Research And Application On Plant-Esterase Inhibition By Organophosphorous Pesticides

With frequent occurring of accident related with pesticide residues in vegetable,more attention is paid to the rapid detection of pesticide residues. Plant-esterase inhibition,a rapid detection method of pesticide residues, with low-cost,easy extraction,convenient preservation,better detection sensitivity,and satisfaction of the popular market demand,became a very important research interesting in rapid detection to organophosphorus pesticide residues.In this paper,the commercial flour was chosen as the raw material of esterase.The flour was firstly mixed with water at a ratio of 1:10(g:mL) and standed for about 15 h,then centrifuged to get the supernatant containing crude plant-esterase.According to the mechanism that the activity of plant-esterase can be inhibited by organophosphorus pesticides, the kinetic constants(k_i,k_d,k_p) and the concentration of inhibitor producing half-inhibition of enzyme activity(IC₅₀) of plant-esterase inhibited by the racemate and enantiomers of malaoxon,isomalathion,and methamidophos were investigated.The inhibition on plant-esterase with optically pure stereoisomers of malaoxon,isomalathion and methamidophos showed stereoselectivity.As for malaoxon,the order of toxicity to tested esterase was R-malaoxon＞RS-malaoxon＞S-malaoxon, and the toxicity of R-malaoxon was about 2-fold as that of S-malaoxon.In contrast,the toxicity of S-methamidophos was 4-8 times as that of R-methamidophos,the inhibitory toxicity of(RS)-methamidophos was between that of R and S forms.The inhibitory potency of individual enantiomer of isomalathion to plant-esterase decreased in the order of (1R,3R)-isomalathion＞(1R,3S)-isomalathion＞(1S,3R)-isomalathion＞(1S,3S)-isomalathion,the inhibition of(1RS,3RS) -isomalathion was weaker than its enantiomers.The spontaneous reactivation rates(k₃) of plant-esterase inhibited by the racemate and enantiomers of malaoxon,isomalathion,and methamidophos were studied.It indicated that plant-esterase inhibited by optically pure stereoisomers of malaoxon,isomalathion and methamidophos reactivates at a measurable rate,except R-methamidophos. However,the plant-esterase inhibited by S-methamidophos spontaneously reactivated more rapidly than the other phosphorylated plant-esterase.The k₃ value of plant-esterase phosphorylated by malaoxon decreased in the order of R-malaoxon＞RS-malaoxon＞S-malaoxon,and the difference between two isomers is not significant.The reactivated potency of plant-esterase inhibited by individual enantiomer of isomalathion was (1R,3R)-isomalathion＞(1RS,3RS)-isomalathion＞(1R,3S)-isomalathion＞(1S,3S)-isomalathion＞(1S,3R)-isomalathion,which show that plant-esterase inhibited by either(1R,3R)- or(1R,3S)-isomalathion spontaneously reactivated 4-10 times more rapidly than the plant-esterase phosphorylated by the(1S)-isomers of isomalathion.The purification process of crude extract of plant-esterase was carried out by salting out with ammonium sulfate and gel column chromatography on Sephadex G-100.Two constitutes were collected,the first and second fraction were purified about 8.3-fold and 7.4-fold from original crude enzyme,respectively.The purity of the crude enzyme,enzyme salted-out by ammonium sulfate and filtrated by Sephadex G-100 gel,was compared by SDS-PAGE electrophoresis.The purity of the plant enzyme which was further purified by Sephadex G-100 column chromatography was significantly improved and the first fraction was more pure than the second fraction.In addition,the limit of detection(LOD) of the crude enzyme to methamidophos,dichlorvos,phoxin,malathion and dimethoate were tested. The LODs of five Organophosphorus Pesticides were in the range of 0.036 to 2.319 mg/kg.Compared the LODs of enzyme salted-out by ammonium sulfate to methamidophos,phoxin and malathion with that of the crude enzyme to these organophosphorus pesticides,the enzyme salted-out by ammonium sulfate was more sensitive than the crude one with the detection sensitivity increased by 2-3 times.