2024 AIChE Annual Meeting
Developing a High-Throughput Fluorometric Assay for Detecting Enzyme Activity on Ldpe
Plastic waste processing continues to be a large ecological issue. The current processes for recycling plastic are insufficient to meet the current demand, meaning most of it ends up as waste that accumulates in the environment. Thus a novel and more efficient plastic recycling method must be developed to reduce this impact. Through the analysis of mealworm gut microbiomes, multiple enzymes have been discovered that are capable of oxidizing low-density polyethylene (LDPE) films. An enzyme capable of facilitating this reaction could be used in a complete biodegradation enzymatic pathway for processing LDPE waste, which could be used as a substitute for current plastic recycling processes. These LDPE-active enzymes belong to a large family with large characteristic differences. Knowing which characteristics facilitate LDPE activity would make it possible to engineer an optimized enzyme. Thus, a high-throughput fluorometric assay was developed to conduct a library search for LDPE-active enzymes. 4-Hydrazino-7-nitro-2,1,3-benzoxadiazole Hydrazine (NBD-H) was used as the fluorometric reagent due to its selective ability to detect aldehydes over other ketones. LDPE films were dosed with purified, previously discovered LDPE-active enzymes at optimal conditions and allowed to dry before reacting with NBD-H. A high fluorometric signal was only observed for samples dosed with an active enzyme. Enzymes that were deactivated with large concentrations of cofactor and samples that did not contain the LDPE-active enzymes had a similar baseline fluorometric signal less than that of the active enzyme samples. The success of this assay paves the way for a library search for other LDPE-active enzymes. The screening results will be used to narrow down candidates for future study and optimization.