Biochemical characterization of a novel β-galactosidase from Lacticaseibacillus zeae and its application in synthesis of lacto-N-tetraose

Lacto-N-tetraose (LNT) is one of the most important components of human milk oligosaccharides, which has various beneficial health effects. beta-Galactosidase is an important enzyme used in dairy processing. The transglycosylation activity of beta-galactosidases offers an attractive approach for LNT synthesis. In this study, we reported for the first time the biochemical charac-terization of a novel beta-galactosidase (LzBgal35A) from Lacticaseibacillus zeae. LzBgal35A belongs to glycoside hydrolases (GH) family 35 and shared the highest iden-tity of 59.9% with other reported GH 35 members. The enzyme was expressed as soluble protein in Escherichia coli. The purified LzBgal35A displayed optimal activity at pH 4.5 and 55 degrees C. It was stable within the pH range of 3.5 to 7.0 and up to 60 degrees C. Moreover, LzBgal35A could catalyze the synthesis of LNT via transferring the ga-lactose residue from o-nitrophenyl-beta-galactopyranoside to lacto-N-triose II. Under optimal conditions, the con-version rate of LNT reached 45.4% (6.4 g/L) within 2 h, which was by far the highest yield of LNT synthesized through a beta-galactosidase-mediated transglycosylation reaction. This study demonstrated that LzBgal35A has great potential application in LNT synthesis.