Heterologous Production, Purification and Characterization of Two Cold-Active β-d-Galactosidases with Transglycosylation Activity from the Psychrotolerant Arctic Bacterium Arthrobacter sp. S3* Isolated from Spitsbergen Island Soil

Cold-adapted microorganisms possess cold-active enzymes with potential applications in different industries and research areas. In this study, two genes encoding β-d-galactosidases belonging to Glycoside Hydrolase families 2 and 42 from the psychrotolerant Arctic bacterium <i>Arthrobacter</i> sp. S3* were cloned, expressed in <i>Escherichia coli</i> and <i>Komagataella phaffii</i>, purified and characterized. The GH2 β-d-galactosidase is a tetramer with a molecular weight of 450 kDa, while the GH42 β-d-galactosidase is a 233 kDa trimer. The Bgal2 was optimally active at pH 7.5 and 22 °C and maintained 57% of maximum activity at 10 °C, whereas the Bgal42 was optimally active at pH 7.0 and 40 °C and exhibited 44% of maximum activity at 10 °C. Both enzymes hydrolyzed lactose and showed transglycosylation activity. We also found that 2 U/mL of the Bgal2 hydrolyzed 85% of lactose in milk within 10 h at 10 °C. The enzyme synthesized galactooligosaccharides, heterooligosaccharides, alkyl galactopyranosides and glycosylated salicin. The Bgal42 synthesized galactooligosaccharides and 20 U/mL of the enzyme hydrolyzed 72% of milk lactose within 24 h at 10 °C. The properties of <i>Arthrobacter</i> sp. S3* Bgal2 make it a candidate for lactose hydrolysis in the dairy industry and a promising tool for the glycosylation of various acceptors in the biomedical sector.