Biocatalysis: Fundamentals and Applications

Long awaited, "Biocatalysis – Fundamentals and Applications" is finally available: Covering the whole range, from a firm grounding in theoretical concepts to in–depth coverage of the practical aspects and future perspectives.
The book not only covers reactions, products and processes using biocatalysts, but also methods of designing and improving them. One unique feature is that chemistry, biology and bioengineering receive equal attention, thus addressing practitioners and students from all three fields.

Spis treści:
Preface.
Acknowledgments.
1 Introduction to Biocatalysis.
1.1 Overview:The Status of Biocatalysis at the Turn of the 21st Century.
1.1.1 State of Acceptance of Biocatalysis.
1.1.2 Current Advantages and Drawbacks of Biocatalysis.
1.1.2.1 Advantages of Biocatalysts.
1.1.2.2 Drawbacks of Current Biocatalysts.
1.2 Characteristics of Biocatalysis as a Technology.
1.2.1 Contributing Disciplines and Areas of Application.
1.2.2 Characteristics of Biocatalytic Transformations.
1.2.2.1 Comparison of Biocatalysis with other Kinds of Catalysis.
1.2.3 Applications of Biocatalysis in Industry.
1.2.3.1 Chemical Industry of the Future: Environmentally Benign Manufacturing, Green Chemistry, Sustainable Development in the Future.
1.2.3.2 Enantiomerically Pure Drugs or Advanced Pharmaceutical Intermediates (APIs).
1.3 Current Penetration of Biocatalysis.
1.3.1 The Past: Historical Digest of Enzyme Catalysis.
1.3.2 The Prese nt: Status of Biocatalytic Processes.
1.4 The Breadth of Biocatalysis.
1.4.1 Nomenclature of Enzymes.
1.4.2 Biocatalysis and Organic Chemistry, or “Do we Need to Forget our Organic Chemistry?”.
2 Characterization of a (Bio–)catalyst.
2.1 Characterization of Enzyme Catalysis.
2.1.1 Basis of the Activity of Enzymes: What is Enzyme Catalysis?
2.1.1.1 Enzyme Reaction in a Reaction Coordinate Diagram.< br>2.1.2 Development of Enzyme Kinetics from Binding and Catalysis.
2.2 Sources and Reasons for the Activity of Enzymes as Catalysts.
2.2.1 Chronology of the Most Important Theories of Enzyme Activity.
2.2.2 Origin of Enzymatic Activity: Derivation of the Kurz Equation.
2.2.3 Consequences of the Kurz Equation.
2.2.4 Efficiency of Enzyme Catalysis: Beyond Pauling’s Postulate.
2.3 Performance Criteria for Catalysts, Processes, and Process Routes.
2.3.1 Basic Performance Criteria for a Catalyst: Activity, Selectivity and Stability of Enzymes.
2.3.1.1 Activity.
2.3.1.2 Selectivity.
2.3.1.3 Stability.
2.3.2 Performance Criteria for the Process.
2.3.2.1 Product Yield.
2.3.2.2 (Bio)catalyst Productivity.
2.3.2.3 (Bio)catalyst Stability.
2.3.2.4 Reactor Productivity.
2.3.3 Links between Enzyme Reaction Performance Parameters.
2.3.3.1 Rate Acceleration.
2.3.3.2 Ratio between Catalytic Constant kcat and Deactivation Rate Constant kd.
2.3.3.3 Relationship between Deactivation Rate Constant kd and Total Turnover Number TTN.
2.3.4 Performance Criteria for Process Schemes, Atom Economy, and Environmental Quotient.
3 Isolation and Preparation of Microorganisms.
3.1 Introduction.
3.2 Screening of New Enzyme Activities.
3.2.1 Growth Rates in Nature.
3.2.2 Methods in Microbial Ecology.
3.3 Strain Development.
3.3.1 Range of Industrial Products from Microorganisms.
3.3.2 Strain Improvement.
3.4 Extremophiles.
3.4.1 Extremophiles in Industry.
3.5 Rapid Screening of Biocatalysts.
4 Molecular Biology Tools for Biocatalysis.
4.1 Molecular Biology Basics: DNA versus Protein Level.
4.2 DNA Isolation and Purification.
4.2.1 Quantification of DNA/RNA.
4.3 Gene Isolation, Detection, and Verification.
4.3.1 Polymerase Chain Reaction.
4.3.2 Optimization of a PCR Reaction.
4.3.3 Special PCR Techniques.
4.3.3.1 Nested PCR.
4.3.3.2 Inve rse PCR.
4.3.3.3 RACE: Rapid Amplification of cDNA Ends.
4.3.4 Southern Blotting.
4.3.4.1 Probe Design and Labeling.
4.3.4.2 Hybridization.
4.3.4.3 Detection.
4.3.5 DNA–Sequencing.
4.4 Cloning Techniques.
4.4.1 Restriction Mapping.
4.4.2 Vectors.
4.4.3 Ligation.
4.4.3.1 Propagation of Plasmids and Transformation in Hosts.
4.5 (Over)expression of an Enzyme Function in a Host.
