Engineering the regiocomplementarity of an epoxide hydrolase from Rhodotorula paludigena by means of computer-aided design for the scale-up enantioconvergent hydrolysis of racemic m-nitrostyrene oxide

E. coli/rpeh, a transformed E. coli strain expressing RpEH, showed higher EH regiocomplementarity for (S)- and (R)-m-nitrostyrene oxide (mNSO). To more effectively prepare (R)-m-nitrophenyl-ethane-1,2-diol (mNPED) via hydrolysis of rac-mNSO, the regioselectivity coefficients of RpEH, especially its alpha(S) for (S)-mNSO, were further elevated by microtuning its substrate-binding pocket (SBP). By means of computer-aided design, eight residues lining the SBP of RpEH were identified, and subjected to valine scanning mutagenesis. Two mutants, RpEH(Y254V) and RpEH(L360V), were selected, hydrolyzing rac-mNSO into (R)-mNPED with enhanced ee(p) values of 92.0% and 94.8%. Then, both Tyr(254) and Leu(360 )were subjected to partial site-saturation mutagenesis. After screening, the best mutant RpEH(L360C) was obtained, whose alpha(S) and beta(R) (96.3% and 98.6%) were larger than those (92.2% and 95.2%) of RpEH. The enantioconvergent hydrolysis of 600 mM rac-mNSO was carried out using 200 mg/mL wet cells of E. coli/rpeh(L360C )at 20 degrees C for 2.5 h in 50 mL 30% Tween-20/phosphate buffer (100 mM, pH 7.0), producing (R)-mNPED with 93.4% ee(p) and 95.7% yield. Furthermore, the analysis by molecular docking (MD) simulation revealed that, compared with RpEH, RpEH(L3)(60C) more regiopreferentially attacks both the C-alpha in the oxirane ring of (S)-mNSO and C-beta of (R)-mNSO.