Study On Dioscin-Glycosidase Protein Post-translational Modification

In the paper,the glycosyl hydrolase-dioscin-glycosidase was obtained by culturing a microorganism.The enzyme was purified by dextran gel column and ion exchange column,and its purity and molecular weight was identified by SDS-polyacrylamide gel electrophoresis and high performance liquid chromatography.The enzymatic properties and the specificity of the enzyme were investigated.Its glycosylation form was identified by chemical methods and enzymatic methods.Its glycosylation sites was found by bio-mass spectrometry analysis.This will provide a theoretical basis for the further study of the enzyme specific hydrolysis mechanism.The dioscin-glycosidase from Absidia sp.DOOd was purified by Sephadex G-75,DEAE-Cellulose DE52 column.The enzyme was tested by SDS-PAGE and HPLC,and was determined that its molecular weight.was 55kDa.The enzyme nature of study showed that:its optimum pH is 5.0,and the pH stability range was 3.0-7.0.Its optimum reaction temperature was 50 ℃,and it was stable between 20-60 ℃.It was not effected by K+,Na+,Mg2+,Ca2+ four kinds of metal ions.It was inhibited by Zn2+,and effect was increase with the increasing of Zn2+ concentration.It was also serious inhibited by Cu2+ and Fe3+.The Vmax was 3.49 mmol/L h.The Km was 4.13 mmol/L.The dioscin-glycosidase was determined the presence of diosgenin glycosidase glycosylation phenomenon by trifluoromethanesulfonic.Its glycosylation form of the N-glycosylation was identified by the peptide N-glycosidase F.It was determine its glycosylation of the peptide by electrospray ionization-quadrupole-time-of-flight tandem mass spectrometry,and it was further analysised the glycosylation sites using tandem mass spectrometry.The results indicated that its glycosylation sites was the N2 of N1EWYDWVGSLVSn2SIDGLR.