Development of Bienzymatic Cascade for Direct Bioelectrochemical Ethanol Oxidation with Alcohol and Aldehyde Dehydrogenases
Authors: Adachi Taiki , Kano Kenji , Sowa Keisei , Kitazumi Yuki , Shirai Osamu 
Published: December 2024

In vivo, a variety of enzymatic cascade reactions are working to efficiently and rapidly metabolize biomolecules. Oxidoreductases especially play key roles in biochemical redox reactions such as fermentation, respiration, and photosynthesis, which might lead to efficient conversion of eco-friendly materials and production of useful compounds. Some enzymatic redox reactions can be artificially coupled with electrode reactions, which is called bioelectrocatalysis. Particularly, an electrical communication without any external electron mediators is called direct electron transfer (DET)-type bioelectrocatalysis, which can be an analytical system evaluating kinetic and thermodynamic characteristics of enzymes, and are expected to be applied to biomimetic devices such as biofuel cells, bioreactors, and biosensors.
We focused on two DET-type enzymes: alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) from acetic acid bacteria (Gluconobacter oxydans). Both enzymes form heterotrimeric structures composed of the catalytic large subunit, the chaperonic small subunit, and the membrane-bound cytochrome c subunit. They physiologically play key roles in the respiration, and the electrons are transferred from substrates to ubiquinone in the periplasmic space. Although DET activities of ADH and ALDH have been already reported, their three-dimensional (3D) structures remain unknown, which prevents the elucidation of detailed electron transfer pathways and improved DET-type bioelectrocatalysis of ADH and ALDH. In this study, we attempted to establish the bienzymatic cascade for bioelectrochemical 4-electron oxidation of ethanol into acetate via acetaldehyde, using ADH and ALDH.
To optimize the surface structure of electrodes for the two enzymes by considering their structural characteristics, we firstly elucidated the 3D structures of ADH and ALDH using cryo-electron microscopy analysis. The 3D structures were reconstructed with a 2.5 Å resolution for ADH and a 2.7 Å resolution for ALDH, respectively. Pyrroloquinoline quinone and four hemes c were resolved in ADH, while a molybdenum cofactor, two iron-sulfur clusters, and three hemes c were resolved in ALDH. Cyclic voltammograms were then recorded at multi-walled carbon nanotube-modified glassy carbon electrodes (CNT/GCs) functionalized with five pyrene derivatives and the enzyme (ADH or ALDH). Regardless of pyrene modification, all prepared electrodes performed clear DET-type ethanol oxidation by ADH and acetaldehyde oxidation by ALDH, respectively, and the 1-pyrenecarboxylic acid (PyCOOH)-functionalized CNT/GC showed the best catalytic properties for both ADH and ALDH.
Based on the electrochemical results, PyCOOH-functionalized CNT/GC was selected as an electrode platform for bienzymatic electrochemical cascade. The electrodes were functionalized with a mixture of ADH and ALDH. The molar concentrations of ADH and ALDH (c
1 and c
2, respectively) were variously controlled under the condition that c
1 + c
2 was constant. The apparent DET-type current density for ethanol oxidation reached a maximum around log (c
1 / c
2) = 0.5, which was higher than that at c
1 / c
2 = ∞ (only ADH). Such increase in the DET-type current density indicates that acetaldehyde produced by ADH was subsequently oxidized by ALDH, which means a bienzymatic cascade. A mathematical model for a bienzymatic DET-type cascade reaction was constructed using the experimental results. These calculations indicated that the ratio of the amounts and catalytic constants of the two enzymes was a key factor controlling the performance of the bienzymatic cascade. In addition, the nanostructure of the enzyme-electrode interface which seems to affect diffusion of the intermediate product was also important for improving cascade efficiency.
An ethanol/air biofuel cell was finally constructed with a bienzyme (ADH and ALDH)-functionalized bioanode and a bilirubin oxidase (BOD)-functionalized biocathode. The open-circuit voltage, the maximum current density, and the maximum power density were 0.75 ± 0.02 V, 2.69 ± 0.09 mA cm–2, and 0.48 ± 0.01 mW cm–2, respectively. Comparing these characteristics with those of a biofuel cell without ALDH, the performances were improved by ALDH co-adsorption. The ethanol/air biofuel cell worked much better than any other ethanol biofuel cells reported to date. The Faraday efficiency for acetate production of the ethanol/air biofuel cell reached 100 ± 4%, which indicates that the bienzymatic DET-type cascade using ADH and ALDH performed highly efficient 4-electron oxidation of ethanol into acetate. A conceptual diagram of the system is shown in the attached figure.
These works will enhance the utilization of biomass fuels and lead to a low-carbon society. These discussions also suggest that the design of the nanostructured interface between the catalysts and electrodes are important for efficient turnover of the intermediate product in the multi-catalytic cascade system.

Figure 1