Introduction to Enzyme Technology (Learning Materials in Biosciences)

Contents
1: Introduction to Enzyme Technology
	What You Will Learn in This Chapter
	1.1 The Field of Enzyme Technology
	1.2 The Development of Enzyme Research
	1.3 Modern Enzyme Research
	1.4 Enzymes as Biocatalysts
	1.5 Industrial Applications of Enzymes
		1.5.1 Enzymes in the Food, Feed, and Textile Industries
		1.5.2 Enzymes in the Chemical and Pharmaceutical Industry
			Take-Home Message
	References
Part I: Fundamentals
	2: Enzyme Structure and Function
		What You Will Learn in This Chapter
		2.1 Structure of Enzymes
			2.1.1 General Architecture
			2.1.2 Primary, Secondary, Tertiary, and Quaternary Structure
			2.1.3 Posttranslational Modifications
			2.1.4 Cofactors
		2.2 Function of Enzymes
			2.2.1 Enzymes as Catalysts
			2.2.2 Active Site
			2.2.3 Activation Energy
			2.2.4 Catalytic Mechanisms
				2.2.4.1 Acid-Base Catalysis
				2.2.4.2 Covalent Catalysis
				2.2.4.3 Metal Ion Catalysis
				2.2.4.4 Proximity and Orientation Effects
				2.2.4.5 Transition State Binding
					Take-Home Message
		References
	3: Enzyme Modeling: From the Sequence to the Substrate Complex
		What Will You Learn in This Chapter?
		3.1 Investigation of the Protein Sequence Space
			3.1.1 Domain Structure of Proteins
			3.1.2 Search for Homologous Proteins
			3.1.3 Clustering of Protein Families
			3.1.4 Outlook: Integration of Sequence Data and Biochemical Data
		3.2 Structural Modeling
			3.2.1 Search for a Suitable Template
			3.2.2 Creation of a Target-Template Alignment
			3.2.3 Creation of a Structural Model
			3.2.4 Sources of Error and Estimation of Model Quality
			3.2.5 Fragment Assembly Methods
			3.2.6 Outlook: Modeling by Molecular Dynamics Simulation
		3.3 Molecular Docking
			3.3.1 Sampling
			3.3.2 Scoring
			3.3.3 Limitations
			3.3.4 Modeling of Protein Complexes
			3.3.5 Outlook: Direct Simulation of Substrate Binding
		3.4 Mechanistic Models of Protein Structure and Dynamics
			3.4.1 Modeling of Conformational Changes
			3.4.2 Modeling of the Biochemical Reaction Mechanism
			3.4.3 Thermodynamic Calculations
		3.5 Outlook: Integration of Mechanistic and Kinetic Modeling
			Take-Home Message
		References
	4: Enzyme Kinetics
		What Will You Learn in This Chapter?
		4.1 Basic Concepts of Chemical Catalysis
		4.2 Homogeneous, Heterogeneous, and Enzymatic Catalysis: Differences and Similarities
		4.3 Enzyme Kinetic Models
			4.3.1 One-Substrate Reactions
			4.3.2 Two-Substrate Reactions
		4.4 Determination of the Kinetic Parameters
			4.4.1 Measurement of the Initial Reaction Rate
			4.4.2 Progress Curve Analysis
			4.4.3 Determination of the Catalytic Constants
		4.5 Enzyme Inhibition
			4.5.1 Competitive Inhibition
			4.5.2 Uncompetitive Inhibition
			4.5.3 Noncompetitive Inhibition
				Take-Home Message
		References
			Further Reading
	5: Enzyme Reactors and Process Control
		What You Will Learn in This Chapter?
