Enzyme Catalysis in Organic Synthesis: A Comprehensive Handbook, Second Edition

1593 Pages·2002·46.79 MB·English

Enzyme Catalysis in Organic Synthesis
Edited by K. Drauz and H. Waldmann
Second Edition
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Enzyme Catalysis in Organic Synthesis
A Comprehensive Handbook
Edited by
Karlheinz Drauz and Herbert Waldmann
Second, Completely Revised and Enlarged Edition
@WILEY-VCH
Editors: This book was carefully produced. Nevertheless
editors, authors and publisher do not warrant the
Prot Dr. Karlheinz Drauz information contained therein to be free of er-
Degussa AG rors. Readers are advised to keep in mind that
1Z N Wolfgang, Bereich FC-TRM statements, data, illustrations, procedural details
Rodenbacher Chaussee 4 or other items may inadvertently be inaccurate.
63457 Hanau-Wolfgang
Germany
Prof. Dr. Herbert Waldmann
Max-Planck-Institut fiir Molekulare Physiologie
Otto-Hahn-Strage 11
44227 Dortmund
Germany
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I'
Foreword
That biological systems are masterful chemists is a fact long appreciated by those
who study how living things build complexity from simple compounds in the
environment. Enzymes catalyze the interconversion of vast numbers of chemical
species, providing materials and energy to fuel cell survival and growth. Enzymes
build the intricate natural products, which, for their potential utility in treating
disease, pose almost unlimited new challenges for ambitious synthetic chemists.
But, unlike most industrial chemical processes, Nature's catalysts generate few
waste products and effect their transformations under mild conditions-in water, at
room temperature and atmospheric pressure. Biocatalysts are models of energy-
efficient, environmentally-consciousc hemistry and will play a prominent role in the
21Stc entury's chemicals industry.
The world of biocatalysis has undergone significant change in the eight years since
the first edition of this handbook appeared. Most of the news is good, with enzymes
showing up in many more organic syntheses and a number of important new
industrial processes coming on line. Apart from continuing clever insights into how
to integrate biocatalysis into synthetic chemistry, several forces are accelerating a
move to biocatalytic processes. In the first place, the search for better, enantiomer-
ically pure drugs has forced many chemists to turn to enzymes for assistance in their
preparation. Ever increasing demands for environmentally acceptable processes
push in the same direction. At the same time, rapidly-developing technologies for
making better catalysts through genetic enginering and for discovering new catalysts
are are offering new process opportunities which in the past were either not
economical or not even conceivable. A plethora of new catalysts to choose from, as
well as a high probability that a catalyst can be further improved during the process
design and engineering phases, means that we can respond rapidly to new synthetic
needs with biocatalybc solutions.
The organization of these volumes into specific technologies and transformations
provides a comprehensive coverage of practical biocatalysis that no other single
source provides. The work of experts in each of the fields, the individual chapters
review vast relevant literature and synthesize it in order to present key concepts and
many illustrative examples. This coverage should give organic chemists immediate
access to the wealth of experience that has accumulated in the biocatalysis world and
allow them to identify the most promising ways to use biocatalysts in their own
I
VI Foreword
syntheses. Biocatalysts should feature prominently in the repertoire of synthetic
chemistry, and this handbook deserves a prominent place in the modern chemist’s
library.
Pasadena, January, 2002 Frances Arnold
Preface
Nearly eight years have passed since we the First Edition of ,,Enzyme Catalysis in
Organic Synthesis“ was issued but much of what we had written in its preface then
still applies today. The application of biocatalysis in organic synthesis is a powerful
technique. It has grown steadily and today this field is well-established in both
academia and industry. With increasing application and acceptance the need for a
comprehensive and up to date overview of the state of the art has grown. In addition
numerous colleagues have approached us and asked for an update of “the Hand-
book”.
In response to these demands and in recognition of the new and groundbreaking
strides taken since the first half of the nineties the Second Edition which is now in
the hand of the reader was prepared. In comparing it with the First edition one
discovers that we have not changed the overall arrangement in the volumes.
Therefore we continue to have a part that addresses general principles (Chapters
1-10) and another one which summarizes the application of enzymes in organic
synthesis according to reaction type (Chapters 11-20). This arrangement was very
well received by the readers before and we hope that it will be for the Second Edition
as well.
However, the entire text was streamlined and in many cases regrouped to ensure
for a better presentation. Also a few chapters which in the long run turned out to be
less relevant to organic synthesis were not included again. In contrast other aspects
were now integrated and attention was given to techniques of enzyme evolution,
bioinformatics and enzymatic reactions in low-water media, areas that have devel-
oped with great pace and that we believe to be of major importance in the time to
come.
We hope that the Second Edition of the “Handbook will be a plentiful source of
information just as valuable as the First Edition was eight years ago.
