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

The global accumulation of plastic waste, particularly persistent polymers like polyurethane (PU), demands urgent solutions. Enzymatic depolymerization offers a viable strategy for PU waste valorization. However, progress has been hindered by the lack of reliable high-throughput screening (HTS) assays capable of precise and quantitative evaluation of enzymatic activity. Current enzyme screening assays face significant limitations due to poor substrate relevance and inadequate quantification methods. Here, we present a novel HTS assay featuring a chemically defined, synthetic poly (ethylene adipate)-based PU substrate that enables unambiguous structure-activity analysis and direct quantification of degradation products (adipic acid and ethylene glycol). Coupled with this substrate is a highly sensitive fluorescence-based detection cascade, in which released ethylene glycol is stoichiometrically converted to resorufin via a two-enzyme system (glycerol dehydrogenase and diaphorase). This assay overcomes key limitations of commercial substrates (e.g., Impranil DLN) by providing quantitative, real-time monitoring of PU hydrolysis with molecular precision. Validation with known PU-degrading enzymes demonstrated that the assay can sensitively distinguish differences in enzymatic activity with high reproducibility and quantitative accuracy. Our platform enables rapid, cost-effective screening of enzyme libraries and engineered variants, significantly advancing enzyme discovery and optimization efforts. By bridging the gap between laboratory research and industrial application, this HTS assay accelerates the development of sustainable PU recycling solutions, offering a critical tool against plastic pollution. Graphical Abstract Legend (A) Workflow of the enzymatic degradation assay. Enzymes are incubated with chemically defined PEA-PU films. The resulting hydrolysate is collected and analysed using a fluorescence-based assay in microplate format (Ex/Em = 535/588 nm). (B) Fluorescence-based detection mechanism. Assay principle based on ethylene glycol (EG), one of the major degradation products. EG is converted through a two-step enzymatic cascade involving glycerol dehydrogenase (GldA) and diaphorase, leading to the generation of fluorescent resorufin from resazurin.