Dynamic Zymography And Secretome Analysis Of Tricho Derma Reesei QM9414Grown On Different Carbon Sources

Due to the increasing use of fossil fuel energy and the supply of them are limited, Human beings are facing energy crisis and serious environmental problems. It hight time to the development of biomass energy. Lignocellulosic biomass is an abundant renewable resource that can be used for the production of alternative transportation fuels. But due to their complex structures and various constituents, it is difficult to microbial to degradate them.The filamentous fungus Trichoderma reesei is one of the most exceptional producers of cellulases and hemicellulases and represents a paradigm for commercial scaling of cellulolytic enzymes. since2008,the gene of Trichoderma reesei has been sequenced. On the basis of this scientists have did a lot of research on genomics,transcriptomics,proteomics,but the regulation of cellulolytic enymes expression by hypercellulolytic Trichoderma reesei is a complex process and our knowledge is still incomplete. In current study, expression of the celluloytic enzymes in Trichoderma reeseican be induced by some disaccharides such as sophorose and D-lactose.but the what was the true inducer,how did the Trichoderma reesei sense the substrate and regulate the secretion of relevant enzymes are not exactly known. In order to better understudy those questions,we did a lot of researchs and got some conclusion as follows:1.Analysis of extracellular enzymes and reducing sugar by dynamic zymography and FACE-PAGEIn combination with Native-PAGE, we can analyze the extracellular cellulolytic enzymes not only by the enzyme activity but also can make a separation of isoenzymes.Native-PAGE can make our research become more deeply.FACE-PAGE is a method that is higher resolution than the determination of reducing sugar method.It can not only charaterize the concentration of the sugar but also the type.The quantitative analysis of the concentration changes in sugar by ImageJ program has been proved to be reliable.2.The dynamic zymography analysis of Trichoderma reesei grown on different carbon sourcesThe regulation of sugar and the production of enzyme is analysis by Native-PAGE in combination with FACE-PAGE. We found that monose do not induce cellulolytic enzymes,disaccharide can induce part of cellulolytic enzymes(such as D-lactose and low concentration of cellobiose). Xylan can induce more cellulolytic enzymes than lactose due to lots of oligosaccharide. Corn stalks and wheat bran(CSWB) can induce the most cellulolytic enzymes including cellulase and hemicellulase. By the analysis of FACE-PAGE, it shows that from the degradation process, a large amount of oligosaccharide has been produced since the48th hour and at the same time a small amount of cellulase and xylanase has been produced. When PHI05mixed with D-lactose and xylose,respectively in a1:1ratio, it shown that it can greatly improve the ability to indce more cellulolytic enzymes than D-latose and xylose only. Focuse on the different carbon sources,we can make a conclusion that oligosaccharide was the true inducer of cellulolytic enzymes. 3.The proteomics analysis of Trichoderma reesei grown on different carbon sourcesThe results of the four carbon sources (1%glycerinum,1%D-lactose,1%xylan,1%CSWB) indicate that CSWB induce the most and total cellulolytic enzymes, followed by D-Lactose and xylan. D-lactose can only induce the cellulase and little xylanase. Xylan can induce a lot of hemicellulase but little cellulase. So according to the results, we found that Trichoderma reesei can produce different cellulolytic enzymes according different carbon sources.4.The proteomics analysis on the time course of Trichoderma reesei grown on CSWBThe express of cellulolytic enzymes ofTrichoderma reeseigrown on CSWB for5days is different. According to the degradation process, in the eatly48h-72h, CBH1,CBH2were the major cellulolytic enzymes and their expression were increased. In the96h-120h,the most major cellulolytic enzymes were xylanase(XYN2,XYN3),acetylxylan esterases,xyloglucan. The results indicate that the cellulolytic enzymes expressed by Trichoderma reesei can penetrate to internal structures of biomass. At the early of the degradation process there are a lot of oligosaccharide which came from a little hemicellulase, as time goes on, a lot of hemicellulase are induced.In the degradation of biomass, cellulose can be completely degradation by Trichoderma reesei, but hemicellulose may not for Trichoderma reesei is lack of pectinase and it only have some main hemicellulase(such as xylanase,xyloglucan,mannase,arabinfuranosidease) that were needed in degradation of hemicellulose.