Abstract
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/pveh3G170E, had the higher EH activity towards racemic pCSO, while both E. coli/pveh3F187L and /pveh3P237L with enhanced regioselectivity coefficient αS values. Secondly, to combine their respective merits, the double- and triple-site variants, pveh3G170E/F187L, pveh3G170E/P237L and pveh3G170E/F187L/P237L, were also constructed. Among all E. coli transformants, E. coli/pveh3G170E/F187L/P237L simultaneously had the highest EH activity of 20.3 U/g wet cell and αS 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, PvEH3G170E/F187L/P237L expressed in E. coli/pveh3G170E/F187L/P237L was purified. Its specific activity and catalytic efficiency towards rac-pCSO were 4.1 U/mg protein and 1.81 mM−1 s−1, which were 3.0- and 3.1-fold those of PvEH3. Finally, the molecular docking simulation analysis indicated that PvEH3G170E/F187L/P237L preferentially attacks the more hindered benzylic carbon of (S)-pCSO over PvEH3, which was consistent with their αS values measured experimentally.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
More From: International Journal of Biological Macromolecules
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.