极端耐热木聚糖酶基因在大肠杆菌和毕赤酵母中的高效表达  

High-level expression of an extreme-thermostable xylanase B from Thermotoga maritima MSB8 in Escherichia coli and Pichia pastoris

作　　者：杨梦华[1] 李颖[1] 关国华[1] 江正强[2] 

机构地区：[1]中国农业大学生物学院,北京100094 [2]中国农业大学食品科学与营养工程学院,北京100083

出　　处：《微生物学报》2005年第2期236-240,共5页Acta Microbiologica Sinica

基　　金：国家"863计划"(2 0 0 1AA2 14 171)~~

摘　　要：以海栖热袍菌 (Thermotogamaritima)MSB8菌株基因组DNA为模板 ,通过PCR扩增出木聚糖酶 (XylanaseB)基因 ,将此基因克隆至大肠杆菌表达载体pET_2 8a(+)和毕赤酵母表达载体pPIC9K ,并分别转化大肠杆菌BL2 1和毕赤酵母GS115。该木聚糖酶在大肠杆菌细胞中表达量高 ,但不能分泌 ;而在毕赤酵母细胞的表达产物可分泌至胞外。酶学性质分析表明 ,此酶分子量约为 4 0kD ,其最适反应温度为 90℃ ,最适反应pH值为 6 6 5 ,且在碱性条件下稳定 ,具有重要的工业应用前景。The family 10 xylanase (XynB) from Thermotoga maritima MSB8 is extremely thermostable and great potential in the applications of various fields of industry. The gene mxynB (64) was amplified by the method of PCR with the template of the genomic DNA of Thermotoga maritima MSB8, and cloned into the expression vectors of Escherichia coli and Pichia pastoris respectively. Xylanase B(40kD)was successfully expressed by the two heterologous protein expression systems with high-level production. The recombinant protein of XynB expressed in Pichia pastoris showed extreme thermostability and pH stability, which was optimally active at 90℃ and quite stable over the pH range of pH 5.0～10.8 at 70℃. After incubation of the enzyme at 100℃ for 30min, XynB retained 70 higher residual activity. The recombinant XynB expressed in Pichia pastoris is of great use in a variety of industrial and agricultural applications.

关 键 词：耐高温木聚糖酶 大肠杆菌 巴斯德毕赤酵母 重组蛋白 异源表达 

分 类 号：Q786[生物学—分子生物学]