		5.1 Parameters for the Description of Ideal Reactors
		5.2 Types of Ideal Enzyme Reactors
		5.3 Mathematical Balancing of Ideal Reactors
			5.3.1 Balancing of an Ideally Mixed Batch Reactor (STR)
			5.3.2 Balancing of a Continuously Operated Ideal Flow Tube Reactor (PFR)
			5.3.3 Balancing of an Ideally Mixed, Continuously Operated Stirred Tank Reactor (CSTR)
			5.3.4 Balancing Under Consideration of Inhibition Phenomena
		5.4 Reactor Selection and Process Control
			5.4.1 Reactor Selection Without Consideration of Inhibition Phenomena
			5.4.2 Reactor Selection for Substrate Surplus Inhibition
			5.4.3 Reactor Selection for Product Inhibition
			5.4.4 Reactor Selection for Parallel Reactions of the Substrate and Subsequent Reactions of the Product
		5.5 Examples of Industrial Enzymatic Processes
			5.5.1 Synthesis of Sitagliptin
			5.5.2 Production of High Fructose Corn Syrup (HFCS)
			5.5.3 Synthesis of Fatty Acid Esters
				Take-Home Messages
		References
Part II: Methods
	6: Enzyme Identification and Screening: Activity-Based Methods
		What You Will Learn in This Chapter
		6.1 Introduction
		6.2 The ``Ideal´´ Enzyme
		6.3 Resources for Enzyme Identification
		6.4 Activity-Based Identification of an ``Ideal´´ Enzyme
			6.4.1 Functional Gene Expression as a Prerequisite for Activity-Based Screening
			6.4.2 Methods for Activity-Based Screening
				6.4.2.1 Fluorescence-Activated Cell Sorting
				6.4.2.2 Complementation and Selection
				6.4.2.3 Determination of Activity on Agar Plates
				6.4.2.4 Determination of Activity in Liquid Assays
				6.4.2.5 Chromatographic Methods
		6.5 Conclusion and Challenges
			Take-Home Message
		References
	7: Bioinformatic Methods for Enzyme Identification
		What You Will Learn in This Chapter
		7.1 From Gene Sequence to Enzyme Function
			7.1.1 Sequence Analysis and Gene Identification
		7.2 Homology Search
			7.2.1 BLAST
		7.3 Multiple Protein Sequence Alignment
			7.3.1 Selection of a Suitable Alignment Program
		7.4 Phylogenetic Analyses
		7.5 Example: Identification of New Halohydrin Dehalogenases
			7.5.1 MSA for the Identification of Specific Sequence Motifs
			7.5.2 Homology Search in Public Databases
			7.5.3 Verification of Correct Gene Annotation
			7.5.4 Phylogenetic Analysis of New Halohydrin Dehalogenases
				Take-Home Message
		References
	8: Optimization of Enzymes
		What You Will Learn in This Chapter
		8.1 Strategies for the Optimization of Enzymes
			8.1.1 Rational Protein Design
			8.1.2 Directed Evolution
			8.1.3 Semi-rational Design
		8.2 Methods for Generating a Mutant Library
			8.2.1 Site-Specific Saturation Mutagenesis
			8.2.2 Iterative Saturation Mutagenesis (ISM)
			8.2.3 ProSAR
			8.2.4 Error-Prone PCR
			8.2.5 DNA Shuffling
			8.2.6 In Vivo Mutagenesis
		8.3 Screening and Selection
		8.4 Successful Examples of Optimized Biocatalysts
			Take-Home Message
		References
	9: Enzyme Production
		What You Will Learn in This Chapter
		9.1 Choice of Host Organism
			9.1.1 Homologous or Heterologous Protein Production
			9.1.2 Limiting Factors in Protein Biosynthesis
		9.2 Production in Eukaryotes or Prokaryotes
			9.2.1 Prokaryotes
				9.2.1.1 Escherichia coli
				9.2.1.2 Bacillus Species
				9.2.1.3 Alternative Prokaryotic Hosts
			9.2.2 Eukaryotes
				9.2.2.1 Yeasts
				9.2.2.2 Filamentous Fungi
				9.2.2.3 Other Eukaryotic Hosts
			9.2.3 Cell-Free Production Systems
		9.3 Choice of Expression and Regulatory System
			9.3.1 Episomal or Chromosomal Recombinant Genes
			9.3.2 Stable Replicating Plasmids Require Selection Pressure
		9.4 Protein Production Can Be Modified and Optimized at any Level of Protein Biosynthesis
