Cloning, Site-directed Mutagenesis And Eukaryotic Expression Of Endoglucanase Gene From Trichoderma Viride

Cellulose is one of the most abundant renewable resources in the earth; efficient use of fiber resources may alleviate the increasingly serious shortage of food and energy all over the world. Cellulose is hydrolyzed into glucose by the cellulase synergy including three components:endoglucanase (EG), cellubiohydralases (CBH) andβ-glucosidases (BG).Cellulase activity affects its industrial application, so the cloning, expression of cellulase gene, and the modification, design of cellulose proteins or the use of other means to obtain cellulase with high activity have become the focus of recent research. Trichoderma viride is one of the fungi that produces the highest cellulase activity and widely distributes in nature, which is often saprophytic on wood, seeds and plant debris. In other case, Coptotermes formoscmus eating dead wood and other cellulose-rich wood substances has several intestinal microbes most of which are cellulose degradation bacteria.In this thesis, a fungal strain G-1 that is able to decompose cellulose was isolated from the rotten wood placed outside for many years, the other fungal strain G-2 that has the same ability was isolated from termite intestinal microbes. Both strains proved to be Trichoderma viride by colony and mycelium morphological observation as well as 18S rDNA DNA sequence analysis. Glucanase gene(EGⅡ) and cellubiohydralases gene(CBHⅡ) was cloned from G-1, twoβ-1,4-glucosidase gene(BGⅠ, BGⅡ) was cloned from G-2.Based on sequence alignment, EGⅡand CBHⅡgene sequences were analyzed and subjected to site-directed mutagenesis in some region to obtain two mutant sequences EGⅡ-mut and CBHⅡ-mut, which led to the mutation of its amino acid sequence in protein for the purpose of increasing the cellulase activity. After the EGⅡ, EGⅡ-mut, CBHⅡwere removed their signal peptide sequences in gene for construction of secreted expression vector pPIC9K-EGⅡ, pPIC9K-EGⅡ-mut, pPIC9K-CBHⅡ, they have been transferred into Pichia pastoris(Pichia pastoris) GS115 by electroporation. Transformants were screened on the MD, MM medium coloured with Congo red by using DNS method for determination of cellulase enzymic activity and recombinant Pichia yeast strains secreting high extracellular enzyme activity were obtained, they were GS115-EGⅡ-7, GS115-EGⅡ-mut-10, and GS115-CBHⅡ-2. The endoglucanase activities of GS115-EGⅡ-7, GS115-EGⅡ-mut-10 were 20.915U/ml, 24.110U/ml. The cellubiohydralases activity of GS115-CBHII-2 was 27.925 U/ml. the enzyme activity of extracellular endoglucanas secreted by the recombinant yeast strains Pichia with mutant sequence(EGⅡ-mut) was higher than that secreted by the recombinant Pichia pastoris with original sequence(EGⅡ), the results indicate that the mutations in some sites can affect the enzyme activity and these sites can be revealed as important domain which is the basis of using molecular biological methods to obtain high activity cellulase.In this paper, six Saccharomyces cerevisiae expression vectors also have been constructed; they are pYES2/CT/lacZ-EGⅡ, pYES2/CT/lacZ-EGⅡ-mut, pYES2/CT/lacZ-BGⅠ,PYES2/CT/lacZ-BⅡ, pYES2/CT-CBHⅡ, pYES2/CT-CBHⅡ-mut. As the basis for subsequent work, the next steps are to obtain recombinant strains of Saccharomyces cerevisiae, to select recombinant S. cerevisiae with the highest endoglucanase, cellubiohydralases, andβ-glucosidases activity, respectively, and to determine the fermentation time in accordance with the highest individual activity of enzyme, and then to mix yeast suspension in proportion to find the maxium rate in degradation of straw cellulose, the highest efficiency of cellulose hydrolysis and fermentation simultaneously.