Molecular modification and heterologous expression of a β-galactosidase from Penicillium sp. in Aspergillusniger for the production of lactose-free and low-lactose dairy products.

A β-galactosidase from Penicillium sp. was molecular modified. Through rational design of the catalytic pocket, a mutant (Y347F/W791F) showing a 24.2% increase in hydrolysis ability was obtained. Unstable amino acids on the protein surface of the mutant were replaced by proline to further enhance its specific activity, and a mutant (Y347F/S619P/W791F) with a 23.7% increase was obtained. Mutant Y347F/S619P/W791F was expressed in Aspergillus niger, and the yield was increased to 1.60 folds by engineering the expression cassette. An enzyme yield of 1, 555.1 U/mL was achieved by high-cell density fermentation. The mutant efficiently hydrolyzed lactose (>90%, w/w) in milk at room temperature (25 °C) as well as low temperatures (10 °C and 4 °C). In addition, it also efficiently hydrolyzed lactose (≥91%, w/w) in whey and enable simultaneous complete lactose hydrolysis during yogurt fermentation. This study demonstrated a food-grade and scalable biotechnological solution for efficient lactose hydrolysis under mild and low-temperature conditions, supporting its application in the industrial production of lactose-free and low-lactose dairy products.