A novel cold-adapted phospho-beta-galactosidase from Bacillus velezensis and its potential application for lactose...

A phospho-β-galactosidase gene (BsGal1332) was cloned from Bacillus velezensis and successfully expressed in Escherichia coli BL21(DE3). The active BsGal1332 was identified to be a homodimer with a combined molecular mass of approximately 113 kDa, and it belonged to the glycoside hydrolase family 1. The BsGal1332 displayed relative strict substrate specificity for galactosyl compounds compared with the other phospho-β-galactosidases. The purified BsGal1332 showed the maximum activity at pH 8.0 and 50 °C for 2-nitrophenyl-β-d-galactopyranoside (oNPGal) and at 40 °C for lactose. BsGal1332 was slightly activated by K⁺ and Na⁺, but not strongly affected by Ca²⁺, and was stable at pH 6.0-7.0 and 40 °C or below it. The activity of BsGal1332 decreased quickly after incubation at 50 °C or higher temperature, suggesting it was a cold-adapted enzyme. Moreover, BsGal1332 could hydrolyze lactose and oNPGal with K_m values of 23.68 and 2.36 mM and k_cat values of 117.55 and 155.61 s^-1 at 4 °C, respectively. Additionally, 1 U of the BsGal1332 could thus be capable of hydrolyzing about 38% of the lactose in 1 mL of milk after incubating at 4 °C for 4 h. Taken together, these properties of BsGal1332 made it a new promising industrial biocatalyst for efficient lactose hydrolysis in milk.