Improvement in the activity and enantioconvergency of PvEH3, an epoxide hydrolase from Phaseolus vulgaris, for p-chlorostyrene oxide by site-saturation mutagenesis

摘要

To further improve the activity and enantioconvergency of PvEH3(G170E) for racemic (rac-) p-chlorostyrene oxide (pCSO), its F187 was randomly substituted by saturation mutagenesis. The double-site variants of pveh3, pET-28a-pveh3(G170E/F187X) (X: any one of 20 residues), were transformed into E. coli BL21(DE3), thereby constructing a mutagenesis library (E. coli/pveh3(G170E/F187X)). E. cohi/pveh3(G170E/F187L) had the highest EH activity of 19.64 U/g wet cell, while /pveh3(G170E/F187I) the highest alpha(s) of 94.5%. The enantioconvergent hydrolysis of 150 mM rac-pCSO by E. cohi/pvelt3(G170E/F187I) produced (R)-p-chlorophenyl-1,2-ethanediol with 92.8% ee(p) and 8.68 mmol/h/L space-time yield. The mechanism of PvEH3(G170E/F187I) with improved enantioconvergency was analyzed by molecular docking simulation.