Cloning, purification and biochemical characterization of two β-N-acetylhexosaminidases from the mucin-degrading gut bacterium Akkermansia muciniphila

Abstract

Two genes encoding the β-N-acetylhexosaminidases Am2301 and Am2446 were cloned successfully from the mucin-degrading bacterium Akkermansia muciniphila. The recombinant enzymes with molecular masses of 61 kDa and 78 kDa were isolated and biochemically characterised. The optimum temperature of both enzymes was 37 °C, and the optimum pH was determined to be pH 5.0 for Am2301 and pH 6.5 for Am2446. The addition of sodium dodecyl sulphate (SDS) reduced the enzymes' activity significantly. Cu²⁺-ions decreased the activity of Am2301 by 70%, while the activity of Am2446 was significantly reduced by Fe³⁺-ions. PugNAc strongly inhibited both enzymes already in the sub-micromolar concentration range. The enzymes catalysed the hydrolysis of β1,4-linked N-acetylgalactosamine and β1,6-linked N-acetylglucosamine from glycan standards, as well as β1,2-linked N-acetylglucosamine units from the non-reducing end of N-glycans. The present study describes the first functional characterisation of β-N-acetylhexosaminidases from this human gut symbiont.

Keywords: Akkermansia muciniphila; Glycan degradation; Intestinal glycosidases; β-N-acetylhexosaminidases.