Introduction: ORGANOPHOSPHORUS compounds are frequently used as pesticides and insecticides in agriculture, livestock and home. Because of the high toxicity, it seems is very important its removal from the environment. An enzyme called ORGANOPHOSPHORUS HYDROLASE (OPH) is responsible for the decomposition of ORGANOPHOSPHORUS compounds in most of the strains. Production of enzymes and strains with more efficiency is frequently performed by genetic engineering techniques. Materials and Methods: In this study, we used PCR-based method for quick and easy improvement in activity of OPH enzyme. We selected 5. 5 mM Mg2+ and 0. 2 mM and Mn2+ concentrations for high PCR product. Results: After one round of error prone PCR (epPCR), The 5 number of screened strains (29%) were shown more ability than the native strains to degrade of diazinon, with more than 25% raising ratio. The E6 strain was found to have highest improvement degradation, with 29. 3% improvement. At 48-hour time point, the E6 strains were able to completely remove of diazinon. Conclusions: The epPCR method has the low complexity than other methods and can provide a diverse library include efficient mutants.

ORGANOPHOSPHORUS compounds are widely used, and their presence in different components of the ecosystem has led to harmful effects on the environment. The use of microorganisms in detoxification of xenobiotic compounds and measurement of pesticide residues is considered as an environmental-friendly and appropriate method. ORGANOPHOSPHORUS HYDROLASE (opd), is a phosphotriester HYDROLASE enzyme which is discovered in some soil microorganisms such as Flavobacterium sp. and has a wide range of substrates and is able to hydrolyze many ORGANOPHOSPHORUS compounds. In the present study, codon optimization was done to express the protein in Escherichia coli. Following omission of the signal peptide sequence and substitution of serine by methionine as the start codon, this fragment was cloned under the control of lac promoter in a promoter-probe vector (pTH1705) as a transcriptional fusion and was used for transformation in two Escherichia coli strains DH5α and XL1-blue. To measure the performance of the transgenic bacteria expressing opd gene (as in trans), they were cultured in M9 mineral medium containing 10 and 50 mg/L diazinon and appropriate available carbon and nitrogen sources. Studying the growth curve of V103 strain revealed that bacterial growth in the presence of 50 mg/L diazinon had been affected while this effect in the presence of 10 mg/L diazinon was minor. Results obtained from high-performance liquid chromatography as a standard method to compare with the performance of obtained transgenic strain showed that after 24 hours, diazinon concentration (initially 10mg/L ) in the bacterial culture medium of V100, V101, V102 and V103 strains reached to 6. 38, 7. 19, 7. 09 and 5. 74, respectively. These results reflected the successful transfer and expression of target gene in the bacterium and this genetically engineered bacterium was able to degrade ORGANOPHOSPHORUS compounds directly.

Objective: ORGANOPHOSPHORUS HYDROLASE (OPH) is a homodimeric enzyme that can hydrolyze phosphoester bonds and reduce the toxicity of ORGANOPHOSPHORUS compounds. This makes OPH a suitable element for the biodegradation of these compounds.Methods: We successfully cloned the OPH gene from Pseudomonas diminuta, after optimization for Pichia pastoris, into a yeast expression vector (pPICZaB). After transformation and induction of recombinant yeasts, the expressed enzyme was investigated for its biochemical and kinetical parameters.Results: The enzyme was purified 7.49-fold to a specific activity of 0.421×103 U/mg protein from the supernatant with a yield of 33%. The purified enzyme was able to degrade organophosphates. It had an optimal activity and stability up to 50oC, and a pH range of 7.0-10.0. The enzyme had a Km of 45.96 mM and a Vmax of 11.23 mM/min (421 mM/min/mg) for paraoxon as a substrate. This enzyme was sensitive to divalent cations and inactivated by denaturing compounds such as SDS. The molecular mass of the purified enzyme as estimated by SDS–PAGE analysis was approximately 40 kDa. Conclusion: In this study, the purified enzyme effectively hydrolyzed paraoxon, an ORGANOPHOSPHORUS compound. The activity and stability of this enzyme at high temperatures and pH, and low Km in comparision with bacterial isolates could make it an attractive biocatalyst for applied bioremediation and bio sensing.

Objective: ORGANOPHOSPHORUS (OPs) compounds are widely used in many pesticides, insecticides and chemical nerve agents. These compounds are hazardous for humans and the environment. Organophosphate HYDROLASE (OPH) is a homodimeric protein initially isolated fromPseudomonas diminuta MG and Flavobacterium species. This enzyme is able to degrade a broad spectrum of toxic OPs compounds. Using immobilized OPH commonly presents a variety of advantages versus the free form of the enzyme.Advantages include an increase in stability, cost reduction by simple recovery and reutilization of the enzyme, quick and easy separation of the reactant and product in the reaction medium.Methods: Plasmid pET-26b (+) was used to generate the OPH protein under the control of the T7lac promoter.E. coli BL21 (DE3) pLysS was used as the host for expression of the OPH enzyme. Recombinant OPH was secreted into the extracellular medium and the purified enzyme was immobilized on the surface ofBacillus subtilis spores by the adsorption method, for the first time.Results: Approximately 42% to 45% enzymatic activity was determined to be associated with spores. Optimal pH and temperature of the enzyme were not altered by the presence of the spores. Thermo and pH stabilities of the immobilized enzyme was higher than the free form of the enzyme.Conclusion: Bacillus subtilis spores are safe for humans and the environment. Therefore this system can be considered an environmentally friendly biocatalyst for degradation of OPs.

