml) Dried pellets in glycerol (ratio 1 : 2) plus Nipasept Na ÷ (0.06%) Nipacombin A (0.01%) Nipaheptyl (0.0125%) PMSF Dipicolinic acid

Storage stability (half-life, hr)

I100 1086 1471

1444 575 125 124 101 250 1550

2500 700 410 556 625 1563 6250

2420 33O

o From Cheetham) 6 Results were usually calculated from the loss in original activity following 500 hr in sealed storage containers at 20°. oo indicates that within the period of the storage trial there was no loss in activity. Note also that a number of compounds related to glycerol such as ethylene glycol, butanediol, hexyleneglycol, 1,3-propanediol, and polyethylene glycol all gave half-lives of less than 300 hr. In these experiments strain NCIB 1578 was used unless otherwise stated.

[40]

[40]


451

contamination were observed even after comparatively short time periods (Table III). These storage methods were useful for all the isomaltuloseproducing organisms tested, including ATCC 29284 which has a relatively poor operational stability. 22,3~ Although drying denatured some enzyme activity, the volumetric activity of the pellets was actually increased due to the reductions in the weight and volume of the pellets caused by the drying. Drying also had the effect of increasing the mechanical strength of the pellets, and of making them smaller, less bulky, and more evenly flowing. Little rehydration occurred even after long periods of operational use. Other common preservation methods either caused excessive loss of activity or caused a loss of pellet strength; these methods included freeze-drying and addition of NaC1 or (NH4)2SO4 (Table II1). 36 Several effects appear to be involved. First, water activity is reduced due to drying and dehydration by the glycerol, together with the associated reduction in oxygen content due to the lower water content. For instance, after storage of dried and undried pellets in a 2-fold excess of glycerol for 500 hr the water activity of the glycerol had increased to aw values of 0.73 and 0.78, respectively. Also the solubility of oxygen was reduced by 45% in a 45% glycerol solution. Second, glycerol may specifically stabilize the enzyme and also act as a carbon source to support the maintenance requirements of the cells. Third, microbial contamination is discouraged by the low oxygen and water concentrations and the high osmotic pressure. An absence of microbial contamination would appear to be especially important, because antibacterial agents such as the Nipa agents or penicillin had such a marked stabilizing effect and because the addition of a saturated solution of the protease inhibitor phenazinemethane sulfonylfluoride (PMSF) alone had a marked stabilizing effect 36 (Table III). Addition of dipicolinic acid, which is present in high concentrations in bacterial spores, had no effect. Other Immobilization Methods Subsequent to Tate and Lyle's patent applications several other companies have also described the use of immobilized cells to produce isomaltulose. The South German Sugar Co. has disclosed a method similar to that used for immobilizing glucose isomerase, involving flocculating Protaminobacter rubrurn (CBS 574.77), such as with Primafloc C7 (1%), extrusion into ropes, followed by drying and cross-linking with 0.1% glutaraldehyde. 37 The immobilized cells are then used in a column main37 M. Munir, European Patent Application 81,105,743.9 (1982).

452


[40]

tained at 45-60 ° through which a concentrated sucrose solution (45-75%, w/w) is passed. Complete conversion of the substrate is not required because a first crop of isomaltulose crystals can be obtained and the resulting mother liquor retreated with the immobilized cells to produce more isomaltulose. An overall yield of about 80% isomaltulose is obtained. The use of Serratia plymuthica, Serratia marcescens and Leuconostoc mesenteroides, flocculation with cationic flocculants and chitosan, and immobilization in cellulose acetates and calcium alginate were also claimed. In a later patent application the use o f P . rubrum cells attached to a polymer support was described? 8 The Mitsui Sugar Co. also entraps cells of S. plymuthica (NCIB 8285) in calcium alginate gels followed by cross-linking with slightly acidified 2% polyethyleneimine for 5 min and then 0.5% glutaraldehyde 39,4°at 5 ° for 30 min. The more involved immobilization procedure is necessary because of the tendency of the enzyme to leak from the cells. The cells are grown at 25° with aeration and agitation on a medium containing 5% sucrose, 3% corn steep liquor, 0.3% Na:HPO4, and 0.2% NaCI. The cell slurry is mixed with an equal volume of 4% sodium alginate and extruded through a dye with pores of 0.6 mm diameter into 0.15 M CaCI2. When used in columns to treat a concentrated sucrose solution (40% w/w) at 25 ° the half-life of the immobilized cells is about 23 days and an average activity of 0.037 g/ml column volume per hour is obtained. Similar results are obtained using Protaminobacter rubrum, for which the average activity is 0.15 g/ml-hr and the half-life is approximately 73 days. The optimum pH of the immobilized enzyme is 5.5, the Km is 0.14 M, and the K~ for isomaltulose is 0.31 M. The operating substrate concentration used is 40% (w/w) and the residual sucrose concentration in the column eluate is controlled at 0.8% (w/w), 85% of the sucrose being converted into isomaltulose. The column eluate is then treated with ion-exchange resin, evaporated, and crystallized as it cooled. Further crystals could be obtained from the mother liquor, or alternatively the column eluate could be solidified and pulverized into a crude product. The Mitsui Sugar Co. has announced that it plans to start production of isomaltulose using initially a 600 ton/year pilot plant in Okayama. The product will be initially sold for use in chewing gum and sugar confectionery. The product is to be supplied as crystals and in a liquid form only 70% as sweet as sucrose. A variant on this method has been carried out by Miles Laboratories, inJ8 W. Haase, P. Egerer, G. Schmidt-Kastner, and H. Perrey, European Patent Appfication 0,160,253 (1985). 39 j. Shimizu, K. Suzuki, and Y. Nakajima, United Kingdom Patent Application 2,082,591 (1982). 4o y. Nakajima, Seito Gijutsu Kenkyu Kaishi 33, 55 (1984).

[40]


453

volving contacting the cells with tannic acid, polyethyleneimine, glutaraldehyde, and a epichlorohydrin/polyamine copolymer? 1 The enzyme from P. rubrurn responsible for converting sucrose into isomaltulose has been isolated by disrupting the cells in a Manton-Gaulin homogenizer. 42 This soluble enzyme was then employed entrapped in semipermeable hollow fibers, adsorbed onto CM-cellulose, titanium dioxide, covalently bound to Sepharose, aldehyde resin, sodium silicate, cyanogen bromide-activated starch, dextran 60, sodium alginate, carrageenan, and maleic anhydride/methylvinyl ether copolymer. Complete conversion of sucrose into isomaltulose together with some trehalulose, glucose and fructose could be obtained when used in reactors. The best half-life reported for the immobilized enzyme was 2075 hr. We have also found that cells can be immobilized in alginate that has been solidified in fibrous form, rather than the conventional gel form. Immobilization is carried out by rapidly mixing the alginate-cell slurry with CaCI2, for instance in a blender or Silversen mixer so that solid calcium alginate is formed virtually instantaneously. However, the particles are irregular and so are liable to abrasion and have poor packing properties in columns. The production of trehaluiose from sucrose or isomaltulose by isomaltulose-producing cells or enzymes has been the subject of a patent applicationfl The trehalulose is purified by ion-exchange chromatography and dried. Like isomaltulose, trehalulose is noncariogenic and of low sweetness; unlike isomaltulose it is more soluble and is only slowly and partially hydrolyzed in the small intestine. This would not appear to be a preferred procedure since the yields of trehalulose are likely to be lower than those obtained for isomaltulose. Conclusion Our immobilized cell process for producing isomaltulose (Fig. 1) is characterized by the use of a new enzyme possessing a novel mechanism and by the high process intensity achieved, that is the high concentration of substrate used, the virtually complete reaction obtained, and the very high operational stabilities reached. The immobilized cells are about 350 times more stable than the free cells, a half-life of about a year being achieved. Thus during one half-life of use, a column has a productivity of 41 O. J. Lantero, European Patent Application 82,109,404.2 (1983). 42 C. Kutzbach, G. Schmidt-Kastner, and H. Schutt, European Patent Application 81,107,714.8 (1982). 43 European Patent Application 109-009-A (1984).

454


[40]

just over 200 kg of product/liter column-volume per year; that is a 10-liter column will process 5.2 m 3 of sucrose substrate in one half-life (per year) and the cells can produce about 1500 times their own weight in crystalline isomaltulose. 22 By comparison GI (glucose isomerase) produces 20003000 times its own weight of high-fructose syrup in its operational lifetime. This integrated continuous process has been successfully operated on a small pilot plant scale in which cells were grown in a 1-m 3 fermenter and using 25-liter columns. On a larger scale, several additional features had to be developed. These included a defined medium for the growth of cells that did not contain peptone and beef extract and that contained a bacteriostat to inhibit the growth of contaminated microorganisms, a method of flocculating cells so as to facilitate their recovery from the fermentation medium, and a large-scale method of extruding the cellalginate slurry to form pellets. This process illustrates the desirability of screening for novel enzyme activities from microorganisms isolated from unusual environments, of using simple, mild immobilization methods and high reactant concentrations, of maintaining a steady state in which high degrees of conversion of substrate into product is achieved, and of developing efficient methods of purifying and recovering products. Desirable improvements to the process include the suppression of trehalulose formation and an increase in the volumetric activity of the columns. The major limitation to improvement is the intrinsic properties of the enzyme. Improvements could possibly be achieved by the discovery of an improved isomaltulose-producing microorganism or possibly by site-directed mutagenisis. However since isomaltulose must be more expensive than sucrose it must be shown to exhibit a significantly improved functionality to be a good commercial product. It is hoped that others will also find some of the approaches and methods adopted in this study useful.

[41]

L-MALIC ACID PRODUCTION BY MICROBIAL CELLS

455

[41] P r o d u c t i o n o f L - M a l i c A c i d b y I m m o b i l i z e d Microbial Cells B y ICHIRO CHIBATA, TETSUYA TOSA, KOZO YAMAMOTO, a n d ISAO TAKATA

L-Malic acid is widely used for medicines and food additives. It has been industrially produced from fumaric acid by the fermentative or enzymatic method employing fumarase (EC 4.2.1.2, fumarate hydratase) as biocatalyst. HOOCCH~---CHCOOH + H20 .

fumarase

• HOOCCH2CHCOOH [

OH

An enzymatic batch process using the enzyme of L a c t o b a c i l l u s b r e v i s was reported in 1960 by Kitahara et al. 1 However, from an industrial standpoint, a continuous enzyme reaction system using immobilized enzyme is considered to be more advantageous than a batch process using enzyme solubilized from microbial cells. 2 Marconi et al. 3 reported that fumarase can be efficiently immobilized into cellulose triacetate, and that the immobilized fumarase makes it possible to develop an economically attractive method to produce L-malic acid. Recently, we have developed an industrially very advantageous method for carrying out the above fumarase reaction? -7 That is, microbial cells having high fumarase activity are immobilized in a polymer gel matrices such as polyacrylamide and Kcarrageenan. The immobilized cells are packed into a column, and a substrate solution (sodium fumarate) is continuously passed through the immobilized cell column. An effluent containing L-malate is obtained without contamination from impurities such as microbial cells, components of cultural medium, and others.

t K. Kitahara, S. Fukui, and M. Misawa, J. Gen. Appl. Microbiol. 6, 108 (1960). z R. A. Messing, "Immobilized Enzymes for Industrial Reactors" (R. A. Messing, ed.), p. 1. Academic Press, New York, 1975. 3 W. Marconi, F. Morosi, and R. Mosti, Agric. Biol. Chem. 39, 1323 (1975). 4 K. Yamamoto, T. Tosa, K. Yamashita, and I. Chibata, Eur. J. Appl. Microbiol. 3, 169 (1976). 5 K. Yamamoto, T. Tosa, K. Yamashita, and I. Chibata, Biotechnol. Bioeng. 19, 1101 (1977). 6 I. Takata, K. Yamamoto, T. Tosa, and I. Chibata, Eur. J. Appl. Microbiol. Biotechnol. 7, 161 (1979). 7 I. Takata, K. Yamamoto, T. Tosa, and I. Chibata, Enzyme Microb. Technol. 2, 30 (1980).



456


[41]

Thus, L-malic acid with high purity is obtained in a good yield by this method compared with the batchwise enzymatic method using cultural broth. The fumarase activity of immobilized cell column is fairly stable, and it can be used for a long period of operation. Therefore, we developed in 1974 an industrial process in which Brevibacterium ammoniagenes with high fumarase activity was immobilized in gel matrices of polyacrylamide. In 1977 we changed the conventional polyacrylamide method to the more economical r-carrageenan method. To increase productivity of L-malic acid in this system, improvement of the K-carrageenan method was attempted. It was found that the addition of polyethyleneimine or tannins to the immobilization medium was effective in increasing the stability of fumarase activity. 8,9 Since 1980 we have been operating this improved immobilized cell system for the industrial production of L-malic acid. These methods are described in this article. Assay Methods

Estimation o f L-Malic Acid. L-Malic acid is measured colorimetrically by the method of Goodman and Stark. l° Assay of Fumarase Activity Native Fumarase. A mixture of 0.1 ml of native fumarase and 1.9 ml of I M sodium fumarate (pH 7.0) is incubated at 37° for 10 min. After the reaction is stopped by the addition of 2 ml of 2 N HCI, the precipitates are removed by centrifugation (3000 g). The L-malic acid formed in the supernatant is determined colorimetrically by the method of Goodman and Stark. i0 Enzyme activity is expressed as micromoles of L-malic acid produced per hour. Free Cells. A mixture of 1.0 g (wet weight) of free cells and 30 ml of 1 M sodium fumarate (pH 7.0) is incubated at 37° for 10 rain. The reaction is stopped by the addition of 2 ml of concentrated HC1, and the L-malic acid formed is determined as in the case of native fumarase. Activity of the free cells is expressed as micromoles of L-malic acid produced per hour. Immobilized Cells. A reaction mixture of immobilized cells (amounts corresponding to 1 g of intact cells) and 30 ml of 1 M sodium fumarate (pH 7.0) is incubated with shaking at 37 °, and L-malic acid in the reaction mixture after 15 and 30 min is determined as in the case of native fumas I. Takata, K. Kayashima, T, Tosa, and I. Chibata, J. Ferment. Technol. 60, 431 (1982). 9 I. Takata, T. Tosa, and I. Chibata, Eur. J. Appl. Microbiol. Biotechnol. 19, 85 (1984). 10A. E. Goodman and J. B. Stark, Anal. Chem. 29, 283 (1957).

[41]

L-MALIC ACID PRODUCTION BY MICROBIAL CELLS

457

rase. The fumarase activity is calculated from the increase in L-malic acid concentration during 15 min. Activity of the cell mixture is expressed as micromoles of L-malic acid produced per hour. Immobilization of Brevibacterium amrnoniagenes with Polyacrylamide

Culture of B. ammoniagenes. Breoibacterium ammoniagenes IAM 1645 is cultured under aerobic conditions at 30° for 24 hr in 100 liters of medium (pH 7.0) containing glucose (2 kg), fumaric acid (0.5 kg), urea (0.2 kg), KH2PO4 (0.2 kg), MgSO4.7H20 (50 g), and corn steep liquor (1 kg). The cells are collected by centrifugation. About 500 g (wet weight) of B. ammoniagenes cells is obtained from 100 liters of broth, and fumarase activity of the immobilized cells is approximately 200/xmol/hr per gram of wet cells in flesh state. Preparation of Immobilized B. ammoniagenes. Cells (1 kg of wet weight) are suspended in 2 liters of physiological saline, and the cell suspension is cooled to 8 °. In 2 liters of water, 750 g of acrylamide monomer and 40 g of N,N'-methylenebisacrylamide are dissolved, and the monomer solution is cooled to 8°. Both are mixed at 8° . To the mixture are added 0.5 liters of 5% (v/v) fl-dimethylaminopropionitrile as an accelerator of polymerization and 0.5 liters of 1% potassium persulfate as an initiator of polymerization, and the reaction mixture is allowed to stand at 20-25 ° for 15 min. The gel formed is cut into cubes (3 × 3 x 3 mm) with a knife, and thoroughly washed with physiological saline. Its fumarase activity is approximately 500/xmol/hr per gram of wet cells in fresh state. Suppression of Formation of Succinic Acid and Enhancement of L-Malic Acid Formation As the immobilized cells have an activity that forms succinic acid as a by-product, succinic acid and unconverted fumaric acid accumulate in the reaction mixture. Although fumaric acid can be easily precipitated by acidifying the reaction mixture with hydrochloric acid, it is very difficult to separate succinic acid from L-malic acid. The key step to a successful process, therefore, is to prevent the formation of succinic acid. As shown in Table I, detergents such as bile extract, bile acid, and deoxycholic acid reduce the amount of succinic acid formed by the immobilized cells. The detergents also remove the permeability barriers for substrate and/or product across the membrane of cells entrapped in the polyacrylamide gel, and thus the yield of L-malic acid is increased. Therefore, for industrial purpose bile extract treatment is economically most suitable.

458


[41]

TABLE I EFFECT OF DETERGENT TREATMENTS ON FORMATION OF L-MALIC ACID AND SUCCINIC ACID"

Detergent

Concentration (%)

Formation of L-malic acid (pmol/hr-g of cells)

No addition CPC CPC SLS SL-10 Triton X-100 Bile acid Bile extract Deoxycholic acid

0.02 0.16 0.02 0.02 0.20 0.20 0.20 0.20

990 4570 3070 6050 1220 5360 6570 7480 7380

Treatment

Formation of succinic acid (mol% of Lmalic acid) 2.5-5.0 2.5-5.0 1.0-2.5 1.0-2.5 2.5-5.0 >5.0 6O >

~401

_=,2o @

10

20

30

Temperature

40

50

60

(*C)

FIG. 2. Effect of temperature on enzyme activity. The activity of intact cells was measured as described in Assay Methods at the temperatures in the figure.

[48]

527

AN ACRYLAMIDE PRODUCTION METHOD

tion of 250 ml 10% (v/v)/3-dimethylaminopropionitrile and 250 ml 10% (w/v) ammonium persulfate. The polymerization reaction starts after about 3-5 min. The temperature of the reaction mixture increases to about 30°, and polymerization is complete after 1 hr. The gel is separated from the reactor and made into granules about 1-2 mm in diameter for industrial use and washed with 0.05 M potassium phosphate buffer solution (pH 7.7). The nitrilase activity thus obtained is almost the same as that of the intact cells. In this process, it is very important to keep the temperature of the gel below 30°. An electron micrograph of the immobilized cells is shown in Fig. 1. Nitrilase Properties of Immobilized Cells Strain S-6

Temperature. The optimal temperature for the formation of acrylamide is 35 ° in intact and immobilized cells, but from the viewpoint of enzyme stability, the reaction temperature should be below I0 °. Figures 2 and 3 show the effect of temperature and the heat stability of the intact cells, respectively, and similar results are also obtained with immobilized cells. The activation energy of the immobilized cells is calculated to be 8.8 kcal/mol (at pH 8.0). pH. The pH activity profile of the nitrilase reaction is different for intact and immobilized cells, as shown in Fig. 4. The immobilized cells

I

I

I

i

!

!

2oo

\ '°ti I

--

" I

I

I

I

10

20

30

40

Treatment

I

50 (rain)

I

60

Fro. 3. Heat stability of the acrylamide-forming enzyme. The acrylamide-forming activity of intact cells was measured as described in Assay Methods at the temperature indicated in the figure. Treating conditions: 0.1% cell suspension incubated in 0.05 M (pH 7.7) potassium phosphate buffer solution. Temperatures: (O) 30°; (0)) 40°; (0) 45°; (®) 50°.

528

ENZYME ENGINEERING (ENZYME TECHNOLOGY) r

i

i

[48]

i

100 80 ~ 6o ~ 4O IX 20

PH FIG. 4. Effect of pH on enzyme activity. The acrylamide-forming activity was measured as described in Assay Methods at the pH values shown in the figure. (O) Intact cells; (0) immobilized cells.

I

,.,1o°

I

I

I

I

\

so

~®60,o

20[ I

0

"\ i

I

I

I

10 20 Acrylamide (,olo)

I

I

30

FIG. 5. Effect of acrylamide concentration on enzyme activity. The acrylamide-forming activity of intact cells was measured as described in Assay Methods. Acrylamide concentrations are indicated in the figure.

