Semirational Design of Glycosyltransferase GT2419 for Efficient Synthesis of Genistein 7- O -Glucoside

Isoflavones from legumes, particularly genistein, are highly valued as functional food ingredients. However, the poor water solubility and low bioavailability hinder its application, which can be improved through glycosylation. The glycosyltransferase GT2419 from Bacillus subtilis ATCC 6633 catalyzes the 7-OH and 4′–OH glycosylation of genistein, but its poor regioselectivity hampers the efficient synthesis of genistein 7- O -glucoside. Here, a semirational design strategy was employed to obtain the F231V mutant, which significantly increased the conversion rate of genistein 7- O -glucoside from 21.1% to 72.2% and improved the 7- O regioselectivity to 73.8%. Kinetic analysis revealed that the F231V mutant exhibited enhanced affinity and catalytic efficiency toward genistein. Computational analysis revealed that the F231V mutant enlarged the substrate-binding pocket and disrupted intermolecular hydrophobic interactions, thereby altering substrate orientation, and enhancing the regioselective formation of genistein 7- O -glucoside. This study provides an efficient biosynthetic method for genistein 7- O -glucoside, with promising applications in the food industry.