Aspergillus Niger Cellulase Isolation, Purification And Characterization Of Its Dynamic Role Mechanism

Two novel cellulases were identified and isolated to homogeneity from a commercial Aspergillus niger cellulase preparation. One was an endo-l,4-β-glucanase(EC. 3.2.1.4) which was prepared by precipitation of the water extract of the culture of Aspergillus niger with ammonium sulfate and desalted by Sephadex G-25, and was further fractionated by two steps of DEAE-TOYOPEARL 650M and one step of POROS 20PI chromatography. The other was a p-glucosidase(EC.3.2.1.21) which was prepared by the above G-25 fractions and was further fractionated by two steps of DEAE-TOYOPEARL 650M chromatography.The specific activity of the endoglucanase with sodium carboxymethyl cellulose was estimated to be 433.38 HJ/mg. Its molecular weight was determined by SDS-PAGE to be 36.0KDa under non-reduced conditions and 38.0KDa under reduced conditions. The enzyme had its optimum activity at pH 3.5 and 55℃ and its Km for sodium carboxymethyl cellulose at 50℃ and pH 5.0 was 7.41mg/mL. Sacilin was inhibited the enzyme. The partial N-terminal amino acid sequences of the purified enzyme were identified as N-XXXFKCVGTSECGAEXG XALPG-22.The specific activity of the p-glucosidase with salicin was 597.12 lU/mg. Its molecular weight was determined by SDS-PAGE to be 117.5KDa under non-reduced and reduced conditions, with the optimum pH at 4.5, and the optimum temperature at 70℃. its Km for sacilin at50℃ andpH5.Owas3.73mg/mL.With the analysis of kinetics, the p-glucosidase showed synergistic action to the endoglucanase and the endoglucanase inhibited the P-glucosidase. The mechanism for their interaction was explained that the endoglucanase was combined with its products and the inhibition of these products to endoglucanase was removed by its hydrolysis by P-glucosidase.