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

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 activities

The 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.

Scientific innovation is essential for the international competitiveness of the UK and the Knowledge Economy, and synthetic biology will be a major driver in the development of new technologies that will have significant impact on world economics. Within this emerging field, the development of new classes of smart biological materials, and the associated synthetic methodologies, will create new opportunities for scientists working at the interfaces of synthetic biology, materials science, and biochemistry. Moreover, the scientific components of the research, which probe fundamental processes such as self-assembly, protein organic solubility and the role of solvent on enzyme structure, dynamics and function, will provide valuable insight which will be made accessible to the commercial, defence and public sectors. The proposed research is closely aligned with current policy priorities across the research councils, especially the EPSRC and BBSRC's thematic area Synthetic Biology. Moreover, the proposed research fall within the remit described by the Dstl for Synthetic Biology Applications for Protective Materials.
The proposed research describes a new approach involving the application of synthetic biology for the rational design of versatile supercharged enzyme-polymer surfactant building blocks (bioblocks). These bioblocks will be used for the preparation of organophosphate (OP) decontaminating materials that span all three phases of matter. The development of these materials is expected to impact on a diverse range of disciplines, including synthetic biology materials science, chemistry, and contribute to the UK's economic competitiveness through the development of new technologies. Moreover, the development of a library of OP-degrading materials that can operate in three phases of matter represents a near-future platform technology that could be readily exploited for a multitude of new defence applications. For example, the generation of high-density enzyme surfactant melts as primary components in disbondable coatings for military hardware or for direct application to personnel during decontamination or preventative procedures. The development of functional enzyme-polymer surfactant bioblock-aerosols could be deployed as a safety precaution in contaminated confined spaces or as an inhalation treatment following OP exposure. The fabrication of porous solids with recyclable catalytic activities has significant technological ramifications, as the hierarchical self-assembly mechanism makes it possible to effectively and cheaply coat a wide range of substrates, including high surface area substrates for OP degradation/disposal. AWP plans to continue to participate in the science innovation training provided by the University of Bristol's Research and Enterprise Development (RED) to fully maximise impact on the commercial sector over the duration of the research programme. This also involves identifying and protecting intellectual property generated from the research, which will be done in conjunction with the University of Bristol and the Dstl.
The new science generated by the discovery and exploration of these new OP-degrading materials will be published in top-tier journals, which will promote the dissemination of knowledge to potential beneficiaries. AWP's recent work on liquid proteins has attracted significant publicity and has been featured in a number of applied engineering and pharmaceutical journals, as well as in the popular media. There will also be participation at international conferences in order to communicate the potential technology to the private sector. It is also likely that the research will impact internationally upon policy-makers and international funding bodies by demonstrating the value of synthetic biology research. The University of Bristol's Research News website will also act as a vehicle to promote the research, and this will raise the university's profile within the UK and internationally.