Enzyme Catalysis in Organic Synthesis A Comprehensive Handbook Volume III Edited by Karlheinz Drauz and Herbert Waldmann Second, Completely Revised and Enlarged Edition ^WILEY-VCH IIX Contents Foreword V Preface VII Volume I 1 Introduction 1 Maria-Regina Kula 1.1 Enzymes as Catalysts 1 1.2 Enzyme Structure and Function 4 (X^ 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 Effectors 26 1.5.3 Influence of pH and Buffers 27 1.5.4 Temperature 28 1.6 Organic Solvents as Reaction Media 31 1.7 Enzyme Handling: Quality Requirements 32 1.8 Biotransformation 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 Biotransformations with Permeabilized or Dried Cells 37 Bibliography 38 2 Production and Isolation of Enzymes 41 Yoshihiko Hirose 2.1 Introduction 41 2.2 Enzyme Suppliers for Biotransformation 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| 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 ExtractionofEnzym.es 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 Salting-out 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 Imanaka 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 Contents XI 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 Catalytic 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 Library 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-Throughput 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 XIII 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-specific Conservation 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 PROSITEea55 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 of 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 Contents XIII 6.2.4 Cross-Linking 175 6.3 Properties of Immobilized 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 Manfred 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 Reactions 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 7.6 Conclusions and Outlook 253 References 254 8 Enzymic Conversions in Organic and Other Low-Water Media 259 Peter Hailing 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 pH Memory 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.