Introduction to Biocatalysis Using Enzymes and Microorganisms

208 Pages·1995·3.98 MB·English

by  S. M. Roberts

INTRODUCTION TO BIOCATALYSIS USING
ENZYMES AND MICRO-ORGANISMS
This is an introductory text intended to give the non-specialist a compre-
hensive insight into the science of biotransformations. The book traces the
history of biotransformations, clearly spells out the pros and cons of
conducting enzyme-mediated versus whole-cell bioconversions, and gives
a variety of examples wherein the bio-reaction is a key element in a reaction
sequence leading from the cheap starting materials to valuable end-
products (such as Pharmaceuticals and agrochemicals, fragrances and
flavours). Biotransformations involving the hydrolysis of esters, amides,
and nitriles, the synthesis of esters and amides, reduction and oxidation
reactions, and carbon-carbon bond-forming systems are discussed. The
book finishes with a discussion of some industrially important large-scale
bioconversions.

INTRODUCTION TO
BIOCATALYSIS USING
ENZYMES AND
MICRO-ORGANISMS
STANLEY M. ROBERTS
University of Exeter
NICHOLAS J.TURNER
University of Exeter
ANDREW J.WILLETTS
University of Exeter
MICHAEL K.TURNER
University College London
CAMBRIDGE
UNIVERSITY PRESS
Published by the Press Syndicate of the University of Cambridge
The Pitt Building, Trumpington Street, Cambridge CB2 1RP
40 West 20th Street, New York, NY 10011-4211, USA
10 Stamford Road, Oakleigh, Melbourne 3166, Australia
© Cambridge University Press 1995
First Published 1995
Library of Congress Cataloging-in-Publication Data
Introduction to biocatalysis using enzymes and micro-organisms / S.M.
Roberts...[et al].
p. cm.
Includes bibliographical references and index.
ISBN 0-521-43070-4. - ISBN 0-521-43685-0 (pbk.)
1. Microbial biotechnology. 2. Enzymes - Biotechnology.
3. Biotransformation (Metabolism) I. Roberts, Stanley M.
TP248.27.M53157 1995
660'.63-dc20 93-48247
CIP
A catalog record for this book is available from the British Library.
ISBN 0-521-43070-4 Hardback
ISBN 0-521-43685-0 Paperback
Transferred to digital printing 2003
Contents
Preface page ix
Abbreviations xii
1 An historical introduction to biocatalysis using enzymes and
micro-organisms 1
1.1 Background . 1
1.2 Kirchhoff and starch hydrolysis 2
1.3 Payen, Persoz and diastase 4
1.4 Berzelius and catalysis 7
1.5 Leibig and Hofmann 8
1.6 Pasteur and organic chemistry 11
1.7 Burton-on-Trent and brewing 15
1.8 Brown and the oxidation of mannitol 16
1.9 Fischer and the specificity of enzyme action 21
1.10 Hill, Pottevin and the reversibility of enzyme action 22
1.11 Reichstein, Howarth and vitamin C 26
1.12 Conclusions and overview 28
References 31
2 The interrelationships between enzymes and cells, with
particular reference to whole-cell biotransformations
using bacteria and fungi 34
2.1 Introduction 34
2.2 Interrelationships between enzymes and cells:
choosing the best biotransformation system 36
2.3 Practical experimental methods for whole-cell
biotransformations using bacteria and fungi 40
2.3.1 What is a suitable micro-organism to act as the
biocatalyst for a particular desired biotrans-
formation? 42
vi Contents
2.3.2 What is the most suitable form of the chosen
biocatalyst to use? 47
2.3.3 What needs to be done to optimize the relevant
enzyme titre(s) of the chosen biocatalyst
preparation? 51
2.3.4 What, if anything, needs to be done to optimize
biocatalyst-substrate interaction? 64
2.3.5 What, if anything, needs to be done to optimize
product yield by preventing alternative
metabolic fates for the substrate and/or
product? 66
2.4 Conclusions and overview 70
Appendix A: Growing and maintaining micro-organisms 71
Appendix B: Details of some useful culture collections 73
Appendix C: Pathogenic bacteria and fungi 74
Appendix D: A typical immobilization protocol:
entrapment in /c-carrageenan 76
Bibliography 77
References 78
3 Useful intermediates and end-products obtained from
whole-cell/enzyme-catalysed hydrolysis and esterification
