Investigation Of The Expression,Purification And Enzymatic Activity Of Glycosidases From Akkermansia Muciniphila

Akkermansia muciniphila is a kind of beneficial bacteria that exists in the human gut and maintains the balance of the host gut microbe by converting mucin on the surface of the gut into beneficial byproducts.There are various of polysaccharide chains in intestinal mucins,and gut microbita can obtain nutrients from intestinal mucins by producing corresponding glycoside hydrolases to act on these polysaccharide chains.A large number of glycosidase genes were found in the genome of Akkermansia muciniphila.In this study,three glycosidases Am0010,Am2148 and Am1924 from Akkermansia muciniphila ATCC BAA-835 were cloned and purified,and the activity of Am1924 was studied.Am0010 is a member of the GH29-A family,Am2148 and Am1924 belong to the GH20 family,they are ?-N-acetylglucosaminosidases.Sequence alignment and catalytic site analysis showed that the Asp-279 of Am0010 could correspond to the conserved nucleophilic sites of GH29-A family enzymes.Sequence alignments and catalytic site analysis of Am2148 and Am1924 with reported GH20 family enzymes showed that Am2148 and Am1924 started from318 to 240 amino acids are highly consistent with the highly conserved H/N-X-A/C/G/M-D-E-A/V/L/V sequence of GH20 ?-N-acetylhexosaminoidase gene sequence.In this study,we have successfully cloned,expressed and purified three glycosidases Am0010,Am2148 and Am1924 and soluble proteins were obtained.p H and salt concentration of the buffer had effects on the solubility and aggregation state of Am2148 protein.The results showed that the buffer ratio of p H=7.5 and 0 m M Na Cl had the best solubility and aggregation state.In the enzyme activity study experiment of Am1924,was found that Am1924 has high specificity for Gal Nac oligomer,and its catalytic activity for p NP-?-Gal Nac is 8 times of p NP-?-Glc Nac.The optimum p H is 5.5.And Am1924 exhibited high hydrolytic activity in a slightly acidic environment(p H 5.5-6.5),which was different from the previously reported N-acetylglucosaminosidases of the GH20 family of Akkermansia muciniphila.