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Enzyme Catalysis in Organic Synthesis A Comprehensive
Handbook 2nd 2nd Edition Karlheinz Drauz Digital
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Author(s): Karlheinz Drauz, Herbert Waldmann
ISBN(s): 9783527299492, 3527299491
Edition: 2
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Enzyme Catalysis in Organic Synthesis

Edited by K. Drauz and H. Waldmann

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Enzyme Catalysis in Organic Synthesis

A Comprehensive Handbook

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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-consciouschemistry and will play a prominent role in the
21Stcentury's chemicals industry.
The world of biocatalysishas 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
VI
I 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

Contents

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 Biotransforrnationswith Permeabilized or Dried Cells 37
Bibliography 38

2 Production and I d a t i o n 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
X
I 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
3.6.4 Thermostability Engineering Based on the Consensus Concept 80
Contents I"'
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
XI1
I Contents

5 Enzyme Bioinformatics 139

Kay Hofmann

5.1 Introduction 139

5.2 Protein Comparison 140
5.2.1 Sequence Comparison versus Structure Comparison 140
5.2.2 Substitution Matrices in Sequence Comparisons 141
5.2.3 Profile Methods 142
5.2.4 Database Searches 144
5.3 Enzyme-specificConservation Patterns 145
5.3.1 General Conservation Patterns 145
5.3.2 Active Site Conservation Patterns 146
5.3.3 Metal Binding Conservation Patterns 146
5.3.4 Making Use of Conservation Patterns 148
5.4 Modular Enzymes 149
5.4.1 The Domain Concept in Structure and Sequence 149
5.4.2 A Classification of Modular Enzymes 150
5.4.3 Inhibitory Domains 151
5.5 Enzyme Databases and Other Information Sources 151
5.5.1 E. C. Nomenclature and ENZYME Database 152
5.5.2 BRENDA 152
5.5.3 KEGG and LIGAND database 153
5.5.4 UM-BBD 153
5.5.5 Structural Databases 153
5.5.6 Metalloprotein Databases 154
5.5.7 Databases for Selected Enzyme Classes 154
5.6 Protein Domain and Motif Databases 154
5.6.1 PROSITE 155
5.6.2 PFAM 156
5.6.3 Other Related Databases 156
5.7 Enzyme Genomics 156
5.7.1 Ortholog Search 157
5.7.2 Paralog Search 157
5.7.3 Non-homology Based methods 159
5.8 Outlook 159
References 161

6 Immobilization o f Enzymes 163

James Lalonde

6.1 Introduction 163

6.2 Methods of Immobilization 164
6.2.1 Non-Covalent Adsorption 165
6.2.2 Covalent Attachment 168
6.2.2.1 Carriers for Enzyme Immobilization 170
6.2.3 Entrapment and Encapsulation 171
6.2.4 Cross-Linking 175
I
Contents xi’i

6.3 Properties of Iminobilized Biocatalysts 175

6.3.1 Mass Transfer Effects 176
6.3.2 Partition 176
6.3.3 Stability 177
6.3.4 Activity of Immobilized Enzymes 177
6.4 New Developments and Outlook 178
6.4.1 Cross-linked Enzyme Crystals (CLEC@) 179
6.4.2 Sol-Gel 181
6.4.3 Controlled Solubility “Smart Polymers” 181
References 182

