Selective Enrichment of α-Glucosylated Trilobatin via Cascade Enzymatic Glycosylation-Hydrolysis in Polysaccharide-Based Aqueous Reaction Systems.

Cyclodextrin glucanotransferase (CGTase)-catalyzed glycosylation is an effective approach to improve flavonoid solubility and bioavailability, while poor substrate solubility and low selectivity often limit its practical application. Herein, a polysaccharide-based sugar-aqueous system coupled with cascade glycosylation-hydrolysis was developed for selective glycosylation of trilobatin. Compared with conventional organic solvents and deep eutectic solvents, the sugar-aqueous system significantly enhanced catalytic performance, achieving 83.62% conversion with α-cyclodextrin. Subsequent amyloglucosidase hydrolysis enabled the selective enrichment of trilobatin 4″-O-α-D-glucopyranoside with a 97.66% purity. Kinetic analysis showed improved enzyme-substrate affinity, while circular dichroism and molecular docking suggested favorable enzyme stability and substrate-polysaccharide interactions that contributed to enhanced glycosylation. These results indicate that polysaccharide-based aqueous systems can promote catalytic efficiency and selectivity beyond their roles as glycosyl donors and solubilizing media, providing a mild and sustainable strategy for producing functional flavonoid glycosides.