Multiple site-directed mutagenesis of a Phaseolus vulgaris epoxide hydrolase to improve its catalytic performance towards p-chlorostyrene oxide based on the computer-aided re-design.

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, had the higher EH activity towards racemic pCSO, while both E. coli/pveh3 and /pveh3 with enhanced regioselectivity coefficient α values. Secondly, to combine their respective merits, the double- and triple-site variants, pveh3, pveh3 and pveh3, were also constructed. Among all E. coli transformants, E. coli/pveh3 simultaneously had the highest EH activity of 20.3 U/g wet cell and α 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 expressed in E. coli/pveh3 was purified. Its specific activity and catalytic efficiency towards rac-pCSO were 4.1 U/mg protein and 1.81 mM s, which were 3.0- and 3.1-fold those of PvEH3. Finally, the molecular docking simulation analysis indicated that PvEH3 preferentially attacks the more hindered benzylic carbon of (S)-pCSO over PvEH3, which was consistent with their α values measured experimentally.

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