Electrochemistry of bilirubin oxidase and its use in preparation of a low cost enzymatic biofuel cell based on a renewable composite binder chitosan

Spherical carbon nanoparticles KetjenBlack (KB) with a high sorption capacity together with conductivity increasing single walled carbon nanotubes (CNTs) were “glued” together by chitosan for the preparation of a composite. A biocathode with bilirubin oxidase (BOD) adsorbed within the composite was characterised and its composition optimised. A renewable biopolymer chitosan present in the composite offered (1) pre-concentration of BOD within the matrix via electrostatic interactions, (2) favourable orientation of BOD for a direct electron transfer (DET) between BOD and the composite, (3) electrochemical visibility of 3 redox sites present in BOD, (4) low charge transfer resistance, (5) high proton conductivity and (6) low overpotential for oxygen reduction. Electrochemical investigation of BOD revealed interesting internal redox communication within the enzyme with some novel insights provided. At least one of tyrosines present in BOD seems to be involved in electron transfer route, as well. The composite used for the biocathode was directly applied for the preparation of a bioanode with fructose dehydrogenase immobilised, working in a DET mode of operation. Finally, integration of the biocathode and the bioanode into a biofuel cell operated in a reagentless and membraneless mode offered a power density of 50 μW cm−2 at 300 mV.

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