Identification and Characterization of a β--Acetylhexosaminidase with a Biosynthetic Activity from the Marine Bacterium S66.

β--Acetylhexosaminidases are glycoside hydrolases (GHs) acting on -acetylated carbohydrates and glycoproteins with the release of -acetylhexosamines. Members of the family GH20 have been reported to catalyze the transfer of -acetylglucosamine (GlcNAc) to an acceptor, i.e., the reverse of hydrolysis, thus representing an alternative to chemical oligosaccharide synthesis. Two putative GH20 β--acetylhexosaminidases, Nah20A and Nah20B, encoded by the marine bacterium S66, are distantly related to previously characterized enzymes. Remarkably, Nah20A was located by phylogenetic analysis outside clusters of other studied β--acetylhexosaminidases, in a unique position between bacterial and eukaryotic enzymes. We successfully produced recombinant Nah20A showing optimum activity at pH 6.0 and 50 °C, hydrolysis of GlcNAc β-1,4 and β-1,3 linkages in chitobiose (GlcNAc) and GlcNAc-1,3-β-Gal-1,4-β-Glc (LNT2), a human milk oligosaccharide core structure. The kinetic parameters of Nah20A for -nitrophenyl-GlcNAc and -nitrophenyl-GalNAc were highly similar: / being 341 and 344 mM s, respectively. Nah20A was unstable in dilute solution, but retained full activity in the presence of 0.5% bovine serum albumin (BSA). Nah20A catalyzed the formation of LNT2, the non-reducing trisaccharide β-Gal-1,4-β-Glc-1,1-β-GlcNAc, and in low amounts the β-1,2- or β-1,3-linked trisaccharide β-Gal-1,4(β-GlcNAc)-1,-Glc by a transglycosylation of lactose using 2-methyl-(1,2-dideoxy-α-d-glucopyrano)-oxazoline (NAG-oxazoline) as the donor. Nah20A is the first characterized member of a distinct subgroup within GH20 β--acetylhexosaminidases.