Directed evolution of a 6-phosphogluconate dehydrogenase for operating an enzymatic fuel cell at lowered anodic pHs

Abstract

Most enzymatic fuel cells (EFCs) are designed for operating at physiological conditions and cannot stand for low pHs due to the enzyme denaturation. Improving the acid tolerance of EFCs represents the increased robustness and diversity of their practical applications. In this study, we successfully constructed a three-enzyme pathway-catalyzed EFC consisting of two dehydrogenases and a diaphorase that can be operated at the anodic pH of 5.4. The acid-intolerance of one enzyme, 6-phosphogluconate dehydrogenase, was overcome by enzyme engineering. After the directed evolution including three rounds of error-prone PCR and a petri-dish-based double-layer screening method, the best 6-phosphogluconate dehydrogenase mutant (named as 3-3) exhibited a 42-fold increase in catalytic efficiency at pH 5.4 compared to the original enzyme. The electrochemical measurements revealed that the EFC equipped with this mutant achieved a maximum power density of 0.13 mW cm−2 at pH 5.4, more than 10-fold higher than that with the same enzyme unit loading but at pH 7.3. Under a high loading amount of enzymes, the EFC yielded a maximum power density of ∼0.5 mW cm−2, and that with the mutant also exhibited an improved stability. This study demonstrates that lowering anodic pH may be a useful strategy for constructing high-performance and robust EFCs and the concerns for enzyme deactivation can be addressed by enzyme engineering.