Biocatalysis : fundamentals and applications

Preface V Acknowledgments VII 1 Introduction to Biocatalysis 1 1.1 Overview:The Status of Biocatalysis at the Turn of the 21st Century 2 1.1.1 State of Acceptance of Biocatalysis 2 1.1.2 Current Advantages and Drawbacks of Biocatalysis 4 1.1.2.1 Advantages of Biocatalysts 4 1.1.2.2 Drawbacks of Current Biocatalysts 5 1.2 Characteristics of Biocatalysis as a Technology 6 1.2.1 Contributing Disciplines and Areas of Application 6 1.2.2 Characteristics of Biocatalytic Transformations 7 1.2.2.1 Comparison of Biocatalysis with other Kinds of Catalysis 8 1.2.3 Applications of Biocatalysis in Industry 9 1.2.3.1 Chemical Industry of the Future: Environmentally Benign Manufacturing, Green Chemistry, Sustainable Development in the Future 9 1.2.3.2 Enantiomerically Pure Drugs or Advanced Pharmaceutical Intermediates (APIs) 10 1.3 Current Penetration of Biocatalysis 11 1.3.1 The Past: Historical Digest of Enzyme Catalysis 11 1.3.2 The Present: Status of Biocatalytic Processes 11 1.4 The Breadth of Biocatalysis 14 1.4.1 Nomenclature of Enzymes 14 1.4.2 Biocatalysis and Organic Chemistry, or “Do we Need to Forget our Organic Chemistry?” 14 2 Characterization of a (Bio-)catalyst 19 2.1 Characterization of Enzyme Catalysis 20 2.1.1 Basis of the Activity of Enzymes: What is Enzyme Catalysis? 20 2.1.1.1 Enzyme Reaction in a Reaction Coordinate Diagram 21 2.1.2 Development of Enzyme Kinetics from Binding and Catalysis 21 Biocatalysis. Andreas S. Bommarius and Bettina R. Riebel Copyright © 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-30344-8 X Contents 2.2 Sources and Reasons for the Activity of Enzymes as Catalysts 23 2.2.1 Chronology of the Most Important Theories of Enzyme Activity 23 2.2.2 Origin of Enzymatic Activity: Derivation of the Kurz Equation 24 2.2.3 Consequences of the Kurz Equation 25 2.2.4 Efficiency of Enzyme Catalysis: Beyond Pauling’s Postulate 28 2.3 Performance Criteria for Catalysts, Processes, and Process Routes 30 2.3.1 Basic Performance Criteria for a Catalyst: Activity, Selectivity and Stability of Enzymes 30 2.3.1.1 Activity 30 2.3.1.2 Selectivity 31 2.3.1.3 Stability 32 2.3.2 Performance Criteria for the Process 33 2.3.2.1 Product Yield 33 2.3.2.2 (Bio)catalyst Productivity 34 2.3.2.3 (Bio)catalyst Stability 34 2.3.2.4 Reactor Productivity 35 2.3.3 Links between Enzyme Reaction Performance Parameters 36 2.3.3.1 Rate Acceleration 36 2.3.3.2 Ratio between Catalytic Constant kcat and Deactivation Rate Constant kd 38 2.3.3.3 Relationship between Deactivation Rate Constant kd and Total Turnover Number TTN 38 2.3.4 Performance Criteria for Process Schemes, Atom Economy, and Environmental Quotient 39 3 Isolation and Preparation of Microorganisms 43 3.1 Introduction 44 3.2 Screening of New Enzyme Activities 46 3.2.1 Growth Rates in Nature 47 3.2.2 Methods in Microbial Ecology 47 3.3 Strain Development 48 3.3.1 Range of Industrial Products from Microorganisms 48 3.3.2 Strain Improvement 50 3.4 Extremophiles 52 3.4.1 Extremophiles in Industry 54 3.5 Rapid Screening of Biocatalysts 56 4 Molecular Biology Tools for Biocatalysis 61 4.1 Molecular Biology Basics: DNA versus Protein Level 62 4.2 DNA Isolation and Purification 65 4.2.1 Quantification of DNA/RNA 66 4.3 Gene Isolation, Detection, and Verification 67 4.3.1 Polymerase Chain Reaction 67 4.3.2 Optimization of a PCR Reaction 69 4.3.3 Special PCR Techniques 71 Contents XI 4.3.3.1 Nested PCR 71 4.3.3.2 Inverse PCR 71 4.3.3.3 RACE: Rapid Amplification of cDNA Ends 71 4.3.4 Southern Blotting 74 4.3.4.1 Probe Design and Labeling 76 4.3.4.2 Hybridization 76 4.3.4.3 Detection 76 4.3.5 DNA-Sequencing 77 4.4 Cloning Techniques 77 4.4.1 Restriction