Journal of Microbiology and Biotechnology

  • 제24권11호
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  • Pages.1516-1524
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  • 2014
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  • 1017-7825(pISSN)
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  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
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Efficient Expression, Purification, and Characterization of a Novel FAD-Dependent Glucose Dehydrogenase from Aspergillus terreus in Pichia pastoris

  • Yang, Yufeng (School of Bioscience and Bioengineering, South China University of Technology) ;
  • Huang, Lei (Department of Chemical and Biological Engineering, Zhejiang University) ;
  • Wang, Jufang (School of Bioscience and Bioengineering, South China University of Technology) ;
  • Wang, Xiaoning (Institute of Life Science, General Hospital of The People's Liberation Army) ;
  • Xu, Zhinan (Department of Chemical and Biological Engineering, Zhejiang University)
  • 투고 : 2014.02.03
  • 심사 : 2014.07.08
  • 발행 : 2014.11.28
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초록

Flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) can utilize a variety of external electron acceptors and also has stricter substrate specificity than any other glucose oxidoreductases, which makes it the ideal diagnostic enzyme in the field of glucose biosensors. A gene coding for a hypothetical protein, similar to glucose oxidase and derived from Aspergillus terreus NIH2624, was overexpressed in Pichia pastoris GS115 under the control of an AOX1 promoter with a level of 260,000 U/l in the culture supernatant after fed-batch cultivation for 84 h. After a three-step purification protocol that included isopropanol precipitation, affinity chromatography, and a second isopropanol precipitation, recombinant FAD-GDH was purified with a recovery of 65%. This is the first time that isopropanol precipitation has been used to concentrate a fermentation supernatant and exchange buffers after affinity chromatography purification. The purified FAD-GDH exhibited a broad and diffuse band between 83 and 150 kDa. The recombinant FAD-GDH was stable across a wide pH range (3.5 to 9.0) with maximum activity at pH 7.5 and $55^{\circ}C$. In addition, it displayed very high thermal stability, with a half-life of 82 min at $60^{\circ}C$. These characteristics indicate that FAD-GDH will be useful in the field of glucose biosensors.

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  2. Production of Industrial Enzymes via Pichia pastoris as a Cell Factory in Bioreactor: Current Status and Future Aspects vol.40, pp.3, 2021, https://doi.org/10.1007/s10930-021-09968-7
  3. Sensitive electrochemical immunosensor using a bienzymatic system consisting of β-galactosidase and glucose dehydrogenase vol.146, pp.12, 2014, https://doi.org/10.1039/d1an00562f