Action pattern of Sulfolobus O-α-glycoligase for synthesis of highly water soluble resveratrol 3,4′-α-diglucoside

• Published in: Enzyme and microbial technology 2024-12, Vol.181, p.110518, Article 110518
• Main Authors: Ahn, Hee-Won, Roy, Jetendra Kumar, Lee, Jaeick, Lee, Mi-Jin, Yoo, Sang-Ho, Kim, Young-Wan
• Format: Article
• Language: English
• Subjects: Animals; Glucosides - biosynthesis; Glucosides - chemistry; Glucosides - metabolism; Glycosylation; Hydrogen-Ion Concentration; O-α-glycoligase; Rats; Resveratrol; Resveratrol - chemistry; Resveratrol - metabolism; Solubility; Stilbenes - chemistry; Stilbenes - metabolism; Substrate Specificity; Transglycosylation; Water - chemistry; Water solubility; α-Glucoside
• ISSN: 0141-0229; 1879-0909; 1879-0909
• DOI: 10.1016/j.enzmictec.2024.110518

This study presents the enzymatic synthesis of resveratrol-3,4′-O-α-diglucoside (RDG) using a hyperactive O-α-glycoligase (MalA-D416R/Q450S) and α-glucopyranosyl fluoride as the donor substrate. The transglycosylation rate for resveratrol by MalA-D416R/Q450S was maximized in 100 mM Tris-HCl (pH 9.5) containing 20 % DMSO at 45°C. Because the pKa of the 4′-OH group of resveratrol is lower than that of the 3-OH group, the 4′-OH group is more nucleophilic at the alkaline pH, leading to a preference for glycosylation at the 4′-OH site rather than the 3-OH site. This preference makes resveratrol 3-O-α-glucoside (R3G) as the more efficient acceptor than resveratrol 4′-O-α-glucoside (R4′G), resulting in negligible production of resveratrol 3-O-α-glucoside (R3G) due to its complete consumption in the second transglycosylation reaction when using a 2:1 ratio of donor to acceptor substrates. From a preparative scale reaction, R4′G and RDG were isolated with yields of 41.2 % and 43.3 %, respectively. The water solubility of RDG exceeded 1.67 M, which represents more than a 9,800-fold improvement compared to resveratrol. In a hydrolysis experiment using intestinal α-glycosidase from rat, the α-glucosides of resveratrol (R4′G and RDG) were completely deglycosylated to the aglycone. [Display omitted] •The MalA-derived engineered O-α-glycoligase produced resveratrol 3,4-O-α-diglucoside (RDG).•The 4’-OH group, with a lower pKa, is preferable to the 3-OH as a glucosylation site.•The conversion yield of RDG was the highest compared to reported enzymatic syntheses.•The water solubility of RDG was improved by 9,800-fold compared to resveratrol.•The facile hydrolysis of RDG by α-glucosidase would lead to efficient absorption in the intestine.