乙偶姻和2,3-丁二醇（2,3-BD）是重要的大宗化學(xué)品,廣泛應(yīng)用于化工、食品,乳、制藥工業(yè);大多數(shù)是化學(xué)合成產(chǎn)品。微生物發(fā)酵糖質(zhì)原料乙偶姻和2,3-丁二醇,不僅具有克服石油原料的短缺,可持續(xù)利用生物質(zhì)資源,而且體現(xiàn)綠色制造的當(dāng)今環(huán)境生態(tài)主題,是工業(yè)生物技術(shù)的前沿?zé)狳c(diǎn)。產(chǎn)生乙偶姻和2,3-BD的微生物較多,芽孢桿菌是主要微生物資源。實(shí)驗(yàn)室前期研究證明Bacillus sp.DL01可高產(chǎn)乙偶姻。論文工作以Bacillus sp.DL01為對(duì)象,圍繞乙偶姻和2,3-丁二醇轉(zhuǎn)化的氧化還原酶的生物信息學(xué)分析及2R,3R-丁二醇的重組表達(dá)及酶學(xué)性質(zhì)開展研究。論文利用Velacensis velezensis SRCM 101413基因組信息,設(shè)計(jì)乙偶姻和2,3-丁二醇轉(zhuǎn)化的相關(guān)氧化還原酶基因引物,以Bacillus sp.DL01基因組為模板通過PCR克隆相關(guān)酶基因,然后連接到T-Vector轉(zhuǎn)化大腸桿菌DH5α,獲得重組質(zhì)粒,測(cè)序獲得2R,3R-BDH（1041 bp）,2S,3S-BDH（843 bp）,meso-BDH（747 bp）,醇脫氫酶（1047 bp）,甘油脫氫酶（1071 b... 

【文章來源】：大連理工大學(xué)遼寧省 211工程院校 985工程院校 教育部直屬院校

【文章頁數(shù)】：74 頁

【學(xué)位級(jí)別】：碩士

【文章目錄】：
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



本文編號(hào)：3442357