ENZYME ENGINEERING

Volume 5

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ENZYME ENGI NEERING Volume 5

Edited by

Howard H. Weetall Corning Glass Works Corning, New York

and

Garfield P. Royer The Ohio State University Columbus, Ohio

PLENUM PRESS · NEW YORK AND LONDON

The Library of Congress cataloged the second volume of this title as follows:

Engineering Foundation Conference on Enzyme Engineering, 2d, Henniker, N. H., 1973. Enzyme engineering; [papers] Edited by E. Kendall Pye and Lemuel B. Wingard, Jr.

New York, Plenum Press [1974]

Called volume 2 in continuation of a volume with the same title published in 1972, which contains the papers of the 1 st Engineering Foundation Conference on Enzyme Engineering.

1. Enzymes - Industrial applications - Congresses. I. Pye, E. Kendall, ed. II. Wingard, L., ed. III. Title. [DNLM: 1. Biomedical engineering - Congresses. 2. En· zymes - Congresses. W3 EN696] TP248.E5E53 1973

ISBN-13: 978-1-4684-3751-5 DOl: 10.1007/978-1-4684-3749-2

660'.63 74·13768

e-ISBN-13: 978-1-4684-3749-2

Proceedings of the Fifth International Enzyme Engineering Conference, held in Henniker, New Hampshire, July 29-August 3,1979.

© 1980 Plenum Press, New York A Division of Plenum Publishing Corporation 227 West 17th Street, New York, N.Y. 10011

Softcover reprint of the hardcover 1st edition 1980

All rights reserved

No part of this book may be reproduced, stored in a retrievai system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher

ORGANIZATION OF THE CONFERENCE

SPONSOR:

The Engineering Foundation 345 East 47th Street New York, N.Y. 10017 U.S.A.

EXECUTIVE COMt:lITTEE:

Howard H. Weeta11. Garfield P. Royer. Lemuel B. Wingard, Jr. Sanford S. Cole. Ichiro Chibata Peter Dunni11. .

ADVISORY BOARD

1. Berezin G. Broun C. Colton D. Fink S. Fukui W. Hornby J. Konecny M. Lilly G. Manecke A. Marconi A. Michaels K. Mosbach

v

Executive Chairman Program Chairman Permanent Member Conference Director Member Member

K. Pye D. Ryu H. Samejima G. Schmidt-Kastner B. Schneider H. Tenoso D. Thomas K. Venkatasubramanian F. Voss M. Weibel o. Zaborsky

vi ORGANIZATION OF THE CONFERENCE

ORGANIZATION OF THE CONFERENCE (CONT'D)

PROGRAM COMMITTEE:

I. Berezin A. Laskin G. Broun G. Manecke R. Chambers G. Royer D. Fink S. Suzuki L. Goldstein W. Veith C. Horvath H. Weetall M. Kula

FINANCIAL CONTRIBUTORS

To The Fifth International Enzyme Engineering Conference

The major contribution received was from:

NOVO Industries, AG

We also received liberal support from the following firms.

Abbott Laboratories Anheuser-Busch Bayer, AG Ciba-Geigy, Ltd. Dow Chemical Company Kraft, Inc. Pfizer, Inc. AHa Laval Astro Lakemidell Boehringer Mannheim, GmbH Corning Glass Works Japanese Society of Enzyme Engineering Miles Laboratories SNAM Progetti, SPA

NEXT ENZYME ENGINEERING CONFERENCE

FALL 1981: Enzyme Engineering VI tentatively

scheduled for September 20 - 25,

1981 at the Hotel Shima Kanko,

Kashikojima, Japan

EXECUTIVE COMMITTEE:

Saburo Fukui. .

Ichiro Chibata.

David Fink.

Jan Konecny

Garfield Royer.

Howard Weetall.

Michael Weibel.

Lemuel Winga~d, Jr.

