化工进展 ›› 2018, Vol. 37 ›› Issue (05): 1933-1939.DOI: 10.16085/j.issn.1000-6613.2017-1287

• 生物与医药化工 • 上一篇    下一篇

环氧化物水解酶PvEH3的表达及手性邻二醇的合成

王瑞1, 许耀辉1, 王克伟2, 邬敏辰2   

  1. 1 江南大学生物工程学院, 江苏 无锡 214122;
    2 江南大学无锡医学院, 江苏 无锡 214122
  • 收稿日期:2017-06-02 修回日期:2017-07-27 出版日期:2018-05-05 发布日期:2018-05-05
  • 通讯作者: 邬敏辰,教授,博士生导师,主要从事酶工程与基因工程研究。
  • 作者简介:王瑞(1989-),女,硕士研究生。E-mail:Wangshen204823@163.com。
  • 基金资助:
    国家自然科学基金项目(21676117)。

Expression of PvEH3,a Phaseolus vulgaris epoxide hydrolase,and synthesis of chiral vicinal diols

WANG Rui1, XU Yaohui1, WANG Kewei2, WU Minchen2   

  1. 1 School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China;
    2 Wuxi Medical School, Jiangnan University, Wuxi 214122, Jiangsu, China
  • Received:2017-06-02 Revised:2017-07-27 Online:2018-05-05 Published:2018-05-05

摘要: 基于环氧化物水解酶(EHs)一级结构的计算机辅助分析,以菜豆(Phaseolus vulgaris)总RNA为模板,采用逆转录PCR和巢式PCR技术扩增了一种新型环氧化物水解酶(PvEH3)的编码基因pveh3。借助表达载体pET-28a(+)将pveh3导入大肠杆菌BL21(DE3)中进行了异源表达。一级和三级结构表明,PvEH3属于α/β水解酶超家族,其催化三联体为环氧化物水解酶Asp101-His297-Asp262的表达及手性邻二醇的合成,两个保守质子供体为Tyr150和Tyr232。在20℃、pH7.0的反应条件下,PvEH3能催化环氧苯乙烷及其4种衍生物的对映会聚水解。实验结果表明,PvEH3对环氧苯乙烷及其对位取代环氧底物(93.2%、86.6%和85.1% eep)的对映会聚性优于其间位取代环氧底物(37.1%和53.3% eep);PvEH3针对5种环氧底物中的环氧苯乙烷具有最高的区域选择性,区域选择性系数αSβR分别为93.9%和99.0%。PvEH3的发现增加了区域选择性高且互补的EHs数目,为单酶催化环氧化物的对映会聚水解提供了更多的选择余地。

关键词: 环氧化物水解酶, 生物催化, 表达, 区域选择性, 对映会聚水解

Abstract: Based on the computer-aided analysis of primary structures of epoxide hydrolases (EHs),a PvEH3-encoding gene,pveh3,was amplified from Phaseolus vulgaris total RNA by RT-PCR and Nested PCR technique.Mediated by expression vector pET-28a (+),pveh3 was heterologously expressed in E. coli BL21 (DE3).Primary and three-dimensional structures indicated that PvEH3 belonged to the α/β-hydrolase superfamily. Its catalytic triad is Asp101-His297-Asp262,and two conservative tyrosine residues served as proton donor are Tyr150 and Tyr232.The enantioconvergent hydrolysis of styrene oxide and its four derivatives were conducted by PvEH3 at a gentle reaction condition,20℃ and pH 7.0.The experimental results indicated that without substitution or at the para positions (93.2%,86.6% and 85.1% eep)of the aromatic ring had a better enantioconvergent than those at the meta position (37.1% and 53.3% eep).PvEH3 possesses the best regioselectivity towards styrene oxide within five substrates and the regioselectivity coefficients,αS and βR,were 93.9% and 99.0%,respectively.The discovery of PvEH3 increased the number of EHs possessing high and complementary regioselectivity,which offered more options for the enantioconvergent hydrolysisof racemic epoxides by single EHs.

Key words: epoxide hydrolase, biocatalysis, expression, regioselectivity, enantioconvergent hydrolysis

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