Researcher(s)
- Joshua Bryan, Chemical Engineering, University of Delaware
Faculty Mentor(s)
- Roman Dickey, Department of Chemical and Biomolecular Engineering, University of Delaware
Abstract
As polymer use continues to increase, so does the carbon footprint created by the resulting plastic waste. The depolymerization process of this plastic waste results in various alcohol-containing molecules that do not have much functionality. However, processes are currently being researched to turn these alcohol functionalities into aldehyde, and eventually amine, functionality. These molecules are more valuable and will provide more incentive for use in monomers for use in other classes of materials. As more processes for deconstructing plastic waste come to light, the potential for a closed-loop process to turn the monomer byproducts into more valuable polymers becomes apparent. The three enzymes responsible for this possible closed-loop process are Alcohol Oxidases (AOs) and ω-Transaminases (TAs). AOs have been shown to provide an irreversible pathway from alcohol to aldehyde functionality. Furthermore, ω-Transaminases can turn these aldehydes into amine groups with a proper amine donor substrate. However, AOs are primarily a fungal genome, meaning they will not express well in E. coli. The gap of knowledge lies within the lack of knowledge in terms of bioprospecting of these AOs, as well as the characterization of AOs from bacterial sources. Not much research has been done to show why some show activity when given an alcohol substrate and why some do not. In addition, it is not known why some of them express well in E. coli as compared to other AOs. Our plan is to use the JGI library of ~30 various Alcohol Oxidases to compare activity in converting alcohol groups to aldehydes. In addition, the expression of each AO in E. coli will be tested using a Western Blot. To test the activity of the enzyme on the alcohol substrates, we will be running resting whole cell assays on our AO library. The alcohol substrate we will be using is vanillyl alcohol for initial screening as a model aromatic compound. After the whole cell plate has run for 24 hours, we will transfer the results to the HPLC to analyze the final product and detect if any conversion to aldehyde was present. This assay will help determine which AOs are most effective. From here, the remaining AOs will be tested on various plastic related alcohol substrates to see their activity. Finally, a whole cell assay is done with co-expression of AOs and a TA to test for complete conversion from an alcohol-based substrate to an amine product. The Western Blot concluded that about 70% of the AOs showed expression. The first screening assay of vanillyl alcohol showed that three AOs showed promising conversion to vanillin. Finally, the screening assay of other alcohol-containing substrates showed that the PET derived alcohols of 1,4-benzenedimethanol and 4-(hydroxymethyl) benzaldehyde were suitable substrates and had a conversion to the dialdehyde counterpart. In the future, the AOs should be purified and ran in an In Vitro setting to test activity, as well as screening for other possible substrates.