氯过氧化物酶降解化学战剂的机制及应用研究

The chemical accidents such as the assassination of Kim Jong-nam and the Ghouta chemical attack occurred during the Syrian Civil War, demonstrate that Chemical Warfare Agents (CWAs) are the potential risk to our country. To solve the problem, rapid decontamination for equipment and human body is essential to control chemical attack, thus, novel CWA decontaminants have been the subject of numerous research by security department of most countries. Enzyme-catalyzed CWA decontaminant is believed to be the next-generation of CWA decontaminant due to its high efficiency, broad-spectrum and friendly to skin and environment. Ocean-origin broad-spectrum chloroperoxidase (CPO) is a versatile enzyme secreted by marine fungus, with the ability of catalyzing a broad spectrum of reactions. Previously, we found that CPO can degrade sulfur mustard (HD) and VX efficiently, which showed potential to become the candidate of the next-generation decontaminant. In this research, we will investigate the degradation spectrum and efficiency of CPO-catalyzed reactions. To identify the mechanism, methods such as accurate mass will be applied in the qualification and quantification of different metabolites. Molecular biology techniques will be carried out to construct CPO mutants to further investigate the mechanism and broader spectrum of CPO. This research will pave way for the development of novel CWA decontaminants.

近年来金正男暗杀、叙利亚化武危机等一系列事件表明，化学战剂依然是我国面临的潜在安全威胁。面对化学战、化学恐怖与化学事故等化学安全问题，首先需要解决的是如何对环境和人体进行高效洗消。化学战剂洗消剂的研发一直是各国安全部门及军队研究的重要领域之一。近年来，以酶促洗消剂为代表的下一代化学战剂洗消装备成为各国研究的重点。CPO（chloroperoxidase）是来源于海洋真菌的氯过氧化物酶，对多种污染物有高效降解效率。在前期研究中，我们发现该酶能够有效降解芥子气、VX等经典化学战剂，有潜力成为下一代化学战剂酶促洗消剂的候选品种。本研究拟进一步探索CPO对化学战剂的降解谱系及降解效率，通过高分辨质谱等手段对其各种降解产物进行准确定性与定量，明确反应机制；在此基础上，利用分子生物学手段对CPO进行突变体设计，对作用机制进行探索，为获得广谱、高效酶促化学战剂洗消剂奠定基础。

氯过氧化物酶（CPO）是一种天然的血红素蛋白，能够在不形成砜的情况下催化硫化物氧化成亚砜，因此其较适合用于降解硫芥子气（HD）。硫芥子气是一种持久性的化学武器，二战后虽处理了大量的HD，但仍然有HD不断泄漏到水生环境中。本文对CPO催化HD降解的条件进行了优化，优化后的Michaelis-Menten参数为Km=0.17 mM、Vmax = 0.06 mM s−1 (R2 = 0.935)、kcat=2717，表明CPO催化降解HD的效率接近超高效酶的催化效率。通过质谱法和核磁共振（NMR）技术鉴定，在最佳反应条件下，通过CPO的催化80%的HD转化为双(2-氯乙基)亚砜与其它代谢产物；并结合NMR滴定实验和分子动力学模拟结果提出了合理的氧化机理。另外，CPO还促进了其他化学战剂，如路易氏剂（L）和乙基毒气（VX）的降解，从而表现出其广泛的底物范围。经本文优化后的反应体系降解HD后的斑马鱼胚胎成功孵化，以及经反应体系降解黄剂(L+HD)后的斑马鱼（Danio rerio，AB株）幼体存活，表明所开发的反应系统在水环境净化方面具有很大的潜力。