Activity Improvement of Organophosphorus Hydrolase Enzyme by Error Prone PCR Method

Document Type : Original Article


1 Molecular Biology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Science, Tehran, Iran

2 Applied Biotechnology Research Center, Baqiyatallah University of Medical Science, Tehran, Iran


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.


  1. Priyadharshini UK, Latha R, Kavitha U, Nirmala N. Effects of Organophosphorus Pesticides on Cardiorespiratory Parameters among the Farmers. J Clin Diagn Res. 2017;11(9):Cc01-cc04. doi:10.7860/jcdr/2017/26724.10590.
  2. Martin-Reina J, Duarte JA, Cerrillos L, Bautista JD, Moreno I. Insecticide reproductive toxicity profile: organophosphate, carbamate and pyrethroids. J Toxins. 2017;4(1):1-7.
  3. Costa LG. Organophosphorus Compounds at 80: Some Old and New Issues. Toxicol Sci. 2018;162(1):24-35. doi:10.1093/toxsci/ kfx266.
  4. Singh BK. Organophosphorus-degrading bacteria: ecology and industrial applications. Nat Rev Microbiol. 2009;7(2):156-164. doi:10.1038/nrmicro2050.
  5. Serdar CM, Gibson DT, Munnecke DM, Lancaster JH. Plasmid Involvement in Parathion Hydrolysis by Pseudomonas diminuta. Appl Environ Microbiol. 1982;44(1):246-249.
  6. Walker AW, Keasling JD. Metabolic engineering of Pseudomonas putida for the utilization of parathion as a carbon and energy source. Biotechnol Bioeng. 2002;78(7):715-721. doi:10.1002/ bit.10251.
  7. Horne I, Sutherland TD, Harcourt RL, Russell RJ, Oakeshott JG. Identification of an opd (organophosphate degradation) gene in an Agrobacterium isolate. Appl Environ Microbiol. 2002;68(7):3371- 3376. doi:10.1128/aem.68.7.3371-3376.2002.
  8. Siddavattam D, Khajamohiddin S, Manavathi B, Pakala SB, Merrick M. Transposon-like organization of the plasmid-borne organophosphate degradation (opd) gene cluster found in Flavobacterium sp. Appl Environ Microbiol. 2003;69(5):2533- 2539. doi:10.1128/aem.69.5.2533-2539.2003.
  9. Singh BK, Walker A, Morgan JA, Wright DJ. Effects of soil pH on the biodegradation of chlorpyrifos and isolation of a chlorpyrifos-degrading bacterium. Appl Environ Microbiol. 2003;69(9):5198- 5206. doi:10.1128/aem.69.9.5198-5206.2003.
  10. Dong YJ, Bartlam M, Sun L, et al. Crystal structure of methyl parathion hydrolase from Pseudomonas sp. WBC-3. J Mol Biol. 2005;353(3):655-663. doi:10.1016/j.jmb.2005.08.057.
  11. Cheng TC, DeFrank JJ, Rastogi VK. Alteromonas prolidase for organophosphorus G-agent decontamination. Chem Biol Interact. 1999;119-120:455-462. doi:10.1016/S0009-2797(99)00058-7.
  12. Cheng TC, Harvey SP, Stroup AN. Purification and Properties of a Highly Active Organophosphorus Acid Anhydrolase from Alteromonas undina. Appl Environ Microbiol. 1993;59(9):3138- 3140.
  13. Farnoosh G, Khajeh K, Latifi AM, Aghamollaei H. Engineering and introduction of de novo disulphide bridges in organophosphorus hydrolase enzyme for thermostability improvement. J Biosci. 2016;41(4):577-588. doi:10.1007/s12038-016-9643-8.
  14. Cho CM, Mulchandani A, Chen W. Bacterial cell surface display of organophosphorus hydrolase for selective screening of improved hydrolysis of organophosphate nerve agents. Appl Environ Microbiol. 2002;68(4):2026-2030. doi:10.1128/AEM.68.4.2026- 2030.2002.
  15. Singh BK, Walker A. Microbial degradation of organophosphorus compounds. FEMS Microbiol Rev. 2006;30(3):428-471. doi:10.1111/j.1574-6976.2006.00018.x.
