Anti-repressor Breeding Of Cellulase-producing Strain And Its Optimization Of Cellulase-producing Conditions

Cellulase is a multi-component enzyme complex, which is generally composed of endo-β-1,4-glucanase, exo-(3-1,4-glucanase andβ-1,4-glucosidase. Cellulose is interested by many scholars, mainly because it has a very wide range of applications, such as food industry, textile industry, animal husbandry, energy and so on. The industrialization process of cellulase is limited by its high production costs. The important ways to raise enzyme yield and reduce enzyme costs are increasing of cellulase producing capability and optimizing of fermentation conditions.In this study, a higher yield cellulase-producting wild strain was screened. Then through UV and microwave irradiation, combining with repressor agent screening, a cellulase-producing mutant was bred, which had higher ability of anti-repressor and enzyme production. Subsequently, the fermentation conditions of mutant were optimized by response surface methodology and parts of enzymatic properties of cellulase was discussed. The main contents and results of this thesis are as follows:Samples were collected from dead branches and rotten soil in Yimeng. A strain of high CMCA and FPA was screened by carboxymethylcellulose congo red medium and fermentation culture. By observing colony morphology, microscopic morphology of mycelium and spores, and based on "Fungal Identification Manual", "ordinary Mycology", it was identified as Trichoderma viride (T. Viride Persex Fx NS90).The effect of different concentrations of glycerol, glucose, cellobiose on Trichoderma viride growth and enzyme production was studied. The results show that, in the concentration range of 4%, the biomass of strain growing showed positive correlation to the concentrations of glycerol, glucose, cellobiose in the medium.When the concentration of glycerol in the medium was 0.4%, the production of enzyme was promoted, but the concentration was 2%, the production was repressed. Cellobiose did not show significant effects on the enzyme production.Except the 0.4% concentration, other concentrations of glucose all repress the enzyme production.After 90s UV radiation and 60s microwave treatment, compared to the original strain in the same concentration of repressor, the CMCA and FPA of the mutant produced had increased 41% and 44.95% respectively. In the growth process of the mutant strain, the color of mutant colony changed from yellow to green, and the spores presented yellow during the cells culture.The fermentation conditions of mutant UW-Ⅲwas optimizated by response surface analysis. According to the model prediction and experimental verification, response surface model for enzyme production fermentation conditions of mutant UW-Ⅲwere optimized that microcrystalline cellulose, wheat bran and fermentation time were the value was 6.1 g/L,8.5 g/L and 96 h, and the CMCA was up to 362.523 U/mL.Parts of the enzymatic properties, including optimum temperature, optimum pH, temperature stability, pH stability and the effect of glycerol, glucose, cellobiose addition was studied. The results showed that:the optimum temperature of cellulase was 50℃. The optimum pH was around 5.0. Cellulose has a relatively good temperature stability and pH stability. Under the low amount of addition, cellobiose has positive effects on cellulose. Glucose has strong inhibition to cellulase.