A Review on Engineering of Organophosphorus Hydrolase (OPH) Enzyme

Document Type : Review Article


Applied Biotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran


Organophosphorus chemicals are compounds which have been used as pesticides and insecticides in agriculture. They’re also used as nervous agents and have raised many problems for human and environment. Among the most important methods of decontamination from these compounds are biodegradation methods. Using OPH enzyme in degradation the mentioned compounds is seen as one of the desirable ways, but low activity and specification and low thermostability are among factors significantly decreasing the optimal application of this enzyme. Using methods of protein engineering based on the alteration of specific protein positions in order to improve the activity, specification and thermostability are some common ways used currently. Numerous studies have been done to increase activity and thermostability of OPH enzyme with alteration of some special amino acids the result of which was an increase against different substrates. OPH enzyme active site connected to substrates that consisted of three large, small and releasing packets were one of the goal areas of changing amino acids used by researchers to improve engineered activities. Among other ways of making enzymes more rigid and stable were bending loops by replacing Proline, creating disulfide bonds, ionic bonds by replacing charged amino acids.


  1. Qiong, L., Qing,T., Xudong, X ., Hong, G., Expression of organophosp horus-degradation gene (opd) in aggregating and non-aggregating filamentous nitrogen-fixing cyanobacteria. Chin J Oceanol Limnol, 2010,   vol .28,pp. 1248-1253.
  2. Singh, B.K., Walker, A., Microbial degradation of organophosphorus compounds. FEMS Microbiol, 2006,vol.30,pp.428–471.
  3. Caceres, T., Fenamiphos and Related Organophosphorus Pesticides. Environ Contam Toxicol, 2010,vol.205, pp.117-162.
  4. Attaway, H., Nelson, J., Baya, A .M., Voll, M J., White,W.E., Grimes, D.J., Colwell, R.R., Bacterial detoxification of diisopropyl fluorophosphate. Appl.  Environ. Microbiol, 1987,vol.53, pp.1685-1689. 
  5. Sorouri Zanjani, R., Mir-Esmaili, S.M., Latifi, A.M., ValiPour, E., Isolation and identification of a type strain bacteria with the highest ability to produce organophosphorus acid anhidrase. J Mazandaran Univ Med Sci, 2009,vol.18,pp. 19-26.
  6. Shimazu, M., Mulchandani, A., Chen,W., Cell Surface Display of Organophosphorus Hydrolase Using Ice Nucleation Protein. Biotechnol program, 2001,vol.17,pp.76-80.
  7. Pinjari. A.B., Pandey, J.P., Kamireddy, S., Siddavattam, D., Expression and subcellular localization of organophosphate hydrolase in acephate-degrading Pseudomonas sp. strain Ind01 and its use as a potential biocatalyst for elimination of organophosphate insecticides. Lett Appl Microbiol,2013,vol.57,pp.63-8.
  8. Harpr, L., McDaniel, S., Miller, W.J., Dissimilar plasmids isolated from Pseudomonas diminuta MG and a Flavobacterium sp. (ATCC 27551) contain identical opd genes. Appl Eviron Microb,1988,vol.54,pp.2586-2589.
  9. Cycon, M., Wójcik, M., Piotrowska-Seget Z., Biodegradation of the organophosphorus insecticide diazinon by Serratia sp and Pseudomonas sp and their use in bioremediation of contaminated soil.Chemosphere,2009,vol.76,pp. 494–501.
  10. Mcdaniel, C., Harper, L., Wild, J., Cloning and sequencing of a plasmid-borne gene (opd) encoding a phosphotriesterase. Bacteriol,1988,vol.170,pp.2306-2311.
  11. Singh, R.K., Tiwari, M.K., Singh, R., Lee, J.K., From Protein Engineering to Immobilization:Promising Strategies for the Upgrade of Industrial Enzymes. Int J Mol,2013,vol.14,pp.1232-1277.
  12. Adamczak, M., Krishna, S.H., Enzyme for Efficient Biocatalysis, Food Technol. Biotechnol,2004,vol.42,pp.251–264.
  13. Goswamy, R., Chaudhuri, A., Study of respiratory failure in organophosphate and carbamate poisoning.Heart & Lung,1994,vol. 23,pp.466-472.
  14. Mulchandani, A., Chen, W., Wang, A., Cho, C., Shimazu, M., ovel biological methods for degradation of organophosphate pesticides .Extended Abstract,2002,vol.42,pp.398-400.
  15. Mulbr, W.,W.Karns , J.S., Parathion hydrolase specified by the Flavobacterium opd gene:relationship between the gene and protein. J Bacteriol,1989,vol.171,pp.6740-6746.
