Distribution And Characterization Of Inulinase On Aspergillus Niger Uγ-2

This experiment have established a set of method of purifying that extracellar and cellwall inulinase on Aspergillus niger U y - 2, and have studied its properties. Cell ferment liquid process exceed to strain , ultrafiltration ammoium sulfate fraction , and the ion exchange of Chromatography, SephadexG-100 Chromatography, by capillary zone electrophoresis appraisal, have gotten pure inulinase of extracell group to divide; The cell broken liquid endures to exceed , ultrafiltration ,ammoium sulfate fraction ,ion-change Chromatography on DEAE-cellulose and the SephadexG-100 Chromatography , the ion-change Chromatography on DEAE-cellulose after, have also gotten pure inulinase of cell wall group .And the extracellar and cellwall are both pure group .The extracellar inulinase was purified to 7.71 folds Its specific activity was 220.5U/mg,the cellwall inulinase purify 70.04 times, The specific activity was 693.4 U/mg.By using SDS-PAGE to measure, The molecular weigh of cellwall inulinase was 108.7 kD , and the extracellar inulinase was 62.3 kD. The extracellar protein has 24.16% sugar and cellwall protein has 12.38% sugar base of sulphuric acid benzol.The surface of electric charge of both prteins are very different, capillary zone electrophorese mensuration the isoelectric point of cellwall inulinase is 5.37, extracellar inulinase the 4.10 of isoelectric point. To be for inulin extracellar inulinase Km value is 0.84375 mmol.L-1, Vmax is 52.08 mmol.L-1, cellwall inulinase Km is 0.633 mmol.L-1, Vmax is 44.64 mmol.L-1; For sucrose, extracellar Km is 5.1515 mmol.L-1, Vmax is 75.75 mmol.L-1 cellwall,inulinase Km is 7.4809 mmol.L-1, Vmax is 76.33 mmol.L-1; In pH value 3-5 scope , the activity of both enzymes is very high, when it is neutral , both are low , the most optimum pH value cellwall inulinase is pH5. 0, extracellar inulinase 4.5.For the cellwall inulinase and extracellar inulinase, the most optimum temperatureare both 60℃, them in 50 ~70℃ enzyme active higher; In pH value 3-7 scope , their stability is very good. Keeping 24 h of room temperature loses basically without activity. It is outside that the summary of thermal stability cellwall inulinase is better than extracellar inulinase, after keeping70 min of high temperature the activity of cellwall inulinase lose level is lower than extracellarinulinase. Cu for extracellar inulinase have more strong inhibition, for Pb have no effect; The cellwall inulinase for Pb2 + is sensitive, for Cu2 + do not have effect.