合理设计来自Anabaena variabilis中的苯丙氨酸解氨酶AvPAL以从低成本的反式肉桂酸生L-苯丙氨酸和β-
发布时间：2021-05-12 13:06
酶在合成有机化学和生物技术的应用上通常受到以下限制:（1）、由于酶作用的底物范围比较窄,许多我们需要的化合物不能被酶催化;（2）、酶的活性高,但是立体选择性较差;（3）、酶在催化条件下不稳定;（4）、产物抑制使得转化率低。在近10-15年间,定向进化技术发展起来。定向进化,有时又被称为实验室进化（试管中的进化）,试图通过反复的基因突变、表达、筛选循环重复自然进化过程,直到筛选（或选择）到符合要求的生物催化剂。这一概念在一定程度上改变了传统采用“合理设计”构建定点突变的蛋白质工程方法。对来自鱼腥藻（Anabaena variabilis）中的苯丙氨酸解氨酶（AvPAL）进行合理设计,以低成本并且市场上可购买的反式肉桂酸（t-CA）为原料,大规模生产L-苯丙氨酸以及β-苯丙氨酸。通过计算机模拟（对接）的方法,得到两个突变体可以提高L-苯丙氨酸的产量,对突变体的氢键分析揭示了催化活性提高的原因。突变体所催化反应的反应速率,底物抑制以及动力学数据与野生型基本一致。根据我们的结果,Arg103突变体的酶活性相对于野生型AvPAL提高了 11倍。我们通过酶法,以低价的肉桂酸为原料,一锅法得到大量光...
【文章来源】：北京化工大学北京市 211工程院校 教育部直属院校
【文章页数】：89 页
【学位级别】：硕士
【文章目录】：
Abstract
摘要
Abbreviations list
Chapter 1 Introduction
1.1.Phenylalanine Ammonia Lyase
1.2.Discovery of phenylalanine ammonia-lyase enzyme
1.3.Sources of Phenylalanine Ammonia Lyase (PAL)
1.4.Structure of Phenylalanine Ammonia Lyase and Phenylalanine Aminomutases Enzymes
1.4.1.Determination the structure of PAM
1.4.1.1.PAM Monomer Structure
1.4.1.2.PAM Tetramer Structure
1.5.Application of Phenylalanine ammonia-lyase
1.6.Rational Designing of PAL
1.7.Our Aim and Objective
Chapter 2 The Production of L-Phenylalanine
2.1 Introduction of L-Phenylalanine and its importance
2.2 Methods used for the synthesis of L-phenylalanine
2.3 Materials and Method used for the Production of L-Phenylalanine
2.3.1.Media Used
2.3.1.1.LB (Luria-Bertani) Liquid Medium
2.3.1.2.LB (Luria-Bertani) Solid Medium
2.3.2.Chemicals
2.4.Strains and Plasmids
2.5.Induced Expression of Recombinant Protein AvPAL
2.5.1.The Pet-28A-Avpal Recombinant Plasmid Was Transferred Into Competent Cells
2.5.2.Inducing Expression of the Target Protein
2.5.3.Ultrasonic Disruption and SDS-PAGE Electrophoresis Protein Detection
2.6.Polyacrylamide Gel Electrophoresis
2.6.1.Stock Solutions
2.6.1.1.Acrylamide/Methylene-bis-acrylamide (30% Acr, 0.8% Bis)
2.6.1.2.Determining Gel Buffer Stock(1.50 M Tris-HCl pH 8.8):
2.6.1.3.Stacking Gel Buffer Stock (0.5 M Tris-HCl pH 6.8)
2.6.1.4.50%(v/v) Glycerol
2.6.1.5.10%(W/V) Sodium Dodecyl Sulfate
2.6.1.6.10% (w/v) Ammonium per Sulfate: (AP)
2.6.1.7.5x Protein Loading Buffer
2.6.1.8.Coomessive Brilliant Blue solution
2.6.1.9.Coomessive Brilliant Blue Decolonization Solution 1L
2.6.2.Separating and Stacking Gel Making Method
2.6.3.Staining and De staining
2.7.Expanding Culture and Induction
2.8.Ultrasonic Crushing
2.9.His-Tag Purification of Target Protein
2.10.Anabaena Variabilis PAL (AvPAL) Optimized DNA Sequence
2.11.Site-Directed Mutagenesis
2.12.PCR (Polymerase Chain Reaction) Magnification
2.12.1 Quick Change Pcr Setup;
2.12.2 Quick Change Pcr Cycling
2.13.Agarose Gel Electrophoresis
2.14.Digestion
2.15.Transformation
2.16.Phe79 and Arg103 Expression and Purification
2.17.L-Phenylalanine Production Reaction Condition
2.18.Enzyme Activity Assay
2.19.Methodology of Molecular Docking Studies
2.20.Results and Discussion of L-Phenylalanine
2.20.1.His-Tag Purification of the Target Protein
2.20.2.Study of Substrates As Well As Yields
2.20.3.Optimization of Bio-Reaction Conditions
2.20.3.1.The Influence of pH on ee and Conversion
2.20.3.2.Effect of Temperature over ee and Conversion
2.20.3.3.Influence of the Incubation Period on ee and Conversion
2.20.4.The Comparison of the Hydromanition Activity of AvPAL, Mutant Arg103 and79 Phe towards Cinnamic Acid for the Production of L-Phenylalanine