Mapping 78 4.4.2 Vectors 78 4.4.3 Ligation 80 4.4.3.1 Propagation of Plasmids and Transformation in Hosts 81 4.5 (Over)expression of an Enzyme Function in a Host 81 4.5.1 Choice of an Expression System 81 4.5.2 Translation and Codon Usage in E. coli 82 4.5.3 Choice of Vector 84 4.5.3.1 Generation of Inclusion Bodies 85 4.5.3.2 Expression of Fusion Proteins 85 4.5.3.3 Surface Expression 87 4.5.4 Expression of Eukaryotic Genes in Yeasts 87 5 Enzyme Reaction Engineering 91 5.1 Kinetic Modeling: Rationale and Purpose 92 5.2 The Ideal World: Ideal Kinetics and Ideal Reactors 94 5.2.1 The Classic Case: Michaelis–Menten Equation 94 5.2.2 Design of Ideal Reactors 96 5.2.3 Integrated Michaelis–Menten Equation in Ideal Reactors 96 5.2.3.1 Case 1: No Inhibition 97 5.3 Enzymes with Unfavorable Binding: Inhibition 97 5.3.1 Types of Inhibitors 97 5.3.2 Integrated Michaelis–Menten Equation for Substrate and Product Inhibition 99 5.3.2.1 Case 2: Integrated Michaelis–Menten Equation in the Presence of Substrate Inhibitor 99 5.3.2.2 Case 3: Integrated Michaelis–Menten Equation in the Presence of Inhibitor 99 5.3.3 The KI –[I]50 Relationship: Another Useful Application of Mechanism Elucidation 103 5.4 Reactor Engineering 105 5.4.1 Configuration of Enzyme Reactors 105 5.4.1.1 Characteristic Dimensionless Numbers for Reactor Design 107 5.4.2 Immobilized Enzyme Reactor (Fixed-Bed Reactor with Plug-Flow) 108 5.4.2.1 Reactor Design Equations 108 5.4.2.2 Immobilization 109 XII Contents 5.4.2.3 Optimal Conditions for an Immobilized Enzyme Reactor 110 5.4.3 Enzyme Membrane Reactor (Continuous Stirred Tank Reactor, CSTR) 110 5.4.3.1 Design Equation: Reactor Equation and Retention 110 5.4.3.2 Classification of Enzyme Membrane Reactors 111 5.4.4 Rules for Choice of Reaction Parameters and Reactors 113 5.5 Enzyme Reactions with Incomplete Mass Transfer: Influence of Immobilization 113 5.5.1 External Diffusion (Film Diffusion) 114 5.5.2 Internal Diffusion (Pore Diffusion) 114 5.5.3 Methods of Testing for Mass Transfer Limitations 116 5.5.4 Influence of Mass Transfer on the Reaction Parameters 118 5.6 Enzymes with Incomplete Stability: Deactivation Kinetics 119 5.6.1 Resting Stability 119 5.6.2 Operational Stability 120 5.6.3 Comparison of Resting and Operational Stability 122 5.6.4 Strategy for the Addition of Fresh Enzyme to Deactiving Enzyme in Continuous Reactors 124 5.7 Enzymes with Incomplete Selectivity: E-Value and its Optimization 126 5.7.1 Derivation of the E-Value 126 5.7.2 Optimization of Separation of Racemates by Choice of Degree of Conversion 128 5.7.2.1 Optimization of an Irreversible Reaction 128 5.7.2.2 Enantioselectivity of an Equilibrium Reaction 129 5.7.2.3 Determination of Enantiomeric Purity from a Conversion–Time Plot 130 5.7.3 Optimization of Enantiomeric Ratio E by Choice of Temperature 130 5.7.3.1 Derivation of the Isoinversion Temperature 130 5.7.3.2 Example of Optimization of Enantioselectivity by Choice of Temperature 131 6 Applications of Enzymes as Bulk Actives: Detergents, Textiles, Pulp and Paper, Animal Feed 135 6.1 Application of Enzymes in Laundry Detergents 136 6.1.1 Overview 136 6.1.2 Proteases against Blood and Egg Stains 138 6.1.3 Lipases against Grease Stains 139 6.1.4 Amylases against Grass and Starch Dirt 139 6.1.5 Cellulases 139 6.1.6 Bleach Enzymes 140 6.2 Enzymes in the Textile Industry: Stone-washed Denims, Shiny Cotton Surfaces 140 6.2.1 Build-up and Mode of Action of Enzymes for the Textile Industry 140 6.2.2 Cellulases: the Shinier Look 141 Contents XIII 6.2.3 Stonewashing: Biostoning of Denim: the Worn Look 143 6.2.4 Peroxidases 144 6.3 Enzymes in the Pulp and Paper Industry: Bleaching of Pulp with Xylanases or Laccases 145 6.3.1 