[48]

AN ACRYLAMIDE PRODUCTION METHOD

529

show an optimal activity at pH 8.5, whereas the optimal pH of the intact cells is 7.7. Acrylonitrile and Acrylamide Concentrations. Acrylonitrile and acrylamide possess a strong toxicity, and would inhibit the enzyme activity and stability. Especially, acrylonitrile influences severely, and so upon reaction it is preferable to keep the concentration of acrylonitrile in the reaction system to a level not exceeding 3% (w/v). Figure 5 shows the effect of acrylamide concentration on enzyme activity at 2.5% (w/v) acrylonitrile. Figure 6 shows the effects of acrylonitrile and acrylamide concentrations on enzyme activity during a semibatchwise reaction in which the acrylonitrile concentration was held almost constant by consecutive additions while acrylamide was accumulating. From these figures, it seems obvious that acrylonitrile (substrate) and acrylamide (product) greatly inhibit the nitrilase enzyme and that it is very important to control the concentrations of them in order to keep a high enzyme activity and stability. In our acrylamide-producing process, we consider that the merits of using the immobilized cells are the prevention of elution of impurities from the cells, increase in the separation of the cells from a reaction mixture, and increase in stability of enzyme toward acrylonitrile and acrylamide.

100;

~ ~

~ 8o ._>

~ 6o ~ 4O ¢Y 2O

0

1~) Acrylamide

i,

I 20 (%)

8

I 30

FIG. 6. Effect of acrylonitrile and acrylamide concentrations on enzyme activity, The acrylamide-accumulating reaction was carried out according to a semibatchwise reaction with a stirred reactor. (Acrylonitrile was added consecutively.) Reaction conditions: temperature, 0-3°; pH, 8.5; immobilized cell concentration, 1%; reaction times, 5-30 hr. Acrylonitrile concentration: (©) 1.0%; (~) 2.0%; (e) 3.0% (w/v).

530


[49]

Acrylamide Production of Immobilized Cells Strain S-6 From these described properties of immobilized cells, we established the acrylamide process conditions at pH 8.0-8.5, temperature below 10°, and acrylonitrile concentration below 3% (w/v). By using our immobilized cells under these conditions, a reaction solution containing more than 20% (w/v) acrylamide can be obtained, i.e., a concentration previously reported to be necessary for an industrial process. I One example of the composition of the reaction solution is as follows: acrylamide, 20% (w/v); acrylonitrile, negligible; acrylic acid, below 0.1% (w/w) per acrylamide. Conclusion As described above, Rhodococcus cells of strain S-6 can be easily immobilized to produce a high nitrilase activity when stabilized by the entrapping method using cationic acrylamide-based polymer gel. We at Nitto Chemical Industry Company, Ltd. have examined the characteristics of such immobilized cells and have also recently designated a suitable bioreactor. We are now producing about 4000 tons of acrylamide per year.

[49] A p p l i c a t i o n o f I m m o b i l i z e d Thiobacillus ferrooxidans for Large-Scale T r e a t m e n t of Acid M i n e Drainage

By T. MURAYAMA, Y. KONNO, T. SAKATA, and T. IMAIZUMI A considerable number of abandoned underground mines still discharge acid mine drainage continuously. It has become necessary to treat the drainage without any complications from an environmental viewpoint. Mine acid is usually caused by oxidation of sulfide minerals, such as pyrite, chalcopyrite, galena, sphalerite, and argentite, which occur in many metalliferous ore deposits, Of these minerals, pyrite is the most important in forming mine acid and releasing toxic concentrations of heavy metals. Thiobacillus ferrooxidans, found naturally in acid mine drainage, increases the oxidation of sulfide minerals and supplies ferrous ion in the acid drainage as the microbial catalyst. Utilizing these characteristics of Thiobacillus ferrooxidans, bacterial oxidation is being adopted in the METHODS IN ENZYMOLOGY, VOL. 136


530


[49]

Acrylamide Production of Immobilized Cells Strain S-6 From these described properties of immobilized cells, we established the acrylamide process conditions at pH 8.0-8.5, temperature below 10°, and acrylonitrile concentration below 3% (w/v). By using our immobilized cells under these conditions, a reaction solution containing more than 20% (w/v) acrylamide can be obtained, i.e., a concentration previously reported to be necessary for an industrial process. I One example of the composition of the reaction solution is as follows: acrylamide, 20% (w/v); acrylonitrile, negligible; acrylic acid, below 0.1% (w/w) per acrylamide. Conclusion As described above, Rhodococcus cells of strain S-6 can be easily immobilized to produce a high nitrilase activity when stabilized by the entrapping method using cationic acrylamide-based polymer gel. We at Nitto Chemical Industry Company, Ltd. have examined the characteristics of such immobilized cells and have also recently designated a suitable bioreactor. We are now producing about 4000 tons of acrylamide per year.

[49] A p p l i c a t i o n o f I m m o b i l i z e d Thiobacillus ferrooxidans for Large-Scale T r e a t m e n t of Acid M i n e Drainage

By T. MURAYAMA, Y. KONNO, T. SAKATA, and T. IMAIZUMI A considerable number of abandoned underground mines still discharge acid mine drainage continuously. It has become necessary to treat the drainage without any complications from an environmental viewpoint. Mine acid is usually caused by oxidation of sulfide minerals, such as pyrite, chalcopyrite, galena, sphalerite, and argentite, which occur in many metalliferous ore deposits, Of these minerals, pyrite is the most important in forming mine acid and releasing toxic concentrations of heavy metals. Thiobacillus ferrooxidans, found naturally in acid mine drainage, increases the oxidation of sulfide minerals and supplies ferrous ion in the acid drainage as the microbial catalyst. Utilizing these characteristics of Thiobacillus ferrooxidans, bacterial oxidation is being adopted in the METHODS IN ENZYMOLOGY, VOL. 136


[49]

APPLICATIONOF IMMOBILIZEDT. ferrooxidans

531

treatment of acid mine drainage at some mines in Japan. In this paper a large-scale treatment of acid mine drainage including a bacterial oxidation system is described as an example of an effective and practical application of the bacteria. Treatment of Acid Mine Drainage To neutralize acid mine drainage and at the same time precipitate heavy metals, lime neutralization has been used for many years, employing calcium hydroxide in many cases. In comparison, neutralization by calcium carbonate has several advantages such as lower cost, less volume, and faster settling of the resultant sludge. However, calcium carbonate simultaneously generates carbonic acid in the course of neutralization, which limits the maximum pH of the drainage to about 6.5. At this pH, the ferrous salt remains in solution and does not precipitate. If the ferrous salt can be oxidized to a ferric salt prior to neutralization, calcium carbonate is made available to neutralize acid mine drainage. Bacterial Oxidation in Treating Acid Mind Drainage To realize the oxidation of the ferrous salt, a bacterial oxidation system was developed at the Yanahara pyrite mine of Dowa Mining Co., Ltd., in 1974, under the guidance of Professor K. Imai of Okayama University, instead of using nitrogen oxide as a catalyst. Thiobacillus ferrooxidans, found naturally in the mine drainage in a concentration of about 106 cells/ml is used for the oxidation. A feature of the bacterial oxidation system developed in Yanahara mine is that the microbes are cultivated and well adsorbed on the surface of basic ferric sulfate, generated by the hydrolysis of ferric sulfate. They are precipitated together with the cell carrier in the next stage, and recycled to the oxidation circuit. In these continuous processes, it is possible to obtain a sufficient concentration of cells, i.e., about 108 cells/ml, and to oxidize 95-98% of the ferrous salt to ferric within 60 min. The mine adopted the improved system in the neutralization of mine drainage, resulting in a considerable saving of the treatment cost--about 320,000 Yen by microbial oxidation compared with 965,000 Yen by nitrogen oxide, based on a monthly treatment of 50,000 m 3 of drainage at pH 2.5 and 2100 mg/liter of ferrous iron. (Patent No. 797734 in Japan.) The bacterial oxidation system was also applied in a partially modified manner to the treatment of acid mine drainage at the abandoned Matsuo sulfur-pyrite mine.

532


[49]

Application of Bacterial Oxidation to the Matsuo Mine 1,2

Background The Matsuo mine, an abandoned mine in the Iwate Prefecture in the northern part of Japan, was a large-scale underground and open-cut mine in which sulfur and pyrite ores had been mined for about 60 years from 1910 through 1971. Total output excavated stood at 29 million tons or 13 million cubic meters. Its mine drainage reached 30 m3/min in the final stage of operation late in the 1960s, at times with strong acidity (pH 1.3 to 1.5). Neutralization by calcium carbonate and calcium hydroxide was being carried out at the mine, but it was difficult to maintain sufficient water quality because of the large quantity and strong acidity of the drainage. This caused acidic pollution and the formation of a red suspension downstream in Kitakami river. The mine was closed in 1971 due to nonprofitability, but mine drainage still discharged continuously from the mine after its closure. The Japanese national government and the Iwate prefectural government have been responsible for the treatment of the drainage after the mine closed, and they decided to apply the bacterial oxidation system developed at the Yanahara mine to the Matsuo mine.

Feasibility Tests for the Application In applying the oxidation system to the neutralization at Matsuo mine, two difficult problems were considered at that time. First, the mine is located in the northern part of Japan in a mountainous area about 1000 m in altitude. Temperatures in winter fall as low as 20° below zero. Thus the problem of whether the cultivation and oxidizing activity of the bacteria could be continuously maintained needed to be examined. Second, basic ferric sulfate, used as the cell carrier at Yanahara mine, cannot be generated in the drainage at Matsuo mine because of its strong acidity level of pH 1.6. Therefore, the National Ministry of Construction entrusted a series of feasibility tests to Dowa Mining Co., Ltd. Under the guidance of Professor K. Imai of Okayama University, Professor J. Shimoiizaka of Tohoku University, and Professor T. Goto of Iwate University, tests were carried out in 1973 and 1974, utilizing Thiobacillus ferrooxidans living naturally in the drainage at Matsuo mine in a concentration of about 2 × 105 cells/ml. The cultivation test of the z E. Yabuuchi and Y. Imanaga, paper presented at the joint Mining and Metallurgy Institute of Japan-American Institute of Mining Engineering Meeting, Denver, Colorado, 1976. 2 T. Ishikawa, T. Murayama, I. Kawahara, and T. Imaizumi, paper presented at the International Symposium on Biohydrometallurgy, Cagliari, Italy, 1983. ("Recent Progress in Biohydrometallurgy." Associazione Mineraria Sarda, 09016 Iglesias, Italy 1983.)

[49]

APPLICATION OF IMMOBILIZEDT. ferrooxidans

533

microbes was carried out by the same method as at Yanahara, i.e., an oxidation-precipitation-recycling system, The results obtained from the tests are summarized as follows: 1. The cultivation and oxidizing activity of Thiobacillus ferrooxidans could be maintained under such severe conditions as those (1.35 °, pH 1.6) of the drainage. 2. Diatomaceous earth was selected as cell carrier because of its strong cell-adsorbing power, acid-resistant properties, and ready settling in tanks which enables it to be recycled to the oxidation circuit. Thus, the researchers succeeded in establishing a new bacterial oxidation system utilizing diatomaceous earth as the cell carrier for treating acid mine drainage at Matsuo mine, converting most ferrous salt to ferric within 60 min after the initial cultivating period of approximately 2 weeks. 3. In the neutralization of the drainage by calcium carbonate, subsequent to the bacterial oxidation, the ferric salt and arsenic compound contained in the drainage were precipitated by raising the pH value to 4. This means that most of heavy metals dissolved in the drainage can be eliminated in the form of precipitates by solid-liquid separation. Applicability of the bacterial oxidization system in the neutralization at Matsuo mine was clarified through these feasibility tests. (Patents No. 4139456 in the United States, No. 1527766 in the United Kingdom, No. 239957 in Canada, and No. 2639045 in West Germany.) Installation o f a N e w Neutralization Plant After the success of the feasibility tests, the Iwate prefectural government decided to install a new neutralization plant including the bacterial oxidation system at Matsuo mine with financial aid from the national Ministry of International Trade and Industry, the authority for the mining TABLE I SPECIFICATIONS FOR WATER TO BE TREATED AND AFTER TREATMENT

Sample

Quantity (mVmin)

Acid drainage Treated water

20 20

pH

Acidity to 8.4 a (mg/liter)

T-Fe b (mg/liter)

A1(mg/liter)

As(mg/liter)

1.6 4.0

4240 786

795 13

189 130

5.77 0.02

S.S c (mg/liter)

30 or less

a Acidity to 8.4 m e a n s an acidity value of water. This is usually indicated by concentrations of calcium carbonate (mg/liter) n e c e s s a r y to raise the pH value of water to 8.4. b T-Fe m e a n s total concentrations o f ferrous and ferric irons in water. c T h e insoluble arsenic contained in 30 mg/liter of S.S (suspended solutions) should be 0.10 mg/ liter.

534


[49]

industry and environmental protection, under the guidance of the Metal Mining Agency of Japan. The fundamental policies for designing the new plant were set as follows and were authorized by the national and prefectural governments. 1. pH. The pH of river water should be maintained, as the target value, within the range 6 to 6.5 at the site of Funata bridge in the Kitakami downstream. 2. Tailings sludge generated by neutralization. The sludge including iron, arsenic, etc. should be stored in a restricted place near the plant, and never be discharged into any river or stream. 3. Capacity of the plant. Specifications for water to be treated and after treatment by the plant are shown in Table I. 4. Form of treating. The drainage should be treated through four lines, each having the treating capacity of 5 m3/min, to treat variable quantities of the drainage. 5. Capacity of sludge dam. The dam for storing hydroxide sludge should be constructed such that its capacity should be sufficient for 20 years' storage. 6. Location of the plant and the sludge dam. The plant should be installed near the portal discharging the drainage and the sludge dam should utilize the nearby marsh. The construction of the plant commenced in August 1977 and was completed in November in 1981. Total installation cost of the plant, including the sludge storage dam, was approximately 9300 million Yen (38 million U.S. dollars) in 1981. After test operation for about 4 months, the plant has been in full operation since April 1982. The Metal Mining Agency of Japan was entrusted with the operation and maintenance of the plant by the Iwate prefectural government. Contents of the Plant The outline of the plant is shown in Figs. I and 2.

Bacterial Oxidation System Bacterial oxidation, the key point of the neutralization system, is shown in Fig. 3. The system includes three processes: oxidation, precipitation, and recycling, as stated. Thiobacillus ferrooxidans is supplied naturally from the flowing drainage upstream, cultivated and well adsorbed on the surface of diatomaceous earth in the oxidation, precipitated and concentrated together with the cell carrier in its settling process subsequent to the oxidation, and

A P P L I C A T I OOF N IMMOBILIZED T. ferrooxidans

[49]

~

535

G 50m ,

#

I

FiG. 1. Layout of principal facilities of the plant. (A) Distribution tank; (B) oxidizing tanks; (C) diatomaceous earth settling tanks; (D) neutralizing tanks; (E) solid-liquid separation tanks; (F) blower house for aeration; (G) pilot plant for metal extraction.

recycled to the oxidation circuit. As was the case with the Yanahara mine, it is possible to maintain sufficient concentrations of cells, about 108 cells/ml, and to oxidize 95-98% of the ferrous salt to ferric within 60 min. However, the behavior of the microbes in these continuous processes is complex and not exactly understood. The oxidation rate of the ferrous salt is observed constantly by an oxidation-reduction potential meter situated at the outlet of oxidizing tank and the information relayed automatically to the control center of facilities. Specifications for the facilities and the materials for the oxidation are shown in Table II.

536


[49]

Acid mine drainage

I Drainage Ireceiving [ tank Nutrient Diatomaceous earth

I 11 DistributiOntank[

I Oxidizing tank I. L Fe'+ ~ F e ' + [

I J

Diatomaceous earth settling tank CaC03 s l u r r y

[ Neutralizing tank

Aeration Coagulant (polymer)

I Aeration Coagulant (polymer) Treated water

Separation tank Sludge to dam

1

Discharge to river FIG. 2. Fundamental flow diagram of the plant.

TABLE II SPECIFICATIONS OF PRINCIPAL FACILITIES AND MATERIALS FOR OXIDATION

Facility and material Oxidizing tank Diatomaceous earth settling tank Diatomaceous earth Coagulant Nutrient

Specifications 580 m 3 of square concrete construction lined with resin; retention time, 60 min 16 m diameter × 5 m height; thickener type; retention time, 100 min Less than the 325 mesh screen; SiO2 83%, A1203 4.8%, Fe203 1.8%, made up in slurry; feeding capacity 21 liters/min Polymer, nonionic; feeding capacity 130 liters/min in solution Ammonium phosphate; feeding capacity 3 liters/min in solution

Number of facilities 4 4

APPLICATION OF IMMOBILIZED T. ferrooxidans

[49]

Nutrient

537

I Diatomace-

ous earth Aeration 11ne

Coagulant

-~Z

J

Distribution tank

Pum For recycling

Oxidizin 9 tank

Diatomaceous earth settling tank

FIG. 3. Flow diagram of bacterial oxidation system.

CaC03 slurry

Diatomaceous earth settling tank

Aeration line

Neutralizing tank

Coagulant

Solid-liquld separating tank

Pumping for sludge transporting

FIG. 4. Flow diagram of neutralization and solid-liquid separation.

538


[49]

Neutralization and Solid-Liquid Separation After bacterial oxidation, the drainage is treated by neutralization and solid-liquid separation. Flow diagrams are shown in Fig. 4. The oxidized drainage is neutralized by CaCO3 slurry in a neutralizing tank to raise its pH value to 4.0, and suspended solids generated by the neutralization are precipitated in a solid-liquid separation tank subsequent to the neutralization. The pH value of water to be maintained after the neutralization is measured with a pH meter at outlet of neutralizing tank, and in the case of failure to reach the settled pH value of neutralized water, an alarm system adjusts the feeding volume of CaCO3 slurry. In solid-liquid separation, the ferric salt and arsenic compound are precipitated as the tailings sludge, extracted from the bottom of the separating tank, and transported by pumping to the sludge storage dam. Treated water, which contains eliminated suspended solids such as ferric salt and arsenic compound, is discharged from the surface of the tank to the river. Specifications for principal facilities and materials for neutralization and solid-liquid separation are shown in Table III.

Sludge Storage Dam The sludge storage dam is constructed near the neutralization plant. The dam is a rock-filled type with a slanting clay core, and is covered T A B L E III SPECIFICATIONS OF PRINCIPAL FACILITIES AND MATERIALS FOR NEUTRALIZATION AND SOLID-LIQUID SEPARATION

Facility and material

Specifications

Neutralizing tank

430 m 3 of square concrete construction lined with

Solid-liquid separation tank CaCO3 slurry Coagulant Blower for aeration

resin; retention time, 45 min 30 m diameter × 4.5 m height; thickener type; retention time, 5 hr Feeding capacity 0.5 mVmin Common to the settling diatomaceous earth Multiple-stage roots blower; 85 N mVmin, ° 1.3 kg/ cm2, b common to the oxidation

Number of facilities 4 4

4

a 85 N mVmin means delivery capacity of air volume of a blower. In this case, " N m TM

means normal m 3, conversion value of air volume under the condition of its pressure, per kg/cm 2. b 1.3 kg/cm 2 means delivery air pressure of blowers.

[49] pH

T. ferrooxidans

OF IMMOBILIZED

APPLICATION

539 Fe z+

Q

ma/min

mg/1

700

\ \

- 600

..'"..

L

•'J"- F e ~ +''' • .

2,0-

".,

i

\

C"'.

\

\

500

/

1.9-

400

•

1.8-

..-

.....

