Research On Rapid Detection Method Of Pesticide Residues Based On Genetic Engineering Esterase

Organophosphorus and carbamate pesticides have been widely used in agricultural production to eliminate pests for many years,and have made important contributions to increasing agricultural production and ensuring production.The use of highly toxic pesticides has been banned or restricted,but there are still violations such as excessive use and hidden addition,resulting in frequent occurrence of agricultural product quality and safety incidents.Achieving the quick and easy detection of the above-mentioned pesticide residues is the key to the quality and safety supervision of agricultural products.Enzyme inhibition method was proposed by Ellman in the 1960s.Over the years,the research on enzyme inhibition method has been intensified.China has successively promulgated 5 relevant national/industry standards since 2001,which can detect 12/18 organophosphorus and carbamate pesticides respectively.Enzyme inhibition has become the main technical means for agricultural product quality and safety supervision.Although there are problems such as low sensitivity and poor accuracy,it has played an important role in regulating the agricultural product market.In the past two decades,our country’s pesticide residue limit standards have become more and more stringent,and the types of high-risk organophosphorus and carbamate pesticides in agricultural products have also undergone great changes.The false-positive and false-negative ratio of the test results of these standard methods exceeds the allowable range,and it is no longer suitable for the needs of the new era of supervision.Therefore,there is an urgent need for improvement and innovation of this method.In this context,this study selected high-risk organophosphorus pesticides such as dichlorvos and trichlorfon.Obtain a microbial esterase with high catalytic efficiency and strong pesticide sensitivity through genetic technology,and carry out separation and purification and characterization of enzymatic properties to optimize reaction conditions.Establish a rapid detection method for organophosphorus pesticide residues based on microbial esterase and fluorescence analysis.Finally,a rapid test kit for dichlorvos residue was developed.The main research contents and results are as follows:1.Purification and characterization of recombinant microbial esterase expressed in Escherichia coli.The recombinant microbial esterase obtained in the early stage was purified by nickel column affinity chromatography,and the protein was removed,and SDS-PAGE electrophoresis analysis was used to verify.The results of electrophoresis showed that the molecular weight of the microbial esterase was 36k Da,which was a single band in the electrophoresis,and a high-purity enzyme solution was obtained.Further study of its enzymatic properties revealed that the enzyme has K_m=0.033 mmol/L,the optimum p H is 7.5,and the optimum temperature is 50℃.The esterase shows poor tolerance to acetonitrile,but can tolerate low concentrations of methanol and ethanol.Fe³⁺,Fe²⁺and Cu²⁺have a certain inhibitory effect on the enzyme activity,and Cu²⁺has the most obvious inhibitory effect.After adding 0.1%BSA as a stabilizer,its activity is still relatively stable after being placed for 240 min.2.Establishment of detection methods for organophosphorus pesticide residues based on microbial esterase and fluorescence analysis.By analyzing the results of the hydrolysis of the four substrates by microorganisms,indole acetate was selected as the most suitable substrate for hydrolysis.The optimal conditions for determining the detection method are:the enzyme concentration is 0.067 U/m L,the substrate concentration is 0.5 mmol/L,and the inhibition reaction time is 15 min.Under the optimal conditions,the inhibition rate of 5 kinds of organophosphorus pesticides including dichlorvos was determined,and a standard curve was established.The results showed that the minimum detection limits of dichlorvos,trichlorfon,methamidophos,profenofos,and paraoxon were 0.0032 mg/L,0.0096 mg/L,0.0990 mg/L,0.0990 mg/L,and 0.0074 mg/L respectively,the R² of the standard curve equation of each pesticide is greater than 0.99,which meets the national standard limit detection requirements.Pears and Chinese cabbage were selected as representative samples to verify the feasibility of this method in the detection of organophosphorus pesticide residues in fruit and vegetable samples.The average recovery rate was 80%-105%,and the RSD was less than 5%.This shows that the method can be applied to the detection of organophosphorus pesticide residues in actual samples.3.The stability of freeze-dried microbial esterase and its application in detecting dichlorvos residues.By studying the concentration of microbial esterase lyophilized solution and the method of lyophilization,the concentration of lyophilized solution was 15%,and liquid nitrogen quick freezing was used as the freezing treatment method.With this scheme,the enzyme activity of the freeze-dried microbial esterase remains above 80%within 30 days.Further optimize the conditions of enzyme concentration,substrate concentration and inhibition reaction time,establish a standard curve for the detection of dichlorvos,and calculate the half inhibitory concentration and detection limit.The fitted equations are R2=0.9903,IC₅₀≈0.026 mg/L and LOD≈0.006 mg/L.Compared with the IC₅₀ proposed in the national standard"GB5009.199-2003 Rapid Detection of Organophosphorus and Carbamate Pesticide Residues in Vegetables",this method has higher sensitivity.On this basis,a rapid test product for dichlorvos residue based on microbial esterase was developed.After the actual samples of pears are tested,the average recovery rate of the product is 80%-90%,which shows that it can meet the rapid detection of dichlorvos pesticide residues in actual samples.