Targeting Polyethylene: How enzymes break down & peptides could detect microplastics
By: Alexander Waldie
The Problem with Polyethylene
Polyethylene (PE) is the most widely produced plastic due to its ease of synthesis, low cost, and favourable properties, including impermeability, durability, and chemical resistance. Due to its popularity, PE is also one of the microplastics most frequently identified through environmental studies. Some examples of where the average Canadian can find PE in their home are shown below in Figure 1. They are broken down by the specific type of PE, defined by the type and number of side chains on the polymer backbone. Multiple studies have now demonstrated that a more branched polymer results in a more environmentally degradable material; however, the exact pathway of degradation is still debated. Specifically, the question of how enzymes aid in degrading PE in the environment has been posed by numerous research groups over the past 30 years. The Honek lab at the University of ݮƵ, led by Dr. John F. Honek (Department of Chemistry), decided to revisit some of these studies and replicate the enzymatic oxidations of PE using commercially available enzymes and standardized PE samples.
Figure 1. You are likely to find three variants of PE in your home, including LDPE, LLDPE, and HDPE. In most cases, the PE is used to contain a product and is therefore likely to have only momentary usefulness before being discarded.
Analyzing Oxidized Polyethylene
By replicating these previous studies, the Honek lab was able to analyze the treated PE using a more chemically specific technique than what was used in the original papers. This technique, known as high-temperature 1H-NMR, examines the hydrogen atom’s chemical environment in the polymer to identify specific forms of oxidation, which present as distinct signals for the oxidized polymer. They also prepared chemically oxidized standards of PE to validate their NMR method before testing the enzyme-treated PE. An example of a 1H-NMR spectrum collected from a validation sample is shown below in Figure 2, with the various peaks colour coded based on their associated functional groups.
Figure 2. High-temperature 1H-NMR provides a unique peak for the types of oxidations found in PE. In contrast, techniques such as FTIR spectroscopy can be fooled by protein residues on the plastic surface.
The enzymes they tested included commercial forms of laccase, manganese peroxidase, and horseradish peroxidase isolated from a variety of plants, fungi, and bacteria using the protocol shown in figure 3. After analyzing over 100 1H-NMR spectra of PE, they were unable to replicate the reported oxidations, with only manganese peroxidase exhibiting some oxidative potential. Though these results do not completely refute the previous studies, they do speak to the need for a carefully designed analysis of treated PE with a thorough cleaning protocol if surface-sensitive techniques like FTIR are to be used to identify oxidation.
Figure 3. The protocol used to test the enzymes included the treatment of PE standards with the enzyme and small molecule mediators, followed by analysis using high-temperature 1H-NMR.
Identifying Plastics with Peptides
Alongside their work with the enzymatic degradation of PE, the Honek lab also worked on a simple and cost-effective method to identify the plastic type of a microplastic sample. This is traditionally performed using FTIR or Raman spectroscopy; however, both methods require expensive equipment and trained personnel. Instead, the Honek lab designed fluorescent probes that use previously identified plastic binding peptides to identify specific plastic surfaces, as shown in figure 4. When they tested these probes, they found that they lacked specificity for their intended plastic targets. Instead, key amino acids such as tryptophan and phenylalanine appeared to impart a general affinity for hydrophobic plastic surfaces.
Figure 4. In theory, the unique amino acid sequence should recognize a specific plastic surface, and the fluorophore should make the plastic glow when excited.
The Honek lab’s work has demonstrated the chemical complexity of working with oxidized plastics and the need for more replication within the enzyme degradation and peptide recognition space. Understanding how plastics degrade and tracking them through the environment could help to find solutions to reduce the socioenvironmental impact of plastics we use on a daily basis. They are now working to publish these results in two papers that they hope will be made available later in the year.
To learn more, Alex’s thesis, “Enzymatic Oxidation of Polyethylene & Peptide-Based Detection of Microplastics” .
Note: This work is currently unpublished. All images included in this article remain the property of the author. The author retains the right to use these images for future publication or use.