Single site mutation of Phaseolus vulgaris epoxide hydrolase improving its enantioselectivity

ZONG Xuncheng; HU Die; KAN Tingting; WANG Tingting; WU Minchen

doi:10.11949/j.issn.0438-1157.20180246

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Single site mutation of Phaseolus vulgaris epoxide hydrolase improving its enantioselectivity

更新时间：2025-12-10

- Single site mutation of Phaseolus vulgaris epoxide hydrolase improving its enantioselectivity
- CIESC Journal Issue 10, Pages: 4335-4341(2018)
- 作者机构：
  
  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
- 作者简介：
- 基金信息：
- DOI：10.11949/j.issn.0438-1157.20180246
  CLC： 814.9
- Online First：05 October 2018，
  
  Published：05 October 2018
- 稿件说明：
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ZONG Xuncheng, HU Die, KAN Tingting, et al. Single site mutation of Phaseolus vulgaris epoxide hydrolase improving its enantioselectivity[J]. CIESC Journal, 2018, (10): 4335-4341.
DOI：

ZONG Xuncheng, HU Die, KAN Tingting, et al. Single site mutation of Phaseolus vulgaris epoxide hydrolase improving its enantioselectivity[J]. CIESC Journal, 2018, (10): 4335-4341. DOI： 10.11949/j.issn.0438-1157.20180246.

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PvEH1W102L、PvEH1P137K及PvEH1I151V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PvEH1W102L对映选择性提高的机理。结果表明，最优突变体PvEH1W102L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；PvEH1W102L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PvEH1W102L对pCSO的对映选择性。

Abstract

For improving the enantioselectivity of Phaseolus vulgaris epoxide hydrolase1 (PvEH1) towards p-chlorostyrene oxide (pCSO)

three mutations (PvEH1W102L

PvEH1P137K and PvEH1I151V) were constructed by site-directed 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.

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