Study On Improving The β-Glucosidase Activity Of Cellulase From Trichoderma

There are quite bright prospects for the application of β-glucosidases in bioenergy,healthy products, food industry and other areas. Especially, as a rate-limiting enzyme,β-glucosidase plays a vital role in the process of cellulose degradation. Trichodermaviride is the world recognized one of cellulase producing strains with higher yield.Owing to the low level of β-glucosidase activity produced by Trichoderma viride, it isof crucial importance to screen the β-glucosidase high-producing strains.Trichoderma viride AS3.3711as a original strain was be induced by compoundmutation including UV-induced mutation，DES-mutation and NaNO2-mutation. MutantTrichoderma viride U3D2Na22was screened with the Congo red screening plates.β-glucosidase activity produced by Trichoderma viride U3D2Na22was3.2times higherthan the original strain.The composition of the enzyme production medium of Trichoderma virideU3D2Na22was analyzed with single factor experiment combined with orthogonaldesign method. The optimal enzyme production of medium is as follows:0.69%straw,0.33%lactose,2%peptone,0.5%(NH4)2SO4,0.3%NaH2PO4and0.2%CaCO3.The optimal fermentation conditions of Trichoderma viride U3D2Na22wereobtained with a large number of experiments. The optimal initial pH is4.0. Theoptimal fermentation temperature is30℃. The optimal incubation time is132hs. Whenat72hours of the fermentation, adding carbon and nitrogen source can obviouslyincrease the yield of β-glucosidase of Trichoderma viride U3D2Na22.Impurity protein in fermentation broth was removered by the method ofammonium sulfate fractionation, and a large number of ions were removered by themethod of dialysis, and the β-glucosidase was purified by the method of sephadexchromatography. Finally with the method of SDS-PAGE the relative molecular mass ofβ-Glucosidase (66.0KDa) was obtained.The optimal temperature of β-Glucosidase from the Trichoderma virideU3D2Na22is60℃. And there still was the excellent thermostability after beingincubated within the limit of40~60℃. The optimum pH for β-glucosidase was5.0,while it was stability under the acidic conditions in which pH was from3.0to6.0. Theβ-glucosidase acticity was easily promoted by metal ions Na+、Mg2+、Mn2+、Fe2+、Co2+and Al3+and inhibited by Ag+、Fe3+、Zn2+、K+and Cu2+, it was also enhenced by the chelant EDTA.