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http://dx.doi.org/10.4014/jmb.1401.01061

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)
Publication Information
Journal of Microbiology and Biotechnology / v.24, no.11, 2014 , pp. 1516-1524 More about this Journal
Abstract
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.
Keywords
Characterization; FAD-GDH; isopropanol precipitation; Pichia pastoris; thermal stability;
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