Enhancing the Enantioselectivity
and Catalytic Efficiency
of Esterase from Bacillus subtilis for
Kinetic Resolution of l‑Menthol through Semirational
Design
Enzymatic kinetic resolution is a promising way to produce l-menthol. However, the properties of the reported biocatalysts
are still unsatisfactory and far from being ready for industrial application.
Herein, a para-nitrobenzylesterase (pnbA) gene from Bacillus subtilis was cloned and expressed to produce l-menthol from d,l-menthyl acetate. The highest enantiomeric
excess (ee) value of the product generated by pnbA was only approximately
80%, with a high conversion rate (47.8%) of d,l-menthyl acetate
with the help of a cosolvent, indicating high catalytic activity but
low enantioselectivity (E = 19.95). To enhance the
enantioselectivity and catalytic efficiency of pnbA to d,l-menthyl acetate in an organic solvent-free system, site-directed
mutagenesis was performed based on the results of molecular docking.
The F314E/F315T mutant showed the best catalytic properties (E = 36.25) for d,l-menthyl acetate, with 92.11%
ee and 30.58% conversion of d,l-menthyl acetate. To further
improve the properties of pnbA, additional mutants were constructed
based on the structure-guided triple-code saturation mutagenesis strategy.
Finally, four mutants were screened for the best enantioselectivity
(ee > 99%, E > 300) and catalytic efficiency
at a
high substrate concentration (200 g/L) without a cosolvent. This work
provides several generally applicable biocatalysts for the industrial
production of l-menthol.