Immobilization of the enzyme GpdQ on magnetite nanoparticles for organophosphate pesticide bioremediation

Abstract

Annually thousands of people die or suffer from organophosphate (pesticide) poisoning. In order to remove these toxic compounds from the environment, the use of enzymes as bioremediators has been proposed. We report here a Ser127Ala mutant based on the enzyme glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes. The mutant, with improved metal binding abilities, has been immobilized using glutaraldehyde on PAMAM dendrimer-modified magnetite nanoparticles. The immobilized system was characterized using elemental analysis as well as infrared, transmission electron and X-ray photoelectron spectroscopies. The amount of GpdQ that was immobilized with the optimized procedure was 1.488 nmol per g MNP. A kinetic assay has been designed to evaluate the activity of the system towards organophosphoester substrates. The specific activity towards BPNPP directly after immobilization was 3.55 μmol mg -1 min -1 , after one week 3.39 μmol mg -1 min -1 and after 120 days 3.36 μmol mg -1 min -1 , demonstrating that the immobilized enzyme was active for multiple cycles and could be stored on the nanoparticles for a prolonged period.3

X-ray Scattering Studies of Protein Structural Dynamics

“…GpdQ is unique among known metallo-phosphodiesterases in that it displays broad substrate specificity, 274 which has made this enzyme the subject of bioengineering efforts for the bioremediation of organophosphate pesticides, including EA 2192, a highly toxic degradation product of the nerve agent VX. 275,276 …”

Organophosphate-degrading metallohydrolases: Structure and function of potent catalysts for applications in bioremediation

“…In particular, mutations in the second coordination sphere (i.e. the replacement of His81, Ser127 and His217 by alanine) led to considerable improvements in metal ion binding affinities without affecting the catalytic performance of the enzyme [137,141]. Furthermore, directed evolution experiments have revealed that the catalytic efficiency (i.e.…”

“…A current endeavor is thus to establish systems that employ both enzymes, preferably in an immobilized state to facilitate continuous use. Preliminary attempts to immobilize the OpdA-related enzyme OPH and GpdQ using mesoporous materials and magnetic nanoparticles, respectively, were encouraging since the process of immobilization led to increased stability and reactivity for both [141,159,160].…”

Utilisation of micro- and nanoscaled materials in microfluidic analytical devices

“…In both studies, it was observed that the immobilised enzymes had higher thermal stability and surprisingly superior activity compared with the free enzymes. Immobilised enzymes on MNPs were also utilised for organophosphate pesticide remediation [55]. A mutant form of the enzyme glycerophosphodiesterase was immobilised on the nanoparticles recovered with poly(amido amine) dendrimer using glutaraldehyde as a linking agent.…”