Introduction to Enzyme Technology (Learning Materials in Biosciences)

✍ Scribed by Karl-Erich Jaeger (editor), Andreas Liese (editor), Christoph Syldatk (editor)

• Publisher: Springer
• Year: 2024
• Tongue: English
• Leaves: 467
• Category: Library

✦ Synopsis

This interdisciplinary textbook provides an easy-to-understand and highly topical introduction to all the specialist areas of modern enzyme technology.

In the first part of this three-part textbook, the reader is introduced to the fundamentals of enzyme structure, reaction mechanisms, enzyme kinetics, enzyme modeling, and process control. In the second part, methods for finding, expressing, optimizing, purifying, immobilizing, and using enzymes in unusual reaction media are presented. In the third part, leading experts use examples to describe current applications of enzymes in the chemical and pharmaceutical industries, for biomass degradation, food production and processing, as additives in detergents and cleaning agents, for constructing biosensors, and as therapeutics.

Students of bachelor and master programs in biology, chemistry, biochemistry, and bioprocess engineering will gain up-to-date access to practical applications and developing industries. However, the fluent writing style makes the work suitable for all readers, who want to gain an easy-to-understand insight into the production and application of enzymes.

This book is a translation of an original German edition. The translation was done with the help of artificial intelligence (machine translation by the service DeepL.com). A subsequent human revision was done primarily in terms of content, so that the book will read stylistically differently from a conventional translation.

✦ Table of Contents

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 anIdeal´´ 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