Introduction: ORGANOPHOSPHORUS (OPs) compounds are chemical compounds used in pesticides that contain synthetic esters, amides, and thiol derivatives of phosphoric, and phosphonic acids. The OPs are harmful to humans and animals because of compounds such as parathion. By acting on nerve cells, parathion creates very dangerous cellular oxidative stresses, which in turn activate programmed cell death.Materials and Methods: In this study, the enzyme ORGANOPHOSPHORUS HYDROLASE (OPH) having esterase activity was selected with the aim of influencing its reaction product with parathion on the viability of human nerve cells. The neuroblastoma SH-SY5Y cell line was exposed to parathion (700 µg/ml) (≈ 40% reduced cell viability) and the product of OPH esterase reaction (1 µg/ml) with the same parathion concentration for two hours to determine their cytotoxicity (≈ 25% reduced cell viability) by MTT, real-time PCR and flow cytometry techniques.Results: The results revealed that parathion (100 µg/ml) inhibited acetylcholinesterase activity by ≈ 65% while OPH-related product reduced acetylcholinesterase activity by ≈ 26%.Conclusions: Considering the widespread use of OPs in modern agriculture, the OPH can be used to reduce the OPs’ destructive effects and the current study could provide new insight into healthy modern agriculture.

ORGANOPHOSPHORUS compounds are one of the important insecticides and pesticides that have been used in chemical warfare as nerve agents. They are a major global clinical problem, with thousands of deaths occurring every year. ORGANOPHOSPHORUS compounds inhibit acetylcholinesterase activity cause to the accumulation of acetylcholine at many synapses in the brain and different parts of neuromuscular junctions. In the cases of intoxication to organophosphours poisons four clinical syndromes have been described; cholinergic crisis, intermediate syndrome, delayed neuropathy and chronic organophosphate inducted neuropsychiatric disorder. Whichever stages has special signs and symptoms. The determination of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity in whole blood and plasma, is a rapid, convenient and benefit screening method of poisoning due to ORGANOPHOSPHORUS compounds but without high sensitivity and specificity, also assess their degradation products in plasma and urine will be feasible but is expensive and is limited to specialized laboratories. The conventional and standard management of poisoned patients include supportive care, detoxification and treatment with antidote atropine sulfate alone or with an oxime. Some of other drugs have been suggested for treatment, e.g. glycopyrrolate, sodium bicarbonate and magnesium sulfate which may in some cases necessary. But still there are many unanswered questions and controversies in the management of ORGANOPHOSPHORUS poisoning. Patients with moderate to severe intoxication who receive first aid and emergency medical treatment may survive. However, the presence of either a cardiac arrhythmia or respiratory failure is associated with a poor prognosis. Finally, to reduce mortality rate, early diagnosis, precise monitoring and selection appropriate treatments are useful. This article presents a review of poisoning with ORGANOPHOSPHORUS compounds.

Background: Organophosphate compound (OPC) poisoning with suicidal intent is common in Indian ICUs. These compounds are the organic derivatives of phosphorous containing acids and their effect on neuromuscular junction and autonomic synapses is clinically important. Organophosphate poisoning can present as acute cholinergic syndrome, intermediate syndrome and delayed neuropathy. Case Presentation: Intermediate syndrome secondary to organophosphate poisoning is a serious health problem leading to increased morbidity and mortality. The incidence of problem varies and ranges from 8%-84% of OPC poisoning cases. After initial recovery from cholinergic crisis, some patients have resurgence of respiratory muscle paralysis requiring continued ventilatory support. This is termed intermediate syndrome (IMS). The factors accounting for this difference is the nature of organophosphate compound, severity of poisoning and inadequate oxime therapy. The recognition of this syndrome is important as if this entity is overlooked it can have disastrous effects. Discussion: Our patient had developed respiratory muscle weakness as evidenced by inadequate respiratory efforts, drop in oxygen saturation, retention of CO2 and need for ventilatory support. There was no evidence of weakness in ocular, neck, bulbar muscles but he had weakness in all 4 limbs more pronounced in proximal muscles. Conclusion: We presented this case of OPC poisoning with intermediate syndrome, which remained for a prolonged time and required mechanical ventilation for 16 days. This case highlights how the timely intervention can save the patient’ s life.