title: Immobilization of organophosphohydrolase OpdA from Agrobacterium radiobacter by overproduction at the surface of polyester inclusions inside engineered Escherichia coli.
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.
authors.affiliation: Institute of Molecular Biosciences, Massey University, Palmerston North, New Zealand.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Blatchford
authors.forename: Colin
authors.initials: C
authors.lastname: Scott
authors.forename: Nigel
authors.initials: N
authors.lastname: French
authors.forename: Bernd H A
authors.initials: BH
authors.lastname: Rehm
mesh_terms: D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics; D017123:Bacteriophage T7 / Q000235:genetics; D020118:Cupriavidus necator / Q000201:enzymology / Q000235:genetics; D004800:Enzymes, Immobilized / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D060847:Metabolic Engineering; D010755:Organophosphates / Q000378:metabolism*; D010575:Pesticides / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D004800:Enzymes, Immobilized; D010755:Organophosphates; D010575:Pesticides; D011993:Recombinant Fusion Proteins; C045290:phosphorylphosphatase; D010744:Phosphoric Monoester Hydrolases