Efficient Expression, Purification, and Characterization of a Novel FAD-Dependent Glucose Dehydrogenase from Aspergillus terreus in Pichia pastoris

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°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.

Structural analysis of fungus-derived FAD glucose dehydrogenase

“…Fungus-derived FADGDHs do not use oxygen as an electron acceptor and are accordingly insensitive to oxygen, their major advantage over GOx 51 . However, differences in substrate specificity between fungus-derived FADGDHs and GOx have been reported 11 12 13 14 15 16 19 . The second preferential substrate of fungus-derived FADGDHs is xylose, and their activity toward xylose is approximately 20% of that toward glucose, whereas GOxs do not react with xylose.…”

“…We previously reported the screening of a fungal genome database using GOx active-site sequence motifs 12 . Recently, several recombinant fungus-derived FADGDHs have been prepared and studied, including enzymes from A. terreus 13 , Glomerella cingulate 14 15 , and Pycnoporus cinnabarinus 16 . With increased demands for more accurate glucose sensing systems that meet the requirements of ISO15197-2013 17 and FDA guidelines 18 , further engineering is still required to improve the enzymatic properties of FADGDH, including stability and substrate specificity 19 .…”

Expression, characterization and mutagenesis of an FAD-dependent glucose dehydrogenase from Aspergillus terreus

“…SEC was employed to further enhance the purity of FAD-GDH, and resulted in a specific activity of 604 U/mg. The final recovery was 44% (Table 3), which is lower than previously reported for protein produced in P. pastoris [26], but is a significant achievement and allows production in the preferred host.…”

“…The enzymatic properties of FAD-GDH produced in E. coli were compared with protein expressed in P. pastoris [26]. The optimum pH and pH stability were the same, the K m value was similar, but the enzyme expressed in E. coli showed a lower optimal temperature and thermal stability.…”

“…Recently, a novel FAD-GDH from Aspergillus terreus NIH2624 was identified and expressed in Pichia pastoris [26]. This enzyme is a good candidate for glucose detection as it is stable over a wide pH range and has a high thermal stability.…”