A high throughput assay to detect enzymatic polyethylene oxidation.

Area of Science:

• Biotechnology
• Polymer Science
• Enzymology

Background:

• Biological plastic deconstruction offers a sustainable alternative to traditional recycling.
• While enzymes for poly(ethylene terephthalate) (PET) recycling exist, enzymes for non-PET plastics remain largely undiscovered.
• A high-throughput screening (HTS) platform is crucial for accelerating the discovery of enzymes for non-PET plastic degradation.

Purpose of the Study:

• To develop a novel HTS liquid-based assay for detecting the initial C-H bond oxidation step in polyolefin degradation.
• To validate the assay's effectiveness in identifying enzymes capable of initiating polyolefin biodeconstruction.

Main Methods:

• Development of a HTS liquid-based assay using 4-hydrazino-7-nitro-2,1,3-benxoxadiozole hydrazine (NBD-H) to detect aldehydes formed during polymer oxidation.
• Testing the NBD-H probe on oxidized low-density polyethylene (LDPE) films and correlating hydrazone generation with carbonyl index.
• Evaluating the assay's ability to identify LDPE-active dye decolorizing peroxidases (DyPs) using receiver operating characteristic analysis.

Main Results:

• Hydrazone generation showed a strong correlation with established carbonyl index metrics for polymer oxidation (R² = 0.97).
• The NBD-H assay reliably identified LDPE-active DyPs, achieving a receiver operating characteristic area under the curve of 0.95.
• The assay enables the screening of thousands of enzymes within 24 hours, significantly faster than existing methods.

Conclusions:

• The developed HTS assay is effective for detecting the initial aldehyde-forming oxidation step in polyolefin degradation.
• This platform accelerates the discovery of novel enzymes crucial for the biological deconstruction of non-PET plastics.
• The assay represents a significant advancement in enzyme discovery for sustainable plastic waste management.