Development And Application Of Organophosphorus Hydrolase Nanogel Biomaterials

Organophosphorus hydrolase (OPH) enzyme that adopts a binuclear metal active center is a member of the amidohydrolase superfamily. They have displayed such a great potency in hydrolyzing a broad range of the extremely toxic organophosphate pesticides and nerve agents with a well characterized stereoselectivity that they have been recognized as a potential candidate for extending the process of biocatalyst organophosphate detoxification. Up to now, great progresses have been made in the synthesis, characterization and theoretics of OPH enzymes. However, the characteristics of environmental unstabilities still limits their practical application. Therefore, modification and immobilization of the native enzymes for the detection and decontamination of organophosphate agents have received considerable attentions.Herein we prepare OPH nanogels through a two-step procedure including surface acryloylation and in situ aqueous polymerization. The OPH nanogels present surface functionalities, environmental stabilities and highly catalytic activities and successfully detoxify organophosphates in different cell lines. With the use of spin-coating, spraying coating or electrostatic spinning techniques, biocatalyst films and fiber composites were built from OPH nanogels, as well as self-assembled porous membranes. The detailed results are described as follows.1. In-situ synthesis of OPH nanogelsOPH was purified from an E. coli BL21 expression system using electro transformation method. The final OPH samples collected after gel filtration and ionic exchange chromatogram exhibited a Km value of 70 mM and catalytic activity of 22,320 U/mL. The dynamic light scattering (DLS) and transmission electro microscopy (TEM) results demonstrated that each OPH nanogel contained a single OPH as core and slightly crosslinked polymer network as shell with an average diameter of 15 nm. Judicious choices of the monomers allow the synthesis of monodispersed nanogels with tunable surface charge and local chemical environment. For example, the use of acrylamide or dimethylaminoethyl methacrylate (DMAEMA) led to neutral or positively charged nanocapsules, respectively.2. Environmental stabilities of OPH nanogelsCatalytic activities of native OPH, OPH nanogel 1 and OPH nanogel 2 were compared under diffent temperatures, pH values and organic solvent contents. Both OPH nanogel 1 and 2 sustained 80% catalytic activity after incubating under 65 oC for 100 min, while native OPH had been denatured completely. OPH nanogels sustained much higher activities against pH decrease than native enzymes, especially nanogel 1 that performed high stability in pH range of 8 to 10. Kinetic parameters (Km and kcat) results suggested that surface modification of OPH maintained the appetency between active site and substrate molucules and displayed no effect on substrate diffusion. Because pH-induced enzyme inactivation usually occurs via unfolding of the protein upon an alteration in surface charge distribution, polyelectrolyte layers helped to maintain active conformation of enzyme molecules. For nanogel 1, peripheral DMAEMA molecules were beneficial to buffer pH variations, and improve substrate affinity through hydrophobic interactions.3. Cellular detoxification of OPH nanogelsLow cytotoxicity of native OPH, OPH nanogel 1 and OPH nanogel 2 in both adherent cell lines (Hela cell and Hek293 cell) and suspension cell line (Jurkat cell) were verified with fluorescence activated cell sorter (FACS) and MTT assay independently. The cellular uptake efficiency of the three biocatalysts turned out to be quite different: native OPH could not pass the plasma membrane by endocytosis; OPH nanogel 2 could enter cytoplasm by endocytosis, but the uptake efficiency was low; OPH nanogel 1 could be uptaken into cells as rapidly as possible. The endoxytosis of OPH nanogel 1 in Hela cells was traced by the use of immunol fluorence staining method. Early endosomes and primary lysosomes were stained with EEA1 and Rab 7 antibody independently. OPH nanogel 1 entered the cytoplasm through endocytosis following the path of plasma membrane adhesion, early endosomes, primary lysosomes and cytoplasm.4. Biocatalysts built from OPH nanogelsOPH nanogel films were conveniently prepared on glass, silica, mica or carbon matrices through spin-coating or spraying coating techniques. Atomic force microscope (AFM) and fluorescence microscopy images demonstrated the uniform distribution of OPH nanogel in densely immobilized OPH nanogel films in contrast with the seriously aggregated native counterparts. The covalent immobilization and homogeneous distribution resulted in biocatalyst reusability, enabling the films sustained certain catalytic ability after hydrolysis cycles. What's more, OPH nanogels were spontaneously absorbed onto the outer surface of polystyrene or polyacrylonitrile fibers. SEM and fluorescence microscopy images verified the continuous immobilization of OPH nanogels on the fiber external surface due to hydrophobic affinity between shell layer of nanogels and polymer fibers. For the composite fibers coated with 13?g OPH nanogel solution, 103?g paraoxon was hydrolyzed within 8 min.5. Porous membrane self-assembled from BSA nanogelsA novel type of porous membrane was self-assembled from single protein nanogels through a self-assembly process. The preparation of single protein nanogels was verified by matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer, transmission electron microscopy (TEM) and dynamic light scattering (DLS) analyses. While the porous morphology was characterized in detail by Fourier transform infrared (FTIR), scanning electro microscopy (SEM), atomic force microscopy (AFM), and fluorescence microscopy. Interestingly, the obtained porous membrane presented rough macro porous morphologies embedded with nano scale pores, providing nano scale spaces and microenvironments similar with nanfiltration membranes. Therefore, porous membranes with nanoscale pores and OPH as biocatalyst would be used in the industry of pesticides waste water treatment.