300

I 4 5 6 7 8 9 1 0 1 1 1 2 1 2 3 4 5 6 7 8 91011121 --

I

I

I

I

I

I

I

1982

I

Ill

I

"l

I

I

I

[

I

I

I

I~L-

1983

Ij I

-]4

2 3 4 I

I

I

1984

FIG. 5. Seasonal variation in quantity and quality of acid mine drainage at Matsuo mine.

partially with asphalt facing. The capacity of the sludge pond impounded by the dam is about 2 million m 3, which is sufficient to maintain 20 years' storage. Current Situation of the New Neutralization Plant

The quantity and quality of acid mine drainage have gradually improved since the neutralization plant began full operation in April 1982, although seasonal variations are observed as shown in Fig. 5. Therefore it

T A B L E IV ANNUAL OPERATING COST OF THE PLANT

Year

Annual cost

Annual quantity of drainage

Cost/m 3 of drainage

1982 1983

701 million Yen 682 million Yen

9.06 million m 3 8.74 million m 3

77 Yen (U.S. $0.32) 78 Y e n (U.S. $0.32)

540


[49]

is now possible to maintain the rapid oxidation rate without any nutrient for the bacteria. The annual operating cost of the plant in 1982 and 1983 is shown in Table IV. Technical Development at Matsuo Mine To defray the treatment cost of acid mine drainage and reduce the tailings sludge volume generated from neutralization, the Metal Mining Agency of Japan is studying a new technical development--converting ferrous and ferric iron contained in the drainage to a marketable commodity as high-grade hematite and magnetite. The Agency commenced the development by bench-scale testing in 1981, and decided to install a pilot plant close to the neutralization plant at Matsuo mine in 1983. This plant was completed in July 1984 and is now operating in the testing stage.

AUTHOR INDEX

541

Author Index Numbers in parentheses are footnote reference numbers and indicate that an author's work is referred to although the name is not cited in the text.

A Abril, O., 263,267(9), 279(9) Adachi, S., 6, 7(9), 443 Adalsteinsson, O., 266 Ado, Y., 395 Ahmed, S. A., 147, 148(20), 149(20) Aiba, S., 385 Aida, K., 517 Aisina, R. B., 69, 70(11) Aizawa, M., 10 Alberts, B., 281 Albertsson, P.-/~., 45,244 Alberty, R. A., 472 Amotz, S., 359 Anantharamaiah, G. M., 158, 162(1, 2) Andersson, E., 55 Andreasen, A. A., 399 Andrews, J. P., 170 Ang, C. Y. W., 494 Angelov, T. I., 326 Annau, E., 480 Anson, M. L., 508 Anteunis, M., 433 Antonini, E., 146, 150, 151, 155(4, 6, 7), 157(2, 3, 4, 5, 6, 7, 8) Aoyagi, H., 513 Arcuri, E. J., 395 Arison, B. H., 130 Asano, Y., 523 Astoor, A., 443 Auriol, D., 240, 241 Austin, M. J., 239 Award, W. M., Jr., 170 Axen, R., 58, 152 B

Baboulene, M., 119 Bacher, A., 310

Bachere, H., 381 Bader, J., 10, 303,304, 305(3, 5, 10), 306(3, 5, 9, 10), 309 Bailley, C., 364 Baker, R. S., 95 Balasubramanian, D., 202(30), 203(30), 204, 205(30), 212(30), 213(30) Baldwin, J. J., 130 Baldwin, T. O., 83 Balny, C., 203(32), 204, 206(32), 212(32), 214(32), 219 Banga, I., 480 Barbaric, S., 190, 201(9), 202(9), 204(9), 205(9), 212(9), 214(9), 222 Barber, J. J., 10 Baret, J. L., 418, 419(7), 420 Barford, J. P., 395 Barnes, C. S., 425 Barry, S., 57, 64 Bartlett, G. R., 28, 49 Baughn, R. L., 266, 276 Beck, J. F., 118 Behal, F. J., 170(12), 171 Behie, L. A., 335,336, 338,339(17), 341(30) Behrens, C. H., 118 Bell, G., 147 Ben Aim, R., 523,530(1) Bender, M. L., 284 Bennett, J. W., 329, 340(1) Berde, B., 324 Berezin, I. V., 118, 119, 291,292(22) Berezin, V. I., 69, 70(11), 147, 197, 201, 202(28), 203(28), 204(28), 205(28), 206, 207(28, 37), 208, 209(42), 210(42, 43), 212(28, 43), 214(28, 43), 219 Berger, S. J., 71 Bergmann, M., 513 Bergmeyer, H. U., 85,270, 278,279(30) Berk, D., 336, 338, 339(17), 341(30) Berke, W., 9 Berkeley, R. C, W., 239

542

AUTHOR INDEX

Bernfeld, P., 395 Bernofsky, C., 56, 61 Bernstein, M., 107 Bethke, H., 319 Biezer, H. J., 366 Bird, B. A., 340 Blain, J. A., 147 Blair, L., 212, 213(53) Blakely, R. L., 238 Blau, N. F., 128, 129(27) Blazo, A., 480 Blumenfeld, H., 276 Blunt, K. W., 442 Bock, R. M., 472 Bogard, M. O., 240, 242 Bohak, Z., 170 Bonner, F. J., 193, 194(16), 198, 200(21), 201(16), 203(16), 204(16), 208(16), 211(16), 212(16), 215(16, 21) Bonse, D., 368 Booth, A. B., 124 Borch, R., 107 Bovara, R., 84, 156 Bowcott, J. E., 227 Bradford, M., 488 Brfinden, C. I., 6 Braunstein, A. E., 480 Bray, D., 281 Bray, R. C., 256 Breddam, K., 159, 162, 163 Bright, H. J., 109 Brockerhoff, H., 129 Brocklehurst, K., 107, 181 Brookes, I. K., 145 Brougham, M. J., 57 Brown, R. T., 350 Bruckner, V., 480 Bryant, G., 240, 242 Buchanan, J. M., 266 Buchel, K. H., 135 Bucke, C., 381, 395, 432, 433(4), 435, 437, 442 Buckland, B. C., 145, 146(6), 147(6), 148(6) Biickmann, A. F., 5, 10, 11, 13(6), 17(6, 10), 21, 39, 45,490, 493 Biihler, M., 304, 305(7) Bullerjahn, R., 179, 184(4) Bulot, E., 498 Bunting, P. S., 62, 64

Burkhard, A., 7 Burris, R. H., 485,489(12) Butler, L. G., 150, 234, 236(3), 237(3)

C Calton, G. J., 464,497, 498(2), 500(1, 2) Cambou, B., 119, 120(14, 15) Campbell, I. M., 340 Campbell, J., 70, 75(16) Canizaro, P. C., 170(12), 171 Carasik, W., 361 Carey, P. R., 134 Carmel, A., 170(9), 171, 174 Carrea, G., 84, 146, 147(14), 150, 151,155(4, 6, 7), 156, 157 Carrier, R. G., 134 Carroll, J. O., 361 Carter, J. A., 71 Casellato, M. M., 150, 157(3, 5) Casmati, G., 201,202(27) Cesti, P., 137 Chang, H. N., 241 Chang, T. M. S., 21, 57, 59(23, 24, 25), 67, 68, 69, 70, 71, 72, 73, 74(9, 12, 13, 22), 75(9, 12, 13, 16), 76(9, 13), 77, 78, 395 Chappelle, E., 84 Charles, M., 10 Cheetham, P. S. J., 432, 435, 437(21, 22), 438(22), 439, 440(25), 441(22), 442, 443(21), 445(21, 22), 446(21, 22), 447(22), 448(22), 449, 450(36), 451(36), 454(22) Chen, W.-P., 356 Chen, Y.-H., 204 Chernyak, V. Y., 197 Chesne, S., 484 Chiba, H., 383 Chibata, I., 331,381,382(11), 395, 455,456, 459, 464, 468, 469, 472, 475,476, 478 Cho, Y. R., 241 Ciegler, A., 329, 340(1) Ciotti, M. M., 36 Clare, D. A., 424 Clark, J., 435, 437(22), 438(22), 439(22), 441(22), 442(22), 445(22), 446(22), 447(22), 448(22), 451 (22), 454(22) Clark, V. M., 268

AUTHOR INDEX Clarke, J. H. R., 225 Cleland, W. W., 265 Cluskey, J. E., 239 Coffey, D. S., 62 Cohen, N., 126 Cohn, M., 265 Coleman, M. H., 148, 149(22), 406 Collet, A., 118 Colowick, S. P., 36 Columbi, F., 146, 147(14), 151, 155(7), 157(7) Connor, W. E., 136 Contaxis, C. C., 236 Cooney, C. L., 498 Corkey, B., 465 Corman, J., 240 Corrieu, G., 395 Costerton, J. W., 334, 335, 339(10) Coton, G., 412 Coughlin, R. W., 10 Cousineau, J., 72 Crans, D. C., 263,267(3) Craven, D. B., 57, 58(19), 64 Cremlin, R., 135 Cremonesi, P., 146, 147(14), 150,151,155(4, 6, 7), 156, 157(2, 3, 4, 5, 6, 7, 8, 12) Crisel, R. M., 95 Crook, E. M., 181 Cross, R. P., 37 Crueger, W., 434 Cuatrecasas, P., 88, 106, 108(6), 494 Cysewski, G., 381,382(6)

543

Delalande, P., 420 Delange, R. J., 170, 178(1) DeLuca, M., 83, 87(11, 12, 13, 14, 15), 88, 89, 90(12), 92 Delz, B., 319 Deming, J. W., 84 Deo, Y. M., 329, 330(2), 331(2), 334, 335, 336, 337, 338, 339(10, 15, 19), 340(21) de Resset, A. J., 366 DesnueUe, P., 129 Detar, C. C., 414 Dickerson, A. G., 324 DiNello, R., 94 Dintzis, H. M., 181 Doerfler, D. L., 340 Dohan, L. A., 420 Doherty, J. V., 225 Donohue, J. A., 192 Dordick, J. S., 137 Dorman, D. E., 95 Dossena, A., 201,202(27) Douzou, P., 203(32), 204, 206(32), 208, 210(46), 212(32), 214(32), 219 Drath, L., 434 Drifford, M., 192 Duarte, J. M. C., 146, 147(12) Dugas, H., 292 Dunhill, P., 64, 75 DunniU, P., 145, 146(6), 147, 148(6), 290 Durand, G., 381,395 Durst, D., 107 Duterrtre, B., 395

E D Daka, N. J., 62, 64(43, 44) Daniel, J. W., 432 Darszon, A., 212, 213(52, 53) D~iumer, H., 181 Davis, B. N., 340 Day, R. A., 225 Dean, P. D. G., 57, 58(19), 64 Debey, P., 208, 210(46) de Bruyn, A., 433 Dehm, P., 170(11), 171, 174(11) Dehnen, W., 186 Delahodde, A., 196, 212(19), 213(19)

Edwards, G., 485 Egerer, P., 452 Eggert, E. W., 319 Egghart, H., 92 Eicke, H. F., 188, 189, 192(7), 217, 219(7), 225(7) Eklund, S. H., 6, 240 Eiiel, E. L., 117 Ellwood, D. C., 239 Elmaleh, S., 523,530(1) EI-Sayed, A. H., 318, 323(3) E1 Seoud, O. A., 201,212 Endo, S., 504

544

AUTHOR INDEX

Enfors, S.-O., 46 Eng, H., 395 Enosono, S., 395 Epton, R., 181 Ergan, F., 73, 74(22) Erjomin, A. N., 208, 210(44) Ernbach, S., 58, 152

F Farr, A. L., 27, 241 Fastrez, J., 180 Fawcett, J. S., 33 Fearon, W. R., 235 Feger, V. H., 240, 242 Feher, G., 212, 213(54) Fendler, J. H., 188, 201(1), 203(1) Ferrara, L., 146, 150, 155(4, 6), 157(4, 6) Ferrari, M., 271 Fersht, A. R., 180, 204 Fields, R., 40, 496 Findeis, M. A., 118, 136(8) Fink, A. L., 284 Fischer, J., 181 Fisher, B. E., 239 Flaks, J. G., 271 Fleminger, G., 170, 171, 173, 174(9) Fletcher, P. D., 192, 200, 203(31), 204, 212, 219 Floss, H. G., 319 Flossdorf, J., 10 Flynn, A., 57 Ford, J., 87(11), 88,413 Frank, C., 304, 305(10) Frank, H., 182 Franks, F., 208, 210(46) Freedman, R. B., 203(31), 204 Friedmann, T. E., 473 Fruton, I. S., 281,503 Fujihashi, T., 163, 167(6) Fujimoto, M., 519 Fujishima, T., 272 Fujita, K., 293 Fukui, S., 145, 146, 147, 148(20), 149(20), 293, 294, 298(3), 299, 301(11), 383,455 Fukushima, S., 293 Fuller, C. W., 109 Furgala, B., 432

Furugren, H., 6 Furui, M., 464, 477 Furukawa, S., 5, 7(8), 21, 34, 35, 45,493 Fusee, M., 464, 498

G Gabert, A., 178 Galunsky, B., 285, 286(12), 292 Ganzinger, D., 343 Garrett, C., 435, 437(22), 438(22), 439(22), 441(22), 442(22), 445(22), 446(22), 447(22), 448(22), 451(22), 454(22) Garrett, P., 268, 271(18) Gattner, H.-G., 163, 168, 186 Gaucher, G. M., 329, 330, 331, 334, 335, 336, 337, 338, 339(10, 15, 17, 19), 340(21), 341,342(5, 24) Gbewonyo, K., 335,340(12) Gehrke, C., 466 Gelfand, D. H., 498 Gellf, G., 383 Geresh, S., 263,267(8) Germeshausen, J., 507 Gestrelius, S., 21, 57, 58(21), 61(21), 63(21) Ghim, Y. S., 241 Ghini, S., 84 Giacomello, A., 271 Gibbons, I., 93, 94 Gibson, K. J., 271 Girotta, S., 84 Gitler, C., 208, 211(51) Glass, J. D., 161, 162 Glazer, A. N., 170, 178(1) Glock, G. E., 56, 61(4) Goldenberg, D., 170(9), 171, 174(9) Goldkamp, A. H., 503 Gooday, G. W., 239 Goodman, A. E., 456, 472 Gorin, G., 235, 236(9) Grandi, C., 194, 196(18), 206(18), 208(18) Grasmick, A., 523, 530(1) Green, E. H., 121 Green, K., 84 Green, S., 56, 61(8) Greenberg, B. D., 170 Greenzaid, P., 119 Grenner, G., 5, 56

AUTHOR INDEX Gricolo, B., 84 Griffin, T., 57 GrootWassink, J. W. D., 329, 330(2), 331 Gross, A., 263,267(8) Gross, E., 178 Griiber, W., 278, 279(30) Grunwald, J., 2l, 57, 59(23, 24, 25), 69, 74(9, 12, 13), 75(9, 12, 13), 76(9, 12, 13), 77, 81(28), 137 Guilbault, G. G., 32 Guilford, M., 64 GOnther, H., I0, 303,304, 305(3, 5, 8, 9, 10, 14), 306(3, 5), 308(6) Gupta, N. K., 56, 61(1, 2, 3) Gutman, I., 44

H Haase, W., 452 Habig, M. C., 523,530(1) Hagi, N., 506 Hagihara, B., 508 Hahn-H~igerdal, B., 55, 381,382(10) Hakney, R. E., 136 Halwachs, W., 55 Hamada, S., 433 Hamada, T., 192 Hamman, J. P., 497, 498(2), 500(2) Hanlon, T. M., 94 H/insler, M., 179, 185 Hanson, H., 178 Harada, T., 506 Harbron, S., 143, 144, 148(4) H/iring, G., 215 Harju, M., 415 Hartman, S. C., 266 Harvey, M. J., 57, 58(19), 64 Hasegawa, E., 383 Hashimoto, H., 440 Hashimoto, K., 6, 7(9) Hashimoto, Y., 406 Hastings, J. W., 83 Hathaway, S., 433 Haufler, U., 284, 285, 290(8), 292, 440 Haugen, G. E., 473 Havewala, N. B., 414, 459 Hayashi, R., 159 Hayes, M. C., 433

545

Haynes, W. C., 239 Haynie, S. L., 263, 267, 279(9) Hedbys, L., 231,233(4) Heier, J., 485 Hellerman, L., 62 Hellman, N. N., 240 Helvin, E. H., 242 Hemmingsen, S. H., 361 Henderson, L. M., 472 Henninger, F., 201,202(27) Hennink, W. E., 256 Henseleit, K., 234 Herbert, J. A. L., 107 Herves, D. V., 364 Hesse, M., 343 Hibino, K., 124 Hidai, M., 124 Hilhorst, R., 208, 211(48, 49), 212(48, 49), 216, 221(1, 2, 3), 225(2, 3), 226(2, 3), 228(1), 229(1) Hill, C., 234 Hinds, J. A., 238 Hinkley, J. E., 87(14), 89, 92 Hippert, B. L., 181 Hirohara, H., 413 Hiromi, K., 7, 22 Hirschbein, B. L., 263,268(4) Hiusjen, J., 93 Ho, T. C.-L., 136 Hoffman, C. A., 240 Hoffman, G., 56, 61(3) Hofmann, A. F., 87(13, 14, 15), 89 Hofmann, H.-J., 180 Hofstee, B. J., 258 Hohansson, A. C., 57 Holcberg, I. B., 381 Holtzmann, E., 170(10), 171, 174(10) Homandberg, G. A., 185, 235, 291,506 Hornby, W. E., 57 Horton, A. A., 484,485(10) Horton, H. R., 423,425(7, 8), 428,429(8) Horwath, R. O., 369 Hoshida, W., 406 Howe, A. M., 212 Howell, S. F., 241 Hsiao, H. Y., 158, 160, 162(1) Hughes, R. D., 507 Hummel, W., 15,498 Hustedt, H., 46

546

AUTHOR INDEX

Ichikawa, T., 292, 504 Igarasi, S., 290 Iida, T., 145, 294, 300(6), 383 Iizuka, T., 21, 35, 45, 493 Imaizumi, T., 532 Imanaga, Y., 532 Imber, C. E., 435, 437(21), 439(21), 443(21), 445(21), 446(21) Imre, V. E., 190, 191(10), 193, 194(17), 200(17), 215 Inaba, Y., 395 Indo, M., 299 Inman, D. J., 57 Inman, J. K., 181 Irino, S., 506 Isherwood, J., 435, 437(21), 439(21), 443(21), 445(21), 446(21) Ishihara, M., 406 Ishikawa, H., 366 Ishikawa, T., 532 Ishiwatari, H., 124 Isowa, Y., 292, 504 Iwamoto, N., 146, 293, 298(3) Izumitani, A., 433 Izumiya, N., 513

J Jablonski, E., 83 Jiickle, H., 193, 194(17), 200(17) Jackson, R. W., 240, 242 Jacques, J., 118 Jakubke, H.-D., 178,179, 180, 181,182, 183, 185, 281 Janolino, V. G., 423,424,425,426,427,428, 429(10, 15), 430(10, 15) Janson, C. A., 265 Jarvis, F. G., 331,336(9) Jeanes, A., 239 Jellet, J. F., 151 Jencks, W. P., 119 Jenkins, W. T., 483 Jensen, R. G., 129 Jin, I.-N., 294 Johansen, J. I., 159, 162, 163 Johansson, G., 39, 46 Johnson, D. B., 57

Johnson, J. W., 128, 129(27) Johnson, M., 331,336(9) Johnson, R. A., 358 Johnson, R. B., 97 Johnston, B. D., 130 Jonczyk, A., 163, 186 Jones, A., 335, 336, 338, 339(17), 341(30) Jones, J. B., 118, 132, 179, 255 Joppich, M., 201,202(27) J6rnvell, H., 6

K Kaboli, H., 241 Kadomura, Y. J., 433 Kakimoto, T., 472 Kalckan, H.M., 273 Kanaya, T., 162, 168(2), 169(2) Kanbayashi, A., 357 Kaneshiro, C. M., 235 Kaninaka, A., 272 Kaplan, N. O., 36 Kapune, A., 234 Karkas, J. D., 507 Karube, I., 21,323 Kasche, V., 234,284, 285,286, 290, 292,440 Katayama, N., 21, 35, 45,490, 493 Kato, J., 381,395,472 Kato, K., 290 Kato, T., 71,513 Kaufman, S., 270 Kawahara, I., 532 Kawahara, K., 290 Kawamoto, T., 395 Kazandjian, R. Z., 137 Kawano, E., 413 Kayashima, K., 456 Kazanskafa, N. F., 69, 70(11) Kazlauskas, R. J., 263, 270(5) Keh, E., 203(32), 204, 206(32), 212(32), 214(32), 219 Kelleher, W. J., 318 Keller, H. W., 366 Kelly, N., 57 Kessler, E., 170, 174 Khaleeluddin, K., 367 Khmel'nitskii, Y. L., 197,206, 208,209(42), 210(42, 43), 211(40), 214(43), 215(40) Khorana, H. G., 266

