Characterization of novel endo-ß-N-acetylglucosaminidase from Bacteroides nordii that hydrolyzes multi-branched complex type N-glycans.

Endo-ß-N-acetylglucosaminidases (ENGases) are enzymes that hydrolyze the N-linked oligosaccharides. Many ENGases have already been identified and characterized. However, there are still a few enzymes that have hydrolytic activity toward multibranched complex-type N-glycans on glycoproteins. In this study, one novel ENGase from Bacteroides nordii (Endo-BN) species was identified and characterized. The recombinant protein was prepared and expressed in Escherichia coli cells. This Endo-BN exhibited optimum hydrolytic activity at pH 4.0. High performance liquid chromatography (HPLC) analysis showed that Endo-BN preferred core-fucosylated complex-type N-glycans, with galactose or α2,6-linked sialic acid residues at their non-reducing ends. The hydrolytic activities of Endo-BN were also tested on different glycoproteins from high-mannose type to complex-type oligosaccharides. The reaction with human transferrin, fetuin, and α1-acid glycoprotein subsequently showed that Endo-BN is capable of releasing multi-branched complex-type N-glycans from these glycoproteins.

Microbial α-L-Rhamnosidases of Glycosyl Hydrolase Families GH78 and GH106 Have Broad Substrate Specificities toward α-L-Rhamnosyl- and α-L-Mannosyl-Linkages.

α-L-Rhamnosidases (α-L-Rha-ases, EC 3.2.1.40) are glycosyl hydrolases (GHs) that hydrolyze a terminal α-linked L-rhamnose residue from a wide spectrum of substrates such as heteropolysaccharides, glycosylated proteins, and natural flavonoids. As a result, they are considered catalysts of interest for various biotechnological applications. α-L-rhamnose (6-deoxy-L-mannose) is structurally similar to the rare sugar α-L-mannose. Here we have examined whether microbial α-L-Rha-ases possess α-L-mannosidase activity by synthesizing the substrate 4-nitrophenyl α-L-mannopyranoside. Four α-L-Rha-ases from GH78 and GH106 families were expressed and purified from Escherichia coli cells. All four enzymes exhibited both α-L-rhamnosyl-hydrolyzing activity and weak α-L-mannosyl-hydrolyzing activity. SpRhaM, a GH106 family α-L-Rha-ase from Sphingomonas paucimobilis FP2001, was found to have relatively higher α-L-mannosidase activity as compared with three GH78 α-L-Rha-ases. The α-L-mannosidase activity of SpRhaM showed pH dependence, with highest activity observed at pH 7.0. In summary, we have shown that α-L-Rha-ases also have α-L-mannosidase activity. Our findings will be useful in the identification and structural determination of α-L-mannose-containing polysaccharides from natural sources for use in the pharmaceutical and food industries.