Novel Organophosphorous Hydrolases for Decontamination

DESCRIPTION (provided by applicant): Organophosphorous (OP) neurotoxins such as paraoxon, sarin, soman, VX, and Russian-VX, are a broad class of chemicals commonly used as pesticides and chemical warfare (CW) agents. Currently, over 100,000 tons of chemical warfare agents are stockpiled worldwide, and thousands-of-tons of OP pesticides are released into the environment annually. Enzymes known as phosphotriesterases, commonly called organophosphorous hydrolases (OPH), are capable of hydrolyzing OP and CW agents into harmless products. Because of their potential for destruction of these neurotoxins, and since most enzymes are environmentally safe and used in commercial products such as laundry detergents, organophosphorous hydrolases have recently become the focus of intense research efforts aimed at developing these enzymes into products for enzyme- based decontamination. Unfortunately, the enzymes currently in development lack many of the desired properties such as solution solubility, thermal and pH stability, and substrate specificity for all of the potential applications of these enzymes. Therefore, we used bioinformatic approaches to identify novel OPH enzymes with unique physical and biochemical properties. We identified, cloned, expressed, purified, crystallized, and determined the x-ray structure of a novel OPH from an extremophile that has significant activity against the chemical warfare agent soman. This enzyme has unique physical properties such as high solubility and thermostability compared to all of the other OPH enzymes under development. Therefore, our version of OPH holds significant promise for technological advancement in both civilian and military applications that require novel OPH enzymes for enzyme-based decontamination. Since large-scale expression and production of our enzyme is necessary to make our enzyme commercially viable, and since our version of OPH has low activity toward V-agents, in Phase I of this project we developed new overexpression vectors for commercial production, and we developed automated screening methods to search for variant enzymes with improved catalytic properties. In Phase II, we propose to develop large-scale production and purification processes for the enzyme, and to further enhance the catalytic activity of the enzyme towards the V-agents using directed-evolution approaches.