Purification of xylanase from Aspergillus niger DFR-5: Individual and interactive effect of temperature and pH on its stability

An extracellular xylanase was purified from Aspergillus niger DFR-5 up to absolute homogeneity using (NH4)2SO4 fractionation (30–65%), size exclusion (Sephadex G-100) and ion-exchange (DEAE-cellulose) chromatography. The preparation yielded a single peak in RP-HPLC confirming its purity. Molecular mass of xylanase as revealed by gel filtration and SDS-PAGE was ∼32 kDa confirming its monomeric nature. Various kinetic parameters of xylanase towards thermo-inactivation were calculated. ΔH°, ΔS° and ΔG° of thermal denaturation suggested that enzyme undergoes significant processes of aggregation instead of unfolding during denaturation. A central composite rotatable design was used to study the interactive effects of temperature, pH and time on xylanase stability which revealed the existence of significant interactions between them. A regression equation was developed to deduce the residual activity of xylanase under any conditions of experimental parameters within the domain. The findings will be useful while applying the enzyme in different juice clarification where pH varies considerably.