声明
Chapter 1 Biochemical investigation of BkToa
1.1 Introduction
1.1.1 Taurine in the human body and its physiological functions
1.1.2 The biosynthesis of taurine in the human body and taurine as
1.1.3 Microbial Taurine metabolism
1.1.4 A key enzyme in taurine metabolism:taurine aminotransferase
1.2Materials
1.2.1 Strains and plasmids
1.2.2 Media and buffers
1.2.3 Important reagents and kits
1.2.4 Equipment
1.3 Methods
1.3.1 Preparation of competent cells
1.3.2 Plasmid construction
1.3.3 Protein expression and purification
1.3.4 Determination of the oligomeric state
1.3.5 Fluorescence-Based Thermal Shift Assay
1.3.6 IsfD-coupled activity assay for BkToa
1.3.7 Glutamate dehydrogenase (GLDH)/alanine dehydrogenase (ALD)-coupled activity assay for BkToa
1.3.8 LC-MS detection of sulfoacetaldehyde formation
1.4 Results and Discussion
1.4.1 Characterization of purified BkToa
1.4.2 BkToa is taurine:2-oxoglutarate aminotransferase
Chapter 2 Structural investigation of BkToa
2.1 Introduction
2.1.1 Classification of transaminases
2.1.2 The features of class III aminotransferases (AT-II)
2.2 Materials
2.2.1 Plasmids
2.2.2 Primers
2.2.3 Important kits
2.3 Methods
2.3.1 Crystallization, data collection and structure determination
2.3.2 Sequence alignment and structure comparison
2.3.3 Docking of the taurine-PLP aldimine
2.3.4 Phylogenetic tree construction
2.3.5 Site-directed mutagenesis
2.3.6 Protein expression and purification of BkToa mutants
2.3.7 IsfD-coupled activity assay of BkToa mutants
2.4 Results and Discussion
2.4.1 Crystal structure of BkToa in complex with PLP and glutamate
2.4.2 Phylogenetic analysis of BkToa
2.4.3 Analysis of the substrate-specificity BkToa
Chapter 3 Conclusions and Prospects
参考文献
Appendices
Notes on publications and participation in scientific research
致谢
天津大学;
