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A Reductive Dehalogenase for Aerobic Applications In the Environment

用于环境中有氧应用的还原脱卤酶

基本信息

批准号：
1803771
负责人：
Steven Rokita
金额：
$ 47.12万
依托单位：
Johns Hopkins University
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2021-08-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1803771&HistoricalAwards=false
关键词：
Reductive Dehalogenase Aerobic Applications Environment

项目摘要

Organic compounds that contain chlorine or fluorine (halogenates), are remarkably difficult to degrade. To improve their degradation, the investigators will modify an enzyme that demonstrates the correct capability but currently lacks the ability to recognize these pollutants. Halogenated phenols will be the model compounds used. If successful, this approach will enable the decontamination of compounds previously found impossible to treat. Graduate students will learn to combine experimental and theoretical approaches to generate new enzymes. Undergraduate students will help design enzymes using an educational game to simulate protein folding and conformation. Underrepresented minorities will be actively recruited to participate in every aspect of the project.Methods for removing halophenol contaminants from our environment are available but unsatisfactory. Strong chemical oxidants can be used in waste water but their residue adversely affects downstream processes. Alternatively, organisms can be used degrade the halophenols, but they require anaerobic conditions. In contrast, the enzyme iodotyrosine deiodinase has the potential to degrade halophenol using a reductive process that is uniquely compatible with atmospheric oxygen. A number of natural examples of this enzyme have been examined, but all are specific for tyrosine derivatives. Consequently, our goal is to develop new variants that accept a broader range of substrates. Three parallel computational approaches will be tested for predicting the necessary mutations to generate stable enzymes with active site geometries favoring halophenol substrates. The highest ranked variants will be expressed and screened for the desired activity. Further optimization will rely on directed evolution using methods of random mutagenesis and gene shuffling. This research effort should define a general approach for modulating active site lids to accommodate chosen substrates in addition to providing a testable product, engineered enzymes for halophenol decontamination.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

含有氯或氟（卤化物）的有机化合物非常难以降解。为了改善它们的降解，研究人员将修改一种酶，这种酶显示出正确的能力，但目前缺乏识别这些污染物的能力。卤代酚将是使用的模型化合物。如果成功，这种方法将使以前发现无法处理的化合物得到净化。研究生将学习结合实验和理论方法来产生新的酶。本科生将使用一个模拟蛋白质折叠和构象的教育游戏来帮助设计酶。将积极招募代表性不足的少数民族参与项目的各个方面。从我们的环境中去除卤代酚污染物的方法是可用的，但不令人满意。强化学氧化剂可用于废水处理，但其残留会对下游工艺产生不利影响。另外，生物也可以用来降解卤代酚，但它们需要厌氧条件。相比之下，碘酪氨酸脱碘酶具有利用与大气氧唯一相容的还原过程降解卤代酚的潜力。这种酶的一些自然例子已经被研究过，但都是针对酪氨酸衍生物的。因此，我们的目标是开发新的变体，接受更广泛的基板。将测试三种并行计算方法，以预测产生具有有利于卤酚底物的活性位点几何形状的稳定酶所需的突变。将对排名最高的变体进行表达并筛选所需的活动。进一步的优化将依赖于使用随机突变和基因重组方法的定向进化。除了提供一种可测试的产品外，这项研究还应定义一种调节活性位点盖以适应选定底物的一般方法，即用于氟氯酚去污的工程酶。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。