Studies On The Selection And Molecular Identification Of High-yield Cellulase Strain And The Fermentation Process

Cellulase has great application value in almost all plants for the processing of raw materials Such as brewing, fruit and vegetable juice processing, food processing, food, feed,paper and extraction of effective components of Chinese herbal medicine, and plant in genetic engineering,cell engineering, oil extraction,textile industry, waste water treatment, bio-energy production, etc.It has many research papers about cellulose,but the serious problem was the strain sush as Trichoderma reesei, Penicillium which produced cellulose had lower the activity of cellulase.So the amout of the cellulase were lagerly, which caused high cost. High-yield cellulase producing strain, the optimum conditions of fermentation and suitable condition of enzyme reaction system can effectively reduce cellulase expenses.In this study, an excellent cellulase-producing strain was screened from the forest floor under rotting wood and soil in Yuelu Mountain. It was mutated sequentially by different physical methods,and microscopic observation and16s rDNA sequence alignment were used for identification mutant strain,besides, the fermentation condition and the conditions enzyme reaction system and enzymatic properties were studied.The main results were as follows:An excellent cellulase-producing strain A24was screened from the forest floor under rotting wood and soil in nature, and the activity of CMCase and FPAase were20.37U/mL,1.20U/mL, respectively.The original strain A24was treated with UV mutagenesis,and the best UV mutation time was7min.In this condition,a mutant strain UV-37had good genetic stability and the activity of CMCase and FPAase were25.35U/mL and1.33U/mL,respectivly.The best60Co-γ ray mutation dose was0.6KGy for the mutant strain UV-37.In this condition, a mutant strain Co-07who had good genetic stability and the activity of CMCase and FPAase were higher than the original strain137.16%and118.80%.The morphological characteristics of colony and mycelium and spore wire were observed by Microscope.From microscopic observation, radial colony, substrate mycelium and aerial hypha helicine spore wire were obtained,and it could be Streptomyces.16s rDNA was amplified and sequenced,and then NCBI database was used for BLAST alignments. Phylogenetic tree was constructed by MEGA5.0,and the mutation strain was Streptomyces misawanensis spp. in Streptomyces at last.The optimized fermentation conditions was:liquid volume50mL, speed of rocking175r/min,culuture temperature40℃,fermentation time120h by single factors and orthogonal experiment,and the activity of CMCase was74.06U/mL.According to the single factors, the best carbon source was straw powder2.5%,Nitrogen source had little effect on cellulase production,and the phosphate concentration was4%. Liquid volume, straw powder, KH2PO4which as the main influence factors,the final result of Response Surface Optimization showed that:three factors had significantly effect on cellulase production. The optimal model was constructed by SAS9.1soft ware, and R2=99.80%,which could fitted well with the factor experiment. Finally, the best formula of medium was determined by the RSM experimental design, and the medium formula was as follows:liquid volume(X1)=60mL, straw powder(X2)=2.8%,KH2PO4(X3)=4.2%, the activity of CMCase was94.16U/mL.CMCase reaction system was studied in mutation strain, and the optimum enzymatic reaction system as follows:enzyme liquid was diluted properly,in pH6.0, substrate concentration1.0%,enzymatic hydrolysis30min,at50℃,then added3.0mLDNS,boiling time10min. Study on the characteristic of cellulase, we concluded that enzyme had good stability when the temperature was between40℃and60℃,at70℃,the enzyme activity descended rapidly.the most favorable pH is6.0,the enzyme also had good stability,and CMCase declined quickly when pH below5or above7.