reactions 80
3.1 Introduction 80
3.2 Hydrolysis of esters 80
3.3 Hydrolysis of amides 87
3.4 Enzyme-catalysed formation of esters and amides 90
3.5 Hydrolysis of nitriles and epoxides 94
3.6 Conclusions and overview 96
Bibliography 97
4 Useful intermediates and end-products obtained from
biocatalysed oxidation and reduction reactions 98
4.1 Introduction 98
4.2 Reduction of ketones using whole cells 98
4.3 Reduction of carbon-carbon double bonds 102
4.4 Reduction of ketones and a,/?-unsaturated carbonyl
compounds using enzymes 103
4.5 Oxidation of alcohols to aldehydes or ketones 108
4.6 Oxidation of ketones to esters or lactones 109
4.7 Hydroxylation of aliphatic and aromatic compounds 111
Contents vii
4.8 Oxidation of alkenes and sulphides 115
4.9 Conclusions and overview 116
Bibliography 117
5 Useful intermediates and end-products obtained from
biocatalysed carbon-carbon, carbon-oxygen, carbon-
nitrogen and carbon-chalcogen bond-forming reactions 118
5.1 Introduction 118
5.2 Enzyme-catalysed asymmetric carbon-carbon
bond formation 118
5.2.1 Aldolases 118
5.2.2 Other C-C bond-forming enzymes 124
5.3 Synthesis of C-X bonds (where X = O, N, S, Hal) using
lyases and haloperoxidases 128
5.4 Application of enzymes in the synthesis of carbo-
hydrate derivatives, including ohgosacchandes 131
5.5 Conclusions and overview 138
Bibliography 139
6 Theapplication of biocatalysis to the manufacture of fine
chemicals 140
6.1 Introduction 140
6.2 Fermentation 140
6.2.1 Organic solvents 142
6.2.2 Carboxylic acids 143
6.2.3 Antibiotics 145
6.3 Steroids 147
6.4 Manufacture of amino acids 153
6.4.1 L-methionine 153
6.4.2 4-hydroxy-D-phenylglycine 154
6.4.3 L-cysteine 157
6.4.4 L-aspartate 158
6.5 /Mactam antibiotics 160
6.6 Isoglucose 164
6.7 Synthesis of (S)-2-chloropropionate and acrylamide:
the influence of cleaner chemistry 168
6.7.1 (S)-2-chloropropionate 168
6.7.2 Acrylamide 169
6.8 The chiral switch 170
6.9 Polymer synthesis 174
6.9.1 Nucleic acids 174
Vlll Contents
6.9.2 Peptides 175
6.9.3 Oligosaccharides 178
6.9.4 Polyesters 179
6.10 New reaction conditions 180
6.11 Genetic engineering 183
6.11.1 Protein production and protein engineering 183
6.11.2 Metabolic pathway engineering and ascorbic
acid manufacture 184
Bibliography 187
Index 188
Preface
Twenty years ago, only a handful of laboratories, some in industry and
some in academia, were interested in using enzyme-catalysed reactions
(biotransformations) in organic synthesis. At best those researchers were
considered to be at the margin of mainstream synthetic organic chemistry;
at worst they were considered to be downright odd.
In the 1980s there was an exponential increase in interest in the area of
biotransformations. That worldwide increase in attention to this field of
research can be assigned to several factors, including the perception and
perseverance of the original researchers in the field, the increased availability
of a wide variety of enzymes, and the realization that many families of
enzymes will transform a wide range of unnatural compounds, as well as
their natural substrates.
The utilization of enzymes in organic synthesis can be advantageous for
several reasons:
1. Enzymes catalyse reactions under mild conditions with regard to
temperature (ca. 37 °C), pressure (1 atm), and pH (ca. 7.0). The trans-
formations are often remarkably energy-efficient when compared with
the corresponding chemical processes.
2. Enzymes often promote highly chemoselective, regioselective, and
stereoselective reactions, and being chiral catalysts, they are often able to
generate optically active compounds. The increased awareness regard-
ing the need to have optically pure compounds for such uses as
Pharmaceuticals and agrichemicals (so as to avoid unnecessary toxicity
and/or ecological damage) has been a significant driving force in
the development and exploitation of non-natural enzyme-catalysed
reactions.
3. Enzymes can promote reactions that are difficult or impossible to
IX