| Cellulose is the most abundant and cheapest recycled resource on the earth. It can be degraded and transformed into ethanol, fuel and other chemical products by fermentation. Cellulose ethanol has been regarded as the best alternative liquid fuel and gradually becomes a research focuse in industrial biotechnology. In this study, three kinds of cellulase from Trichoderma viride AS3.3711 were integrated into Saccharomyces cerevisiae genome simultaneously to obtain transformants which can directly digest the cellulose into glucose and further transform glucose into alcohol. Fermentation conditions for the production of ethanol from natural cellulose bagasse were investigated and optimized.After the Nhe I site on P-glucosidase I (bgl1) gene was synonymously mutated by PCR, the bgl1 was cloned into the high-copy integrative expression vector pScIKP, generating a recombinant plasmid named pScIKP-bgll. Then it was digested by a pair of isocaudamer Nhe I and Xba I, and the bgl1 expression cassete was ligated into a Nhe I digested plasmid pScIKP-ec, constructed the recombinant plasmid pScIKP-ecb. The positive recombinant clones were identified by colony PCR and restriction endonuclease digestion. After the recombinant plasmid pScIKP-ecb was linearized and transformed into S. cerevisiae by electroporation, high G418-resistant colony ecb3000 was obtained.We used filter paper activity (FPA) method to detect the total enzyme activity of cellulases in recombinant S.cerevisiae. To analyze the enzyme activity of EGⅢand CBHⅡ, the DNS method was employed using CMC as the substrate. Using pNPC and pNPG as specificities substrate, the CBHⅡand EGIII activity could be detected respectively. The results showed that the highest enzyme activity of recombinant cellulases appeared after 84 h of cultivation. The maximum enzyme activity of FPA and CMC was 0.583 u/mL and 3.70 u/mL respectively, and the specific activity of CBH II and EGIII could be achieved at 0.02 u/mL and 0.58 u/mL respectively.Based on orthogonal test, the factors influencing the growth of recombinant s.cerevisiae, such as glucose levels, corn steep liquor (CSL) levels and urea levels of culture medium, were investigated. The optimum condition was as follows:250 rpm of the shaker rotate speed,100 g/L of glucose,12 g/L of CSL and 1 g/L of urea. The effect degrees from high to low were glucose, CSL, rotate speed, and urea. In order to further optimize the nitrogen sources of the culture, single-factor experiment was studied and showed that the optimal conditions was 100 g/L of glucose,12 g/L of CSL and 7 g/L of (NH4)2SO4 at 30℃and 250 rpm.Drying bagasse was crushed to powder and sifted through a 35 mesh sieve to be used as sole carbon source. Through orthogonal experiments, the fermentation conditions of recombinant s.cerevisiae in flask were investigated. The results showed that the best conditions is:20% total inoculation concentration,28 g/L of CSL and 7 g/L of (NH4)2SO4 After fermentation condition of flask was determined, it was enlarged to a 5 L fermentor. In early period of fermentation, the fermentation condition was similar as the culture condition. In the primary and secondary period of fermentation, the main parameter is:fermentation temperature at 32℃, pH 4.5, anaerobic and unstirred. The results indicated that the highest concentration of ethanol could achieve 4.6% (v/v), which was 77.46% of the theoretical values.
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