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Asp^(101)-His^(297)-Asp^(262),两个保守质子供体为Tyr^(150)和Tyr^(232)。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。
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 theα/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 andβR,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.
作者
王瑞
许耀辉
王克伟
邬敏辰
WANG Rui;XU Yaohui;WANG Kewei;WU Minchen(School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China;Wuxi Medical School,Jiangnan University,Wuxi 214122,Jiangsu,China)
出处
《化工进展》
EI
CAS
CSCD
北大核心
2018年第5期1933-1939,共7页
Chemical Industry and Engineering Progress
基金
国家自然科学基金项目(21676117)
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
epoxide hydrolase
biocatalysis
expression
regioselectivity
enantioconvergent hydrolysis
作者简介
第一作者:王瑞(1989—),女,硕士研究生。E-mail:Wangshen204823@163.com。;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。E-mail:biowmc@126.com。
