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KMID : 0545120140240111516
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Journal of Microbiology and Biotechnology
2014 Volume.24 No. 11 p.1516 ~ p.1524
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Efficient Expression, Purification, and Characterization of a Novel FAD-Dependent Glucose Dehydrogenase from Aspergillus terreus in Pichia pastoris
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Yufeng Yang
Lei Huang
Jufang Wang
Xiaoning Wang
Zhinan Xu
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Abstract
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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°C. In addition, it displayed very high thermal stability, with a half-life of 82 min at 60°C. These characteristics indicate that FAD-GDH will be useful in the field of glucose biosensors.
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KEYWORD
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characterization, FAD-GDH, isopropanol precipitation, Pichia pastoris, thermal stability
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