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

High‐redox potential laccases (HRPLs) from white‐rot fungi are versatile biocatalysts whose practical use is highly dependent on their thermostability. In this work, an evolved HRPL variant was subjected to structure‐guided evolution to improve its thermostability. We first selected several surface flexible loops in the laccase structure by inspecting them through molecular dynamics and an analysis of B‐factors. The resulting segments were grouped into three MORPHING (Mutagenic Organized Recombination Process by Homologous In vivo Grouping) blocks, which were constructed in Saccharomyces cerevisiae and explored at high temperatures. This evolution process gave rise to a double mutant that showed a half‐life at 70°C enhanced by 31 min with an optimum temperature for activity of 75°C and similar kinetic parameters. The Ser264Lys and Ser356Asn mutations modified the contacts established between these residues and those that surround them, altering the surface loops and thereby the enzyme properties. [ABSTRACT FROM AUTHOR]

Plastic waste is a major threat in our industrialized world and is driving research into bioplastics. The success of biobased polyethylene furanoate (PEF) as a viable alternative to polyethylene terephthalate (PET) of fossil origin will depend on designing effective enzymes to break it down, aiding its recycling. Here, a panel of fungal and bacterial cutinases were functionally expressed in a tandem yeast expression system based on Saccharomyces cerevisiae and Pichia pastoris . The activity of the enzyme panel was tested with soluble PEF model scaffolds, observing a correlation with the degradation of real PEF powder. A high-throughput colorimetric screening assay based on the PEF scaffold diethyl furan-2,5-dicarboxylate was developed, establishing the basis for future directed evolution campaigns of PEFases., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)