Mu Jing-Yui, Wang Qiao, Yang Daniel, Wang En-Si, Wang Qing, Huang Yue
School of Pharmaceuticals, Jilin University, Changehun 130021, China.
Sheng Wu Gong Cheng Xue Bao. 2006 Jan;22(1):82-6.
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.
预期重组黑曲霉葡萄糖氧化酶能够在里氏木霉中表达并具有稳定的活性。通过PCR技术和基因克隆方法,在大肠杆菌DH5α中构建了里氏木霉CBHI启动子-CBHI信号肽基因-黑曲霉葡萄糖氧化酶基因-里氏木霉CBHI终止子-构巢曲霉gpd启动子-大肠杆菌潮霉素B磷酸转移酶基因-构巢曲霉trpC终止子-pUC19(pCBHGOD)载体。用Kpn I酶切pCBHGOD得到的里氏木霉CBHI启动子-CBHI信号肽基因-黑曲霉葡萄糖氧化酶基因-里氏木霉CBHI终止子-构巢曲霉gpd启动子-大肠杆菌潮霉素B磷酸转移酶基因-构巢曲霉trpC终止子线性DNA片段(CBHGOD片段)转化里氏木霉QM9414原生质体。通过PCR方法筛选出具有同源重组黑曲霉葡萄糖氧化酶基因的里氏木霉突变体克隆。突变的里氏木霉菌株在小麦秸秆诱导下培养5天产生重组葡萄糖氧化酶。Western blot分析表明,重组葡萄糖氧化酶的分子量与Sigma公司的天然黑曲霉葡萄糖氧化酶标准品相同。培养基中重组葡萄糖氧化酶的活性为25u/mL。与Sigma公司葡萄糖氧化酶标准品相比,产量为0.5g/L。Western blot分析显示,在里氏木霉培养过程中没有重组葡萄糖氧化酶的降解。里氏木霉有能力成为生产黑曲霉葡萄糖氧化酶的新型重组宿主。