声明
1 Introduction
1.1 Research background and significance
1.1.1 3R/3S-Acetoin
1.1.2 3R/3S-Acetoin production
1.1.3 2,3-Butanediol
1.1.4 2,3-Butanediol usage
1.1.5 Economical value of 3R/3S-AC and 2,3-BD
1.1.6 Mechanism of 3R/3S-AC and 2,3-BD production
1.1.7 Butanediol dehydrogenase group enzymes
1.1.8 Mechanism of production from microorganisms
1.2 Domestic progress and overseas progress
1.2.1 meso-Butanediol dehydrogenase (budC)
1.2.2 (2R,3R)-Butanediol dehydrogenase (R-BDH)
1.2.3 Bacillus species Producing 3R/3S-AC and 2,3-BD
1.2.4 Bacillus sp. DL01
1.2.5 DA production from Bacillus sp. DL01
1.2.6 Characterization of ALDC from Bacillus sp. DL01
1.3 Major content and methodology of the Dissertation.
2 Bioinformatic analysis of enzymes responsible for 3R/3S-AC and 2,3-BD conversion
2.1 Preface
2.2 Material and methods
2.2.1 Strains, plasmids, enzymes, and culture conditions
2.2.2 Primers design and PCR conditions
2.2.3 Competent cell
2.2.4 Vector ligation
2.2.5 Transformation
2.2.6 T vector sequencing
2.2.7 Gene cloning
2.2.8 Softwares
2.3 Results
2.3.1 Cloning genes encoding enzymes for AC and BD conversion
2.3.2 16S Ribosomal DNA of Bacillus sp. DL01
2.3.3 2R,3R-Butanediol dehydrogenase (R-BDH)
2.3.4 Meso-Butanediol dehydrogenase (budC)
2.3.5 2S,3S-Butanediol dehydrogenase (S-BDH)
2.3.6 Alcohol dehydrogenase
2.3.7 Glycerol dehydrogenase (gdh)
2.4 Discussion
3 Recombinant expression and characterization of 2R,3R-Butanediol dehydrogenase
3.1 Preface
3.2 Material and methods
3.2.1 Strains, plasmids, primers and culture conditions
3.2.2 Total crude lysate preparation
3.2.3 Crude lysate activity of Bacillus sp. DL01
3.2.4 Bradford protein assay
3.2.5 Construction of vector for expression R-bdh from Bacillus sp. DL01
3.2.6 Recombinant expression of R-BDH protein
3.2.7 Purification of R-BDH (AKTA)
3.2.8 SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide agarose gel electrophoresis)
4.2.9 Enzyme activity of R-BDH
3.2.10 Gas chromatography (GC)
3.2.11 Bioinformatic tools
3.3 Results
3.3.1 Total crude lysate activity
3.3.2 Isolation of R-bdh
3.3.3 3D modelling of R-BDH
3.3.4 Cloning of R-BDH
3.3.5 Expression and purification of R-BDH
3.3.6 Effect of temperature on R-BDH activity
3.3.7 Effect of pH on R-BDH activity
3.3.8 Effect of metal ions on R-BDH activity
3.3.9 The kinetic parameters of R-BDH
3.3.10 Gas chromatography
3.4 Discussion
Conclusions
Prospection
参考文献
Research Projects and Publications in Master Study
致谢
大连理工大学;
