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Updated: Jan 30, 2026

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
Published on: March 16, 2011
Directed Evolution of a Bacterial Laccase (CueO) for Enzymatic Biofuel Cells
Lingling Zhang1, Haiyang Cui1, Zhi Zou1,2
1Institute of Biotechnology, RWTH Aachen University, Worringer Weg 3, 52074, Aachen, Germany.
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 (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.
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