Immobilization of organophosphohydrolase OpdA from Agrobacterium radiobacter by overproduction at the surface of polyester inclusions inside engineered Escherichia coli.

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  • Author(s): Blatchford PA;Blatchford PA; Scott C; French N; Rehm BH
  • Source:
    Biotechnology and bioengineering [Biotechnol Bioeng] 2012 May; Vol. 109 (5), pp. 1101-8. Date of Electronic Publication: 2011 Dec 26.
  • Publication Type:
    Journal Article; Research Support, Non-U.S. Gov't
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: Wiley Country of Publication: United States NLM ID: 7502021 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1097-0290 (Electronic) Linking ISSN: 00063592 NLM ISO Abbreviation: Biotechnol Bioeng Subsets: MEDLINE
    • Publication Information:
      Publication: <2005->: Hoboken, NJ : Wiley
      Original Publication: New York, Wiley.
    • Subject Terms:
      Gene Expression*; Agrobacterium tumefaciens/*enzymology ; Enzymes, Immobilized/*metabolism ; Escherichia coli/*enzymology ; Organophosphates/*metabolism ; Phosphoric Monoester Hydrolases/*metabolism; Agrobacterium tumefaciens/genetics ; Bacteriophage T7/genetics ; Cupriavidus necator/enzymology ; Cupriavidus necator/genetics ; Escherichia coli/genetics ; Metabolic Engineering ; Pesticides/metabolism ; Promoter Regions, Genetic ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/metabolism
    • Abstract:
      Organophosphorus pesticides (OP) are highly toxic and are widely used as insecticides. Bacterial organophosphohydrolases which hydrolyze a variety of OPs have been considered for the clean-up of polluted environments. This study describes the engineering of Escherichia coli towards the overproduction of the organophosphohydrolase (OpdA) from Agrobacterium radiobacter at the surface of polyester inclusions. The OpdA was N-terminally fused via a designed linker region to the C-terminus of polyester inclusion-forming enzyme PhaC of Ralstonia eutropha. The PhaC-L-OpdA fusion protein was overproduced by using the strong T7 promoter and when coexpressed with genes phaA (encoding β-ketothiolase) and phaB (encoding acetoacetyl-CoA reductase) from R. eutropha this led to formation of polyester inclusions abundantly displaying OpdA. These OpdA beads showed organophosphohydrolase activity of 1,840 U/g wet polyester beads or 4,412 U/g protein. Steady state kinetics revealed that when compared with free OpdA the k(cat) (s(-1)) of 139 of immobilized OpdA was reduced by about 16.5-fold while the K(M) (M) of 2.5 × 10(-4) was increased by 1.6-fold. The immobilized OpdA showed increased temperature stability. Moreover, the stability of OpdA immobilized to polyester beads was assessed by incubating OpdA beads at 25°C for up to 11 days and no significant loss in enzyme activity was detected. The application performance of the OpdA beads with respect to hydrolysis of OPs in contaminated environments was demonstrated in wool scour spiked with fluorescent coumaphos. This study demonstrated a new strategy toward the efficient recombinant production of immobilized organophosphohydrolase, the OpdA, suitable for bioremediation applications.
      (Copyright © 2011 Wiley Periodicals, Inc.)
    • Accession Number:
      0 (Enzymes, Immobilized)
      0 (Organophosphates)
      0 (Pesticides)
      0 (Recombinant Fusion Proteins)
      EC 3.1.3.- (phosphorylphosphatase)
      EC 3.1.3.2 (Phosphoric Monoester Hydrolases)
    • Publication Date:
      Date Created: 20111216 Date Completed: 20120723 Latest Revision: 20161125
    • Publication Date:
      20240829
    • Accession Number:
      10.1002/bit.24402
    • Accession Number:
      22170266