Introduction 145 6.3.2 Wood 146 6.3.2.1 Cellulose 146 6.3.2.2 Hemicellulose 147 6.3.2.3 Lignin 147 6.3.3 Papermaking: Kraft Pulping Process 149 6.3.4 Research on Enzymes in the Pulp and Paper Industry 150 6.3.4.1 Laccases 150 6.3.4.2 Xylanases 151 6.3.4.3 Cellulases in the Papermaking Process 152 6.4 Phytase for Animal Feed: Utilization of Phosphorus 152 6.4.1 The Farm Animal Business and the Environment 152 6.4.2 Phytase 153 6.4.3 Efficacy of Phytase: Reduction of Phosphorus 154 6.4.4 Efficacy of Phytase: Effect on Other Nutrients 155 7 Application of Enzymes as Catalysts: Basic Chemicals, Fine Chemicals, Food, Crop Protection, Bulk Pharmaceuticals 159 7.1 Enzymes as Catalysts in Processes towards Basic Chemicals 160 7.1.1 Nitrile Hydratase: Acrylamide from Acrylonitrile, Nicotinamide from 3-Cyanopyridine, and 5-Cyanovaleramide from Adiponitrile 160 7.1.1.1 Acrylamide from Acrylonitrile 160 7.1.1.2 Nicotinamide from 3-Cyanopyridine 162 7.1.1.3 5-Cyanovaleramide from Adiponitrile 162 7.1.2 Nitrilase: 1,5-Dimethyl-2-piperidone from 2-Methylglutaronitrile 163 7.1.3 Toluene Dioxygenase: Indigo or Prostaglandins from Substituted Benzenes via cis-Dihydrodiols 163 7.1.4 Oxynitrilase (Hydroxy Nitrile Lyase, HNL): Cyanohydrins from Aldehydes 167 7.2 Enzymes as Catalysts in the Fine Chemicals Industry 170 7.2.1 Chirality, and the Cahn–Ingold–Prelog and Pfeiffer Rules 170 7.2.2 Enantiomerically Pure Amino Acids 172 7.2.2.1 The Aminoacylase Process 172 7.2.2.2 The Amidase Process 174 7.2.2.3 The Hydantoinase/Carbamoylase Process 174 7.2.2.4 Reductive Amination of Keto Acids (l-tert-Leucine as Example) 177 7.2.2.5 Aspartase 180 7.2.2.6 l-Aspartate-β-decarboxylase 180 7.2.2.7 l-2-Aminobutyric acid 181 7.2.3 Enantiomerically Pure Hydroxy Acids, Alcohols, and Amines 182 7.2.3.1 Fumarase 182 XIV Contents 7.2.3.2 Enantiomerically Pure Amines with Lipase 182 7.2.3.3 Synthesis of Enantiomerically Pure Amines through Transamination 183 7.2.3.4 Hydroxy esters with carbonyl reductases 185 7.2.3.5 Alcohols with ADH 186 7.3 Enzymes as Catalysts in the Food Industry 187 7.3.1 HFCS with Glucose Isomerase (GI) 187 7.3.2 AspartameÒ, Artificial Sweetener through Enzymatic Peptide Synthesis 188 7.3.3 Lactose Hydrolysis 191 7.3.4 “Nutraceuticals”: l-Carnitine as a Nutrient for Athletes and Convalescents (Lonza) 191 7.3.5 Decarboxylases for Improving the Taste of Beer 194 7.4 Enzymes as Catalysts towards Crop Protection Chemicals 195 7.4.1 Intermediate for Herbicides: (R)-2-(4-Hydroxyphenoxypropionic acid (BASF, Germany) 195 7.4.2 Applications of Transaminases towards Crop Protection Agents: l-Phosphinothricin and (S)-MOIPA 196 7.5 Enzymes for Large-Scale Pharma Intermediates 197 7.5.1 Penicillin G (or V) Amidase (PGA, PVA): β-Lactam Precursors, Semi-synthetic β-Lactams 197 7.5.2 Ephedrine 200 8 Biotechnological Processing Steps for Enzyme Manufacture 209 8.1 Introduction to Protein Isolation and Purification 210 8.2 Basics of Fermentation 212 8.2.1 Medium Requirements 213 8.2.2 Sterilization 214 8.2.3 Phases of a Fermentation 214 8.2.4 Modeling of a Fermentation 215 8.2.5 Growth Models 216 8.2.6 Fed-Batch Culture 216 8.3 Fermentation and its Main Challenge: Transfer of Oxygen 218 8.3.1 Determination of Required Oxygen Demand of the Cells 218 8.3.2 Calculation of Oxygen Transport in the Fermenter Solution 219 8.3.3 Determination of kL, a, and kLa 220 8.3.2.1 Methods of Measurement of the Product kLa 221 8.4 Downstream Processing: Crude Purification of Proteins 223 8.4.1 Separation (Centrifugation) 223 8.4.2 Homogenization 225 8.4.3 Precipitation 226 8.4.3.1 Precipitation in Water-Miscible Organic Solvents 228 8.4.3.2 Building Quantitative Models for the Hofmeister Series and