Enzymatic biofuel cells are artificial metabolic networks that are designed to extract electrical current from ambient fuel sources. In the simplest biofuel cell design, a single immobilized oxidoreductase enzyme is incorporated in each electrode of the cell; an enzyme that oxidizes a fuel is at the anode and a different enzyme that reduces oxygen to water is at the cathode. These two enzymes evolved separately in nature and when linked together in-vitro, they may or may not possess the desired features to create an optimized metabolic pathway.

Metabolic Control Analysis (MCA) is an analytical technique that aims to quantify the distribution of control that the enzymes in a metabolic network exhibit over the steady-state fluxes through a given system. In a biofuel cell, the flux of interest is the electrical current generated by the cell. Regardless of transport limitations and other constraints, kinetic limitations can become a potential bottleneck in the operation of a biofuel cell. In this presentation, MCA is used to identify optimal operating conditions so that the control of the metabolic flux is equally distributed between the anode and the cathode.

One of the requirements of MCA is having kinetic models of each enzyme participating in the system. Full kinetic models of the biofuel cell enzymes are presented along with kinetic parameter values for the enzymes obtained from the literature. The most interesting kinetic feature of the biofuel cell network is that substrate for the cathodic enzyme (oxygen) is an alternative and non-productive substrate for the anodic enzyme. Comparisons between experimental and theoretical results suggest that the kinetic models are able to capture the behavior that is observed in simple operating biofuel cells. Results of the MCA analysis are used to define an envelope of optimal operating conditions based upon given enzyme loadings on an electrode. Moreover, the kinetic properties of enzymes, such as their affinity for substrates especially electron transfer mediators, can not only alter the control distribution between the nodes of the metabolic network but can also offer insights to engineer more efficient enzymes for this application.

Biocatalytic electrodes are being increasingly employed in systems requiring catalytic selectivity in applications such as biofuel cells and biosensors. The theoretical treatment provided by MCA will be easily extended to multi-enzymatic electrodes as these complex networks will require further optimization to continue achieving higher catalytic activities and currents.