芽孢杆菌乙偶姻和丁二醇转化酶分析及2R,3R-BDH表征

发布时间：2021-10-17 20:19

　　乙偶姻和2,3-丁二醇（2,3-BD）是重要的大宗化学品,广泛应用于化工、食品,乳、制药工业;大多数是化学合成产品。微生物发酵糖质原料乙偶姻和2,3-丁二醇,不仅具有克服石油原料的短缺,可持续利用生物质资源,而且体现绿色制造的当今环境生态主题,是工业生物技术的前沿热点。产生乙偶姻和2,3-BD的微生物较多,芽孢杆菌是主要微生物资源。实验室前期研究证明Bacillus sp.DL01可高产乙偶姻。论文工作以Bacillus sp.DL01为对象,围绕乙偶姻和2,3-丁二醇转化的氧化还原酶的生物信息学分析及2R,3R-丁二醇的重组表达及酶学性质开展研究。论文利用Velacensis velezensis SRCM 101413基因组信息,设计乙偶姻和2,3-丁二醇转化的相关氧化还原酶基因引物,以Bacillus sp.DL01基因组为模板通过PCR克隆相关酶基因,然后连接到T-Vector转化大肠杆菌DH5α,获得重组质粒,测序获得2R,3R-BDH（1041 bp）,2S,3S-BDH（843 bp）,meso-BDH（747 bp）,醇脱氢酶（1047 bp）,甘油脱氢酶（1071 b... 

【文章来源】：大连理工大学辽宁省 211工程院校 985工程院校 教育部直属院校

【文章页数】：74 页

【学位级别】：硕士

【文章目录】：
摘要
abstract
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 of3R/3S-AC and2,3-BD
        1.1.6 Mechanism of3R/3S-AC and2,3-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(bud C)
        1.2.2 2R,3R-Butanediol dehydrogenase(R-BDH)
        1.2.3 Bacillus species producing3R/3S-AC and2,3-BD
        1.2.4 Bacillus sp.DL
        1.2.5 DA production from Bacillus sp.DL
        1.2.6 Charcterization od ALDC from Bacillus sp.DL
    1.3 Major content and methodology of dissertation
2 Bioinformatic analysis of enzymes responsible for 3R/3S-AC and 2,3-BD conversion
    2.1 Preface
    2.2 Materials 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 Software
    2.3 Results
        2.3.1 Cloning genes encoding enzymes for AC and BD conversion
        2.3.2 16S ribosomal DNA of Bacillus sp.DL
        2.3.3 2R,3R-Butanediol dehydrogenase(R-BDH)
        2.3.4 Meso-Butanediol dehydrogenase(bud C)
        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-BDHprotein
        3.2.7 Purification of R-BDH(AKTA)
        3.2.8 SDS-PAGE
        3.2.9 Enzyme activity of R-BDH
        3.2.10 Gas chromatography
        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 Kinetic parameters of R-BDH
        3.3.10 Gas chromatography
    3.4 Discussion
Conclusions
Prospection
Reference
Research Projects and Publications in Master Study
Acknoledgement



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