Study On Selection, Fermentation And Enzymatic Characteristic Of A Cellulase Producing Strain

In the present thesis, microbe with cellulase activity was found in dark and humid environment, insect digestive tract and soil sample. By primary screening of crystal violet medium and secondary screening of Congo red cellulose choice medium, about 30 strains which could use cellulose as only carbon source was found. By contrasting to Aspergillus niger, Trichoderma virde, Aspergillus gansu preserved by Anhui Provincial Key lab of Microbial Control, the strain RCEF4093 was chosen as target strain. RCEF4093 was identified as Penicillium, Moniliaceae, Moniliales, Hyphomycetes, Deuteromycotina,by morphology and physiological characteristics. The strain RCEF4093 of penicillium with the highest cellulase activity was chose to study the optimum of fermentation conditions for cellulase, Glucanase purification and enzymological character.Cellulase was induced when basic carbon source was depleted, the medium was covered with only insoluble cellulose. All the shake flask and the slant medium without any reducing sugar except elicitor experiment in the thesis.Cellulase production research was carried out by fermentation with strain RCEF-4093.The results showed that 0.3% rice straw powder and 0.2% bran as carbon source,2 g?L-1ammonium sulfate as nitrogen source, 0.01%arginine,the initial pH is 5.5,the temperature is 30℃,under the above conditions and growing 90h,the strain got the highest activities.The activities of CMCase and FPase in the fermented broth are 293.74IU/mL and 67.31IU/mL.Then, further purification of CMCase component, Glucanase was made. Some other impurities was removed through metal mesh filting, centrifuging and rotary evaporator. With the process of enzyme purification the optimal concentration was 40%-60% saturation determined by two ammonium sulfate precipitation experiments, the lowest specific activity of cultural supernatant, the most specific activity of precipitation, no protein denaturation and the most yield. Salt ions was removed through dialysis. A main enzyme component of CMCase activity was purified by Sephadex G-75, the purified enzyme shows high activity on CMC, but little hybrid protein still exists. The purification multiple was 2.54 Basic enzymatic characteristic of obtained CMCase was studied. 3% of concentration gel and 10% of separation gel was chose as electrophoresis plastic through literature search and comparison, low-molecular-weight standard protein as marker for electrophoresis. A relative molecular weight of 39.2kD was detected by SDS-PAGE and the protein is composed of one subunit of peptide strain. The kinetics method shows its Km of optimal substrate CMC is6.07×10-2g/mL,the optimal pH is 5, and it is relatively stable within pH 4-6.The optimal temperature is 50℃, and it is stable under 60℃.