ABSTRACT
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 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. 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.