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.
Lyu et al. (2026) studied this question.