Cloning and secretory expression of functional diisopropylfluorophosphatase (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 broadsubstrate from OPs. In this study, for the first time, the secretory expression of DFPase in Bacillus subtilis was investigated in order toaccelerate the biodegradation rate of OPs.Methods: DFPase gene was amplified using polymerase chain reaction (PCR) from the pET۲۸-inaV/N-dfpase plasmid. The PCR productwas subcloned in the pWB۹۸۰ plasmid. Competent B. subtilis WB۶۰۰ were transformed with recombinant plasmid. SDS PAGE techniquewas used to study the expression of protein secreted in superrich medium.Results: Appearance of the ۹۴۶ bp band in agarose gel after digestion of transformed plasmid confirmed the presence of DFPase gene inthis construct. Approximately, ۳۵ kDa protein band was shown in culture medium after incubating at ۳۵°C for ۷۲ hours and ۱۵۰ 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 ۳۳۳۳ U/L.Conclusion: Bacillus subtilis is a suitable host for production of secretory and active form of DFPase.

Keywords:

Organophosphorus compounds , B. subtilis WB۶۰۰ , Secretory expression , DFPase