Sanford Cole. .

vii

Executive Chairman

Program Chairman

U.S.A. Member

European Member

U.S.A. Member

U.S.A. Member

U. S .A. Member

Permanent Member

Conference Director

WORKSHOPS HELD AT EE V

During the five days of the conference, a series of workshops were held for the participants. These workshops were as follows:

Chemistry of Immobilization (L. Goldstein and G. Manecke, Chairmen)

Enzymes in Organic Synthesis (J. Konecny and H. Samejima, Chairmen)

Biomedical Applications: Enzyme Stabilization and Therapy (T. Chan?, and L. Wingard, Jr., Chairmen)

Analytical Applications (B. Mattiasson, Chairman)

Kinetics and Reactor Design (W. Vieth and D. Thomas, Chairmen)

viii

PREFACE

Enzyme technology continues to maintain a high degree of interest both in the academic and industrial communities. Since the last Enzyme Engineering Conference held in Bad Neuenahr, Federal Republic of Germany, two years ago, an increasing emphasis has been placed on the study and application of immobilized whole cells and organelles. This new emphasis has been reflected in the number of presentations directed to this area.

The Fifth International Enzyme Engineering Conference was held in Henniker, New Hampshire, July 29 to August 3, 1979. The organizers of this conference are especially grateful for the generous support received from a number of industrial organizations.

The conference was attended by 183 participants representing over 22 countries making this truly an international conference.

During this conference, emphasis was placed on a wide variety of areas including: enzyme production, energy transduction, co­factor modification, biomass conversion, immobilized enzymes, cells and organelles, and enzymatic synthesis of chemicals and pharma­ceuticals.

This volume contains most of the presentations and posters presented at the Fifth Conference. The names of the session co­chairmen, workshop chairmen, committee members and sponsoring organizations are included as an appreciation of their efforts in making this a successful conference.

The preparation of this volume was carried out by the editors including editing and proofing of the individual manuscripts and the final copy of this volume. The editors are indebted to Ms. S. Harrau of Corning Glass Works, Biomedical Research Department, for her job in retyping and proofreading all the manuscripts and put­ting them in their final form for reproduction by the publisher.

ix

x PREFACE

Our special thanks go to Mr. Sanford Cole of the Engineering Foundation without whose help this conference could not have taken place.

Howard H. Wee tall Garfield P. Royer

March, 1980

CONTENTS

KEYNOTE PAPER . . 1

Achievements and Predicted Developments in Enzyme Engineering. 3

E. Katcha1ski-Katzir

SESSION I. ENZYME PRODUCTION 31 Chairmen: R. Kula and M. Weibel

Comparative Quantitative Physiology of High Cellulase 33 Producing Strains of Trichoderma Reesei

D. D. Y. Ryu, R. Andreotti, J.Medeiros and M. Mande1s

Bacterial Exo-Enzyme and Exo-Toxin Export. . . . 41 E. A. Pepper, J. Melling and R. C. W. Berkeley

Enzyme Purification by Liquid-Liquid Extraction. 45 H. Hustedt, K. H. Kroner, U. Menge and M.-R. Kula

Continuous Isolation of Yeast-Lytic Enzymes from Citophaga. . . . • • . . • . . . . . • • • 49

J. A. Asenjo

Characterization of Membranes for Enzyme Retention • 57 E. F1asche1

Cloning of the Penicillin G-Acy1ase Gene of Escherichia coli ATCC 11105 on Mu1ticopy P1asmids. 61

H. Mayer, J. Collins and F. Wagner

Immobilized Tannin as Protein Adsorbent-Preparation, Characteristics, and Applications. • . • . . . 71

I. Chibata, T. Tosa, T. Mori and T. Watanabe

Aminoa1ky1po1ysaccharide Sorbents in Purification of Enzymes. • . . . . . . . . . . . • • . . . . • . • • 75

G. B. Gerasimiene, A. A. G1emza, D. A. Kazlauskas, D. P. Karpaviciene, V. V. Ku1iene

xi

CONTENTS

Immunochemical Identification of a New Thermostable S-galactosidase from a Bacillus Species • • . . . . • 79

M. Schulein

Mass Spectrometer Monitoring of a Yeast Fermentation. 85 J. C. Weaver, C. R. Perley and C. L. Cooney

Affinity Chromatographic Investigations on Enzyme Coenzyme Complexes of CoA-Dependent Enzymes • . . 89

B. Limbach and H. -L. Schmidt

SESSION II. ENZYMES AND ENERGY TRANSDUCTION .•.. 93 Chairmen: I. V. Berezin and S. Suzuki

Principles of Bioelectrocatalysis 95 I. V. Berezin, S. D. Varfolomeev and M. V. Lomonosov

Cofactor Modified Electrodes for Energy Transfer. .• 101 L.B. Wingard, Jr.

