A novel cold-active GH1 β-galactosidase from psychrotolerant Microbacterium phyllosphaerae LW106 suitable for lactose hydrolysis and galactooligosaccharides synthesis in milk at low temperature

Cold-active β-galactosidase offers great potential for application in biocatalysis-based processes in the food industry. Here, a novel β-galactosidase, MpGal1, was isolated from the psychrotolerant Microbacterium phyllosphaerae strain LW106 and characterized. MpGal1, a new member of the glycoside hydrolase family 1 (GH1), is a monomer enzyme of molecular weight approximately 52 kDa, possessing cold-activity-related structural features. This enzyme demonstrated optimal catalytic activity at pH 7.0 and 40 °C, with a half-life ( t 1/2 ) of 15.6 min at 45 °C. MpGal1 preferentially hydrolyzes 4-nitrophenyl-β-D-fucopyranoside over its primary substrate, 2-nitrophenyl-β-D-galactopyranoside ( o NPβGal). Kinetic analysis at 4 °C demonstrated a K m value of 142.85 mmol L −1 for lactose, with a corresponding kcat value of 5.35 s −1 at 4 °C. Notably, MpGal1 exhibited exceptional transgalactosylation activity, achieving a galactooligosaccharide (GOS) yield of 38 g/100 g when incubated with a 40 g/L lactose solution supplemented with 160.0 nkat of enzyme at 40 °C for 24 h. Furthermore, MpGal1 reduced the lactose content in milk by 30.8 % while producing 3.3 g/100 g of GOS during a 36-h incubation at 4 °C. These findings cumulatively establish MpGal1 as a promising biocatalyst for lactose hydrolysis and GOS synthesis in the dairy industry, particularly applicable for low-temperature processes. • A GH1 β-galactosidase MpGal1 originates from the Microbacterium phyllosphaerae LW106. • MpGal1 shows broad substrate specificity. • MpGal1 displays marked thermolability at 45 °C and higher temperatures. • MpGal1 efficiently hydrolyzes lactose and produces GOS in milk at 4 °C.