Research On Expression In E.coli And Molecular Evolution Of Endoglucanase III From Trichoderma Viride

The cellulose is one of the most abundant reproducible material in the earth, so if is used to produce ethanol for fuel the crisis of energy sources and environmental pollution etc. will be settled. Beacause of this, the cellulose ethanol has been considered to be the future fuel by people. From now on, the cellulosic biomass has been regarded as the greatest potential raw material for the fuel ethanol production. However,the cellulose hasn't been fully utilized because of its dense structure which is difficulty to hydrolysis to be the saccharide for microorganisms using, which is the first difficulty of industrialization. So far as we know, cellulase can hydrolyze the cellulose to simple sugars for fermentation to ethanol,whilethe low activity of cellulase and high cost of production have seriously hampered its industrial application. Now, more and more people carry out the researches of cellulase in the world.In order to solve the problem that the activity was too low to determine when the cellulase gene expressed in the E.coli, and improve the cellulases' activity, the following researches were done in this paper. EndoglucanseⅢgene egl3 was subcloned into expression vector pET-22b (+) and a recombinant plasmid pET-egl3 was constructed then transformed into E.coli BL21 (DE3). Recombinant protein EndoglucanseⅢwas purified by metal chelating affinity chromatography, the moleculer weight of EGⅢwas 42kDa by the SDS-PAGE analyzing. And the specific activity of EGⅢwas 6U/mg. Its optimal temperature and pH were 60℃and 4.0, respectively.From now on, the three dimensional structure of endoglucanseⅢof T. viride is unknown. In order to obtain mutants with higher activity, site-saturation mutagenesis of irrational design was performed. Based on the crystal structure of endoglucanase from Thermoascus aurantiacus , 3D model of EGⅢfrom T.viride was constructed by the method of homologous modeling protein structure prediction on SWISS-MODEL program. Analysing the key sites of the model combined with energy calculation by the computer-aided design software Prosa2003, four mutants R130,E218,Q246 and E329 were selected in the catalytic domain of EGⅢ. What's a pity, we didn't selct any mutants whose activity increased from Q246 and E329. However, the mutants R130P and E218F were obtained using site-saturation mutagenesis, whose enzyme activities were improved obviously. At the same time characteristics of the two mutations were investigated. Their specific activities were 2.8 and 3.45 times of the wild type. Furthermore, Km and Kcat of E218F mutation were increased one time and 5.4 times more than the wild type. While, Km and Kcat of R130P mutation had not any change. Compared with wild type, optimal temperature and pH of the two mutated enzymes were both enhanced to 65℃and 4.5, respectively.