Immobilized Thylakoids and Chromatophores: Hydrogen Production and ATP Regeneration . • .

V. Larreta Garde, M. F. Cocquempot, J. N. Barbotin, B. Thomasset and D. Thomas •....

Some Aspects of Direct Bioelectrocatalytic Regeneration of Cofactors . . • • . • • . •

I. V. Osipov, S. D. Varfolomeev

A New Enzyme Transducer Combination: The Enzyme

109

119

Transistor. . . . . . • • . . . 123 B. Danielsson, K. Mosbach, I. Lundstrom, L. Stiblert

A Simple, General Method for Preparation of Hydro-Soluble Polymeric Adenine Nucleotide Coenzymes. 127

F. Le Goffic, S. Sicsic and C. Vincent

Cofactor Reaction-Diffusion Kinetics for an Alcohol Dehydrogenase Membrane. . . • . . . . • • • • 133

L. B. Wingard, Jr. and J. R. Millis

Approaches to Stabilization of Hydrogenase and Nitrogenase Against Oxygen Inactivation . . . • 135

A. M. Klibanov, N. O. Kaplan and M. D. Kamen

CON~N~ •

Biochemical Energy Conversion System. . • . . . . • 143 S. Suzuki, I. Karube, T. Matsunaga and H. Kayano

SESSION III: BIOMASS CONVERSION . 147 Chairman: G. Tsao

Utilization of B-Glucosidase from Aspergillus Species in the Hydrolysis of Cellulose. • • . • . . . . • •• 149

P. M. A. Nybergh and M. J. Bailey

The Comparative Role of Exoglucosidase and Cellobiase in Glucose Formation from Cellulose . . . • .. 153

A. A. Klyosov, A. P. Sinitsyn and M. L. Rabinowitch

Novel Enzymes from Methylotrophic Microorganisms. 167 A. I. Laskin, C. T. Hou and R. N. Patel

Ethanol - Butanediol Fermentation of Xylose 171 S. Veeraraghavan, Y. Y. Lee, R. P. Chambers, T. A. McCaskey

Fluorimetric Monitoring of Methanogenesis in'Anaerobic Digesters . . • • . . • . . . . . . . . . • 175

E. J. Nyns, H. P. Naveau and M. J. Delafontaine

SESSION IV: BIOMEDICAL AND ANALYTICAL APPLICATIONS OF ENZYMES AND ANTIBODIES ....

Chairmen: M. Wilchek and P. Carr

Enzyme Channelling Immunoassay. Enzyme Immunoassay Technique. .

E. F. Ullman and D. J. Litman

A New Homogeneous

Human Renal Renin: Purification, Production of

177

179

Specific Antibodies. Clinical Applications • . 187 F. X. Galen, C. Devaux, P. Sicard, T. Guyene, J. Bariety, J. Menard and P. Corvol

Competitive Binding Studies of Carbohydrate-Lectin Interactions Using a Lectin Electrode . . . • . . . 193

C. Borrebaeck and B. Mattiasson

Potentiometric Enzymatic Measurement of Glucose: Possible In Vivo Use. . . . . . . . . . . . . . 197 ---L. B. Wingard, Jr., S. K. Wolfson, Jr., C. C. Liu,

S. J. Yao, J. G. Schiller and A. L. Drash

xiv CONTENTS

Immobilized Enzyme Pipette - "Impette". • • • . . •• 201 P. V. Sundaram

Electrochemical Determination of Lipids in Serum. •. 205 S. Suzuki and I. Karube

Immobilized Enzymes for Medical Application . . 209 E. I. Chazov, V. N. Smirnov, V. P. Torchilin, I. M. Tereshin, B. V. Moskvichev

Photoenzography: Using Enzymes for Photographic Aims. . . . . . .

