Theses and Dissertations at Montana State University (MSU)
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Item Biochemical and biophysical characterization of plastic degrading aromatic polyesterases(Montana State University - Bozeman, College of Letters & Science, 2019) Topuzlu, Ece; Chairperson, Graduate Committee: Valerie Copie; Brandon C. Knott and Mark D. Allen were authors and Japheth Gado, Harry P. Austin, Erika Erickson, Bryon S. Donohoe, Nicholas A. Rorrer, Fiona L. Kearns, Graham Dominick, Christopher W. Johnson, Valerie Copie, Christina M. Payne, H. Lee Woodcock, Gregg T. Beckham and John E. McGeehan were co-authors of the article, 'Structural and biochemical characterization of MHETASE' submitted to the journal 'Proceedings of the National Academy of Sciences of the United States of America' which is contained within this dissertation.As the world is producing more plastics than it can recycle, accumulation of manmade polymers in the environment is becoming one of the greatest global threats humanity is facing today. One of the major contributors to the plastics pollution problem is polyethylene terephthalate (PET), an aromatic polyester widely used in the packaging, beverage, garment and carpeting industries. As a response to the onslaught of plastics in the environment, fungi and bacteria are evolving metabolic pathways to convert plastics into useable energy sources. One of these organisms, a bacterium, Ideonella sakaiensis 201-F6, has recently been identified to convert PET into its monomers, terephthalic acid (TPA) and ethylene glycol (EG), and to use these compounds for energy and growth. I. sakaiensis' ability to convert PET is made possible by two enzymes, named PETase and MHETase. As a first step, PETase breaks down the insoluble substrate PET into a soluble major hydrolysis product - mono-(2- hydroxyethyl) terephthalate (MHET), which is then further hydrolyzed by MHETase into TPA and EG. Crystal structure of PETase, as well as some of its biochemical features, have been reported several times to date, but MHETase has remained largely uncharacterized. This work focuses on further discovery-driven biophysical and biochemical characterization of PETase, visualization of PETase activity on various polyester surfaces, as well as the structural and biochemical characterizations of the MHETase enzyme. We have found that several aspects of PETase-mediated substrate surface modification hydrolysis mechanisms differ depending on the specific mechanical and material characteristics of the substrate. We have also found that PETase is inhibited by BHET. Additionally, we have solved the crystal structure of MHETase. MHETase consists of an alpha/beta hydrolase domain, and a 'lid' domain, commonly seen in lipases. Molecular dynamics simulations revealed the mechanism of MHETase action. Through bioinformatics approaches, we have also identified mutants of interest for improved MHETase activity. Coincubation of MHETase with PETase affects PET turnover in a synergistic fashion. Taken together, this work provides additional insights into the mechanisms of action of the PETase and MHETase enzymes, which may open new avenue for bioremediation and removing plastics from the environment in a sustainable manner.Item The non-classical crystallization of CeO 2 nanoparticles(Montana State University - Bozeman, College of Letters & Science, 2017) Pettinger, Natasha Wren; Chairperson, Graduate Committee: Patrik R. Callis; Robert E. A. Williams, Jinquan Chen and Bern Kohler were co-authors of the article, 'Crystallization kinetics of cerium oxide nanoparticles formed by spontaneous, room-temperature hydrolysis of cerium(IV) ammonium nitrate in light and heavy water' in the journal 'Physical chemistry chemical physics' which is contained within this thesis.Over the past couple of decades, new in situ characterization techniques such as liquid-cell TEM have revitalized efforts to understand the mechanisms of crystal formation. The spontaneous, room-temperature crystallization of CeO 2 from mM concentrations of cerium(IV) ammonium nitrate (CAN) in water was studied using UV-Vis absorption spectroscopy, transient absorption spectroscopy, x-ray diffraction, and high-resolution transmission electron microscopy. Characterization of the final nanoparticles revealed polycrystalline CeO 2 nanoparticles that are stable from aggregation over a period of months. Most of the nanoparticles are between 3 and 9 nm, although a small proportion of larger particles between 10 and 150 nm were also detected. Crystallization is accompanied by a large change in absorption which can be modeled by the presence of just two species. These species are argued to be an amorphous, hydrated intermediate that is converted to nanocrystalline CeO 2 over a period of minutes to hours. The rate-limiting step of the amorphous to crystalline transition involves a proton transfer reaction, as evidenced by a solvent kinetic isotope effect of ~10. Ultrafast transient absorption measurements show a drastic difference between the optical properties of the crystalline nanoparticles and the amorphous precursors. This system is an excellent model system for studying non-classical crystallization because the minutes-to-hours time scale and the small sizes of the nanoparticles and precursors allow for in situ observation of crystallization using steady-state absorption spectroscopy. This system would also lend itself well to characterization by other techniques such as liquid-cell TEM or x-ray absorption spectroscopy.Item Ptyalin action on insoluble acid-treated cellulose residues(Montana State University - Bozeman, College of Letters & Science, 1949) Sanborn, Eldon N.Item An analysis of dendritic cooperativity in protein hydrolysis(Montana State University - Bozeman, College of Letters & Science, 2005) O'Dell, Jacob Webb; Chairperson, Graduate Committee: Mary J. CloningerCatalysts are compounds that increase the rate of a chemical reaction by lowering the activation energy while not being permanently altered. Adding a catalyst to a reaction, while increasing the rate, complicates purification because the catalyst must be separated from the product(s) of the reaction. If the catalyst is a drastically different size than the product then seperation can be achieved as easily as filtering over a membrane. Dendrimers are becoming popular scaffolding for tethering catalysts. Attaching a catalyst to a dendrimer makes a bigger catalytic unit that is easily separated from reaction product(s) of similar size to the untethered catalyst. However, attaching a catalyst to a dendritic framework usually results in a decrease in the catalystαs activity. Previous work reported that attaching three salicylic acid residues in close proximity to each other on a linear PPI polymer catalyzed the hydrolysis of the protein immunoglobulin Gαs peptide backbone. Attaching three salicylic acid residues at random locations on the polymer showed significantly less catalytic activity. Salicylic acid functionalized generations 1-5 PPI dendrimers were synthesized and characterized. Rate enhancement of IgG hydrolysis by the functionalized dendrimers was studied with SDS-PAGE. Generation 5 salicylic acid functionalized PPI dendrimers catalyzed the hydrolysis of IgG while lower generations and 5-nitrosalicylic acid did not. Generation 5 salicylic acid functionalized dendrimers catalyzing IgG hydrolysis is another in a small number of examples of catalytic systems enhanced by dendritic scaffolding.