[Coenzyme-free in vitro biotransformation for the synthesis of galacto-N-biose]

Galacto-N-biose (GNB), a core structural unit of O-glycan and a key component of gastrointestinal mucins, plays important physiological roles in regulating intestinal microecology and maintaining the mucosal barrier. Traditional enzymatic routes for the synthesis of GNB rely on the supplies of ATP and UDP-sugars. To address this disadvantage, we designed an in vitro ATP-free and UDP-sugar-free enzymatic pathway to synthesize GNB directly from lactose and N-acetylgalactosamine (GalNAc). This pathway consisted of two core enzymes, lactose phosphorylase (LacP) and lacto-N-biose phosphorylase (LnbP), as well as one auxiliary enzyme, polyphosphate glucokinase (PPGK). By incorporating PPGK to remove the byproduct glucose, this pathway not only pushed the reaction equilibrium toward the biosynthesis of GNB, but also achieved the regeneration of inorganic phosphate. Through the systematic optimization of key parameters such as reaction temperature, pH, and enzyme dosage, a molar yield of 76% was achieved with 10 mmol/L GalNAc. This in vitro biotransformation pathway provides a technical basis for the low-cost, large-scale green production of GNB and other high-value functional oligosaccharides.