UV Mutagenesis In Trichoderma Reesei And Selective Breeding Of Its Hypercellulolytic Mutant Strains

Biomass in the form of cellulose, hemicellulose and lignin exists in large quantities. It is an effective way to achieve sustainable development of economic and society by developing and utilizing biomass energy. On conversion and utilization of biomass raw material, Trichoderma reesei performs one of important microorganism that produce cellulases, its enzymes are used widely in many field such as energy, biocontrol, food et al. In this study, Trichoderma reesei was mutated by UV lights, for the purpose of selecting mutant strain can produe high yields of cellulase, and increasing the enzyme yields further, decreasing the production cost.Based on selecting high-yielding cellulolase mutants in this thesis, T.reesei Rut C30 mutant strains were selected by using UV mutagenesis, and fermented condition for the mutant strain was optimized. Genomic DNA of Rut C30 and its mutant strains were analysed by Random amplified polymorphic DNA (RAPD).The spore suspension of T.reesei Rut C30 was mutated by UV irradiation,40s was confirmed as the optimize irradiated time, the death proportion of strains was 83.500% after 40s treatment by UV, and the positive mutation rate was 8.679%. A mutant strain A13 was finally obtained after twice selection, taking ratio of hydrolyzed ring diameter and colony diameter (H/d) on the Congo-red selective plate as indicator for the first time selection, then the filter paper activity was measured by fluid fermentation for the second time. The filter paper activity (FPA) of this mutant strain A13 was 4.328U/ml, increasing 55.350% compared to its original strain Rut C302.786U/ml. The genetic stability was determined by measuring the levels of enzyme production for 6 successive generations, and the mutant strain A13 was quite stable at 4.160U/ml for FPA.Optimized study was made on carbon source, complicated nitrogen source, and fermented temperature this three single factor, and the best condition was confirmed. Under the condition of 1.000% avicel cellulose as carbon source,0.150%peptone and 0.050%(NH4)2SO4 as complicated nitrogen source, fermented temperature 28℃, the FPA of A13 was 4.915U/ml, increasing 18.150% than 4.160U/ml before. Adding appropriate amount malt extract to fermented substrate including avicel cellulose, and study showed that malt extract can indeed improve T.reesei Rut C30 mutant strain to produce more cellulases. When added 1.000% malt extract on the base of 1.000% avicel cellulose, the FPA increased to 5.720U/ml. The best ratio of avicel cellulose and malt extract was performed by using Central Composite Design (CCD). Analysis of response surface was made against the experiment result of CCD, which showed that under the optimized fermented condition, as the concentration of avicel cellulose and malt extract was 13.19g/L and 11.03g/L respectively, the measured FPA can achieve 5.986U/ml, close to the top value 6.082U/ml predicted by soft ware.RAPD analysis was performed against genomic DNA of original and four mutant strains, also the RAPD-PCR reaction was optimized. After having selected six arbitrary primers, their amplified bands were performed to cluster analysis, the result showed that similarity coefficient of the five strains was between 0.6046512-0.8837209 which represented their kinship quite close but still of certain genetic variation.