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Abstract
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, PhNah20A and PhNah20B, encoded by the marine bacterium Paraglaciecola hydrolytica S66T, are distantly related to previously characterized enzymes. Remarkably, PhNah20A 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 PhNah20A 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 PhNah20A for p-nitrophenyl-GlcNAc and p-nitrophenyl-GalNAc were highly similar: kcat/KM 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 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. PhNah20A is the first characterized member of a distinct subgroup within GH20 β-N-acetylhexosaminidases.
Originalsprog | Engelsk |
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Artikelnummer | 417 |
Tidsskrift | International Journal of Molecular Sciences (Online) |
Vol/bind | 21 |
Udgave nummer | 2 |
Antal sider | 22 |
ISSN | 1661-6596 |
DOI | |
Status | Udgivet - 9 jan. 2020 |
Fingeraftryk
Dyk ned i forskningsemnerne om 'Identification and Characterization of a β-N-Acetylhexosaminidase with a Biosynthetic Activity from the Marine Bacterium Paraglaciecola hydrolytica S66T'. Sammen danner de et unikt fingeraftryk.Projekter
- 1 Afsluttet
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OliGram: Design and gram scale enzymatic synthesis of human milk oligosaccharides
Stougaard, P., Svensson, B., Bertelsen, H., Seo, J. H. & Pedersen, L. H.
01/08/2014 → 31/12/2018
Projekter: Projekt › Forskning