Abstract
Indiscriminate use of organophosphorus (OP)-based insecticides is a great concern to human health because of bioaccumulation-induced health hazards. Potentially fatal consequences and limited treatment methods of OP poisoning necessitate the need for the development of reliable, selective, cost-effective, and sensitive methods of OP detection. To tackle this issue, the development of effective devices and methods is required to sensitively detect as well as degrade OPs. Enzymatic sensor systems have gained popularity due to high catalytic activity, enhanced detection limits, and high sensitivity with the environmentally benign operation. Organophosphorus acid anhydrolase (OPAA) from Alteromonas sp. JD6.5 is capable of hydrolyzing the P-F, P-O, P-S, and P-CN bonds, in OPs, including nerve agents of the G/V-series. Several mutants of OPAA are reported which have greater activity against various OPs. In this study, recombinant expression of the OPAA-FL variant in Escherichia coli was performed, purified, and subsequently tested for activity against ethyl paraoxon. OPAA-FL variant showed its optimum activity at pH 8.5 and 50 °C. Colorimetric and fluorometric assays were used for estimation of ethyl paraoxon based on p-nitrophenol and fluorescein isothiocyanate (FITC) fluorescence intensity, respectively. Colorimetric and fluorometric assay estimation indicates that ethyl paraoxon can be estimated in the linear range of 0.01 to 1 mM and 0.1 to 0.5 mM, with LOD values 0.04 mM and 0.056 mM, respectively. Furthermore, the OPAA-FL variant was immobilized into alginate microspheres for colorimetric detection of ethyl paraoxon and displayed a linear range of 0.025 to 1 mM with a LOD value of 0.06 mM.
Key points
• Biosensing of paraoxon with purified and encapsulated OPAA-FL variant.
• Colorimetric and fluorometric biosensing assay developed using OPAA-FL variant for paraoxon.
• First report on alginate encapsulation of OPAA-FL variant for biosensing of paraoxon.
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Acknowledgments
The authors would like to thank Prof. S. Harvey, US Army Edgewood Biological Center, MD, USA, for a kind gift of the pSE420-OPAA-FL vector. A. Joshi would like to thank the SERB-Early career award. M. Jain thanks the Ministry of Education (MoE), Government of India, for the Ph.D. fellowship, and B. Joshi would like to acknowledge the DST-INSPIRE fellowship (IF170325).
Funding
Funding for this work was provided by the Department of Biotechnology, Government of India, for a research grant (BT/PR20319/BBE/117/189/2016 and BT/PR24652/NER/95/795/2017).
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P. Kodgire, A. Joshi, and M. Jain designed the experiments; M. Jain and P. Yadav performed the experiments and data analysis; B. Joshi assisted in the experiments and data analysis; P. Kodgire, A. Joshi, and M. Jain wrote the manuscript.
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Jain, M., Yadav, P., Joshi, B. et al. A novel biosensor for the detection of organophosphorus (OP)-based pesticides using organophosphorus acid anhydrolase (OPAA)-FL variant. Appl Microbiol Biotechnol 105, 389–400 (2021). https://doi.org/10.1007/s00253-020-11008-w
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DOI: https://doi.org/10.1007/s00253-020-11008-w