AUTHOR INDEX Kierstan, M., 381,395 Kihara, K., 234, 292,503,504, 505, 506(6) Kimble, B. K., 240, 241(6) Kimura, K., 395 Kimura, T., 146,293,298(3) King, C. K., 9 King, E. J., 443 Kinoshita, S., 517 Kirby, A. J., 268 Kirchner, G., 137 Kirtane, J. G., 130 Kitahara, A., 188 Kitahara, K., 455 Kito, M., 206, 207(39) Klein, J., 313,395 Klesov, A. A., 284 Klibanov, A. M., 118, 119, 120(14, 15), 137, 147 Klyacbko, N. L., 197,201,202(28), 203(28), 204(28), 205(28), 206, 207(28), 208, 209(42), 210(43), 211(40), 212(28, 43), 214(28, 43), 215(40), 219, 220 Kobayashi, T., 62 Koelsch, R., 178 Koepsell, H. J., 240, 242 Kolot, F. B., 381 Komatsu, A., 293,298, 299 KOnnecke, A., 178, 179, 180, 181, 182, 183, 184(4), 185, 187(20) Koob, R., 7 Kopp, B., 318, 320(la), 323(3) Kornberg, H. L., 484, 485(10) Koshiro, S., 299, 301(11) Kosogi, Y., 357 Kramer, D. N., 32 Krampitz, O. L., 485 Krebs, H. A., 234,499 Kricka, L. J., 84, 87(15), 89, 92 Kroner, K. H., 10, 46 Krutzsch, H. C., 178 Kuhl, P., 178, 181, 185,281 Kuhimann, W., 55 Kiihn, I., 55 Kuhn, R. W., 159 Kula, M.-R., 5, 7, 9, 10, 11, 13(6), 15, 17(6), 21, 45, 46, 244,490, 493,498 Kulys, J., 21, 33(5) Kumar, C., 202(30), 203(30), 204, 205(30), 212(30), 213(30) Kuninaka, A., 519

547

Kunitz, M., 47 Kuno, S., 310 Kunze, H. E., 238 Kurganov, B. I., 206, 207(37) Kutzbach, C., 453 Kuwayama, H., 408

L Laane, C., 208,211(48, 49), 212(48, 49), 216, 221(1, 2), 225(2), 226(2), 228(1), 229(1) Laidler, K. J., 7, 21, 57, 59(26), 60, 62, 63(31), 64, 65(26, 31), 66(31) Laki, K., 480 Lam, K. S., 329, 330, 331(2), 342(5) Lamed, R., 64 Landis, D. A., 241 Lantero, O. J., 453 Lantz, L., 497,498(2), 500(2) Laraway, J. W., 366 Lardy, H. A., 485 Larsson, P. O., 3, 5, 7, 10, 21, 25, 34, 48, 57, 58(20), 59, 63, 64, 75, 103, 108, 231,395 Lasch, J., 178 Laskowski, M., Jr., 185, 235, 291,506 Lattes, A., 119 Lee, J., 85 Lee, S.-L., 343 Lee, S, S., 485,489(12) Le Goffic, F., 22 Lesser, B. H., 336, 338, 339(17), 341(30) Leuchtenberger, W., 10, 493 Levasbov,A. V., 197, 201,202(28), 203(28), 204(28), 205(28), 206, 207(28, 37), 208, 209(42), 210(42, 43), 211(40), 212(28), 214(28, 43), 215(40), 219, 220 Levich, V. G., 309 Levin, Y., 64 Levner, M., 436 Lewis, J. M., 263,267, 281 Lewis, L. T., 358 Libby, R. M., 97 Liberatori, F. A., 160, 162 Lilly, M. D., 64, 75, 138, 143, 144, 145, 146, 147, 148(4, 6), 290 Lin, Y. Y., 132 Lindberg, M., 5, 59, 64(30), 108 Lindley, M. G., 433 Lindman, B., 189, 192(4)

548

AUTHOR INDEX

Lindsley, J., 171 Link, M. L., 304, 305(9), 306(9) Linsmaler, E. M., 344 Lis, H., 230 Litman, D. J., 94 Llor, A., 192 Lloyd, N. E., 358, 369 Lodi, R., 156 Logan, R. M., 358 Lopez, A., 240, 241,242(19), 243(19) L6pez-Levia, M., 55 Lopresti, R. J., 126 Loss, von F., 433 Loucks, A., 92 Lowe, C., 25, 34, 48, 56, 57, 58(19, 20), 64, 75 Lowry, O. H., 27, 29, 71,241 Luddy, F. E., 405 Lugaro, G., 150, 157(3) Luger, P., 433 Luisi, P. L., 189, 190, 192(5), 193, 194, 196(18), 198,200,201,202(27), 203(33), 204, 206, 207(26, 36), 208, 210(41), 211(16), 212(16, 26, 36, 41), 214(9, 26, 36, 41), 215, 216, 217, 220(6), 221(6), 222 Lukasheva, E. V., 69, 70(11) Lfithi, P., 216 Lynn, K. R., 236

M MacAllister, R. V., 367 McCaskill, D. G., 330, 341(4) MacClement, B. A. E., 134 McClure, D. E., 130 McCoy, C. J., 145 McDonald, A., 57 Macdonald, I. A., 87(14), 89, 432 Macdonald, J. A., 151, 156 McDonald, M. R., 47 McDougall, B., 290 McElroy, W. D., 83 Maclntosh, F. C., 67, 68(1) Mclssac, J. E., Jr., 160, 162(6) McLean, P., 56, 61(4) McMullen, W. H., 361 Macrae, A. R., 147, 148, 149(22), 406, 409

Maeda, H., 7, 21, 22, 25, 26(16), 45 Magid, L. J., 200 Mahmoud, W., 318, 323(3) Mahony, D. E., 151 Malakhova, E. A., 206, 207(37) Malhorta, O. P., 284 Maliarik, M. J., 368 Malinauskas, A., 21, 33(5) Malouf, C., 71, 81(19) Malthouse, J. P. G., 107 Mamiya, G., 235, 236(9) M~nsson, M. O., 6, 7, 10, 21, 57, 58(21), 61(21), 63(21), 104 Mantle, P. G., 324 March, S. C., 88, 106, 108(6) Marconi, W., 455 Margalith, P., 381 Margolin, A. L., 284 Marguardt, J., 178 Mariella, R. P., 37 Marietta, M. A., 137 Marshall, R. O., 357 Marshall, W. E., 56, 61(3) Martin, L. D., 130 Martinek, K., 118, 119, 147, 185, 197, 201, 202(28), 203(28), 204(28), 205(28), 206, 207(37), 208, 209(42), 210(42, 43), 212(28, 43), 214(28, 43), 215(40), 219, 220, 291,292(22) Martinez, H. M., 204 Marring, S. C., 69 Masaki, T., 163, 165, 167(6) Mason, S. G., 67, 68(2) Massey, V., 108 Masuda, S., 134 Matsmara, Y., 124 Matsuo, T., 406 Mattiasson, B., 55, 181 Mattis, J. A., 185, 235, 291,506 Mauch, W., 440 Mavrides, C., 482,484, 486(8) Maxwell, E. S., 273 Mayhew, S. G., 229 Mazenod, F. P., 263,268(4) Mazid, M. A., 7, 21, 57, 59(26), 60, 62, 63(31), 64(31), 65(26, 31), 66(31) Mazzola, G., 146, 151, 155(7), 156, 157(7) Mazur, R. H., 503 Mead, J., 203(31), 204

AUTHOR INDEX Meienhofer, J., 178 Meier, P., 190, 191(10), 208, 210(41), 212(41), 214(41), 215(10, 41) Melvin, E. H., 239 Menger, F. M., 192, 201, 202(29), 204(29), 212(29), 214(29), 219 Mensd6rfer, F., 215 Mensler, K., 130 Messina, E., 271 Messing, R. A., 247,413,455 Metelitza, D. J., 208, 210(44, 45) Meyer, B., 368 Michal, G., 465 Mii, S., 272 Mildvan, A. S., 265 Misawa, M., 455 Misiorowski, R. L., 206, 207(38) Mitsuhashi, H., 344 Mitsunobu, O., 493 Mittal, K. L., 189, 192(4) Miwa, N., 31 Miyawaki, S., 124 Mizukami, H., 343 Moll, M., 381,395 M611ering, H., 278,465 M6nch, W., 186 Monnier, N., 484 Monsan, P., 119,240, 241,242(19), 243(19), 246, 247(27) Montal, M., 208, 211(51), 212, 213(52, 53, 54) Mori, K., 126, 134, 292,504 Mori, T., 464,469 Morihara, K., 162, 163, 165, 168(2), 169(1, 2), 291 Morikawa, Y., 21 Moroe,T., 293,298 Morosi, F., 455 Morr, M., 9, 39 Morris, C. J. O. R., 33 Morris, J. F., 121 Morrison, J. D., 117, 118 Morrison, J. F., 483 Mosbach, K., 3, 5, 6, 7, 10, 21, 25, 34, 48, 56, 57, 58(20, 21), 59, 61(21), 63, 64, 75, 103,104, 108, 152, 181,231,233(4), 381, 382(10), 395 Mosbach, R., 395 Mosher, H. S., 118

549

Mosti, R., 455 Miiller, F., 256, 257, 258 Muneyuki, R., 165 Munir, M., 434, 451 Muramatsu, M., 22, 34, 56 Murayama, T., 532 Myers, C., 161 N Nagai, T., 293 Nagakura, M., 408 Nagakura, N., 343 Nagase, T., 413 Nagata, S., 304, 305(9), 306(9) Nakajima, Y., 452 Nakamura, K., 163, 165, 167(6) Nakamura, R. M., 97 Nakane, P., 96 Nagel, A., 256 Narendranathan, T. J., 143, 144(3), 148(4) Narinesingh, D., 62, 65(37) Narita, H., 206, 207(39) Navarro, J. M., 381,395 Needham, D. M., 480 Neely, W. B., 239 Neidleman, S. L., 369 Neukom, C., 126 Neumann, S., 10, 303, 304, 305(3, 5, 14), 306(3, 5) Neurath, H., 159 Neway, J., 329, 330, 331(2) Ngo, T. T., 62 Nibley, D. A., 84 Nicoli, M. Z., 83 Nicot, C., 196, 212(19), 213(19) Nielsen, T. K., 359 Nikolova, N., 326 Nilsson, K., 104, 152, 231 Nishida, Y., 464 Nishimura, S., 292, 504 Nisselbaum, J. S., 56, 61(8) Nonaka, Y., 504, 505 Nordl6v, H., 343 Nordwig, A., 170(11), 171, 174(11) Norris, R., 107 Novak, T., 192 Novaka, Y., 292 Nozaki, H., 130

550

AUTHOR INDEX O

O'Carra, P., 64 Ochoa, S., 272 Odawara, H., 366 Offord, R. E., 234 Ogata, K., 517 Ogata, M., 6, 7(9) Ogawa, Y., 524 Ogura, Y., 508 Ohmori, M., 292, 504 Ohno, M., 513 Ohroff, G., 124 Ohta, Y., 508 Oka, T., 162, 163,165, 168(2), 169(1, 2), 291 Okada, H., 5, 9, 21, 22, 34, 35, 45, 56, 490, 493 Okahashi, N., 433 Okuda, K., 9 Okuso, M., 366 Oldfield, C., 203(31), 204 Omachi, A., 56, 61(3) Omata, T., 145, 146, 147, 148(20), 149(20), 293,294, 298(3), 300(6), 383 Ono, E., 406 Onozawa, K., 124 Ooshima, T., 433 Orentos, D. G., 446 Oriol, E., 240, 241 Oritani, T., 293,298 Orr, W., 482, 484,486(8) Oshima, A., 206, 207(39) Ostergaard, J. C. W., 69 Osumi, M., 294, 383 Otillio, N. F., 258 Otsuka, S., 117 Ottesen, M., 159 Overbeek, J. Th. G., 216 Oyama, K., 234, 292,503,504, 505, 506 P Pain, S., 420 Paknikar, S. K., 130 Palmer, D. N., 132 Pande, A., 190, 191(10), 193, 194(17), 200(17), 215(10), 216 Pantin, V. I., 201,202(28), 203(28), 204(28), 205(28), 206, 207(28), 208(40), 211(40), 212(28), 214(28), 215(40)

Papaconstantinou, S., 523,530(1) Pape, H., 323 Parikh, I., 88, 106, 108(6) Passonneau, J. V., 29 Patel, D. N., 358 Patterson, J. D. E., 147 Paul, F., 240, 241 Pazoutova, S., 326 Pellegrini, A., 193,194(16), 201(16), 203(16), 204(16), 208(16), 211(16), 212(16), 215(16) Pelmont, J., 484 Pelzig, M., 162 Penney, C., 292 Perrey, H., 452 Perrins, N. F., 200 Pfitzner, U., 343, 344(8), 349 Phelps, D. J., 134 Picciolo, G. L., 84 Pickett, D. J., 309 Pileni, M. P., 208, 210(47) Pillay, G., 170(10), 171, 174(10) Pisano, J. J., 178 Pitcher, W. H., 413,414, 459 Plaut, G. W. E., 485 Playne, M. J., 146 Pollak, A., 55, 263,276 Porath, J., 58, 152 Portino, A. D., 381 Poulsen, P. B., 255, 363 Powell, J. T., 483 Prenosil, J., 216 Prince, I. G., 395 Puc, A., 325

R Raab, A. W., 130 Radhakrishnan, R., 266 Raft, M., 281 Randall, R. J., 27, 241 Ranki, J. C., 239 Rao, A. S., 130 Rauschenbach, P., 304 Re, L., 5 Reents, A. C., 366 Rehecek, Z., 320, 321(7), 324(7), 325 Rehm, H. J., 318, 320(1a), 323(3) Reichenbach, H., 498

AUTHORINDEX Reilly, P. J., 241 Reithel, F. J., 234, 236, 237(3) Rekker, R. F., 226 Remaley, A. T., 340 Resurreccion, E., 71, 81(19) Riechmann, L., 284, 290(7), 440 Rios-Mercadillo, V. M., 263 Rist, C. E., 239 Robbers, J. E., 319 Roberts, K. R., 281,433 Robinson, B. H., 192,200,203(31), 204,212, 219, 225 Robinson, W. G., 56, 61(1) Robson, R. J., 266 Robyt, J. F., 240, 241(6, 7) Roda, A., 84, 87(15), 89 Roe, J. H., 49 Roels, J. A., 356 Rogovin, S. P., 240 Rohrbach, R. P., 366, 368 Rokugawa, K., 272, 519 Rosato, L. M., 340 Rosebrough, N. J., 27, 241 Rosenbusch, J. P., 212, 213(55) Rosenthal, A. M., 78 Rosenthal, T., 170(10), 171, 174(10) Rossodivita, A., 5 Roth, P., 178 Rothbart, H. L., 405 Roussel, D. J., 499 Rowley, G. L., 93 Royal, K. M., 61 Royer, G. P., 158, 160, 162, 170 Rozniewska, T., 335, 336, 339(17) Rozzell, J. D., 485 Rubenstein, K. E., 93 Rubin, B. A., 436 Rubin, J. R., 109 Rueffer, M., 343

S Sahm, H., 10 Sakashita, K., 524 Salermo, C., 271 Samokhin, G. P., 118, 119(10), 147 Sano, T., 493 Sasaki, M., 134 Sato, T., 331,464, 468,469,475

551

Satoh, A., 7, 22 Satoh, H., 504 Satoh, M., 292 Satoh, Y., 524 Saucy, G., 126 Sawamura, N., 406 Sawyer, B., 56, 61(6, 7) Schaefer, H. G., 7 Schellenberger, V., 179, 180, 183, 185(5) Schild, H. O., 324 Schiweck, H., 434 Schlatter, J. M., 503 Schmid, R. D., 55 Schmidt, H. L., 5, 56, 303 Schmidt-Berg-Lorenz, S., 440 Schmidt-Kastner, G., 452, 453 Schmitt, E. W., 168 Schneider, R. S., 93 Schoelmerich, J., 87(13, 14), 89 Sch6nfeld, M., 212, 213(54) Schuetz, H. J., 304, 305(9, 10), 306(9) Schiigerl, K., 55 Schulman, J. H., 227 Schulman, M. P., 56, 61(3) Schurs, A. H. W. M., 93 Schtitte, H., 10, 15 Schutt, H., 453 Schwartz, R. D., 145 Schwartz, W. E., 162 Schwarz, M. K., 235 Scollar, M. P., 137 Scott, A. J., 343 Scott, C., 126 Scott, T. A., 242 Sczyrbak, C. A., 324 Sedlmaier, H., 304 Segel, I. H., 42, 336 Sekiguchi, J., 341,342(24) Sekiguichi, M., 440 Semenov, A. N., 119, 185, 291,292(22) Senti, F. R., 240 Seto, T., 192 Shahani, K. M., 129 Shaked, Z., 10 Shallenberger, R. S., 440 Sharon, N., 230 Sharpe, E. S., 240 Sharpless, K. B., 118 Shaw, C. E., 147 Shimizu, J., 452

552

AUTHOR INDEX

Shipton, M., 107 Shoda, M., 385 Shono, T., 124 Shumate, S. E., 395 Shvyadas, V.-Y. K., 284 Sicsic, S., 22 Siddiqui, I. R., 432 Sidebotham, R. L., 239, 240(3) Sidrowicz, W., 170(12), 171 Siegbahn, N., 21,104 Siegel, M., 268, 271(18) Sigman, D. S., 170, 178(1) Silk, D. B. A., 507 Simmons, W. H., 170 Simon, H., 10, 303,304, 305(3, 5, 7, 8, 9, 10, 11, 14), 306(3, 5, 9), 308(6), 309, 310 Siqueira, R., 336, 339(17) Sizer, I. W., 483 Skold, C., 93 Skoog, F., 344 Skopan, H., 304, 308(6) Skudder, P. J., 424, 425(10), 427(10), 429(10), 430(10) Slater, T. F., 56, 61(6, 7) Slesser, K. N., 130 Sliwkowski, M. B., 423,425(7, 8), 429(8) Sliwkowskl, M. X., 423, 424, 425(8, 10), 427, 428, 429(8, 10, 15), 430(10, 15) Slokoska, L. S., 326 Slonekar, J. H., 446 Smeds, A.-L., 46 Smith, B. R., 146 Smith, R. E., 194, 196(18), 201, 206(18), 208(18), 212(25) Snell, E. E., 472 Sobue, S., 433 Socic, H., 325 Soejima, M., 163, 165, 167(6) Sonomoto, K., 294, 299, 301(11) Souzu, J., 344 Spector, L. B., 119 Speziale, V., 119 Sportoletti, G., 150, 157(2) Spruijt, R., 216, 221(2), 225(2), 226(2) Stach, W., 10 Stalling, D., 466 Stanley, P. E., 84 Stark, J. B., 456, 472 Staub, F. B., 480

Steinberg, R. A., 498 Steinmann, B., 201, 206(26), 207(26), 208(26), 212(26), 214(26) Stempel, G. H., 37 Stevenson, R. W., 405 Steytler, D. C., 212 Stich, K., 319 Stier, T. J. B., 399 Stille, J. K., 130 Stiso, S. N., 94 StOcklgt, J., 343, 349(3) Stodda, F. H., 240 Strasser, R., 212,213(52) Straub, B., 189 Strauli, U., 56, 61(7) Stringer, C. S., 240 Strominger, J. L., 273 Stuchburg, T., 107 Stuckwisch, C. G., 128 Suckling, C. J., 256 Suckling, K. E., 256 Sugimoto, Y., 34 Suguro, T., 126, 134 Sumi, A., 464, 470 Sumner, J. B., 235, 241 Sunamoto, J., 192 Susckitzky, H., 107 Sutthoff, R. F., 367 Suzuki, H., 21, 45 Suzuki, K., 432,452 Suzuki, S., 21,323 Svensson, S., 231 Swaisgood, H. E., 423, 424, 425, 426, 427, 428,429(8, 10, 15), 430 Swan, M., 56, 61(9) Swann, W., 464 Switzer, R. L., 271,447, 451(35) Szechinski, J., 170(12), 171 Szent-Gy6rgyi, A., 480 T Tabony, J., 192 Tajima, N., 298 Takagaki, Y., 266 Takahashi, T., 290 Takamatsu, S., 470, 475,476, 478 Takano, T., 524

