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Eric Williamsen and Anthony Lobo
The global accumulation of plastic waste is an environmental disaster, with one of the most common plastics being poly(ethylene terephthalate) (PET). Cutin is one of the two polymers that makes up the waxy surface of many plants and shares many structural similarities with PET. Additionally, it is believed that the hydrolysis of cutin via cutinase produced by microbes occurs more rapidly than that of PETase. The objective of this study was to culture thermophilic bacteria found in compost to break down cutin, which closely resembles PET, and evaluate the process. A sterilized broth solution, including cutin isolated from apple skins, was inoculated with compost. Samples were taken regularly from an experimental group and control group inoculated with sterile compost. We performed extraction techniques that were necessary to isolate pure cutin, followed by gas chromatography-mass spectrometry (GCMS) and high-performance liquid chromatography (HPLC) analysis of extracted cutin inoculated with various bacteria to determine if present bacteria were capable of enzymatic degradation of cutin into suspected monomer units. Due to this research being preliminary, the focus was primarily on fine-tuning GCMS and HPLC methods that resulted in quality retention and separation on chromatograms, which were used on the cutin samples to detect cutinase activity. This research provides efficient cutin extraction and isolation, and the chromatographic methods will be useful in further detection of cutinase activity and subsequent quantitation of breakdown monomers.
Cutinase is a highly versatile efficient catalyst that can both hydrolyze and synthesize organic material. Additionally, it functions in water as well as organic solvents. Its lack of thermostability is the reason it has not been widely commercialized. Discovering more stable cutinase enzymes could have large implications for manufacturing, chemical, and purification industries. Cutinase from leaf branch compost has been shown to breakdown commercially used plastics around the globe. Identifying efficient cutinase species requires quick, detailed, and highly precise results from qualitative separations techniques. Specifically, testing the capability of cutinases to enzymatically degrade polymers through detection of broken-down monomer units is a viable approach when using GCMS because of its high sensitivity selected ion monitoring and ability to reveal qualitative information through fragmentation. However, due to the similar nature in test sample monomer units, the HPLC method could not provide efficient separation. But the increased definition of shoulder peaks could allow for the use of HPLC in combination with another more qualitative technique such as GCMS or NMR.
Zubler, Samantha; Miller, Addisen; and Beckman, Samantha, "Extraction of Cutin and Examination of Possible Breakdown to Monomer Units by Thermophilic Compost Enrichments" (2021). Biochemistry and Molecular Biology Presentations. 23.
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