Novel Organophosphorous Hydrolases for Decontamination

用于净化的新型有机磷水解酶

基本信息

批准号：
7173455
负责人：
ANDREW D MESECAR
金额：
$ 34.49万
依托单位：
LYBRADYN, INC.
依托单位国家：
美国
项目类别：
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/7173455
关键词：
Acids Bacteria Bandage Biochemical Bioinformatics Biological Assay Bioremediations Biosensor Chemical Warfare Chemical Warfare Agents Chemicals Class Clothing Coupled Decontaminant Decontamination Deinococcus radiodurans Detection Detergents Development Devices Drug Formulations Engineering Environment Enzymes Exposure to Fermentation Genetic Engineering Hour Hydrolase Hydrolysis Insecticides Iowa Laboratories Lotion Methods Military Personnel Neurotoxins Numbers Paint Paraoxon Pesticides Phase Phosphoric Triester Hydrolases Process Production Property Protective Clothing Protein Overexpression Pseudomonas Research Research Personnel Sarin Screening Result Screening procedure Series Small Business Technology Transfer Research Solubility Solutions Soman Squid Structure Substrate Specificity System Testing Time United States Universities Variant base commercial application cost desire directed evolution enzyme activity expression vector genetic technology high throughput screening improved mutant novel physical property prevent programs research and development tabun thermostability vector

项目摘要

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（由申请人提供）：有机磷（OP）神经毒素，例如副毒素，沙林，索曼，VX和俄罗斯-VX，是一类广泛的化学物质，通常用作农药和化学战（CW）代理。目前，全世界已储存了100,000吨化学战的代理商，每年将数千千万的OP农药释放到环境中。称为磷酸三酯酶的酶，通常称为有机磷酸水解酶（OPH），能够将OP和CW剂水解为无害产物。由于它们可能会破坏这些神经毒素，并且由于大多数酶在环境上是安全的，并且用于洗衣清洁剂等商业产品，因此有机磷酸化水解酶最近已成为旨在将这些酶开发为基于酶消毒的产品的强烈研究工作的重点。不幸的是，目前正在开发的酶缺乏许多所需的特性，例如溶液溶解度，热和pH稳定性以及这些酶的所有潜在应用的底物特异性。因此，我们使用生物信息学方法来鉴定具有独特物理和生化特性的新型OPH酶。我们确定，克隆，表达，纯化，结晶，并确定了对化学战剂Soman具有重要活性的新型OPH的X射线结构。与正在开发的所有其他OPH酶相比，该酶具有独特的物理特性，例如高溶解度和热稳定性。因此，我们版本的OPH版本在需要新颖的OPH酶来进行基于酶的净化的新型OPH酶方面对技术进步具有巨大的希望。由于我们的酶的大规模表达和生产是使我们的酶在商业上可行的必要条件，而且由于我们的OPH版本对V-Agents的活性较低，因此在该项目的第一阶段中，我们开发了用于商业生产的新过表达向量，我们开发了自动筛选方法，以搜索具有改善催化性能的变体酶。在第二阶段，我们建议使用定向进化的方法来开发酶的大规模生产和纯化过程，并进一步增强酶对V-Agent的催化活性。