(69d) A Self-Assembling Protein Hydrogel for Enzyme Incorporation Onto Electrodes in Biofuel Cells

We developed a self-assembling protein hydrogel for enzyme immobilization on electrodes for enzymatic biofuel cell applications.  The building blocks of this hydrogel are two protein block copolymers (PC1 and PC2). PC1 contains a triangular trimer protein fused to a recombinant PDZ protein, and PC2 contains the same trimer protein and the peptide ligand of PDZ, as well as a repetitive non-structural S peptide [((AG)₃PEG)₁₀] and a docking station peptide SH3_lig. The S peptide facilitates the retention of water in the hydrogel and SH3_lig enables stable non-covalent immobilization of the SH3 (Src homology 3 domain from the adaptor protein Crk)-modified target protein. Individually, PC1 and PC2 exist as viscous solutions composed of trimeric proteins. Mixing solutions of PC1 and PC2 triggers the physical association of PDZ with its peptide ligand, joining individual PC1 and PC2 trimers and giving rise to the formation of a highly crosslinked protein network (hydrogel).  Rheological measurements confirmed the formation of a protein hydrogel and shear-thinning properties reflective of a physical hydrogel. This hydrogel is stable in solution with a very low erosion rate. To demonstrate the utility of this hydrogel for enzyme immobilization, two recombinant proteins, PTDH (phosphite dehydrogenase) and Slac (laccase), were purified as fusions to the SH3 protein and immobilized individually in the hydrogel. A very low leaching rate of the target protein from the hydrogel in solution was observed. The Slac-containing hydrogel was subsequently rendered conductive for application in a direct electron transfer enzymatic electrode. The potential of this hydrogel for power generation will be discussed.