Engineered β-galactosidase catalyzes lactose to prebiotics in situ in raw milk.

The enzymatic conversion of lactose to galactooligosaccharides (GOS) within raw milk offers a promising avenue for reducing lactose content while enhancing its prebiotic benefits. This process hinges on utilizing enzymes with high transglycosylation activity compatible with current milk processing methods. In the present studies, Bacillus circulans β-galactosidase (BglD) was identified as an effective enzyme, achieving a lactose conversion rate of 73.61% in milk. However, BglD's resistance to pasteurization underscored the need for enzyme modifications. Through molecular directed evolution, a mutant T473L/R484P was engineered. It exhibited improved catalytic performance at lower temperatures, with optimal activity at pH 6.5 and 55 °C. In contrary to the wild-type, mutant T473L/R484P exhibited reduced thermostability, and its activity diminished rapidly above 50 °C. Remarkably, in simulated dairy environments, mutant T473L/R484P converted over 78% to 82% of lactose at low temperatures (5 to 10 °C), reducing lactose to around 10.4 to13.0 g/L while generating approximately 30 to 34 g/L of GOS. These findings highlight the mutant's potential in producing low-lactose, GOS-enriched milk, and pave the way for innovative in situ lactose-to-GOS conversion processes in raw milk.