title: Identification and characterization of thermostable glucose dehydrogenases from thermophilic filamentous fungi.
abstract: FAD-dependent glucose dehydrogenase (FAD-GDH), which contains FAD as a cofactor, catalyzes the oxidation of D-glucose to D-glucono-1,5-lactone, and plays an important role in biosensors measuring blood glucose levels. In order to obtain a novel FAD-GDH gene homolog, we performed degenerate PCR screening of genomic DNAs from 17 species of thermophilic filamentous fungi. Two FAD-GDH gene homologs were identified and cloned from Talaromyces emersonii NBRC 31232 and Thermoascus crustaceus NBRC 9129. We then prepared the recombinant enzymes produced by Escherichia coli and Pichia pastoris. Absorption spectra and enzymatic assays revealed that the resulting enzymes contained oxidized FAD as a cofactor and exhibited glucose dehydrogenase activity. The transition midpoint temperatures (Tm) were 66.4 and 62.5 °C for glycosylated FAD-GDHs of T. emersonii and T. crustaceus prepared by using P. pastoris as a host, respectively. Therefore, both FAD-GDHs exhibited high thermostability. In conclusion, we propose that these thermostable FAD-GDHs could be ideal enzymes for use as thermotolerant glucose sensors with high accuracy.
authors: Nanomaterials Research Institute, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8565, Japan.
authors: Kazumichi
authors: K
authors: Ozawa
authors: Nanomaterials Research Institute, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8565, Japan.
authors: Hisanori
authors: H
authors: Iwasa
authors: Nanomaterials Research Institute, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8565, Japan.
authors: Noriko
authors: N
authors: Sasaki
authors: Nanomaterials Research Institute, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8565, Japan.
authors: Nao
authors: N
authors: Kinoshita
authors: Nanomaterials Research Institute, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8565, Japan.
authors: Atsunori
authors: A
authors: Hiratsuka
authors: Nanomaterials Research Institute, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8565, Japan. yokoyamakj@stf.teu.ac.jp.|School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1 Katakura, Hachioji, Tokyo, 192-0982, Japan. yokoyamakj@stf.teu.ac.jp.
authors: Kenji
authors: K
authors: Yokoyama
mesh_terms: D003001:Cloning, Molecular; D003067:Coenzymes / Q000032:analysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D005658:Fungi / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D005948:Glucose Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006358:Hot Temperature; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013057:Spectrum Analysis; D032901:Talaromyces / Q000201:enzymology* / Q000235:genetics; D055327:Thermoascus / Q000201:enzymology* / Q000235:genetics
chemical_list: D003067:Coenzymes; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D005948:Glucose Dehydrogenases
keywords: FAD-dependent glucose dehydrogenase; Gene cloning; Recombinant protein production; Thermostability