Study On Cellulase Production For Non-specific Chitosan Hydrolysis By Trichoderma Viride

The specific enzymes and the non-specific enzymes can both degrade chitosan to chitooligosaccharides. Cellulase produced by Trichoderma viride is an excellent non-specific enzyme preparation. And it can degrade chitosan to chitooligosaccharides of polymeric degree 5⁸ with high biological activites. So cellulase produced by Trichoderma viride can be used to prepare chitooligosaccharides with high biological activites.The mutant strain Trichoderma viride XY-131 with stable genetic speciality, which had been treated with UV and NTG, was isolated with effectual screening method from the acidity-cellulase-producing strain Y-19. The chitosanase activity of the initial strain was 0.200 IU/mL. The chitosanase activity of the mutant was 0.403 IU/mL.Response Surface Methodology and Design Expert 7.1.3 were employed to optimize the fermentation medium based on single factor test. The obtained optimal medium were as follows: Avicel 2.56%, corn cob 1.03%, cottonseed cake meal 4.79%, wheat bran 0.5%, KH₂PO₄ 0.2%, (NH₄)2SO₄ 0.2%, CaCl₂ 0.03%, MgSO₄ 0.03%. The optimal fermentation conditions were culture temperature 30℃, initial pH 5.5, volume of medium 75mL/250mL, the speed of shaker 160 r/min, inoculum size 10%(v/v), adding 0.1g/100mL Tween-60 at the 48h hour. Under these conditions, the chitosanase activity could be achieved 2.38IU/mL. The chitosanase activity was 2.68IU/mL for 30L fermenter.The concentrate enzymes were prepared by plate filter,ultrafiltration technology,ethanol precipitation and freezing centrifugation. The total yield of enzymes was 41%. And the chitosanase activity of the concentrate liquid enzymes was 14.06IU/mL . The chitosanase activity of enzyme power was 130.04IU/g.The enzyme properties were investigated preliminarily. The optimum pH of cellulase acting on chitosan was 5.0. The optimum temperature was 60℃. The stable pH ranger, the thermal stability were obtained. The enzyme could stay steady at the temperature under 50℃, pH4.0⁵.0. Chitosanase activity was activated by metal ions Mn²⁺,Co²⁺ and Mg²⁺. Chitosanase activity was inhibited by metal ion Fe²⁺.