4.5.1 Choice of an Expression System.
4.5.2 Translation and Codon Usage in E. coli.
4.5.3 Choice of Vector.
4.5.3.1 Generation of Inclusion Bodies.
4.5.3.2 Expression of Fusion Proteins.
4.5.3.3 Surface Expression.
4.5.4 Expression of Eukaryotic Genes in Yeasts.
5 Enzyme Reaction Engineering.
5.1 Kinetic Modeling: Rationale and Purpose.
5.2 The Ideal World: Ideal Kinetics and Ideal Reactors.
5.2.1 The Classic Case: Michaelis–Menten Equation.
5.2.2 Design of Ideal Reactors.
5.2.3 Integrated Michaelis–Menten Equation in Ideal Reactors.
5.2.3.1 Case 1: No Inhibition.
5.3 Enzymes with Unfavorable Binding: Inhibition.
5.3.1 Types of Inhibitors.
5.3.2 Integrated Michaelis–Menten Equation for Substrate and Product Inhibition.
5.3.2.1 Case 2: Integrated Michaelis–Menten Equation in the Presence of Substrate Inhibitor.
5.3.2.2 Case 3: Integrated Michaelis–Menten Equation in the Presence of Inhibitor.
5.3.3 The KI –[I]50 Relationship: Another Useful Application of Mechanism Elucidation.
5.4 Reactor Engineering.
5.4.1 Configuration of Enzyme Reactors.
5.4.1.1 Characteristic Dimensionless Numbers for Reactor Design.
5.4.2 Immobilized Enzyme Reactor (Fixed–Bed Reactor with Plug–Flow).
5.4.2.1 Reactor Design Equations.
5.4.2.2 Immobilization.
5.4.2.3 Optimal Conditions for an Immobilized Enzyme Reactor.
5.4.3 Enzyme Membrane Reactor (Continuous Stirred Tank Reactor, CSTR).
5.4.3.1 Design Equation: Reactor Equation and Retention .
5.4.3.2 Classification of Enzyme Membrane Reactors.
5.4.4 Rules for Choice of Reaction Parameters and Reactors.
5.5 Enzyme Reactions with Incomplete Mass Transfer: Influence of Immobilization.
5.5.1 External Diffusion (Film Diffusion).
5.5.2 Internal Diffusion (Pore Diffusion).
5.5.3 Methods of Testing for Mass Transfer Limitations.
5.5.4 Influence of Mass Transfer on the Reaction Parameters.
5.6 Enzymes with Incomplete Stability: Deactivation Kinetics.
5.6.1 Resting Stability.
5.6.2 Operational Stability.
5.6.3 Comparison of Resting and Operational Stability.
5.6.4 Strategy for the Addition of Fresh Enzyme to Deactiving Enzyme in Continuous Reactors.
5.7 Enzymes with Incomplete Selectivity: E–Value and its Optimization.
5.7.1 Derivation of the E–Value.
5.7.2 Optimization of Separation of Racemates by Choice of Degree of Conversion.
5.7.2.1 Optimization of an Irreversible Reaction.
5.7.2.2 Enantioselectivity of an Equilibrium Reaction.
5.7.2.3 Determination of Enantiomeric Purity from a Conversion–Time Plot.
5.7.3 Optimization of Enantiomeric Ratio E by Choice of Temperature.
5.7.3.1 Derivation of the Isoinversion Temperature.
5.7.3.2 Example of Optimization of Enantioselectivity by Choice of Temperature.
6 Applications of Enzymes as Bulk Actives: Detergents, Textiles, Pulp and Paper, Animal Feed.
