Improving the Performance of Methanol Biofuel Cells Utilizing an Enzyme Cascade Bioanode with DNA-Bridged Substrate Channeling

The development of enzymatic biofuel cells has been plagued by the high cost of enzyme purification and low efficiency of fuel oxidation. Here, we demonstrate a protein purification-free approach to assemble an alcohol dehydrogenase and aldehyde dehydrogenase enzyme cascade-based bioanode for use in a methanol biofuel cell. Each enzyme was fused to a different sequence-specific zinc finger DNA-binding protein. The zinc finger domains serve as both tags to isolate the enzymes from crude cell lysates as well as anchors to immobilize the enzymes on DNA-modified multiwalled carbon nanotubes. The biofuel cells based on the enzyme cascade bioanodes show a maximum power output of 24.5 ± 3.2 μW cm –2, which is comparable to fuel cells utilizing purified enzymes. Further analysis of kinetic behavior revealed a significant increase in the reactivity of the complexes due to substrate channeling of the aldehyde intermediate.