Characterization and Molecular Modification of a Cold-Adapted β-Galactosidase from Flavobacterium algicola for Preparation of Lactose-Free Milk.

β-Galactosidases are widely used to produce lactose-free dairy products, but efficient lactose hydrolysis under refrigerated conditions remains limited by the low activity of many commercial enzymes. In this study, a cold-adapted β-galactosidase OUC-JST from Flavobacterium algicola was characterized and engineered. OUC-JST exhibited maximal activity at 20 °C and pH 7.0. Structure-guided mutagenesis identified R110I, which shifted the optimal temperature to 15 °C, while K1023D increased activity approximately 3-fold. The combined variant R110I/K1023D (OUC-JST-N) retained the optimal temperature of 15 °C and exhibited 2.80-fold higher specific activity than the wild type. Molecular dynamics simulations suggested that OUC-JST-N may modulate correlated motions and active-site flexibility, providing a possible explanation for its improved performance. In pasteurized whole milk, OUC-JST-N hydrolyzed 98.37% of lactose at 10 °C within 48 h, leaving 812.25 mg/L residual lactose. This study highlights the potential of the cold-adapted β-galactosidase OUC-JST and its variant in the dairy processing industry as a solution to lactose intolerance.