A high throughput assay to detect enzymatic polyethylene oxidation

Area of Science:

• Biotechnology
• Polymer Science
• Environmental Science

Background:

• Biological plastic recycling offers sustainable alternatives to traditional methods.
• While poly(ethylene terephthalate) (PET) recycling is advancing, enzymes for other plastics, like low-density polyethylene (LDPE), are scarce.
• LDPE constitutes a significant portion of plastic waste, necessitating new deconstruction strategies.

Purpose of the Study:

• To develop a high-throughput screening method for identifying enzymes capable of deconstructing non-PET plastics.
• To detect the initial oxidation of the C-H bond to an aldehyde in polymers.
• To accelerate the discovery of enzymes for polyolefin deconstruction.

Main Methods:

• A novel high-throughput screen was developed using 4-hydrazino-7-nitro-2,1,3-benxoxadiozole hydrazine (NBD-H) to detect aldehydes formed during polymer oxidation.
• The probe reacts with aldehydes to form a fluorescent hydrazone on plasma-oxidized LDPE films.
• Fourier Transform Infrared Spectroscopy (FTIR) and receiver operating characteristic (ROC) analysis were used to validate the assay's effectiveness.

Main Results:

• Hydrazone generation showed a strong correlation with aldehyde peaks detected by FTIR (R² = 0.92).
• The NBD-H probe successfully identified LDPE-active dye decolorizing peroxidases (DyPs) that generate aldehydes on LDPE films (1.7–3.0 fold change).
• The assay demonstrated high reliability with an ROC area under the curve of 0.95.

Conclusions:

• The developed assay provides an effective platform for screening LDPE oxidation.
• This method can be parallelized and automated to accelerate the discovery of enzymes for polyolefin deconstruction.
• The findings pave the way for advancing biological recycling of non-PET plastics.