Expression And Molecule Evolution Of The Gene Of Endoglucanase Ⅰ In Escherichia Coli

Cellulose can be degraded to glucose by cellulase system,and glucose is easily transformed to a kind of new and clear energy source:fuel ethanol.So Construction of genetic engineering bacteria which can effectively express high activity cellulase would be helpful to the development of cellulose fermentation industry in future.In this research,EGⅠwas effectivitly expressed with endoglucanse activity from Escherichia coli,Then EGI was purified by metal affinity chromatography and was measured the characteristics of enzyme.When using CMC-Na as substract,The specific activity of EGⅠis 3.8U/mg, the value of Km is 18.62mg/mL,the value of Vmax is 32.53μmol/min,the value of catalytic efficiency constant Kcat/Km is 1.03 ml/mg.s,the optimum temperature and optimum pH were 48℃and 5.2,respectively.The value of Tm is approximate to 60℃. In this research,by using multiple sequnce homology alignment and choosing the crystal structure of endoglucanseⅠfrom Trichoderma reseei whose amino acid sequence has 99%similarity to endoglucanseⅠfrom Trichoderma viride as reference,the positions of Glu217,Glu222 were predicted to be the active sites of EGⅠfrom Trichoderma viride and were selected to be mutated to Asp.the position Asp219 which is speculated to be another active site was selected to perform to the site-directed saturation mutagenesis.Pro366,Asp242,Pro160 which are in conservative domain were also selected to process to site-directed random mutagenesis.And then by using protein structure analysis software prosa2003 to assist to calculate the stucture energy,The result showed that if Pro366,Asp242,Pro198 are mutated to other amino acids,the energy which is responsible to maintain nature configuration of enzyme was predicted to descend.And the position of Gly291 which is confirmed to be in non-conservative domain by multiple sequence homology alignment was selected to perform to site-directed random mutagenesis.Finally,only one mutant G291S which is retain the enzyme activity was screened,The amino acid residue of Gly in position 291 was confirmed to be transformed to Ser by sequencing.The mutant G291S was purified and measured the characteristics of this mutated enzyme and was used to compare to the wild type enzyme.When using CMC-Na as substract,the specific activity of mutant G291S is 8.3U/mg which is 2.2 times than that of wid type,the value of Km declined to 12.34mg/mL which is 0.66 times than that of wild type.The value of Vmax is 40.04μmol/min,it has not distinctly change when compare to that of wild type. The value of catalytic efficiency constant Kcat/Km is reach up to 2.03 ml/mg.s, which is 1.97 times than that of wild type.The optimum temperature and optimum pH of mutant G291S are not change when compare to that of wild type. The value of Tm is fall down into the range of between 55℃and 60℃.