Journal
JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY
Volume 24, Issue 11, Pages 1516-1524Publisher
KOREAN SOC MICROBIOLOGY & BIOTECHNOLOGY
DOI: 10.4014/jmb.1401.01061
Keywords
Characterization; FAD-GDH; isopropanol precipitation; Pichia pastoris; thermal stability
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Funding
- National High Technology Research and Development Program of China [2011AA02A114]
- New Century Excellent Talents in University [NCET-10-0399]
- National Natural Science Foundation of China [21276093, 21006088, 21176214]
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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/1 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 degrees C. In addition, it displayed very high thermal stability, with a half-life of 82 min at 60 degrees C. These characteristics indicate that FAD-GDH will be useful in the field of glucose biosensors.
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