Identification and Characterization of a β-N-Acetylhexosaminidase with a Biosynthetic Activity from the Marine Bacterium Paraglaciecola hydrolytica S66T

beta-N-Acetylhexosaminidases are glycoside hydrolases (GHs) acting on N-acetylated carbohydrates and glycoproteins with the release of N-acetylhexosamines. Members of the family GH20 have been reported to catalyze the transfer of N-acetylglucosamine (GlcNAc) to an acceptor, i.e., the reverse of hydrolysis, thus representing an alternative to chemical oligosaccharide synthesis. Two putative GH20 beta-N-acetylhexosaminidases, PhNah20A and PhNah20B, encoded by the marine bacterium Paraglaciecola hydrolytica S66(T), are distantly related to previously characterized enzymes. Remarkably, PhNah20A was located by phylogenetic analysis outside clusters of other studied beta-N-acetylhexosaminidases, in a unique position between bacterial and eukaryotic enzymes. We successfully produced recombinant PhNah20A showing optimum activity at pH 6.0 and 50 degrees C, hydrolysis of GlcNAc beta-1,4 and beta-1,3 linkages in chitobiose (GlcNAc)(2) and GlcNAc-1,3-beta-Gal-1,4-beta-Glc (LNT2), a human milk oligosaccharide core structure. The kinetic parameters of PhNah20A for p-nitrophenyl-GlcNAc and p-nitrophenyl-GalNAc were highly similar: k(cat)/K-M being 341 and 344 mM(-1)s(-1), respectively. PhNah20A was unstable in dilute solution, but retained full activity in the presence of 0.5% bovine serum albumin (BSA). PhNah20A catalyzed the formation of LNT2, the non-reducing trisaccharide beta-Gal-1,4-beta-Glc-1,1-beta-GlcNAc, and in low amounts the beta-1,2- or beta-1,3-linked trisaccharide beta-Gal-1,4(beta-GlcNAc)-1,x-Glc by a transglycosylation of lactose using 2-methyl-(1,2-dideoxy-alpha-d-glucopyrano)-oxazoline (NAG-oxazoline) as the donor. PhNah20A is the first characterized member of a distinct subgroup within GH20 beta-N-acetylhexosaminidases.