title: Enhancing enzyme stability by construction of polymer-enzyme conjugate micelles for decontamination of organophosphate agents.
abstract: Enhancing the stability of enzymes under different working environments is essential if the potential of enzyme-based applications is to be realized for nanomedicine, sensing and molecular diagnostics, and chemical and biological decontamination. In this study, we focus on the enzyme, organophosphorus hydrolase (OPH), which has shown great promise for the nontoxic and noncorrosive decontamination of organophosphate agents (OPs) as well as for therapeutics as a catalytic bioscavanger against nerve gas poisoning. We describe a facile approach to stabilize OPH using covalent conjugation with the amphiphilic block copolymer, Pluronic F127, leading to the formation of F127-OPH conjugate micelles, with the OPH on the micelle corona. SDS-PAGE and MALDI-TOF confirmed the successful conjugation, and transmission electron microscopy (TEM) and dynamic light scattering (DLS) revealed ∼100 nm size micelles. The conjugates showed significantly enhanced stability and higher activity compared to the unconjugated OPH when tested (i) in aqueous solutions at room temperature, (ii) in aqueous solutions at higher temperatures, (iii) after multiple freeze/thaw treatments, (iv) after lyophilization, and (v) in the presence of organic solvents. The F127-OPH conjugates also decontaminated paraoxon (introduced as a chemical agent simulant) on a polystyrene film surface and on a CARC (Chemical Agent Resistant Coating) test panel more rapidly and to a larger extent compared to free OPH. We speculate that, in the F127-OPH conjugates (both in the micellar form as well as in the unaggregated conjugate), the polypropylene oxide block of the copolymer interacts with the surface of the OPH and this confinement of the OPH reduces the potential for enzyme denaturation and provides robustness to OPH at different working environments. The use of such polymer-enzyme conjugate micelles with improved enzyme stability opens up new opportunities for numerous civilian and Warfighter applications.
authors.affiliation: Molecular Sciences and Engineering Team, Natick Soldier Research, Development and Engineering Center , 15 Kansas Street, Natick, Massachusetts 01760, United States.
authors.forename: Nisaraporn
authors.initials: N
authors.lastname: Suthiwangcharoen
authors.forename: Ramanathan
authors.initials: R
authors.lastname: Nagarajan
mesh_terms: D043303:Aryldialkylphosphatase / Q000737:chemistry* / Q000378:metabolism; D003666:Decontamination; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D008823:Micelles; D046529:Microscopy, Electron, Transmission; D010755:Organophosphates / Q000737:chemistry*; D010261:Paraoxon / Q000737:chemistry; D020442:Poloxamer / Q000737:chemistry*; D011137:Polystyrenes / Q000737:chemistry; D012996:Solutions; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
chemical_list: D008823:Micelles; D010755:Organophosphates; D011137:Polystyrenes; D012996:Solutions; D020442:Poloxamer; D043303:Aryldialkylphosphatase; D010261:Paraoxon