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dc.contributor.advisorChairperson, Graduate Committee: Valerie Copieen
dc.contributor.authorTopuzlu, Eceen
dc.contributor.otherBrandon 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.en
dc.description.abstractAs 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.en
dc.publisherMontana State University - Bozeman, College of Letters & Scienceen
dc.titleBiochemical and biophysical characterization of plastic degrading aromatic polyesterasesen
dc.rights.holderCopyright 2019 by Ece Topuzluen, Graduate Committee: Brian Bothner; Martin Teintze; Jennifer DuBois; Gregg T. Beckhamen & Biochemistry.en

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