绿色木霉葡聚糖内切酶EGIII基因在大肠杆菌中的表达及其分子进化的研究
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摘要
纤维素是地球上最丰富的可再生性物质之一,如果能够利用纤维素来生产燃料乙醇将缓解人类面临的能源和环境污染等危机,因此纤维素乙醇已被人们看作未来能源。纤维素原料是最有发展潜力的燃料乙醇生产原料,但因其结构致密很难被分解成微生物利用的糖类,是目前利用其工业化生产乙醇首要面临的困难。纤维素酶是能够水解纤维素的一类酶的总称,它可以将纤维素水解成单糖,进而发酵产生乙醇,然而目前发现纤维素酶活力低,成本高,严重制约了其工业化应用。
针对纤维素酶活力低,以及纤维素酶基因在大肠杆菌中表达无活力的问题,本论文做了以下研究:将绿色木霉EGⅢ基因亚克隆到表达载体pET-22b(+),构建重组质粒pET-egl3,转化大肠杆菌BL21(DE3),检测到EGⅢ酶活,利用金属亲和层析对重组EGⅢ进行纯化,SDS—PAGE显示纯化蛋白的分子量约为42kD,纯化后的酶比活力为6U/mg,最适反应温度为60℃,最适pH为4.0。
由于对绿色木霉内切葡聚糖酶EGⅢ的三维结构尚不清楚,本研究采用了非理性设计进行内切葡聚糖酶EGⅢ分子进化的研究,利用定点饱和突变的方法来获得高活力突变酶。以Thermoascus aurantiacus内切酶的晶体结构,通过SWISSMODEL(http://www.expasy.org/)同源建模绿色木霉EGⅢ的3D结构,分析模板关键位点,结合计算机辅助设计软件Prosa2003能量计算确定了EGⅢ催化区内的R130、E218、Q246和E329为突变位点。利用刚果红染色法进行筛选,从Q246和E329突变库中没能筛选到有活力提高的突变体,而R130和E218各筛选到一株酶活有提高的突变子R130P和E218F,比活为野生型EGⅢ的2.8倍和3.45倍。对其酶学性质的研究发现,突变酶E218F的Km提高了一倍,催化效率Kcat提高了5.4倍;而R130P的Km和Kcat没有明显变化。两个突变酶的最适酶解温度和pH分别都提高至65℃和4.4。
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.
针对纤维素酶活力低,以及纤维素酶基因在大肠杆菌中表达无活力的问题,本论文做了以下研究:将绿色木霉EGⅢ基因亚克隆到表达载体pET-22b(+),构建重组质粒pET-egl3,转化大肠杆菌BL21(DE3),检测到EGⅢ酶活,利用金属亲和层析对重组EGⅢ进行纯化,SDS—PAGE显示纯化蛋白的分子量约为42kD,纯化后的酶比活力为6U/mg,最适反应温度为60℃,最适pH为4.0。
由于对绿色木霉内切葡聚糖酶EGⅢ的三维结构尚不清楚,本研究采用了非理性设计进行内切葡聚糖酶EGⅢ分子进化的研究,利用定点饱和突变的方法来获得高活力突变酶。以Thermoascus aurantiacus内切酶的晶体结构,通过SWISSMODEL(http://www.expasy.org/)同源建模绿色木霉EGⅢ的3D结构,分析模板关键位点,结合计算机辅助设计软件Prosa2003能量计算确定了EGⅢ催化区内的R130、E218、Q246和E329为突变位点。利用刚果红染色法进行筛选,从Q246和E329突变库中没能筛选到有活力提高的突变体,而R130和E218各筛选到一株酶活有提高的突变子R130P和E218F,比活为野生型EGⅢ的2.8倍和3.45倍。对其酶学性质的研究发现,突变酶E218F的Km提高了一倍,催化效率Kcat提高了5.4倍;而R130P的Km和Kcat没有明显变化。两个突变酶的最适酶解温度和pH分别都提高至65℃和4.4。
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.
引文
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