Preface ......................................................... V
Acknowledgments ............................................... VII
1. Introduction to Biocatalysis ................................ 1
1.1. OverviewrThe Status of Biocatalysis at the Turn of the
21st Century .......................................... 1
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 Biocatalitic
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 fcd 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: l,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-p-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. AspartameO, 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
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/SL-YS 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):
Brensted 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 Enantio-
selectivity 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 Reaction M: 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. P-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
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. a/P-Barrel Proteins as a Model for the
Investigation of Evolution .......................... 474
16.4.1. Why Study a/P-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: AG < 0,
y(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 1-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-
Griissner 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
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