Using multiple site-directed modification of epoxide hydrolase to significantly improve its enantioselectivity in hydrolysis of <i>rac</i>-glycidyl phenyl ether

Yao Li; Xiaoyang Ou; Zewang Guo; Minhua Zong; Wenyong Lou

doi:10.1016/j.cjche.2020.03.025

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Using multiple site-directed modification of epoxide hydrolase to significantly improve its enantioselectivity in hydrolysis of rac-glycidyl phenyl ether

Biotechnology and Bioengineering | Updated：2026-01-06

- Using multiple site-directed modification of epoxide hydrolase to significantly improve its enantioselectivity in hydrolysis of rac-glycidyl phenyl ether
- Chinese Journal of Chemical Engineering Vol. 28, Issue 8, Pages: 2181-2189(2020)
- Affiliations：
  
  Lab of Applied Biocatalysis, School of Food Science and Engineering, South China University of Technology,Guangzhou,China,510640
- Author bio：
- Funds：
- DOI：10.1016/j.cjche.2020.03.025
  CLC：
- Published：2020
- Accepted：
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-glycidyl phenyl ether[J]. 中国化学工程学报, 2020, 28(8): 2181-2189.
DOI：

-glycidyl phenyl ether[J]. 中国化学工程学报, 2020, 28(8): 2181-2189. DOI： 10.1016/j.cjche.2020.03.025.

摘要

Abstract

The epoxide hydrolase gene (

EH) from

Sphingomonas

sp. HXN-200 was synthesized and expressed in robust

Escherichia coli

cells that had a dual protection system. The enantioselectivity (

-value) of the recombinant

EH was 7.7 and the yield of the remaining (

)-PGE was 24.3% for the hydrolysis of racemic phenyl glycidyl ether (

rac

-PGE). To improve the catalytic properties of

the site-directed mutagenesis was carried out based on homology modeling

sequence alignment and molecular docking. Six residues (V195

V196

F218

N226

Q312

and M332) near the active site were mutated to hydrophobic amino acids and the positive mutations were selected for combinatorial mutation. The optimal mutant

V196A/N226A/M332A

had an enhanced

-value of 21.2 and a specific activity of 4.57 U·mg

-1

-wet cells

which were 2.8-

and 2.3-fold higher than those of wild-type

EH. The optimal temperature and pH for purified

V196A/N226A/M332A

to catalyze the hydrolysis of

rac

-PGE were 25 ℃ and 7.0 with 200 U·mg

-1

. The

enantioselectivity and yield of the remaining (

)-PGE of

E. coli

V196A/N226A/M332A

increased from 7.7 to 21.2 and 24.3% to 40.9%

respectively. The molecular docking and kinetic parameter analyses showed that

V196A/N226A/M332A

has a greater affinity toward (

)-PGE than (

) - PGE

and that it was more difficult for the

-atom of ASP170 to achieve the nucleophilic attack on the Cα of (

)-PGE

resulting in its improved enantioselectivity.

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⁰

Using multiple site-directed modification of epoxide hydrolase to significantly improve its enantioselectivity in hydrolysis of rac-glycidyl phenyl ether

DOI：10.1016/j.cjche.2020.03.025

摘要

Abstract

关键词

Keywords

references