Developing an electrochemical assay for the directed evolution of enzymes for electrosynthesis Rokas Gerulskis, Dylan Boucher, Shelley Minteer University of Utah, USA Traditional high-throughput assays for enzyme directed evolution aim to detect mutant “hits”, high activity mutants, through semiquantitative spectrochemical methods. These assays typically employ a secondary catalyst or reagent which produces a colorimetric signal proportional to the product generated by the enzyme of interest, with the aim of screening hundreds of mutants for high activity in short order. These assays are not only laborious to optimize, but are extremely product-specific, offering little utility in the realm of substrate-scope determination. A similar kinetic method in the context of enzymatic electrochemistry is mediated electrocatalysis, wherein the redox of electron mediating species such as ferrocene, viologens, or quinones occurs in proximity of enzyme active sites proportional to enzymatic activity and is consequently independent of the specific redox reaction catalyzed by the enzyme. The rate of mediator turnover can then be quantified as a catalytic current thus providing a readout of enzymatic activity. Electrochemical literature has focused on precise quantification of electroenzymatic activity requiring laborious purification of target enzymes, while little attention has been directed to maximizing reaction throughput in the realm of mutant and substrate-scope screening. This work introduces preliminary investigations into a method to rapidly screen enzyme mutants and substrate specificity using a 96-well electrochemical cell.
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© The Author(s), 2023
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