An enzymatic BioBattery based on multicopper oxidases with reduced substrate diffusion constraints for sustainable energy harvesting.

The development of efficient enzymatic energy conversion systems is often limited by restricted substrate transport to the electrode surface and instability of immobilized biocatalysts. In this work, we report the design and experimental validation of an enzymatic fuel cell, referred to as a BioBattery, aimed at reducing substrate diffusion constraints through the co-immobilization of catalytic components within a structured electrode matrix. The system employs multicopper oxidases as anodic biocatalysts, and three enzymes-multicopper oxidase from Pyrobaculum aerophilum, laccase from Trametes versicolor, and bilirubin oxidase from Myrothecium verrucaria-were systematically evaluated to identify the most effective anodic configuration. In parallel, different cathodic architectures were investigated with emphasis on oxygen reduction as the terminal electron-accepting reaction. The optimal configuration consisted of the multicopper oxidase preparation from Pyrobaculum aerophilum at the anode coupled with an amine-reactive phenazine ethosulfate-modified bovine serum albumin (arPES-BSA) matrix at the cathode, where BSA acts as a structural scaffold for mediator immobilization. The resulting BioBattery was further integrated with a previously reported BioCapacitor system and demonstrated reproducible charging of a 470 μF capacitor. The obtained results highlight the potential of structured enzyme-mediator architectures to improve the operational stability and electrochemical performance of enzymatic energy devices. Overall, the proposed BioBattery configuration provides a promising platform for sustainable energy harvesting in low-power applications such as wearable and implantable electronic systems.