PURIFICATION AND KINETIC STUDIES OF ORGANOPHOSPHORUS HYDROLASE FROM B. DIMINUTA
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 Lowryâ€™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 Î¼M and 50 Î¼M/min respectively. At optimum pH (7.5) and incubation temperature (35Â°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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