A two-step high-throughput screening platform for engineering enzymes to cure unsaturated polyester resins

Abstract

Unsaturated polyester resins are conventionally cured using cobalt accelerators, which have recently been classified as potentially carcinogenic. Peroxygenase-based enzymatic systems offer a sustainable accelerator alternative if re-engineered to function efficiently under polymerisation conditions in organic solvents. In this work, we present a two-step high-throughput screening platform designed to evolve the peroxygenase's tolerance to organic solvents, followed by the evaluation of the curing performance in polyester resins under application-relevant conditions. Curing was monitored by measuring the consumption of styrene monomer during cross-linking using a 96-well plate Raman spectrometer. The two-step screening platform was validated by screening three site-saturation mutagenesis libraries at positions L61, F157, and L209, located in the substrate tunnel of the unspecific peroxygenase HspUPO. The variant F157L exhibited a 2.5-fold improvement in tolerance to the organic cosolvent dimethyl sulfoxide (increased residual activity from 26.2 to 66.1% in 67 vol% DMSO) and a 1.2-fold improvement in curing performance (increased conversion rate from 8.22 to 10.14% h⁻¹). The screening platform and obtained results demonstrate that HspUPO is a promising alternative to toxic cobalt accelerators.