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

摘要

Two genes encoding the beta-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 degrees 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 beta 1,4-linked N-acetylgalactosamine and beta 1,6-linked N-acetylglucosamine from glycan standards, as well as beta 1,2-linked N-acetylglucosamine units from the non-reducing end of N-glycans. The present study describes the first functional characterisation of beta-N-acetylhexosaminidases from this human gut symbiont. (c) 2017 Elsevier Ltd. All rights reserved.