Regio-Stereoselective Synthesis of Galactosyl- <i>sn</i> -2-glycerol by <i>Thermotoga naphthophila</i> Glycosidase-Catalyzed Transglycosylation in a Cosolvent-Mediated System: Kinetic and Thermodynamic Insights

Galactosides are a class of molecules widely occurring in plants and animals that play an irreplaceable role in a variety of physiological functions. Since galactosyl-sn-2-glycerol is a key intermediate in the biosynthesis of galactoside compounds, its synthesis represents the first milestone toward the sustainable production of galactosides. This work reports a novel pathway for regio-stereoselective in vitro biosynthesis of galactosyl-sn-2-glycerol using a thermophilic glucosidase from Thermotoga naphthophila RKU-10 (Tn0602). By leveraging biowaste lactose and glycerol, we achieved a yield of 416.9 mmol/L in an optimized tert-butanol/water system (30–40% water)–an 8- (the same enzyme load by mass) or 2.6- (by unit of specific activity) fold increase over commercial Kluyveromyces lactis β-galactosidase. These results are in strong agreement with the COSMO-RS predictions, providing a thermodynamic rationale and demonstrating the model’s reliability as a predictive tool for solvent engineering. Structural confirmation of the galactosyl-sn-2-glycerol products was achieved using LC-MS and 2D-NMR (COSY 1H–1H and HSQC 1H–13C), demonstrating that the reaction yielded a diastereomeric mixture of α- and β-galactosyl-sn-2-glycerols in a 1:2 ratio. We further evaluated previously reported mutants with improved oligogalactoside synthesis activity; screening showed that the N222Y variant achieved a galactosylglycerol yield of 81.4% within 12 h, a substantial improvement over wild-type Tn0602 (42.1%) when compared at the same enzyme loading. Molecular docking suggests that the N222Y mutation optimizes the binding pocket to favor glycerol transglycosylation while suppressing competing hydrolysis and oligogalactose synthesis.