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Research on Expression in E.coli and Molecular Evolution of Endoglucanase III from Trichoderma Viride

Author: HuangYanYan
Tutor: HuangRiBo
School: Guangxi University
Course: Fermentation Engineering
Keywords: Trichoderma viride Endoglucanase Ⅲ E. coli Sentinel saturation mutagenesis
CLC: Q78
Type: Master's thesis
Year: 2008
Downloads: 181
Quote: 0
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Abstract


Cellulose is one of the earth's most abundant renewable material , if we can use cellulose to produce fuel ethanol will ease the energy crisis and environmental pollution , human face cellulosic ethanol has been seen as a future energy . Cellulosic feedstock is the most potential for development of fuel ethanol production of raw materials , but difficult because of its compact structure and is broken down into sugars microorganisms using ethanol primary difficulties facing the current use of its industrial production . Cellulase is a general term for a class of enzymes capable of hydrolyzing cellulose , it can be cellulose hydrolysis into single sugar, then fermented to produce ethanol , however , found that low cellulase activity , high cost , has seriously hampered its industrial application . For low cellulase activity , as well as dynamic cellulase gene expression in E. coli , the papers do the following research : the Trichoderma viride EG III was subcloned into the expression vector pET- 22b ( ) to construct the recombinant plasmid pET the -egl3, was transformed into E. coli BL21 (DE3) , detected the activity of EG Ⅲ metal affinity chromatography purified recombinant EG Ⅲ , SDS-PAGE showed the purified protein has a molecular weight of approximately 42 kD , the purified enzyme specific activity 6U / mg, the optimum reaction temperature is 60 ℃, the optimum pH was 4.0 . Trichoderma viride endoglucanase EG III three-dimensional structure is not clear , this study uses the irrational design endoglucanase EG Ⅲ molecular evolution research , the use of fixed-point saturation mutagenesis method to obtain high vitality mutation enzymes. Thermoascus aurantiacus within enzyme crystal structure, homology modeling green Trichoderma EG III through SWISSMODEL (http://www.expasy.org/) 3D structure analysis template key sites , combined with computer - aided design software Prosa2003 energy calculation determine the EG Ⅲ catalytic region of R130 , E218 , Q246 and E329 mutation site . Congo red staining method for screening, from Q246 and E329 mutation screening the library could not improve vitality mutant R130 and E218 screening to an enzyme activity improved mutants R130P and E218F than Activity for wild type EG Ⅲ 2.8 -fold and 3.45 -fold . The study found that its enzymatic properties doubled the Km of the mutant enzyme E218F improve catalytic efficiency Kcat increased 5.4 times ; R130P Km and Kcat no significant change . The optimum hydrolysis temperature and pH of the two mutant enzymes increased to 65 ° C and 4.4 , respectively .

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