Supercharged enzyme-polymer surfactant bioblocks for the preparation of organophosphate decontaminating materials

由于有机磷作为杀虫剂在发展中国家的农村地区被广泛使用，据估计，全世界每年约有300万人被有机磷中毒。OPs还被用于化学战剂配方，包括2013年叙利亚Ghouta Sarin袭击事件以及1994年和1995年日本地铁袭击事件。根据法国2013年9月发布的一份情报评估，仅叙利亚的库存就包括数百吨沙林和数十吨VX。因此，本研究方案描述了应用合成生物学合理设计多功能增压酶-聚合物表面活性剂积木(生物块)，以制备跨越所有三个物质相的有机磷去污材料。在这里，有机磷降解酶有机磷水解酶(OPH)的合成增压变体的表面将被彻底重新设计，以产生粘附性酶-聚合物熔体、稳定的生物气溶胶和分级组装的固体膜。这种新方法的多功能性高度依赖于通过在增压酶表面构建静电组装的聚合物表面活性剂电晕来操纵蛋白质-蛋白质相互作用的能力，这是基于AWP最近开创的合成方法。该方法包括两个关键步骤：(I)放大蛋白质表面的正电荷密度，然后(Ii)阴离子聚合物表面活性剂链与蛋白质表面的阳离子位置的静电偶联。值得注意的是，由此产生的聚合物表面活性剂分子的表面结合电晕增加了具有吸引力的分子间蛋白质-蛋白质相互作用的范围，这反过来又允许在无水条件下形成熔体所需的粒子运动。或者，可以调节电晕的亲水性-亲脂性平衡(HLB)，以便为气溶胶的生成提供有机溶剂兼容性，或者促进表面活性物质介导的自组装来生成纳米孔固体。因此，本研究方案的总体目标是合理设计和合成可用于制备这三类有机磷去污材料的有机磷降解酶-聚合物表面活性生物块。这项研究计划将在四个主要研究目标上按顺序实施：-在硅胶启发下设计、表达和纯化增压有机磷水解酶(ScOPH)文库-高密度scOPH-聚合物熔体的合成-使用有机溶剂兼容性产生活性生物气溶胶-表面活性剂介导的scOPH组装获得具有可回收催化活性的多孔固体。研究计划描述了一种结合合成生物学、生物物理学和材料科学的内部技术以及大规模设施中可用的技术的科学方法。由于该计划有一个强烈的应用重点，新的方法描述了重组增压酶的开发，它将进行优化，以实现最大的催化性能。总之，可在所有三个阶段运行的OP降解酶-聚合物表面活性材料库的开发代表着一种不久的将来的平台技术，可容易地用于多种新的国防应用，包括用于军事硬件或人员的可剥离涂层、用于OP污染的密闭空间或吸入治疗的基于生物气雾剂的对策，以及用于OP降解/处置的高效酶基反应堆。

Since their widespread application as pesticides in rural areas of developing countries, it is estimated that approximately 3 million people worldwide are poisoned by organophosphates (OPs) every year. OPs have also been used in chemical warfare agent formulations, in incidents including the Ghouta Sarin Attack in Syria, 2013, and the Japanese subway attacks in 1994 and 1995. According to a French intelligence assessment published in September 2013, stockpiles in Syria alone include several hundreds of tonnes of sarin and several tens of tonnes of VX. Accordingly, this research proposal describes the application of synthetic biology for the rational design of versatile supercharged enzyme-polymer surfactant building blocks (bioblocks) for the preparation of organophosphate decontaminating materials that span all three phases of matter. Here, the surfaces of synthetic supercharged variants of the organophosphate-degrading enzyme organophosphate hydrolase (OPH) will be radically re-engineered to produce adhesive enzyme-polymer melts, stable bioaerosols, and hierarchically assembled solid membranes.The versatility in this new methodology is highly dependent on the ability to manipulate protein-protein interactions through the construction of an electrostatically-assembled polymer surfactant corona at the surface of a supercharged enzyme, which is based on the synthetic methodology recently pioneered by AWP. The approach involves the reengineering of a protein surface in two key steps: (i) amplification of the positive charge density on the protein surface, followed by (ii) electrostatic coupling of anionic polymer surfactant chains to the cationic sites on the protein surface. Significantly, the resulting surface-bound corona of polymer surfactant molecules increases the range of the attractive intermolecular protein-protein interactions, which in turn allows the particle motions required for melt formation under anhydrous conditions. Alternatively, the hydrophilic-lipophilic balance (HLB) of the corona can be tuned to either provide organic solvent compatibility for aerosol generation or to promote surfactant mediated self-assembly to produce nanoporous solids. Accordingly, the global aim of this research proposal is the rational design and synthesis of the organophosphate-degrading enzyme-polymer surfactant bioblocks that can be used for the preparation of these three classes of organophosphate decontaminating materials. The research program will be implemented sequentially across four primary research objectives: - In silico inspired design, expression and purification of a supercharged organophosphate hydrolase (scOPH) library - The synthesis of high-density scOPH-polymer melts - Active bioaerosol generation using organic solvent compatibility - Surfactant-mediated assembly of scOPH to give porous solids with recyclable catalytic activitiesThe research programme describes a scientific approach that combines in-house techniques for synthetic biology, biophysics and materials science, as well as techniques available at large-scale facilities. As there is a strong application focus in the programme, the new methodology describes the development of recombinant supercharged enzymes, which will be optimised for maximum catalytic performance. In conclusion, the development of a library of OP-degrading enzyme-polymer surfactant materials that can operate in all three phases represents a near-future platform technology that could be readily exploited for a multitude of new defence applications, including disbondable coating for military hardware or personnel, bioaerosol-based countermeasures for OP contaminated confined airspaces or for inhalation treatments, and high efficiency enzyme-based reactors for OP degradation/disposal.