A novel thermostable beta-galactosidase for low-lactose and galacto-oligosaccharide-rich milk

Abstract Β-galactosidases (EC.3.2.1.23), which hydrolyze lactose to glucose and galactose, have two main applications in the food industry: the production of low-lactose milk and dairy goods for lactose intolerant people and the generation of galacto-oligosaccharides (GOS) by transgalactosylation reactions. Owing to their thermostability, β-galactosidases derived from thermophilic microorganisms are highly attractive for industrial applications, as elevated temperatures can enhance initial enzymatic productivity, improve substrate solubility, and reduce the risk of microbial contamination. In this study, a novel thermostable β-galactosidase (BWbg1) was obtained through functional screening of a metagenomic library from As Burgas hot spring (Ourense, Spain). The analysis of the protein amino acid sequence reveals that the enzyme belongs to GH35 family. The purified enzyme shows maximum activity at 80 °C and pH 7 and retains more than 72% of its activity after incubation at 55 °C for 6 h. BWbg1 can produce up to 48% (w/w) of GOS from a solution of 40% (w/v) of lactose at 70 °C following a 4 h reaction. Furthermore, remarkable heat activation, detected after 2 h of incubation at 65 °C, enhances its ability to hydrolyze lactose and produce GOS after HTST and VAT pasteurization of commercial milk. The results of this study reveal that BWbg1 is a versatile enzyme, capable of hydrolyzing lactose from commercial milk and producing GOS even at 8 °C, making it a strong candidate for industrial application. Key points • Novel thermostable GH35 β-galactosidase (BWbg1) mined from a hot spring metagenome. • BWbg1 high activity at 80 °C enables efficient GOS synthesis from lactose and milk. • BWbg1 retains activity after pasteurization and remains active at 8 °C.