AUTHORINDEX Takasaki, Y., 357 Takata, I., 331,381,456, 459 Takinami, K., 406 Tamada, S., 134 Tamamushi, B., 189, 190(8) Tanabe, H., 366 Tanabe, M,, 165 Tanaka, A., 145, 146, 147, 148(20), 149(20), 293,294, 298(3), 299, 300(6, 7), 301(11), 383 Tanaka, E., 124 Tanaka, T., 406 Tani, Y., 523 Taniguchi, H., 240, 241(7) Thanos, J., 10, 303,304, 305(3, 5, 8), 306(3, 5) Theorell, H., 58 Therisod, M., 137 Thiesen, N. O., 359 Thomas, D., 73, 74(22) Thomas, J. K., 192 Thomas, T. H., 181 Thompson, K. N., 358 Thorpe, G. H. G., 84 Tiemeyer, W., 304, 305(11) Tischer, W., 304, 305(11) Tobita, H., 6, 7(9) Tochino, Y., 162, 168(2), 169(1, 2) Todd, J. R., 147 Tomka, I., 216 Ton, H. Y., 507 Toprakcioglu, C., 200 Tosa, T., 331, 381,455, 456, 459, 464, 468, 469, 475, 476, 478 Tramper, J., 255, 256, 257, 258, 262, 335 Treimer, J. F., 343 Troll, W., 171 Tsuchiya, H. M., 239, 240, 242 Tsunoda, T., 517 Tsuzuki, H., 162, 168(2), 169(1, 2)

U Uchida, K., 130 Uchida, M., 126 Uchida, T., 381 Uchida, Y., 124 Ullman, E. F., 93, 94

553

Umemura, I., 475 Updike, M. H., 497, 498(2), 500(2) Urab¢, I., 5, 9, 21, 22, 34, 35, 45, 56, 490, 493 Usanov, S. A., 208, 210(44) Ushiro, S., 395 Usui, H., 408 Usui, K., 366 Utimoto, K., 130

V Vacher, M., 196, 212(19), 213(19) van Beeumen, J., 433 van Berge Henegouwen, G. P., 87(13), 89 Vandamme, E. J., 290 van der Plas, H. C., 256, 257, 258 Van der Westen, H. M., 229 Vann, W. P., 291,414 van Tilburg, R., 356, 361 van Velzen, A. G., 361 Van Weeman, B. K., 93 Veeger, C., 208, 211(48, 49), 212(48, 49), 216, 221(1, 2), 225(2), 226(2), 228(1), 229(1), 229 Veelken, M., 323 Veide, A., 46 Vellom, D., 92 Verhegge, G., 433 Vidaluc, J. L., 119 Villee, C. A., 56, 61(5) Vincent, C., 22 von Dreissig, W., 433 yon Rienacker, R., 124 Vosbeck, K. D., 170 Vowinkel, E., 168

W Wada, A., 508 Wada, M., 381,395, 493 Wagner, F., 313 Wahlefeld, A. W., 44, 465 Waks, M., 137, 196, 212(19), 213(19) Wallenfells, K., 230, 284 Walseth, T. F., 240, 241(6) Walsh, K. A., 159

554

AUTHOR INDEX

Walter, R., 170 Wan, J., 395 Wandrey, C., 5, 7, 9, 10, 11, 13(6), 17(6, 10), 19, 21, 45,490, 493 Wang, D. I. C., 335, 340(12) Wang, S. S., 9 Warthon, C. W., 181 Watanabe, I., 524 Watanabe, N., 189, 190(8) Watson, J. D., 281 Wax, M., 276 Weatherburn, M. W., 339 Webb, E. C., 238 Weber, G., 126 Weetall, H.-H., 181,291,413,414, 486 Weidenbach, G., 367, 368 Weii, R., 230 Welch, G. R., 104 Wells, M. W., 206,207(38) Welsch, T., 182 Weng, L., 94 Werkman, C. H., 485 White, F. H., 381 Whitehead, T. P., 84 Whitesides, G. M., 10, 55, 118, 263, 266, 267, 268, 270(5), 271(18), 276, 279(9), 303 Wichmann, R., 5, 10, 11, 13(6), 17(6, 10), 19, 21, 45,490, 493 Widmer, F., 162, 163 Wiener, F. P., 436 Wienhausen, G., 86(12), 87(12), 89, 90(12), 92 Wiget, P., 193, 194(16), 201(16), 203(16), 204(16), 208(16), 211(16), 212(16), 215(16) Wilcheck, M., 64 Wilen, S. H., 118 Wilham, C. A., 239 Wilke, C., 381,382(6) Wiilemot, R. M., 240 Williams, R. F., 192, 507 Williamson, J., 465 Wilson, M. B., 96 Wilson, P. W., 485,489(12) Winer, A. D., 58 Wirz, B., 137 Wirz, J., 212,213(55) Witiak, D. T., 136 Woenckhaus, C., 7

Wfhler, F., 234 Wolf, R., 193,194(16), 198, 200(21), 201(16), 203(16), 204, 206, 207(36), 208(16), 211(16), 212(16, 36), 214(36), 215(16, 21, 36), 217, 220(6), 221(6), 222(6) Wong, C.-H., 118, 136(7), 263, 267(9), 279(9), 303 Wong, J. W., 330, 341(4) Wong, M., 192 Wood, D. N., 335 Wood, L. L., 497,498(2), 500(1) Woodley, C. L., 56, 61(2) Worden, R. M., 395 Wykes, J. R., 64, 75

Y Yabuuchi, E., 532 Yagi, H., 124 Yam, C. F., 62 Yamada, H., 523 Yamada, K., 201,202(29), 204(29), 212(29), 214(29), 219, 220(9) Yamada, T., 383 Yamada, Y., 22, 34, 56, 341,342(24) Yamaga, M., 130 Yamaguchi, Y., 293,298 Yamamoto, H., 413 Yamamoto, K,, 455,475 Yamamoto, L. A., 336 Yamamoto, S., 192 Yamanaka, S., 406 Yamashita, K., 293,455,464, 470, 477 Yamazaki, T., 366 Yamazaki, Y., 7, 21, 22, 25, 26(16), 45 Yang, J. T., 204 Yaron, ,~., 170, 171, 173, 174 Yasuda, T., 523 Yasuhara, S., 294, 383 Yaverbaum, S., 414 Yoshida, T., 299 Yoshii, H., 383 Yoshimoto, T., 170 Yoshimura, J., 440 Yoshino, H., 272, 519 Yoshino, M., 395 Young, E. G., 47 Yu, Y. T., 68, 69, 77, 78(14)

AUTHOR INDEX

Z Zaks, A., 137 Zappelli, P., 5 Zenk, M. H., 343,344(8), 349 Zerner, B., 238

Zervas, L., 513 Ziehr, H., 498 Zittan, L. E., 363 Z611ner, R., 284, 285,290(8), 292 Zulauf, M., 189, 192(7)

555

SUBJECT INDEX

557

Subject Index

A ABTS. See 2,2'-Azinodi-(3-ethylbenzothiazoline-6-sulfonic acid) Acetate kinase assay, 271 partition, in aqueous two-phase systems containing dextran T500, PEG plus PEG sulfate and substrate, 52 specific activity, 264 stability in immobilized form, 264 Acetic acid sanitizers, 418 N-Acetylgalactosamine, 230-231 Acetyl phosphate properties, 265 synthesis, 263, 267-268 Achromobacter protease I covalent coupling to poly(L-glutamic acid), 165-166 covalent coupling to silica gel containing immobilized poly(L-glutamic acid), 166-167 in enzymatic conversion of porcine insulin to human insulin, 162163 in enzymatic semisynthesis of human insulin, 163-170 immobilization, 165-167 immobilized, assay, 163-164 Acid mine drainage. See also Matsuo Mine ferrous and ferric iron in, conversion to marketable commodities, 540 treatment, 531 bacterial oxidation in, 530-531 with immobilized T. ferrooxidans, 530-540 Ac-Phe-Ala-NH2, chymotrypsin-catalyzed synthesis, 182-183 Acrylamide

measurement, 525 production, 523 using immobilized nitrilase-containing microbial cells, 523-530 use, 523 Acryionitrile, measurement, 525 N-Acryloxysuccinimide, 274 N*-AcryloyI-L-lysinemethyl ester, 37-38 Actinoplanes missouriensis, 361 ADP, bioluminescent assay using coimmobilized enzymes, 87 Aerosol OT, 216 HPLC, 190-191 purification, 189-192 sources, 189 Aerosol OT/water/isooctane system phase diagram, 189 use for enzymes, 189 Agroclavine production, 320 retention time on HPLC, 320 D-Alanine, estimation of, 473 L-Alanine bioluminescent assay using coimmobilized enzymes, 86 continuous production using two immobilized microbial cells, 472-479 crystallization from reactor column effluent, 479 estimation of, 472 industrial production, 478-479 production from ammonium fumarate, elimination of side reactions, 475-476 bioreactors for, 477-478 in closed column reactor, 477 sequential reactions in, 477-478 Alanine aminotransferase. See Glutamicpyruvic aminotransferase

558

SUBJECT INDEX

Alanine dehydrogenase, kinetic parameters for native NADH and PEG-NADH, 12 Alanine racemase, assay, in free and immobilized microbial cells, 474 Albumin, partition, in aqueous two-phase systems containing dextran T500 and PEG plus PEG sulfate, 51 Alcohol. See also Ethanol continuous fermentation conventional batch, 393 by immobilized yeast cells, features, 393 Melle Boinet batch, 393 process flow diagram, 390 fermentation, basic technology, 385386 production, for power, 380 Alcohol:NAD÷ oxidoreductase. See Alcohol dehydrogenase Alcohol dehydrogenase. See also Site-tosite enzyme systems assay, 108-109 coimmobilization with coenzyme materials, 57-58 methods, 58-59 coimmobilization with NAD, 57 activity, 63, 66 analysis of data, 62-63 dimensionless parameters 6 and p definition, 63 at different ethanol concentrations and flow rates, 65-66 double-logarithmic plots of product concentrations at exit against flow rates with different concentrations of substrate ethanol, 64-65 efficiency, 63, 66 kinetic studies, 61-63 results, 63-66 coimmobilized with coenzyme, 56-67 coimmobilized with NAD, 56 free and immobilized, Michaelis constants for, 153 horse liver coimmobilization with NAD(H) analog, 57 to Sepharose 4B, 58-59 in coupled two-enzyme reactor, steady-state analysis, 42-45

immobilized site-to-site enzyme system with lactate dehydrogenase, 104-106 kinetic constants, 43 in reverse miceUes, 208-212 soluble site-to-site enzyme system with lactate dehydrogenase, 106108 source, 26 immobilization, 56-57 on CNBr-activated Sepharose 4B, 152 immobilized, recovery of activity, 153 and NAD analog, attachment procedure inside nylon tube, 59-61 PEG-NADH as coenzyme, 13 in reverse miceUes, kinetic parameters, 214 reversibly immobilized, preparation, 106 sources, 57-58 yeast covalent attachment, with NAD, to interior of partially hydrolyzed nylon tube, 57-67 and malate dehydrogenase, microencapsulation with soluble dextranNAD, 57 microencapsulated with malate dehydrogenase, recycling of NAD + and NADH, 70-71 source, 151 specific activity, 151 stability in microcapsules, 77 Alcohol dehydrogenase-NAD(H)Sepharose complex, 57 Alginate fibers, cell immobilization on, 453 Alginate-immobilized microbial cell pellets, mechanical strength, 442-443 Alkaline phosphatase, in reverse micelles, 207 Alkaloids. See also Strictosidine analytical methods for determination of, 319-320 production by C. purpurea, improved capacity, with application of immobilization technique, 320-329 retention on HPLC, 320 Alkanal monooxygenase. See Bacterial luciferase Amidase, assay, 524-525 Amino-acid dehydrogenase + formate

SUBJECT INDEX dehydrogenase, immobilized, application, 354 L-Amino acids, production using aspartic aminotransferase from E. coli, 484 Aminoacylase immobilized, application, 354 reaction, bioreactor for, based on aqueous two-phase systems, 55 N~-[(6-Aminohexyl)carbamoylmethyl]NADH, coimmobilization with alcohol dehydrogenase, to Sepharose 4B, 5759 N-(3-Amino-2-hydroxypropyl)-2-(tritylamino)acetamide, 24 Aminopeptidase, immobilized application, 354 sequential hydrolysis of peptides with, procedure, 174-177 Aminopeptidase P application to sequential hydrolysis of proline-containing polypeptides, 170-178 assay, 171 colorimetric assay, 174 definition of unit, 174 fluorimetric assays, 174 glass-bound absence of endopeptidase activity, 173 adsorption, 172-173 enzyme activity measurement, 174 metal ion requirement, 173 pH dependence, 173 stability, 172 temperature dependence, 173 immobilization, 172 soluble, enzyme activity measurement, 173 Aminotransferase in biocatalysts advantages, 481 disadvantages, 481-482 discovery, 480 immobilization, 485-492 immobilized application, 354 laboratory-scale processes using, 481 long-term stability, measurement, 491492

559

Ping-Pong kinetics, 480 pyridoxal 5'-phosphate cofactor, 480 attached to soluble polyethylene glycol derivative, 496 macromolecularized synthesis of, 494-495 use in continuous flow membrane reactor, 496-497 retention in membrane reactor, 492497 reaction catalyzed, 479-480 driving to completion, 482-483 half-reactions, 480 sources, 483-485 substrate specificity, 482 Amoxycillin, synthesis, in hydrolasecatalyzed condensation reaction, 282 Ampicillin, synthesis, in hydrolase-catalyzed condensation reaction, 282 a-Amylase-treated starch, treatment with glucoamylase, 376 Amyloglucosidase, immobilized, applications, 354 Androsterone, bioluminescent assay using coimmobilized enzymes, 87 Angiotensin II, sequential hydrolysis with immobilized aminopeptidase, 175-176 Antibiotic fermentations, longevity, 330331 increasing, 342 Apomyoglobin, 177 Aromatic-amino-acid aminotransferase, 484 Artificial cells recyling of NAD(P) by multienzyme systems microencapsulated within, 67-82 retention of NAD + within, 81-82 Aspartame, 432 enzymatic production, 503-504, 516 raw materials, 504-505 selection of enzymes, 504 selection of organic cosolvent, 506507 selection of protecting group, 504 and isomaltulose, combination, 433 preparation, by conventional organic synthesis, 503 production by immobilized thermoase, 503-516 batchwise operation, 514-516

560

SUBJECT INDEX

in continuous column operation, 513514 operation, 513-516 substrate preparation, 513 sweetness, 503 synthesis, 234 in hydrolase-catalyzed condensation reaction, 282 Aspartase assay in free and immobilized microbial cells, 473 in immobilized E. coli, 465 immobilized, application, 355 Aspartase + L-aspartic acid, immobilized, application, 355 Aspartate aminotransferase. See Glutamate pyruvate transaminase L-Aspartate ammonia-lyase. See Aspartase L-Aspartate/3-decarboxylase activity of P. dacunhae, stabilization by glutaraldehyde treatment, 476-477 assay, in free and immobilized microbial cells, 473 L-Aspartic acid estimation of, 465-466, 472 industrial production, using polyurethane-immobilized cells containing aspartase, 463-471 production, by immobilized E. coli cells in batch reactor, 466-467 Aspartic aminotransferase, E. coli, relative rates of production of L-amino acids, 484 Aspergillus niger, lipase, 299 ATP analogs, synthesis, 5 with carboxymethyl dextran carrier, partition coefficient in aqueous twophase system, 55 dextran-bound partition in aqueous two-phase systems containing dextran T500, PEG plus PEG sulfate, and substrate, 52 partition in aqueous two-phase systems containing dextran T500 and PEG plus PEG sulfate, 51 preparation, 47-49 enzymatic regeneration

with acetyl phosphate/acetate kinase, 263-264, 265 from ADP and AMP, 263-280 application to enzyme-catalyzed syntheses, 273-280 enzymatic assays, 270-273 with methoxycarbonyl phosphate/ acetate kinase, 263-264 with phosphoenolpyruvate/pyruvate kinase, 263-264, 265 phosphorylating agents used in, 263 properties, 265 phosphoryl group donors, synthesis, 267-270 procedures, 263-264 synthesis of phosphoryl donors, 265266 in enzyme reactor studies, 9 immobilized, as active coenzyme, 3 modification in N-6 position, substituents used for, 4 partition, in system containing dextran T500, PEG, and PEG sulfate, 55 Azaheterocyclic chemistry, 256 2,2'-Azinodi-(3-ethylbenzothiazoline.6. sulfonic acid), 94-95 Azotobacter vinelandii, oxaloacetate decarboxylase, 485

B Bacillus subtilis, menthyl ester hydrolysis, in two-liquid phase biocatalytic reaction, 139-140, 145 Backward transfer, in solubilization of enzymes in reverse micellar media, 193 Bacterial cells, solubilized in hydrocarbon miceUar solutions, 215 Bacterial luciferase activity recovered on Sepharose, 89 analytical usefulness, in bioluminescent assays, 83 assay, 85 reaction catalyzed, 83 source, 83-84 Baker's yeast, production, 422 Benzylpenicillin, penicillin amidase-catalyzed synthesis, 287

SUBJECT INDEX fl-lactam antibiotics, enzyme-catalyzed semisynthesis, 281-292 Bifidobacteria, utilization of isomaltulose, 433 Biocatalysts, in organic solvents, 137 uses, 137 Bioluminescent assay, using coimmobilized enzymes, 82-93 advantages, 83 automation, 91-93 enzyme sources, 83-84 immobilization procedure, 85-88 instrumentation, 84 kinetics, 90-92 metabolites assayed, 86-90 stability of immobilized enzymes, 88 storage of immobilized enzymes, 88 Biphasic aqueous-organic mixtures, enzymatic esterifications in, 118-119 Bis(2-ethylhexyl)sodium sulfosuccinate. See Aerosol OT bis-NAD, as directing aid for orientation of site-to-site enzyme systems, 103-104 DL-Borneol, optical resolution, 302 Bovine serum albumin, in reverse micellar media, 213 Bradykinin, sequential hydrolysis with immobilized aminopeptidase, 176-177 Breoibacteriurn immobilization using K-carrageenan and polyethyleneimine, 460-461 using K-carrageenan and tannin, 461 using K-carrageenan modified with amines, 461-462 nitrilase activity, 523 Brevibacterium ammoniagenes culture of, 457 fumarase activity, 456 immobilization with polyacrylamide, 457 immobilized cells continuous enzymatic production of Lmalic acid, 458 productivity of L-malic acid, 460 Brevibacterium flavum culture of, 459 immobilization, with K-carrageenan, 459 immobilized cells continuous enzymatic production of Lmalic acid, 459-460

561

productivity of L-malic acid, 460 immobilized with r-carrageenan, improvements, 461-463 immobilized with modified K-carrageenan, 461-462 1,2-Butanediol alkaline hydrolysis of esters, 128 yeast lipase-catalyzed resolution of, via transesterification, 127-134 2-Butanol alkaline hydrolysis of esters, 128 yeast lipase-catalyzed resolution of, via transesterification, 127-132 Bz-Arg-VaI-NH2, trypsin-catalyzed synthesis, 287 Bz-Gly-Lys-Leu-OMe, trypsin-catalyzed formation, 185-186

C Cacao butterlike fat, production, 411 by interesterification with carrier-adsorbed lipase, 406 Calcium alginate, as yeast entrapping agent, 396 Candida boidinii, 10 Candida cylindrica, lipase. See Lipase, C. cylindrica Carbohydrates, biologically active, 230 N6-(2-Carboxyethyl)-NAD, 35-37 Carboxylesterase pig liver preparation, 122 preparative resolution of racemic primary alcohols catalyzed by, 121-126 source, 121 stereoselective transesterification in biphasic systems, for preparation of racemic alcohols and racemic esters, 120-137 transesterification reaction, 119 Carboxypeptidase Y assay, 159-160 in conversion of porcine insulin to human insulin, 163 deblocking in peptide synthesis with, 157-162 procedures, 159-160 deblocking of peptide esters, 160

