Development and Characterization of Cellulolytic Enzymatic Fuel Cells with DNA-Organized Multi-Enzyme Cascade
Authors: Yu Sooyoun , Chen Qi , Chen Wilfred , Myung Nosang Vincent 
Published: February 2020

As fossil fuel is depleting, an alternative source of energy to accommodate the current standard of living is of great interest in research. Out of many methods that have been studied, fuel cells have been shown to be both environmentally friendly and flexible in types of fuels, such as hydrogen and alcohols. Enzymatic fuel cells (EFCs), more specifically, use isolated enzymes as a catalyst to redox reactions instead of noble metal catalysts commonly used in other fuel cells, which are nonrenewable. EFCs are also attractive because they can operate under mild conditions such as room temperature, atmospheric pressure, and neutral pH.1Along with microbial fuel cells (MFCs), EFCs are the only type of fuel cell that can use biologically derived materials – such as glucose – as fuels. This eliminates the additional steps to obtain pure hydrogen typically used as fuel.
Early works with enzymatic fuel cells only involved a single enzyme as biocatalyst, usually glucose oxidase (GOx) to convert glucose to electricity via glucose-oxygen redox reactions.2This results in incomplete oxidation of fuel and thus a lower power density output. We plan to mimic the structured multi-enzyme scaffold in anaerobic bacteria called cellulosome to develop a biological approach for fuel cell applications via enzyme organization to create multi-enzyme cascade for cellulose hydrolysis and glucose oxidation. We hypothesized that the substrate channeling effect of organized enzyme cascade can enhance the efficiency in power generation.
Previously, protein scaffolds have been used as scaffold to organize cellulases, but the problem of scaffold truncation occurred when the scaffold size increased.3 On the other hand, the length of DNA can be increased easily by technique such as rolling circle amplification.4 It also provides high binding specificity and affinity with flexible design.5DNA was therefore selected as scaffolds and immobilization tags in our design. In the fully assembled cellulolytic biofuel cell, the anode was fabricated by simple method of electrospinning a nanofiber mat of nickel coated with a few monolayers of gold via galvanic displacement. The main DNA scaffold was modified with a thiol group for high gold-binding affinity to maximize the density of the multi-enzyme scaffold on the anode. Three cellulases - endoglucanase, exoglucanase, and β-glucosidase -, which synergistically break down cellulose, a cellulose-binding domain (CBD), glucose oxidase, and a mediator were site-specifically bound to the DNA scaffold in an appropriate order for efficient conversion of cellulose to electricity (Fig 1).
The cellulases and the CBD were expressed in E. coli. The purification was based on the thermally induced reversible inverse phase transition property of the fusion partner, elastin-like polypeptide (ELP), and conjugation with DNA was enabled by its fusion partner, HaloTag. The glassy carbon cathode was functionalized with oxygen reducing agent, bilirubin oxidase, adsorbed onto a carbon nanotube. Binding of each enzyme on DNA scaffolds was confirmed and the surface density of electrode surface was quantified by Quartz Crystal Microbalance (QCM). Cyclic voltammetry and electrochemical impedance spectroscopy were performed to characterize the electrochemical properties of the assembled enzymatic fuel cell.
Acknowledgements
This work was made possible by the catalysis and biocatalysis division of the National Science Foundation (NSF).
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Figure 1