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

作　　者：Yao Li Xiaoyang Ou Zewang Guo Minhua Zong Wenyong Lou 

机构地区：[1]Lab of Applied Biocatalysis,School of Food Science and Engineering,South China University of Technology,Guangzhou 510640,China

出　　处：《Chinese Journal of Chemical Engineering》2020年第8期2181-2189,共9页中国化学工程学报（英文版）

基　　金：the National Natural Science Foundation of China(21676104,21878105,21908070);the National Key Research and Development Program of China(2018YFC1603400,2018YFC1602100);the Key Research and Development Program of Guangdong Province(2019B020213001);the Science and Technology Program of Guangzhou(201904010360);the Fundamental Research Funds for the Central Universities(2019PY15,2019MS100);the China Postdoctoral Science Foundation(BX20180102)for partially funding this work。

摘　　要：The epoxide hydrolase gene(SpEH) from Sphingomonas sp. HXN-200 was synthesized and expressed in robust Escherichia coli cells that had a dual protection system. The enantioselectivity(E-value) of the recombinant SpEH was 7.7 and the yield of the remaining(R)-PGE was 24.3% for the hydrolysis of racemic phenyl glycidyl ether(rac-PGE). To improve the catalytic properties of SpEH, 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 SpEH^(V196A/N226A/M332A) had an enhanced E-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 SpEH. The optimal temperature and p H for purified Sp EHV196 A/N226 A/M332 Ato catalyze the hydrolysis of rac-PGE were 25 ℃ and 7.0 with 200 U·mg^-1. The enantioselectivity and yield of the remaining(R)-PGE of E. coliSpEH^(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 SpEH^(V196A/N226A/M332A) has a greater affinity toward(S)-PGE than(R)-PGE, and that it was more difficult for the O-atom of ASP170 to achieve the nucleophilic attack on the Cα of(R)-PGE, resulting in its improved enantioselectivity.

关 键 词：Epoxide hydrolase Phenyl glycidyl ether ENANTIOSELECTIVITY Directed modification 

分 类 号：TQ426.97[化学工程]