562

SUBJECT I N D E X

deblocking of peptides attached to PEG handle, 161 immobilization, methods, 160 immobilized, uses, 162 preparation, 159 properties, 158 K-Carrageenan beads casting, 333 production, 335 commercial preparations, variations in, 334 diffusivity of nutrients and secondary metabolites into and out of, 334-335 immobilization of microbial cells with, 474-475 immobilization of PeniciUium with, 332334 improved immobilization of microbial cells, 460-462 modification with amines, 461-462 as yeast entrapping agent, 396 Catharanthus roseus, strictosidine synthase, 343 Celite beads, entrapment of Penicillium on, 335 Cellulose nitrate membrane microcapsules containing dextran-NAD and multienzyme systems, preparation, 75 preparation, 68-69 Cephalexin, synthesis, in hydrolase-catalyzed condensation reaction, 282 Cetyltrimethylammonium bromide reverse micellar media, 205 preparation, 216-217 Chanoclavine production, 320 retention time on HPLC, 320 Chiral chemicals, synthesis, 117-118 dehydrogenases in, 9-10 Chiral compounds, preparation, 302-317 1-Chloro-2-propanol alkaline hydrolysis of esters, 128 epoxidation, 129 Cholic acid methyl ester, oxidation to 12ketochenodeoxyxholic acid methyl ester, in two-phase system, 156 Chymosin, milk clotting with, 424 Chymotrypsin immobilization, to Enzacryl AH, 181

immobilized, in peptide synthesis, 182184 a-Chymotrypsin immobilized, assay, 181 in reverse micelles, 201-205 CD spectra, 204-205 kinetic parameters, 214 superactivity, 216 Citronellol hog liver carboxylesterase-catalyzed resolution of, via transesterification reactions, 121, 126 propionic esters, alkaline hydrolysis, 122 Clauiceps, saprophytic culturing, 317 Claviceps purpurea alkaloid production effect of nitrogen concentration, 325326 in various alginate concentrations, 320-322 alkaloid production capacity, improvement, 320-329 alkaloid yields, increasing, by increasing particle amount in fermentation broth, 327-328 biomass development, in various alginate concentrations, 320-322 culture, 318 immobilization, methods, 319 immobilization technique, simulation of natural alkaloid production conditions, 329 immobilized ceils, 317-329 alkaloid production in l-liter air-lift fermenter, 327 morphological aspects, 320 preparation, 318 immobilized mycelia morphology, 324-325 semicontinuous fermentation, 323 mycelium wet weight in calcium alginate beads, determination, 319 Claoiceps sclerotia, 317 Clostridial aminopeptidase assay, 171 colorimetric assay, 174 definition of unit, 174 fluorimetric assays, 174 glass-bound absence of endopeptidase activity, 173

SUBJECT INDEX adsorption, 172-173 application to sequential hydrolysis of proline-containingpolypeptides, 170-178 enzyme activity measurement, 174 metal ion requirement, 173 pH dependence, 173 stability, 172 temperature dependence, 713 immobilization, 172 immobilized, properties, 172-173 isolation, 171-172 soluble, enzyme activity measurement, 173 Clostridium histolyticum, aminopeptidase. See Clostridial aminopeptidase CM-PEG, preparation, 160-161 CoA, immobilized, as active coenzyme, 3 Coenzyme modified, synthesis, 12 regeneration, 7 simultaneous regeneration, 10 Coenzyme-dependent enzymes, immobilized, activity, 3 Coimmobilization, 56 of NAD with dehydrogenase, 21-34 activity of immobilized dehydrogenases, assay, 28 applications, 29-33 advantages, 33 continuous production of L-malate with, 29-31 disadvantages, 33 enzyme leakage from gel, 33-34 ethanol analyzer using, 31-33 FDH-MDH-NAD gel, 27 immobilized proteins, determination, 28 LADH-diaphorase-NAD gel, 27 methods, 21 NAD content, determination, 28 preparation of gel, 26-27 spatial arrangement in, 56 Condensation products enzyme-catalyzed synthesis, equilibriumcontrolled, vs. kinetically controlled, 280-292 kinetically controlled synthesis with enzymes

563

compared to equilibrium-controlled synthesis, 285-292 effect of enzyme concentration, 286287 effect of enzyme properties, 287-290 effect of substrate properties, 291-292 effect of water content, 290-291 factors affecting yield, 286 kinetically controlled maximum, 286287 procedure, 285-286 substrates, 285 Condensation reactions, hydrolase-catalyzed, mechanisms, 281-284 Cortisone, stereospecific reduction, in twophase systems, 154-155 Cortisone reductase. See 20/3-Hydroxysteroid dehydrogenase Corvire, 422-423 Corynebacterium glutamicum, phenylalanine production using, 498 Creatine kinase, activity recovered on Sepharose, 89 Creatine phosphate, bioluminescent assay using coimmobilized enzymes, 87 Cyclodextrin, 432 synthesis, in hydrolase-catalyzed condensation reaction, 283 1-Cyclohexyl-3(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate, 58-59 Cytochrome-cs hydrogenase, in carriermediated multienzymatic system in reverse micellar medium, 228-229 Cytochrome-c hydrogenase, in reverse micelles, 211 Cytochrome P-450, in reverse micelles, 210 Cytochrome P-450, -b5 reductase, in reverse micelles, 210

D Deblocking reactions, 157-158 in peptide synthesis, with carboxypeptidase Y, 157-162 Dehydrogenase, 56 coimmobilization with NAD. See Coimmobilization, of NAD with dehydrogenases

564

SUBJECT I N D E X

for native NADH and PEG-NADH, 1113 Dextran branching, 239 clinical, synthesis, 254 concentration, determination, 242 in dental caries, 239 high molecular weight, synthesis, 251252, 254 intermediate molecular weight, synthesis, 254 low molecular weight, synthesis, 254 molecular weight, control of, during synthesis, 240, 251,254 produced by immobilized dextransucrase, characterization, 251-254 uses, 239 Dextran-NAD, recycling within cellulose nitrate membrane microcapsules, 76-77 within microcapsules, measurement, 7576 within polyamide membrane microcapsules, 77 Dextran-NADH preparation, 75 retained within microcapsules, recycling, 74-77 Dextransucrase action mechanism, 240 assay, 241 free and immobilized behavioral differences between, 249253 initial reaction rate, effect of initial sucrose concentration, 249- 251 immobilization, 241 effect of maltose addition on, 248-249 effect of support specific area on, 247-248 efficiency, effect of amount of enzyme on, 248 procedure, 245-247 immobilized assay, 247 dextran produced by, characterization, 251-254 kinetic characterization, 249-251 kinetics of fructose production catalyzed by, 249-250

from L. mesenteroides dextran synthesis, 239-240 immobilization, 241 inductor, 239 low-branched dextran polymer produced by, 239 production, by batch-fed L. mesenteroides culture, 242-243 protein determination, 241 purification, 240, 242-245 by ultrafiltration and gel chromatography, 242-245 reaction catalyzed, 239 Dextran synthesis by immobilized L. mesenteroides dextransucrase, 239-254 in vitro, 239-240 in presence of acceptors, 251-254 a,to-Diaminopoly(ethylene glycol), 39-40 Diaminopolyethylene glycol, synthesis, 493-496 Diaphorase assay, 85 pig heart, source, 26 source, 83-84 a,to-Dichloropoly(ethylene glycol), 39 2,3-Dichloropropanol alkaline hydrolysis of esters, 128 epoxidation, 129 Diethylenetriamines, substituted, as enzyme reactor disinfectants, 418-419 Dihydrolipoamide dehydrogenase. See also Diaphorase in carrier-mediated multienzymatic system in reverse micellar medium, 228-229 Dihydroxyacetone phosphate enzyme-catalyzed synthesis, 263,277 synthesis, 266 3,7-Dimethyl-l-octanol, propionic esters, alkaline hydrolysis, 122 3,7-Dimethyl-l-octyl propionate, hog liver carboxylesterase-catalyzed resolution of, via transesterification reactions, 121, 126 Dipeptidyl aminopeptidase I, in sequencing of proline-containing peptides, 178 Dipeptidyl aminopeptidase IV, in sequencing of proline-containing peptides, 178

SUBJECT INDEX Dipeptidyl peptidase I. See Dipeptidyl aminopeptidase I Dipeptidyl peptidase IV. See Dipeptidyl aminopeptidase IV Disaccharide synthesis in hydrolase-catalyzed condensation reaction, 282 with immobilized fl-galactosidase, 230233 Disinfectants, for enzyme reactor, 418-419 Double transfer, in solubilization of enzymes in reverse micellar media, 193

E Electrochemical cell, design, 308-309 Eiectroenzymatic reduction reaction sequence, 303 scheme, 304 Electromicrobial reduction reaction sequence, 303 scheme, 304 ELISA. See Enzyme-linked immunosorbent assay Elymoclavine production, 320 retention time on HPLC, 320 [Leu]-Enkephalin, sequential hydrolysis with immobilized aminopeptidase, 175 2-Enoate reductase assay, 312-313 reduced mediator, preparation, 312 from C. tyrobutyricum, 303 immobilization, in calcium alginate gels, 313-314 immobilized, repeated use of, 315 kinetic parameters, 304, 306-307 purification, 309-310 reaction catalyzed, 305 stereoselectivity, 304 substrate specificity, 304, 305-306 Enterobacter agglomerans. See Erwinia rhapontici Enzymatic esterifications, in biphasic systems, 117-118 Enzymatic reaction, Michaelis-Menten kinetics, 223 Enzymatic transesterifications, for preparative production of optically active compounds, 119-137

565

experimental design, 120 rationale, 119-120 Enzyme channeling immmunoassay, 94, 98-103 coated surface, 98-100 soluble reagent microtiter plate protocol, 99-102 rapid protocol, 102-103 Enzyme immunoassay, 93 homogeneous, 93-94 Enzyme-linked immunosorbent assay, 93 Enzyme reactors, 7, 414. See also Membrane reactor; Open tubular heterogeneous enzyme reactor aqueous two-phase systems, applications, 54-55 continuous reaction system reaction rates of coenzymes, 41 theoretical analysis, 40-42 countercurrent partitioning using aqueous two-phase systems, 45-46 with coupled enzyme regeneration, 7-8 coupled two-enzyme steady-state analysis, 40-45 theoretical analysis, 40-42 fixed bed, 415-417 fluidized bed, 415-417 industrial operations with, strategy, 419422 model, steady-state analysis, 42-45 plug flow, 415 retention of coenzyme in, 13-15 stirred tank, 415-417 ultrafiltration system, 45 Enzymes, in organic syntheses, application of, 254-255 Epichlorohydrin, yeast fipase-catalyzed resolution of, via transesterification, 133 Ergometrine production, 320 retention time on HPLC, 320 Erwinia rhapontica, immobilized cells, isomaltulose production, 436-437 Erwinia rhapontici cells immobilized by various methods, activity and stability, 441 immobilized cells, isomaltulose production, 439 immobilized preparations, isomaltulose-

566

SUBJECT INDEX

forming activity, operational stabilities of, 445-448 isomaltulose-synthesizing activity, storage stability under various conditions, 450 Escherichia coli

aminotransferases, 483-484 aspartase activity effect of ammonium fumarate concentration, 470-471 effect of pH, 469-470 effect of temperature, 468-469 in various preparations, 467-468 cell immobilization, 464-465 culture, 474 immobilization with K-carrageenan, 475 in polyazetidine, 500 immobilized, in L-alanine production, 472-479 immobilized cells L-alanine production, elimination of side reactions, 475-476 aspartase activity, 465 immobilized cells containing aspartase, for L-aspartic acid production, 463471 immobilized with K-carrageenan, activities with and without pH treatment, 476 phenylalanine production, free cell studies, 498-499 polyurethane-immobilized cells containing aspartase activity, properties, 467-471 transaminases, 498-499 Esterase, immobilized, application, 355 Ethanol. See also Alcohol continuous fermentation, 394 using yeast immobilized in synthetic resin, 380-394 fermentation, using immobilized yeast cells, types of vessels for, 386-389 oxidation by NAD, with production of acetaldehyde and NADH, in tubular flow reactor system, 61-63 production bioreactor for, based on aqueous twophase systems, 55 yeast systems tested for, 382

Ethanol analyzer, using coimmobilized LADH-diaphorase-NAD gel, 31-33 Ethyl 2-(tritylamino)ethanoate, 24 F

Fatty acid esters, synthesis, in hydrolasecatalyzed condensation reaction, 283 FDH. See Formate dehydrogenase Fermentation vessel, 386-389 Finnsugar/Fermco IGI product, 367-368 Firefly luciferase activity recovered on Sepharose, 89 analytical usefulness, in bioluminescent assays, 82-83 assay, 84-85 reaction catalyzed, 82 source, 83-84 FMNH2, production, 83 Folch-Pi proteolipid, in reverse micellar media, 213 Formate:NAD ÷ oxidoreductase. See Formate dehydrogenase Formate dehydrogenase from C. boidinii, 305 as NADH-regenerating enzyme, 1011 for native NADH and PEG-NADH, kinetic parameters, 12-13 source, 10, 151 specific activity, 151 yeast, source, 26 Forward transfer, in solubilization of enzymes in reverse micellar media, 194, 195 6-O-a-D-Glucopyranosyl-D-fructofuranose. See Isomaltulose Fructose corn syrup, 55%, 364-366 Fumarase activity assays, 456-457 assay, in free and immobilized microbial cells, 473-474 immobilized application, 355 L-malic acid production, 455 Fumarate hydrase. See Fumarase Fumaric acid, estimation of, 465-466, 472 Fungi, immobilized cells, in fundamental studies, 340-341

SUBJECT INDEX

G fl-Galactosidase assay, 232 immobilization to Sepharose 4B, 231232 immobilized, disaccharide synthesis with, 230-233 transgalactosylation reaction, 230-23 l Galactosyl-N-acetylgalactosamine formation, 230-231 preparation via reversal of hydrolysis reaction, 231,233 via transferase reaction, 232-233 structure, 233 yield, 233 Glucagon, in reverse micelles, 211 Glucoamylase hydrolysis, 376 immobilized, saccharification, flow rate for, 377-378 liquid, saccharification, 374 liquid hydrolysis, costs, 378 6-P-Gluconate, bioluminescent assay using coimmobilized enzymes, 86 D-Glucose, bioluminescent assay using coimmobilized enzymes, 86 time course of light output, 90, 92 Glucose-6-phosphate dehydrogenase, microencapsulated, for multistep enzyme reaction of conversion of urea into glutamic acid, 72-73 Glucose dehydrogenase from B. megaterium, 13 immobilized in solution in semipermeable microcapsules, for multistep enzyme reaction in conversion of urea to ammonia to glutamic acid to alanine, 71-72 Glucose/galactose syrups, production, 376 Glucose isomerase batch isomerization, cycle duration, 360 cross-linked, for batch isomerization, 359-360 immobilization, methods, 356 industrial, first, 357-358 purified reusable, 358-359 Glucose isomerase, immobilized, 356-370 applications, 354

567

comparison of batch and fixed bed processes, 362-363 for fixed bed isomerization, 370 for fixed bed operation, 361-362 industrial-scale production-application, 353 development of products and processes, 356-364 effect of glucose substrate purity, 363 first, 357-358 first continuous isomerization process, 358 future developments, 368-369 impurities, 363-364 industrial development, 358-363 initial phase, 357-358 isomerization process, bottleneck model, 364-365 new products, 366-367 new syrup refining processes, 368 process optimization, 363-365 products, 364 second-generation isomerization processes and products, 364-368 products, 356-357 Glucose oxidase and anti-PRP, microtiter plates coated with, 98 derivatization with amino groups, 97-98 and horseradish peroxidase, reaction catalyzed together, 94 Glucose 6-phosphate enzyme-catalyzed synthesis, 263,279 repeated production, in aqueous twophase system, 52-54 L-Glutamate, bioluminescent assay using coimmobilized enzymes, 86 L-Glutamate:NAD(P)÷ oxidoreductase. See Glutamate dehydrogenase Glutamate dehydrogenase coimmobilization with 12a-hydroxysteroid dehydrogenase, on Sepharose CL-4B activated with tresyl chloride, 152-153 free and immobilized, Michaelis constants for, 153 immobilized, recovery of activity, 153 immobilized in solution in semipermeable microcapsules, for multistep enzyme reaction in conversion of

568

SUBJECT INDEX

urea to ammonia to glutamic acid to alanine, 71-72 microencapsulated, for multistep enzyme reaction of conversion of urea into glutamic acid, 72-73 PEG-NADH as coenzyme, 13 source, 151 specific activity, 151 Glutamate pyruvate transaminase, immobilized in solution in semipermeable microcapsules, for multistep enzyme reaction in conversion of urea to ammonia to glutamic acid to alanine, 71-72 Glutamic-oxaloacetic aminotransferase E. coli, 483-484 assays, 486-487 immobilization for L-phenylalanine production, 486 mixed with immobilized oxaloacetate decarboxylase from P. putida, 492 immobilization, on porous diatomaceous earth, 491 pig heart, 483 assay, 488 immobilization, 488 Glutamic-pyruvic aminotransferase immobilization, on porous diatomaceous earth, 490-491 immobilized, long-term operational stability, 492 pig heart assay, 488-489 immobilization, 488 porcine, 483 Glutamic-pyruvic transaminase, 480 Glycerides, 1,3-substituted, synthesis in hydrolase-catalyzed condensation reaction, 283 sn-Glycerol 3-phosphate enzyme-catalyzed synthesis, 263, 276277 synthesis, 266 Godo-AGI, 370 Gramicidin S synthetases, 330

H Haemophilus influenzae, capsular antigen, 95

Hexokinase and acetate kinase, enzyme reactor based on aqueous two-phase system, 46 activity recovered on Sepharose, 89 partition in aqueous two-phase systems conraining dextran T500, PEG plus PEG sulfate, and substrate, 52 in aqueous two-phase systems conmining dextran T500 and PEG plus PEG sulfate, 51 High dextrose equivalent syrup, 412 High-fructose corn syrup, 356, 432 High-fructose syrup, 376 Horseradish peroxidase chromagen substrates, 94 and glucose oxidase, reaction catalyzed together, 94 Hydantoinase, immobilized, application, 354, 355 Hydrogenase, in reverse micelles, 211-212 Hydrolase, products synthesized using, 280, 283-283 Hydro-lyase, transesterification reaction, 119 Hydrolytic reactions, reversal, 234, 503 in disaccharide synthesis, 233 urease as model for investigation of, 238 N-[2-Hydroxy-3-[2-(tritylamino)acetamido]propyl] methacrylamide, 25 (2R)-Hydroxycarboxylate-viologen oxidoreductase, 303-304 assay, 313 reduced mediator, preparation, 312 enrichment procedure, for P. vulgaris, 311 kinetic parameters, 304, 307-308 partially purified, use of, 315-316 purification, 311-312 reaction catalyzed, 306 reduction of 2-oxocarboxylate, 316 reduction of enoate, 315-316 stereoselectivity, 304 substrate specificity, 304, 306-307 in whole cells, 315-316 D-Hydroxyisocaproate dehydrogenase, kinetic parameters for native NADH and PEG-NADH, 12 L-Hydroxyisocaproate dehydrogenase,

SUBJECT INDEX

kinetic parameters for native NADH and PEG-NADH, 12 [N-(2-Hydroxy-3-methacrylamidopropyl)carbamoyl]methylammonium ptoluene sulfonate, 25 Nr- [N-[N-(2-Hydroxy-3-methacrylamido propyl)carbamoylmethyl]carbamoylmethyl]-NAD, 25-26 fl-Hydroxysteroid:NAD(P) ÷ oxidoreductase. See 3 (or 17)/3-Hydroxysteroid dehydrogenase 3a-Hydroxysteroid dehydrogenase microencapsulated, with L. mesenteroides with NADH oxidase, in stereospecific steroid oxidation, 7374 steroid transformation with, 156-157 7a-Hydroxysteroid dehydrogenase, activity recovered on Sepharose, 89 12ct-Hydroxysteroid dehydrogenase coimmobilization with glutamate dehydrogenase, on Sepharose CL-4B activated with tresyl chloride, 152153 free and immobilized, Michaelis constants for, 153 immobilized, recovery of activity, 153 source, 151 specific activity, 151 steroid transformation with, 156-157 20/3-Hydroxysteroid dehydrogenase on aqueous and reverse micellar media, Michaelis-Menten parameters for, 223-224 in carrier-mediated multienzymatic system in reverse micellar medium, 228-229 encapsulation in reverse micelles, method, 221 free and immobilized, Michaelis constants for, 153 immobilization on CNBr-activated Sepharose 4B, 152 immobilized, recovery of activity, 153 reverse micellar enzymology of, 216 in reverse micelles activity, determination, 221-222 parameters regulating activity, 226 source, 151 specific activity, 151

