Industrial Starch Debranching Enzymes

Preface
Contents
Contributors
Chapter 1: An Overview on Starch Processing and Key Enzymes
1.1 Introduction
1.2 Enzymes Involved in Starch Conversion
1.2.1 Endo- and Exoamylases
1.2.1.1 α-Amylases
1.2.1.2 β-Amylases
1.2.1.3 Glucoamylases
1.2.1.4 α-Glucosidases
1.2.2 Debranching Enzymes
1.2.2.1 Pullulanases
1.2.2.2 Isoamylases
1.2.3 Transferase
1.2.3.1 Cyclodextrin Glucanotransferases (CGTAs, EC 2.4.1.19)
1.3 Conclusion and Future Perspectives
References
Chapter 2: Classification and Enzyme Properties of Starch Debranching Enzymes
2.1 Introduction
2.2 Classification of Microbial SDBEs
2.2.1 Glucoamylases
2.2.2 Pullulanases
2.2.3 Isoamylases
2.3 Sources and Biochemical Properties
2.3.1 Temperature Optimum and Thermostability
2.3.2 pH Optimum
2.3.3 Specific Activity
2.4 Commercial SDBEs
References
Chapter 3: Sequence, Structure, and Engineering of Microbial Starch Debranching Enzymes
3.1 Introduction
3.2 Sequence Classifications
3.3 Structural Features
3.4 Protein Engineering
References
Chapter 4: Production and the Applications in Preparation of Branched Sugar Products of Starch Debranching Enzymes
4.1 Introduction
4.2 Production of SDBEs
4.2.1 Heterologous Expression of SDBEs in E. coli Strains
4.2.2 Heterologous Expression of SDBEs in Bacillus Strains
4.2.3 Heterologous Expression of SDBEs in Yeast Strains
4.3 Conclusions
References
Chapter 5: Recombinant Expression of Starch Debranching Enzymes in Escherichia coli
5.1 Introduction
5.2 Effect of Fermentation Conditions on Soluble Expression of Recombinant Pullulanase
5.2.1 Recombinant B. deramificans Pullulanase Easily Formed Active Protein Aggregates
5.2.2 Effect of Induction Temperature on Fermentation of the Recombinant Strain
5.2.3 Effect of Inducer Concentration on Fermentation of the Recombinant Strain
5.2.4 Effect of Osmotic Pressure Regulator on Fermentation of Recombinant Strain
5.2.4.1 Effects of Osmotic Pressure Regulators on the Fermentation of the Recombinant Strain
5.2.4.2 Effects of Betaine Concentration and Addition Time on Fermentation of the Recombinant Strain
5.2.5 Optimization of High-Density Fermentation Conditions of the Recombinant Strain
5.2.5.1 Effect of Fermentation Temperature on High-Density Fermentation of the Recombinant Strain
5.2.5.2 Effect of Betaine on High-Density Fermentation of the Recombinant Strain
5.2.5.3 Comparison of Fermentation Parameters of Recombinant Strain in Shake Flask and 3-L Fermenter
5.3 Effect of N-Terminal Domain Excision on the Thermostability and Secretory Efficiency of Pullulanase
5.3.1 Construction, Recombinant Expression, and Purification of the N-Terminal Truncated Mutant of Pullulanase
5.3.1.1 Construction and Recombinant Expression of the N-Terminal Truncated Mutant
5.3.1.2 Isolation and Purification of the N-Terminal Truncated Mutant
5.3.2 Construction, Expression, and Purification of Superimposed Mutants
5.3.2.1 Construction of Superimposed Mutants
5.3.2.2 Isolation and Purification of Superimposed Mutants
5.3.3 Analysis of Enzymatic Properties of Mutants
5.3.3.1 Determination of Optimum pH for Mutants
5.3.3.2 Optimum Temperature and Temperature Stability
5.3.3.3 Determination of the Kinetic Parameters of Mutants
5.3.3.4 Substrate Specificity Analysis of Mutants
5.3.4 Application of Mutants in Starch Saccharification
5.4 Surfactant Promotes the Soluble Secretory Expression of Pullulanase in E. coli
5.4.1 Depolymerization of Pullulanase Active Aggregates by Surfactants
5.4.2 The Influence of Surfactant Species on the Growth and Enzyme Production of E. coli
5.4.3 Effect of Triton X-100 Concentration on Growth and Enzyme Production of E. coli
5.4.4 Effect of Triton X-100 Addition Time on the Growth and Enzyme Production of E. coli
5.4.5 Effect of Triton X-100 on High-Density Fermentation of E. coli in a 3-L Fermenter
5.5 Optimization of the Induction Method Combined with the Glycine Feeding Strategy to Promote the Extracellular Expression of...
