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Applied Microbiology and Biotechnology

, Volume 101, Issue 1, pp 173–183 | Cite as

Identification and characterization of thermostable glucose dehydrogenases from thermophilic filamentous fungi

  • Kazumichi Ozawa
  • Hisanori Iwasa
  • Noriko Sasaki
  • Nao Kinoshita
  • Atsunori Hiratsuka
  • Kenji YokoyamaEmail author
Biotechnologically relevant enzymes and proteins

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 (T m) 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.

Keywords

FAD-dependent glucose dehydrogenase Gene cloning Recombinant protein production Thermostability 

Notes

Compliance with ethical standards

Conflict of interests

The authors declare that they have no competing interests.

Ethical statement

This articles does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2016_7754_MOESM1_ESM.pdf (846 kb)
ESM 1 (PDF 846 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Kazumichi Ozawa
    • 1
  • Hisanori Iwasa
    • 1
  • Noriko Sasaki
    • 1
  • Nao Kinoshita
    • 1
  • Atsunori Hiratsuka
    • 1
  • Kenji Yokoyama
    • 1
    • 2
    Email author
  1. 1.Nanomaterials Research Institute, National Institute of Advanced Industrial Science and TechnologyTsukubaJapan
  2. 2.School of Bioscience and BiotechnologyTokyo University of TechnologyHachiojiJapan

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