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

Escherichia coli's Copper efflux oxidase (CueO), a bacterial laccase, 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 more positive onset potential was performed by sampling random and site-saturation mutagenesis libraries in a robust electrochemical screening system. Two positions D439 and L502 were found to be involved in improving the onset potential. Combination of two beneficial substitutions led to a further improved CueO variant (D439T/L502K) with a significantly increased onset potential (0.54 V), making it comparable to high redox potential fungal laccases. Coupling with a glucose dehydrogenase (anodic compartment), the EBFC exhibited an open circuit voltage (Voc) of 0.56 V and a 1.72-fold increase of maximum power output. Directed enzyme evolution by tailoring enzymes to application conditions in EBFCs has been validated and might in combination with a molecular understanding enable breakthroughs in EBFC performances (i.e., power output, lifetime, harsh-environment tolerance, etc.) in the future.