			9.4.1 Constitutive Promoters
			9.4.2 Controllable Promoters
				9.4.2.1 The Lac Promoter
				9.4.2.2 The T7 Expression System
				9.4.2.3 Other Inducible Promoters
			9.4.3 Modifications at Gene and Protein Level
				9.4.3.1 Adaptation of a Recombinant Gene to the Host Organism
				9.4.3.2 Secretion of a Recombinant Enzyme
				9.4.3.3 Toxicity and Solubility of a Recombinant Enzyme
					Take-Home Message
		References
	10: Enzyme Purification
		What You Will Learn in This Chapter
		10.1 Introduction
		10.2 Parameters
		10.3 Process Development
			10.3.1 Technical Enzymes
			10.3.2 Diagnostic Enzymes
			10.3.3 Therapeutic Enzymes
				10.3.3.1 Cell Harvesting or Cell Separation and Solid/Liquid Separation
				10.3.3.2 Precipitation Steps
				10.3.3.3 Product Isolation and Concentration
				10.3.3.4 Further Chromatography Steps and Affinity Chromatography
				10.3.3.5 Affinity Chromatography
				10.3.3.6 Virus Removal
				10.3.3.7 Formulation and Freeze-Drying
				10.3.3.8 Requirements for Purified Therapeutic Enzymes
			10.3.4 Application Example: Industrial Purification of Factor VII
				Take Home Message
		References
	11: Enzyme Immobilization
		What You Will Learn in This Chapter
		11.1 Importance, Definition and Goals
		11.2 Methodological Principles
			11.2.1 Noncovalent Bonding to Support Materials
				11.2.1.1 Novozym 435
			11.2.2 Covalent Bonding to Carrier Materials
				11.2.2.1 Plexazyme AC
			11.2.3 Entrapment in Polymer Matrices
				11.2.3.1 Lentikats: β-Galactosidase
			11.2.4 Inclusion in Membranes
				11.2.4.1 Amicon: Stirring Cell
			11.2.5 Carrier-Free Cross-Linking
				11.2.5.1 CLEA 301: Penicillin Acylase
		11.3 Molecular and Physicochemical Effects
			11.3.1 Molecular Modification
			11.3.2 Limitations of Mass Transfer
		11.4 Performance Characteristics
			11.4.1 Characteristics of the Immobilization Process
			11.4.2 Characteristics of Immobilizates
		11.5 Choice of Methods
			Take Home Message
		References
	12: Enzymatic Reactions in Unusual Reaction Media
		What You Will Learn in This Chapter
		12.1 Introduction
		12.2 Enzymatic Reactions Using Organic Solvents
			12.2.1 Classification of Organic Solvents
			12.2.2 Effects of Organic Solvents on Enzyme Activity
			12.2.3 Enzymatic Reactions with Addition of Water-Miscible Solvents
			12.2.4 Enzymatic Reactions in Two-Phase Systems with Little or Non-Water-Miscible Solvents
			12.2.5 Enzymatic Reactions in Almost Anhydrous Organic Solvents
			12.2.6 Basic Rules for Working with Enzymes in Almost Anhydrous Organic Solvents
		12.3 Ionic Liquids, Deep Eutectic Solutions, and Supercritical Fluids as Reaction Media for Enzymatic Reactions
			12.3.1 Ionic Liquids
			12.3.2 Enzymatic Reactions in Ionic Liquids
			12.3.3 Deep Eutectic Solutions
			12.3.4 Enzymatic Reactions in Deep Eutectic Solutions
			12.3.5 Supercritical Fluids
			12.3.6 Enzymatic Reactions in Supercritical Fluids
		12.4 Enzymatic Reactions Under Complete Solvent-Free Conditions
			Take Home Message
		References
Part III: Applications
	13: Principles of Applied Biocatalysis
		What You Will Learn from This Chapter
		13.1 Cofactor-Dependent Biotransformations
			13.1.1 Substrate-Coupled Systems
			13.1.2 Enzyme-Coupled Systems
			13.1.3 Self-Sufficient Cascades
		13.2 Approaches to Substrate Dosing
			13.2.1 Fed-Batch Processes
			13.2.2 Use of Adsorber Materials
			13.2.3 Multiphasic Systems
			13.2.4 Substrate Supply from the Gas Phase
		13.3 Approaches to Product Removal
			13.3.1 Stripping of By-Products
			13.3.2 Multiphasic Systems
