Cell surface display of organophosphorus hydrolase for sensitive spectrophotometric detection of p-nitrophenol substituted organophosphates

•OPH displayed on the surface of Escherichia coli using INP display system.•OPH whole cell biocatalyst showed excellent enzyme activity and stability.•OPH strain capable of detecting PNP substituted OPs sensitively. Organophosphates (OPs) widely exist in ecosystem as toxic substances, for which sensitive and rapid analytical methods are highly requested. In the present work, by using N-terminal of ice nucleation protein (INP) as anchoring motif, a genetically engineered Escherichia coli (E. coli) strain surface displayed mutant organophosphorus hydrolase (OPH) (S5) with improved enzyme activity was successfully constructed. The surface location of INP-OPH fusion was confirmed by SDS-PAGE analysis and enzyme activity assays. The OPH-displayed bacteria facilitate the hydrolysis of p-nitrophenol (PNP) substituted organophosphates to generate PNP, which can be detected spectrometrically at 410nm. Over 90% of the recombinant protein present on the surface of microbes demonstrated enhanced enzyme activity and long-term stability. The OPH activity of whole cells was 2.16U/OD600 using paraoxon as its substrate, which is the highest value reported so far. The optimal temperature for OPH activity was around 55°C, and suspended cultures retained almost 100% of its activity over a period of one month at room temperature, exhibiting the better stability than free OPH. The recombinant E. coli strain could be employed as a whole-cell biocatalyst for detecting PNP substituted OPs at wider ranges and lower detection limits. Specifically, the linear ranges of the calibration curves were 0.5–150μM paraoxon, 1–200μM parathion and 2.5–200μM methyl parathion, and limits of detection were 0.2μM, 0.4μM and 1μM for paraoxon, parathion and methyl parathion, respectively (S/N=3). These results indicate that the engineered OPH strain is a promising multifunctional bacterium that could be used for further large-scale industrial and environmental applications.