定点突变提高菜豆环氧水解酶的对映选择性  

Single site mutation of Phaseolus vulgaris epoxide hydrolase improving its enantioselectivity

作　　者：宗迅成 胡蝶 阚婷婷 王婷婷 邬敏辰 ZONG Xuncheng;HU Die;KAN Tingting;WANG Tingting;WU Minchen(School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China;Wuxi Medical School,Jiangnan University, Wuxi 214122,Jiangsu,China;School of Pharmaceutical Sciences,Jiangnan University,Wuxi 214122,Jiangsu,China)

机构地区：[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 [3]江南大学药学院,江苏无锡214122

出　　处：《化工学报》2018年第10期4335-4341,共7页CIESC Journal

基　　金：国家自然科学基金项目(21676117);2017年江苏省研究生实践创新计划项目(SJCX17_0467)~~

摘　　要：为了提高菜豆环氧水解酶(PvEH1)催化对氯环氧苯乙烷(pCSO)的对映选择性,利用定点突变构建了PvEH1^(W102L)、PvEH1^(P137K)及PvEH1^(I151V)三种突变体,分别研究了突变体全细胞动力学拆分rac-pCSO的催化特性,优化了PvEH1W102L动力学拆分rac-pCSO的初始底物浓度及反应时间,通过分子模拟分析了PvEH1^(W102L)对映选择性提高的机理。结果表明,最优突变体PvEH1^(W102L)的E值为25.5,相对酶活为212.6%,分别是PvEH1的11.6倍与2.1倍;PvEH1^(W102L)动力学拆分150 mmol·L^(-1) rac-pCSO,反应4 h后,可获得(R)-pCSO(产率为45.62%,ees为96.30%)和(R)-对氯苯基乙二醇(产率为50.91%,eep为90.26%)。分子模拟结果分析表明,PvEH1的102位色氨酸(W)突变为亮氨酸(L)降低了酶与(R)-pCSO的结合能力,从而提高了PvEH1^(W102L)对pCSO的对映选择性。For improving the enantioselectivity of Phaseolus vulgaris epoxide hydrolase1(PvEH1)towards pchlorostyrene oxide(pCSO),three mutations(PvEH1W102L,PvEH1P137K and PvEH1I151V)were constructed by sitedirected mutagenesis.Firstly,catalytic characteristics of the enzymatic hydrolysis of rac-pCSO by the whole cell of E.coli expressing PvEH1W102L,PvEH1P137K or PvEH1I151V were studied respectively.After then,the rac-pCSO initial concentration in the kinetic resolution of rac-pCSO by PvEH1W102L were optimized,in additional the course was monitored to determine the reaction time.At last,the mechanism of PvEH1W102L’s improvement in enantioselectivity towards pCSO was analyzed by molecular simulation.The result indicated that,the E value and relative activity of the best mutation PvEH1W102L were 25.5 and 212.6%,which were 11.6 and 2.1 times of PvEH1,respectively.After four-hour reaction,the yield of(R)-pCSO in the kinetic resolution was 45.62%(ees=96.30%),when the yield of(R)-p-chlorophenyl-1,2-ethanediol was 50.91%(eep=90.26%).Molecular simulation analysis showed that the mutation of the tryptophan(W)at position 102 of PvEH1 to leucine(L)reduced the binding ability of the enzyme to(R)-pCSO,thereby increasing the enantioselectivity of PvEH1W102L to pCSO.

关 键 词：生物催化 定点突变 环氧水解酶 对氯环氧苯乙烷 对氯苯基乙二醇 分子模拟 

分 类 号：Q814.9[生物学—生物工程]