title: 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.
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, P.R. China.
authors.forename: Yufeng
authors.initials: Y
authors.lastname: Yang
authors.forename: Lei
authors.initials: L
authors.lastname: Huang
authors.forename: Jufang
authors.initials: J
authors.lastname: Wang
authors.forename: Xiaoning
authors.initials: X
authors.lastname: Wang
authors.forename: Zhinan
authors.initials: Z
authors.lastname: Xu
mesh_terms: D001230:Aspergillus / Q000201:enzymology / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D005182:Flavin-Adenine Dinucleotide; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D042843:Glucose 1-Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D042843:Glucose 1-Dehydrogenase