Spectrophotometric-Based Assay to Quantify Relative Enzyme-Mediated Degradation of Commercially Available Bioplastics

• Published in: Polymers Vol. 15; no. 11; p. 2439
• Main Authors: Hoekstra, Matthew, Smith, Myron L
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 24.05.2023; MDPI
• Subjects: Amino acids; Analysis; assay development; Assaying; Bacteria; Biodegradation; bioplastic; Bioplastics; biopolymer degradation; Chemical bonds; Composting; Decomposition (Chemistry); Degradation; enzyme degradation; Enzymes; Hydrolysis; Identification and classification; Identification methods; Landfill; Mathematical analysis; Methods; NMR; Nuclear magnetic resonance; Peptides; PLA depolymerase; Plastics; Polyester resins; Polyethylene terephthalate; Polylactic acid; Polymers; Properties; Protein expression; Proteinase; proteinase K; Proteins; Scanning electron microscopy; Scanning microscopy; Seawater; Spectrophotometry; Temperature; Canada; Japan; enzyme degradation; proteinase K; bioplastic; biopolymer degradation; PLA depolymerase; assay development
• ISSN: 2073-4360; 2073-4360
• DOI: 10.3390/polym15112439

We present a spectrophotometric-based assay to identify enzymes that degrade commercially available bioplastics. Bioplastics comprise aliphatic polyesters with hydrolysis-susceptible ester bonds and are proposed as a replacement for petroleum-based plastics that accumulate in the environment. Unfortunately, many bioplastics can also persist in environments including seawater and waste centers. Our assay involves an overnight incubation of candidate enzyme(s) with plastic, followed by A610 spectrophotometry using 96-well plates to quantify both a reduction in residual plastic and the liberation of degradation by-products. We use the assay to show that Proteinase K and PLA depolymerase, two enzymes that were previously shown to degrade pure polylactic acid plastic, promote a 20-30% breakdown of commercial bioplastic during overnight incubation. We validate our assay and confirm the degradation potential of these enzymes with commercial bioplastic using established mass-loss and scanning electron microscopy methods. We show how the assay can be used to optimize parameters (temperature, co-factors, etc.) to enhance the enzyme-mediated degradation of bioplastics. The assay endpoint products can be coupled with nuclear magnetic resonance (NMR) or other analytical methods to infer the mode of enzymatic activity. Overall, the screening capacity of the spectrophotometric-based assay was demonstrated to be an accurate method to identify bioplastic-degrading enzymes.