Texas A&M University LibrariesTexas A&M University LibrariesTexas A&M University Libraries
    • Help
    • Login
    OAKTrust
    View Item 
    •   OAKTrust Home
    • Colleges and Schools
    • Office of Graduate and Professional Studies
    • Electronic Theses, Dissertations, and Records of Study (2002– )
    • View Item
    •   OAKTrust Home
    • Colleges and Schools
    • Office of Graduate and Professional Studies
    • Electronic Theses, Dissertations, and Records of Study (2002– )
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Directed evolution of phosphotriesterase for detoxification of the nerve agent VX

    Thumbnail
    View/Open
    etd-tamu-2006B-CHEM-Ghanem.pdf (1.476Mb)
    Date
    2006-10-30
    Author
    Ghanem, Eman Mohamed
    Metadata
    Show full item record
    Abstract
    Phosphotriesterase (PTE) isolated from the soil bacterium Flavobacterium sp. is a member of the amidohydrolase superfamily. PTE catalyzes the hydrolysis of a broad spectrum of organophosphate triesters including the insecticide paraoxon, and the chemical warfare agents; GF, sarin, and soman. In addition, PTE has been shown to catalytically hydrolyze the lethal nerve agent, VX. However, the rate of VX hydrolysis is significantly slower. PTE was subjected to directed evolution studies to identify variants with enhanced activity towards VX hydrolysis. First generation libraries targeted amino acid residues in the substrate binding site. The H254A mutation displayed a 4-fold enhancement in kcat and a 2-fold enhancement in kcat/Km over wild type PTE. The double mutant H254Q/H257F was isolated from the second generation libraries and displayed a 10-fold enhancement in kcat and a 3-fold enhancement in kcat/Km. In addition, H254Q/H257F displayed a 9-fold enhancement in kcat/Km for the hydrolysis of the VX analog, demeton-S. An in vivo selection approach utilizing organophosphate triesters as the sole phosphorus source is discussed. The selection is based on co-expressing PTE with the phosphodiesterase (GpdQ) from E. aerogenes. Substrate specificity of GpdQ was investigated using a small library of structurally diverse organophosphate diesters and phosphonate monoesters. Results obtained from the in vivo growth assays showed that GpdQ enabled E. coli to utilize various organophosphate diesters and phosphonate monoesters as the sole phosphorus source. Cells co-expressing PTE and GpdQ were tested for their ability to utilize two different organophosphate triesters as the sole phosphorus source. The results from this experiment indicate that the growth rate is limited by the phosphotriesterase activity. Protein translocation to the periplasm was proven advantageous for in vivo selection since it overcomes the limitation of intercellular delivery of the substrate of interest. Translocation of PTE to the periplasmic space of E. coli was examined. Two signal peptides were tested; the native leader peptide from Flavobacterium sp. and the signal sequence of alkaline phosphatase. The results obtained from cellular fractionation indicated that neither signal peptides were able to translocate PTE to the periplasm and that the protein remained in the cytoplasm.
    URI
    http://hdl.handle.net/1969.1/4330
    Subject
    Phosphotriesterase
    organophosphates
    Directed evolution
    Collections
    • Electronic Theses, Dissertations, and Records of Study (2002– )
    Citation
    Ghanem, Eman Mohamed (2006). Directed evolution of phosphotriesterase for detoxification of the nerve agent VX. Doctoral dissertation, Texas A&M University. Texas A&M University. Available electronically from http : / /hdl .handle .net /1969 .1 /4330.

    DSpace software copyright © 2002-2016  DuraSpace
    Contact Us | Send Feedback
    Theme by 
    Atmire NV
     

     

    Advanced Search

    Browse

    All of OAKTrustCommunities & CollectionsBy Issue DateAuthorsTitlesSubjectsDepartmentThis CollectionBy Issue DateAuthorsTitlesSubjectsDepartment

    My Account

    LoginRegister

    Statistics

    View Usage Statistics
    Help and Documentation

    DSpace software copyright © 2002-2016  DuraSpace
    Contact Us | Send Feedback
    Theme by 
    Atmire NV