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立体选择性芴酮还原菌株的筛选及其关键酶的分离

The Screening of a Stereoselective Reduction Strain of Fluorenone and the Isolation of Its Key Reductase

作者: 专业:生物化学与分子生物学 导师:杨青 年度:2010 学位:硕士  院校: 大连理工大学

Keywords

Biotransformation, Asymmetric reduction, Streptomyces sp, Carbonyl reductase, Fluorenol

        手性醇是合成医药品、农用化学品、食品和其它精细化学品的重要中间体和原料。羰基不对称还原是制备手性醇的重要途径。本研究筛选获得能够有效催化潜手性芴酮高立体选择性还原的链霉菌菌株,初步分离纯化获得其中起关键作用的羰基还原酶,为芴酮的不对称还原提供了新的生物催化剂来源及技术基础。本论文主要研究内容如下:(1)从天然土壤放线菌库中筛选获得能够有效催化2-氯芴酮还原的链霉菌菌株Streptomyces resistomycificus DUT002。菌株转化获得对映体纯的右旋2-氯芴醇,绝对构象为R型。(2)确定了链霉菌DUT002转化潜手性芴酮的特性。菌株驯化培养4代,还原能力提高到2.4倍。菌株的最适反应条件为30℃、pH 7.5。以10%(V/V)DMSO作为有机助溶剂,羰基还原活力提高43%。以5%(W/V)麦芽糖作为辅助底物,为体系提供氢供体和能量,促进还原型辅酶NAD(P)H再生,活力可提高30%。金属离子Al3+、Zn2+和阴离子NO3-、H2PO4-、HPO42-也能够有效提高反应活力。链霉菌DUT002的底物谱包括2-取代芴酮。取代基为F、Cl、Br、I、CH3时,产率分别是92%、91%、87%、81%和93%,e.e.值分别为>99%、100%、>99%、92%和74%。(3)对链霉菌DUT002羰基还原酶进行了初步的分离纯化。Tris缓冲液溶解的细胞裂解液具有芴酮还原活力,其活性组分为依赖于还原辅酶NADP(H)的蛋白质。通过(NH4)2SO4沉淀、Sephadex G-25脱盐、SOURCE 30Q阴离子交换层析、2’,5’-ADP-Sepharose 4B亲和分离和SDS聚丙烯酰胺凝胶电泳,获得分子量为31kDa的蛋白亚基,分离倍数为366倍。
    Chiral alcohols are important intermediates and raw materials of pharmaceuticals, agrochemicals, food products and other fine chemicals. Asymmetric reduction of carbonyl compounds is the efficient way to produce chiral alcohols. In this study, a Streptomyces strain with highly prochiral fluorenone reducing activity was obtained and a preliminary trial on isolation of the relevant reductase was carried out. This work may provide a new path for fluorenone asymmetric reduction.The main findings of this dissertation include:(1) Strain Streptomyces resistomycificus DUT002, which was screened from soil, showed efficient carbonyl reducing activity and could be used as a biocatalyst for the asymmetric reduction of 2-chloro-fluorenone. The product 2-chloro-fluorenol was enantiomerically pure. The optical rotation exhibited dextrorotation, and the absolute configuration was deduced to be R.(2) The characteristics of prochiral compounds reduction by the strain DUT002 were determined. The fourth generation of strain DUT002 by domestication increased the activity of reduction by 2.4 folds. The optimal temperature and pH was 30℃and pH 7.5, respectively. 10%(V/V) of organic co-solvent DMSO increased the carbonyl reductive capacity by 43%. 5%(W/V) of co-substrate maltose could be used as energy and hydrogen sources to up the reductive capacity by 30%. Metallic ions Al3+, Zn2+ and anions NO3-, H2PO4-, HPO42- increased the reaction yield rate. The substrate spectrum of strain DUT002 included 2-substituted-fluorenone. The strain could reduce 2-fluoro-, chloro-, bromo-, iodo-and methyl-fluorenone with yields of 92%,91%,87%,81%,93%and e.e. values of>99%,100%, >99%,92%,74%.(3) The carbonyl reductase from the strain DUT002 were isolated. The fluorenone reductive activity was in cell lysates dissolved with Tris buffer. The biocatalyst showed NADP(H)-dependent activity, and was isolated through ammonium sulfate recipitation, Sephadex G-25 desalting chromatography, SOURCE 30Q anion exchange chromatography,2’, 5’-ADP-Sepharose 4B isolation and SDS polyacrylamide gel electrophoresis. The isolated subunit was 31kDa accordind to SDS-PAGE analysis and purified by 336 folds based on activity measurement.
        

