Novel Organophosphorous Hydrolases for Decontamination

描述(申请人提供)：有机磷(OP)神经毒素，如对氧磷、沙林、梭曼、VX和俄罗斯-VX，是一类广泛的化学物质，通常用作杀虫剂和化学战剂(CW)。目前，全球储存着超过10万吨的化学战剂，每年有数千吨有机磷农药释放到环境中。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水解剂水解成无害的产品。由于具有破坏这些神经毒素的潜力，有机磷水解酶最近已成为密集的研究努力的焦点，旨在将这些酶开发成基于酶的去污染产品。然而，这些酶缺乏许多所需的性质，如溶液溶解性，热稳定性和pH稳定性，以及底物专一性，这些酶的所有潜在应用。我们最近从一种极端细菌中鉴定、克隆、表达、纯化、结晶和确定了一种新的OPH的X射线结构，该OPH对化学战剂梭曼具有显著的活性。该酶可以大量过度表达，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版本在民用和军用应用中都具有重大的技术进步前景，这些应用需要用于基于酶的去污的新型OPH酶。由于使用我们现有的表达载体和宿主菌株进行大规模表达和生产在经济上是不可行的，而且我们的OPH版本对V-试剂的活性很低，我们建议开发一种经济上可行的、工业化的耐辐射葡萄球菌OPH酶生产系统，并利用定向进化基因技术来提高D-Radiodurans OPH对V-试剂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.