Directed modification of a novel epoxide hydrolase from Phaseolus vulgaris to improve its enantioconvergence towards styrene epoxides
2016
Abstract To improve the enantioconvergence of an epoxide hydrolase from Phaseolus vulgaris ( Pv EH1) towards styrene epoxides, its directed modification was performed based on the rational design by using the molecule docking simulation and the multiple alignment. The single- and multi-site mutation genes of pveh1 were constructed as designed theoretically by PCR, and expressed in E. coli BL21(DE3), respectively. Among all Pv EH1 mutants tested, a three-site mutant, Pv EH1 L105I/M160A/M175I , was selected having the highest activity of 10.66 U/g wet cell and the best regioselectivity (α S > 99%, β R = 86.4%), by which rac - 1a was transformed into ( R )- 1b with 87.8% enantiomeric excess ( ee p ), much higher than that (33.6% ee p ) by Pv EH1. Furthermore, it completely hydrolyzed rac - 2a – 5a into ( R )- 2b – 5b with 52.3–70.9% ee p .
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