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

Objective The construction of a novel bioanode based on l -proline oxidation using a cascade reaction pathway comprised of thermostable dehydrogenases. Results A novel multi-enzymatic cascade pathway, containing four kinds of dehydrogenases from thermophiles (dye-linked l -proline dehydrogenase, nicotinamide adenine dinucleotide (NAD)-dependent Δ 1 -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/cm 2 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. Conclusions 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.