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.

Promoter engineering for efficient production of sucrose phosphorylase in Bacillus subtilis and its application in enzymatic synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid.

2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G), a stable glucoside derivative of L-ascorbic acid (L-AA), can be one-step synthesized by sucrose phosphorylase (SPase). In this study, we attempted to produce extracellular SPase in Bacillus subtilis WB800 for the food-grade production of AA-2G. The results showed that the secretion of SPases did not require signal peptide. Promoter and its compatibility to target SPase gene were proved to be the key factors for high-level secretion. The strong promoter P43 and synthetic SPase gene derived from Bifidobacterium longum (BloSPase) were selected due to generate a relatively high extracellular activity (0.94 U/mL) for L-AA glycosylation. A highly active dual-promoter system PsigH-100-P43 was further constructed, which produced the highest extracellular and intracellular activity were 5.53 U/mL and 6.85 U/mL in fed-batch fermentation, respectively. Up to 113.58 g/L of AA-2G could be achieved by the supernatant of fermentation broth and a higher yield of 146.42 g/L was obtained by whole-cells biotransformation. Therefore, the optimal dual-promoter system in B. subtilis is suitable for the food-grade scale-up production of AA-2G.