作　　者： ; ; ; ; ; ; ;

机构地区： 广西大学生命科学与技术学院

出　　处： 《工业微生物》 2009年第5期30-38,共9页

摘　　要： 提取了台湾家白蚁总RNA并反转录获得eDNA，PCR扩增出白蚁内切葡聚糖酶的基因，并将目的基因分别克隆到大肠杆菌和酿酒酵母载体中，构建了产内切-β-1，4-葡聚糖酶的基因工程菌。由于大肠杆菌会有少量的泄漏表达，而所用的酿酒酵母表达载体是本实验室构建带有INU信号肽的表达载体，故都可采用刚果红平板染色法筛选具有羧甲基纤维素酶（CMCase）活性的重组转化子。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。 The total RNA of Coptotermes formosanus was extracted and the eDNA was obtained by reverse transcription. The gene encoding endo-β-1,4-glucanase was isolated by PCR, and then it was ligated to an E. coli vector and a yeast vector to construct the cell~ases-produeing gene engineering strains. Leak expression could be done by recombinant E. coli strains, while endo-β-1, 4-glucanase could be secreted out by the guide of the INU signal peptide in recombinant yeast strains. So the transformants could be screened by activity plate assays with Congo Red method, respectively. The endo-β-1,4-glucanase activity was assayed as CMCase activity with CMC-Na as a substrate. The endo-β-1,4-glucanase of recombinant E. coli strains was purified by nickel affinity chromatography. The results showed that the optimal temperature and pH of purified endoglucanase were 42 t and 6.5, respectively; The Vmax and Km for endo-β-1,4-glucanase were shown to be 0. 071 mg/mL＇min and 80. 2712 mg/mL, respectively.

关 键 词： 内切 葡聚糖酶 台湾家白蚁 酿酒酵母

领　　域： [生物学] [农业科学]