Gene Cloning,Expression And Characterization Of The ?-Galactosidase From Akkermansia Muciniphila

?-galactosidase is an exoglycosidase that cleaves galactosyl groups from the non-reducing ends of glycoconjugates and releases free galactose,so it is an important tool for the analysis of glycan structures.The existing ?-galactosidase lack the substrate specificity to meet the development of glycobiology,so we need to find some special galactosidases.Akkermansia muciniphila is a gram-negative stringent anaerobe from the human intestinal mucin.There have been no reports in the previous study about the strain to express ?-galactosidase.A novel ?-galactosidase gene was discovered from Akkermansia muciniphila in this study.The recombinant ?-galactosidase was obtained after gene cloning,the expression vector constructing and recombinant expression.Enzymatic activity and substrate specificity were studied after purification.1.The gene AmBGa10874 encoding ?-galactosidase was cloned in this study from A.muciniphila.After the construction of the recombinant expression vector,E.coli BL21(DE3)was chosen to work as expression host.The crude enzyme extractions were purified by using a nickel column(Ni2+-NTA).The result shown by SDS-PAGE,and the enzyme activity was tested by using the substrate 4MU-?-Gal.The results indicated that the purification of the recombinant ?-galactosidase was quite good and the enzyme has activity to 4MU-?-Gal.2.The activity and substrate specificity of recombinan ?-galactosidase were probed with various pNP-glycosides(pNP-?/?-Gal,pNP-?/?-Fuc,pNP-?/?-Man,pNP-?/?-Glc,pNP-?/?-GlcNAc).The results indicated that the recombinant ?-gaIactosidase could only hydrolyze pNP-?-Gal.The enzyme AmBGal0874 works best at an optimum pH of 3.5 and the optimum temperature of 45?,without dependence on metal ions,and the addition of Mg2+reduced the activity of Am0874 significantly,whereas other tested metal ions showed little effect on the activity of the enzyme.SDS strongly inhibited Am0874 already at the lowest tested concentration(0.1%V/V),while the addition of 2-mercaptoethanol,urea,or Triton X-100 had no or little effect on the enzyme's activity.The specific activity of enzyme is 83.33 mU/mg,the measured Km value is 1.73 mM and the Vmax is 0.54?mol/min/mg.3.The substrate specificity of the recombinant ?-galactosidase was tested using different disaccharides.The result showed that the enzyme AmBGal0874 was able to hydrolyze the?1,3-and ?1,6-linked galactose of the non-reducing ends of different substrates,while the hydrolysis efficiency of the ?1,4-linked galactose was extremely low.The efficiency of substrates with the same linkage but different adjacent sugar molecules also differs greatly,indicating that the aglycone moiety also affects the hydrolysis activity of Am0874.Overall,the ?-galactosidase gene from Akkermansia muciniphila was successfully cloned and expressed in E.coli.It is the first enzyme to be isolated and characterized from human intestinal mucin.The enzyme has a strong temperature tolerance and the pH environment of the reaction is acidic,which expands the range of application conditions and enriches the library of ?-galactosidase gene.At the same time,the ?-galactosidase has a strong hydrolysis ability and a strict substrate specificity,it can hydrolyze the ?1,3-and ?1,6-linked galactose of non-reducing ends for various substrates,and it will will be promising candidates for bioanalytical applications.