  16. Fletterick, R.J., Buchbinder, J.L., Stephenson, R.C., Dresser, M.J., Pitera, J.W., Scanlan, T.S., Biochemical characterization and crystallographic structure of an Escherichia coli protein from the phosphotriesterase gene family. Biochem,1998,vol.37,pp. 5096–5106.
  17. Hill, C.M., Li, W.S., Thoden, J.B., Holden, H.M., Raushel, F.M., Enhanced degradation of chemical warfare agents through molecular engineering of the phosphotriesterase active site. J Am Chem Soc,2003,vol.125,pp.8990-8991.
  18. Kolakowski, J.E., DeFrank , J.J., Harvey, S.P., Szafraniec, L.L., Beaudry ,W.T., Lai, K.,Wild, J.R., Enzymatic hydrolysis of the chemical warfare agent VX and its neurotoxic analogues by organophosphorus hydrolase. J Bcatal Biotrasform,­1997,vol.14,pp.297-312.
  19. Russell, A.J., Berberich ,J.A., Drevon, G.F., Koepsel, R.R., Biomaterials for mediation of chemical and biological warfare agents. Annu Rev Biomed Eng,2003,vol.5,pp.1-27.
  20. Lenz, D.E., Broomfield, C.A., Maxwell, D.M., Cerasoli, D.M., Nerve Agent Bioscavengers: Protection Against High- and Low-dose Organophophorus Exposure. CRC Press, Boca Raton, FL,2001,vol.56,pp.215-243.
  21. Kawahara, K., Tanaka, A., Yoon, J., Yokota, A., Reclassification of parathione-degrading Flavobacterium sp. ATCC 27551 as Sphinogobium fuliginis. General Application,2010,vol.59,pp.249-255.
  22. Dong ,Y.J.,Bartlam , M.,Sun, L.,Zhou, Y.F., Zhang, Z.P., Zhang, C.G., Rao, Z.Z., hang , X.E., Crystal structure of methyl parathion hydrolase from Pseudomonas sp. WBC-3. J Mol Biol, 2005,vol.353,pp.655-63.
  23. Turanli-Yildiz, B., Alkim, C.,Cakar, Z.P., Protein Engineering Methods and Applications. In Tech,2012,vol.978,pp.953-51.
  24. Vieille   ,C., ZEIKUS, G.J., Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability.Microbiol Mol Biol Rew ,2001,vol.65,pp.1–43.
  25. Sekhar, Talluri., Advances in engineering of proteins for thermal stability.Int J Adv Biotech Res,2011,vol.2,pp.190-200.
  26. Cambillau, C., Claverie, J.M., Structural and Genomic Correlates of Hyperthermostability. J Biol Chem,2000,vol.275,pp.32383-32386.
  27. Singh, A., Lal, R., Sphingobium ummariense sp. nov., a hexachlorocyclohexane (HCH)-degrading bacterium, isolated from HCH-contaminated soil. Int J Syst Evol Micr,2009,vol.59,pp.162–166. 
  28. Nazarian, A., Amini, B., Detection of  Pseudomonas and Flavobacterium Species Harbouring Organophosphorus Degrading Elements from Environment. Iran J Med Sci,2008,vol.10,pp.239 – 244.
  29. Ghanem, E., Raushel, F.M., Toxicol Appl Pharm,­2005,vol.207,pp.459–S470.
  30. Aubert, D.S., Li,Y., Raushel, F.M., Mechanism for the hydrolysis of organophosphates by the Bacterial Phosphotriesterase. Biochem,2004,vol.56,pp.5707–5715.
  31. Zhang, X.Z., hong - Cui, Z.L., Hong, Q.L., High-Level Expression and Secretion of Methyl Parathion Hydrolase in Bacillus subtilis WB800. Appl Environ Microb,2005,vol.71,pp.4101–4103.
  32. Zhang, H.,Li, Q.,Ye, T., Zhang, Z., Lin, L.,Optimization of the whole-cell catalytic activity of recombinant Escherichia coli cells with surface-immobilized organophosphorus hydrolase.J Environ Biol,2013,vol.34,pp.315-319.
  33. Khodi, S., Latifi1, A.M., Mojtaba, S., Morteza, M., Aghamollaei, H., Surface Display of Organophosphorus Hydrolase on E. coli Using N-Terminal Domain of Ice Nucleation Protein InaV. J Microbiol Biotechnol,2012,vol.22,pp.234–238.
  34. Kanga, D.G., Limb, G.B., Cha, J.H., Functional periplasmic secretion of organophosphorous hydrolase using the twin-arginine translocation pathway in Escherichia coli.Journal of Biotechnology,2005,vol.118,pp.379–385.
