Studies On The Methods Of Phytoesterase Immobilization And Its Application

For the past few years,the toxicosis incidents of human and animal resulted from taking food which contains high level of pesticide residues are happened usually,so people has think highly to the security of foods.At present,GC and HPLC is the common method for detecting pesticide residues,but they are too expensive and complex to use as a method for fieldwork detection.Because the organophosphorus and carbamate pesticides can inhibit the activity of AChE,so people use this principle to develop some researches of pesticide rapid detection.But the expensive and the low extraction of AchE make it can not satisfy the requirement of pesticide rapid detection.Phytoesterase has the low cost and easily to store,so people use phytoesterase to replace the AChE.this articles use soybean as material to study the sensitivity of phytoesterase to pesticide,and the feasibility of develop a production.The results are as follow:1.Study on extraction of phytoesterase from soybeans as follows:soybean flour was surged for 120 min with phosphate buffer(0.3mol/L,pH 7.0),then,centrifugalize the filtrate at 5000rpm,supernate is the enzyme of phytoesterase.2.Optimize the optimal reaction of phytoesterase,the optimal reaction as follows:0.1mL enzyme solution,0.1mL substrate,1.9 mL pH7.5 phosphate buffer solution(PBS)and 0.25mg/mL developer0.9 mL, bulk volume is 10 mL,reaction time is 5min,reaction temperature is 30℃.3 Through evaluated the total enzyme activity of 26 different soybeans,Tong nong 10 was selected as the best phytoesterase source.Through investiged the the sensitivity to pesticides of 8 pesticides,we know the sensitivity is dichlorvos＞trichlorphon＞parathion＞acephatemet＞dimethoate＞omethoate＞durshan＞tricarnam.4 The thick laminar analysis filter paper was selected from the 3 materials to immobilize phytoesterase. The optimized parameters was as follows:the concentration of enzyme is 80%,the pH value is 7.5,the temperature is 4℃and the time of immobilisation is 24h,the time of dry blast is 2h and the temperature of dry blast is 40℃.5.Study on Nitrocellulose membrane of immobilization.The optimized parameters was as follows:dip the Nitrocellulose membrane in PBS(0.1mol/L pH7.0) for 48h to activation,the concentration of enzyme is 70%,the pH value is 7.5,the temperature is 4℃and the time of immobilisation is 48h,the glutaraldehyde concentration was 0.25%,bovine serum albumin(BSA) concentration was 0%.6.Study on chitosan and chitosan membrane of immobilization.The optimized parameters of chitosan membrane was as follows:the concentration of enzyme is 80%,the pH value is 7.5,the temperature is 4℃ and the time of immobilisation is 24h,the glutaraldehyde concentration was 0.5%.7.The characteristic of 3 immobilized enzyme was researched and compared with dissociative enzyme. The result was that the temperature of chitosan membrane of immobilization was 30℃,the pH value is 7.5. The temperature of thick laminar analysis filter paper of immobilization was 30℃,the pH value is 7.5,the storage life is better than dissociative enzyme.The temperature of Nitrocellulose membrane of immobilization was 40℃,the pH value is 8.0,both higher than dissociative enzyme.8.Compared with these immobilized enzyme,determine the thick laminar analysis filter paper was the best carrier of immobilization.Use this immobilized enzyme to check the pesticide,the result shows:it is very sensitivity to dichlorvos,the LDCs of dichlorvos,trichlorphon,parathion,acephatemet,dimethoate,omethoate, durshan,tricarnam were 0.01mg/L,0.1 mg/L,0.5 mg/L,0.5 mg/L,1 mg/L,10 mg/L,10 mg/L,50 mg/L respectively.