Identification and Characterization of a β-<i>N</i>-Acetylhexosaminidase with a Biosynthetic Activity from the Marine Bacterium <i>Paraglaciecola hydrolytica</i> S66<sup>T</sup>
oleh: Triinu Visnapuu, David Teze, Christian Kjeldsen, Aleksander Lie, Jens Øllgaard Duus, Corinne André-Miral, Lars Haastrup Pedersen, Peter Stougaard, Birte Svensson
| Format: | Article |
|---|---|
| Diterbitkan: | MDPI AG 2020-01-01 |
Deskripsi
β-<i>N</i>-Acetylhexosaminidases are glycoside hydrolases (GHs) acting on <i>N</i>-acetylated carbohydrates and glycoproteins with the release of <i>N</i>-acetylhexosamines. Members of the family GH20 have been reported to catalyze the transfer of <i>N</i>-acetylglucosamine (GlcNAc) to an acceptor, i.e., the reverse of hydrolysis, thus representing an alternative to chemical oligosaccharide synthesis. Two putative GH20 β-<i>N</i>-acetylhexosaminidases, <i>Ph</i>Nah20A and <i>Ph</i>Nah20B, encoded by the marine bacterium <i>Paraglaciecola hydrolytica</i> S66<sup>T</sup>, are distantly related to previously characterized enzymes. Remarkably, <i>Ph</i>Nah20A was located by phylogenetic analysis outside clusters of other studied β-<i>N</i>-acetylhexosaminidases, in a unique position between bacterial and eukaryotic enzymes. We successfully produced recombinant <i>Ph</i>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)<sub>2</sub> and GlcNAc-1,3-β-Gal-1,4-β-Glc (LNT2), a human milk oligosaccharide core structure. The kinetic parameters of <i>Ph</i>Nah20A for <i>p</i>-nitrophenyl-GlcNAc and <i>p</i>-nitrophenyl-GalNAc were highly similar: <i>k</i><sub>cat</sub>/<i>K</i><sub>M</sub> being 341 and 344 mM<sup>−1</sup>·s<sup>−1</sup>, respectively. <i>Ph</i>Nah20A was unstable in dilute solution, but retained full activity in the presence of 0.5% bovine serum albumin (BSA). <i>Ph</i>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,<i>x</i>-Glc by a transglycosylation of lactose using 2-methyl-(1,2-dideoxy-α-<span style="font-variant: small-caps;">d</span>-glucopyrano)-oxazoline (NAG-oxazoline) as the donor. <i>Ph</i>Nah20A is the first characterized member of a distinct subgroup within GH20 β-<i>N</i>-acetylhexosaminidases.