Function and Structure of Lacticaseibacillus casei GH35 β-Galactosidase LBCZ_0230 with High Hydrolytic Activity to Lacto-N-biose I and Galacto-N-biose

• Published in: Journal of applied glycoscience : JAG Vol. 70; no. 2
• Main Authors: Saburi, Wataru, Ota, Tomoya, Kato, Koji, Tagami, Takayoshi, Yamashita, Keitaro, Yao, Min, Mori, Haruhide
• Format: Journal Article
• Language: Japanese
• Published: Tsukuba Japan Science and Technology Agency 01.01.2023
• Subjects: Bacteria; Binding; Crystal structure; D-Galactose; E coli; Galactose; Galactosidase; Glucosamine; Glycosidases; Glycoside hydrolase; Hydrophobicity; Lactobacilli; N-Acetylglucosamine; Phosphotransferase; Probiotics; Residues; Structural analysis; Structure-function relationships; Substrates; β-Galactosidase
• ISSN: 1344-7882; 1880-7291
• DOI: 10.5458/jag.jag.JAG-2022_0014

β-Galactosidase (EC 3.2.1.23) hydrolyzes β-D-galactosidic linkages at the non-reducing end of substrates to produce β-D-galactose. Lacticaseibacillus caseiis one of the most widely utilized probiotic species of lactobacilli. It possesses a putative β-galactosidase belonging to glycoside hydrolase family 35 (GH35). This enzyme is encoded by the gene included in the gene cluster for utilization of lacto-N-biose I (LNB; Galβ1-3GlcNAc) and galacto-N-biose (GNB; Galβ1-3GalNAc) viathe phosphoenolpyruvate: sugar phosphotransferase system. The GH35 protein (GnbG) from L. caseiBL23 is predicted to be 6-phospho-β-galactosidase (EC 3.2.1.85). However, its 6-phospho-β-galactosidase activity has not yet been examined, whereas its hydrolytic activity against LNB and GNB has been demonstrated. In this study, L. caseiJCM1134 LBCZ_0230, homologous to GnbG, was characterized enzymatically and structurally. A recombinant LBCZ_0230, produced in Escherichia coli, exhibited high hydrolytic activity toward o-nitrophenyl β-D-galactopyranoside, p-nitrophenyl β-D-galactopyranoside, LNB, and GNB, but not toward o-nitrophenyl 6-phospho-β-D-galactopyranoside. Crystal structure analysis indicates that the structure of subsite −1 of LBCZ_0230 is very similar to that of Streptococcus pneumoniaeβ-galactosidase BgaC and not suitable for binding to 6-phospho-β-D-galactopyranoside. These biochemical and structural analyses indicate that LBCZ_0230 is a β-galactosidase. According to the prediction of LNB's binding mode, aromatic residues, Trp190, Trp240, Trp243, Phe244, and Tyr458, form hydrophobic interactions with N-acetyl-D-glucosamine residue of LNB at subsite +1.