Anticancer and antifungal efficiencies of purified chitinase produced from Trichoderma viride under submerged fermentation

Abstract

Trichoderma viride AUMC 13021 isolated from Mangrove soil of Ras Mohammed protected area at Sharm El-Sheikh, Egypt, was optimized to promote chitinase activity under submerged fermentation. The maximum enzyme yield (38.33 U/mg protein) was obtained at 1.4% of colloidal chitin, 96 h of incubation, 35°C, pH 6.5 and 125, rpm and using maltose (1%) and yeast extract (1%) as supplementation of salt basal medium. The enzyme has been purified with an overall yield of 73.1% and 5.48 purification fold, and a specific activity of 210.16 U/mg protein. The molecular mass of the purified chitinase was 62 kDa. Maximal activity of chitinase was recorded at pH 6.5 and 40°C. The highest activity was recorded in the case of colloidal chitin, with an apparent K_m value of 6.66 mg/ml and V_max of 90.8 U/ml. The purified chitinase was activated by Ca²⁺ and Mn²⁺ while the activity was inhibited by Hg²⁺, Zn²⁺, Cu²⁺, Co²⁺, dodecyl sulphate and EDTA. In vivo, the median lethal dose (LD₅₀) was approximately 18.43 mg/kg body weight of Sprague Dawley rats. MTT assay showed that the purified chitinase has a toxic effect to MCF7 with an IC50 value 20 μg/ml, and HCT-116 cell lines with an IC50 value 44 μg/ml. Moreover, the purified enzyme showed significant antifungal activity against Fusarium oxysporum f. sp. lycopersici race 3 the causal agent of tomato wilt.