Purification And Characterization Of Bifunctional Xylanase, Isolated From Aspergillus Niger

Three types of xylanase (EC 3.2.1.8) were detected in the strain Aspergillus niger A-25 , one of which, designated as XynIII, also displayed a β-(1,3-1,4)-glucanase (EC 3.2.1.73) activity, as determined by zymogram analysis and cutting gel.XynIII was purified by ultrafiltration and ion-exchange chromatography methods. Its apparent molecular weight was about 27.9 kDa, estimated by SDS-PAGE. The purified XynIII could hydrolyze birchwood xylan, oat spelt xylan, lichenin, and barley β-glucan, but not CMC, avicael cellulose, or soluble starch under the assay conditions in this study. It is a kind of glycoprotein with 21.7% neutral sugars. The optimum pHs of XynIII for xylanase and β-(1,3-1,4)-glucanase both were 4.6. The xylanase activity was stable at pH 2.6-10.6, while, the β-(1,3-1,4)-glucanase activity was stable at pH 3.6-10.6. The optimum temperatures for both activities of Xynlll were about 50 ℃. Thermostabilities were investigated by measuring the residual activity of the enzyme after 1 h of incubation of the enzymes at temperatures ranging from 20 to 80 ℃. Both enzymes were very stable below 50℃. Both activities of Xynlll were completely inhibited by the addition of Hg²⁺. The other ions Ca²⁺, Co²⁺, Cu²⁺, Mn²⁺, Pb²⁺, K⁺, Zn²⁺ and EDTA had different inhibitive effects on both activities of XynIII, while Fe²⁺ and Mg²⁺ showed a slight promotion role on both the activities.The xylanase and β-(1,3-1,4)-glucanase activities of XynIII both had a similar optimal pH and pH stability, as well as the similar optimal temperature and temperature stability. Moreover, the effects of metal ions on the two enzymatic activities were also similar.The V_max values for xylanase and β-(1,3-1,4)-glucanase were 0.258 and 0.149 a A/min/ml, respectively, and the K_m values were 4.54 and 10.02 mg/ml, respectively. The overall hydrolytic rates of XynIII in different mixtures of xylan and lichenin with various ratios coincided with those calculated using the Michaelis-Menten model when assuming for two substrates competing for the same active site in the enzyme. Cellulose binding test reveal that XynIII has a cellulose binding domain.Accordingly, the results indicates that XynIII was a novel bifunctional enzyme and...