  16. Yang H, Carr PD, McLoughlin SY, et al. Evolution of an organophosphate-degrading enzyme: a comparison of natural and directed evolution. Protein Eng. 2003;16(2):135-145. doi:10.1093/ proeng/gzg013.
  17. van Loo B, Spelberg JH, Kingma J, Sonke T, Wubbolts MG, Janssen DB. Directed evolution of epoxide hydrolase from A. radiobacter toward higher enantioselectivity by error-prone PCR and DNA shuffling. Chem Biol. 2004;11(7):981-990. doi:10.1016/j. chembiol.2004.04.019.
  18. Keshtvarz M, Salimian J, Yaseri M, et al. Bioinformatic prediction and experimental validation of a PE38-based recombinant immunotoxin targeting the Fn14 receptor in cancer cells. Immunotherapy. 2017;9(5):387-400. doi:10.2217/imt-2017-0008.
  19. Rezaee E, Miri E, Salimian J, Olad G, et al. Survey and comparison of immunization scale of the recombinant proteins of attachment subunit of tetanus and botulinom (A) toxins. Journal of Ilam University of Medical Sciences. 2013;21(5):109-114. [Persian].
  20. Rezaee E, Saadati M, Salimian J, et al. Evaluating and Comparing Immunization Level of the Recombinant Proteins, Binding Domain of Tetanus Neurotoxin and B Subnnit of Heat Labile Toxin of Escherichia coli, and their Relation to Immunological Memory. Journal of Ilam University of Medical Sciences. 2014;21(7):266- 273.
  21. Mao S, Gao P, Lu Z, et al. Engineering of a thermostable beta- 1,3-1,4-glucanase from Bacillus altitudinis YC-9 to improve its catalytic efficiency. J Sci Food Agric. 2016;96(1):109-115. doi:10.1002/jsfa.7066.
  22. Cui B, Zhang L, Song Y, et al. Engineering an enhanced, thermostable, monomeric bacterial luciferase gene as a reporter in plant protoplasts. PLoS One. 2014;9(10):e107885. doi:10.1371/ journal.pone.0107885.
  23. Nakaniwa T, Tada T, Takao M, Sakai T, Nishimura K. An in vitro evaluation of a thermostable pectate lyase by using error-prone PCR. J Mol Catal B Enzym. 2004;27(2):127-131. doi:10.1016/j. molcatb.2003.10.005.
  24. Pritchard L, Corne D, Kell D, Rowland J, Winson M. A general model of error-prone PCR. J Theor Biol. 2005;234(4):497-509. doi:10.1016/j.jtbi.2004.12.005.
  25. Yang J, Ruff AJ, Arlt M, Schwaneberg U. Casting epPCR (cepPCR): A simple random mutagenesis method to generate high quality mutant libraries. Biotechnol Bioeng. 2017;114(9):1921-1927. doi:10.1002/bit.26327.
  26. Minamoto T. Random Mutagenesis by Error-Prone Polymerase Chain Reaction Using a Heavy Water Solvent. Methods Mol Biol. 2017;1498:491-495. doi:10.1007/978-1-4939-6472-7_33.
  27. Rasila TS, Pajunen MI, Savilahti H. Critical evaluation of random mutagenesis by error-prone polymerase chain reaction protocols, Escherichia coli mutator strain, and hydroxylamine treatment. Anal Biochem. 2009;388(1):71-80. doi:10.1016/j.ab.2009.02.008.
  28. Cadwell RC, Joyce GF. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 1992;2(1):28-33. doi:10.1101/ gr.2.1.28.
  29. Drummond DA, Iverson BL, Georgiou G, Arnold FH. Why high-error-rate random mutagenesis libraries are enriched in functional and improved proteins. J Mol Biol. 2005;350(4):806-816. doi:10.1016/j.jmb.2005.05.023.
  30. Liao Y, Zeng M, Wu ZF, et al. Improving phytase enzyme activity in a recombinant phyA mutant phytase from Aspergillus niger N25 by error-prone PCR. Appl Biochem Biotechnol. 2012;166(3):549- 562. doi:10.1007/s12010-011-9447-0.
  31. Zuo ZY, Zheng ZL, Liu ZG, Yi QM, Zou GL. Cloning, DNA shuffling and expression of serine hydroxymethyltransferase gene from Escherichia coli strain AB90054. Enzyme and Microbial Technology. 2007;40(4):569-577. doi:10.1016/j. enzmictec.2006.05.018.