Novel Organophosphorous Hydrolases for Decontamination

描述（由申请人提供）：有机磷（OP）神经毒素，如对氧磷，沙林，索曼，VX和俄罗斯-VX，是一类广泛的化学品，通常用作杀虫剂和化学战（CW）剂。目前，全球储存的化学战剂超过10万吨，每年有数千吨的有机磷农药被释放到环境中。被称为磷酸三酯酶的酶，通常被称为有机磷水解酶（OPH），能够将OP和CW试剂水解成无害产品。由于有机磷水解酶具有破坏这些神经毒素的潜力，因此近年来已成为研究的焦点，旨在将这些酶开发成酶基去污产品。然而，这些酶缺乏许多理想的性质，如溶液溶解度、热稳定性和pH稳定性以及底物特异性，这些都是这些酶的潜在应用。我们最近鉴定、克隆、表达、纯化、结晶，并确定了一种对化学战剂索曼具有显著活性的极端微生物的新型OPH的x射线结构。该酶可以大量过表达，并且与所有其他正在开发的OPH酶相比，具有理想的物理性质，如高溶解度和热稳定性。因此，我们的OPH版本在民用和军事应用中都具有重大的技术进步前景，这些应用需要新型的OPH酶来进行酶基净化。鉴于利用现有表达载体和宿主菌株进行大规模表达和生产在经济上不可行，且我们的OPH对v型病原体的活性较低，我们建议开发一种经济上可行的、工业规模生产系统，并利用定向进化基因技术增强D. radiodurans OPH对v型病原体和其他有机磷化合物的催化潜力。

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, organophosphorous hydrolases have recently become the focus of intense research efforts aimed at developing these enzymes into products for enzyme-based decontamination. However, these enzymes 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. We have recently 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. The enzyme can be overexpressed in large quantities, and has desirable 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 using our current expression vectors and host strains would not be economically viable, and since our version of OPH has low activity toward V-agents, we propose to develop an economically viable, industrial scale production system for D. radiodurans OPH enzymes, and to enhance the catalytic potential of D. radiodurans OPH towards V-agents and other organophosphorous compounds using directed evolution genetic technology.