瑞氏木霉表达黑曲霉葡萄糖氧化酶

利用高表达分泌纤维素酶的真菌瑞氏木霉表达重组的黑曲霉葡萄糖氧化酶。在大肠杆菌DH5α中构建瑞氏木霉纤维素酶CBHI启动子和CBHI信号肽基因黑曲霉葡萄糖氧化酶基因瑞氏木霉纤维素酶CBHI终止子构巢曲霉的甘油醛3磷酸脱氢酶启动子大肠杆菌抗潮霉素B磷酸转移酶基因构巢曲霉色氨酸C终止子pUC19(命名为pCBHGOD)质粒,线性化后用瑞氏木霉纤维素酶CBHI启动子和CBHI信号肽基因黑曲霉葡萄糖氧化酶基因瑞氏木霉纤维素酶CBHI终止子构巢曲霉的甘油醛3磷酸脱氢酶启动子大肠杆菌抗潮霉素B磷酸转移酶基因构巢曲霉色氨酸C终止子(命名为CBHGOD)核酸片段转化瑞氏木霉QM9414原生质体。用PCR扩增方法筛选出同源重组葡萄糖氧化酶基因的瑞士木霉突变株。用麦杆诱导瑞氏木霉突变株,生产黑曲霉葡萄糖氧化酶,Westernblot分析重组的葡萄糖氧化酶分子量与Sigma公司的天然黑曲霉葡萄糖氧化酶一致,生产的重组酶活性25umL,相当于Sigma公司葡萄糖氧化酶标准品的产量为0.5gL。瑞氏木霉可用于生产黑曲霉葡萄糖氧化酶。

Recombinant Aspergillus niger Glucose Oxidase Expressed in Trichoderma reesei

It was expected that recombinant Aspergillus niger glucose oxidase could be expressed in Trichoderma reesei with stable activity. T. reesei CBHI promoter--CBHI ss. gene--A. niger glucose oxidase gene--T. reesei CBHI terminator--A. nidulans gpd promoter--E. coli Hygromycin B phosphotransferase gene--A. nidulans trpC terminator--pUC19 (pCBHGOD) vector was constructed in E. coli DH5alpha by PCR application and gene cloning methods. T. reesei QM9414 protoplast was transformed by T. reesei CBHI promoter-CBHI ss. Gene--A. niger glucose oxidase gene--T. reesei CBHI terminator-A. nidulans gpd promoter--E. coli Hygromycin B phosphotransferase gene--A. nidulans trpC terminator linear DNA fragment (CBHGOD fragment) that was made by digestion of pCBHGOD with Kpn I. T. reesei mutant clone with homologous recombinant A. niger glucose oxidase gene was selected by PCR method. Recombinant glucose oxidase was produced by mutant T. reesei strain under induction of wheat straw for 5 days. Recombinant glucose oxidase molecular mass was showed the same as native A. niger glucose oxidase standard from Sigma company by Western blot analysis. Recombinant glucose oxidase activity was 25u/mL in medium. The yield was 0.5 g/L in comparison with Sigma company glucose oxidase standard. There was no recombinant GOD degradation during Trichoderma reesei cultivation that was showed in Western blot analysis. Trichoderma reesei has capability to be a new recombinant host for Aspergillus niger GOD production.