Directed Evolution of a Bacterial Laccase (CueO) for Enzymatic Biofuel Cells

Abstract

Escherichia coli's copper efflux oxidase (CueO) has rarely been employed in the cathodic compartment of enzymatic biofuel cells (EBFCs) due to its low redox potential (0.36 V vs. Ag/AgCl, pH 5.5) towards O₂ reduction. Herein, directed evolution of CueO towards a more positive onset potential was performed in an electrochemical screening system. An improved CueO variant (D439T/L502K) was obtained with a significantly increased onset potential (0.54 V), comparable to that of high-redox-potential fungal laccases. Upon coupling with an anodic compartment, the EBFC exhibited an open-circuit voltage (V_oc ) of 0.56 V. Directed enzyme evolution by tailoring enzymes to application conditions in EBFCs has been validated and might, in combination with molecular understanding, enable future breakthroughs in EBFC performance.