Characteristics And Preparation Of Chlorpyrifos-Degrading Enzymes From Pseudomonas Stutzeri

Chlorpyrifos is one of most important organophosphorus pesticides (OPs). It is high efficient, broad-spectrum and widely used in pest control of vegetables and rice. Chlorpyrifos is becoming widely used after the highly toxic OPs were banned. The OPs residue of food and environment is one of the worst problems in the world.Enzymic degradation was studied for the pollution control of OPs. Using Pseudomonas stutzeri JHY01 strain as degradation bacteria, the present research was focusing on: locating the degrading enzymes and optimizing its extraction technology, investigating the enzymes characteristics, formulating the enzymes preparation and evaluating its degrading effect. The results are as follows:1)The degradation rate of 40 mg/L chlorpyrifos in 30 minutes by intracellular enzymes is 79.85%, but by extracellular enzymes is only 8.41%. It indicates that the degrading enzymes are typical intracellular enzymes.2)The L9 (34) orthogonal experiments were designed to optimize the ultrasonic extraction conditions of the degrading enzymes. The optimal culture time of the bacteria is 12 hours, at which time the degradation rate of enzymes extracted is biggest. And the degrading activity of the crude enzymes is maximal (with the degradation rate 84.47%) when the ultrasonic conditions are as follows: the biomass of the strain to extraction buffer volume 1∶4 (g/ml), with total radiate 30 times, 4 seconds each time, 4 seconds interval (UAS,Sonics-VCX500, 500W, 25KHz).3)The enzymes are connatural and have nicer activity at pH6.0~10.0, and the optimum pH is 7.2; The favorable temperature range for the enzymes is 30~50℃, and the optimum temperature is 40℃; Cu2+, Fe3+, Mn2+ can inhibit the enzymes activity; The enzymes have no activity when diluted to 100 times.4) Through detecting the appearance and the activity, the suitable wetting agent and stability agent were screened out. And with orthogonal experiments, the best formula of the crude enzymes preparation was designed, which is: enzymes content 50%, SL as stabilizing agent and content 2%, WS as wetting agent and content 7.5%. After stored for 7 days at 4℃, the degradation rate of the preparation is 77.65%. The surface tension of the preparation is about 53.07mN/m2, and the pH is 7.60. The decomposition rate of the preparation which stored for 7 days at 4℃is less than 5%.5) The crude enzymes preparation can degrade chlorpyrifos which residued on the leaves of tobacco effectively, after they were diluted 5~10 times the effect was obvious also. The crude enzymes preparation have obvious degrading effect to the chlorpyrifos residued in the soil, the degradation rate of the preparation can over 89% after reacting for 7 days. And the degradation rate can reach 57%~65% after the preparation was diluted 5~10 times.