定点突变提高菜豆环氧水解酶的对映选择性

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PvEH1^W102L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%，ee_s为96.30%）和（R）-对氯苯基乙二醇（产率为50.91%，ee_p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。

Single site mutation of Phaseolus vulgaris epoxide hydrolase improving its enantioselectivity

For improving the enantioselectivity of Phaseolus vulgaris epoxide hydrolase1 (PvEH1) towards p-chlorostyrene oxide (pCSO), three mutations (PvEH1^W102L, PvEH1^P137K and PvEH1^I151V) were constructed by site-directed mutagenesis. Firstly, catalytic characteristics of the enzymatic hydrolysis of rac-pCSO by the whole cell of E. coli expressing PvEH1^W102L, PvEH1^P137K or PvEH1^I151V were studied respectively. After then, the rac-pCSO initial concentration in the kinetic resolution of rac-pCSO by PvEH1^W102L were optimized, in additional the course was monitored to determine the reaction time. At last, the mechanism of PvEH1^W102L'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 PvEH1^W102L 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% (ee_s=96.30%), when the yield of (R)-p-chlorophenyl-1,2-ethanediol was 50.91% (ee_p=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 PvEH1^W102L to pCSO.