Cloning, expression, and enzymatic characterization of a cold-adapted β‐galactosidase from a marine source and its transglycosylation potential

BACKGROUND: Cold-adapted β‐galactosidases are valuable for low-temperature applications in biotechnology, food and pharmaceutical industries. OBJECTIVE: The study aimed to isolate novel cold-active β‐galactosidase-producing strains from marine sediments and develop more efficient and stable cold-active β‐galactosidase. MATERIALS AND METHODS: A cold-adapted stain ( Bacillus sp. K3) was isolated from deep-sea sediments. The enzyme gene was cloned and expressed in Escherichia coli BL21 (DE3). The enzyme was purified and characterized for its biochemical properties and transglycosylation potential. RESULTS: The recombinant β‐galactosidase exhibited optimal activity at 20°C and maintained stability from −20°C to 50°C, with peak efficiency at pH 6.0. Kinetic analysis confirmed high catalytic efficiency at low temperatures. Its activity was enhanced by Triton X‐100, Mg 2+ , and Mn 2+ , while strongly inhibited by Hg 2+ , Cu 2+ , EDTA, and DTT. Notably, the enzyme achieved a 13.7% galacto-oligosaccharide (GOS) conversion at 20°C. CONCLUSION: A cold-adapted stain ( Bacillus sp. K3) was isolated from deep-sea sediments, and it produces a β‐galactosidase that functions at low temperatures. The study highlights the potential of the K3 β‐galactosidase for low-temperature applications in biotechnology, food, pharmaceutics and environmental industries.