Cohn– Edsall and Setschenow Equations 228 8.4.4 Aqueous Two-Phase Extraction 229 Contents XV 8.5 Downstream Processing: Concentration and Purification of Proteins 231 8.5.1 Dialysis (Ultrafiltration) (adapted in part from Blanch, 1997) 231 8.5.2 Chromatography 233 8.5.2.1 Theory of Chromatography 233 8.5.2.2 Different Types of Chromatography 235 8.5.3 Drying: Spray Drying, Lyophilization, Stabilization for Storage 236 8.6 Examples of Biocatalyst Purification 237 8.6.1 Example 1: Alcohol Dehydrogenase [(R)-ADH from L. brevis (Riebel, 1997)] 237 8.6.2 Example 2: l-Amino Acid Oxidase from Rhodococcus opacus (Geueke 2002a,b) 238 8.6.3 Example 3: Xylose Isomerase from Thermoanaerobium Strain JW/SLYS 489 240 9 Methods for the Investigation of Proteins 243 9.1 Relevance of Enzyme Mechanism 244 9.2 Experimental Methods for the Investigation of an Enzyme Mechanism 245 9.2.1 Distribution of Products (Curtin–Hammett Principle) 245 9.2.2 Stationary Methods of Enzyme Kinetics 246 9.2.3 Linear Free Enthalpy Relationships (LFERs): Brønsted and Hammett Effects 248 9.2.4 Kinetic Isotope Effects 249 9.2.5 Non-stationary Methods of Enzyme Kinetics: Titration of Active Sites 249 9.2.5.1 Determination of Concentration of Active Sites 249 9.2.6 Utility of the Elucidation of Mechanism: Transition-State Analog Inhibitors 251 9.3 Methods of Enzyme Determination 253 9.3.1 Quantification of Protein 253 9.3.2 Isoelectric Point Determination 254 9.3.3 Molecular Mass Determination of Protein Monomer: SDS-PAGE 254 9.3.4 Mass of an Oligomeric Protein: Size Exclusion Chromatography (SEC) 256 9.3.5 Mass Determination: Mass Spectrometry (MS) (after Kellner, Lottspeich, Meyer) 257 9.3.6 Determination of Amino Acid Sequence by Tryptic Degradation, or Acid, Chemical or Enzymatic Digestion 258 9.4 Enzymatic Mechanisms: General Acid–Base Catalysis 258 9.4.1 Carbonic Anhydrase II 258 9.4.2 Vanadium Haloperoxidase 260 9.5 Nucleophilic Catalysis 261 9.5.1 Serine Proteases 261 9.5.2 Cysteine in Nucleophilic Attack 265 XVI Contents 9.5.3 Lipase, Another Catalytic Triad Mechanism 266 9.5.4 Metalloproteases 268 9.6 Electrophilic catalysis 269 9.6.1 Utilization of Metal Ions: ADH, a Different Catalytic Triad 269 9.6.1.1 Catalytic Mechanism of Horse Liver Alcohol Dehydrogenase, a Medium-Chain Dehydrogenase 269 9.6.1.2 Catalytic Reaction Mechanism of Drosophila ADH, a Short-Chain Dehydrogenase 271 9.6.2 Formation of a Schiff Base, Part I: Acetoacetate Decarboxylase, Aldolase 274 9.6.3 Formation of a Schiff Base with Pyridoxal Phosphate (PLP): Alanine Racemase, Amino Acid Transferase 275 9.6.4 Utilization of Thiamine Pyrophosphate (TPP): Transketolase 277 10 Protein Engineering 281 10.1 Introduction: Elements of Protein Engineering 282 10.2 Methods of Protein Engineering 283 10.2.1 Fusion PCR 284 10.2.2 Kunkel Method 285 10.2.3 Site-Specific Mutagenesis Using the QuikChange Kit from Stratagene 287 10.2.4 Combined Chain Reaction (CCR) 288 10.3 Glucose (Xylose) Isomerase (GI) and Glycoamylase: Enhancement of Thermostability 289 10.3.1 Enhancement of Thermostability in Glucose Isomerase (GI) 289 10.3.2 Resolving the Reaction Mechanism of Glucose Isomerase (GI): Diffusion-Limited Glucose Isomerase? 