Novel Organophosphorous Hydrolases for Decontamination

用于净化的新型有机磷水解酶

• 批准号: 6834554
• 负责人: ANDREW D MESECAR
• 金额: $ 9.62万
• 依托单位: LYBRADYN, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-20 至 2005-02-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6834554
• 关键词: bacterial proteins; bioreactors; biotechnology; bioterrorism /chemical warfare; biotransformation; directed evolution; environmental toxicology; enzyme activity; enzyme structure; esterase; gram positive bacteria; hazardous substances; mass tissue /cell culture; nerve gas; neurotoxins; organophosphorus insecticide; technology /technique development; transfection /expression vector

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.

描述（由申请人提供）：有机磷（OP）神经毒素，如对氧磷、沙林、梭曼、VX和Russian-VX，是一类广泛的化学品，通常用作杀虫剂和化学战剂。目前，全世界储存的化学战剂超过100 000吨，每年有数千吨OP杀虫剂释放到环境中。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试剂水解成无害的产物。由于它们对这些神经毒素的破坏潜力，有机磷水解酶最近已成为旨在将这些酶开发成用于基于酶的去污的产品的密集研究努力的焦点。然而，这些酶缺乏许多所需的性质，如溶液溶解度，热和pH稳定性，以及这些酶的所有潜在应用的底物特异性。我们最近已经确定，克隆，表达，纯化，结晶，并确定了一种新的OPH的X-射线结构从极端微生物，具有显着的活性对化学战剂梭曼。该酶可以大量过表达，并具有理想的物理性质，如高溶解度和热稳定性相比，所有其他OPH酶的发展。因此，我们的OPH版本在民用和军事应用中具有重大的技术进步前景，这些应用需要新型OPH酶进行酶基去污。由于使用我们目前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，并且由于我们的OPH版本对V-因子具有低活性，我们建议开发一种经济上可行的、工业规模的D. radiodurans OPH酶，并提高D.利用定向进化遗传技术，研究抗放射性铀OPH对V剂和其他有机磷化合物的影响。