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.