Studies On The Breeding Of New Cellulase Strain By Protoplast Fusion And The Fermentation Process

Cellulase which can degrade cellulose into carbohydrate has been widely used in industry. There are many reports about cellulase, and the studies are given priority to Trichoderma viride fungi, Penicillium fungi and Aspergillus fungi. The composition of different sources of cellulase is not alike. Penicillium fungi can secrete comprehensive enzymatic system and produce more beta glycosidase enzymes than Trichoderma viride. But the enzymatic activity produced by Penicillium fungi is low, and the adaptability to pH and temperature is not strong enough. Bacillus subtilis has strong resistance with heat, acid alkali resistance, drought resistance, ultraviolet, and organic solvents. A new strain with high cellulose enzyme activity and strong resistance had been screened by protoplast fusion between Penicillium Q5and Bacillus subtilis K3for parents. The main results as follows:The conditions of protoplast formation and regeneration were optimized by measuring the dried mycelia, counting the protoplasts with microscope and calculating the regeneration rate. It turned out that the conditions including the pretreatment of strains, the content of cellulase and time for reaction were optimized by enzymatic hydrolytic methods. The amount of protoplast from Penicillium Q5gained a maximum,7.35×10cfu/mL, and the corresponding above used values were0.5%Glycolic,10%sugar,10mg/mL lysozyme and snail enzyme (1:1),3h, and35＞. The amount of protoplast from K3maximized,1.46X107cfu/mL, and the corresponding above used values were4U/mL penicillin,1mg/mL lysozyme,1h, and35℃.Inactivating conditions of strains Q5&K3were determined by survival rate. In65℃, strains Q5&K3protoplasts were treated for1h and2.5h respectively. It was suitable for strains Q5&K3protoplast to receive15min and25min radiation treatments under30W UV-light in the distance of20cm.The conditions of fusion were optimized by orthogonal analyses:the fusion temperature38℃, the fusion time10min, the concentration of PEG40%, the fusion rate was3.08×10"7. Based on the colony characteristics of strains from regeneration media and the transparent ring size on the Congo red culture medium, five recombinant strains had been selected. Recombinant strains R8, R17, R35, R62, R158were obtained.Through morphology observation, analysis of biochemical properties, genetic stability and resistance research, a recombinants which gained higher resistance ability and cellulase activity was attained by screening.Cellulase of the fusant R62in pH8reserved74.01%of the cellulase activity at pH6. From pH6to pH10, it reserved57.35%of the enzymatic activity. After15minutes’heat treatment under60℃, the cellulase produced by R62enhanced37.66%compared with strain Q5which was treated under room temperature, and it was six times with strain Q5under60℃.The optimal culture medium formula was obtained by monofactorial experiments and response surface methodology:starch2.99%, medicine medium3.02%, microcrystalline cellulose4.04%, ammonium sulfate1.5%. The optimal enzyme production conditions were speed220r/min, temperature30℃, pH5.5, fermentation time108h. Under the optimal conditions, the cellulose activity which had improved6%higher than the initial enzyme activity were189.39U/mL.