N. F. Kazanskaya

The Design of Peroxide Enzyme Membrane Polarographic

213

Sensors for Clinical and Industrial Analysis. • . 217 T. A. Grooms, L. C. Clark, Jr. and B. J. Weiner

New Approaches Using Immobilized Enzymes for the Removal of Urea and Ammonia . . • . . • . . • . . 225

T. M. S. Chang

Enzyme-Collagen Reactor Designed from an Hemodialysis Module. . • . • . . •. ...••.••••••. 231

P. R. Coulet, F. Paul, D. Dupret and D. C. Gautheron

Immobilization and Characterization of Enzymes on Hollow Fibers for a Possible Use in the Biomedical Field . . . . . . • . . • . . • • . . • 235

P. G. Pietta, D. Agnellini, G. Mazzola, G. Vecchio, S. Colombi and G. Bianchi

Magnetic Microspheres for Targeting of Drugs. . . •• 239 K. Mosbach and U. Schroder

Modification of Enzymes with Water-Soluble Polymers . 243 I. M. Tereshin and B. V. Moskvichev

Detoxication of Ammonia by Immobilized Urea Cycle Enzymes . . • . • •. ...•. •....• 247

Y. Miura, H. Urabe, K. Miyamoto and M. Okazaki

Use of the Enzyme Thermistor for Continuous Monitoring and Control in Biotechnology. • . • . • • • . . 251

B. Mattiasson, B. Danielsson and F. Winquist

Distribution of Damkohler Number of Spherical Matrix Particles by Image Analysis

M. Sernetz, M. Chun, R. Kindt and B. Gelleri 255

CONTENTS

On-line Determination of Reaction Rate Versus Substrate Concentration for an Enzymatically Catalysed Reaction by Means of a Microcomputer System. • • • .•• • • • • • • • • • • • •

R. Wichmann and C. Wandrey

Microbial Sensors for Ethyl Alcohol, Acetic Acid and Ammonia • • • •

I. Karube and S. Suzuki

SESSION V: LARGE-SCALE TRANSFORMATIONS USING BOUND ENZYMES. • • • • • • .• ••••• •

Chairmen: H. Samejima and W. Pitcher

xv

259

263

267

Improved Whey Treatment by Immobilized Lactase. 269 W. Marconi, F. Bartoli, F. Morisi and A. Mariani

Lactose Hydrolysis by Immobilized Lactase: Semi-industrial Experience • • • • . • • 279

L. A. Dohan, J. L. Baret, S. Pain and P. Delalande

Production of Useful Nucleotides with Immobilized Microbial Cells .• ••• • •• •••••• 295

Y. Ado, K. Kimura and H. Samejima

Heterogeneous Biocatalysis in the Degradation of S-Glucan Polymers • • . • . • • • • • • 305

P. Linko and Y. Y. Linko

Absorption Into a Liquid Film in Laminar Flow Coupled with an Enzyme Catalyzed Reaction at the Solid Surface 309

J. E. Prenosil, T. Buhlmann and H. Pedersen

Catalyst Deactivation and Optimal Operation Policy: Immobilized S-galactosidase • • • • • • • • • • • • • 317

J. E. Prenosil, J. Peter and J. R. Bourne

Stability Studies on the Immobilized Glucose Oxidase/ Catalase Enzyme System. • • . • . • . • • • • • 321

R. S. Carter, J. E. Prenosil and J. R. Bourne

Hydrodynamics and Reactors .••••

J. M. Engasser, M. Garnier

Kinetics of Hollow Fiber Enzyme

J. Caumon, A. Marc, J. Lede and 325

Isomerose Syrups Containing Greater than 90% Fructose 329 S. A. Barker, H. A. Bowes and P. J. Somers

xvi

SESSION VI: IMMOBILIZED CELLS AND ORGANELLES •• Chairmen: S. Fukui and I. Chibata

CONTENTS

333

Inunobilization of Whole Hicrobial Cells for the Production of 6-Amino Penicillanic Acid 335

J. Klein and F. Wagner

Application of Inunobilized Biocatalysts to Bio-conversion in Hydrophobic Conditions. . . . . 347

S. Fukui, T. Ornata, T. Yamane and A. Tanaka

Alginate Bead Entrapped Yeast Cells for Continuous Inversion of Sucrose and Holasses • • . • • . 355

