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

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 (Voc) 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 Einflussreiche Hinterbänkler: Die gelenkte Evolution der bakteriellen Laccase CueO durch elektrochemisches Screening ergab, dass Substitutionen in der zweiten Koordinationssphäre des T1‐Cu‐Zentrums zu einer drastischen Abnahme der Überspannung um 0.12 V führen. Diese Abnahme macht CueO konkurrenzfähig gegenüber pilzlichen Laccasen und anwendbar in enzymatischen Biobrennstoffzellen.