292 10.4 Enhancement of Stability of Proteases against Oxidation and Thermal Deactivation 293 10.4.1 Enhancement of Oxidation Stability of Subtilisin 293 10.4.2 Thermostability of Subtilisin 295 10.5 Creating New Enzymes with Protein Engineering 295 10.5.1 Redesign of a Lactate Dehydrogenase 295 10.5.2 Synthetic Peroxidases 297 10.6 Dehydrogenases, Changing Cofactor Specificity 298 10.7 Oxygenases 300 10.8 Change of Enantioselectivity with Site-Specific Mutagenesis 302 10.9 Techniques Bridging Different Protein Engineering Techniques 303 10.9.1 Chemically Modified Mutants, a Marriage of Chemical Modification and Protein Engineering 303 10.9.2 Expansion of Substrate Specificity with Protein Engineering and Directed Evolution 304 11 Applications of Recombinant DNA Technology: Directed Evolution 309 11.1 Background of Evolvability of Proteins 310 Contents XVII 11.1.1 Purpose of Directed Evolution 310 11.1.2 Evolution and Probability 311 11.1.3 Evolution: Conservation of Essential Components of Structure 313 11.2 Process steps in Directed Evolution: Creating Diversity and Checking for Hits 314 11.2.1 Creation of Diversity in a DNA Library 315 11.2.2 Testing for Positive Hits: Screening or Selection 318 11.3 Experimental Protocols for Directed Evolution 319 11.3.1 Creating Diversity: Mutagenesis Methods 319 11.3.2 Creating Diversity: Recombination Methods 319 11.3.2.1 DNA Shuffling 320 11.3.2.2 Staggered Extension Process (StEP) 321 11.3.2.3 RACHITT (Random Chimeragenesis on Transient Templates) 322 11.3.3 Checking for Hits: Screening Assays 323 11.3.4 Checking for Hits: Selection Procedures 324 11.3.5 Additional Techniques of Directed Evolution 325 11.4 Successful Examples of the Application of Directed Evolution 325 11.4.1 Application of Error-prone PCR: Activation of Subtilisin in DMF 325 11.4.2 Application of DNA Shuffling: Recombination of p-Nitrobenzyl Esterase Genes 326 11.4.3 Enhancement of Thermostability: p-Nitrophenyl Esterase 328 11.4.4 Selection instead of Screening: Creation of a Monomeric Chorismate Mutase 329 11.4.5 Improvement of Enantioselectivity: Pseudomonas aeruginosa Lipase 329 11.4.6 Inversion of Enantioselectivity: Hydantoinase 330 11.4.7 Redesign of an Enzyme’s Active Site: KDPG Aldolase 331 11.5 Comparison of Directed Evolution Techniques 331 11.5.1 Comparison of Error-Prone PCR and DNA Shuffling: Increased Resistance against Antibiotics 331 11.5.2 Protein Engineering in Comparison with Directed Evolution: Aminotransferases 332 11.5.2.1 Directed Evolution of Aminotransferases 332 11.5.3 Directed Evolution of a Pathway: Carotenoids 333 12 Biocatalysis in Non-conventional Media 339 12.1 Enzymes in Organic Solvents 340 12.2 Evidence for the Perceived Advantages of Biocatalysts in Organic Media 341 12.2.1 Advantage 1: Enhancement of Solubility of Reactants 341 12.2.2 Advantage 2: Shift of Equilibria in Organic Media 342 12.2.2.1 Biphasic Reactors 342 12.2.3 Advantage 3: Easier Separation 343 12.2.4 Advantage 4: Enhanced Stability of Enzymes in Organic Solvents 344 12.2.5 Advantage 5: Altered Selectivity of Enzymes in Organic Solvents 344 XVIII Contents 12.3 State of Knowledge of Functioning of Enzymes in Solvents 344 12.3.1 Range of Enzymes, Reactions, and Solvents 344 12.3.2 The Importance of Water in Enzyme Reactions in Organic Solvents 345 12.3.2.1 Exchange of Water Molecules between Enzyme Surface and Bulk Organic Solvent 345 12.3.2.2 Relevance of Water Activity 346 12.3.3 Physical Organic Chemistry of Enzymes in Organic Solvents 347 12.3.3.1 Active Site and Mechanism 347 12.3.3.2 Flexibility of Enzymes in Organic Solvents 347 12.3.3.3 Polarity and Hydrophobicity of Transition State and Binding Site 348 12.3.4 Correlation of Enzyme Performance with Solvent Parameters 349 12.3.4.1 Control through Variation of Hydrophobocity: log P Concept 350 12.3.4.2 Correlation of Enantioselectivity with Solvent Polarity and Hydrophobicity 350 12.4 Optimal Handling of Enzymes in Organic Solvents 351 12.4.1 Enzyme Memory in Organic Solvents 352 12.4.2 Low Activity in Organic Solvents Compared to Water 353 12.4.3 Enhancement of Selectivity of Enzymes in Organic Solvents 354 12.5 Novel Reaction Media for Biocatalytic Transformations 355 12.5.1 