Abstract

Plastics are a useful and necessary material in the modern world. However, the methods of extraction and the finite nature of petroleum necessitates divesting from traditional petroleum-derived plastics. One avenue being pursued is sustainably sourced plastics; namely plastics made from a biorenewable starting source. Enzyme catalysis and Old Yellow Enzymes (OYEs) in particular are of note in pursuing the generation of plastics from biorenewable sources. Engineering OYEs in pursuit of expanded substrate scope, improved efficiency, and other traits is growing increasingly popular and tools towards this aim remain valuable. In this work, I characterized, optimized, and utilized a novel fluorescence-based enzyme cascade in the investigation of nine candidate OYEs and their activities on four biorenewable plastic precursors. This enzyme cascade couples the redox activity of OYEs to the release of 4-methylumbelliferone, a fluorophore with a fluorescence intensity maximum at 445 nm. First the cascade was verified using Malate Dehydrogenase. This was followed by the optimization of the enzymatic cascade through the mutation of supporting enzyme GapA to utilize NADPH instead of its native NADH and the adjustment of NADPH concentrations to limit off-target fluorescence. Once optimized, the assay was utilized in a screen of nine OYEs with four biorenewable plastic precursors. Hits from the screen were investigated and the assay itself was investigated in the pursuit of enzymatic characterization. With a successful use-case demonstrated for this robust fluorescence-based activity assay, I was successfully able to add a tool to the development of OYEs towards generating sustainable plastics.