Li_2019_Int.J.Biol.Macromol_121_326

To improve the activity and regioselectivity of a Phaseolus vulgaris epoxide hydrolase (PvEH3) towards p-chlorostyrene oxide (pCSO), the site-directed mutagenesis was conducted based on the computer-aided re-design. Firstly, seven single-site variants of a PvEH3-encoding gene (pveh3) were constructed as designed theoretically and expressed in E. coli BL21(DE3), respectively. One transformant, E. coli/pveh3(G170E), had the higher EH activity towards racemic pCSO, while both E. coli/pveh3(F187L) and /pveh3(P237L) with enhanced regioselectivity coefficient alphaS values. Secondly, to combine their respective merits, the double- and triple-site variants, pveh3(G170E/F187L), pveh3(G170E/P237L) and pveh3(G170E/F187L/P237L), were also constructed. Among all E. coli transformants, E. coli/pveh3(G170E/F187L/P237L) simultaneously had the highest EH activity of 20.3U/g wet cell and alphaS value of 95.2%, by which the hydrolysis of rac-pCSO enantioconvergently produced (R)-p-chlorophenylethane-1,2-diol with an enantiomeric excess of 93.2%. Furthermore, PvEH3(G170E/F187L/P237L) expressed in E. coli/pveh3(G170E/F187L/P237L) was purified. Its specific activity and catalytic efficiency towards rac-pCSO were 4.1U/mg protein and 1.81mM(-1)s(-1), which were 3.0- and 3.1-fold those of PvEH3. Finally, the molecular docking simulation analysis indicated that PvEH3(G170E/F187L/P237L) preferentially attacks the more hindered benzylic carbon of (S)-pCSO over PvEH3, which was consistent with their alphaS values measured experimentally.