Trichoderma Viride Endoglucanase Iii In Saccharomyces Cerevisiae Surface Display And Characterization Of Within

Cellulose, the major component of the cell wall of plants, is the most abundant and renewable carbohydrate. And as such, cellulose degradation is of considerable ecological, agricultural and commercial importance. The filamentous fungus Trichoderma viride is well known as strongly cellulolytic and xylanolytic microorganisms. Trichoderma viride produces a cellulosome complex consisting of endoglucanases[EG(EC3.2.1.4)], cellobiohydrolases [CBH(EC3.2.1.91)] and (3-glucosidases[BG (EC 3.2.1.21)].Yeast cell surface display system has so many advantages. Among the many advantages of the system, in which proteins are genetically displayed on the cell surface, are easy reproduction of the displayed biocatalysts and easy separation of product from catalyst. As proteins and peptides of various kinds can be displayed on the yeast cell surface, the system is expected to allow the preparation of functional proteins. With its ability to express many of the functional proteins necessary for post-translational modification and in a range of different sizes, the yeast based molecular display system appears uniquely useful among the various display systems so far developed.The Trichoderma viride Gold No.1, stored in our lab, has the ability to produce high level of cellulases. Therefore, it is necessary to study the cellulases. EndoglucanaseⅢwhich plays a very important role in cellulose degradation, is also the main goal of this paper. We get eg3 gene by PCR from T. viride genomic DNA, and clone eg3 gene into pMD18-T vecter. The recombined plasmid pMD18T-eg3 transform into E.coli DH5a, so the eg3 gene can store and replicate in the E.coli. The eg3 gene is sequenced. Compared the sequence of eg3 gene and the data from NCBI, we find that there is no interon in eg3 gene. However, there is no reference.The recombined pAJ401-eg3 transform into S. cerevisiae H158. So the EGⅢis expressed in S. cerevisiae H158. The EGⅢis detected by Congo-Red Staining method. The molecular mass of EGⅢis about 54 kDa by SDS-PAGE. Enzymatic activity assay shows that there is no significant difference between EGⅢand the native EGⅢfrom T. viride. The EGⅢreaches the maximum 297IU/mL. The optimum pH is 6.0, and the optimum temperature is 55℃.The eg3 gene is cloned into the cell-surface display vector pYD1, generating a recombinant plasmid named pYD1-eg3, which is then transformed into S. cerevisiae EBY100. The cell-surface displayed EGⅢis detected by Congo-Red Staining method and enzyme activity assay. The results show that the cell-surface displayed EGIII can produced hydrolysis halos on the Congo-Red-CMC plate; The enzyme activity of the cell-surface displayed EGⅢreaches the maximum 257 IU/g after induced for 48 h; The optimum pH is 7.0, and enzyme activity is stable in the pH range 6.0～8.0; The optimum temperature is 65℃, and incubated for 1h and 3h at 65℃,87% and 45% of the cell-surface displayed EGⅢactivity are remained respectively; Mg2+, Fe2+ and Mn2+ are the activators, Cu2+, Fe3+, Co2+ and Ag+ are the inhibitors of the cell-surface displayed EGⅢ.