Organophosphorous compounds (OPs) form the basis of many biocides and nerve agents, affecting millions of people each year. Current treatments for acute OP poisoning use atropine to prevent neurotransmitter overaccumulation, and oximes for rescuing phosphorylated acetylcholinesterase (AChE) prior to an irreversible ageing event, but medical care may be required for weeks. Hydrolysis of the OP phosphoester bond prevents the inhibitive phosphorylation of AchE, thus enzymes are an obvious candidate for novel treatments. Enzyme-based therapies for OP poisoning have been shown to delay mortality, but leaching of lipophilic OPs from fat deposits leads to prolonged toxicity, superseding the detoxifying effects of the enzyme due to systemic clearance. Furthermore, these same enzymes may be used for the bioremediation of waterways affected by OP pesticide runoff from crops. Here, an artificial membrane-binding protein from the OP-degrading enzyme OpdA is produced that spontaneously partitions into membranes to enhance enzyme-based therapies for OP poisoning. OpdA was expressed in Escherichia coli with high yields, and cationised with high efficiency to homogenise the surface charge for subsequent anionic surfactant engraftment, giving a supercationic enzyme, cOpdA, which maintained structure and activity, but rapidly penetrated cells, resulting in cytotoxicity. Electrostatic engraftment of an anionic polymer surfactant to the surface of the cOpdA mitigated the cytotoxicity, and the constructs were shown to remain active at the cell surface for 5 days. Crucially, the surfactant conjugation has no deleterious effects on the enzyme, and surprisingly causes a significant increase in its turnover rate and substrate affinity. Small angle X-ray scattering and molecular dynamics simulations show that the surfactant forms a highly-dynamic compact corona surrounding the enzyme. Finally, the high-yield expression system for OpdA by Escherichia coli is used in conjunction with a new bioprinting methodology to produce a 3D bacterial microreactor for OP degradation. The novel cell-laden bioink is printable at high resolutions, is not cytotoxic, and maintains its structure over several weeks. Crucially, the printed structures maintain OP-degrading activity.
|Date of Award||6 Nov 2018|
- The University of Bristol
|Supervisor||J L R Anderson (Supervisor) & Adam W Perriman (Supervisor)|