7 Reaction Engineering for Enzyme-Catalyzed Biotransformations 185

Manfed Biselli, Udo Kragl and Christian Wandrey

7.1 Introduction 185

7.2 Steps of Process Optimization 186
7.3 Investigation of the Reaction System 190
7.3.1 Properties of the Enzyme 190
7.3.2 Properties of the Reaction System 193
7.3.2.1 Thermodynamic Equilibrium of the Reaction 193
7.3.2.2 Complex Reaction Systems: The Existence of Parallel and Consecutive
Reactions 195
7.3.2.3 Other Properties of the Reaction System 204
7.3.2.4 Application of Organic Solvents 204
7.4 Investigation of Enzyme Kinetics 208
7.4.1 Methods of Parameter Identification 209
7.4.2 The Kinetics of One-Enzyme Systems 210
7.4.2.1 THE Michaelis-Menten Kinetics 210
7.4.2.2 Competitive Inhibition 214
7.4.2.3 Non-Competitive Inhibition 215
7.4.2.4 Uncompetitive Inhibition 216
7.4.2.5 Reversibility of One-Substrate Reactions 217
7.4.2.6 Two-Substrate Re,ictions 218
7.4.2.7 Kinetics of Aminoacylase as Example of a Random
Uni-Bi Mechanism 223
7.4.3 Kinetics of Multiple Enzyme Systems 230
7.5 Enzyme Reactors 232
7.5.1 Basic Reaction Engineering Aspects 232
7.5.2 Reactors for Soluble Enzymes 238
7.5.2.1 Reactor Optimization Exemplified by the Enzyme
Membrane Reactor 241
7.5.2.2 Control of Conversion in a Continuously Operated EMR 249
7.5.3 Reactor Systems for Immobilized Enzymes 250
7.5.4 Reaction Techniques for Enzymes in Organic Solvent 251
XIV
I Contents

Conclusions and Outlook 253

7.6
References 254

8 Enzymic Conversions in Organic and Other Low-Water Media 259

Peter Halling

8.1 Introduction 259

8.2 Enzyme Form 260
8.2.1 Lyophilized Powders 260
8.2.2 Immobilized Enzymes 261
8.2.3 Cross-Linked Crystals 261
8.2.4 Direct Precipitation in Organic Solvents 262
8.2.5 Additives in Catalyst Powders 262
8.2.6 Solubilized Enzymes 263
8.3 Residual Water Level 264
8.3.1 Fixing Initial Water Activity of Reaction Components 266
8.3.2 Control of Water Activity During Reaction 269
8.3.3 “Water Mimics” 273
8.4 Temperature 274
8.5 Substrate (Starting Material) Concentrations 274
8.6 Solvent Choice 276
8.6.1 Effects on Equilibrium Position 276
8.6.2 “Solvent Effects” that Really are Not 276
8.6.3 Solvent Polarity Trend and Recommended Choices 277
8.6.4 Solvent Parameters 279
8.6.5 Solvent Effects on Selectivity 280
8.6.6 No Solvent or Little Solvent Systems 280
8.7 Acid-Base Conditions 281
8.7.1 pHMemory 281
8.7.2 Processes Erasing pH Memory 282
8.7.3 Systems for Acid-Base Buffering 283
References 285

9 Enzymatic Kinetic Resolution 287

Jonathan M.J. Williams, RebeccaJ. Parker, and Claudia Neri

9.1 Introduction 287

9.2 Alcohols and their Derivatives 288
9.2.1 Cyanohydrins 289
9.2.2 Other Readily Racemized Substrates 290
9.2.3 Enzyme and Metal Combinations 293
9.3 Carboxylic Acids and their Derivatives 297
9.3.1 Readily Enolized Carboxylic Acid Derivatives 297
9.3.2 Amino-Esters and Related Compounds 301
9.3.3 Reactions of cyclic amino acid derivatives 302
9.4 Reduction of fi-Ketoesters 307
9.5 Conclusion 309
‘Ontents Ixv
References 310

10 Enzymes from Extreme Therrnophilic and HyperthermophilicArchaea and

Bacteria 313
Costanzo Bertoldo and Carabed Antranikian

10.1 Introduction 31 3
10.2 Starch-ProcessingEnzymes 315
10.2.1 Thermoactive Annylolyhc Enzymes 316
10.2.1.1 Heat-StableAmylases and Glucoamylases 316
10.2.1.2 a-Glucosidases 317
10.2.1.3 Thermoactive Pullulanases and CGTases 317
10.3 Cellulose-Hydro1yzing Enzymes 0321
10.3.1 Thennostable Cellulases 321
10.4 Xylan-Degrading Enzymes 324
10.4.1 Thermostable Xylanases 324
10.5 Chitin Degradation 325
10.6 Proteolytic Enzyrnes 326
10.6.1 Stable Proteases 327
10.7 Intracellular Enzymes 329
References 331

