COmbining artificial intelligence and high-thrOughput electrOchemical micrOfluidic screening to engineer muLti copper oxidase – COOOL

The expected market for multicopper oxidases (MCOs), in particular laccases, is one of the fastest growing in the world with 2954 patents filed between 1998 and 2022. The industrial sector has filed 1548 patents, including L'oréal, Dupond, Novozymes,... But, the inability of laccases to operate at neutral/alkaline pH limits their use. Our group has pioneered the use of a different class of MCOs, Bilirubin oxidase (BOD), more stable in the presence of chloride and in a wider range of pH than any other MCOs, increasing their range of applications. We used BODs for the valorization of lignin and as a cathode in biofuel cells. BODs may find other applications due to their ability to convert a wide range of compounds but unlike laccases, BODs suffer from a low redox potential (+0.34V vs. Ag/AgCl at pH 7).
Our challenge is therefore the production of high redox potential BOD (?+0.5V vs. Ag/AgCl at pH 7) that will be used more efficiently in lignin valorization, in biofuel cells, and for unexplored applications where low enzymes redox potential has been a constraint. To achieve this goal, we will combine electrochemistry and microfluidics to develop a high-throughput microfluidic system (achieving throughputs ?100-1000 events per second) that can be used to screen libraries of ?1 million enzyme variants in a day, providing the redox potential of all the enzymes screened.
We envision this strategy as a generic tool for the design of enzymatic systems whose applications are dependent on the redox potential of enzymes.