Site-directed Mutagenesis of <i>Pv</i>EH1 to Improve Its Catalytic Properties towards <i>ortho</i>-Methylphenyl Glycidyl Ether

doi:10.13523/j.cb.20190602

China Biotechnology

2019, Vol. 39

Issue (6): 9-16 DOI: 10.13523/j.cb.20190602

Site-directed Mutagenesis of PvEH1 to Improve Its Catalytic Properties towards ortho-Methylphenyl Glycidyl Ether

Ting-ting KAN¹,Xun-cheng ZONG²,Yong-jun SU¹,Ting-ting WANG¹,Chuang LI²,Die HU^3,^**(

),Min-chen WU^3,^**(

)

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

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Abstract

Epoxide hydrolases can catalyze the kinetic resolution of racemic epoxides,retaining enantiopure single enantiomers of epoxides. The catalytic properties of Phaseolus vulgaris epoxide hydrolase (PvEH1) towards phenyl glycidyl ether and its methyl derivates were assayed.Seven residues of PvEH1 were then selected for site-directed mutagenesis based on the results of molecular docking simulation and multiple sequence alignment,followed by single-site and combinatorial mutagenesis to obtain mutants possessing enhanced catalytic properties towards ortho-methylphenyl glycidyl ether (1a). The substrate spectrum analysis showed that PvEH1 displayed both the highest activity (157.2U/g wet cell) and enantioselectivity (E=5.6)towards 1a.Thus, 1a was selected as the model substrate.Among the constructed seven E.coli transformants expressing single-site mutant of PvEH1,E.coli/pveh1 ^L105I and E. coli/pveh1 ^V106I exhibited notably improved EH activity and E value.Compared with E.coli/pveh1, the EH activity and E value of E.coli/pveh1 ^L105I/V106I were improved by 2.1 times and 50%, respectively.Additionally,the specific activity (17.6U/mg) and the catalytic efficiency [17.7L/(mmol·s)]of purified PvEH1 ^L105I/V106I were 1.5-and 2.1-fold those of PvEH1. SDS-PAGE analysis indicated that the soluble expression level of target protein was enhanced by the combinatorial mutagenesis.The kinetic resolution of 100mmol/L 1a by E.coli/pveh1 ^L105I/V106I whole cells afforded (R)-1a (ee>96%)with 31.2% yield and a space-time yield of 5.12g/(L·h). Therefore, the superior enzymatic properties will make E.coli/pveh1 ^L105I/V106I a promising biocatalyst for the preparation of optically pure (R)-1a.

Key words： Epoxide hydrolase Site-directed mutagenesis Catalytic activity Kinetic resolution Ortho-methylphenyl glycidyl ether

Received: 17 November 2018 Published: 12 July 2019

ZTFLH:

Q814.9

Corresponding Authors: Die HU,Min-chen WU E-mail: Butterflystudy@163.com;biowmc@126.com

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	Ting-ting KAN
	Xun-cheng ZONG
	Yong-jun SU
	Ting-ting WANG
	Chuang LI
	Die HU
	Min-chen WU

Cite this article:

Ting-ting KAN,Xun-cheng ZONG,Yong-jun SU,Ting-ting WANG,Chuang LI,Die HU,Min-chen WU. Site-directed Mutagenesis of PvEH1 to Improve Its Catalytic Properties towards ortho-Methylphenyl Glycidyl Ether. China Biotechnology, 2019, 39(6): 9-16.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20190602 OR https://manu60.magtech.com.cn/biotech/Y2019/V39/I6/9

Table 1 Analysis conditions

Table 2 Sequences of primers for mutagenesis

Table 3 The activity and enantioselectivity of PvEH1

Fig.1 Molecular docking simulation and multiple sequencealignment (a)The locally magnified 3-D configuration of PvEH1 docking with (S)-1a (b) Spatial locations of mutation sites (c) Multiple sequence alignment of PvEH1 with three plant-derived EHs.Thirty residues of PvEH1 in proximity to (S)-1a within 6? are marked, among which seven ones selected for site-directed mutagenesis are highlighted in triangles

Table 4 Catalytic properties of mutants towards 1a

Fig.2 SDS-PAGE analysis of the purified PvEH1 and PvEH1^L105I/V106I Lane M: Protein marker; Lane 1: Supernatant extract of E.coli/pveh1; Lane 2:Supernatant extract of E.coli/pveh1^L105I/V106I;Lane 3: Purified PvEH1; Lane 4: Purified PvEH1^L105I/V106I

Table 5 The kinetic parameters of PvEH1 and PvEH1^L105I/V106I

Table 6 Kinetic resolution of 1a at high concentrations using E.coli/pveh1^L105I/V106I

Fig.3 Hydrolysis progress curves of 1a by E.coli/pveh1^L105I/V106I


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