2.20.5.Docking Study
2.21.Conclusion
Chapter 3 Engineer the Phenylalanine Ammonia Lyase of AnabaenaVariabilis (AvPAL) for the Production of β-Phenylalanine from LowCost Trans-Cinnamic Acid
3.1.Introduction and Brief History of β-phenylalanine
3.2.Application of β-Phenylalanine
3.3.Synthetic Ways of β-Phenylalanine
3.4.Materials and Method used for the Production of b-Phenylalanine
3.4.1.Materials
3.4.2.Extraction of Plasmids and Genomes
3.4.2.1.Extraction of Plasmid
3.4.2.2 Extraction of Genomic DNA
3.4.3.Strains and Plasmids
3.4.4.Induced Expression and Purification of Recombinant Protein AvPAL
3.4.5.Bioinformatics Tools
3.4.6.Site-Directed Mutagenesis
3.4.7.PCR (Polymerase Chain Reaction) Amplification
3.4.7.1.PCR System
3.4.7.2.Setting PCR Machine Programmed
3.4.8.Agarose Gel electrophoresis
3.4.9.Val165Ser, Leu358 and Ile105 Expression and Purification
3.4.10.β-Phenylalanine Production Reaction Condition
3.4.11.Enzyme Assay
3.5.Result and Discussion of b-Phenylalanine
3.5.1.Sequence and Structure Alignment
3.5.1.1.Aligning of Sequence
3.5.1.2.Structure Alignment
3.5.2.His-Tag Purification of the Target Protein
3.5.3.Investigation of Substrates and Products
3.5.4.Optimizations of Bio Reaction Conditions
3.5.4.1.Effect of pH over ee and Conversion
3.5.4.2.Effect of Temperature over ee and Conversion
3.5.4.3.Effect of Incubation Period over ee and Conversion
3.5.5.Comparison of the Hydromanition Activity of Aminomutases and Ammonia Lyasetowards Cinnamic Acid for the Production of β-Phenylalanine
3.6 Conclusions
Chapter 4 Summary
4.1.Conclusion
4.2.Rational designing of AvPAL mutant for L-phenylalanine and β-PhenylalanineProduction
4.2.1.L-Phenylalanine Production
4.2.2.β-Phenylalanine Production
4.3.Expression and Protein Purification of AvPAL and their Mutant
4.4.Innovation Points
4.5.Outlook
Chapter 5 Reference
ACKNOWLEDGEMENTS
About the author and mentor
附件
本文编号：3183470
【文章来源】：北京化工大学北京市 211工程院校 教育部直属院校
【文章页数】：89 页
【学位级别】：硕士
【文章目录】：
Abstract
摘要
Abbreviations list
Chapter 1 Introduction
1.1.Phenylalanine Ammonia Lyase
1.2.Discovery of phenylalanine ammonia-lyase enzyme
1.3.Sources of Phenylalanine Ammonia Lyase (PAL)
1.4.Structure of Phenylalanine Ammonia Lyase and Phenylalanine Aminomutases Enzymes
1.4.1.Determination the structure of PAM
1.4.1.1.PAM Monomer Structure
1.4.1.2.PAM Tetramer Structure
1.5.Application of Phenylalanine ammonia-lyase
1.6.Rational Designing of PAL
1.7.Our Aim and Objective
Chapter 2 The Production of L-Phenylalanine
2.1 Introduction of L-Phenylalanine and its importance
2.2 Methods used for the synthesis of L-phenylalanine
2.3 Materials and Method used for the Production of L-Phenylalanine
2.3.1.Media Used
2.3.1.1.LB (Luria-Bertani) Liquid Medium
2.3.1.2.LB (Luria-Bertani) Solid Medium
2.3.2.Chemicals
2.4.Strains and Plasmids
2.5.Induced Expression of Recombinant Protein AvPAL
2.5.1.The Pet-28A-Avpal Recombinant Plasmid Was Transferred Into Competent Cells
2.5.2.Inducing Expression of the Target Protein
2.5.3.Ultrasonic Disruption and SDS-PAGE Electrophoresis Protein Detection
2.6.Polyacrylamide Gel Electrophoresis
2.6.1.Stock Solutions
2.6.1.1.Acrylamide/Methylene-bis-acrylamide (30% Acr, 0.8% Bis)
2.6.1.2.Determining Gel Buffer Stock(1.50 M Tris-HCl pH 8.8):
2.6.1.3.Stacking Gel Buffer Stock (0.5 M Tris-HCl pH 6.8)
