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

β- 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 β- N -acetylhexosaminidases, Ph Nah20A and Ph Nah20B, encoded by the marine bacterium Paraglaciecola hydrolytica S66 ^T , are distantly related to previously characterized enzymes. Remarkably, Ph Nah20A was located by phylogenetic analysis outside clusters of other studied β- N -acetylhexosaminidases, in a unique position between bacterial and eukaryotic enzymes. We successfully produced recombinant Ph 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) ₂ and GlcNAc-1,3-β-Gal-1,4-β-Glc (LNT2), a human milk oligosaccharide core structure. The kinetic parameters of Ph Nah20A for p -nitrophenyl-GlcNAc and p -nitrophenyl-GalNAc were highly similar: k _cat / K _M being 341 and 344 mM ^-1 s ^-1 , respectively. Ph Nah20A was unstable in dilute solution, but retained full activity in the presence of 0.5% bovine serum albumin (BSA). Ph 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, x -Glc by a transglycosylation of lactose using 2-methyl-(1,2-dideoxy-α-d-glucopyrano)-oxazoline (NAG-oxazoline) as the donor. Ph Nah20A is the first characterized member of a distinct subgroup within GH20 β- N -acetylhexosaminidases.