5.5.1 Effect of Inducer Type and Concentration on Growth and Enzyme Production of E. coli
5.5.2 Effect of Glycine Concentration on Growth and Enzyme Production of E. coli
5.5.3 Effect of Lactose Flow Acceleration on Pullulanase Production by High-Density Fermentation of E. coli in a 3-L Fermenter
5.5.4 Effect of Induction Time on Pullulanase Production by High-Density Fermentation of E. coli in a 3-L Fermenter
5.5.5 Effect of Glycine Feeding Strategy on Pullulanase Production by High-Density Fermentation of E. coli in a 3-L Fermenter
5.5.6 ``Mixed Conformation´´ Model of Pullulanase Expression in E. coli
5.6 Recombinant Expression of Thermobifida fusca Isoamylase in E. coli BL21(DE3)
5.6.1 Cloning and Sequence Analysis of the T. fusca Isoamylase Gene
5.6.2 Construction of Recombinant Plasmid and Strain
5.6.3 Shake Flask Fermentation of the Recombinant Strain and Purification
5.6.4 High-Density Fermentation of the Recombinant Strain in a 3-L Fermenter
5.7 Recombinant Expression of T. Fusca Isoamylase in E. coli MDS42
5.7.1 Construction of the Recombinant Strain E. coli MDS42/Tfu_1891-pSX2
5.7.2 Expression of Recombinant Strain
5.7.3 Optimization of Fermentation Conditions for Recombinant Strain E. coli MDS42/Tfu_1891-pSX2 by Shake Flask Cultivations
5.7.3.1 Effect of Induction Temperature on the Growth and Enzyme Production of the Recombinant Strain
5.7.3.2 Effect of Inducer Type and Concentration on the Growth and Enzyme Production of the Recombinant Strain
5.7.4 Optimization of Fermentation Conditions for Recombinant Strain E. coli MDS42/Tfu_1891-pSX2 by 3-L Fermenter Cultivation
5.7.4.1 Effect of Induction Temperature on the Growth and Enzyme Production of the Recombinant Strain
5.7.4.2 Effect of Induction Time on the Growth and Enzyme Production of the Recombinant Strain
5.7.4.3 Effect of Inducer Concentration on the Growth and Enzyme Production of the Recombinant Strain
5.7.5 Comparison of Fermentation Parameters of Recombinant Isoamylase Production in Shake Flasks and 3-L Fermenters
References
Chapter 6: Production of Starch Debranching Enzymes in Bacillus Strains
6.1 Introduction
6.2 Recombinant Expression of Pullulanase in B. choshinensis
6.2.1 Construction of the Recombinant Strain B. choshinensis (pNCMO2/pulA-d2)
6.2.2 Optimization of Shake-Flask Cultivations
6.2.2.1 Original Fermentation Medium Optimization
6.2.2.2 Carbon Source Optimization
6.2.2.3 Nitrogen Source Optimization
6.2.2.4 Metal Ion Optimization
6.2.2.5 Key Factor Optimization Using Response Surface Methodology
6.2.2.6 Seed Growth Curve and Seed Cultivation Time Optimization
6.2.2.7 Inoculation Ratio Optimization
6.2.2.8 Original pH Optimization
6.2.2.9 Cultivation Temperature Optimization
6.2.3 Optimization of 3-L Fermenter Cultivations
6.2.3.1 Batch Fermentation in a 3-L Fermenter
6.2.3.2 Effect of pH on Fermentation of Recombinant B. choshinensis
6.2.3.3 Effect of Dissolved Oxygen on Fermentation of Recombinant B. choshinensis
6.2.3.4 Effect of Inorganic Nitrogen Source on Fermentation of Recombinant B. choshinensis
6.2.3.5 Effect of Beef Extract Concentration on Fermentation of Recombinant B. choshinensis
6.2.4 The Mechanism by which Magnesium Ions Promote the Expression of ``High Activity´´ Pullulanase in Recombinant B. choshine...
6.2.5 Magnesium Ions Promote the Expression of ``High Activity´´ Pullulanase in Recombinant B. choshinensis
6.2.5.1 Effect of Magnesium Ions on the Expression of Other Heterologous Proteins in B. choshinensis
6.2.5.2 Effect of Magnesium Ions on the Specific Activity and Aggregation of Pullulanase Pure Enzyme Expressed by B. choshinen...
