Improving the Enantioselectivity of an Epoxide Hydrolase towards p-Methylphenyl Glycidyl Ether by Site-directed Mutagenesis

doi:10.13523/j.cb.1907025

China Biotechnology

2020, Vol. 40

Issue (3): 88-95 DOI: 10.13523/j.cb.1907025

Orginal Article

Improving the Enantioselectivity of an Epoxide Hydrolase towards p-Methylphenyl Glycidyl Ether by Site-directed Mutagenesis

SU Yong-jun¹,HU Die²,HU Bo-chun³,LI Chuang³,WEN Zheng³,ZHANG Chen¹,WU Min-chen^2,^**

1 School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, China
2 Wuxi School of Medicine, Jiangnan University, Wuxi 214122, China
3 School of Biotechnology, Jiangnan University, Wuxi 214122, China

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Abstract

Epoxide hydrolase can be used in the kinetic resolution or enantioconvergent hydrolysis of racemic epoxides for prepare optically pure epoxides or vicinal diols, which has broad application prospects. To improve the enantioselectivity of Aspergillus usamii epoxide hydrolase (AuEH2) towards racemic p-methylphenyl glycidyl ether (rac-pMPGE). According to the protein-ligand finger-print (IFP) in the molecular dynamics simulation, the key residue site A250 with the highest interaction frequency towards (R)-pMPGE was selected, and then replaced by other 19 residues by site-directed mutagenesis. A mutant with the improved enantioselectivity was obtained and purified by affinity chromatography. Furthermore, the kinetic parameters and regioselectivity coefficients towards (R)-and (S)-pMPGE of the purified mutant were measured, respectively, and the recombinant E. coli whole cells was applied to the kinetic resolution of rac-pMPGE. The mutant AuEH2^A250H possessed the highest E value of 38.4, which was remarkably higher than that of AuEH2 (12.7). The specific activity of E. coli/aueh2^A250H was determined to be 51.9U/g wet cells. The k_cat/K_m for (S)-pMPGE of the purified AuEH2^A250H was increased from 10.0mmol/(L·s) to 12.8mmol/(L·s), while decreased from 1.13mmol/(L·s) to 0.35mmol/(L·s) for (R)-pMPGE. Furthermore, using the whole cells of E. coli/aueh2^A250H as biocatalyst, the kinetic resolution of 20mmol/L rac-pMPGE was performed at 25℃ for 1h, obtaining (R)-pMPGE with > 99% ee_s and 40.7% yield. The results indicated that the mutations at A250 played an essential role in regulating the activity and enantioselectivity of AuEH2. The mutant with the improvement of enantioselectivity of AuEH2, which has potential for industrial application on the preparation of (R)-pMPGE.

Key words： Epoxide hydrolase Rational design Site-directed mutagenesis Enantioselectivity p-Methyl phenyl glycidyl ether

Received: 12 July 2019 Published: 18 April 2020

ZTFLH:

Q814.9

Corresponding Authors: Min-chen WU

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	Yong-jun SU
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	Bo-chun HU
	Chuang LI
	Zheng WEN
	Chen ZHANG
	Min-chen WU

Cite this article:

SU Yong-jun,HU Die,HU Bo-chun,LI Chuang,WEN Zheng,ZHANG Chen,WU Min-chen. Improving the Enantioselectivity of an Epoxide Hydrolase towards p-Methylphenyl Glycidyl Ether by Site-directed Mutagenesis. China Biotechnology, 2020, 40(3): 88-95.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.1907025 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I3/88

Table 1 PCR primers for the site-directed mutagenesis

Fig.1 Analysis of molecular docking and molecular dynamics simulation(a) The 3D structure model of AuEH2 (b) Molecular docking model of AuEH2 with (R)-pMPEG (c) The IFP plot between AuEH2 with (R)-pMPEG in the MD simulation

Fig.2 Relative activity (a) and enantioselectivity (b) of AuEH2 and its mutants

Fig.3 SDS-PAGE analysis of the expressed AuEH2 and the mutant AuEH2^A250HM: Protein marker; Lane 1: E. coli/pET-28a; Lane 2: E. coli/aueh2; Lane 3: Purified AuEH2; Lane 4: Purified AuEH2^A250H

Table 2 Determination of kinetic parameters of WT and variants^*

Fig. 4 Regioselectivity of AuEH2^A250H towards (R)-and (S)-pMPGE

Fig.5 The hydrolytic course curve of rac-pMPGE catalyzed by E. coli/aueh2 (a) and E. coli/aueh2^A250H (b)

Table 3 Comparison of resolution of rac-pMPGE to prepare (R)-pMPGE with reported EHs

Fig.6 The hydrolytic course curve of preparation of (R)-pMPGE by whole cells of E. coli/aueh2^A250H


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