Volume I1

11 Hydrolysis and Formation of C - 0 Bonds 335

11.1 Hydrolysis and Fmnation of Carboxylid Acid Esters 335
Hans-joachim Cars and Fritz Theil
11.1.1 Hydrolysis and Formation of Carboxylic Acid Esters 351
11.1.1.1 Hydrolysis of Carboxylic Acid Esters 351
11.1.1.2 Formation of Carboxylic Esters 472
11.1.1.3 Inter- and Intramolecular Alcoholysis 545
References 574
11.2 Hydrolysis of Epoxides 579
Kurt Faber and Romano V: A. Orru
11.2.1 Epoxide Hydrolases in Nature 581
11.2.1.1 Isolation and Characterization of Epoxide Hydrolases 582
11.2.1.2 Structure and Mechanism of Epoxide Hydrolases 584
11.2.1.3 Screening for Microbial Epoxide Hydrolases 587
11.2.2 Microbial Hydrolysis of Epoxides 588
11.2.2.1 Fungal Enzymes 588
11.2.2.2 Bacterial Enzymes 590
11.2.2.3 Yeast Enzymes 591
11.2.3 Substrate Specificity and Selectivity 592
11.2.3.1 Asymmetrization of meso-Epoxides 592
XVI
I Contents

11.2.3.2 Resolution of Racemic Epoxides 592

11.2.3.3 Deracemization Methods 596
11.2.4 Use of Non-Natural Nucleophiles 599
11.2.5 Applications to Asymmetric Synthesis GOO
11.2.6 Summary and Outlook 604
References 605
11.3 Hydrolysis and Formation of Glycosidic Bonds 609
Chi-Huey Wong
1.3.1 Introduction 609
11.3.2 Glycosyltransferases of the Leloir Pathway 611
11.3.2.1 Synthesis of Sugar Nucleoside Phosphates 613
11.3.2.2 Substrate Specificity and Synthetic Applications of
Glycosyltransferases 619
11.3.2.3 In Situ Cofactor Regeneration 626
11.3.2.4 Cloning and Expression of Glycosyltransferases 628
11.3.3 Non-Leloir Glycosyltransferases:Transfer of Glycosyl donors from
Glycosyl Phosphates and Glycosides 631
11.3.4 Glycosidases 633
11.3.4.1 Equilibrium-controlledSynthesis 633
11.3.4.2 Kinetically Controlled Synthesis 634
11.3.4.3 Selectivity 634
11.3.5 Synthesis of N-glycosides 637
11.3.5.1 Nucleoside Phosphorylase 638
11.3.5.2 NAD Hydrolase 639
11.3.6 Biological Applications of Synthetic Glycoconjugates 639
11.3.6.1 Glycosidase and Glycosyl Transferase Inhibitors 639
11.3.6.2 Glycoprotein Remodeling 641
11.3.7 Future Opportunities 642
References 643
11.4 Natural Polysaccharide-degrading Enzymes 653
Constanzo Bertoldo and Carabed Antranikian
11.4.1 Introduction 653
11.4.2 Starch 6 5 3
11.4.2.1 Classification of Starch-degrading Enzymes 654
11.4.2.2 a-Amylase (1,4-a-~-Glucan,4-G1ucanhydrolase, E. C. 3.2.1.1) 655
11.4.2.3 P-Amylase (1,4-a-~-Glucan Maltohydrolase, E. C. 3.2.1.2) 656
11.4.2.4 Glucoamylases (1,4-a-~-glucan glucohydrolase,E. C. 3.2.1.3) 656
11.4.2.5 a-Glucosidase (a-6-GlucosideGlucohydrolase, E. C. 3.2.1.20) 657
11.4.2.6 Isoamylase (Glycogen 6-Glucanohydrolase,E. C. 3.2.1.68) 657
