PURIFICATION AND KINETIC STUDIES OF ORGANOPHOSPHORUS HYDROLASE FROM B. DIMINUTA
Keywords:
Organophosphorus pesticides, Brevundimonas diminuta, Organophosphorus hydrolase, chromatographyAbstract
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.
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