A Study On The Selection And Immobilization Of Phytoesterases Used In Detecting The Residue Of The Pesticides

In this thesis, through employment of the enzyme inhibition mechanism of enzymatic analysis, the phytoesterases used in detecting pesticides residue were studied. The classical phytoesterases wheat seeds were used as experimental material to select the kinds of buffer solution, the ratio of material and solution, extracting time and the centrifugal velocity. So the best extracting techniques was confirmed. Then the general esterase activity and the specific activity of seeds from eleven species of plants and of two botanic materials for processing were studied and compared. The proper phytoesterase was obtained, which was compared with houseflies of general esterase activity and the specific activity, to estimate its exploitation value. The esterase was then applied to analyze sensitivity of six pesticides (Dichlorvos, Phoxim, Trichlorfon, Metolcarb, Carbofuran and Methomyl) to estimate its exploitation feasibility. Finally, the esterase was primary purified and immobilized, setting foundation for its exploitation and utilization. The results showed as following:1. The optimal extracting conditions were showed. Fresh seeds and botanic materials were ground into 100 meshes, mixed with PB buffer at 1:5, kept in 4℃for 16 hours, filtrated with gauze, centrifuged at 10000r/min for 10 minutes.2. The general esterase activity and the specific activity of seeds from Vigna sp. were significantly higher than that from other botanic materials and houseflies, which were 14.695U and 1.219U/mg. In addition, this enzyme resource was easily obtained, simply extracted and cheap, so the enzyme obtained from Vigna sp. was an optimal enzyme source for detecting pesticides residue.3. The esterase obtained from Vigna sp. is applied to analyze sensitivity of six pesticides (Dichlorvos, Phoxim, Trichlorfon, Metolcarb, Carbofuran and Methomyl). The result showed that the sensitivity to Trichlorfon is the highest, followed by Dichlorvos and Phoxim. However, it is less sensitive to Carbamate pesticides. The determination coefficient to pesticides concentration and inhibition rate was 0.9423, which meets the national standard of detection. This estimated its exploitation feasibility for detecting the pesticide residue.4. The specific activity and multiple of purification of primary purified enzyme were both increased. The specific activity of enzyme obtained from Vigna sp. was 2.743 times than before and 2.420-fold after deposited by 60% ammonia sulfate, while it was 4.214 times and 3.460-fold after dialyzed and concentrated.5. The best immobilization conditions were: 4℃,8h. The enzyme membranes were stable for at least 60 days in preservative films at -20℃in dark, which can remain 80% of the activity.