High Throughput Bioengineering of Detoxification Enzymes

解毒酶的高通量生物工程

• 批准号: 6693486
• 负责人: MARGARET K BRADLEY
• 金额: $ 10万
• 依托单位: MODULAR GENETICS, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6693486
• 关键词: aminoacid; bioengineering /biomedical engineering; biosensor device; biotechnology; bioterrorism /chemical warfare; chemical registry /resource; chemical substitution; combinatorial chemistry; detoxification; diisopropylfluorophosphate; environmental contamination; enzyme activity; enzyme linked immunosorbent assay; enzyme substrate; hydrolase; organic chemicals; phosphates; polymerase chain reaction; protein engineering; protein quantitation /detection; toxin metabolism; transfection /expression vector

DESCRIPTION (provided by applicant): Our revised project High Throughput Bioengineering of Detoxification Enzymes focuses on organophosphate hydrolases, which have immediate value for mediation or detection of chemical warfare agents (CW; i.e., nerve gases), and long term value for dealing with contaminating pesticides in humans and the environment. Two candidate organophosphate hydrolases have been structurally determined; a TIM barrel-like, dimeric organophosphate hydrolase (OPH) from Pseudomonas diminuta, and a 13-propeller peptide diisopropylfluoro-phosphatase (DFPase) from Lo/igo vu/garis (squid). Each has some proven, but inefficient, effect against organophosphate CW agents and pesticides, and modification of certain residues has been shown to increase their ability to hydrolyze some of these agents [1, 3, 3b, 11-15]. We propose to use our patented technologies for seamless gene assembly (TOPPs) to generate a large library of designed, substrate-specific substitutions (DPSSs). By making multiple modifications in 24-25 residues shown lining the active sites of both OPH and DFPase [1,2,3], we will generate at least 1,000 modified genes for each in 6 months. By screening the expressed genes for their enzyme kinetics with 3-4 substrates (including pesticides and surrogate CW agents), data will be available for designing new modifications in these sets. Preliminary data have already been integrated into our iterative, primer design, and we are preparing to automate the whole process. Our Phase I work will be to expand enormously the number of mutants available for both enzymes, and we believe this approach is a novel way to produce the desired improvements. Phase II aims will likely include i) verification of enzyme activities in the resource library, ii) further analyses of their stability, optimal conditions of assay and other substrates (by us or by collaborators), and iii) finally adding adaptors and modifications to these enzymes for use as biosensors or as a detoxification and/or decontamination tools.

描述(由申请人提供)：我们的修订项目高通量解毒酶生物工程专注于有机磷水解酶，它对化学战剂(CW；即神经毒气)的调解或检测具有直接价值，并对处理人类和环境中的污染农药具有长期价值。已经确定了两种候选的有机磷水解酶：一种是来自小型假单胞菌的TIM桶状二聚体有机磷水解酶(OPH)，另一种是来自LO/igo vu/Garis(Squid)的13个螺旋肽的二异丙基氟磷酸酶(DFPase)。每种药物对有机磷CW制剂和杀虫剂都有一些已证实但效率不高的效果，对某些残留物的修饰已被证明增强了它们对其中一些制剂的水解力[1，3，3b，11-15]。我们建议使用我们的无缝基因组装(TOPS)专利技术来生成大量设计的底物特定替换(DPSS)文库。通过对OPH和DFP酶[1，2，3]活性部位所示的24-25个残基进行多重修饰，我们将在6个月内为每个基因产生至少1,000个修饰基因。通过用3-4种底物(包括农药和替代CW制剂)筛选表达基因的酶动力学，将为在这些集合中设计新的修饰提供数据。初步数据已经整合到我们的迭代、入门设计中，我们正准备将整个过程自动化。我们的第一阶段工作将是极大地扩大可用于这两种酶的突变体数量，我们相信这种方法是一种产生所需改进的新方法。第二阶段的目标可能包括：i)验证资源库中的酶活性；ii)进一步分析它们的稳定性、分析和其他底物的最佳条件(由我们或合作者)；以及iii)最后为这些酶添加接头和修饰，以用作生物传感器或作为解毒和/或去污工具。