Substrate as Solvent (Neat Substrates): Acrylamide from Acrylonitrile with Nitrile Hydratase 355 12.5.2 Supercritical Solvents 356 12.5.3 Ionic Liquids 356 12.5.4 Emulsions [Manufacture of Phosphatidylglycerol (PG)] 357 12.5.5 Microemulsions 358 12.5.6 Liquid Crystals 358 12.5.7 Ice–Water Mixtures 359 12.5.8 High-Density Eutectic Suspensions 361 12.5.9 High-Density Salt Suspensions 362 12.5.10 Solid-to-Solid Syntheses 363 12.6 Solvent as a Parameter for Reaction Optimization (“Medium Engineering”) 366 12.6.1 Change of Substrate Specificity with Change of ReactionM: Specificity of Serine Proteases 366 12.6.2 Change of Regioselectivity by Organic Solvent Medium 367 12.6.3 Solvent Control of Enantiospecificity of Nifedipines 367 13 Pharmaceutical Applications of Biocatalysis 373 13.1 Enzyme Inhibition for the Fight against Disease 374 13.1.1 Introduction 374 13.1.2 Procedure for the Development of Pharmacologically Active Compounds 376 13.1.3 Process for the Registration of New Drugs 377 13.1.4 Chiral versus Non-chiral Drugs 379 XIX 13.2 Enzyme Cascades and Biology of Diseases 380 13.2.1 β-Lactam Antibiotics 380 13.2.2 Inhibition of Cholesterol Biosynthesis (in part after Suckling, 1990) 382 13.2.3 Pulmonary Emphysema, Osteoarthritis: Human Leucocyte Elastase (HLE) 385 13.2.4 AIDS: Reverse Transcriptase and HIV Protease Inhibitors 389 13.3 Pharmaceutical Applications of Biocatalysis 393 13.3.1 Antiinfectives (see also Chapter 7, Section 7.5.1) 393 13.3.1.1 Cilastatin 393 13.3.2 Anticholesterol Drugs 393 13.3.2.1 Cholesterol Absorption Inhibitors 395 13.3.3 Anti-AIDS Drugs 396 13.3.3.1 Abacavir Intermediate 396 13.3.3.2 Lobucavir Intermediate 397 13.3.3.3 cis-Aminoindanol: Building Block for Indinavir (Crixivan®) 397 13.3.4 High Blood Pressure Treatment 398 13.3.4.1 Biotransformations towards Omapatrilat 398 13.3.4.2 Lipase Reactions to Intermediates for Cardiovascular Therapy 400 13.4 Applications of Specific Biocatalytic Reactions in Pharma 402 13.4.1 Reduction of Keto Compounds with Whole Cells 402 13.4.1.1 Trimegestone 402 13.4.1.2 Reduction of Precursor to Carbonic Anhydrase Inhibitor L-685393 404 13.4.1.3 Montelukast 404 13.4.1.4 LY300164 404 13.4.2 Applications of Pen G Acylase in Pharma 406 13.4.2.1 Loracarbef® 406 13.4.2.2 Xemilofibran 406 13.4.3 Applications of Lipases and Esterases in Pharma 407 13.4.3.1 LTD4 Antagonist MK-0571 407 13.4.3.2 Tetrahydrolipstatin 407 14 Bioinformatics 413 14.1 Starting Point: from Consequence (Function) to Sequence 414 14.1.1 Conventional Path: from Function to Sequence 414 14.1.2 Novel Path: from Sequence to Consequence (Function) 414 14.2 Bioinformatics: What is it, Why do we Need it, and Why Now? (NCBI Homepage) 415 14.2.1 What is Bioinformatics? 415 14.2.2 Why do we Need Bioinformatics? 416 14.2.3 Why Bioinformatics Now? 416 14.3 Tools of Bioinformatics: Databases, Alignments, Structural Mapping 418 14.3.1 Available Databases 418 14.3.2 Protein Data Bank (PDB) 418 Contents XX Contents 14.3.3 Protein Explorer 419 14.3.4 ExPASy Server: Roche Applied Science Biochemical Pathways 419 14.3.5 GenBank 419 14.3.6 SwissProt 420 14.3.7 Information on an Enzyme: the Example of dehydrogenases 420 14.3.7.1 Sequence Information 421 14.3.7.2 Structural Information 422 14.4 Applied Bioinformatics Tools, with Examples 422 14.4.1 BLAST 422 14.4.2 Aligning Several Protein Sequences using ClustalW 425 14.4.3 Task: Whole Genome Analysis 427 14.4.4 Phylogenetic Tree 427 14.5 Bioinformatics for Structural Information on Enzymes 429 14.5.1 The Status of Predicting Protein Three-Dimensional Structure 430 14.6 Conclusion and Outlook 431 15 Systems Biology for Biocatalysis 433 15.1 Introduction to Systems Biology 434 15.1.1 