Biochemical characterisation of the neuraminidase pool of the human gut symbiont Akkermansia muciniphila

•Four putative neuraminidases from a human gut bacterium were cloned and recombinantly expressed in active form.•Functional characterisation revealed that the enzymes had similar temperature optima, but distinct metal requirements and pH optima.•The enzymes showed different substrate specificities towards C5-modified sialic acid analogues.•Simple expression and purification procedure make these enzymes potentially valuable for bioanalytical and industrial applications Since the isolation and identification of Akkermansia muciniphila one decade ago, much attention has been drawn to this gut bacterium due to its role in obesity and type 2 diabetes. This report describes the discovery and biochemical characterisation of all four putative neuraminidases annotated in the A. muciniphila genome. Recombinantly expressed candidate genes, which were designated Am0705, Am0707, Am1757 and Am2085, were shown to cover complementary pH ranges between 4.0 and 9.5. Temperature optima of the enzymes lay between 37 and 42 °C. All four enzymes were strongly inhibited by Cu2+ and Zn2+, and loss of activity after the addition of EDTA suggests that all neuraminidases, with the exception of Am0707, require divalent metal ions for their catalytic function. Chemoenzymatically synthesised α2,3- and α2,6-linked indoyl-sialosides were utilised to determine the regioselectivity and substrate promiscuity of the neuraminidases towards C5-modifications of sialic acids with N-acetyl-, N-glycolyl-, N-propionyl-, or hydroxyl-groups. The combination of simple purification procedures and good activities of some of the characterised neuraminidases makes them potentially interesting as tools in bioanalytical or industrial applications.