Immobilization Of Methyl Parathion Hydrolase And Its Application In Detection And Degradation

Methyl parathion（MP）is a kind of organophosphorus pesticide which was widely used in farm crops.MP could inhibit the activity of cholinesterase in the nervous system of pests and possess wide range of insecticidal spectrum.MP was often processed into emulsifiable oil or powder which could control many pests in rice,cotton,fruit trees,tea,vegetables,and other crops.However,the strong neurotoxicity of methyl parathion is also toxic to human body which could lead to memory impairment,headache,speech difficulties and other symptoms.At present,degrading methyl parathion by methyl parathion hydrolase is one of the most effective ways.However,the free methyl parathion hydrolase has the disadvantages of poor stability,difficult to achieve the complete eliminating of MP and could not be reused.The immobilized methyl parathion enzyme has been widely concerned since it can not only solve the disadvantages of the free enzyme,but also reduce the cost of industrial application.In this study,the methyl parathion hydrolase Azau-MPH preserved by our laboratory was selected as the immobilization target,three kind of immobilization carriers were chosen to achieve the immobilization.Also,the possibility of immobilization Azau-MPH in practical application were explored.The specific research contents are as follows:（1）The metal-organic framework MIL-88A was prepared by hydrothermal method and characterized by SEM,XRD and FT-IR.Subsequently,the prepared MIL-88A was used to selectively immobilize the target protein from Azau-MPH crude enzyme solution and got the Azau-MPH@MIL-88A.The results showed that the specific enzyme activity of Azau-MPH@MIL-88A towards MP was 14.9 U/mg,and higher than that of free Azau-MPH.Moreover,the storage stability of Azau-MPH@MIL-88A was significantly improved.In specific,the Azau-MPH@MIL-88A still has about 80%activity retention after 60 days storage at 4℃.Finally,in the present of 7-amino-4-methylcoumarin,the Azau-MPH@MIL-88A was selected as a catalyst to prepare a fluorescent sensor.The detection limit of the fluorescent sensor is 0.038μM.Linear detection range is 0.038-189μM.（2）Another MOFs of ZIF-8 and Azau-MPH@ZIF-8 were prepared by coprecipitation method and the SEM,FT-IR and CLSM were used to determine the successful load of Azau-MPH within ZIF-8.For immobilized Azau-MPH@ZIF-8,the catalysis activity was 1.2 U/mg under the most comfortable condition.But the thermal stability,resistance to organic reagents and resistance to SDS were significantly improved.This showed that Azau-MPH@ZIF-8 could realize the efficient degradation of MP by Azau-MPH under hush environment.In addition,the Azau-MPH@ZIF-8 were used for cyclic degradation in actual cherry samples.After 6 cycles,about 60%of the initial enzyme activity was retained.（3）In order to improve the recovery convenience of immobilized Azau-MPH,ZIF-8@Fe₃O₄ nanoparticles were introduced and successfully prepared ZIF-8@Fe₃O₄@Azau-MPH.The successful preparation of ZIF-8@Fe₃O₄@Azau-MPH was verified by TEM,FT-IR and XRD.After that,The properties of ZIF-8@Fe₃O₄@Azau-MPH and free Azau-MPH were determined.The results showed that the specific enzyme activity of ZIF-8@Fe₃O₄@Azau-MPH towards MP was 3.07 U/mg,and it has excellent storage stability.Besides,the ZIF-8@Fe₃O₄@Azau-MPH could retain about 90%of its initial enzyme activity after six times of recycling use,which indicated that it has certain application prospects in practical industrial applications.