Isolation of Aspergillus niger glucose oxidase gene and cloning of that prokaryotic host

Introduction: Glucose oxidase (GO) is an enzyme, which use in food, chemical and personal care industries as well as glucose diagnostic kits. The aim of this study was PCR-mediated amplification of GO gene from Aspergillus niger genome and cloning of it in E. coli as a basis for production of recombinant GO in Iran. Methods: A. niger (2029) was cultured in media containing peptone, glucose and malt extract in 24’C for 48-72 h. Genomic DNA was extracted by sonication and freeze-thaw in liquid nitrogen in a lysis buffer containing EDTA and SDS. GO gene was amplified with designed primers under optimized PCR condition. The PCR product was cloned in pTZ57R plasmid using InsT/AcloneTM (Fermentas) and the constructed plasmid was transformed into E. coli DH5. Map of this plasmid was confirmed by restriction analysis and named pTZ57RGO. Results: Our data showed the methods used in this study was adequate for culture and extraction of DNA from filamentous fungi such as A. niger. We showed amplified DNA fragment has expected size in agarose gel electrophoresis and also the cloned GO has a correct size after restriction analysis. Conclusion: The GO gene isolated successfully from A. niger via optimized PCR conditions and was cloned in prokaryotic host. This is the first report of isolation and cloning of GO gene in Iran that can be used for further cloning of that gene in expression vectors for production of recombinant enzyme.