Enzyme Inhibition Determination Method Of Organophosphorus And Carbamate Pesticides With Esterase From Wheat Bran

The problem of pesticide resticides in the farm produces had increasingly attracted the attention of government and the public. It is necessary to control the use of pesticides, and to strengthen efforts to detect so that the products of excessive pesticide residues are not allowed to enter the market. Due to its simple pre-treatment, low cost, less detection time, Enzyme-inhibition methods are very suitable for China's small-scale, decentralized sales system in regularly on-site rapid detection. Because the plant esterase has some advantages such as wide sources, low cost, easy to extract and save, et.cl, so it has caused the attention of many scholars, in order to be able to replace acetylcholinesterase. Wheat bran is the by-product of wheat processing, production accounts for about 20% of wheat processing capacity, the source of a wider, cheaper. In the present work, Wheat bran was chosen as the resource of plant esterase, the sensitivity of purified and unpurified esterase to organophosphorus and carbamate pesticides, the optimization of pre-treatment, and the elimination of false positive in the spicy vegetables were demonstrated. The main results as follows:1. The sensitization effect of surfactants on the fast blue B salt chromogenic reaction system and the inhibitory times were determined by wheat bran esterase-a-naphthol-fast blue B salt reaction. On the basis of the optimum determination of enzyme activity to the anionic surfactant SDS as best sensitization agent, in the absorption wavelength of 535 nm, could make the reaction system of relative absorbance value increased to 152.76%. The most restricted time of isoprocarb on the esterase for was 40 minutes, while other pesticides restricted time were 30 minutes.2. Under the best detection system, the sensitivity and limit of detection ?LOD? of 6 different pesticides detection by purified and unpurified wheat bran esterase were studied. The results showed that the range of LOD of unpurified wheat bran esterase on 6 different pesticides is 0.006?0.45mg/L. Compared with the maximum residue levels specified in GB 2763-2014, wheat bran esterase could meet the need of detection of dichlorvos, isoprocarb, carbaryl, carbosulfan and rogor, except methyl parathion. And by different methods purified wheat bran esterase, then preceded sensitivity experiments with them. The results showed that the LOD's range of wheat bran esterase purified by molecular sieve column chromatography on 6 different pesticides is 0.003?0.193mg/L, its sensitivity was 1.1?2.3 times higher than crude enzyme. The LOD's range of wheat bran esterase purified by aqueous two-phase extraction on 6 different pesticides is 0.005?0.116mg/L, its sensitivity was 1.35?2.2 times higher than crude enzyme. But the LOD's range of esterase segmented by ammonium sulfate precipitation on 6 different pesticides is 0.039?0.386mg/L, its sensitivity lower than crude enzyme significantly.3. The kinetic constants ?K_i?K_a?K₂? and the concentration of inhibitor producing half-inhibition of enzyme activity (IC₅₀) were calculated by bimolecular reaction rate constant.The results showed that the inhibition on wheat bran esterase with 6 kinds of pesticides showed stereoselectivity. And the kinetic constants reflected the ability that esterase was inhibited by pesticides were consistent with the above experiment. The order of inhibition ability to tested esterase was dichlorvos> isoprocarb> carbaryl>methyl parathion> carbosulfan> rogor.4. The pesticide residue extraction solution, extracting methods, extraction time and the treatment of sample solution were separately optimizatied by using Enzyme-inhibition method with standard addition recovery rate as index, The results showed that the optimal way to extract pesticides residues in vegetables was to use 5% acetone/PBS as extracting solution, and extract for 5 min with the method of ultrasonic extracting, then filter by 0.45 ?m organic film. With the above pre-treatment methods, the response rate ranged from 83.61% to 102.81%, with coefficient of variation at the ranged from 1.2% to 7.23%. Therefore, the pre-treatment can meet the need of rapid detection of pesticide residues by enzyme inhibition method.5. In this experiment, activity inhibition rate as the index, different heating treatment to deal with 4 kinds of spicy vegetables were carried out to elimanited or reduce the false positive in enzyme inhibition method. The results indicated that under the appropriate heating temperature, which was 80?,90?,80?,80?, for the materials of scallion, onion, garlic and green pepper respectively, the inhibition rate of enzyme activity from 37.06%?9.65%?17.02%? 50.34% down to 12.69%?2.95%?5.79%, 33.61%. In addition to green pepper, to some extent, this method could eliminate or greatly reduce the false positive that brought by spicy vegetables in the enzyme-inhibition method, which could improving the accuracy in enzyme-inhibition method to detect the spicy vegetables.