Cloning and secretory expression of functional diisopropyl-fluorophosphatase (DFPase) in Bacillus subtilis

Background and aims: Synthetic organophosphates (OPs) inhibit acetylcholinesterase resulting in the accumulation of acetylcholine, failure of organs, and eventually death. Diisopropyl-fluorophosphatase (DFPase) is one of the OPs degrading enzymes that has broad substrate from OPs. In this study, for the first time, the secretory expression of DFPase in Bacillus subtilis was investigated in order to accelerate the biodegradation rate of OPs. Methods: DFPase gene was amplified using polymerase chain reaction (PCR) from the pET28-inaV/N-dfpase plasmid. The PCR product was subcloned in the pWB980 plasmid. Competent B. subtilis WB600 were transformed with recombinant plasmid. SDS PAGE technique was used to study the expression of protein secreted in superrich medium. Results: Appearance of the 946 bp band in agarose gel after digestion of transformed plasmid confirmed the presence of DFPase gene in this construct. Approximately, 35 kDa protein band was shown in culture medium after incubating at 35°C for 72 hours and 150 rpm. Measurement of enzyme’s activity was done by monitoring the release of fluoride from diisopropyl fluorophosphate (DFP), using ion-meter. Results showed that enzyme’s activity was 3333 U/L. Conclusion: Bacillus subtilis is a suitable host for production of secretory and active form of DFPase.

Immobilized enzymatic biocatalysts and their application for destruction of organophosphorus compounds in water, soil and air systems

Undecomposed residues of organophosphorus compounds (OPC) after treatment with pesticides of plants or animals often fall into natural objects (water, soil and air). Modern approaches to the immobilization of enzymes allowing obtaining of stable biological products are described, as well as the possible using of immobilized enzymes for the decomposition of different OPC: paraoxone, methyl and ethyl paraoxone, coumaphos, parathion, methyl and ethyl parathion, chlorpyrifos, soman, VX, methylphosphonic acid and its isobutyl and diisobutyl ethers, diisopropyl fluorophosphate.

Main steps of developing chemical organophosphorus agents abroad

Organophosphorus compounds (OPC) occupy a special place among chemical warfare agents (CWA). High level of toxicity, a wide range of physicochemical properties, polyapplication of action already in the 1930s attracted the close attention of foreign military experts. In 1936, the German chemist Gerhard Schrader for the first time synthesized O-ethyl-dimethylamidocyanophosphate, known today as a herd. By the beginning of the Second World War, the staff of his laboratory synthesized over two thousand new OPC. Some of these compounds were selected for further study as CW agents and subsequently were adopted as weapons by the German army. In 1938 the same Gerhard Schrader have synthesized the organophosphorus compound, closed to tabun, but more toxic: O-isopropyl methyl fluorophosphate, called sarin. In 1944 the German chemist, the 1938 Nobel laureate in chemistry Richard Kuhn synthesized soman and revealed the damaging effect of organophosphorus CWA’s. In 1941 the British chemist Bernard Saunders synthesized diisopropyl fluorophosphate. During World War II the industrial production of organophosphorus CWA’s was organized in Germany, Great Britain and in the USA. Germany produced tabun, sarin and soman, the western allies: diisopropyl fluorophosphate. Till the end of World War II the leadership in the sphere of the development of nerve agents belonged to Nazi Germany. After the end of the war the German scientists, many of whom were devoted Nazis, continued their work under the auspices of military departments of the USA and Great Britain. Subsequently phosphorylated thiocholine esters: V-series substances (VG, VM, VR, VX, EA 3148, EA3317 agents etc.) were synthesized with their participation. The wide range of organophosphorus compounds was tested on volunteers in Porton Down (Great Britain) and in the Edgewood arsenal (USA). But after the synthesis of V-series agents the work on organophosphorus CWA’s did not stop. In recent years there appeared the tendency of the transformation of real threats connected with the chemical weapons use, to propaganda sphere. In recent years, there has been a tendency toward the transformation of real threats associated with the use of chemical weapons into provocation and an advocacy field, but this does not mean that the search for new CWA in Western countries has been stopped.

