Performance evaluation of soil microorganism producing organophosphate hydrolase enzyme in detoxification of organophosphorus compounds (diazinon) and its kinetic properties assessment

Organophosphorus compounds, are widely used as pesticides in agriculture. Organophosphate Hydrolase (OPH) enzyme hydrolyses a wide variety of organophosphate esters. In the present study, by using both enrichment culture and maximum dilution frequency techniques, the diazinon degradation bacteria was isolated from treated samples and was identified by morphological and biochemical tests. The enzyme was extracted from its bacterial strain and then purified using ammonium sulfate and ion exchange chromatography. The purity of the enzyme was confirmed by electrophoresis (SDS-PAGE), then its molecular weight was determined by specific marker proteins and its kinetic properties was evaluated.Using enrichment culture technique and the increasing factor of the biodegradability diazinon, enzymatic hydrolysis time decreased significantly with less than 10 hours. The main metabolite produced from diazinon hydrolysis was identified as 2-isopropyl-6-methyl-4-Hydroxy pyrimidine (IMHP, with less toxicity) through both thin layer chromatography (TLC) and spectrophotometry techniques. Purified OPH enzyme specific activity was found to be 57.48 fold of 16.44 U/mg of protein with a yield of 11.4 percent and approximate molecular weight of 40 KD. Maximum activity enzyme was at optimum pH 8.5 and temperature of 37 ͦ C. Substrate concentration up to 0.05 M and Co+2, Ca+2 metal ions had positive effects on activity enzyme. Michaelis constant (Km) and maximal velocity (Vmax) values were calculated as 434.2 μM and 107.3 μM/min, respectively. The results showed that OPH enzyme with its activity gradual and effective in addition to detoxification, could be played a key role in recovery of phosphate for the production of biofertilizer.