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The Trichoderma viride endoglucanase III in Saccharomyces cerevisiae surface display and enzymatic properties

Author: ZhangFuLiang
Tutor: YangGe
School: Qufu Normal University
Course: Biochemistry and Molecular Biology
Keywords: Endoglucanase III Trichoderma viride Saccharomyces cerevisiae surface display
CLC: Q814
Type: Master's thesis
Year: 2011
Downloads: 49
Quote: 1
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Abstract


Cellulose (cellulose) is one of the main components of the plant cell wall, is the Earth's most abundant number but do not get full use of renewable resources. Therefore, the degradation of cellulose has important environmental, agricultural and commercial significance. Filamentous fungus Trichoderma viride is known to decompose cellulose and hemicellulose, containing the complete degradation of cellulose Enzymes: endoglucanases, exoglucanases, and β-glucosidase. Yeast display system has many advantages: the protein was demonstrated to the surface of the cell, the biocatalysis process can be repeated and catalytic products are easily separated; because of a variety of proteins and peptides can be displayed to the surface of the cell, so that the system can be used for the pre-preparation of the functional protein; yeast display system of functional protein can be a series of different sizes of the modification of the post-transcriptional processing. So, compared to other display systems, yeast display system has a unique advantage, to obtain a significant development. Starting from the the Trichoderma viride gold kept in our laboratory to efficiently degrade cellulose No. 1 (Trichoderma viride Gold No.1) strains, genomic DNA was extracted, amplified by PCR to obtain the endoglucanase Ⅲ (eg3) gene This gene is transformed into E. coli saved. Preliminary judgment of the gene does not contain introns by sequencing combined with data from the NCBI database. Internal the endoglucanase Ⅲ (eg3) gene of Saccharomyces cerevisiae heterologous expression. With yeast S. cerevisiaeHl58 heterologous Table Da Neiqie glucanase Ⅲ (eg3) gene expression product EG III and nature study. Congo red staining qualitative detection EG Ⅲ hydrolysis circle by Congo red tablet judge eukaryotic expressed EG Ⅲ is to have the biological activity of; With SDS-PAGE determination EG Ⅲ the relative molecular quality, found that the relative molecular weight for 54KDa this than the experimental is expected to be slightly larger; measured by the enzymatic properties of EG Ⅲ most suitable reaction temperature of 55 ° C. The optimum pH was 6.0, heterologous expression of EG Ⅲ maximal activities 297IU/mL. These basic research by the Surface Display EG III laid a solid foundation. Internally endoglucanase Ⅲ (EG Ⅲ) carried out the study of yeast surface display. With Saccharomyces cerevisiae the surface display technology systems S.cerevisiaeEBY100, the EG Ⅲ displayed to the yeast cell surface, the EG III immobilized in the yeast cell surface by the hydrolysis of ring of the Congo red staining to determine the activity of the cell surface display of EG III; through Enzymology The measured properties of the surface display EG Ⅲ galactose induced 48h enzyme activity up to 257IU / g, the optimum reaction temperature of 65 ° C at 65 ℃ for 1h and 3h, the enzyme activity is still maintained at 87% and 45%, respectively; The optimum pH is 7.0, and the high activity between pH6.0 ~ 8.0; studied the influence of metal ions on the surface display of EG III, found Mg2, Fe2, Mn2 activator CU2, Fe3, CO2 and Ag is inhibited agent.

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