| Sialic acids,or neuraminic acids,are a family of nine-carbon monosaccharic acids.In nature,sialic acids are normally linked to galactose through α 2,3 or α 2,6 linkages,to N-acetylgalactosamine through α 2,6 linkages,or to other sialic acids through α 2,8 linkages.Sialidases are a class of exo-alpha-hydrolytic enzymes which are responsible for the release of non-reducing end sialic acids from glycoconjugates,and used as the tool enzymes in glycan structural analysis.Akkermansia muciniphila is a human intestinal probiotics,which was discovered in recent years.It is gram-negative and strictly anaerobic.They were reported to be capable of digesting mucin to provide the source of carbon,nitrogen and energy for their growth and reproduction.The whole genome sequence of this bacterium has been decoded recently.The genes encoding sialidases were found by using the bioinformatic method,and sialidase activity was reported in some literatures.The aim of this study was to discover novel sialidases from Akkermansia muciniphila based on the information above.Four sialidase genes from Akkermansia muciniphila were cloned and designated as Am0705,Am0707,Am1757 and Am2085 respectively.The four sialidase genes were then expressed in the E.coli BL21(DE3)cells that β-galactosidase gene was knocked out.The enzyme was successfully expressed in E.coli evidenced by the observation of one SDS-PAGE band after Nickel-NTA purification.Proper substrate is essential for enzymatic activity detection and charactrisation.In order to mimic the natural format of sialic acid containing glycoconjugate,one-pot enzymatic synthesis was carried out in this study to obtain X-gal-based sialosides.The enzymes involved for the the synthesis included:sialyltransferase PdST6 from Photobacterium damsel,sialyltransferase CjST8 from Campylobacter jejuni,CMP-Sia synthetase NmCTT from Neisseria meningitidis and sialic acid aldolase DfNeuly from Dyadobacter fementans.Eight sialosides,including X-Gal α 2,3 Neu5Ac,X-Gal α 2,6 Neu5Ac,X-Gal α 2,3 Neu5Gc,X-Gal α 2,6 Neu5Gc,X-Gal α 2,3 KDN,X-Gal α 2,6 KDN,X-Gal α 2,3 Neu5Prop and X-Gal α 2,6 Neu5Prop were successfully synthesized and purified.X-Gal α 2,3 Neu5Ac and X-Gal α 2,6 Neu5Ac were used as substrates to detect the recombinant enzymes activity,and all four recombinant sialidases were active.According to the result of substrate specificity using the eight sialosides synthesized before,all four sialidases were active to both α 2,3 linked and α 2,6 linked glycosidic bonds and showed higher activity towards X-Gal α 2,3 Neu5Ac.All four enzymes showed the lowest enzymatic activities towards X-Gal α 2,3 KDN and X-Gal α 2,6 KDN.Therefore,X-Gal α 2,3 Neu5Ac was used as the substrate for enzymatic characterization of the four recombinant sialidases.The optimum pH values of four recombinant sialidases(Am0705,Am0707,Am1757 and Am2085)were 8.0、6.0、7.5 and 7.0 respectively;The optimum temperatures were 42℃,42℃,37℃ and 37℃ respectively.Km values of Am0705,Am0707,Am1757 and Am2085 were determined to be 106 μmol/L,80 μmol/L,121μmol/L and 101 μmol/L respectively.Vmax values were 50.6 μmol/min,6.6 μmol/min,27.5 μmol/min and 10.4 μmol/min respectively.EDTA completely inhibited the activities of Am0705,Am 1757 and Am2085,whereas Am0707 possessed 57.9%of full enzymatic activity.Cu2+ and Zn2+ totally inhibit four sialidase activities.Fe2+ significantly increased the activity of Am0705 to 179.9%,and completely inactivated Am2085.However,Fe3+dramatically increased the activity of Am2085 to 145.1%.In the study,four sialidase isoenzyme genes were cloned from the human intestinal probiotics Akkermansia muciniphila,and all four sialidase were expressed and purified successfully in E.coli BL21(DE3)that β-galactosidase gene was knocked out.X-Gal was used as sialic acids acceptor to synthesize 8 sialosides which have structure closer to the natural sialilated conjugates,using one-pot multiple enzymes method.Eight sialosides were utilized as substrates for the substrate characterization.All four recombinant enzymes showed broad substrate specificity,and could hydrolyse both α 2,3 and α 2,6 linked sialic acids.The results of this study provided four potential enzymes for structure analysis of glycans containing sialic acids.In addition,the 8 X-Gal based sialosides synthesized by one-pot enzymatic method can be used as ideal substrates for sialidase characterization. |
