| Endoglucanase I (EGI) is an important enzyme of Trichoderma reesei, it cleaves internal glycosidic bonds in cellulose chains. EGI consists of two domains joined together by a glycosylated interdomain linker peptide. The larger catalytic domain contains the active site, which carries out general acid catalyzed hydrolysis of the β-1,4-glycosidic bonds in cellulose. The smaller cellulose-binding domain (CBD) improves binding of enzymes onto cellulose and enhances its enzymatic degradation.Although the hydrolysis mechanism and the substrate specificity of EGI, while it is cleaving the β-l,4-glycosidic bonds in cellulose, have been basically clarified, how CBD module cooperates with CD and Linker modules in the process of cellulose hydrolysis has not been reasonably explained yet.In this thesis, we hope generate a series of hybrid endoglucanases, which have the same CD module and Linker module with EGI except CBD module. An Error-PCR library of the gene of CBD from EGI has been constructed. It would be very hard to use the traditional screening method to obtain the genes of CBD of different cellulose-binding ability. Compared with the traditional screening method, phage display has more advantages.Phage display is a powerful method for selecting and engineering polypeptides with desired binding specificities. The method relies on the fact that if gene fragments encoding polypeptides are fused to M13 coat protein genes, these 'fusion genes' can be incorporated in bacteriophage particles that also display the heterologous proteins on their surfaces. In this way, a physical linkage is established between phenotype and genotype. Using simple molecular biology techniques, large and diverse phage-displayed polypeptide libraries can be generated. Phage displaying polypeptides with a desired binding specificity can be selected from library pools by binding to an immobilized ligand, and the sequences of selected polypeptides can be deduced from the sequence of the encapsulated DNA.In addition, phage display application to the directed evolution of enzymes is beginning to emerge. Until now, the research of phage-display cellulase has not beenpublished yet. If a cellulase is successfully displayed on the surface of phage, the result will be clearly helpful to the research of phage display as a tool for the directed evolution of cellulase. This is another emphasis in this paper.A phage enzyme of EGI has been constructed for proving the feasibility of phage display in cellullolytic enzymes. The phage enzyme EGI has proved to be active, which not only has the ability of binding cellulose by enzyme -linked immunosorbent assay (ELISA), but also has catalytic efficiency on insoluble substrate. The value of CMCase activity is 7.0 * 10"16IU /pfu. The results showed the phage display used in cellullolytic enzymes was feasibile.Despite considerable efforts, the exact role and function of the CBDs in the enzymatic action of cellulases are not well understood. In this study, CBD has been displayed on phage surface, which proved to have the ability of binding cellulose by ELISA. Then a 5xl05 size phage library displaying the CBDs derived from the gene of CBDEGiby Error-prone PCRhas been constructed. This library will be screened for obtaining different CBDs of binding ability to cellulose by phage display, then a series of recombinant enzymes will be constructed. Compare their performances with the corresponding wild-type enzyme on soluble and cystalline substrates, and then discuss the role of CBD in cellulose degradation.Furthermore, the EGI and CBD recombinant phages infected E.coll HB2151 cells. IPTG induced expression of EGI and CBD in E.coll' HB2151 cells. After assaying the supernantant, periplasmic extract, and whole cell extract, that soluble EGI and CBD were found to be produced and concentrated in the supernantant and confirmed by SDS-PAGE. Some efforts of purifying CBD protein have also been done. The purified CBD will be used to measure the kinetic parameters for getting more useful information. |
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