Directed evolution of metalloenzymes through electrochemical droplet microarrays
The goal of this Marie Curie Individual fellowship proposal is to establish directed evolution of redox enzymes by means of electrochemical microarrays (DEMED) to enable the direct screening of the enzyme properties desired for their application in electrochemical devices. An O2 reducing metalloenzyme for implementation in biocathodes of H2/O2 enzymatic fuel cells will serve as model system to demonstrate that directed evolution of such redox enzymes screened by electrochemical droplet microarray is advantageous to specifically improve biofuel cell performances. The selected metalloenzyme is rubredoxin: oxygen oxidoreductase (ROO), which has never been applied to H2/O2 enzymatic fuel cells so far. First, ROO gene will be cloned and its random mutagenesis library will be synthesized. Second, the electrochemical droplet microarray will be adapted to enable the screening of the desired properties of the metalloenzyme. Third, electrochemical directed evolution of ROO will be carried out. Finally, the interface of ROO and electrode based on redox active polymers will be co-evolved with ROO to achieve high electron transfer rates to the enzyme and thus enable the fabrication of a high performance biocathode. It is expected that this project will have a groundbreaking on directed evolution of metalloenzymes for their practical implementation in electrochemical devices.