• Ashish Gothwal Centre for Biotechnology, Maharshi Dayanand University, Rohtak 124001
  • Monika Dahiya Centre for Biotechnology, Maharshi Dayanand University, Rohtak 124001
  • Puneet Beniwal Centre for Biotechnology, Maharshi Dayanand University, Rohtak 124001
  • Vikas Hooda Centre for Biotechnology, Maharshi Dayanand University, Rohtak 124001


Organophosphorus pesticides, Brevundimonas diminuta, Organophosphorus hydrolase, chromatography


Objective: Extraction and purification of Organophosphorus hydrolase (OPH) enzyme from Brevundimonas diminuta and to study kinetic properties of the purified enzyme.

Methods: The enzyme was extracted from bacteria and purified by using a combination of gel filtration and ion-exchange chromatography and the purity of an enzyme was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The activity of the purified enzyme was monitored by enzyme assay and total protein content was determined by using Lowry's method. The kinetic properties of the enzyme were also studied.

Results: A 72 kDa organophosphorus hydrolase (OPH) enzyme was extracted and purified. The purified enzyme was homodimer and showed a single band on SDS-PAGE. The Michaelis constant (Km) and maximal velocity (Vmax) values of free OPH enzyme for methyl parathion as substrate was 285.71 μM and 50 μM/min respectively. At optimum pH (7.5) and incubation temperature (35°C), free enzyme showed maximum activity with incubation time of 8 min.

Conclusion: The bacteria contain OPH enzyme with high potential to detoxify OP pesticides, attractive for bioremediation due to good pH & temperature conditions, were also useful in development of bio analytical techniques such as biosensors for OP pesticide detection.


Download data is not yet available.


Brown KA. Phosphotriesterases of Flavobacterium sp. Soil Bio Biochem 1980;12:105-12.

Raushel FM. Bacterial detoxification of organophosphate nerve agents. Curr Opin Microbiol 2002;5:288-95.

Mulbry WW, Karns JS. Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene and protein. J Bacteriol 1989;171:6740-6.

Gorla P, Pandey JP, Parthasarathy S, Merrick M, Siddavattam D. Organophosphate Hydrolase in Brevundimonas diminuta is targeted to the periplasmic face of the inner membrane by the twin arginine translocation pathway. J Bacteriol 2009;191:6292-9.

Benning MM, Kuo JM, Raushel FM, Holden HM. Three-Dimensional structure of phosphotriesterase: an enzyme capable of detoxifying organophosphate nerve agents. Biochem 1994;33:15001-7.

Benning MM, Kuo JM, Raushel FM, Holden HM. Three-dimensional structure of the binuclear metal center of phosphotriesterase. Biochem 1995;34:7973-8.

Omburo GA, Kuo JM, Mullins LS, Raushel FM. Characterization of the zinc binding site of bacterial phosphotriesterase. J Biol Chem 1992;267:13278-83.

Lewis VE, Donarski WJ, Wild JR, Raushel FM. Mechanism and steriochemical course at phosphorus of the reaction catalyzed by a bacterial phosphotriesterase. Biochem 1988;27:1591-7.

Donarski WJ, Dumas DP, Heitmeyer DP, Lewis VE, Raushel FM. Structure-activity relationships in the hydrolysis of substrates by the phosphotriesterase from Pseudomonas diminuta. Biochem 1989;28:4650-55.

Dumas DP, Caldwell SR, Wild JR, Raushel FM. Purification and properties of the phosphotriesterase from Pseudomonas diminuta. J Biol Chem 1989;264:19659-65.

Lai K, Dave KI, Wild JR. Bimetallic binding motifs in organophosphorus hydrolase are important for catalysis and structural organization. J Biol Chem 1994;269:16579-84.

McDaniel CS, McDaniel J, Wales ME, Wild JR. Biocatalytic Coatings. Paint Coatings Ind 2005;26-33.

Simonian AL, Flounders AW, Wild JR. FET-based biosensors for the direct detection of organophosphate neurotoxins. Electroana 2004;16:1896-6.

Rainina EI, Efremenco EN, Varfolomeyev SD, Simonian AL, Wild JR. The development of a new biosensor based on recombinant E. coli for the direct detection of organophosphorus neurotoxins. Biosens Bioelectro 1996;11:991-1000.

Mulchandani A, Chen W, Mulchandani P, Wang J, Rogers KR. Biosensors for the direct determination of organophosphate pesticides. Biosens Bioelectro 2001;16:225-30.

Petrikovics I, Papahadjopoulos D, Hong K, Cheng TC, Baskin SI, Jiang J, et al. Comparing therapeutic and prophylactic protection against the lethal effect of paraoxon. Toxico Sci 2004;77:258-62.

Herzlinger D, Viitanen P, Carrasco N, Kaback HR. Biochem 1984;23:3688-93.

Bernhardt TG, de Boer PAJ. The Escherichia coli amidase AmiC is a periplasmic septal ring component exported via the twin-arginine transport pathway. Mol Microbio 2003;48:1171-82.

Liu YH, Liu Y, Chen ZS, Lian J, Huang X, Chung YC. Purification and characterization of a novel organophosphorus pesticide hydrolase from Penicillium lilacinum BP303. Enz Microbial Tech 2004;34:297-3.

Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nat 1970;227:680-85.

Chaudhry GR, Ali AN, Wheeler WB. Isolation of a methyl parathion-degrading Pseudomonas sp. that possesses DNA homologous to the opd gene from a Flavobacterium sp. Appl Env Microbio 1988;54:288-93.

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin-Phenol reagents. J Biol Chem 1951;193:265-75.

Najavand S, Lotfi AS, Noghabi KA, Mohsenifar A, Milani MM, Mota-Kalvanagh A, et al. A high potential organophosphorus pesticide-degrading enzyme from a newly characterized, Pseudomonas aeruginosa NL01. Afr J Microbiol Res 2012;6:4261-9.

Votchitseva YA, Efremenko EN, Aliev TK, Varfolomeyev SD. Properties of hexahistidine tagged organophosphate hydrolase. Biochem 2006;71:216-22.

Rowland SS, Speedie MK, Pogell BM. Purification and characterization of a secreted recombinant phosphotriesterase (parathion hydrolase) from streptomyces lividans. Appl Env Microbio 1991;57:440-4.

Dhull V, Gahlaut A, Dilbaghi N, Hooda V. Acetylcholinesterase Biosensors for electrochemical detection of organophosphorus compounds: a review. Biochem Res Int 2013;1-18.



How to Cite

Gothwal, A., M. Dahiya, P. Beniwal, and V. Hooda. “PURIFICATION AND KINETIC STUDIES OF ORGANOPHOSPHORUS HYDROLASE FROM B. DIMINUTA”. International Journal of Pharmacy and Pharmaceutical Sciences, vol. 6, no. 10, Oct. 2014, pp. 341-4, https://www.innovareacademics.in/journals/index.php/ijpps/article/view/2849.



Original Article(s)