| Organophosphorus pesticides are widely used in agricultural production, but it is very harmful for the health of people for its highly toxicity and residual heavily and incorrectly use which leading to the organic phosphorus pesticide residues exceeding used on agricultural products. The traditional detection methods, such as gas chromatography, etc, are unable to meet the needs of the site-rapid detection for its complexity process and long testing cycle. Therefore, one of the important means to control pesticide residues is to develop a convenient, sensitive and rapid cetection technology. The enzyme inhibition assay is generally welcomed by the grass-roots organizations for its characteristic such as fast detection, simple equipment and easily operation which are suitable for the rapid detection of fruits and vegetables. Enzyme inhibition assay was divided into animal cholinesterase inhibition and plant esterase inhibition depending on the enzyme source, animal cholinesterase method was limited for its narrow range of enzyme source and highly costs, while the phytoesterase was highly concerned in recent years for its rich enzyme source, low costs and convenient material.In this study, we made a further study beyond the existing research, we expanded filter range of the enzyme source, choose the large white beans for the enzyme source, then we developed the extraction process of, made a study on the method of purification on large white beans esterase, systematic discussed the feature of large white beans esterase, found the system of detection method on organophosphorus pesticides.The main results are as follows:(l)Both the total esterase activity and specific activity of large white beans were higher than the phytoesterase extracted from other9kinds of plant seeds, the sensitivity to different toxic organic phosphorus pesticides (omethoate, dichlorvos and trichlorfon) was also very strong. The minimum detection limit of large white beans esterase on seven kinds of commonly used organophosphorus pesticides, were lower than the maximum residue limits (MRL). The large white beans esterase can fully meet the needs of the rapid detection of organophosphorus pesticide residues.(2)Through single factor experiment and response surface optimization, the optimum extraction process of large white beans esterase was as follows:Solid-liquid ratio of1:5.04, phosphate buffer (pH7.04,0.043mol/L) as the extraction solvent, stirring95.51min,4℃for overnight, then8000r/min centrifugal l0min, the total esterase activity of large white beans was9.84U/mL.(3) After2OOOr/min to8OOOr/min differential centrifugation,60%ammonium sulfate fractionation, DEAE-32ion exchange chromatography, and Sephadex G-200gel filtration chromatography, large white beans esterase was purified111.73times, and its esterase activity recovery rate was43.9%.(4)The nature of the large white beans esterase studies have shown that the optimum temperature of large white beans esterase was30℃, the optimum pH was7.5, and the large white beans esterase was relatively stable when the temperature was between25℃to40℃and pH was between6.5to8.0. Hg2+,Cu2+,Zn2+,Fe3+,Ag+strongly inhibited the large white beans esterase, especially Cu2+, it can make the enzyme inhibition rate up to58.29%. Ca2+, Mn2+, Mg2+had little effect on white esterase activity, while Na+had certain stimulative effect. The optimum substrate concentration of large white beans esterase was16mmol/L and Km is0.280mmol/L.(5)We established the method system of detecting organophosphorus pesticide residues by large white beans esterase, such as follows:put20mL/L enzyme solution and a-naphthyl acetate solution into test tubes, mixed and made it reaction for10minutes in water at30℃, the add in solid blue B salt solution, mixed and made it color for10minutes in water at30℃, and determine the absorbance at595nm wavelength within10minutes. |
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