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

ss-Galactosidase (EC 3.2.1.23) hydrolyzes ss-D-galactosidic linkages at the non-reducing end of substrates to produce ss-D-galactose. Lacticaseibacillus casei is one of the most widely utilized probiotic species of lactobacilli. It possesses a putative ss-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; Galss1-3GlcNAc) and galacto-N-biose (GNB; Galss1-3GalNAc) via the phosphoenolpyruvate: sugar phosphotransferase system. The GH35 protein (GnbG) from L. casei BL23 is predicted to be 6-phospho-ss-galactosidase (EC 3.2.1.85). However, its 6-phospho-ss-galactosidase activity has not yet been examined, whereas its hydrolytic activity against LNB and GNB has been demonstrated. In this study, L. casei JCM1134 LBCZ_0230, homologous to GnbG, was characterized enzymatically and structurally. A recom-binant LBCZ_0230, produced in Escherichia coli, exhibited high hydrolytic activity toward o-nitrophenyl ss-D-galactopyranoside,p-nitrophenyl ss-D-galactopyranoside, LNB, and GNB, but not toward o-nitrophe-nyl 6-phospho-ss-D-galactopyranoside. Crystal structure analysis indicates that the structure of subsite -1 of LBCZ_0230 is very similar to that of Streptococcus pneumoniae ss-galactosidase BgaC and not suit-able for binding to 6-phospho-ss-D-galactopyranoside. These biochemical and structural analyses indicate that LBCZ_0230 is a ss-galactosidase. According to the prediction of LNB & apos;s binding mode, aromatic resi-dues, Trp190, Trp240, Trp243, Phe244, and Tyr458, form hydrophobic interactions with N-acetyl-D-glu-cosamine residue of LNB at subsite +1.

Automated summary

Lacticaseibacillus casei is a widely used probiotic species that possesses a putative ss-galactosidase. This enzyme hydrolyzes LNB and GNB, but its activity against 6-phospho-ss-galactosidase has not been investigated.