Purification, physicochemical and thermodynamic studies of antifungal chitinase with production of bioactive chitosan-oligosaccharide from newly isolated Aspergillus griseoaurantiacus KX010988

Abstract

In our search for chitinase and chitosanase producer from unconventional sources, the marine-derived fungus Aspergillus griseoaurantiacus KX010988 was obviously the best producer of the highest chitinase and chitosanase activities by solid state fermentation of potato shells. Chitinase was purified in three steps involving ammonium sulphate precipitation, DEAE-cellulose ion-exchange chromatography and Sephacryl S-300 gel chromatography. 12.55 fold increase in purity with a recovery of 17.6 was obtained. The molecular mass of the purified chitinase was found to be 130kDa. It was optimally active at pH 4.5 and 40°C. Km and Vmax values were 0.22mgmL−1 and 19.6μmolemin−1mg−1 respectively. Mn2+ and Zn2+ ions lead to increased chitinase activity. While Fe2+and Cu2+ions strongly inhibited the chitinase activity. The thermodynamics of pure chitinase including activation energy for thermal denaturation (Ea,d), change of free energy (ΔGd), enthalpy(ΔHd), entropy(ΔSd) and half life values (T1/2) at 40, 50 and 60°C were determined. Chitinase showed antifungal activity against pathogenic fungus Fusarium solani. Chitosanase was partially purified by acetone precipitation (50–75%) v/v concentration. The hydrolytic products of moderate molecular weight of chitosan by chitosanase were analyzed by thin layer chromatography (TLC) after 12 and 24h respectively. Chitosan-oligosaccharides showed good antibacterial and antioxidant activities.