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 Analysis of fish tissue for trace amounts of lead by furnace atomic absorption spectroscopy(Montana State University - Bozeman, College of Letters & Science, 1972) Neuman, Dennis R.Item Application of the O 2-doped ECD to isomer differentiation(Montana State University - Bozeman, College of Letters & Science, 1984) Campbell, James AllenThe addition of oxygen to the nitrogen carrier gas of a constant current electron capture detector (BCD) is shown to provide increased sensitivity and isomer distinction for environmentally important compounds such as polychlorinated biphenyls, polycyclic aromatic amines and hydroxides with appropriate EC-enhancing tags, chloroanthracenes and chlorophenanthrenes, methylanthracenes and methylphenanthrenes, and 2,3,7-trichlorodibenzo-p-dioxin. In most cases, the isomers of these particular compounds can be distinguished from the other isomers based solely on the measured response enhancements. The oxygen doped BCD has been applied to compound identification where only partial resolution with capillary column gas chromatography is obtained. In addition, in instances where several compounds coelute and have the exact same retention times, the mole fraction of each component in the unresolved peak can be determined. Atmospheric pressure ionization mass spectrometry (APIMS) gives an indication of the ions formed with and without the presence of oxygen in the source. For the chloroanthracenes, methylanthracenes, and methylphenanthrenes, actual oxygen incorporation is observed when oxygen is present. In contrast, for the polycyclic aromatic amines derivatized with trifluoroacetic anhydride (TFAA), the reaction with oxygen to produce an induced response involves a charge transfer between the oxygen anion and the analyte molecule. Several TFAA derivatives of the aminoanthracenes and aminophenanthrenes were examined using electron impact mass spectrometry and chemical ionization mass spectrometry. Negative chemical ionization mass spectrometry with methane and isobutane as the reagent gases was evaluated as a method of isomer differentiation of these compounds and shows considerable promise. In order to improve the precision involved in the measurement of response enhancements, two methods with parallel and series arrangement of the ECDs were utilized. The parallel arrangement with dual columns represents a significant improvement in the reproducibility of response enhancement measurements. The series detector arrangement seems to be confusing in view of possible additional reactions in the detectors. The addition of ethyl chloride to an electron capture detector increased the response of anthracene and similar molecules with a low normal BCD response and those that react with the gaseous electron through a resonance type of reaction mechanism. The addition of ethyl chloride to the detector does not significantly increase the baseline frequency, in contrast to the addition of oxygen. The negative ions formed in this reaction have been identified.Item Separate and simultaneous bioconcentration in fathead minnows of five organic chemicals(Montana State University - Bozeman, College of Letters & Science, 1984) Tischmak, Dale JohnItem The application of furnace atomic absorption to the solution of problems in environmental analysis(Montana State University - Bozeman, College of Letters & Science, 1973) Lech, Jerome FrancisItem A molecular basis for uranium toxicity(Montana State University - Bozeman, College of Letters & Science, 2014) Burbank, Katherine Ann; Chairperson, Graduate Committee: Brent M. Peyton; Robert K. Szilagyi was a co-author of the article, 'Development of a computational model to describe U(VI) and Pyrroloquinoline quinone interactions' which is contained within this thesis.; Robert K. Szilagyi and Brent M. Peyton were co-authors of the article, 'The effect of CA 2+ displacement by UO 2 2+ on the biological funtion of methanol dehydrogenase' which is contained within this thesis.; Robert A. Walker and Brent M. Peyton were co-authors of the article, 'A molecular basis of metal toxicity by U(VI) in PQQ dependent bacterial dehydrogenase' which is contained within this thesis.Environmental and health problems associated with uranium extend well beyond its radioactive properties. Hexavelent uranium is a common environmental contaminant that reacts with water to form the dioxo-uranium cation, UO 2 2+. Environmental uranium contamination is the result of a number of activities including uranium mining, production and use of depleted uranium for military purposes, storage and disposal of nuclear weaponry, and fuel for nuclear power plants. Despite the potential importance of the interaction of UO 2 2+ with biologically relevant molecules, only limited molecular insight is available. In a recent publication, the presence of UO 2 2+ in submicromolar concentrations was shown to affect ethanol metabolism in Pseudomonas spp. by displacing the Ca 2+ of the pyrroloquinoline quinone (PQQ) cofactor. Accordingly, the interaction of UO 2 2+ with PQQ is used here as a starting point to carry out both an in vitro and in silico analysis of UO 2 2+ and its interactions with biologically relevant cofactors and metabolites. This work represents a proposed molecular mechanism of uranium toxicity in bacteria, and has relevance for uranium toxicity in many living systems. The structural insights from modeling allow us to expand the scope of potential uranium toxicity to other systems by considering the favorable coordination mode to pyridine nitrogen adjacent to carboxylic and/or carbonyl groups. Consequently, the recent discovery of uranium toxicity at submicromolar levels in bacteria provides relevance to serious environmental and public health issues in the light of current EPA regulation of 0.13 micron uranium limit in drinking water.