Dortmund and Hanau, February 2002 Karlheinz Drauz, Herbert Waldmann
I
IX
Cont ents
Foreword V
Preface VII
Volume I
1 Introduction 1
Maria- Regina Kulo
1.1 Enzymes as Catalysts 1
1.2 Enzyme Structure and Function 4
1.3 Cofactors and Coenzymes 12
1.4 Enzyme Nomenclature 21
1.5 Enzyme Kinetics 23
1.5.1 Reaction Rate and Substrate Concentration 23
1.5.2 Inhibitors and Efl’ectors 26
1.5.3 Influence of pH and Buffers 27
1.5.4 Temperature 28
1 .G Organic Solvents as Reaction Media 31
1.7 Enzyme Handling: Quality Requirements 32
1.8 Biotransforrnation Using Whole Cells 33
1.8.1 General Aspects 33
1.8.2 Biotransformation with Growing Cells 36
1.8.3 Biotransformation with Resting Cells 37
1.8.4 Biotransforrnations with Permeabilized or Dried Cells 37
Bibliography 38
2 Production and Idation of Enzymes 41
Yoshihiko Hirose
2.1 Introduction 41
2.2 Enzyme Suppliers for Biotransforrnation
44
2.3 Origins of Enzymes 45
2.3.1 Microbial Enzymes 45
2.3.2 Plant Enzymes 46
2.3.3 Animal enzymes 46
I
X Contents
2.4 Fermentation of Enzymes 46
2.4.1 Liquid Fermentation 46
2.4.2 Solid Fermentation 47
2.4.3 Extraction of Enzymes 47
2.5 Extraction of Enzymes 47
2.5.1 Microbial Enzymes 47
2.5.2 Plant Enzymes 48
2.5.3 Animal Enzymes 48
2.6 Concentration 48
2.7 Purification of Enzymes 49
2.7.1 Chromatography 49
2.7.1.1 Ion Exchange Chromatography (IEX) 49
2.7.1.2 Hydrophobic Interaction Chromatography (HIC) 54
2.7.1.3 Gel Filtration (GF) 56
2.7.1.4 Reversed Phase Chromatography 58
2.7.1.5 Hydrogen Bond Chromatography 59
2.7.1.6 Affinity Chromatography 59
2.7.1.7 Saltingout Chromatography 62
2.7.2 Precipitation 62
2.7.2.1 Precipitation by Salting out 62
2.7.2.2 Precipitation by Organic Solvents 63
2.7.2.3 Precipitation by Changing pH 63
2.7.2.4 Precipitation by Water-Soluble Polymer 63
2.7.3 Crystallization 64
2.7.4 Stabilization During Purification 64
2.7.5 Storage of Enzymes 64
2.7.5.1 Storage in Liquids 64
2.7.5.2 Storage in Solids 65
2.8 Commercial Biocatalysts 65
References 66
3 Rational Design of Functional Proteins 67
Tadayuki lmanaka and Haruyuki Atomi
3.1 Protein Engineering 67
3.2 Gene Manipulation Techniques in Enzyme Modification 68
3.3 Protein Crystallization 70
3.4 Comparative Modeling of a Protein Structure 73
3.5 What is Needed to Take a Rational Approach? 75
3.6 Examples of Protein Engineering 76
3.6.1 Protein Engineering Studies: Providing a Rational Explanation
for Enzyme Specificity 76
3.6.2 Enhancing the Thermostability of Proteases 78
3.6.3 Contribution of Ion Pairs to the Thermostability of Proteins from
Hyperthermophiles 79
I"'
Contents
3.6.4 Thermostability Engineering Based on the Consensus Concept 80
3.6.5 Changing the Optimal pH of an Enzyme 81
3.6.6 Changing the Cofactor Specificity of an Enzyme 82
3.6.7 Changing the Substrate Specificity of an Enzyme 84
3.6.8 Changing the Product Specificity of an Enzyme 85
3.6.9 Combining Site-directed Mutagenesis with Chemical Modification 86
3.6.10 Changing the Catalyhc Activity of a Protein 087
3.7 Conclusions 89
References 90
4 Enzyme Engineering by Directed Evolution 95
Oliver May, Christopher A. Voigt and Frances H. Arnold
4.1 Introduction 95
4.2 Evolution as an Optimizing Process 96
4.2.1 The Search Space of Chemical Solutions 97
4.2.2 The Directed Evolution Algorithm 98
4.3 Creating a Librarr of Diverse Solutions 99
4.3.1 Mutagenesis 99
4.3.1.1 Random Point Mutagenesis of Whole Genes 99
4.3.1.2 Focused Mutagenesis 104
4.3.1.3 Calculation of Mutagenesis Hot-Spots 105
4.3.2 Recombination 107
4.3.2.1 In Vitro Recombination 107
4.3.2.2 In vivo Recombination 110
4.3.2.3 Family Shuffling 111
4.4 Finding Improved Enzymes: Screening and Selection 112
4.4.1 You Get What You Screen For 113
4.4.2 Screening Strategies 113
4.4.2.1 Low-Throughput Screening 114
4.4.2.2 High-Throughpu t Screening 115
4.4.2.3 Choosing Low versus High Throughput 116
4.4.2.4 Analyzing the Mutant Fitness Distribution 117
4.4.3 Selection and Methods to Link Genotype with Phenotype 119
4.5 Applications of Directed Evolution 121
4.5.1 Improving Functional Enzyme Expression and Secretion 122
4.5.2 Engineering Enzymes for Non-natural Environments 127
4.5.3 Engineering Enzyme Specificity 129
4.5.3.1 Substrate Specificity 129
4.5.3.2 Enantioselectivity 131
4.6 Conclusions 132
References 133