Significant improvement in catalytic activity and enantioselectivity of a Phaseolus vulgaris epoxide hydrolase, PvEH3, towards ortho-cresyl glycidyl ether based on the semi-rational design

The investigation of substrate spectrum towards five racemic (rac-) aryl glycidyl ethers (1a–5a) indicated that E. coli/pveh3, an E. coli BL21(DE3) transformant harboring a PvEH3-encoding gene pveh3, showed the highest EH activity and enantiomeric ratio (E) towards rac-3a. For efficiently catalyzing the kinetic resolution of rac-3a, the activity and E value of PvEH3 were further improved by site-directed mutagenesis of selected residues. Based on the semi-rational design of an NC-loop in PvEH3, four single-site variants of pveh3 were amplified by PCR, and intracellularly expressed in E. coli BL21(DE3), respectively. E. coli/pveh3(E134K) and /pveh3(T137P) had the enhanced EH activities of 15.3 ± 0.4 and 16.1 ± 0.5 U/g wet cell as well as E values of 21.7 ± 1.0 and 21.2 ± 1.1 towards rac-3a. Subsequently, E. coli/pveh3(E134K/T137P) harboring a double-site variant gene was also constructed, having the highest EH activity of 22.4 ± 0.6 U/g wet cell and E value of 24.1 ± 1.2. The specific activity of the purified PvEH3(E134K/T137P) (14.5 ± 0.5 U/mg protein) towards rac-3a and its catalytic efficiency (k(cat)/K(m) of 5.67 mM(−1) s(−1)) for (S)-3a were 1.7- and 3.54-fold those (8.4 ± 0.3 U/mg and 1.60 mM(−1) s(−1)) of PvEH3. The gram-scale kinetic resolution of rac-3a using whole wet cells of E. coli/pveh3(E134K/T137P) was performed at 20 °C for 7.0 h, producing (R)-3a with 99.4% ee(s) and 38.5 ± 1.2% yield. Additionally, the mechanism of PvEH3(E134K/T137P) with remarkably improved E value was analyzed by molecular docking simulation.