Y. Y. Linko, L. Weckstrom and P. Linko

Physical Characterization of Biocatalyst Particles Obtained form Polymer Entrapment of Whole Cells 359

J. Klein, P. Washausen, H. Kluge and H. Eng

The Use of Free and Inunobilized Cells in the Presence of Organic Solvents: The Oxidation of Cholesterol by Nocardia Rhodochrous • . . . . . . . . . .. 363

J. H. C. Duarte and H. D. Lilly

Properties of Enzymes Solubilized in Hydrocarbons via Reversed Hicelles . . . . . . . . . . 369

P. Luisi, P. Heyer and R. Wolf

The Potential Use of Inunobilized Plant Cells for the Production and Transformation of Natural Products . 373

P. Brodelius, B. Deus, K. Hosbach and H. H. Zenk

Inunobilized Whole Cells of the Yeast Trigonopsis variabilis Containing D-Amino Acid Oxidase for the Production of a.-Keto Acids. .....•.... 383

P. Brodelius, B. Hagerdal and K. Hosbach

Enzymatic Synthesis of Pantothenic Acid by Escherichia coli Cells. . • . . • . . • . . . . . . .. 389

Y. Kawabata and A. L. Demain

Production of Useful Chemicals Using Cells Inunobilized with Polyacrylamide and Carrageenan . . • • . . • •. 393

I. Chibata

Affinity Chromatographic Purification of Proteins Using lnunobilized Cells . • . • . • . . . . 401

B. Hattiasson and H. Ramstorp

CONTENTS

Synthesis of Coenzymes by Immobilized Cell System H. Yamada, S. Shimizu, Y. Tani and T. Hino

SESSION VII: BOUND ENZYMES AND THE SYNTHESIS OF FINE CHEMICALS AND PHARMACEUTICALS •••••

Chairmen: J. Konecny and K. Mosbach

xvii

405

414

Continuous Deacetylation of Cephalosporins. • • 415 J. Konecny and M. Sieber

Thermostability of Soluble and Immobilized Subtilisins After Their Modification by Dextrans and Dextrins • • 423

L. A. Nakhapetyan and V. Kh. Akparov

Isolation and Immobilization of Porcine Ligandin with Glutathione Transferase Activity • • • • • • 427

L. Callegaro and A. Fontana

Studies on the Stability of Soluble and Immobilized Alcohol Dehydrogenase from Yeast Mitochondria 431

M. J. Brougham and D. B. Johnson

Distribution of Staphylococcal Nuclease Insolublizied on Sepharose. • . • • • • • • • • • • 435

J. M. Guisan, V. M. Fernandez and A. Ballesteros

Immobilized Penicillin Acylase for Production of 6-APA From Penicillin-V. • • • • • • • • • • • • . ..• •• 439

S. Gestrelius

Preparation and Characterization of Metal Oxide-coated Supports for Oxidase Enzymes • • • •

B. R. Allen and D. J. Fink

~nzyme Immobilization on Pyridine Containing Polymers. • • • • • • • • • • • • • • • •

F. Pittner, T. Miron, G. Pittner and M. Wilchek

Cross-linked Pectate. • M. A. Vijayalakshmi, D. Picque and G. B. Broun

Immobilization of Biocatalysts Using Ultrafiltration

443

447

451

Techniques. • • • • • • • • • • • • • • • • • • 453 C. Wandrey, R. Wichmann, A. F. Bueckmann and M. R. Kula

xviii CONTENTS

B-Galactosidase Immobilized on Benzoquinone­Activated Bead Cellulose and on Oxirane-Acrylic Beads .• •..•.....•..•••.. 457

M. Chun and M. Sernetz

Application of Immobilized Carboxypeptidase Y for Deblocking in Peptide Synthesis . . . • . . . . . 461

G. P. Royer, H. Y. Hsiao and G. M. Anantharamaiah

Adsorption and Distribution of Enzymes in Carriers. 465 K. Buchholz, S. K. Duggal and A. Borchert

Application of Photosensitive Immobilized a­Chymotrypsin to Synthetic Reaction. . . .

I. Karube and S. Suzuki

APPENDIX

List of Participants.

Subject Index • . . •

469

473

483