Organohalide pollutant detoxification using engineered reductive dehalogenases

使用工程还原脱卤酶对有机卤化物污染物进行解毒

• 批准号: 2627607
• 金额: --
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2627607
• 关键词: Organohalide; pollutant; detoxification; using; engineered

The reductive dehalogenases (RDases) are key enzymes in organohalide respiration, which is performed by a unique subset of bacteria. The RDases are cobalamin-dependent and able to selectively cleave carbon-halogen bonds through reduction. As a result, RDase containing organisms have been targeted as a potential bioremediation tool for the clean-up of contaminated anaerobic sites where organohalides have accumulated, often due to improper disposal. The RDases can be split into two classes: i) the canonical respiratory dehalogenases, which use a halogenated compound as a final electron acceptor during organohalide respiration and ii) the catabolic respiratory dehalogenases that occur in the catabolic pathways of non-organohalide respiring bacteria [3]. The catabolic RDase enzymes tend to be oxygen-tolerant, suggesting these are more desirable targets for future mechanistic studies and bioremediation applications. A range of mechanisms have been proposed including long range versus short range/inner sphere mechanisms on the basis of substrate/ligand complex crystal structures. It thus remains unclear what are the fundamental drivers of appropriate substrate positioning and B12 reduction. Making use of a range of catabolic reductive dehalogenases we will determine what governs substrate specificity and explore the scope to rationally repurpose this. We will establish both the mechanism of B12-mediated substrate reduction (using in crystallo approaches) and NAD(P)H mediated B12-reduction (using spectroscopic approaches) to provide a complete mechanistic understanding of catalysis. We will use fundamental understanding gained at Manchester to guide in vivo bioremediation of environmentally relevant organohalides at Toronto. The more fundamental aspects of the study will be carried out at Manchester in the MIB taking advantage of the local strength in enzymology, protein engineering and biotechnology. The in vivo bioremediation studies will be carried out in Toronta at BioZone taking advantage of the local expertise in chemical engineering. We propose the student will spend a ~12 month placement in the 3rd year at the Edwards lab in BioZone. The project fits both within the world class bioscience as well as industrial biotechnology areas.

还原性脱卤酶（RDases）是有机卤化物呼吸的关键酶，由一个独特的细菌亚群进行。RDases是钴胺素依赖性的，并且能够通过还原选择性地裂解碳-卤素键。因此，含有RDase的生物体已被作为一种潜在的生物修复工具，用于清理受污染的厌氧场所，其中有机卤化物已积累，通常是由于处置不当。RDases可以分为两类：i）典型的呼吸脱卤酶，其在有机卤化物呼吸过程中使用卤代化合物作为最终电子受体，以及ii）在非有机卤化物呼吸细菌的分解代谢途径中发生的分解代谢呼吸脱卤酶[3]。分解代谢RDase酶往往是耐氧的，这表明这些是未来的机制研究和生物修复应用更理想的目标。已经提出了一系列的机制，包括长距离与短距离/内球机制的基础上基板/配体复合物的晶体结构。因此，仍然不清楚适当的基板定位和B12减少的基本驱动因素是什么。利用一系列的分解代谢还原脱卤酶，我们将确定是什么支配底物特异性，并探索合理地重新利用这一范围。我们将建立B12介导的底物还原（使用晶体方法）和NAD（P）H介导的B12还原（使用光谱方法）的机制，以提供对催化的完整机理理解。我们将使用在曼彻斯特获得的基本理解，以指导在多伦多的环境相关的有机卤化物的体内生物修复。这项研究的更基本的方面将在曼彻斯特的MIB进行，利用当地在酶学，蛋白质工程和生物技术方面的优势。体内生物修复研究将在多伦多的BioZone进行，利用当地的化学工程专业知识。我们建议学生将在第三年在BioZone的Edwards实验室进行约12个月的实习。该项目既适合世界级的生物科学，也适合工业生物技术领域。