Enzymatic Properties And Rapid Detection Of Pesticide Residues Using Immobilized Wheat Esterase

Pesticides were contributed to increase crop production and ensure food supply,organic phosphorus and carbamate pesticides usage in China is much higher than other pesticides.However,due to long-term accumulations in environment,pesticides contaminations have raised serious public concern regarding the food safety and human health.Although numerous analysis methods including chromatography and mass spectroscopy could quantitatively provide an accurate evaluation of the health risk of integrated OPs exposure,they still suffer from some intrinsic disadvantages of expensive analysis settings entailing well-trained personnel and inconvenience for field applications.Therefore,it is essential to develop the rapid and sensitive screening methods to ascertain the presence of these compounds in food safety management.In the present work,wheat esterase was immobilized,the immobilization conditions and enzymatic properties of immobilized enzymes were investigated.A pesticide residues rapid detection card was developed which is more convenience and cheaper.The main results are as follows:1.Taking the plant-esterase from wheat bran as the enzyme source,the physical adsorption method was selected by comparing with cross-linking method.Glass fiber film as adsorption carrier was used to explore the optimum conditions for immobilization with the evaluation index of enzyme activity recovery.On the basis of single-factors experiments,the Box-behnken center-united experimental design principles were used to design the experiment.The response surface analysis of 3 factors(buffer's pH,immobilized time and immobilized temperature)and the interactive effect on immobilization were demonstrated.The optimum immobilized conditions were confirmed as follow:phosphate buffer's pH 7.0,immobilized time 4 h,and immobilized temperature 29 ?.Based on these conditions,the detection value of the enzyme recovery rate was 28.21%,and its verified value was 27.51%.It is proved that the response surface method can effectively optimize the immobilization conditions of wheat bran esterase.2.Enzymatic properties of immobilized wheat esterase were studied,the results show that the optimum pH for immobilized wheat esterase on glass fibre membranes is 7.0,optimum temperature for immobilized enzymes is 40 ?,when the enzyme was immobilized on glass fibre membranes,its Km is observed to be 6.73 mg/mL,its Vmax is 1.01 mg/mL min.It has less effect of loss of enzyme activity when storage at 4 ?.And there are no significant difference between initial activities and pH incubation activities on different pH values.After 8 hours of water incubation,immobilized enzyme still retained about 90%of the enzyme activity,while the free wheat bran esterase only retained about 70%.After storage at 4 ? for 60 days,immobilized enzyme still retained about 60%enzyme activity.In addition,stability of wheat esterase were increased as a result of membrane immobilization.These results also riched the fundamental knowledge for using wheat esterase to detection pesticides.3.Inhibition effect of immobilized enzyme on different pesticides were performed a good linear relationship with different concentrations with a index of activity inhibition rate,the sensitivity and limit of detection(LOD)of 6 different pesticides were studied.The limit of detection of Dimethoate?Parathion-methyl?Dichlorvos?Carbaryl?Isoprocarb?Carbofuran are 0.009 1?0.009 4?0.0015?0.015?0.017?0.009 4 mg/L,respectively.Detection time to pesticides with immobilized enzyme is usually 15 min.While,the limit of detection with free enzyme of Dimethoate?Parathion-methyl?Dichlorvos?Carbaryl?Isoprocarb?Carbofuran are 0.35?0.453?0.006?0.043?0.035?0.014 mg/L,respectively.Detection time to pesticides with free enzyme is usually 30 min.Compared with free enzyme,inhibition time of immobilized enzyme is shorter than that of free enzyme,the limit of detection of immobilized enzyme is lower that of free enzyme.Thus,immobilized enzyme can use to detecting pesticides.4.Based on the principle of colorimetric,the new disposable detection card was developed with substrate membrane,enzyme membrane and chromogenic membrane for rapid detection of pesticide residues,which was based on the inhibition of plant esterase.The work of rapid detect card only needs to adding extraction of samples,detection time is just 15 min.The limit of detection of Dimethoate?Parathion-methyl?Dichlorvos?Carbaryl?Isoprocarb?Carbofuran is 0.5?0.02?0.2?2?0.5?0.1 mg/L,respectively.While,the limit of detection with national standard's rapid detection card based on AChE inhibition of Dimethoate?Parathion-methyl?Dichlorvos?Carbaryl?Isoprocarb?Carbofuran is 1.3?0.3?2.5 mg/L,respectively,which are all higher than that of our card.Verification test for pesticide and vegetable samples have proved that our card has the advantages of high sensitivity and accuracy,lower cost,easy operation,that can detecting most organic phosphorus and carbamate pesticides residues,suitable for large-scale screening of samples.