6.1 Application of Enzymes in Laundry Detergents.
6.1.1 Overview.
6.1.2 Proteases against Blood and Egg Stains.
6.1.3 Lipases against Grease Stains.
6.1.4 Amylases against Grass and Starch Dirt.
6.1.5 Cellulases.
6.1.6 Bleach Enzymes.
6.2 Enzymes in the Textile Industry: Stone–washed Denims, Shiny Cotton Surfaces.
6.2.1 Build–up and Mode of Action of Enzymes for the Textile Industry.
6.2.2 Cellulases: the Shinier Look.
6.2.3 Stonewashing: Biostoning of Denim: the Worn Look.
6.2.4 Peroxidases.
6.3 Enzymes in the Pulp and Paper Indu stry: Bleaching of Pulp with Xylanases or Laccases.
6.3.1 Introduction.
6.3.2 Wood.
6.3.2.1 Cellulose,
6.3.2.2 Hemicellulose,
6.3.2.3 Lignin,
6.3.3 Papermaking: Kraft Pulping Process.
6.3.4 Research on Enzymes in the Pulp and Paper Industry.
6.3.4.1 Laccases.
6.3.4.2 Xylanases.
6.3.4.3 Cellulases in the Papermaking Process.
6.4 Phytase for Animal Feed: Utilization of Phosphorus.
6.4.1 The Farm Animal Business and the Environment.
6.4.2 Phytase.
6.4.3 Efficacy of Phytase: Reduction of Phosphorus.
6.4.4 Efficacy of Phytase: Effect on Other Nutrients.
7 Application of Enzymes as Catalysts: Basic Chemicals, Fine Chemicals, Food, Crop Protection, Bulk Pharmaceuticals.
7.1 Enzymes as Catalysts in Processes towards Basic Chemicals.
7.1.1 Nitrile Hydratase: Acrylamide from Acrylonitrile, Nicotinamide from 3–Cyanopyridine, and 5–Cyanovaleramide from Adiponitrile.
7.1.1.1 Acrylamide from Acrylonitrile.
7.1.1.2 Nicotinamide from 3–Cyanopyridine.
7.1.1.3 5–Cyanovaleramide from Adiponitrile.
7.1.2 Nitrilase: 1,5–Dimethyl–2–piperidone from 2–Methylglutaronitrile.
7.1.3 Toluene Dioxygenase: Indigo or Prostaglandins from Substituted Benzenes via cis–Dihydrodiols.
7.1.4 Oxynitrilase (Hydroxy Nitrile Lyase, HNL): Cyanohydrins from Aldehydes.
7.2 Enzymes as Catalysts in the Fine Chemicals Industry.
7.2.1 Chirality, and the Cahn–Ingold–Prelog and Pfeiffer Rules.
7.2.2 Enantiomerically Pure Amino Acids.
7.2.2.1 The Aminoacylase Process.
7.2.2.2 The Amidase Process.
7.2.2.3 The Hydantoinase/Carbamoylase Process.
7.2.2.4 Reductive Amination of Keto Acids (l–tert–Leucine as Example).
7.2.2.5 Aspartase.
7.2.2.6 l–Aspartate–â–decarboxylase.
7.2.2.7 l–2–Aminobutyric acid.
7.2.3 Enantiomerically Pure Hydroxy Acids, Alcohols, and Amines.
7.2.3.1 Fumarase.
7.2.3.2 Ena ntiomerically Pure Amines with Lipase.
7.2.3.3 Synthesis of Enantiomerically Pure Amines through Transamination.
7.2.3.4 Hydroxy esters with carbonyl reductases.
7.2.3.5 Alcohols with ADH.
7.3 Enzymes as Catalysts in the Food Industry.
7.3.1 HFCS with Glucose Isomerase (GI).
7.3.2 AspartameÒ, Artificial Sweetener through Enzymatic Peptide Synthesis.
7.3.3 Lactose Hydrolysis.
7.3.4 “Nutraceuticals”: l–Carnitine as a Nutrient for Athletes and Convalescents (Lonza).
