| | Contents | |
| | | |
| |
| | 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 |
| 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 |
| 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 |
| 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 |
| 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- -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 |
| 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): -Lactam Precursors, Semi-synthetic -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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 13.2 | Enzyme Cascades and Biology of Diseases | 380 |
| 13.2.1 | -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(r) | 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 |
| 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 |
| 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 |  /-Barrel Proteins as a Model for the Investigation of Evolution | 474 |
| 16.4.1 | Why Study /-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:  G < 0,  (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 |
| 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 |
| 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 |
