Study On Purification And Pesticide Sensitivity Of Esterase From Snow-peas And Kidney-bean

Pesticide plays an extremely important role in the development of Chinese agriculture,but the abuse of pesticide leads to the increasingly striking problem of residues. So it is an urgent affix to develop a simple, convenient, economic and rapid detection technique of pesticide residues. This research filtrates out the most efficient enzymes from several kinds of plant enzymes, then separates and purifies them, at the same time studies the enzymology character of plant esterase and finally detect the sensitivity of plant esterase to 23 pesticides. The results of this research are as follows:1. The optimal concentration for Snow-pea and Kidney-bean esterase are 0.33μg/mL and 0.45μg/mL respectively, the optimal reaction temperature is 40℃, the optimal PH is 6.5. 1-Naphthylacetate is a specific substrate, of which is sensitive to serine and BNPP. Their km value are 0.085mmol/L and 0.119mmol/L.2.By applying the techniques of Ion Exchange(IX) and gel-filtration to purify the two plant esterase, the specific activity of Snow-pea and Kidney-bean esterase has been increased 56 times and 41 times compared with previous result respectively, and the recovery rate of enzyme activity are 45.75% and 41.53%.3. By applying SDS-PAGE to identify the purity and determine the molecular weight of Snow-pea and Kidney-bean’s purified enzymes, the results indicates that there is a relatively obvious protein band and the relative molecular of Snow-pea and Kidney-bean esterase is 26 kDa.4. Among 16 organophosphorus pestcides, Snow-pea esterase is relatively sensitive to dichlorvos, moncrotophos, phoxim, dipterex, triazophos, profenofos, methamidophos, chlorpyrifos, dimethoate and omethoate, on the contrary, it’s little sensitive to malathion, acephate, fenitrothion, anilofos and glufosinate, and it’s not sensitive to methidathion. Among 7 carbamates pesticides, Snow-pea is relatively sensitive to methomyl, carbofuran, propoxur and aldicarb, it’s little sensitive to pirimicarb, monosultap and carbosulfan.5. The sensibility to 8 organophosphorus pestidides of Snow-pea is increased overall after being purified, it’s IC50 is among 10-6~10-8mol/L. After being purified, the sensibility of Snow-pea esterase to dipterex, chlorpyrifos and omethoate are increased 2 times, and the sensibility to phoxim, triazophos, dimethoate and dipterex are not significantly increased. The IC50 of the four kinds of carbamates pesticides(propoxur, methomyl, aldicarb, carbofuran) to the purified Snow-pea esterase is among 10-6~10-7mol/mL. The sensibility of enzyme to pesticides is increased by purified Snow-pea esterase,that is 4 times to carbofuran,2 times to methomyl and not is increased to propoxur.6. Among 16 organophosphorus pesticides, Kidney-bean esterase is relatively sensitive to dichlorvos, moncrotophos, phoxim, dipterex,triazophos, profenofos, methamidophos, chlorpyrifos, dimethoate omethoate,fenitrothion and acephate, on the contrary, it’s little sensitive to malathion, dimethoate and anilofos, and it’s not sensitive to glufosinate and methidathion. Among 7 carbamates pesticides, Kidney-bean esterase is relatively sensitive to methomyl, carbofuran, propoxur and aldicarb, it’s little sensitive to pirimicarb and carbosulfan.7. The IC50 of 8 organophosphorus pesticides to purified Kidney-bean esterase is among 10-6~10-8mol/L. After being purified, the sensibility of Kidney-bean esterase to dichlorvos, moncrotophos, phoxim, omethoate are increased 2 times, and the sensibility to triazophos, chlorpyrifos, dimethoate and dipterex are not significantly increased. The IC50 of the four kinds of carbamates pesticides(propoxur, methomyl, aldicarb, carbofuran) to the purified Kidney-bean esterase is among 10-6~10-8mol/mL. The sensibility of enzyme to pesticides is increased by purified colored Kidney-bean esterase, that is 4 times to propoxur, 2 times to aldicarb and not significantly increased to methomyl and carbofuran.8. Snow-pea and Kidney-bean esterase are very sensitive to several commonly used pesticide. Their sensibility are increased compared to the existent enzymatic inhibition method and they have the advantage of low price, simple extraction, easy detection and so on. The Snow-pea and Kidney-bean esterase are promising in the detection of pesticide residue.