7.3.5 Decarboxylases for Improving the Taste of Beer.
7.4 Enzymes as Catalysts towards Crop Protection Chemicals.
7.4.1 Intermediate for Herbicides: (R)–2–(4–Hydroxyphenoxypropionic acid (BASF, Germany).
7.4.2 Applications of Transaminases towards Crop Protection Agents: l–Phosphinothricin and (S)–MOIPA.
7.5 Enzymes for Large–Scale Pharma Intermediates.
7.5.1 Penicillin G (or V) Amidase (PGA, PVA): â–Lactam Precursors, Semi–synthetic â–Lactams.
7.5.2 Ephedrine.
8 Biotechnological Processing Steps for Enzyme Manufacture.
8.1 Introduction to Protein Isolation and Purification.
8.2 Basics of Fermentation.
8.2.1 Medium Requirements.
8.2.2 Sterilization.
8.2.3 Phases of a Fermentation.
8.2.4 Modeling of a Fermentation.
8.2.5 Growth Models.
8.2.6 Fed–Batch Culture.
8.3 Fermentation and its Main Challenge: Transfer of Oxygen.
8.3.1 Determination of Required Oxygen Demand of the Cells.
8.3.2 Calculation of Oxygen Transport in the Fermenter Solution.
8.3.3 Determination of kL, a, and kLa .
8.3.2.1 Methods of Measurement of the Product kLa.
8.4 Downstream Processing: Crude Purification of Proteins.
8.4.1 Separation (Centrifugation).
8.4.2 Homogenization.
8.4.3 Precipitation.
8.4.3.1 Precipitation in Water–Miscible Organic Solvents.
8.4.3.2 Building Quantitative Models for the Hofme ister Series and Cohn–Edsall and Setschenow Equations.
8.4.4 Aqueous Two–Phase Extraction.
8.5 Downstream Processing: Concentration and Purification of Proteins.
8.5.1 Dialysis (Ultrafiltration) (adapted in part from Blanch, 1997).
8.5.2 Chromatography.
8.5.2.1 Theory of Chromatography.
8.5.2.2 Different Types of Chromatography.
8.5.3 Drying: Spray Drying, Lyophilization, Stabilization for Storage.
8.6 Examples of Biocatalyst Purification.
8.6.1 Example 1: Alcohol Dehydrogenase [(R)–ADH from L. brevis (Riebel, 1997)].
8.6.2 Example 2: l–Amino Acid Oxidase from Rhodococcus opacus (Geueke 2002a,b).
8.6.3 Example 3: Xylose Isomerase from Thermoanaerobium Strain JW/SLYS 489.
9 Methods for the Investigation of Proteins.
9.1 Relevance of Enzyme Mechanism.
9.2 Experimental Methods for the Investigation of an Enzyme Mechanism.
9.2.1 Distribution of Products (Curtin–Hammett Principle).
9.2.2 Stationary Methods of Enzyme Kinetics.
9.2.3 Linear Free Enthalpy Relationships (LFERs): Brønsted and Hammett Effects.
9.2.4 Kinetic Isotope Effects.
9.2.5 Non–stationary Methods of Enzyme Kinetics: Titration of Active Sites.
9.2.5.1 Determination of Concentration of Active Sites.
9.2.6 Utility of the Elucidation of Mechanism: Transition–State Analog Inhibitors.
9.3 Methods of Enzyme Determination.
9.3.1 Quantification of Protein.
9.3.2 Isoelectric Point Determination.
9.3.3 Molecular Mass Determination of Protein Monomer: SDS–PAGE.
9.3.4 Mass of an Oligomeric Protein: Size Exclusion Chromatography (SEC).
9.3.5 Mass Determination: Mass Spectrometry (MS) (after Kellner, Lottspeich, Meyer).
9.3.6 Determination of Amino Acid Sequence by Tryptic Degradation, or Acid, Chemical or Enzymatic Digestion.
9.4 Enzymatic Mechanisms: General Acid–Base Catalysis.
9.4.1 Carbonic Anhydrase II.
9.4.2 Vanadium Haloperoxidase.
9.5 Nucleophilic Ca talysis.
9.5.1 Serine Proteases.
9.5.2 Cysteine in Nucleophilic Attack.
9.5.3 Lipase, Another Catalytic Triad Mechanism.
9.5.4 Metalloproteases.
9.6 Electrophilic catalysis.
9.6.1 Utilization of Metal Ions: ADH, a Different Catalytic Triad.
9.6.1.1 Catalytic Mechanism of Horse Liver Alcohol Dehydrogenase, a Medium–Chain Dehydrogenase.
9.6.1.2 Catalytic Reaction Mechanism of Drosophila ADH, a Short–Chain Dehydrogenase.
9.6.2 Formation of a Schiff Base, Part I: Acetoacetate Decarboxylase, Aldolase.
9.6.3 Formation of a Schiff Base with Pyridoxal Phosphate (PLP): Alanine Racemase, Amino Acid Transferase.
9.6.4 Utilization of Thiamine Pyrophosphate (TPP): Transketolase.
10 Protein Engineering.
10.1 Introduction: Elements of Protein Engineering.
10.2 Methods of Protein Engineering.
10.2.1 Fusion PCR.
10.2.2 Kunkel Method.
10.2.3 Site–Specific Mutagenesis Using the QuikChange Kit from Stratagene.
10.2.4 Combined Chain Reaction (CCR).
10.3 Glucose (Xylose) Isomerase (GI) and Glycoamylase: Enhancement of Thermostability.
10.3.1 Enhancement of Thermostability in Glucose Isomerase (GI).
10.3.2 Resolving the Reaction Mechanism of Glucose Isomerase (GI): Diffusion–Limited Glucose Isomerase?
10.4 Enhancement of Stability of Proteases against Oxidation and Thermal Deactivation.
10.4.1 Enhancement of Oxidation Stability of Subtilisin.
10.4.2 Thermostability of Subtilisin.
10.5 Creating New Enzymes with Protein Engineering.
10.5.1 Redesign of a Lactate Dehydrogenase.
10.5.2 Synthetic Peroxidases.
10.6 Dehydrogenases, Changing Cofactor Specificity.
10.7 Oxygenases.
10.8 Change of Enantioselectivity with Site–Specific Mutagenesis.
10.9 Techniques Bridging Different Protein Engineering Techniques.
10.9.1 Chemically Modified Mutants, a Marriage of Chemical Modification and Protein Engineering.
10.9.2 Expansion of Substrate Specificity with Protein Eng