立体选择性芴酮还原菌株的筛选及其关键酶的分离

摘要4-5
Abstract5
引言10-11
1 文献综述11-30
    1.1 生物不对称还原法制备手性醇11-14
        1.1.1 手性醇的应用11-12
        1.1.2 手性醇的制备方法12-13
        1.1.3 生物法不对称还原制备手性醇13-14
    1.2 羰基还原酶14-25
        1.2.1 醛酮还原酶(AKR)15-20
        1.2.2 短链脱氢/还原酶(SDR)20-25
    1.3 手性芴醇的应用及合成25-28
        1.3.1 手性芴醇的应用25
        1.3.2 手性芴醇的合成25-28
    1.4 羰基还原酶的筛选和重组表达28
        1.4.1 生物催化剂筛选28
        1.4.2 重组表达28
    1.5 论文的研究内容及意义28-30
2 立体选择性芴酮还原菌株筛选及特性30-51
    2.1 引言30
    2.2 实验材料与器材30-31
        2.2.1 供试菌株30
        2.2.2 实验仪器30-31
        2.2.3 实验试剂31
    2.3 菌株筛选策略31-32
    2.4 实验方法32-38
        2.4.1 2-取代芴酮还原分析方法32-34
        2.4.2 数据计算34
        2.4.3 培养基配方34-35
        2.4.4 菌体培养和细胞收集35
        2.4.5 2-氯芴酮转化菌株的筛选35
        2.4.6 产物鉴定35
        2.4.7 2-氯芴酮高立体选择性还原菌株的筛选及鉴定35-36
        2.4.8 产物旋光度测定36
        2.4.9 菌株驯化36
        2.4.10 温度和pH对还原反应的影响36
        2.4.11 反应的时间、浓度依赖性36
        2.4.12 有机助溶剂对反应的影响36-37
        2.4.13 辅助底物对反应的影响37
        2.4.14 金属离子及阴离子对反应的影响37
        2.4.15 菌株转化底物谱扩展37-38
    2.5 实验结果38-49
        2.5.1 2-氯芴酮转化菌株的筛选38
        2.5.2 产物鉴定38-40
        2.5.3 2-氯芴酮高立体选择性还原菌株的筛选40-41
        2.5.4 产物旋光度测定41-42
        2.5.5 菌株还原底物2-氯芴酮能力驯化42
        2.5.6 温度和pH对还原反应的影响42-43
        2.5.7 反应的时间、浓度依赖性43-45
        2.5.8 有机助溶剂对反应的影响45
        2.5.9 辅助底物对反应的影响45-46
        2.5.10 金属离子及阴离子对反应的影响46-47
        2.5.11 菌株转化底物谱扩展47-49
    2.6 讨论49-50
    2.7 小结50-51
3 芴酮还原酶分离纯化51-66
    3.1 引言51
    3.2 实验材料与器材51-52
        3.2.1 供试菌株51-52
        3.2.2 实验仪器52
        3.2.3 实验试剂52
    3.3 实验方法52-57
        3.3.1 2-氯芴酮还原检测方法52
        3.3.2 数据计算52
        3.3.3 培养基配方52-53
        3.3.4 蛋白质浓度测定方法53-54
        3.3.5 蛋白溶液还原活力检测方法54
        3.3.6 SDS聚丙烯酰胺凝胶电泳方法54-55
        3.3.7 菌体培养和细胞收集55
        3.3.8 细胞破碎55
        3.3.9 发酵液、细胞裂解液的辅酶依赖性55
        3.3.10 (NH_4)_2SO_4沉淀蛋白55-56
        3.3.11 蛋白溶液Sephadex G-25脱盐56
        3.3.12 SOURCE 30Q阴离子交换层析56
        3.3.13 2’,5’-ADP-Sepharose 4B亲和分离56-57
    3.4 实验结果57-65
        3.4.1 发酵液、细胞裂解液的辅酶依赖性57-58
        3.4.2 (NH_4)_2SO_4沉淀58-60
        3.4.3 Sephadex G-25脱盐60-61
        3.4.4 SOURCE 30Q阴离子交换层析61-63
        3.4.5 2’,5’-ADP-Sepharose 4B亲和分离63-65
    3.5 讨论65
    3.6 小结65-66
结论66-67
参考文献67-73
附录A 专有名词缩写73-75
附录B 常用仪器一览表75-77
附录C 常用试剂一览表77-80
附录D HPLC谱图及标准曲线80-88
附录E 产物鉴定88-96
攻读硕士学位期间发表学术论文情况96-97
致谢97-99
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