11.4.2.7 Pullulanase Type I (a-Dextrin 6-Glucanohydrolase,E. C. 3.2.1.41) 657
11.4.2.8 Pullulanase Type I1 or Amylopullulanase 658
11.4.2.9 Pullulan Hydrolases (Type I, Neopullulanase;Trpe 11, Isopullulanase,
E. C. 3.2.1.57, Pullulan Hydrolase Type 111) 659
11.4.2.10 Cyclodextrin Glycolsyltransferase (1,4-a-~-Glucan 4-a-~-(1,4-a-~-
G1ucano)-Transferase,E. C. 2.4.1.19) 659
11.4.2.11 Biotechnological Applications of Starch-degrading Enzymes 659
=Ontents Ixv''
11.4.3 Cellulose 661
11.4.3.1 Cellulose-degrading Enzyme Systems 663
11.4.3.2 Endoglucanase (I,4-P-~-Glucan-Glucanohydrolase, E.C. 3.2.1.4) 663
11.4.3.3 Cellobiohydrolase (1,4-P-~-Glucan Cellobiohydrolase, E. C. 3.2.1.91) 663
11.4.3.4 P-Glucosidase (P-D-GlucosideGlucohydrolase, E. C. 3.2.1.21) 664
11.4.3.5 Fungal and Bacterial Cellulases 664
11.4.3.6 Structure and Synergistic Effect of Cellulases 665
11.4.4 Xylan 667
11.4.4.1 The Xylanolytic Elnzyme System 668
11.4.4.2 Endoxylanase (1;l-P-D-Xylan Xylanohydrolase, E. C. 3.2.1.8) 670
11.4.4.3 P-Xylosidase (P-c-XylosideXylohydrolase, E. C. 3.2.1.37) 670
11.4.4.4 a-L-Arabinofuranosidase (E. C. 3.2.1.55) 671
11.4.4.5 a-Glucuronidase (E.C. 3.2.1.136) 671
11.4.4.6 Acetyl Xylan Esterase (E.C. 3.1.1.6) 672
11.4.4.7 Mechanism of Action of Endoxylanase 672
11.4.4.8 Biotechnological Applications of Xylanases 672
11.4.5 Pectin 673
11.4.5.1 Classification of Pectic Substances 675
11.4.5.2 Pectolpc Enzymes 675
11.4.5.3 Classification of Pectolytic Enzymes 676
11.4.5.4 Protopectinase 676
11.4.5.5 Pectin Methylesttxase 677
11.4.5.6 Pectin and Polygalacturonate Depolymerizing Enzymes 677
11.4.5.7 Pectin and Polygalacturonate Hydrolase 678
11.4.5.8 Pectin and Polygalacturonate Lyase 679
11.4.5.9 Biotechnological Applications of Pectolytic Enzymes 680
References 681
11.5 Addition of Water to C=C Bonds 686
Marcel Wubbolts
11.5.1 Addition of Water to Alkenoic Acids 686
11.5.2 Addition of Water to Alkene-Dioic Acids 687
11.5.2.1 L- and D-Malic A'cid 687
11.5.1.2 Substituted Malic Acids 688
11.5.3 Addition of Water to Alkene-TricarboxylicAcids 688
11.5.3.1 Citric Acid and Derivatives 688
11.5.4 Addition of Water to Alkynoic Acids 690
11.5.5 Addition of Water to Enols 690
11.5.5.1 Carbohydrates: Addition of Water to 2-Keto-3-Deoxysugars 690
11.5.5.2 Addition/Elimination of Water with Other Enols 691
11.5.6 Addition of Water to Unsaturated Fatty Acids 693
11.5.6.1 CoA and ACP Coupled Fatty Acid Hydratases 693
11.5.6.2 Hydratases Acting on Free Fatty Acids 695
11.5.7 Addition of Water to Steroids 695
References 690
XVlll
I Contents

