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Site-Directed Mutagenesis of Multicopper Oxidase from Hyperthermophilic Archaea for High-Voltage Biofuel Cells

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Abstract

Enzymes from hyperthermophilic archaea are potential candidates for industrial use because of their superior pH, thermal, and long-term stability, and are expected to improve the long-term stability of biofuel cells (BFCs). However, the reported multicopper oxidase (MCO) from hyperthermophilic archaea has lower redox potential than MCOs from other organisms, which leads to a decrease in the cell voltage of BFCs. In this study, we attempted to positively shift the redox potential of the MCO from hyperthermophilic archaeon Pyrobaculum aerophilum (McoP). Mutations (M470L and M470F) were introduced into the axial ligand of the T1 copper atom of McoP, and the enzymatic chemistry and redox potentials were compared with that of the parent (M470). The redox potentials of M470L and M470F shifted positively by about 0.07 V compared with that of M470. In addition, the catalytic activity of the mutants towards 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) increased 1.2–1.3-fold. The thermal stability of the mutants and the electrocatalytic performance for O2 reduction of M470F was slightly reduced compared with that of M470. This research provides useful enzymes for application as biocathode catalysts for high-voltage BFCs.

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Authors and Affiliations

  1. Department of Frontier Fiber Technology and Science, Graduate School of Engineering, University of Fukui, 3-9-1 Bunkyo, Fukui, Fukui, Japan

    Eiichiro Takamura, Shunsuke Taki, Hiroaki Sakamoto & Shin-ichiro Suye

  2. Department of Applied Chemistry and Biotechnology, Graduate School of Engineering, University of Fukui, 3-9-1 Bunkyo, Fukui, Fukui, Japan

    Takenori Satomura & Shin-ichiro Suye

  3. Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Kita-gun, Miki-cho, Kagawa, Japan

    Haruhiko Sakuraba

  4. Department of Biomedical Engineering, Faculty of Engineering, Osaka Institute of Technology, Omiya, 5-16-1 Asahi-ku, Osaka, Japan

    Toshihisa Ohshima

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  1. Eiichiro Takamura
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  2. Shunsuke Taki
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  3. Hiroaki Sakamoto
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  4. Takenori Satomura
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  5. Haruhiko Sakuraba
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  6. Toshihisa Ohshima
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  7. Shin-ichiro Suye
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Correspondence to Eiichiro Takamura.

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Takamura, E., Taki, S., Sakamoto, H. et al. Site-Directed Mutagenesis of Multicopper Oxidase from Hyperthermophilic Archaea for High-Voltage Biofuel Cells. Appl Biochem Biotechnol 193, 492–501 (2021). https://doi.org/10.1007/s12010-020-03440-5

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