Biotechnology Letters

, Volume 41, Issue 4–5, pp 605–611 | Cite as

Construction of a novel bioanode for amino acid powered fuel cells through an artificial enzyme cascade pathway

  • Takenori SatomuraEmail author
  • Kousaku Horinaga
  • Shino Tanaka
  • Eiichiro Takamura
  • Hiroaki Sakamoto
  • Haruhiko Sakuraba
  • Toshihisa Ohshima
  • Shin-ichiro Suye
Original Research Paper



The construction of a novel bioanode based on l-proline oxidation using a cascade reaction pathway comprised of thermostable dehydrogenases.


A novel multi-enzymatic cascade pathway, containing four kinds of dehydrogenases from thermophiles (dye-linked l-proline dehydrogenase, nicotinamide adenine dinucleotide (NAD)-dependent Δ1-pyrroline-5-carboxylate dehydrogenase, NAD-dependent l-glutamate dehydrogenase and dye-linked NADH dehydrogenase), was designed for the generation of six-electrons from one molecule of l-proline. The current density of the four-dehydrogenase-immobilized electrode, with a voltage of + 450 mV (relative to that of Ag/AgCl), was 226.8 μA/cm2 in the presence of 10 mM l-proline and 0.5 mM ferrocene carboxylate at 50 °C. This value was 4.2-fold higher than that of a similar electrode containing a single dehydrogenase. In addition, about 54% of the initial current in the multi-enzyme cascade bioanode was maintained even after 15 days.


Efficient deep oxidation of l-proline by multiple-enzyme cascade reactions was achieved in our designed electrode. The multi-enzyme cascade bioanode, which was built using thermophilic dehydrogenases, showed high durability at room temperature. The long-term stability of the bioanode indicates that it shows great potential for applications as a long-lived enzymatic fuel cell.


Enzymatic fuel cell Thermophilic enzyme Enzyme cascade reaction Dye-linked dehydrogenase 



We thank Ms. Yumi Sugamura for technical assistance. We would like to thank Editage ( for English language editing.

Supplementary information

Supplementary Figure 1—SDS-PAGE of purified enzymes making up the l-proline multi-step oxidation pathway. A, Ap-LPDH; B, ThP5CDH; C, GkNADHDH; D, Pi-GDH; M, protein marker; E, purified enzyme.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

10529_2019_2664_MOESM1_ESM.pptx (8.7 mb)
Supplementary material 1 (PPTX 8907 kb)


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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Division of Engineering, Faculty of EngineeringUniversity of FukuiFukuiJapan
  2. 2.Organization for Life Science Advancement ProgramsUniversity of FukuiFukuiJapan
  3. 3.Department of Applied Chemistry and Biotechnology, Graduate School of EngineeringUniversity of FukuiFukuiJapan
  4. 4.Department of Frontier Fiber Technology and Science, Graduate School of EngineeringUniversity of FukuiFukuiJapan
  5. 5.Department of Applied Biological Science, Faculty of AgricultureKagawa UniversityKagawaJapan
  6. 6.Department of Biomedical Engineering, Faculty of EngineeringOsaka Institute of TechnologyOsakaJapan

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