High-Efficiency Enzymatic Fuel Cell Via Multienzyme Cascade on DNA Scaffold
Authors: Yu Sooyoun , Chen Qi , Chen Wilfred , Myung Nosang Vincent 
Published: February 2020

Even though enzymatic fuel cells (EFCs) are considered as a promising green alternative method of power generation, much of their research has been limited to the use of a single enzyme as biocatalyst, leading to incomplete oxidation as well as limited range of simple fuels. In this work, enzymatic fuel cell utilizing multienzyme cascade system immobilized on DNA scaffold as anodic biocatalyst as well as high-surface area, electrically conductive carbonaceous nanofibers (CNFs) as electrodes was built for the first time to accommodate a complex molecule such as cellulose as fuel. Three cellulases and cellulose-binding domain (CBD) were expressed in E.Coli, purified using elastin-like polypeptide (ELP) and then conjugated with DNA linker using a fusion partner called HaloTag. All four enzymes and commercial glucose oxidase (GOx) were site-specifically immobilized on customized DNA template via hybridization for efficient cellulose hydrolysis and subsequent glucose oxidation. Varied number of enzymes were combined on the DNA scaffold and resulting reaction rate was compared to the same number of enzymes freely suspended in solution. Successful immobilization of enzymes on the DNA template increased the reaction rate up to 10-fold compared to when they were in solution, confirming the synergistic effect of the multiple enzymes immobilized on a scaffold. Investigation of temperature and pH effect revealed imitating the enzymes’ typical habitat (i.e. 37 oC and pH5) further increased the reaction rate of the multienzyme system. Carbonaceous nanofibers (CNFs) were used as the electrodes for both anode and cathode, which were first produced as polyacrylonitrile (PAN) nanofibers via electrospinning method. Three sets of design of experiments (DOEs) were used to systematically vary the solution, electrospinning, and environmental conditions. Analyses of the effect of the conditions on nanofiber diameter and bead density allowed for minimization of PAN nanofiber diameter down to 38 ± 7 nm with negligible bead density. PAN nanofibers were then converted to CNFs via a two-step heat treatment, and their resulting molecular structure confirmed by FT-IR and XRD was graphitic. Electrical conductivity characterized by 4-point probe measurement supported that CNFs were suitable for electrode applications, ranging from 1.2 to 7.4 S/cm. Electrochemical characterization of CNF mat electrode functionalized with GOx exhibited direct electron transfer from GOx cofactor to the CNF mat electrode at E=-0.63 V vs. Ag/AgCl. Anodic performance of CNF mat electrode functionalized with multienzyme system was electrochemically characterized by cyclic voltammetry, chronoamperometry and electrochemical impedance spectroscopy. Finally, cellulolytic enzymatic fuel cell was assembled with the multienzymatic CNF anode and CNF cathode, which was functionalized with bilirubin oxidase for oxygen reduction.
Figure 1. Schematic representation of the cellulolytic enzymatic fuel cell with multienzyme cascade on DNA scaffold immobilized on carbonaceous nanofiber mat electrode. Components not drawn to scale.

Figure 1