Cloning And Expression For Kitchen Garbage EGⅢ Cellulase Degradation

Big founder mark song simplified trichoderma viride produces cellulose enzyme can degrade cellulose, cellulose enzyme can break down cellulose to produce glucose compound enzyme known collectively, including the endoglucanase(endo-glucanase), cellobiose hydrolysis enzyme(cellobihydrolase) and beta-glycosidase enzymes (betal,4-glueosidase), these three enzymes synergy to convert cellulose into glucose. Endoglucanases play an important role in non-crystalline cellulose and soluble substrate, such as carboxymethyl cellulose and hydroxyethyl cellulose, Random hydrolysis of the cellulose chain beta-glucosidase key to its viscosity decreased rapidly and reducing groups increased, and produce glucose, cellobiose, and a variety of different size of short-chain fiber glucan.In this study, using molecular biology means to build engineered bacteria,which can efficiently degrade food waste. The study results are as follows:1.using the CTAB method to extract the DNA, T.viride DNA as a template, the gene fragment obtained after PCR; PCR product was purified and ligated vector, and transformed into E. coli extract transforming plasmid in order to T.viride DNA as a template, the gene fragment obtained after PCR, and its single restriction enzyme digestion and double-digested authentication, identified by endoglucanase EGⅢ successfully expressed in E. coli, and Successfully obtained gene fragment.2. Bioinformatic analysis of the gene fragment, including the restriction sites, the genetic characteristics of the gene analysis, amino acids, nucleotides and codons, analysis showed that:shield with116kinds of enzymes found44recognitionsites, the full-length sequence of length1849bp, encoding616amino acids.3. Recombinant Pichia pastoris expression product for SDS-PAGE analysis and enzyme activity studies have shown that effective secretion and efficient expression of endoglucanase, the highest enzyme activity reached681U/mL culture medium size of approximately61kDa proteinstrip, the expression product having a normal biological activity is consistent with the the natural the endoglucanase nature of the basic. 4. Induction time, inoculum size, pH value by setting different gradient to explore the recombinant yeast to optimize the expression conditions. Optimal induction time for60h, the highest activity is530U/mL; starting inoculum size and select the most reasonable when OD600=4; Pichia pastoris has the ability to self-adjust the pH=5.8case.