A coupled fluorescence assay for high-throughput screening of polyurethane-degrading enzymes

The global accumulation of plastic waste, including polyurethane (PU), demands urgent solutions. Enzymatic depolymerization offers a viable strategy for PU waste valorization. However, the lack of reliable high-throughput screening (HTS) assays for PU-degrading enzymes has hindered progress. Conventional methods are limited by either poor substrate relevance or inadequate quantification, limiting their industrial applicability and predictive accuracy. Here, we developed a HTS assay integrating a chemically defined, synthetic poly(ethylene adipate)-based PU (PEA-PU) substrate with a sensitive fluorescence-based detection cascade. Upon enzymatic hydrolysis, PEA-PU releases adipic acid and ethylene glycol, the latter converted to resorufin via a two-enzyme system (glycerol dehydrogenase and diaphorase). This assay enables rapid quantification, molecular-level monitoring of PU degradation with high reproducibility and sensitivity. Validation using known PU-degrading enzymes (ICCM and BaCut1) demonstrated strong correlation between fluorescence-derived ethylene glycol levels and chromatographic measurements, confirming quantitative accuracy. Unlike commercial substrates, our assay establishes clear structure-activity relationships while enabling direct product quantification. Demonstrating the assay's utility, we screened a focused library of BaCut1 variants and rapidly identified mutants with superior efficacy against both synthetic substrates and commercial PU foam. Ultimately, this work bridges the gap between high-throughput capacity and material relevance, providing a scalable pipeline to accelerate the discovery and engineering of enzymes for real-world PU plastic valorization.