Improving the activity and enantioselectivity of PvEH1, a Phaseolus vulgaris epoxide hydrolase, for o-methylphenyl glycidyl ether by multiple site-directed mutagenesis on the basis of rational design

• Published in: Molecular catalysis 2019-10, Vol.476, p.110517, Article 110517
• Main Authors: Li, Chuang, Kan, Ting-Ting, Hu, Die, Wang, Ting-Ting, Su, Yong-Jun, Zhang, Chen, Cheng, Jian-Qing, Wu, Min-Chen
• Format: Article
• Language: English
• Subjects: Catalytic property; Kinetic resolution; PvEH1; Rational design; Site-directed mutagenesis
• ISSN: 2468-8231; 2468-8231
• DOI: 10.1016/j.mcat.2019.110517

[Display omitted] •PvEH1 had the highest specific activity and E value for rac-1 among tested rac-1–5.•Catalytic properties of PvEH1 for rac-1 were improved by site-directed mutagenesis.•The maximum concentration of rac-1 by E. coli/pveh1L105I/V106I/L196D was 100 mM.•Scale-up kinetic resolution of rac-1 retained (R)-1 with 95.0% ees and 32.1% yield. Substrate spectrum assay exhibited that PvEH1, which is an epoxide hydrolase from P. vulgaris, had the highest specific activity and enantiomeric ratio (E) for racemic o-methylphenyl glycidyl ether (rac-1) among tested aryl glycidyl ethers (1–5). To produce (R)-1 via kinetic resolution of rac-1 efficiently, the catalytic properties of PvEH1 were further improved on the basis of rational design. Firstly, the seven single-site variants of PvEH1-encoding gene (pveh1) were PCR-amplified as designed, and expressed in E. coli BL21(DE3). Among all expressed single-site mutants, PvEH1L105I and PvEH1V106I had the highest specific activities of 17.6 and 16.4 U/mg protein, respectively, while PvEH1L196D had an enhanced E value of 9.2. Secondly, to combine their respective merits, one triple-site variant, pveh1L105I/V106I/L196D, was also amplified, and expressed. The specific activity, E value, and catalytic efficiency of PvEH1L105I/V106I/L196D were 23.1 U/mg, 10.9, and 6.65 mM−1 s−1, respectively, which were 2.0-, 1.8- and 2.4-fold higher than those of wild-type PvEH1. The source of PvEH1L105I/V106I/L196D with enhanced E value for rac-1 was preliminarily analyzed by molecular docking simulation. Finally, the scale-up kinetic resolution of 100 mM rac-1 was conducted using 5 mg wet cells/mL E. coli/pveh1L105I/V106I/L196D at 25 °C for 1.5 h, producing (R)-1 with 95.0% ees, 32.1% yield and 3.52 g/L/h space-time yield.