386248 Design of Self-Assembles Biocomplexesfor Bio/Nano-Devic Integration
Enzymatic biofuel cells, part of the rapidly growing are of bioelectochemistry, use enzymes as catalysts in order to harvest energy from alternative fuels such as glucose and ethanol. Protein engineering research in this area has largely focused on increasing the activity and stability the enzyme catalysts. In order to further improve enzymatic biofuel cells, research must be performed to optimize the bio/nano interface. By designing self-assembling biocomplexes that integrate electrode nanomaterials, more efficient electron transfer and higher power densities are expected.
Small laccase (SLAC) of Streptomyces coelicolor was chosen as a single enzyme system in order to study the bio/nano interface. SLAC has been previously used for oxygen reduction on an enzymatic cathode due to its high redox potential, neutral pH of operation, direct electron transfer and recombinant expression in E. coli. The sequential Kreb’s cycle enzyme malate dehydrogenase (MDH), citrate synthase (CS) and aconitase (Aco) was also studied as a multi-enzyme system. Several methods were employed to design bio/nano self-assembled complexes for electrochemical device integration.
In nature, many proteins have evolved to self-assemble into complex structures. There has been significant interest in bioengineering to design protein-protein interfaces that drive the self-assembly of proteins into ordered, complex structures. Strategies include introducing amino acids that produce contacts along the protein-protein interface such as disulfide bonds and hydrogen bonds to enhance the stability of native interfaces of the creation of novel interfaces. A new variant of SLAC was designed by introducing two non-native cysteine residues on the surface of the enzyme. In an oxidative environment, these cysteine residues form disulfide bonds with the corresponding cysteine residues of another unit of SLAC In this manner, multiple units of SLAC form stable, self-assembled protein aggregates. Carbon nanotubes and other nanomaterials can be trapped within the aggregates. Active, stable SLAC aggregates are potentially useful for device integration, since the tightly packed enzyme aggregates could be trapped on the electrode in materials such as carbon felt or a polymer matrix without being released into the bulk solution, resulting in longer lifetimes and increased performance.
Methods of attaching enzymes directly to the electrode material were also explored. Peptides were previously computationally designed to self-assemble around single walled carbon nanotubes in a virus-like fashion. A fusion protein was constructed from SLAC and the binding peptide to create a bifunctional enzyme with oxygen reduction activity and carbon nanotube binding capability.
DNA has been shown to wrap around carbon nanotubes, and it can be used as a scaffold for enzyme self-assembly while directly interacting with the electrode material. Zinc finger DNA binding domains bind site-specifically to DNA, and enzyme-zinc finger fusions can be employed to bind along a predetermined sequence of DNA. This strategy is particularly useful when extended to the multi-enzyme system of MDH, CS and Aco. By arranging sequential enzymes closely together in an ordered fashion, the active sites of the enzymes in the pathway can be physically closer to each other, minimizing the distance that intermediates have to travel. This will reduce transport limitations and improve the efficiency of the system. When applied to an electrochemical device, the DNA is expected to wrap around the carbon nanotubes, tethering the enzymes close to the electrode.