569

steroid transformation with, 156-157 3 (or 17)/3-Hydroxysteroid dehydrogenase source, 151 specific activity, 151 steroid transformation with, 156-157 3a-Hydroxysteroids, bioluminescent assay using coimmobilized enzymes, 87 7a-Hydroxysteroids, bioluminescent assay using coimmobilized enzymes, 87 12a-Hydroxysteroids, bioluminescent assay using coimmobilized enzymes, 87

I Immobilized active coenzymes, 3-9 applications, 7-9 preparation, 3-6 degree of substitution of polymers, 56 preassembly approach, 3-6 solid-phase modular approach, 3-5 special requirements, 5 water-soluble polymers in, 5-6 properties, 6-7 Immobilized cell systems, industrial applications, 353 Immobilized enzyme hydrolysis, 377-379 flow rate, 377 productivity, 377-378 running expenses, 377-378 Immobilized enzymes activity/productivity, 372-373 criteria for use as catalyst in organic chemistry, 255-256 industrial applications, 255, 353 industrial operation, 371-379 manufacturing cost, 371-372 minimum commercial viability, calculated criteria, 373 for organic syntheses, 117 Immobilized microbial ceils applications, 354 r-carrageenan immobilization, 455-456, 474-475 improvement, 456 continuous production of L-alanine, 472479 isomaltulose production using, 432-454 L-malic acid production, 455-463

570

SUBJECT INDEX

polyazetidine immobilization, 497 reactor configuration, for isomaltulose production, 443-444 Indole alkaloids, 342-343 Insulin human preparation using immobilized Achromobacter protease I, 168-170 synthesis in hydrolase-catalyzed condensation reaction, 282 tert-butyl ester, trypsin-catalyzed formation from DAI and threonine tert-butyl ester, 186 porcine enzymatic conversion to human insulin, 162 hydrolysis using immobilized Achromobacter protease I, 167-168 Interesterification lipase-catalyzed, set-up for, 410-411 TES buffer used as activator, 409-410 of triglyceride, 405 using olive oil and stearic acid, 407-409 Invertase hydrolysis, 376 immobilized, applications, 354 liquid hydrolysis, costs, 378 Iron-cytochrome-c reductase, in reverse micelles, 210, 211 Isomaltose, synthesis, in hydrolase-catalyzed condensation reaction, 282 Isomaltulose as calorific bulking agent, 432 calorific value, 432 crystallization, 448-449 in food, drink, and medicine formulations, 433 noncariogenic nature, 433 physical properties, 433 production by fermentation, 433-435 by immobilized microbial cells, 432454 alginate-immobilized cell pellets used in, mechanical strength of, 442-443 ceil-free isomaltulose-forming enzyme in, 439-440 cell immobilization, 437-439 choice of immobilization method, 441-442

choice of microorganism, 435-437 desirable improvements, 454 immobilization methods, 451-453 large-scale method, 454 pilot plant, 454 potential advantages of, 435 productivity, 453-454 reactor configuration, 443-444 regeneration of immobilized cell activity, 447-448 stabilization, 445-448 storage stability of immobilized cells, 449-451 by solid-state fermentation of sugar cane, 435 properties, 432-433 resistance to acidic hydrolysis, 433 sweetness, 432 Isomaltulose synthease characterization, 439-441 from E. rhapontici, 440-441 from P. rubrum, 440-441 Isopropanol dehydrogenase, PEG-NADH as coenzyme, 13 Isosyrup, 356 with 55% fructose, 369 global production, 364 Isosyrup industry, development of, 356369

K Ketomax GI-100, 366-367, 370 20-Ketosteroids, reduction to 20/3-hydroxysteroids, in two-phase systems, 154155 Kinase, enzyme reactor based on aqueous two-phase system, 46 design, 46-47 determination of partition coefficients, 50-52 optimal partition, investigation of, 50-52 repeated production of glucose 6-phosphate, 52-54 Kobayashi-Laidler theory, 62

L Laccase, steroid transformation with, 156157 Lactase

SUBJECT INDEX A. niger, properties, 413-414 A. oryzae

pH optimum, 414 properties, 413-414 acid fungal, 413 deactivation during hydrolysis of whey, factors controlling, 419-420 hydrolysis, 376 immobilized applications, 354 engineering considerations for processes using, 414-417 hydrolysis of whey, and cleaningsanitation cycles, 420 industrial developments with, 422423 kinetic behavior, 413 lactose hydrolysis, 412-423 microbial contamination of reactor, 417-419 operational life, 414 production-application, 411-423 properties, 413-414 reactor design, 415-416 reactor using, activity, 419 reactor using, stability, 419 sanitation, 418-419 semiindustrial operations with, 421423 stability, effect of cleaning-sanitation cycles in reactor, 420-421 temperature of operations with, and operational life of enzyme, 421422 immobilized composites, 412 preparation, 413 liquid hydrolysis, costs, 378 thermal deactivation, 419 L-Lactate bioluminescent assay using coimmobilized enzymes, 86 production, in coupled two-enzyme reactor, steady-state analysis, 4245 L-Lactate:NAD + oxidoreductase. See LLactate dehydrogenase Lactate dehydrogenase. See also Site-tosite enzyme systems activity recovered on Sepharose, 89 assay, 108 beef heart

571

immobilized site-to-site enzyme system with alcohol dehydrogenase, 104-106 soluble site-to-site enzyme system with alcohol dehydrogenase, 106108 Lactate dehydrogenase kinetic parameters for native NADH and PEG-NADH, 12 rabbit muscle in coupled two-enzyme reactor, steady-state analysis, 42-45 kinetic constants, 43 source, 151 specific activity, 151 Lactobacillus brevis, enzymatic batch process for L-malic acid production using, 455 Lactose hydrolysis reaction, 412 hydrolyzed, 411-412 applications, 412 Lactose-containing feedstocks, 415-416 LADH. See Alcohol dehydrogenase, horse liver L-Leucine, production, in enzyme-membrane reactor, 18-20 Leucine dehydrogenase, kinetic parameters for native NADH and PEGNADH, 12 Leuconostoc mesenteroides dextransucrase. See Dextransucrase

immobilization, 452 as source of NADH oxidase, microencapsulated with 3a-hydroxysteroid dehydrogenase, for stereospecific steroid oxidation, 73-74 Leucrose, 240 Lima bean trypsin inhibitor, 177 Lipase from A. niger, 299 adsorbed to Celite coated with buffer solution, preparation, 408-409 adsorbed to Celite coated with glycerol, preparation, 406-407 from C. cylindracea, 299 Celite-adsorbed, interesterification reaction, 409-410 preparative resolution of racemic esters catalyzed by, 134-136 preparative resolution of racemic

572

SUBJECT INDEX

secondary alcohols catalyzed by, 125, 127-134 stereoselective transesterification in biphasic systems, for preparation of racemic alcohols and racemic esters, 120-137 Celite-adsorbed preparation, 406-407 regiospecific interesterification of triglyceride with, 405-411 immobilized applications, 354-355 properties of, 302 interesterification with, 405 from R. delemar, 299 Celite-adsorbed, interesterification reaction, 409-410 Lipid-polyamide membrane artificial cell, applications, 82 Lipid-polymer membrane microcapsules assaying using ammonia, 79 assaying using urea, 79 cofactor recycling with different amounts of NAD ÷ retained within, 79-80 conversion of urea into glutamic acid with NADH recycling in, 80-81 permeability, 78 preparation, 78-79 recycling of free NADH retained within, 77-81 results, 79-81 Lipoamide dehydrogenase. See also Siteto-site enzyme systems assay, 108 scavenger assay with, as enzyme competing with LDH for NADH formed by ADH, 109-110, 112-113 Lipophilin, solubilization in reverse micellar media, 194-197 Lipoxygenase, in reverse micelles, 211212 kinetic parameters, 214 Liquid enzyme hydrolysis costs, 378 time course, 374 total saccharification cost, 374 Liquid enzymes, vs. immobilized, comparative economics, 379 Luciferase. See also Bacterial luciferase; Firefly luciferase

coimmobilized with other enzymes, analytical usefulness, 82 coimmobilized with oxidoreductase, on nylon tubing, 84 coupled to oxidoreductase, 83 Luciferase/oxidoreductase preparation assay, coupled reaction, 85 source, 84 Luciferin, source, 84 Lyase, immobilized, application, 355 Lylose. See Isomaltulose Lysozyme in reverse micelles, 206-209 kinetic parameters, 214 solubilization in reverse micellar media, 194-197 superactivity, 216

M L-Malate bioluminescent assay using coimmobilized enzymes, 86 continuous production, by coimmobilized FDH-MDH-NAD gel, 29-31 Malate dehydrogenase activity recovered on Sepharose, 89 microencapsulated, with malate dehydrogenase, recycling of NAD + and NADH, 70-71 PEG-NADH as coenzyme, 13 stability in microcapsules, 77 Thermus

molecular weight, 33-34 source, 26 L-Malic acid estimation of, 456, 465-466, 472 industrial production, 455,462-463 production by immobilized microbial cells, 455-463 comparison of various preparations, 461,463 continuous enzyme reaction, 458 effect of detergent treatments, 457458 enhancement of, 457-458 suppression of succinic acid formation, 457-458 uses, 455 Malolactic enzyme, immobilized, applications, 354

SUBJECT INDEX Maltose, synthesis, in hydrolase-catalyzed condensation reaction, 282 Maltose syrup, 432 Mammalian cell culture, using microcartiers and immobilized systems, 353 Matsuo Mine application of bacterial oxidation to, 532 feasibility tests, 532-533 neutralization and solid-liquid separation, 538 neutralization plant bacterial oxidation system, 534-537 neutralization plant contents, 534-535 neutralization plant current situation, 539 neutralization plant installation, 533534 neutralization plant operating costs, 539 sludge storage dam, 538 background, 532 technical developments at, 540 Maxazyme, 361,370 MDH. See Malate dehydrogenase Membrane reactor, 7-9, 11 advantages of, 20 continuous enzymatic transformation in, with simultaneous NADH regeneration, 9-21 experimental setup, 15-19 flow diagram of continuous process in, 16 laboratory model, 16-17 optimal ratio of enzyme activities, 20 performance, 19-21 polarization control, 16-17 pulse-free piston pump for, 19 simple, 16 oL-Menthol isomers, 293 optical resolution of, by entrapped biocatalysts, 293-302 stereoselective esterification by gelentrapped lipase, 293, 299-302 analytical methods, 300-301 effect of organic solvents, 301 enzyme, 299 materials, 299 methods, 299 prepolymer, 299 reaction conditions, 300-301

573

L-Menthol uses, 293 DL-Menthyl succinate selective hydrolysis by gel-entrapped yeast cells, 293-299 analytical methods, 296 cultivation of yeast, 294 effect of solvents, 295 materials, 293-294 methods, 293-294 prepolymers, 293-294 production of L-menthol, 298-299 properties of gel-entrapped cells, 297298 reaction conditions, 295-296 synthesis, 296-297 Metalloproteinases, 504 Nr-[N-(6-Methacrylamidohexyl) carbamoylmethyl]-NAD, 25-26 3-Methoxy-l-butanol hog liver carboxylesterase-catalyzed resolution of, via transesterification reactions, 121, 125-126 propionic esters, alkaline hydrolysis, 122 Methoxycarbonyl phosphate properties, 265 synthesis, 263,270 6-Methylacrylamidohexylammonium chloride, 23-24 6-Methyl-5-hepten-2-ol alkaline hydrolysis of esters, 128 yeast lipase-catalyzed resolution of, via transesterification, 127-133 3-Methyl-l-pentanol, propionic esters, alkaline hydrolysis, 122 Methyl-l-pentyl propionate, hog liver carboxylesterase-catalyzed resolution of, via transesterification reactions, 121, 126 6-Methylsalicylic acid synthetase, half-life, 330 Micelles, filled and unfilled characterization, 197 molecular weight determination, 197-199 Microbial immobilization, requirements for supports, 396-397 Micrococcus luteus, oxaloacetate decarboxylase, 484-485 Microemulsion, 192 Microencapsulation, of multienzyme system, 57, 67-82

574

SUBJECT INDEX

applications, 67, 81-82 methods, 68-70 Microorganisms, immobilized, application, 355 Milk, ultrahigh-temperature sterilized. See UHT milk Milk permeates microbial contamination, 414-415 utilization, 411 Milk xanthine oxidase, 255 coimmobilization with superoxide dismutase and catalase, 257 commercial, 257 immobilization experimental procedure, 258-259 procedure, 257-258 specific activity, 257 storage stability, 257 Mine acid. See also Acid mine drainage cause, 530 Monoglyceride, fatty acid composition, analysis, 408 Myoglobin, in reverse miceUar media, 213

N NAD analogs, synthesis, 5 as analytical reagent, repeated use of, 33 bioluminescent assay using coimmobilized enzymes, 86 coimmobilization with dehydrogenases. See Coimmobilization, of NAD with dehydrogenases coupled to polyethyleneimine, 75 free recycling, by microencapsulated yeast alcohol dehydrogenase and malate dehydrogenase, 70-71 retained inside microcapsules, methods for, 81-82 immobilized, as active coenzyme, 3 immobilized active, uses, 7 immobilized derivatives, kinetic properties, 6-7 modification in N-6 position, substituents used for, 4 polymeric, 38-39 coenzymatic properties, 34-35 properties, 38-39

polymerizable derivative, 34 coimmobilization with dehydrogenases in polyacrylamide gel, 22 polymerization, 39 preparation, 22-26, 35-39 synthesis, 35 recycling, 21, 35, 56 in microcapsules, 81-82 in stereospecific steroid oxidation, 7374 sources, 57-58 NAD(H) as coenzyme for dehydrogenase, 9 immobilized, coenzyme activity, 3 polyethylene glycol-bound. See PEGNAD(H) regeneration, 10 NAD(P), recycling, by multienzyme systems microencapsulated in artificial cells, 67-82 NAD(P)H electroenzymatic regeneration, 303-304 practical aspects, 314 regeneration, 303 NAD(P)+ reductase, methyl viologendependent, 304 NAD-N6-[N-(N-acryloy1-1-methoxycarbonyl-5-aminopentyl)propionamide], 34, 38 NAD+-N6-[N-(6-aminohexyl)acetamide] coupled to dextran T70, 75 immobilized with soluble dextran T70 activated with cyanogen bromide, 59 NADH dehydrogenases for, 11-13 free, recycling by microencapsulated yeast alcohol dehydrogenase and malate dehydrogenase, 70-71 by multienzyme systems microencapsulated in artificial cells, 70-74 free, retained within lipid-polymer membrane microcapsules, recycling, 77-81 H2-driven regeneration of, and subsequent reduction of apolar steroid, in reverse micellar medium, 228-229 preparation, kinetic parameters of dehydrogenases for, 12

SUBJECT INDEX recycling, in conversion of urea and ammonia into amino acid, by multienzyme system in semipermeable microcapsules, 71-72 NADH:FMN oxidoreductase, activity recovered on Sepharose, 89 NADP analogs, synthesis, 5 bioluminescent assay using coimmobilized enzymes, 87 time course of light output, 90, 92 in enzyme reactor studies, 9 immobilized, as active coenzyme, 3 partition, in aqueous two-phase system containing Ficoll and UCON, 55 NADP(H) as coenzyme for dehydrogenase, 9 free, recycling, by multienzyme systems microencapsulated in artificial cells, 70-74 recycling, in sequential conversion of urea and ammonia to amino acid, in multistep enzyme reaction in microcapsules, 72-73 regeneration, I0 Nicotine adenine dinucleotide. See NAD Nitrilase assay, 524 immobilized, application, 355 Nocardia erythropolis, 3/3-dehydrogenation of cholesterol, in two-liquid phase biocatalytic reaction, 140-141 Nocardia rhodochrous, conversion of cholesterol to cholestenone, in twoliquid phase biocatalytic reaction, 139-141, 145-148 Nonseparation immunoassay advanatges of, 103 compared to ELISA, 94, 103 principle, 94 sensitivity, 94, 103 Nuclease P~, assay, 272 Nucleic acid, solubilized in hydrocarbon micellar solutions, 215 Nucleoside triphosphate conversion of RNA to mixture of, 278279 enzymatic regeneration, 266 Nutrisearch Company, 422 Nylon tubing

575

covalent attachment of alcohol dehydrogenase and NAD analog inside, procedure, 59-61 covalent attachment of alcohol dehydrogenase and NAD to, 59 oxidoreductase and luciferase coimmobilized on, 84

O 2-Octanol alkaline hydrolysis of esters, 128 yeast lipase-catalyzed resolution of, via transesterification, 127-132 Oligonucleotides, synthesis, in hydrolasecatalyzed condensation reaction, 282 Open tubular heterogeneous enzyme reactor, 59, 67 Optically active alcohols and esters, enzymatically prepared, chemical conversion to other optically active compounds, 130 Optically active compounds enzymatic production in biphasic aqueous-organic systems, 117-137 enzymatic production in nonaqueous organic systems, 137 function, 117-118 Optisweet 22, 367, 370 OTHER. See Open tubular heterogeneous enzyme reactor Oxaloacetate, decarboxylation, 482-483 Oxaloacetate decarboxylase, 483,499 P. putida assay, 490 immobilization, 489-490 sources, 484-485 Oxaloacetic acid, decarboxylation, 499500 Oxidoreductase assay, 85 coimmobilized with luciferase, on nylon tubing, 84 coupled to luciferase, 83 in reverse micelles, 208-212 source, 83 2-Oxocarboxylate reductase, from Proteus, 303

576

SUBJECT INDEX

P Packed bed fermentation vessel, 387 Palatinose. See Isomaltulose Palmitate-enriched glyceride, interesterification with pancreatic lipase to obtain, 405 Pancreatic lipase porcine, monophasic system, in nearly anhydrous organic solvents, 137 in reverse micelles, 206 Pancreatic ribonuclease, in reverse micelles, 206-207 kinetic parameters, 214 Papain, immobilized assay, 181 in peptide synthesis, 183 Parallel flow reactor, 386-388 Patulin assay, in Penicillium culture filtrates, 339-340 biosynthetic reaction, 329-330 production by immobilized Penicilliurn, 329-342 PEG carboxylated, elution in membrane reactor, 14-15 elution in membrane reactor, 14-15 water-soluble handle, in peptide synthesis, 158-159 PEG-NAD cofactor activity, 35 preparation, 34, 40 synthesis, 35 PEG-NAD(H) preparation, 39-40 elution in membrane reactor, 13, 14-15 preparation, kinetic parameters of dehydrogenases for, 12 properties, 11 synthesis, 11-12 PEG sulfate, preparation, 49-50 Penicillin assay, in Penicillium culture filtrates, 339-340 biosynthetic reaction, 330 production by immobilized Penicillium, 329-342 semisynthesis, 280-292 Penicillin (G or V) acylase, immobilized, application, 355

Penicillin amidase from E. coli, 284 synthesis of semisynthetic penicillins and peptides catalyzed by, mechanism, 284 Penicillium chrysogenurn antibiotic-producing activity establishment, 335-337 maintenance, 337-339 antibiotic productivity, determination, 339 batch cultivations, cessation of antibiotic production, 330 cell growth, determination, 339 culture filtrates, assays, 339-340 immobilization equipment, 331-335 on K-carrageenan, 332-334 procedure, 331-335 immobilized, sampling procedures, 339 immobilized ceils, in fundamental studies of secondary metabolism, 340342 Penicillium urticae antibiotic-producing activity establishment, 335-337 maintenance, 337-339 antibiotic productivity, determination, 339 batch cultivations, cessation of antibiotic production, 330 cell growth, determination, 339 culture filtrates, assays, 339-340 immobilization equipment, 331-335 on K-carrageenan, 332-334 procedure, 331-335 immobilized, sampling procedures, 339 immobilized cells, in fundamental studies of secondary metabolism, 340342 patulin-producing, effect of harsh immobilization, 342 PEP, bioluminescent assay using coimmobilized enzymes, 87 Pepsin A, in reverse micelles, 203 Peptidase, immobilized, application, 355 Peptide semisynthesis, 162, 280-292 Peptide synthesis chain elongation and release, carboxypeptidase Y in, 159

