摘要
纤维素(Cellulose)是地球上最丰富的有机物质,它是光合作用最主要的产物,同时也是最丰富的可再生资源,地球上每年光合作用可产生大于100亿吨的植物干物质,其中一半以上是纤维素和半纤维素。但纤维素的利用目前尚未完全开发,随着地球石化能源短缺和枯竭的日趋严峻,人类把眼光转向了对纤维素的利用,利用纤维素酶将纤维素降解为可发酵的单糖,进而发酵生产乙醇。然而目前发现的纤维素酶都存在活力低和反应速度慢的缺点,因此寻找新的或者通过改造现有的纤维素酶基因获得活力更高纤维素酶是解决纤维素开发利用的关键问题。
本研究提取了台湾家白蚁总RNA通过RT-PCR扩增出白蚁内切-β-1,4-葡聚糖酶基因,并分别构建了表达内切-β-1,4-葡聚糖酶的大肠杆菌和酿酒酵母表达载体。利用金属镍亲和层析对大肠杆菌表达的内切-β-1,4-葡聚糖酶进行纯化,CMC酶活检测显示纯化酶的最适温度和最适pH值分别为42℃、6.5;内切-β-1,4-葡聚糖酶的Vmax为0.071mg/ml~*min,Km值为80.2712mg/ml,比活力为13.544U/mg。利用定点突变对白蚁内切-β-1,4-葡聚糖酶基因进行改造,首先是借助计算机根据白蚁内切-β-1,4-葡聚糖酶的同源建模三维结构信息,利用ProSA2003软件根据能量(knowledge-based potentials)最低化原则,计算机辅助设计确定白蚁内切-β-1,4-葡聚糖酶氨基酸饱和突变位点,通过同源建模分析表明53D、56D和411E三个位点都是活性中心位点。利用兼并引物对53,56,411位点的氨基酸分别进行饱和突变,筛选到三个有活力的突变子C53、E53和C56。对突变酶进行酶学性质的研究表明,C53的最适温度、最适pH、米氏常数、Vmax和比活力分别为42℃、6.5、37.683 mg/mL、0.0334umol/mL~*min、9.912 U/mg;突变酶E53的最适温度、最适pH、米氏常数、Vmax和比活力分别为37℃、6.5、93.819mg/mL、0.124umol/mL~*min、16.421U/mg;突变酶C56的最适温度、最适pH、米氏常数、Vmax和比活力分别为42℃、8.0、26.809mg/mL、0.0324umol/mL~*min、16.470U/mg。
Cellulose(Cellulose)is the most organic material and renewable resources,which is the main product of photosynthesis.There are more than 10 billion tons of biomass plants on Earth,Over half of which is cellulose and hemicellulose.However,the cellulose has not been used fully.As the shortage of petrochemical energy on the Earth,people start to use cellulose to fermente ethanol.But now the activity of cellulose are low and fermentation are very slow,so searching for new cellulases,or mutating the existing cellulase gene to get higher cellulase become a hot research.
In this research,we extracted Coptotermesformosanus' total RNA,and obtained the cDNA by reverse transcription.The gene encoding endo-β-1,4-glucanase was isolated by PCR,and then it was ligated to an Ecoli vector and a yeast vector to construct the cellulases-producing gene engineering strains.The endo-β-1,4-glucanase of recombinant Ecoli strains was purified by nickel affinity chromatography.The results showed that the optimal tempreture and pH of purified endoglucanase were 42℃/6.5 respectively;The Vmax and Km for endo-β-1,4-glucanase were shown to be 0.071 umol/mL~*min /80.271mg/mL/13.544U/mg.In this research,we determined three endo-β-1,4-glucanase amino acid mutation sites(C53,E53 and C56)by three-dimensional model of endo-β-1,4-glucanase and ProSA2003 software(knowledge-based potentials).The optimum temperature,optimum pH,Km,Vmax and activity of mut-C53 were shown to be 42℃/6.5/37.683mg/mL/0.0334umol/mL~*min/9.912 U/mg respectively; The optimum temperature,optimum pH,Km,Vmax and activity of mut-E53 were shown to be 37℃/6.5/93.819mg/mL/0.124umol/mL~*min/16.421U/mg respectively;The optimum temperature,optimum pH,Km,Vmax and activity of mut-C56 were shown to be 42℃/8.0/26.809mg/mL/ 0.0324umol/mL~*min/16.470U/mg respectively.
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