Bravo, Joseph2024-08-222024-08-222022-06https://hdl.handle.net/11299/265108University of Minnesota M.S. thesis. June 2022. Major: Microbial Engineering. Advisor: Mikael Elias. 1 computer file (PDF); vi, 61 pages.Quorum sensing refers to bacterial communication systems based on a signal molecule that is produced and diffuses into the environment. The concentration of this signal in the environment allows for the behavior to be controlled in a cell-density dependent manner. Quorum sensing systems utilizing Acyl Homoserine Lactones (AHLs) are prominent in many microbial organisms and communities of high impact to human interests. These include certain agricultural and human pathogens, biofilm production, and aquatic biofouling. The structure of AHLs is variable and these structural changes determine the specificity of the signal. Several strategies that interfere with quorum sensing are naturally used by various organisms. One such strategy is to use enzymes that degrade the AHL signal, known as Quorum Quenching (QQ). Previously characterized QQ enzymes have exhibited a broad range of AHL specificity. To broaden the ability of QQ approaches, we decided to create QQ enzymes with increased AHL specificity. This presents a challenge, as we wish to avoid altering the active site catalysis while altering the specificity of the hydrophobic acyl chain of the substrate. We used the broad spectrum, thermostable lactonase from Parageobacillus caldoxylosilyticus, dubbed GcL, as a starting point of our rational design approach. Previously determined crystal structures of GcL uncovered key positions in substrate binding cleft that interact with the AHL substrate’s acyl tail. These positions were used as targets for site saturation mutagenesis, and several mutants with altered substrate preference were identified in a screening step and confirmed with purified enzyme and kinetic characterization. Several mutations were combined, and the best mutants obtained thus far have shifted substrate preference by up to 200-fold in favor of short-chain AHL. These mutants were crystallized, and their structures revealed a reshaping of the active site binding cleft, as well as an alternate substrate binding conformation for a prominent active site loop, which explains the changes in kinetic properties.enThesis or Dissertation