Substantially improving the enantioconvergence of PvEH1, a Phaseolus vulgaris epoxide hydrolase, towards m-chlorostyrene oxide by laboratory evolution.

Background: Epoxide hydrolase can regioselectively catalyze the oxirane ring-opening hydrolysis of rac-epoxides producing the corresponding chiral diols. In our laboratory, a gene named pveh1 encoding an EH from Phaseolus vulgaris was cloned. Although the directed modification of PvEH1 was carried out, the mutant PvEH1 showed a limited degree of enantioconvergence towards racemic (rac-) m-chlorostyrene oxide (mCSO).

Results: PvEH1 and PvEH1 were combinatively subjected to laboratory evolution to further enhance the enantioconvergence of PvEH1 towards rac-mCSO. Firstly, the substrate-binding pocket of PvEH1 was identified using a CAVER 3.0 software, and divided into three zones. After all residues in zones 1 and 3 were subjected to leucine scanning, two E. coli transformants, E. coli/pveh1 and /pveh1, were selected, by which rac-mCSO was transformed into (R)-m-chlorophenyl-1,2-ethanediol (mCPED) having 55.1% and 27.2% ee. Secondly, two saturation mutagenesis libraries, E. coli/pveh1 and /pveh1 (X: any one of 20 residues) were created at sites Y149 and P184 of PvEH1. Among all transformants, both E. coli/pveh1 (65.8% α and 55.1% ee) and /pveh1 (66.6% α and 59.8% ee) possessed the highest enantioconvergences. Finally, the combinatorial mutagenesis was conducted by replacements of both Y149L and P184W in PvEH1, constructing E. coli/pveh1, whose α reached 97.5%, higher than that (75.3%) of E. coli/pveh1. In addition, the enantioconvergent hydrolysis of 20 mM rac-mCSO was performed by E. coli/pveh1, giving (R)-mCPED with 95.2% ee and 97.2% yield.

Conclusions: In summary, the enantioconvergence of PvEH1 was successfully improved by laboratory evolution, which was based on the study of substrate-binding pocket by leucine scanning. Our present work introduced an effective strategy for the directed modification of enantioconvergence of PvEH1.