Production and Characterization of crude chitinase from Trichoderma virens

Abstract

Chitinase hydrolyzes chitin, a biopolymer of N-acetylglucosamine which is being widely used in biological and agricultural research. Optimization of chitinase production by Trichoderma virens was conducted using batch cultures of Trichoderma Minimal Medium (TM medium) that consists of 2.0% (w/v) colloidal chitin as substrate. Fermentation was performed at agitation speed of 200 rpm and incubated at 30ºC in 250 ml shake flasks. Optimum chitin concentration was obtained at 4.0% (w/v). The addition of a stimulant, methanol, at 0.2% (v/v) enhances chitinase production by 71.4%. Higher concentrations of methanol would inhibit chitinase production. Works for optimizing agitation speed was carried out in a 2.0 L batch culture using B.Braun Biostat-B bioreactor. The optimum conditions for chitinase productions were performed in the same bioreactor using disc turbine impellers (diameter of 5.5 cm) with air flow rate of 1.0 l/m and agitation speed of 250 rpm resulting in an increased chitinase activity of 0.447 U/ml compared to 0.052 U/ml in shake flask fermentation. This improvement of 88.4% might be due to efficient mixing and high oxygen transfer rate which is essential for fungal growth. Characterization of crude chitinase was carried out to determine the optimal conditions for the crude chitinase activity to hydrolyze chitin and the crude enzyme stability. The crucial physical factors affecting the optimum and stability of chitinase activity were pH and temperature. pH optimum was achieved at pH 4.0 in citrate phosphate buffer and chitinase was very stable at pH 3.0-7.0, with 350% of residual activity is retained at pH 7.0. Optimum temperature for chitinase activity was achieved at 50ºC while its stability decreases with the increment of temperature. Only 37.5% of residual chitinase activity was retained at 70ºC.