绿色木霉葡聚糖内切酶Ⅰ基因在大肠杆菌中的表达与分子改造
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摘要
纤维素可以在纤维素酶系的作用下降解成葡萄糖,而葡萄糖又能用于生产一种新型清洁能源:燃料乙醇。在这个转化途径中,纤维素酶降解纤维素成葡萄糖是最大的瓶颈,因此构建高效表达纤维素酶的基因工程菌并获得高酶活的纤维素酶,对今后纤维素的发酵生产应用具有重要的意义。本研究使绿色木霉内切葡聚糖酶Ⅰ顺利地在大肠杆菌中获得表达,对该酶进行纯化并测定其酶学性质,研究发现在大肠杆菌中表达出的重组酶在以羧甲基纤维素纳(CMC-Na)作为底物时,其比活为3.8U/mg、Km为18.62mg/mL、Vmax为32.53μmol/min、其催化效率Kcat/Km为1.03 ml/mg.s、其最适反应温度约为48℃、最适反应pH值约为5.2、其Tm值在60℃附近。
本研究还通过同源比比对法,确定了以与绿色木霉内切葡聚糖酶Ⅰ氨基酸序列相似性达到99%的瑞氏木霉内切葡聚糖酶Ⅰ的蛋白质3D结构为参考,预测并选择了绿色木霉内切葡聚糖酶Ⅰ的酶活性中心位点Glu217、Glu222进行定点突变,将其定向突变为Asp,而对活性中心位点Asp219位点进行定点饱和突变,并辅助结合蛋白质结构分析软件prosa2003对其进行能量计算,通过分析计算结果,选择了保守区的Pro366、Asp242、Pro198三个氨基酸位点进行定点随机突变。而后又通过多序列同源比对法,选择了非保守区的Gly291氨基酸位点进行定点随机突变通过筛选,最终只筛训揭恢暧忻富畹耐槐涿窯291S,即Gly突变为Ser,纯化并测定了该突变酶的酶学性质并与野生型酶进行了比较,该突变酶以羧甲基纤维素(CMC-Na)为底物时,其比活为野生型酶的2.2倍,达到8.3U/mg;Km值下降至12.34mg/mL,为野生型酶的0.66倍;其Vmax为40.04μmol/min,与野生型酶相比较没有明显的提高;其催化效率Kcat/Km达到2.03 ml/mg.s,为野生型酶的1.97倍;其最适反映温度和最适反应pH值没有发生改变;而其Tm值降至55℃至60℃之间。
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℃.
本研究还通过同源比比对法,确定了以与绿色木霉内切葡聚糖酶Ⅰ氨基酸序列相似性达到99%的瑞氏木霉内切葡聚糖酶Ⅰ的蛋白质3D结构为参考,预测并选择了绿色木霉内切葡聚糖酶Ⅰ的酶活性中心位点Glu217、Glu222进行定点突变,将其定向突变为Asp,而对活性中心位点Asp219位点进行定点饱和突变,并辅助结合蛋白质结构分析软件prosa2003对其进行能量计算,通过分析计算结果,选择了保守区的Pro366、Asp242、Pro198三个氨基酸位点进行定点随机突变。而后又通过多序列同源比对法,选择了非保守区的Gly291氨基酸位点进行定点随机突变通过筛选,最终只筛训揭恢暧忻富畹耐槐涿窯291S,即Gly突变为Ser,纯化并测定了该突变酶的酶学性质并与野生型酶进行了比较,该突变酶以羧甲基纤维素(CMC-Na)为底物时,其比活为野生型酶的2.2倍,达到8.3U/mg;Km值下降至12.34mg/mL,为野生型酶的0.66倍;其Vmax为40.04μmol/min,与野生型酶相比较没有明显的提高;其催化效率Kcat/Km达到2.03 ml/mg.s,为野生型酶的1.97倍;其最适反映温度和最适反应pH值没有发生改变;而其Tm值降至55℃至60℃之间。
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℃.
引文
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