  35. Latifi, A.M., Khodi, S., Mirzaei, M., Miresmaeili, M.,­Babavalian, H., Isolation and characterization of five chlorpyrifos degrading bacteria. African J BIOTECH,2012,vol.11,pp.3140-3146. 
  36. Roa, L.B.,Timperley, C.M., Griffiths, A.D., Fersht, A.R., Phosphotriesterase variants with high methylphosphonatase activity and strong negative trade-off against phosphotriesters.PROTEIN ENG,2011,vol.24,pp.151–159.
  37. Roa, L.B., Oliynyk, Z., Timperley, C.M.,Griffiths ,A.D.Fersht, A.R.,Highest paraoxonase turnover rate found in a bacterialphosphotriesterase variant. Protein Eng,2011­,vol.24,pp.209–211.
  38. Hawwa, R., Larsen, D.S., Ratia, K., Mesecar, D.A., Structure-Based and Random Mutagenesis ApproachesIncrease the Organophosphate-Degrading Activityof a Phosphotriesterase Homologue from Deinococcus radiodurans. J Mol Biol,2009,vol.393,pp.36–57.
  39. DiSioudi, B., Grimsley, J.K., Lai, K.,Wild, J.R., Modification of near active site residues in organophosphorus hydrolase reduces metal stoichiometry and alters substrate specificity. Biochem,1999,vol.38,pp.2866-72.
  40. Li, W.S., Lum, K.T., Goodspeed, M.C., Sogorb, A.M., Raushel, M.F., Stereoselective Detoxification of Chiral Sarin andSoman Analogues by Phosphotriesterase.Bioorgan Med Chem,2001,vol.9,pp. 2083–2091.
  41. Janet, K., Grimsley, A., Barbara,C.B., James, R.,Wild, A., Andrew, D.M., Structural and mutational studies of organophosphorushydrolase reveal a cryptic and functional allosteric-binding site.Arch Bioch Bioph,2005,vol.442,pp.169–179.
  42. Gopal, S., Rastogi, V., Ashman,W., Mulbry,W., Mutagenesis of Organophosphorus Hydrolaseto Enhance Hydrolysis of the Nerve Agent VX. Biochem Bioph Res Co,2000,vol.279,pp.516–519.
  43. Yang, H., Carr, P.D., McLoughlin, S.Y., Liu, J.W., Horne, I., Qiu, X., Jeffries, C.M.J., Russe, R.J., Oakeshott, J.G., Ollis,D.L., Evolution of an organophosphate-degrading enzyme:a comparison of natural and directed evolution. Protein Eng, 2003,vol.16,pp.135–145.
  44. Theriot, M.C., Semcer, R.L., Shah, S.S., Grunden, A.M. Improving the Catalytic Activity of Hyperthermophilic Pyrococcus horikoshii Prolidase for Detoxification of Organophosphorus Nerve Agents over a Broad Range of Temperatures. Appl microbiol biotechnol,2011,vol.89,pp.35-43.
  45. Grimsley, J.K., Structural and mutational studies of organophosphorus hydrolase reveal a cryptic and functional allosteric-binding site. .Arch Bioch Bioph,2005,vol.442,pp.169–179.
  46. Walker, J.P., Kimble, K.W., Asher, S.A., Photonic crystal sensor for organophosphate nerve agents utilizing the organophosphorus hydrolase enzyme. Anal Bioanal Chem, 2007,vol..389,pp.2115–2124.
  47. Lu, H.D., Wheeldon, I.R., Banta, S., Catalytic biomaterials:engineering organophosphate hydrolase to form self-assembling enzymatic hydrogels. Protein Eng Des Sel,2010,vol.23,pp.559–566.
  48. Chu, X., Tian, J., Wu, N., Fan, Y., An intramolecular disulfide bond is requiredfor the thermostability of methyl parathion hydrolase, OPHC2. Appl Microbiol Biotechnol, 2010,vol.88, ,pp.125–131.
  49. Tian, J., Wang, P., Huang, L., Chu, X., Wu, N., Fan, Y., Improving the thermostability of methyl parathion hydrolase from Ochrobactrum sp.M231 using a computationally aided method. Appl Microbiol Biotechnol, 2013,vol.97,pp.2997–3006.
  50. Su,Y., Tian, J., Wang, P., Chu, X., Liu, G., Wu, N., Fan, Y., Improving the Thermostability of a Methyl Parathion Hydrolase by Adding the Ionic Bond on Protein Surface. Appl Biochem Biotechnol,2011,vol.165,pp.989–999.