Systems Approach versus Reductionism 434 15.1.2 Completion of Genomes: Man, Earthworm, and Others 435 15.2 Genomics, Proteomics, and other -omics 435 15.2.1 Genomics 435 15.2.2 Proteomics 436 15.3 Technologies for Systems Biology 438 15.3.1 Two-Dimensional Gel Electrophoresis (2D PAGE) 438 15.3.1.1 Separation by Chromatography or Capillary Electrophoresis 439 15.3.1.2 Separation by Chemical Tagging 440 15.3.2 Mass Spectroscopy 441 15.3.2.1 MALDI-TOF-MS (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight MS) 444 15.3.2.2 ESI-triple-quadrupole MS 444 15.3.2.3 ESI-MS Using an Ion Trap Analyzer 445 15.3.3 DNA Microarrays 446 15.3.4 Protein Microarrays 447 15.3.5 Applications of Genomics and Proteomics in Biocatalysis 448 15.3.5.1 Lactic Acid Bacteria and Proteomics 448 15.4 Metabolic Engineering 449 15.4.1 Concepts of Metabolic Engineering 449 15.4.2 Examples of Metabolic Engineering 451 16 Evolution of Biocatalytic Function 457 16.1 Introduction 458 16.1.2 Congruence of Sequence, Function, Structure, and Mechanism 460 16.2 Search Characteristics for Relatedness in Proteins 461 16.2.1 Classification of Relatedness of Proteins: the -log Family 461 XXI 16.2.2 Classification into Protein Families 464 16.2.3 Dominance of Different Mechanisms 465 16.3 Evolution of New Function in Nature 466 16.3.1 Dual-Functionality Proteins 469 16.3.1.1 Moonlighting Proteins 469 16.3.1.2 Catalytic Promiscuity 469 16.3.2 Gene Duplication 470 16.3.3 Horizontal Gene Transfer (HGT) 471 16.3.4 Circular Permutation 474 16.4 α/β-Barrel Proteins as a Model for the Investigation of Evolution 474 16.4.1 Why Study α/β-Barrel Proteins? 474 16.4.2 Example of Gene Duplication: Mandelate and a-Ketoadipate Pathways 475 16.4.2.1 Description of Function 480 16.4.3 Exchange of Function in the Aromatic Biosynthesis Pathways: Trp and His Pathways 481 17 Stability of Proteins 487 17.1 Summary: Protein Folding, First-Order Decay, Arrhenius Law 488 17.1.1 The Protein Folding Problem 488 17.1.2 Why do Proteins Fold? 489 17.2 Two-State Model: Thermodynamic Stability of Proteins (Unfolding) 491 17.2.1 Protein Unfolding and Deactivation 491 17.2.2 Thermodynamics of Proteins 491 17.3 Three-State Model: Lumry–Eyring Equation 493 17.3.1 Enzyme Deactivation 493 17.3.2 Empirical Deactivation Model 494 17.4 Four-State Model: Protein Aggregation 496 17.4.1 Folding, Deactivation, and Aggregation 496 17.4.2 Model to Account for Competition between Folding and Inclusion Body Formation 498 17.4.2.1 Case 1: In Vitro – Protein Synthesis Unimportant 498 17.4.2.2 Case 2: In Vivo – Protein Synthesis Included 499 17.5 Causes of Instability of Proteins: ΔG < 0, γ(t), A 501 17.5.1 Thermal Inactivation 502 17.5.2 Deactivation under the Influence of Stirring 503 17.5.3 Deactivation under the Influence of Gas Bubbles 504 17.5.4 Deactivation under the Influence of Aqueous/Organic Interfaces 505 17.5.5 Deactivation under the Influence of Salts and Solvents 505 17.6 Biotechnological Relevance of Protein Folding: Inclusion Bodies 505 17.7 Summary: Stabilization of Proteins 506 17.7.1 Correlation between Stability and Structure 507 Contents XXII 18 Artificial Enzymes 511 18.1 Catalytic Antibodies 512 18.1.1 Principle of Catalytic Antibodies: Connection between Chemistry and Immunology 512 18.1.2 Test Reaction Selection, Haptens, Mechanisms, Stabilization 514 18.1.2.1 Mechanism of Antibody-Catalyzed Reactions 516 18.1.2.2 Stabilization of Charged Transition States 517 18.1.2.3 Effect of Antibodies as Entropy Traps 517 18.1.3 Breadth of Reactions Catalyzed by Antibodies 518 18.1.3.1 Fastest Antibody-Catalyzed Reaction in Comparison with Enzymes 518 18.1.3.2 Antibody-Catalyzed Reactions without Corresponding Enzyme Equivalent 518 18.1.3.3 Example of a Pericyclic Reaction: Claisen Rearrangement 518 18.1.3.4 Antibody Catalysts with Dual Activities 518 18.1.3.5 Scale-Up of an Antibody-Catalyzed Reaction 520 18.1.3.6 Perspective for Catalytic Antibodies 520 18.2 Other Proteinaceous Catalysts: Ribozymes and Enzyme Mimics 521 18.2.1 Ribozymes: RNA World before Protein World? 