High Throughput Bioengineering of Detoxification Enzymes

描述（由申请人提供）：我们修订的项目解毒酶的高通量生物工程侧重于有机磷酸酯水解酶，它具有调解或检测化学战剂（CW，即神经毒气）的直接价值，以及处理人类和环境中的污染农药的长期价值。两个候选的有机磷水解酶已被结构确定；来自小假单胞菌的TIM桶状二聚体有机磷酸酶（OPH）和来自Lo/igo vu/garis（鱿鱼）的13螺旋桨肽二异丙基氟磷酸酶（DFPase）。每一种都有一些被证实的，但效率不高的，对有机磷化学剂和农药的作用，并且对某些残留物的改性已被证明可以提高它们水解某些有机磷化学剂的能力[1,3,3b, 11-15]。我们建议使用我们的专利技术无缝基因组装（TOPPs）来生成一个设计的，底物特异性替代（dpss）的大型文库。通过对OPH和DFPase活性位点上显示的24-25个残基进行多次修饰[1,2,3]，我们将在6个月内分别产生至少1000个修饰基因。通过筛选表达基因与3-4种底物（包括农药和替代CW剂）的酶动力学，将为设计这些组的新修饰提供数据。初步数据已经集成到我们的迭代，初级设计，我们正准备自动化整个过程。我们第一阶段的工作将是极大地扩大这两种酶可用的突变体的数量，我们相信这种方法是一种产生预期改进的新方法。II期的目标可能包括i)验证资源库中的酶活性，II)进一步分析其稳定性，测定和其他底物的最佳条件（由我们或合作者），以及iii)最终添加适配器和修饰这些酶，以用作生物传感器或解毒和/或去污工具。

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.