Directed modification of a novel epoxide hydrolase from Phaseolus vulgaris to improve its enantioconvergence towards styrene epoxides

摘要

To improve the enantioconvergence of an epoxide hydrolase from Phaseolus vulgaris (PvEH1) 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 pvehl were constructed as designed theoretically by PCR, and expressed in E. coli BL21 (DE3), respectively. Among all PvEH1 mutants tested, a three-site mutant, PvEH1(L1051/M160A/M1751), was selected having the highest activity of 10.66 U/g wet cell and the best regioselectivity (alpha(s) > 99%, beta(R) = 86.4%), by which rac-la was transformed into (R)-lb with 87.8% enantiomeric excess (ee(p)), much higher than that (33.6% ee(p)) by PvEHl. Furthermore, it completely hydrolyzed rac-2a-5a into (R)-2b-5b with 523-70.9% ee(p). (C) 2016 Elsevier B.V. All rights reserved.