Colorimetric High-Throughput Screening Assay to Engineer Fungal Peroxygenases for the Degradation of Thermoset Composite Epoxy Resins

At present, the end-of-life management of thermoset composite epoxy resins is limited to incineration and landfill storage, highlighting demand for development more sustainable measures. Due their broad spectrum C-H oxyfunctionalization reactions, fungal unspecific peroxygenases (UPOs) are becoming important biotechnological tools in organic synthesis while potential use biodegradation processes should not be underestimated. Here, we present a colorimetric screening assay aimed at engineering UPOs degradation resins. We based our study on Hexflow ® RTM-6, commercial resin used extensively aeronautics sector. UPO mutants from short long families were initially benchmarked by GC/MS determine N -dealkylation activity N,N-bis(2-hydroxypropyl)- p -toluidine (NNBT), main structural scaffold RTM-6. A reliable high-throughput method was developed quantify lactaldehyde released attack tertiary amine NNBT. Based an evolved Psathyrella aberdarensis that expressed yeast, small subset mutant libraries with different mutational loadings constructed screened NNBT -dealkylation, thereby establishing directed evolution platform as vehicle engineer degrading variants.