12 Hydrolysis and Formation of C-N Bonds 699

12.1 Hydrolysis of Nitriles 699

Birgit Schulze
12.1.1 Introduction 699
12.1.2 Types of Nitrile Hydrolyzing Enzymes 700
12.1.2.1 Enzymatic Hydrolysis of Organic Nitriles 700
12.1.2.2 Enzymatic Hydrolysis of Cyanide 702
12.1.3 Examples of Enzymatic Nitrile Hydrolysis 703
12.1.3.1 Enantioselective Hydrolysis of Nitriles 703
12.1.3.2 Monohydrolysis of Dinitriles 705
12.1.3.3 Substrate and Product Inhibition of Nitrile Hydrolysis 708
12.1.3.4 Activation and Stabilization of Nitrile Hydratases 710
12.1.3.5 Nitrile Hydrolysis in Organic Solvents 710
12.1.3.6 Large Scale Production of Acrylamide 711
12.1.4 Availability and Industrial Future of Nitrile Hydrolyzing Biocatalysts 713
References 713
12.2 Formation and Hydrolysis of Amides 716
Birgit Schulze and Erik de Vroom
12.2.1 Introduction 716
12.2.2 Enzymatic Formation of Amides 716
12.2.3 Enzymatic Enantioselective Hydrolysis of Amides 719
12.2.3.1 Hydrolysis of Carboxylic Amides 719
12.2.3.2 Hydrolysis of Amino Acid Amides 720
12.2.3.3 Hydrolysis of Cyclic Amides 727
12.2.4 Selective Cleavage of the C-Terminal Amide Bond 728
12.2.5 Amidase Catalyzed Hydrolytic and Synthetic Processes in the Production
of Semi-syntheticAntibiotics 729
12.2.5.1 Enzymatic Production of 6-APA, 7-ADCA and 7-ACA Using Amidases:
Hydrolytic Processes 730
12.2.5.2 A New Fermentation-based Biocatalytic Process for 7-ADCA 735
12.2.5.3 Enzymatic Formation of Semi-syntheticAntibiotics:
Synthetic Processes 735
12.2.6 Conclusions and Future Prospects 737
References 738
12.3 Hydrolysis of N-Acylamino Acids 741
Andreas 5.Bommarius
12.3.1 Introduction 741
12.3.2 Acylase I (N-AcylaminoAcid Amidohydrolase, E. C. 3.5.1.4.) 742
12.3.2.1 Genes, Sequences, Structures 743
12.3.2.2 Substrate Specificity 744
12.3.2.3 Stability of Acylases 746
12.3.2.4 Thermodynamics and Mechanism of the Acylase-catalyzed Reaction 748
12.3.3 Acylase I I (N-Acyl-L-Aspartate Amidohydrolase,Aspartoacylase,
E.C. 3.5.1.15.) 749
12.3.4 Proline Acylase 1:N-Acyl-L-ProlineAmidohydrolase) 752
Contents I
12.3.5 Dehydroamino k i d Acylases 753
12.3.6 D-Specific Aminoacylases 754
12.3.7 Acylase Process on a Large Scale 757
References 758
12.4 Hydrolysis and Formation of Hydantoins 761
Markus Pietzsch m d Christoph Syldatk
12.4.1 Classification and Natural Occurrence of Hydantoin Cleaving and Related
Enzymes 761
12.4.2 D-Hydantoinases- Substrate Specificity and Properties 773
12.4.3 DN-Carbamoylases - Substrate Specificity and Properties 777
12.4.4 L-Hydantoinases- Substrate Specificity and Properties 784
12.4.5 L-N-Carbamoylases- Substrate Specificityand Properties 786
12.4.6 Hydantoin Raceinases 792
12.4.7 Conclusions 794
References 796
12.5 Hydrolysis and Formation of Peptides 800
Hans-Dieterjakulike
12.5.1 Introduction 800
12.5.2 Hydrolysis of Peptides 801
12.5.2.1 Peptide-CleavingEnzymes 801
12.5.2.2 Importance of Proteolysis 813
12.5.3 Formation of Peptides 818
12.5.3.1 Tools for Peptide Synthesis 818
12.5.3.2 Choice of the Ideal Enzyme 822
12.5.3.3 Principles of Enzymatic Synthesis 823
12.5.3.4 Manipulations to Suppress Competitive Reactions 831
12.5.3.5 Approaches to Irreversible Formation of Peptide Bond 840
12.5.3.6 Irreversible C-N Ligations by Mimicking Enzyme Specificity 842
12.5.3.7 Planning and Process Development of Enzymatic Peptide Synthesis 851
12.5.4 Conclusion and Outlook 858
References 859
12.6 Addition of Amines to C=C Bonds 866
Marcel Wubbolts
12.6.1 Addition of Ammonia to Produce Amino Acids 866
12.6.1.1 Aspartic Acid 866
12.6.1.2 Aspartic Acid Derivatives 868
12.6.1.3 Histidine Ammonia Lyase 869
12.6.1.4 Phenylalanine, Tyrosin and L-DOPA 870
12.6.1.5 Serine and Threonine Deaminases 871
12.6.1.6 Ornithine Cyclocleaminase 871
12.6.2 Ammonia Lyases that Act on Other Amines 871
12.6.2.1 Elimination of Ammonia from Ethanolamine 871
References 872
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 Mathematics - Mind Map
First 2024 - University

