Doctoral Dissertations
Date of Award
8-2024
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Major
Microbiology
Major Professor
Todd B. Reynolds
Committee Members
Elizabeth Fozo, Juan Jurat-Fuentes, Erik Zinser, Jennifer DeBruyn
Abstract
Poly-L-lactic acid (PLLA) is the most widely used bioplastic with production expected to greatly increase over the next several years. While considered to be biodegradable, PLLA biodegrades at a slow rate in the environment and can take several years to fully disintegrate. As such, a thorough understanding of the biological mechanisms contributing to the environmental breakdown of these plastics is crucial for ensuring stewardship of plastics containing PLLA. Furthermore, the identification of novel, biochemically diverse PLLA depolymerizing enzymes may be exploited for biotechnological applications in the remediation and recycling of PLLA. As synthetic polymers such as PLLA are a relatively novel invention, few enzymes have been discovered that specifically target these substrates. Enzymes that have activity against plastic typically occur as promiscuous side reactions as these are not the intended substrates of the enzymes. The typically low-level activity of these enzymes against plastic substrates often necessitates protein engineering strategies to enhance depolymerization activity.
In this dissertation, we identify, characterize, and engineer several PLLA depolymerizing protease enzymes originating from bacteria. In Chapter II, we identify the AprE protein from Bacillus pumilus (BpAprE) as an enzyme with some PLLA depolymerization activity, and through a comparative, mutational analysis with a non-degrading homolog originating from Bacillus subtilis (BsAprE), both enzymes were engineered to have substantially improved PLLA depolymerization activities. While this chapter focused mainly on the substrate specificity of the enzymes, Chapter III expands this comparative analysis to look at the surface-associated residues and how they contribute to PLLA adsorption. The key findings of this study demonstrate that increased adsorption is associated with greater ability to depolymerize PLLA independent of substrate specificity. Chapter IV focuses on a novel trypsin-like protease (Tlp) originating from Kibdelosporangium aridum, the use of AlphaFold to determine the putative binding pocket of Tlp, and site directed mutagenesis to engineer the protein to have higher substrate specificity for high molecular weight PLLA. In the appendices, we discuss additional experimentation with BsAprE and BpAprE regarding reaction condition optimization and thermostable protein engineering (Appendix I) as well as the identification of a novel polyester degrading fungus, Aspergillus sydowii (Appendix II).
Recommended Citation
Cannon, Jordan Alexander, "Protein Engineering of Bacterial Proteases for Enhanced Poly-L-lactic Acid Depolymerization. " PhD diss., University of Tennessee, 2024.
https://trace.tennessee.edu/utk_graddiss/10440