Main steps of developing chemical organophosphorus agents abroad

Organophosphorus compounds (OPC) occupy a special place among chemical warfare agents (CWA). High level of toxicity, a wide range of physicochemical properties, polyapplication of action already in the 1930s attracted the close attention of foreign military experts. In 1936, the German chemist Gerhard Schrader for the first time synthesized O-ethyl-dimethylamidocyanophosphate, known today as a herd. By the beginning of the Second World War, the staff of his laboratory synthesized over two thousand new OPC. Some of these compounds were selected for further study as CW agents and subsequently were adopted as weapons by the German army. In 1938 the same Gerhard Schrader have synthesized the organophosphorus compound, closed to tabun, but more toxic: O-isopropyl methyl fluorophosphate, called sarin. In 1944 the German chemist, the 1938 Nobel laureate in chemistry Richard Kuhn synthesized soman and revealed the damaging effect of organophosphorus CWA’s. In 1941 the British chemist Bernard Saunders synthesized diisopropyl fluorophosphate. During World War II the industrial production of organophosphorus CWA’s was organized in Germany, Great Britain and in the USA. Germany produced tabun, sarin and soman, the western allies: diisopropyl fluorophosphate. Till the end of world war ii the leadership in the sphere of the development of nerve agents belonged to Nazi Germany. After the end of the war the German scientists, many of whom were devoted Nazis, continued their work under the auspices of military departments of the USA and Great Britain. Sub consequently phosphorylated thiocholine esters: V-series substances (VG, VM, VR, VX, EA 3148, EA3317 agents etc.) were synthesized with their participation. The wide range of organophosphorus compounds was tested on volunteers in Porton Down (Great Britain) and in the Edgewood arsenal (USA). But after the synthesis of V-series agents the work on organophosphorus CWA’s did not stop. In recent years there appeared the tendency of the transformation of real threats connected with the chemical weapons use, to propaganda sphere. In recent years, there has been a tendency toward the transformation of real threats associated with the use of chemical weapons into provocation and an advocacy field, but this does not mean that the search for new CWA in Western countries has been stopped.

Applying In Silico Approaches for Designing a Chimeric InaV/N-DFPase Protein and Evaluating its Binding with Diisopropyl-Fluorophosphate

The N-terminal domain of the ice-nucleation protein InaV (InaV-N) ofPseudomonas syringaewas applied to display the DFPase on the cell surface.In silicotechniques were used to generate a model in order to examine the possibility of DFPase exhibition on the cell surface. The secondary and tertiary structures of a chimeric protein were determined and then, the predicted model was subjected to several repeated cycles of stereochemical evaluation and energy minimization. The homology-modeled structure of the InaV/N-DFPase protein was docked to DFP. The optimizedinaV/N-dfpasegene was translated to 519 amino acids. The minimum free energy of the best-predicted secondary structures was formed by RNA molecules (-215.45 kcal/mol). SOPMA analysis results showed that the main helix peak corresponded to the anchor fragment. Validation of the 3D model indicated that 86.1% of amino acid residues were incorporated into the favored regions. The moldock score was 360.22 for DFP. Results of this study indicated that according toin silicoanalysis, all of these findings were effective in targeting DFPase.

Chemical Weapons: History of the Study of Organophosphorus Toxic Agents Abroad

Organophosphorus compounds occupy a unique positon among all chemical warfare agents (CWA's). Since the 1930-s their high toxicity, wide range of physical-chemical properties and complex action attracted close attention of foreign military experts. In 1936 a German chemist, Dr. Gerhard Schrader, synthesized O-ethyl-dimethyl amidocyanophosphate, known as tabun, for the first time. By the beginning of World War II, more than two thousand new organophosphorus and phosphorus containing compounds were synthesized by his laboratory's stuff. Some of these compounds were selected for further study as CW agents and subsequently were adopted as weapons by the German army. In 1938 the same Gerhard Schrader have synthesized the organophosphorus compound, closed to tabun, but more toxic: О-isopropyl methyl fluorophosphate, called sarin. In 1944 the German chemist, the 1938 Nobel laureate in chemistry Richard Kuhn synthesized soman and revealed the damaging effect of organophosphorus CWA's. In 1941 the British chemist Bernard Saunders synthesized diisopropyl fluorophosphate. During World War II the industrial production of organophosphorus CWA's was organized in Germany, Great Britain and in the USA. Germany produced tabun, sarin and soman, the western allies: diisopropyl fluorophosphate. Till the end of World War II the leadership in the sphere of the development of nerve agents belonged to Nazi Germany. After the end of the war the German scientists, many of whom were devoted Nazis, continued their work under the auspices of military departments of the USA and Great Britain. Subsequently phosphorylated thiocholine esters: V-series substances (VG, VM, VR, VX, EA 3148, EA3317 agents etc.) were synthesized with their participation. The wide range of organophosphorus compounds was tested on volunteers in Porton Down (Great Britain) and in the Edgewood arsenal (USA). But after the synthesis of V-series agents the work on organophosphorus CWA's did not stop. In recent years there appeared the tendency of the transformation of real threats connected with the chemical weapons use, to propaganda sphere. The provocation which the «Novichok» agent, arranged primitively by the British intelligence, is the perfect example of such a transformation. But it does not mean that the research in the sphere of new organophosphorus CWA's in the West is stopped