Characterization and improvement of substrate-binding affinity of d-aspartate oxidase of the thermophilic fungus Thermomyces dupontii

  • Shouji TakahashiEmail author
  • Kohei Osugi
  • Yuya Shimekake
  • Akira Shinbo
  • Katsumasa Abe
  • Yoshio Kera
Biotechnologically relevant enzymes and proteins


d-Aspartate oxidase (DDO) is a valuable enzyme that can be utilized in the determination of acidic d-amino acids and the optical resolution of a racemic mixture of acidic amino acids, which require its higher stability, higher catalytic activity, and higher substrate-binding affinity. In the present study, we identified DDO gene (TdDDO) of a thermophilic fungus, Thermomyces dupontii, and characterized the recombinant enzyme expressed in Escherichia coli. In addition, we generated a variant that has a higher substrate-binding affinity. The recombinant TdDDO expressed in E. coli exhibited oxidase activity toward acidic d-amino acids and a neutral d-amino acid, d-Gln, with the highest activity toward d-Glu. The Km and kcat values for d-Glu were 2.16 mM and 217 s−1, respectively. The enzyme had an optimum pH and temperature 8.0 and 60 °C, respectively, and was stable between pH 5.0 and 10.0, with a T50 of ca. 51 °C, which was much higher than that in DDOs from other origins. Enzyme stability decreased following a decrease in protein concentration, and externally added FAD could not repress the destabilization. The mutation of Phe248, potentially located in the active site of TdDDO, to Tyr residue, conserved in DDOs and d-amino acid oxidases, markedly increased substrate-binding affinity. The results showed the great potential of TdDDO and the variant for practical applications.


d-Aspartate oxidase Thermomyces dupontii Thermophilic fungus Thermostable E. coli expression Site-directed mutagenesis 



This study was supported by the Grant-in-Aid for Scientific Research (C) (23580106) to S. Takahashi from the Japan Society for the Promotion of Science.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Research involving human participants

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


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© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of BioengineeringNagaoka University of TechnologyNagaokaJapan

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