Novel Organophosphorous Hydrolases for Decontamination

用于净化的新型有机磷水解酶

• 批准号: 7056338
• 负责人: ANDREW D MESECAR
• 金额: $ 37.31万
• 依托单位: LYBRADYN, INC.
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7056338
• 关键词: Bacillus subtilis; Escherichia coli; bacterial proteins; bioreactors; biotechnology; bioterrorism /chemical warfare; catalyst; directed evolution; environmental contamination; enzyme activity; enzyme biosynthesis; fermentation; gram positive bacteria; high throughput technology; hydrolase; nerve gas; neurotoxins; organophosphorus insecticide; protein purification; technology /technique development; thermostability; 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, 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.

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。由于它们具有破坏这些神经毒素的潜力，而且大多数酶对环境是安全的，并用于洗衣粉等商业产品，有机磷水解酶最近已成为密集研究的焦点，旨在将这些酶开发成基于酶的去污产品。遗憾的是，目前正在开发的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，以满足这些酶的所有潜在应用。因此，我们使用生物信息学的方法来鉴定具有独特物理和生化特性的新型OPH酶。我们鉴定、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来自一种极端细菌，对化学战剂梭曼具有显著的活性。与所有其他正在开发的OPH酶相比，该酶具有独特的物理性质，如高溶解性和热稳定性。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。由于我们的酶的大规模表达和生产是使我们的酶具有商业可行性所必需的，并且由于我们的OPH版本对V-试剂的活性较低，在该项目的第一阶段，我们开发了用于商业生产的新的过表达载体，并且我们开发了自动筛选方法来寻找具有更好催化性能的变异酶。在第二阶段，我们建议开发大规模生产和纯化该酶的工艺，并利用定向进化方法进一步提高该酶对V-试剂的催化活性。