2.6.1.4.50%(v/v) Glycerol
2.6.1.5.10%(W/V) Sodium Dodecyl Sulfate
2.6.1.6.10% (w/v) Ammonium per Sulfate: (AP)
2.6.1.7.5x Protein Loading Buffer
2.6.1.8.Coomessive Brilliant Blue solution
2.6.1.9.Coomessive Brilliant Blue Decolonization Solution 1L
2.6.2.Separating and Stacking Gel Making Method
2.6.3.Staining and De staining
2.7.Expanding Culture and Induction
2.8.Ultrasonic Crushing
2.9.His-Tag Purification of Target Protein
2.10.Anabaena Variabilis PAL (AvPAL) Optimized DNA Sequence
2.11.Site-Directed Mutagenesis
2.12.PCR (Polymerase Chain Reaction) Magnification
2.12.1 Quick Change Pcr Setup;
2.12.2 Quick Change Pcr Cycling
2.13.Agarose Gel Electrophoresis
2.14.Digestion
2.15.Transformation
2.16.Phe79 and Arg103 Expression and Purification
2.17.L-Phenylalanine Production Reaction Condition
2.18.Enzyme Activity Assay
2.19.Methodology of Molecular Docking Studies
2.20.Results and Discussion of L-Phenylalanine
2.20.1.His-Tag Purification of the Target Protein
2.20.2.Study of Substrates As Well As Yields
2.20.3.Optimization of Bio-Reaction Conditions
2.20.3.1.The Influence of pH on ee and Conversion
2.20.3.2.Effect of Temperature over ee and Conversion
2.20.3.3.Influence of the Incubation Period on ee and Conversion
2.20.4.The Comparison of the Hydromanition Activity of AvPAL, Mutant Arg103 and79 Phe towards Cinnamic Acid for the Production of L-Phenylalanine
2.20.5.Docking Study
2.21.Conclusion
Chapter 3 Engineer the Phenylalanine Ammonia Lyase of AnabaenaVariabilis (AvPAL) for the Production of β-Phenylalanine from LowCost Trans-Cinnamic Acid
3.1.Introduction and Brief History of β-phenylalanine
3.2.Application of β-Phenylalanine
3.3.Synthetic Ways of β-Phenylalanine
3.4.Materials and Method used for the Production of b-Phenylalanine
3.4.1.Materials
3.4.2.Extraction of Plasmids and Genomes
3.4.2.1.Extraction of Plasmid
3.4.2.2 Extraction of Genomic DNA
3.4.3.Strains and Plasmids
3.4.4.Induced Expression and Purification of Recombinant Protein AvPAL
3.4.5.Bioinformatics Tools
3.4.6.Site-Directed Mutagenesis
3.4.7.PCR (Polymerase Chain Reaction) Amplification
3.4.7.1.PCR System
3.4.7.2.Setting PCR Machine Programmed
3.4.8.Agarose Gel electrophoresis
3.4.9.Val165Ser, Leu358 and Ile105 Expression and Purification
3.4.10.β-Phenylalanine Production Reaction Condition
3.4.11.Enzyme Assay
3.5.Result and Discussion of b-Phenylalanine
3.5.1.Sequence and Structure Alignment
3.5.1.1.Aligning of Sequence
3.5.1.2.Structure Alignment
3.5.2.His-Tag Purification of the Target Protein
3.5.3.Investigation of Substrates and Products
3.5.4.Optimizations of Bio Reaction Conditions
3.5.4.1.Effect of pH over ee and Conversion
3.5.4.2.Effect of Temperature over ee and Conversion
3.5.4.3.Effect of Incubation Period over ee and Conversion
3.5.5.Comparison of the Hydromanition Activity of Aminomutases and Ammonia Lyasetowards Cinnamic Acid for the Production of β-Phenylalanine
3.6 Conclusions
Chapter 4 Summary
4.1.Conclusion
4.2.Rational designing of AvPAL mutant for L-phenylalanine and β-PhenylalanineProduction
4.2.1.L-Phenylalanine Production
4.2.2.β-Phenylalanine Production
4.3.Expression and Protein Purification of AvPAL and their Mutant
4.4.Innovation Points
4.5.Outlook
Chapter 5 Reference
ACKNOWLEDGEMENTS
About the author and mentor
附件
本文编号：3183470
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