Directed evolution of bacterial laccase CueO for enzymatic biofuel cells
Lingling Zhang, Haiyang Cui, Zhi Zou, Tayebeh Mirzaei Garakani, Catalina Novoa-Henriquez, Bahareh Jooyeh, and Ulrich Schwaneberg, Angew. Chem. Int. Ed, 2019. DOI: 1002/ange.201814069
Directed evolution of CueO via electrochemical screening found substitutions that led to the dramatic overpotential decrease of 0.12 V, making CueO competitive to fungal laccases and applicable in enzymatic biofuel cells.
In this study, the bacterial laccase CueO from Escherichia coli was chosen to perform directed evolution towards lowering the overpotential of cathodic oxygen reduction. A robust and efficient 8-channel electrochemical screening platform was developed for the first time to evaluate CueO variants generated by random mutagenesis and subsequent site-saturation mutagenesis (SSM). The enzyme immobilization could be accomplished in 20 seconds and directly from crude cell lysates. Two positions adjacent to the coordinated ligands of the T1 copper site, was identified as a main region that contribute to improvements in the onset potential. A remarkable increased onset potential of 0.54 V was obtained with two amino acid substitutions. Finally, the cathode generated open circuit potential of 0.56 V as well as 1.72-fold enhanced power output for the enzymatic biofuel cell comprising the generated CueO variant coupled with a glucose dehydrogenase. The developed directed evolution protocol offers a promising methodology beyond chemistry and materials science in improving laccase properties such as substrate specificity, turnover efficiency, and tolerance to harsh-environment conditions.
This work was supported by the Alexander von Humboldt Foundation.