• Login
    View Item 
    •   Home
    • Massey Documents by Type
    • Theses and Dissertations
    • View Item
    •   Home
    • Massey Documents by Type
    • Theses and Dissertations
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Polyester synthases and polyester granule assembly : a thesis presented to Massey University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology

    Icon
    View/Open Full Text
    2whole.pdf (2.275Mb)
    1front.pdf (197.1Kb)
    Export to EndNote
    Abstract
    PHAs are a class of biopolymers consisting of (R)-3-hydroxy-fatty acids and are produced by the majority of eubacteria and some archaeal bacteria as carbon storage material. In general, PHA is synthesised when a carbon source is available in excess while another essential nutrient is limited. The key enzyme of PHA biosynthesis, the PHA synthase, catalyses the polymerisation of the substrate (R)-3-hydroxyacyl-CoA to PHA accompanied by the release of coenzyme A. PHA is stored intracellularly as inclusions, the so-called PHA granules. When the external carbon source becomes exhausted, bacteria can metabolise these carbon inclusions by degradation of the polymer. PHA granules are water-insoluble, spherical inclusions of approximately 50-500 nm in diameter which consist of a hydrophobic polyester core surrounded by a phospholipid layer with embedded and attached proteins. One could consider isolated PHA granules as bio-beads due to their structure and size. In this study we tested if the PHA synthase can be used as an anchor molecule in order to display proteins of interest at the PHA granule surface. Furthermore, these modified PHA granules were analysed for their potential applicability as bio-beads in biotechnological procedures. The concept of using the PHA synthase as granule-anchoring molecule for display of proteins of interest was established by the functional display of the ß- galactosidase at PHA granules. This “proof of concept” was followed by the display of biotechnologically more interesting proteins. The IgG binding domain of protein A as well as streptavidin, which is known for its biotin binding ability, were fused to the PHA synthase, respectively, and therefore localised at the PHA granule surfaces during PHA granule assembly, resulting in functional bio-protein A -beads and bio-streptavidin-beads. Moreover, their applicability in biotechnological assays was demonstrated. Recently, we fused the green fluorescent protein (GFP) to the PHA synthase and demonstrated that the PHA granule assembly does not start randomly distributed in the cytoplasm but occurred localised at or near the cell poles. To further investigate if the localisation of the PHA granule formation process is due to polar positional information inherent to the PHA synthase, different mutated versions of the PHA synthase of Cupriavidus necator were created and analysed for a potential alteration in localisation. Furthermore, the phasin protein PhaP1 of C. necator was fused to HcRed, a far-red fluorescent protein, and localisation studies were accomplished when the fusion protein was expressed under different conditions in Escherichia coli.
    Date
    2008
    Author
    Peters, Verena
    Rights
    The Author
    Publisher
    Massey University
    URI
    http://hdl.handle.net/10179/808
    Collections
    • Theses and Dissertations
    Metadata
    Show full item record

    Copyright © Massey University
    | Contact Us | Feedback | Copyright Take Down Request | Massey University Privacy Statement
    DSpace software copyright © Duraspace
    v5.7-2020.1-beta1
     

     

    Tweets by @Massey_Research
    Information PagesContent PolicyDepositing content to MROCopyright and Access InformationDeposit LicenseDeposit License SummaryTheses FAQFile FormatsDoctoral Thesis Deposit

    Browse

    All of MROCommunities & CollectionsBy Issue DateAuthorsTitlesSubjectsThis CollectionBy Issue DateAuthorsTitlesSubjects

    My Account

    LoginRegister

    Statistics

    View Usage Statistics

    Copyright © Massey University
    | Contact Us | Feedback | Copyright Take Down Request | Massey University Privacy Statement
    DSpace software copyright © Duraspace
    v5.7-2020.1-beta1