Isolation And Optimization Of Its Cellulase Fermentation Of Trichoderma Viride, And Enzymatic Property Study Of Partial Cellulase Component

Microbe with cellulase activity was found in soil sample collected from Jinyan, Leshan. Sichuan. By primary and subtle screening, a mildew with relatively high cellulase activity was isolated, and identified Trichoderma viride by morphology. The isolated sample was fermented in liquid state after isolation, and its fermentation conditions were optimized by the author. The optimization result through experiment is described as below: 0.75% CMC-Na of total medium volume as the carbon source, 0.4% (NH4)2SO4 as the nitrogen source, 20% triangle flask bulk for medium volume, 5% inoculating volume, initializing medium pH to 4, 30℃, 180 rev/min to ferment for 96 hours. The leaven's utilities upon various substrates of different types of cellulose were studied, coming with the conclusion that it has lower activity on higher crystallized native cellulose such as absorbent carton while it keeps relatively high sensitivity on lower crystallized amorphous cellulose such as cellulose ramification. The activity of the main component, endo-l,4-B-glucanase, was enhanced, and its hydrolysable efficiency upon CMC reached 1.548U/mlleaven.Then further purification of CMCase component in cellulase leaven was made. The leaven was concentrated by transpiration under 50℃ after remove the thalli and some impurities through centrifuging, then coarse enzymatic component was isolated followed by salting out with 20-80% ammonium sulfate. A single enzyme component with CMCase activity was purified by a procedure including ion-exchange chromatography with DEAE Sepharose FF, gel chromatography with Sephacryl S-200, and displayed one single band on PAGE. The purified enzyme shows relatively high activity on CMC, while other crystalline cellulose such as cotton fiber and Avicel is not attacked to a significant extent. The purification multiple was 43.8, and the specific activity was 23.7 unit/mg protein with CMC as its substrate.Basic enzymatic characteristic of obtained CMCase was studied. A relative molecular weight of 59.2KD was detected by SDS-PAGE demonstrating one single strain shows that the protein is composed of one subunit of peptide strain. The protein's PI is 6.2 confirmed by isoelectrofocusing, and kinetics method shows its Km of optimal substrate CMC is 4.86 × 10-2g/ml. The optimal pH condition is 5.0, and it is relatively stable within pH4~6. The optimal temperature is 50℃, and it has relatively good stability under 65℃. Affluences on enzymatic activity of different ions were studied, under a condition of 1mmol/L ion concentration, some metal ions could promote its catalysis activity, such as Cu2+, Mn2+, Ca2+, especially the former gives a additional 50% rise of activity. The others works as inhibitor, and they are Hg2+, Ag+, Cd2+, K+, Ba2+. After mixed for 30min with Hg2+, the enzyme lost almost all its activity on CMC.