Expression Of Cellobiase Gene Of Aspergillus Niger In Yeast Cells

Cellobiase(CB) is a primary constituent of cellulase, it plays an important part in the hydrolysis of cellulose. The present cellobiase activity of cellulase preparation is so low that couldn't hydrolyze cellulose efficiently. In order to elevate enzyme efficiency, increa se the cellobiase activity is meaningful. In this thesis, the gene of cellobiase(bgl) which comes from Aspergillus niger was expressed in Saccharomyces cerevisiae and Pichia pas toris respectively and the fermentation parameters were optimized in shake flask. The sy nergistic effect of cellobiase and cellulase of Trichoderma reesei was investigated in cell ulose hydrolysation.The bgl gene was expressed in Saccharomyces cerevisiae by use of integrative plasmi d.7 strains of recombinant Saccharomyces cerevisiae were selected with G418 Geneticin and validated by PCR experiment. However, the transformants couldn't utilize cellobiose to produce ethanol and the cellobiase activity wasn't detected in fermentation broth. Thi s may be caused by inactivity of cellobiase which were high glycosylated.In Pichia pastoris, the bgl gene was expressed by pPIC9K. A recombinant Pichia pas toris with high copies of bgl gene were selected and validated by PCR experiment. Besi des, PCR result showed that foreign gene inserted into genome of Pichia pastoris which means the transformant could utilize methanol efficiently. The transformant could produ ce active cellobiase. The molecular weight of cellobiase produced by Pichia pastoris was higher than that produced by Aspergillus niger. But they resembled with each other in the optimal temperature and pH value.The expression level of CB in BMMY media and YG media were discussed in shake flask and the parameters were optimized. When BMMY media was used, the cellobiase activity reached 11.8 IU/mL afer 8 days'fermentation under optimized conditions which as 1.37 times as optimized before. While YG media was used, the cellobiase activity r eached 9.01 IU/mL. Through the production of cellobiase was relatively low in YG med ia, the advantages of low cost and simple operation make YG media suitable for applica tion in industry.The capability of cellobiase cooperated with cellulase to hydrolyze cellulose was resea rched by use of acid pretreated corn cobs as substrate. The results suggested that no di fference lies between the two kinds of cellobiase which have different origins and the yi eld of enzymatic hydrolysis increased from 62.4% to 80.7% by addition of cellobiase. T he parameters of enzymatic hydrolysis were optimized. The yield of enzymatic hydrolysi s reached 93.1% after 72 hours under optimized conditions. But the optimal time of hyd rolysis was 48 hours.In this thesis, the bgl gene of Aspergillus niger was transformed into Pichia pastoris and the high expression of active cellobiase was realized. The results are valuable in pr oducing cellobiase by the way of submerged fermentation and promoting the course of i ndustrialization of ethanol produced from cellulosic material.