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

Abstract To further improve the activity and enantioconvergency of Pv EH3 G170E for racemic ( rac -) p -chlorostyrene oxide ( p CSO), 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. coli / pveh3 G170E/F187L had the highest EH activity of 19.64 U/g wet cell, while / pveh3 G170E/F187I the highest α S of 94.5%. The enantioconvergent hydrolysis of 150 mM rac - p CSO by E. coli / pveh3 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 Pv EH3 G170E/F187I with improved enantioconvergency was analyzed by molecular docking simulation.