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.
Aspergillus niger ; enzymology ; genetics ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Fungal Proteins ; genetics ; metabolism ; Glucose Oxidase ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Trichoderma ; genetics ; metabolism