			13.3.3 Direct Crystallization of the Product
			13.3.4 Membrane Process
			13.3.5 Use of Ion Exchange Resins
		13.4 Approaches to Deracemization
		13.5 Conclusion and Prospects
			Take Home Message
		References
	14: Enzymes in the Chemical and Pharmaceutical Industry
		What You Will Learn in This Chapter
		14.1 Origins of Enzyme Use in the Chemical and Pharmaceutical Industry
		14.2 Enzymes in the Chemical Industry
			14.2.1 Lipase (EC 3.1.1.3)
				14.2.1.1 Preparation of (R)-Phenylethylamine
			14.2.2 d-Hydantoinase (EC 3.5.2.2)
				14.2.2.1 Preparation of D-p-Hydroxyphenylglycine
			14.2.3 Nitrile Hydratase (EC 4.2.1.84)
				14.2.3.1 Production of Acrylamide
				14.2.3.2 Preparation of Nicotinamide
			14.2.4 Alkane Monooxygenase (EC 1.14.15.3) and ω-Transaminase (EC 2.6.1.62)-Based Multienzyme Process
				14.2.4.1 Preparation of ω-Amino Lauric Acid
		14.3 Enzymes in the Pharmaceutical Industry
			14.3.1 Enzymes for the Synthesis of Precursors and Building Blocks
				14.3.1.1 Preparation of Hydroxynitrile
				14.3.1.2 Preparation of Cipargamine
				14.3.1.3 Production of Antibiotics
			14.3.2 Enzymes in the Synthesis of APIs
				14.3.2.1 Preparation of Sitagliptin Phosphate
			14.3.3 Enzymes for Drug Metabolism Studies
				14.3.3.1 Isoenzymes
				14.3.3.2 Inhibition
				14.3.3.3 Metabolite Identification
				14.3.3.4 Prodrugs
			14.3.4 Enzymes in the Synthesis of Natural Products
				14.3.4.1 Preparation of Artemisinin
				14.3.4.2 Enzymes for the Modification of Complex Structures
			14.3.5 Enzymes as Drugs
		14.4 Conclusion
			Take Home Message
		References
	15: Enzymes for the Degradation of Biomass
		What You Will Learn in This Chapter
		15.1 Composition of Biomass
			15.1.1 Cellulose
			15.1.2 Hemicellulose
			15.1.3 Pectin
			15.1.4 Lignin
		15.2 Cellulases
			15.2.1 Cellobiohydrolases (CBHs)
			15.2.2 Endoglucanases (EGs)
			15.2.3 β-Glucosidases (BG)
			15.2.4 Polysaccharide Monooxygenases (PMOs)
		15.3 Additional Enzymes for Biomass Degradation
			15.3.1 Hemicellulases
			15.3.2 Pectinases
			15.3.3 Ligninases
		15.4 Pretreatment of Biomass
			15.4.1 Physical Pretreatment
			15.4.2 Chemical Pretreatment
			15.4.3 Biological Pretreatment Methods
		15.5 Process Overview
			15.5.1 Biomass Cultivation
			15.5.2 Transport
			15.5.3 Pretreatment
			15.5.4 Enzymatic Hydrolysis
			15.5.5 Ethanol Production
				Take Home Message
		References
	16: Enzymes in Food Production
		What You Will Learn in This Chapter
		16.1 Determining Factors of Enzymes in the Food Industry
			16.1.1 Endogenous and Exogenous Enzymes in Foodstuffs
			16.1.2 Requirements for the Use of Exogenous Enzymes in Food Processing
			16.1.3 Recombinant Production of Enzymes for the Food Industry
		16.2 Oxidoreductases
			16.2.1 Importance to the Food Industry
			16.2.2 Applications in the Food Industry
		16.3 Transferases
			16.3.1 Importance to the Food Industry
			16.3.2 Applications in the Food Industry
		16.4 Hydrolases
			16.4.1 Importance to the Food Industry
			16.4.2 Lipases/Esterases: Applications in the Food Industry
			16.4.3 Glycosidases: Applications in the Food Industry
			16.4.4 Proteases/Peptidases: Applications in the Food Industry
		16.5 Lyases
			16.5.1 Importance to the Food Industry
			16.5.2 Areas of Application in the Food Industry
		16.6 Isomerases
			16.6.1 Importance to the Food Industry
			16.6.2 Applications in the Food Industry
		16.7 Ligases
			16.7.1 Importance to the Food Industry
			16.7.2 Application in the Food Industry
		16.8 Conclusion
		Further Reading
			References
	17: Enzymes in Detergents and Cleaning Agents
		What Do You Learn in This Chapter?