6.2.5.3 Heat Treatment Distinguishes the Active and Inactive Forms of Pullulanase
6.2.5.4 Effect of Magnesium Ions on the Secondary Structure of Pullulanase Expressed by B. choshinensis
6.2.5.5 Effect of Magnesium Ions on the Cell Morphology of B. choshinensis
6.2.5.6 Effect of Magnesium Ion on Transcription and Protein Expression of pulA-d2 and HWP Genes
6.2.6 ``Folding Master´´ Mechanism of Pullulanase Expression in B. choshinensis
6.3 Recombinant Expression of Pullulanase in B. subtilis
6.3.1 Construction of CRISPR/Cas9 Gene Editing System Disruption Plasmid
6.3.2 Disruption of the srfC Gene by the CRISPR/Cas9 Gene Editing System
6.3.3 Disruption of the spoIIAC Gene by the CRISPR/Cas9 Gene Editing System
6.3.4 Disruption of nprE and aprE Genes by the CRISPR/Cas9 Gene Editing System
6.3.5 Disruption of the amyE Gene by the CRISPR/Cas9 Gene Editing System
6.3.6 Transformation of Screened Wild-Type B. subtilis
6.3.7 Improved Pullulanase Expression Through Promoter Optimization
6.3.7.1 The Expression of β-CGTase Was Enhanced by Single Promoter Optimization
6.3.7.2 The Expression of β-CGTase Was Enhanced by Dual-Promoter Optimization
6.3.7.3 Expression of Pullulanase and α-CGTase Using the Dual Promoter PHpaII-PamyQ
6.3.7.4 Fermentation Culture of Recombinant Strain WS5PUL in a 3-L Fermenter
Protease Disruption and Optimization of Pullulanase Fermentation Conditions
Knockout of Six Extracellular Proteases
Expression of Pullulanase in WS11
Fermentation of Recombinant Strain WS11PUL in a 3-L Fermenter
6.3.7.5 Optimization of Fermentation Conditions of Recombinant Strain WS11PUL
6.3.8 The Effect of Protease on the Expression of Pullulanase and the Optimization of Fermentation Feed Solution
6.3.8.1 Effect of Protease on the Expression of Pullulanase in Shake Flask Fermentation
6.3.8.2 Effect of Protease on the Expression of Pullulanase in a 3-L Fermenter
6.3.8.3 Purification of Pullulanase Samples, Determination of Kinetic Parameters and Aggregation State under Different Proteas...
6.3.8.4 Thermal Stability of Pullulanase Samples at Different Protease Concentrations
6.3.8.5 Improve the Activity of Pullulanase by Optimizing the 3-L Fermenter Feeding Solution
6.3.9 Increasing Cell Wall Negative Charge and Optimizing Signal Peptide to Improve the Expression of Pullulanase
6.3.9.1 Effect of Chaperone Protein on the Expression of Pullulanase in WS11
6.3.9.2 Effect of hrcA Gene Knockout on the Expression of Pullulanase
6.3.9.3 The Chaperone Gene Was Coexpressed on Plasmid
6.3.9.4 The Chaperone Protein Gene Was Expressed on the Pullulanase Expression Plasmid
6.3.9.5 Effect of Chaperone GrpE on the Expression of Pullulanase in WS4, WS5, WS9, and WS10
The Chaperone Protein Gene Was Expressed on the Pullulanase Expression Plasmid
6.3.9.6 The Effect of Chaperone Gene Integration into the Genome on the Expression of Pullulanase
6.3.10 Effect of dltB Gene Knockout on the Expression of Pullulanase
6.3.11 The Effect of lytC Gene Knockout on the Expression of Pullulanase
6.3.12 Effect of Signal Peptide on the Expression of Pullulanase
6.3.12.1 Screening of Signal Peptides
6.3.12.2 Expression of Different Signal Peptides in WS9D and WS9DL
6.3.13 Fermentation of Recombinant Strains WS9DPUL/ywtF and WS9DLPUL/ywtF in a 3-L Fermenter
References
Chapter 7: Applications of Starch Debranching Enzymes in Starch Processing
7.1 Introduction
7.2 Glucose and Oligosaccharides
7.2.1 Glucose and Malto-Oligosaccharides
7.2.1.1 Two-Step Liquefaction-Saccharification
7.2.1.2 One-Step Liquefaction-Saccharification
7.2.2 Trehalose
7.2.3 Isomalto-oligosaccharides
7.3 Cyclodextrins
7.4 Cycloamyloses
7.5 Isomalto/Malto-Polysaccharides
7.6 Slowly Digesting Starch and Resistant Starch
7.6.1 Production of RS and SDS by Debranching Enzyme Treatment
7.6.1.1 Pullulanases
7.6.1.2 Isoamylase
7.6.1.3 Amylopullulanase
7.6.2 Production of RS and SDS in Combination with Debranching Enzymes and Other Treatments
7.6.2.1 Dual Treatment by Amylase and Pullulanase
7.6.2.2 Dual Treatment by Pullulanase and Amylosucrase
7.6.2.3 Dual Treatment by Pullulanase and Ultrasound
7.7 Complex Starch
7.7.1 Production of Starch Complexes by Debranching Enzyme Treatment
7.7.2 Production of Starch Complexes in Combination with Debranching Enzymes and Other Treatments
References