A high throughput assay to detect enzymatic polyethylene oxidation

Biological plastics deconstruction and upcycling have emerged as a sustainable alternative to traditional recycling technologies for plastics waste. The discovery and engineering of efficient thermostable poly(ethylene terephthalate) (PET) hydrolases has made biological PET recycling possible at scale; however, enzymes for non-PET plastics, which account for approximately 70% of all plastics produced, remain largely undiscovered. To accelerate the discovery of such enzymes, a high throughput screening (HTS) platform is needed. Here, we develop a HTS liquid-based assay to detect one of the first committed steps of polyolefin degradation, oxidation of the C-H bond to an aldehyde. We test 4-hydrazino-7-nitro-2,1,3-benxoxadiozole hydrazine (NBD-H), which reacts with generated aldehydes to form a fluorescent hydrazone, on oxidized low-density polyethylene (LDPE) films. Hydrazone generation correlated well with established carbonyl index metrics for polymer oxidation (R² = 0.97). Moreover, we demonstrate that the probe reliably identifies LDPE-active dye decolorizing peroxidases (DyPs) that generate aldehydes on LDPE films, serving as effective screen as demonstrated by a receiver operating characteristic area under the curve of 0.95. Due to the rapid fluorescent readout and parallelization in microarray plates, this assay enables screening thousands of enzymes in 24 hours compared to time-consuming established approaches, accelerating discovery of enzymes that catalyze the first step of polyolefin biodeconstruction.