Screening Fungus Strain For High Yield Of Alkaline Cellulase And Cloning, Expression Of Its Cellulase Gene

Cellulose is the most abundant renewable sources in the world. Many researches showed that it had been technically feasible to obtain fermentable carbohydrate by the enzymatic degradation of plant biomass for ethanol production or else. During this process, cellulase is necessarily needed. To obtain one gene engenering cellulase strain with high production, the following research work has been done in this paper.1. Bacillus sp.CYl-3 which had high CMCase producing ability was isolated from waste pulp and identified by Congo red dying combined with filtrates cultured enzyme activity measuring. Optimal growing conditions of the strain were 34-46℃, pH 6.2—8.8. Results in shaking flask culture showed that the maximum cellulase activity was reached at 34-43℃ and pH 7.8-8.8 when cultured for 36h.The optimal conditions for the enzymatic reaction were 60℃ and pH 5.5-8.The cellulase isolated from the strain charactered as high alkaline and temperature stability. The Cu²⁺, Mn(2+), Mg(2+) and Ca(2+) can promote enzyme activity, while Co(2+) and Zn(2+) inhibit it. The strain could produce endo-1,4-β -glucanase and cellulase activity reaches 13.6U/mL under the most suitable condition. This cellulase preparation had a good potential application in the textile biofinishing and detergent industry.2. A genomic library of Bacillus sp.CYl-3 was constructed in Escherichia coli strain DH_5α by ligating BamH I-digested pBluSKM vector with 2-7kb Sau3A I-digested genomic DNA from Bacillus sp.CYl-3. Six clones expressing carboxymethylcellulase(CMCase) activity were isolated from nearly 100,000 clones by screening this library with Congo red dying. These clones showed the identification when they were digested by restriction endonuclease, they were thought to be the same clone.3. The nucleotide sequence of a 2189 bp fragment was found in plasmid pGJc-1, which contain a 1593 bp open reading frame(ORF), named celC. It is preceded by a potential...