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

doi:10.16085/j.issn.1000-6613.2017-1287

Abstract

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% ee_p)of the aromatic ring had a better enantioconvergent than those at the meta position (37.1% and 53.3% ee_p).PvEH3 possesses the best regioselectivity towards styrene oxide within five substrates and the regioselectivity coefficients,α_S and β_R,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

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% ee_p）的对映会聚性优于其间位取代环氧底物（37.1%和53.3% ee_p）；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α_S和β_R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

CLC Number:

Q556.9

WANG Rui, XU Yaohui, WANG Kewei, WU Minchen. Expression of PvEH3,a Phaseolus vulgaris epoxide hydrolase,and synthesis of chiral vicinal diols[J]. Chemical Industry and Engineering Progress, 2018, 37(05): 1933-1939.

王瑞, 许耀辉, 王克伟, 邬敏辰. 环氧化物水解酶PvEH3的表达及手性邻二醇的合成[J]. 化工进展, 2018, 37(05): 1933-1939.

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