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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，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。由于它们具有破坏这些神经毒素的潜力，并且由于大多数酶是环境安全的并且用于商业产品如洗衣洗涤剂中，有机磷水解酶最近已经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。不幸的是，目前正在开发的酶缺乏许多所需的性质，如溶液溶解度，热和pH稳定性，以及底物特异性，这些酶的所有潜在应用。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。我们鉴定，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。这种酶具有独特的物理性质，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-剂具有低活性，因此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搜索具有改进的催化性质的变体酶。在第二阶段，我们建议开发该酶的大规模生产和纯化工艺，并使用定向进化方法进一步增强该酶对V因子的催化活性。