Extraction and Purification of Organophosphorus hydrolase Enzyme from Soil Microorganism Pseudomonas diminuta

Abstract

Synthetic organophosphorus compounds are highly toxic hence, widely used as pesticides, insecticides and chemical warfare agents. Organophosphorus hydrolase (OPH) is an organophosphotriester hydrolysing enzyme; effectively hydrolyse a range of organophosphate esters. The objective of the present study was extraction and purification of OPH enzyme from Pseudomonas diminuta bacteria (soil microorganism) and to study kinetic properties of the purified enzyme. Enzyme was extracted and purified from bacteria by ammonium sulphate precipitation and ion exchange chromatography. Purity of an enzyme was determined by sodium dodecyl sulphate-polyacryamide gel electrophoresis (SDS-PAGE). Purified OPH enzyme specific activity was found to be 27.7 fold of 32.8 U/mg protein, molecular weight of 72 Kda and it is a homodimer since it has shown a single band in SDS-PAGE separation. Maximum activity of the free OPH enzyme was found at Optimum pH 7.5 and temperature 35 o C with the incubation time of 10 min. Michaelis constant (K m ) and maximum velocity (V max ) values of free OPH enzyme for methyl parathion as substrate was found to be 286.2 µM and 2.5 µM/min respectively.