		17.1 History of Detergent Enzymes
		17.2 Production of the Enzymes
		17.3 Production of Detergents and Cleaning Agents Containing Enzymes
			17.3.1 Manual Dishwashing Detergents
		17.4 Proteases
			17.4.1 Molecules
			17.4.2 Enzyme Action
			17.4.3 Stability, Toxicity, and Environmental Aspects of Proteases
		17.5 Amylases
			17.5.1 Molecules
			17.5.2 Enzyme Action
		17.6 Cellulases
			17.6.1 Molecules
			17.6.2 Enzyme Action
		17.7 Mannanases
			17.7.1 Molecules
			17.7.2 Enzyme Action
		17.8 Lipases
			17.8.1 Molecules
			17.8.2 Enzyme Action
		17.9 Pectate Lyases
			17.9.1 Molecules
			17.9.2 Enzyme Action
		17.10 Sustainability Aspects
		17.11 Conclusion and Outlook
			Take Home Message
		References
	18: Enzymes and Biosensor Technology
		What You Will Learn in This Chapter
		18.1 Chemo- and Biosensors
			18.1.1 Definition: Chemo/Biosensor
			18.1.2 Classification of Biosensors
			18.1.3 Sensor Parameters
		18.2 Electrochemical Enzyme Biosensors
			18.2.1 Potentiometry
				18.2.1.1 Definitions
				18.2.1.2 Potentiometric Enzyme Electrodes
			18.2.2 Amperometry
				18.2.2.1 Definitions
				18.2.2.2 Amperometric Enzyme Electrodes
			18.2.3 Application Examples
		18.3 Looking Beyond the ``End of One´s Nose´´: Alternative Transductor Principles and Biomolecules
			18.3.1 Optical Biosensors
			18.3.2 Mass-Sensitive Biosensors
		18.4 Conclusion and Outlook
			18.4.1 Microsystems
			18.4.2 Molecular Gates with Enzyme Sensors
				Take-Home Message
		References
	19: Therapeutic Enzymes
		What You Will Learn in This Chapter
		19.1 External Applications of Therapeutic Enzymes
		19.2 Oral Applications of Enzymes
		19.3 Intravenous Use of Therapeutic Enzymes
			19.3.1 Urokinase
			19.3.2 Thrombin
			19.3.3 Asparaginase and Pegasparagase
			19.3.4 Therapeutic Enzymes for the Treatment of Rare Diseases
			19.3.5 The PEGylation of Proteins
		19.4 Conclusion and Outlook
			Take Home Message
		References
	20: Enzymes in Molecular Biotechnology
		What You Will Learn in This Chapter
		20.1 Nucleic Acid Synthesis
			20.1.1 DNA Polymerases
			20.1.2 RNA Polymerases
			20.1.3 Reverse Transcriptase
		20.2 Nucleic Acid-Cutting Enzymes
			20.2.1 Deoxyribonucleases
				20.2.1.1 DNA-Specific Endonuclease
					Deoxyribonucleases I
				20.2.1.2 Structure-Specific DNA Endonucleases
					Apurinic/Apyrimidinic (AP) Endonuclease 1, APE1
					Endonuclease IV, Endo IV
					Endonuclease V, Endo V
					Phage T7 Endonuclease I, T7EI
				20.2.1.3 Restriction Endonucleases, REases
					Naming REases
					Classification of REase
				20.2.1.4 Sugar-Nonspecific Nucleases
					Benzonase
					Mung Bean Nuclease
					P1 Nuclease
					S1 Nuclease
					Micrococcal Nuclease, MNase
				20.2.1.5 Exonucleases
					Exonuclease I (EXOI)
					Exonuclease III, (ExoIII)
					Exonuclease VII (EXOVII)
					Lambda Exonuclease (λ Exo)
					Bal 31 Nucleases
			20.2.2 Ribonucleases
				20.2.2.1 Endoribonuclease
					RNase A
					RNase H
		20.3 Programmable Nucleases
		20.4 DNA/RNA Modifying Enzymes
			20.4.1 Ligases
			20.4.2 Phosphatases
			20.4.3 Kinases
			20.4.4 Terminal Transferases
			20.4.5 Methyltransferase
			20.4.6 Capping Enzyme
			20.4.7 Glycosylases
			20.4.8 Cre-Recombinase
			20.4.9 Single-Strand Binding Proteins
			20.4.10 RecA-Type Proteins
			20.4.11 Single-Strand Binding Proteins
			20.4.12 DNA Helicases
		20.5 Questions
			Take Home Message/Conclusion
		References
			Further Reading-Books
			Further Reading-Online Resources
			Specific References
				To Polymerases in General
				To DNA Polymerases for PCR
				To DNA Polymerases for Sequencing
				To Non-coding RNA
				To Reverse Transcription and Retrotransposons
				To Nucleases
				To Programmable Nucleases
				To Capping Enzyme
				To Cre-Recombinase
				To Helicase
Index