Abstract
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.
Highlights
Organophosphorus compounds were widely used in the world as an insecticide and additives in oil and plastic industry from the end of World War II [1]
The results showed the growth of recombinant strain on the medium containing kanamycin, which indicates the accuracy of transformation because this ability is related to pWB980-dfpase plasmid
For the detection of recombinant bacteria expressing DFPase, the colonies harboring the recombinant plasmid were inoculated on the plates containing Mineral Salts Medium (MSM) with 50 mg/L diisopropyl fluorophosphate (DFP) as the sole source of carbon and energy, which was only observed in five out of seven transformants
Summary
Organophosphorus compounds were widely used in the world as an insecticide and additives in oil and plastic industry from the end of World War II [1]. Nervous system is one of the major biological systems of animals on which OPs are effective and impair its function in many ways. 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. Results: Appearance of the 946 bp band in agarose gel after digestion of transformed plasmid confirmed the presence of DFPase gene in this construct. Conclusion: Bacillus subtilis is a suitable host for production of secretory and active form of DFPase.
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