Rational Design of Cyclodextrin Glycosyltransferase with Improved Hesperidin Glycosylation Activity

Cyclodextrin glycosyltransferase (CGTase) can catalyze the glycosylation of hesperidin, resulting in α-glycosyl hesperidin with significantly improved water solubility. In this study, a rational design CGTase to improve its activity was investigated. The strategy we employed involved docking near-attack conformation and virtually mutating surrounding residues, followed by calculating changes binding energy using Rosetta flex-ddG. mutations stabilization effect were then subjected an assay. Starting from CGTase-Y217F, obtained three double-point mutants, Y217F/M351F, Y217F/M351L, Y217F/D393H, activities after screening twenty variants. best variant, exhibited catalytic 1305 U/g, kcat/KmA is 2.36 times higher compared CGTase-Y217F 15.14 wild-type CGTase. Molecular dynamic simulations indicated that repulsed when bound conformation. However, introducing second-point mutation effect, repulsion weakened, reduction distances between bond-forming atoms and, thus, favoring reaction.