Prepared by: Instructor Johnson

Date: August 12, 2025

Results 1: Statistical analysis and interpretation

Learning Objective 1: Interdisciplinary approaches
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 1: Diagram/Chart/Graph]
Learning Objective 2: Comparative analysis and synthesis
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 3: Literature review and discussion
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Learning Objective 4: Critical analysis and evaluation
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 4: Diagram/Chart/Graph]
Learning Objective 5: Interdisciplinary approaches
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Example 5: Research findings and conclusions
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 6: Problem-solving strategies and techniques
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Key Concept: Statistical analysis and interpretation
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 8: Ethical considerations and implications
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 9: Fundamental concepts and principles
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Exercise 2: Critical analysis and evaluation
Key Concept: Fundamental concepts and principles
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Key terms and definitions
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Key Concept: Case studies and real-world applications
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 13: Diagram/Chart/Graph]
Practice Problem 13: Theoretical framework and methodology
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Literature review and discussion
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Important: Practical applications and examples
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 16: Diagram/Chart/Graph]
Note: Research findings and conclusions
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 17: Learning outcomes and objectives
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Note: Best practices and recommendations
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 19: Key terms and definitions
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Discussion 3: Comparative analysis and synthesis
Key Concept: Interdisciplinary approaches
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Key Concept: Comparative analysis and synthesis
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Theoretical framework and methodology
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 23: Learning outcomes and objectives
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 24: Diagram/Chart/Graph]
Key Concept: Experimental procedures and results
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Ethical considerations and implications
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Remember: Learning outcomes and objectives
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
[Figure 27: Diagram/Chart/Graph]
Definition: Research findings and conclusions
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 28: Diagram/Chart/Graph]
Note: Problem-solving strategies and techniques
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 29: Diagram/Chart/Graph]
Note: Statistical analysis and interpretation
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Exercise 4: Ethical considerations and implications
Note: Research findings and conclusions
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Comparative analysis and synthesis
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 32: Comparative analysis and synthesis
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 33: Problem-solving strategies and techniques
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Learning outcomes and objectives
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 35: Diagram/Chart/Graph]
Note: Best practices and recommendations
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Practice Problem 36: Problem-solving strategies and techniques
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 37: Historical development and evolution
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 38: Diagram/Chart/Graph]
Important: Current trends and future directions
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Study tips and learning strategies
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 40: Diagram/Chart/Graph]
Discussion 5: Comparative analysis and synthesis
Important: Interdisciplinary approaches
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 41: Diagram/Chart/Graph]
Example 41: Current trends and future directions
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 42: Research findings and conclusions
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Practice Problem 43: Comparative analysis and synthesis
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 44: Diagram/Chart/Graph]
Remember: Assessment criteria and rubrics
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 45: Diagram/Chart/Graph]
Example 45: Fundamental concepts and principles
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Practice Problem 46: Research findings and conclusions
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 47: Current trends and future directions
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 48: Diagram/Chart/Graph]
Definition: Comparative analysis and synthesis
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 49: Theoretical framework and methodology
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Results 6: Research findings and conclusions
Note: Experimental procedures and results
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Important: Current trends and future directions
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 52: Interdisciplinary approaches
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 53: Diagram/Chart/Graph]
Important: Fundamental concepts and principles
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 54: Key terms and definitions
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Fundamental concepts and principles
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
[Figure 56: Diagram/Chart/Graph]
Key Concept: Ethical considerations and implications
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 57: Assessment criteria and rubrics
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 58: Diagram/Chart/Graph]
Definition: Comparative analysis and synthesis
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Practice Problem 59: Interdisciplinary approaches
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Results 7: Experimental procedures and results
Practice Problem 60: Fundamental concepts and principles
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Current trends and future directions
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 62: Practical applications and examples
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Definition: Interdisciplinary approaches
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Experimental procedures and results
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Key Concept: Ethical considerations and implications
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 66: Literature review and discussion
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Remember: Practical applications and examples
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 68: Diagram/Chart/Graph]
Remember: Literature review and discussion
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Historical development and evolution
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Chapter 8: Ethical considerations and implications
Note: Assessment criteria and rubrics
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Definition: Statistical analysis and interpretation
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 72: Diagram/Chart/Graph]
Practice Problem 72: Critical analysis and evaluation
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Theoretical framework and methodology
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Key Concept: Research findings and conclusions
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Note: Study tips and learning strategies
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Study tips and learning strategies
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Remember: Research findings and conclusions
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 78: Diagram/Chart/Graph]
Example 78: Learning outcomes and objectives
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Research findings and conclusions
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
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