521 18.2.2 Proteinaceous Enzyme Mimics 521 18.3 Design of Novel Enzyme Activity: Enzyme Models (Synzymes) 523 18.3.1 Introduction 523 18.3.2 Enzyme Models on the Basis of the Binding Step: Diels–Alder Reaction 523 18.3.3 Enzyme Models with Binding and Catalytic Effects 525 18.4 Heterogenized/Immobilized Chiral Chemical Catalysts 526 18.4.1 Overview of Different Approaches 526 18.4.2 Immobilization with Polyamino Acids as Chiral Polymer Catalysts 526 18.4.3 Immobilization on Resins or other Insoluble Carriers 527 18.4.4 Heterogenization with Dendrimers 528 18.4.5 Retention of Heterogenized Chiral Chemical Catalysts in a Membrane Reactor 529 18.4.6 Recovery of Organometallic Catalysts by Phase Change: Liquid–Liquid Extraction 531 18.5 Tandem Enzyme Organometallic Catalysts 532 19 Design of Biocatalytic Processes 539 19.1 Design of Enzyme Processes: High-Fructose Corn Syrup (HFCS) 540 19.1.1 Manufacture of HFCS from Glucose with Glucose Isomerase (GI): Process Details 540 19.1.2 Mathematical Model for the Description of the Enzyme Kinetics of Glucose Isomerase (GI) 541 19.1.3 Evaluation of the Model of the GI Reaction in the Fixed-Bed Reactor 543 19.1.4 Productivity of a Fixed-Bed Enzyme Reactor 547 Contents XXIII 19.2 Processing of Fine Chemicals or Pharmaceutical Intermediates in an Enzyme Membrane Reactor 549 19.2.1 Introduction 549 19.2.2 Determination of Process Parameters of a Membrane Reactor 550 19.2.2.1 Case 1: Leakage through Membrane, no Deactivation 551 19.2.2.2 Case 2: Leakage through the Membrane and Deactivation of Enzyme 552 19.2.2.3 Design Criterion for EMRs 552 19.2.3 Large-Scale Applications of Membrane Reactors 553 19.2.3.1 Enantiomerically Pure l-Amino Acids for Infusion Solutions and as Building Blocks for New Drugs 553 19.2.3.2 Aqueous–Organic Membrane Reactors 554 19.2.3.3 Other Processes in Enzyme Membrane Reactors 554 19.3 Production of Enantiomerically Pure Hydrophobic Alcohols: Comparison of Different Process Routes and Reactor Configurations 556 19.3.1 Isolated Enzyme Approach 556 19.3.2 Whole-Cell Approach 559 19.3.3 Organometallic Catalyst Approach 561 19.3.4 Comparison of Different Catalytic Reduction Strategies 563 20 Comparison of Biological and Chemical Catalysts for Novel Processes 569 20.1 Criteria for the Judgment of (Bio-)catalytic Processes 570 20.1.1 Discussion: Jacobsen’s Five Criteria 570 20.1.2 Comment on Jabobsen’s Five Criteria 572 20.2 Position of Biocatalysis in Comparison to Chemical Catalysts for Novel Processes 575 20.2.1 Conditions and Framework for Processes of the Future 575 20.2.2 Ibuprofen (Painkiller) 577 20.2.3 Indigo (Blue Dye) 578 20.2.4 Menthol (Peppermint Flavoring Agent) 580 20.2.4.1 Separation of Diastereomeric Salt Pairs 580 20.2.4.2 Homogeneous Catalysis with Rh-BINAP 580 20.2.4.3 Lipase-Catalyzed Resolution of Racemic Menthol Esters 582 20.2.5 Ascorbic Acid (Vitamin C) 583 20.2.5.1 The Traditional Reichstein–Grüssner Synthesis 584 20.2.5.2 Two-Step Fermentation Process to 2-Ketogulonic Acid with Chemical Step to Ascorbic Acid 584 20.2.5.3 One-Step Fermentation to 2-Ketogulonic Acid with Chemical Step to Ascorbic Acid 585 20.3 Pathway Engineering through Metabolic Engineering 586 20.3.1 Pathway Engineering for Basic Chemicals: 1,3-Propanediol 586 20.3.2 Pathway Engineering for Pharmaceutical Intermediates: cis-Aminoindanol 588 Index 593