Deep oxidation of glucose in enzymatic fuel cells through a synthetic enzymatic pathway containing a cascade of two thermostable dehydrogenases

A synthetic enzymatic pathway was designed for the deep oxidation of glucose in enzymatic fuel cells (EFCs). Polyphosphate glucokinase converts glucose to glucose-6-phosphate using low-cost, stable polyphosphate rather than costly ATP. Two NAD-dependent dehydrogenases (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) that were immobilized on the bioanode were responsible for generating two NADH per glucose-6-phosphate (i.e., four electrons were generated per glucose via a diaphorase-vitamin K3 electron shuttle system at the anode). Additionally, to prolong the enzyme lifetime and increase the power output, all of the recombinant enzymes that originated from thermophiles were expressed in Escherichia coli and purified to homogeneity. The maximum power density of the EFC with two dehydrogenases was 0.0203 mW cm−2 in 10 mM glucose at room temperature, which was 32% higher than that of an EFC with one dehydrogenase, suggesting that the deep oxidation of glucose had occurred. When the temperature was increased to 50 °C, the maximum power density increased to 0.322 mW cm−2, which was approximately eight times higher than that based on mesophilic enzymes at the same temperature. Our results suggest that the deep oxidation of glucose could be achieved by using multiple dehydrogenases in synthetic cascade pathways and that high power output could be achieved by using thermostable enzymes at elevated temperatures.

► A novel synthetic enzymatic pathway in enzymatic fuel cells (EFCs) can generate four electrons per glucose.
► We used polyphosphate glucokinase for converting glucose to glucose-6-phosphate without ATP.
► Thermostable enzyme-based EFCs can work well at elevated temperatures.
► Maximum power density was 0.322 mW cm−2 at 50 °C, eight times higher than that of mesophilic enzyme-based EFC.