SUBJECT INDEX deblocking, with immobilized carboxypeptidase Y, 157-162 enzymatic approaches, 179 protease-catalyzed advantages, 187 future of, 187-188 requirements, 187 thermodynamic approach, 179, 184187 protease-mediated kinetic approach, 179-184, 187 immobilization procedures, 180-182 materials, 180-182 strategies, 179 using immobilized proteases, 178-188 water-solube PEG handle in, 158-159 Peroxidase in reverse micelles, 210, 212 kinetic parameters, 214 steroid transformation with, 156-157 sec-Phenethyl alcohol, alkaline hydrolysis of esters, 128 L-Phenylalanine production, using immobilized aminotransferase, 484, 486 production via E. coli equilibrium studies, 499-500 optimization of cell loading, 501-503 production via polyazetidine-immobilized E. coli, 497-503 Phenylalanine dehydrogenase, for native NADH and PEG-NADH, kinetic parameters, 12 sec-Phenylethyl alcohol, yeast lipasecatalyzed resolution of, via transesterification, 127-132 Phenylpyruvate conversion to phenylalanine, effect of increasing aspartic acid concentration, 498 as starting material for phenylalanine production, 497-498 DL-Phe-OMe, preparation, 513 5'-Phosphodiesterase assays, 518 immobilization, 518 immobilized activity, and pH, 518-519 application, 355 enzymatic properties, 518-519 in industrial operation, stability, 519

577

Phosphoenolpyruvate properties, 265 synthesis, 263, 268-269 Phospholipase, in reverse micelles, 206207 Phospholipase A2, in reverse micelles, 207 5-Phospho-a-D-ribosyl pyrophosphate concentration, determination, 271-272 synthesis, 266-267 from ribose 5-phosphate, 278 5-Phospho-a-D-ribosyl pyrophosphate synthetase assay, 271 source, 270 Photinus-luciferin 4-monooxygenase. See Firefly luciferase Photinus pyralis, luciferase. See Firefly luciferase Photobacterium fischeri, luciferase/oxidoreductase preparation, 84 Photo-cross-linkable resin. See also Yeast, photo-cross-linkable resin-immobilized structure, 383 Phyllostine, production by Penicillium urticae mutant P3,341 Plasmid, solubilized in hydrocarbon micellar solutions, 215 Poly(acrylamide-co-N-acryloxysuccinimide), 274-275 assay for active ester content, 275 Polyamide membrane microcapsules containing dextran-NAD and multienzyme systems, preparation, 75 preparation, by interfacial polymerization, 69-70 Polyethylene glycol. See PEG Polynucleotide phosphorylase, assay, 272273 Polynucleotides, synthesis, in hydrolasecatalyzed condensation reaction, 282 Polyribose phosphate enzyme-channeling assays, 95-103 equipment, 95-96 materials used, 95-98 measurement of protein concentrations, 96 methods, 98-103 reagent preparation, 95-98 glucose oxidase-labeled, preparation, 97 HRP-labeled, preparation, 96-97 Porin, in reverse micellar media, 213

578

SUBJECT INDEX

(Pro-Gly-Pro)10, sequential hydrolysis with immobilized aminopeptidase, 176-177 Progesterone reduction, in enzymatic reactions in reverse micelles, 229 stereospecific reduction, in two-phase systems, 154-155 Proline-containing pelbtide, sequencing, 170, 178 (2R)-Propanediol, preparation, by combination of Candida utilis and AIcaligenes eutrophus, 316-317 Propylene oxide, yeast lipase-catalyzed resolution of, via transesterification, 133 Protaminobacter immobilization, 451-453 isomaltulose production, 439 isomaltulose-synthesizing activity, storage stability under various conditions, 450 Protaminobacter rubrum enzyme responsible for isomaltulose production immobilization, 453 isolation, 453 isomaltulose production, 434 Protease immobilized application, 355 peptide synthesis using, 178-188 in peptide synthesis, 161-162 in reverse micellar media, 201-206 Proteus vulgaris growth, 311 (2R)-hydroxycarboxylate-viologen oxidoreductase, enrichment procedure, 311 Pseudomonas chlororaphis, nitrilase activity, 523 Pseudomonas dacunhae L-aspartate fl-decarboxylase activity, stabilization, 476 culture, 474 immobilization with r-carrageenan, 475 immobilized, in L-alanine production, 472-479 immobilized cells, L-alanine production, elimination of side reactions, 476 immobilized with r-earrageenan, activi-

ties with and without pH treatment, 476 Pseudomonas oleovorans, epoxidation activity, in two-liquid phase biocatalytic reaction, 139-140, 145 Pseudomonas putida conversion of 1,7-octadiene to 7,8epoxy-l-octene, in two-liquid phase biocatalytic reaction, 139, 144-145 oxaloacetate decarboxylase, 484-485, 489-490 transaminase, 497-498 Pyrite, in formation of mine acid, 530 Pyrophosphatase, inorganic, in reverse micelles, 206-207 Pyruvate kinase specific activity, 264 stability in immobilized form, 264

R Racemic alcohols, resolution, using esterase-catalyzed transesterification, 131 Racemic esters, yeast lipase-catalyzed preparative resolution of, via transesterification, 134-136 Racemic primary alcohols, hog liver carboxylesterase-catalyzed resolution of, via transesterification reactions, 121126 Racemic secondary alcohols, preparative resolution catalyzed by yeast lipase, 125, 127-134 Raffinate, treatment with glucoamylase, 376 Reaction center from R. sphaeroides, in reverse micellar media, 213 Reactors. SeeEnzyme reactors Reductase, 302 Research Association for Petroleum Alternatives Development, 380 research activities, 395 Retention, definition of, 14 Reverse micellar media concentration, expression, 200-201 enzymes in, 188-216 assay, 199-200 conformation and activity of, 199-212 intrinsic rate parameters, determination, 222-224

SUBJECT INDEX intrinsic rate parameters for, 200-201 HEPES-hexanol-CTAB-octane system, phase diagram, 217-219 hydrocarbon-soluble substrates added to, 215 multienzymatic reactions in, 228-229 pH determination, 201 solubilization of enzymes in, 192-197, 219-221 inje%tion method, 192-193,219-220 methods, 192, 219 phase-transfer method, 193-195, 220221 stability diagrams, 192-193 by transfer from solid state, 194-197, 220 Reverse micelles enzymatic conversion of apolar substrates in log P definition of, 226 determination of, 226-227 mole fraction of cosuffactant in interphase and its amount in continuous phase, determination, 227228 parameters regulating, determination of, 225-228 for enzymatic synthesis of apolar compounds, 216 enzymes in activity of, determination, 221-222 conformational aspects, 215 discrepancies of results between laboratories, 215 relationship between water of water pool and guest enzyme, 215 and suffactant purity, 215 vs. normal aqueous solutions, 200 water shell model, 198-199, 215 optimal solubility of proteins in, 215-216 physical characterization, before enzyme uptake, 192 preparation, 189-192 protein-containing characterization, 197-199 ultracentrifugation, 197-198 use to solubilize proteins in aprotic media, 212-215 Rhizopus arrhizus

579

immobilized lipase, continuous hydrolysis of triglyceride, in two-liquid phase biocatalytic reactors, 147 lipase, immobilization on Celite, 148 Rhizopus de&mar, lipase, 299 Rhizopus niveus, lipase, 149 Rhodococcus Strain 6 culture, 525 immobilized cells acrylamide production, 530 electron micrograph, 526, 527 enzyme activity effect of acrylonitrile and acrylamide concentrations, 529 effect of pH, 527-529 effect of temperature, 526-527 nitrilase properties, 527-529 preparation, 525-527 nitrilase activity, 523-524 Rhodopsin, in reverse micellar media, 213 Rhodotorula minuta, conversion of menthyl succinate to menthol, in twoliquid phase biocatalytic reaction, 140-141, 146 Rhodotorula minuta var. texensis culture, 294 gel-entrapped cells activity, 297 effect of gel hydrophobicity, 297 effect of reaction temperature, 297 properties, 297 stability of hydrolytic activity, 297298 immobilization, 294 Ribonuclease, superactivity, 216 5'-Ribonucleotides preparation, by enzymatic hydrolysis of RNA, 517 for preparation of food additives and drugs, 517 produced by immobilized 5'-phosphodiesterase concentration, 522 isolation, 522 purification, 522 separation, 522 production by-products, 517 effect of Zn2+ ions during continuous operation, 519-520

580

SUBJECT INDEX

enzyme support, 518 material, 518 methods, 518-521 nucleotides for, 518 preparation of substrate solution, 519521 process, 520-522 using immobilized 5'-phosphodiesterase, 517-522 Ribose 5-phosphate, synthesis, by acidcatalyzed hydrolysis of AMP, 278 RNase, reduced and carboxymethylated, 177

S Saccharification cost comparisons, 378-379 minumum cost for immobilized enzymes, 379 for liquid hydrolysis, 379 total cost, 374 Saccharification tank system, 375-377 running costs, 375-376 saccharification time, 375-376 Saccharomyces. See Yeast Secologanin source, 344 in strictosidine formation, 342 Secondary metabolism immobilized cells in fundamental studies of, 340-342 longevity, 340-341 Secondary metabolites, commercial production, 329 Semisynthesis catalyzed by penicillin amidase and serine protease, mechanisms, 281282 of human insulin, 162-170 of peniciUins and peptides, 280-292 of peptide, 162 of proteins, 234 Serine carboxypeptidase. See Carboxypeptidase Y Serine protease, synthesis of semisynthetic penicillins and peptides catalyzed by, mechanism, 284 Serratia isomaltulose production, 439

isomaltulose-synthesizing activity, storage stability under various conditions, 450 Serratia marcescens, immobilization, 452 Serratia plymuthica immobilization, 452 isomaltulose production, 434 Silane-glutaraldehyde immobilization, of lactase, 413 Site-to-site enzyme systems ADH activity, 110-112 characterization, 110-113 enzyme assays, 108-110 immobilized system, 104-106 orientation, 104-108 procedure, 104 scavenger enzyme assay, 109-110, 112113 soluble system, 106-108 Skim milk membrane vesicle fractions, preparation, 424 Sodium alginate, from L. hyperborea, for cell immobilization, 437 Soybean trypsin inhibitor, 177 Specialist Dairy Ingredient company, 422 Spezyme IGI, 370 Spherosil, porous silica supports, characteristics, 246 Steroid enzyme-catalyzed transformations in water-organic solvent two-phase systems, 150-157 assays, 151-152 enzyme immobilization, 152-153 materials, 151 methods, 154-157 parameters affecting, 157 two-liquid phase biocatalytic reactions for, 139-141 Steroid isomerase, steroid transformation with, 156-157 Streptomyces murinus, in glucose isomerase production, 361 Streptomyces olivochromogenes, in glucose isomerase production, 366 Streptomyces phaechromogenes, in glucose isomerase production, 362 Streptomyces rubiginosus, 367 in glucose isomerase production, 358

SUBJECT INDEX

Streptomyces wedmorensis, in glucose isomerase production, 357 Strictosidine formation, 342 preparative synthesis, 349-350 Strictosidine synthase, 342-350 activity, determination, 346 activity, effect of secologanin concentration, 347-348 activity, pH profile, 346-347 assay, 344 immobilization, materials, 343-344 immobilized preparation, 345-349 stability, 348-349 thermostability, 348 isoenzymes, 343 isolation, 344-346 preparation, 344 reaction catalyzed, 342-343 Substance P octapeptide, sequential hydrolysis with immobilized aminopeptidase, 175 Subtilisin, immobilized, application, 354 Sucrose syrup, invertase treatment of, 376 Sulfhydryl oxidase adsorbed directly onto Spherosil QMA, 426-427 covalent attachment to controlled-pore glass beads, 425-426 immobilization, 425-427 conditions, 425 pore volume of matrices for, 425 immobilized activities in various preparations, 426 applications, 354 stability, 428 storage, 428 isolation of milk proteins with increased activity of, 423--425 purification, 425 reactors activity, 428-429 characteristics, 427-430 normalized residence time, 429-430 operational protocol, 428 Suspended bed fermentation vessel, 387 Sweetase, 362, 370 Sweet-protein syrup, 422 Sweetzyme, 361-362, 370

58 1

T Takasweet, 361,370 Tego-Diocto BS, 419 Testosterone bioluminescent assay using coimmobilized enzymes, 87 oxidation to androstenedione, in twophase system, 155 Theorell-Chance mechanism, 42 Thermoase for aspartame production, selection, 504 immobilization, 507-5 ! 1 by cyanuric chloride, 511 by glutaraldehyde, 510-511 immobilized assay, 512-513 for condensation between Z-Asp and Phe-OMe, 505-506 determination of amounts on support materials, 511-512 substrate preparation, 513 Thermolysin, 504 immobilization to Enzacryl AH, 181 via diazo coupling, 508 immobilized application, 355 in peptide synthesis, 185 Thiobacillus ferrooxidans, immobilized application, 355 in treatment of acid mine drainage, 530540 Thiolproteinases, 504 Thiols, and food flavors, 423 Toyopearl activation, 510 as support material for thermoase immobilization, 507-510 Transaminase. See Aminotrarlsferase Transamination reaction, 480-481 equilibrium constant, 481-482 Trehalulose production, 453 in immobilized microbial cells, 440 properties, 453 Triacylglycerol lipase. See also Lipase, C. cylindrica in reverse micelles, 207 Triglyceride

582

SUBJECT INDEX

fatty acid in 2-position of, analysis, 408 isolation, 407 oleic safflower oil interesterified, fatty acid composition, 410 reformed, isolation from interesterification reaction mixture, 406 regiospecific interesterification, reaction scheme, 406 total fatty acid composition, analysis, 407-408 17,20fl,2l-Trihydroxysteroid:NAD+ oxidoreductase. See 20fl-Hydroxysteroid dehydrogenase Trisaccharides, synthesis, in hydrolasecatalyzed condensation reaction, 282 6-(Tritylamino)hexylamine dihydrochloride, 22-23 N-[6-(Tritylamino)hexyl]methacrylamide, 24 Trypsin in enzymatic conversion of porcine insulin to human insulin, 162-163 immobilization, to carboxymethylcellulose, 181 immobilized assay, 181 in peptide synthesis, 183-186 in reverse micelles, 203,205 kinetic parameters, 214 side-chain cleavage with, 161-162 Tuftsin, sequential hydrolysis with immobilized aminopeptidase, 176 Two-liquid phase biocatalytic reactions aqueous phase, 138-142 classification, 138-139 discrete aqueous continuous phase, 138140 discrete aqueous discontinuous phase, 138-140 discrete aqueous phase, 143 at interface, 138, 142-143 kinetic considerations, 143-144 no discrete aqueous phase, 140-142 steady-state conditions, 143-144 types of, 138-143 Two-liquid phase biocatalytic reactors, 138-149 advantages, 138 choice of, 146-147 concentration profiles of reactant at steady state in, 142-143

effect of organic solvent, 145-146 experimental methods, 147-149 future prospects, 149 immobilization technique on Celite, 148 using hydrophilic photo-cross-linkable resin prepolymers, 148 using hydrophobic photo-cross-linkable resin prepolymers, 147-148 using Urethane prepolymers, 148 packed bed, 146-147, 149 performance, effect of phase ratio and biocatalyst concentration on, 144 shaken flask, 149 stirred tank, 146-149 U UDPglucose activity, assay, 273 enzyme-catalyzed synthesis, 279-280 UDPglucose pyrophosphorylase, assay, 273 UHT milk cooked flavor of, 423 flavor modification, with immobilized sulfhydryl oxidase, 423,430-431 Urea hydrolysis, 234 effect of organic solvents on, measurement, in determination of urease reaction rate, 236-237 measurement, 236 products, 238 Urease immobilized in solution in semipermeable microcapsules, for multistep enzyme reaction in conversion of urea to ammonia to glutamic acid to alanine, 71-72 jack bean source, 235 stability, effect of organic solvents on, 236 microencapsulated, for muitistep enzyme reaction of conversion of urea into glutamic acid, 72-73 Urea synthesis from ammonium carbonate, 234 nonenzymatic, 234 urease-catalyzed

SUBJECT INDEX measurement, 235-236 reaction mechanism, use of organic solvents to study, 238 yield effect of hydrogen ion concentration, 237 effect of nature of organic solvent, 237 effect of organic solvent, 237 effect of substrate, 237 with urease in water-organic solvent mixtures, 234-238 Uridine-5'-diphosphoglucose. See also UDPglucose enzymatic synthesis from glucose and RNA, 267-268 enzyme-catalyzed synthesis, 263

V Vibrio harveyi, luciferase. See Bacterial luciferase

W Washout, 14 Whey clarified and demineralized, hydrolysis with immobilized lactase, 420 lactase treatment of, 376 microbial contamination, 414-415 obtained by clotting with chymosin, 424 raw, hydrolysis with immobilized lactase, 419 ultrafiltration, 411 utilization, 411 Whey syrups, lactolyzed, uses, 412

X Xanthine oxidase, 256-258. See also Milk xanthine oxidase commercial, immobilization onto Sepharose 4B, 258-259 immobilized oxidation of 1-methylxanthine to 1methyluric acid, 259-262 oxidation of xanthine to uric acid by, 259

583

preparation, using milk as starting material, 257-260 reaction specificity, 261-262 potential substrates, 256-257 reaction catalyzed, 256

Y Yeast calcium alginate-immobilized, 394-405 bench-scale studies, 397-398 cell viability for stable long-run operations, 394 contamination prevention, 398-399 design of reactor, 397-398 improvement of cell viability, 399 maximum cell concentration in carrier, 394 pH of inlet substrate solution, 399 pilot plant, 394 pilot plant operations, 399-402 process, 398 productivity, 395, 402-404 semicommercial plant, 402-403 continuous immobilizer, 383-384 conventional batchwise fermentation with, 405 compared to immobilized cells, 402 immobilization, 395 entrapping materials, requirements, 381-383 entrapping methods, 381 in photo-cross-linkable resin, 381-385 immobilized alcohol production, in various preparations, 396 fermentation characteristics, 385-387 fermentation system, contamination prevention, 389 life span, 394 maintenance of cell viability, 399-400 microbiocidal sterilization, 389-390 selection of carriers, 396 sludge removal measures, 388-389 immobilized cells, applications, 354-355 photo-cross-linkable resin-immobilized, 380-394 alcohol concentration, 392-393 alcohol productivity, 393 bench-scale plant, 389-391

584

SUB.IECT INDEX

continuous operation of experimental plants, 389-392 ethanol productivity, 380 fermentation system, 394 immobilization process, 383-384 pilot plant, 389, 391-392 properties, 384-385 theoretical alcohol yield, 393 yeast concentration, 392 strains, selection and improvement for ethanol production, 399 vacuum fermentation technique, 405

Z Z-Arg-AIa-NH2, papain-catalyzed formation, 183 Z-Asp, and Phe-OMe, condensation between, 505-506, 513-514 Z-L-Asp, preparation, 513 Z-GIy-Phe-Leu-NH2, chymotrypsin-catalyzed synthesis, 184 Z-Lys-Leu-NH2, trypsin-catalyzed synthesis, 183-184 Z-Phe-Leu-NH2, thermolysin-catalyzed synthesis, 185

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Microbial Enzymes and Biotransformations

Electrochemistry of Immobilized Particles and Droplets

Immobilized pH Gradients: Theory and Methodology

Neurotransmitter Enzymes

Enzymes Everywhere

Artificial Enzymes

Industrial Application of Immobilized Biocatalysts

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Oral Cells and Tissues

Mast Cells and Basophils

ANIMAL CELLS AND TISSUES

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