Preparation And Application Of Magnetic Nanocrystalline Enzyme Preparation

With the rapid development of agricultural production and pesticide economy,organophosphorus pesticides have been widely used as an indispensable means of production in modern agriculture.A lot of organophosphorus pesticides are used but the utilization rate is low.A large number of pesticide residues eventually enter the water body,which seriously damages the water environment,threatens the safety of drinking water and endangers human health.In this study,organophosphorus hydrolase(OPH)was used as the model enzyme,and chlorpyrifos(CP)was used as the representative organophosphorus pesticide pollutant.In an aqueous solution at room temperature,coprecipitate method was used to embed OPH into the porous zeolite imidazole framework(ZIF-8).According to this method,magnetic nanocrystalline enzyme composites OPH@mZIF-8 were prepared.The main contents of the characterization of OPH@mZIF-8 composite materials include:SEM,X-ray diffraction and nitrogen adsorption were used to analyze the structure of OPH@mZIF-8 enzyme preparation.The localization of OPH@mZIF-8 enzyme was analyzed by FT-IR.The quantitative analysis of OPH@mZIF-8 enzyme was carried out by TGA,SDS polyacrylamide gel electrophoresis and Bradford method.The heat stability,acid-base tolerance and ion tolerance of OPH@mZIF-8 enzyme preparation were determined.The degradation effect of OPH@mZIF-8 on chlorpyrifos in different water samples was determined.This study provides a green,environmental,economical and efficient immobilization scheme for the degradation of pesticide residues in water environment.Specific research results are as follows:1.Prepare OPH hydrolase and OPH@mZIF-8 enzyme preparation.The oph gene cloned in this study is 100%similar to the methylparathion hydrolase gene(AAP06948.1)of Pseudomonas sp.WBC-3 reported in this study.The expression strain was successfully constructed,and about 80 mg OPH with a size of38 k Da could be extracted from 4 L culture.OPH can hydrolyze parathion and other organophosphorus compounds containing P-O bonds,and degrade pesticides such as methyl parathion,chlorpyrifos and parathion.OPH@mZIF-8 enzyme preparation was prepared by coprecipitation method at room temperature,and about 180 mg enzyme preparation could be prepared by 20 mg OPH.Due to the addition of Fe₃O₄ magnetic nanoparticles(MNP),the enzyme preparation powder appears brown.2.Characterization analysis of OPH@mZIF-8 enzyme preparation.By scanning electron microscopy(SEM),the size of OPH@mZIF-8 enzyme preparation was about 200-300 nm,presenting a clear cube structure.The addition of OPH and MNP has no significant effect on the structure and crystallinity of ZIF by X-ray diffraction and nitrogen adsorption.The microporous structure of OPH@mZIF-8enzyme preparation was verified by nitrogen adsorption method,indicating that OPH enzyme and MNP were successfully embedded in the crystal.Fourier transform infrared spectroscopy(FT-IR)was used to analyze the location of OPH@mZIF-8,which proved that OPH@mZIF-8 successfully encapsulated the enzyme in the crystal structure of ZIF-8.The enzyme preparation OPH@mZIF-8 was quantitatively analyzed by thermogravimetric analysis,Bradford method and SDS polyacrylamide gel electrophoresis.According to thermogravimetric data,OPH enzyme mass ratio was 7.09%.According to Bradford method and electrophoresis calculation,the loading capacity of OPH@mZIF-8 enzyme preparation is 11.06%,and the loading efficiency is 80%.Magnetic detection results show that the saturation magnetization(Ms)value of OPH@mZIF-8 is 26.3 emu·g^-1.3.Analysis of enzyme properties of OPH@mZIF-8 enzyme preparation.Under the same enzyme concentration,the relative activity of OPH@mZIF-8was 90%.Michaelis-menten kinetic parameters showed that the K_m value of OPH was17.8?M and that of OPH@mZIF-8 was 26.6?M.The coating of ZIF-8 resulted in a decrease in the affinity of the enzyme to the substrate and the maximum reaction rate of the enzyme.OPH@mZIF-8 enzyme preparation has good thermal stability:after being treated at 70?for 1 h,OPH@mZIF-8 enzyme preparation retains 85%of its initial activity,while free enzyme activity is only 13%of its initial activity.In alkaline condition(p H=10),OPH@mZIF-8 enzyme preparation showed good stability.In terms of ion tolerance,OPH@mZIF-8 enzyme preparation showed better stability than free enzyme.It was noted that the free enzyme activity decreased by 66%in the presence of Fe³⁺,but the enzyme preparation OPH@mZIF-8 still maintained a high activity.The enzyme preparation OPH@mZIF-8 retained 53.9%relative activity after10 cycles.4.Degradation analysis of OPH@mZIF-8 enzyme preparation to chlorpyrifos poisoning in water.In different water samples,OPH@mZIF-8 enzyme preparation showed better activity and stability than free enzyme.The activity and stability of OPH@mZIF-8enzyme preparation in surface water are basically not affected by water environment.In the sewage free enzyme affected by the sewage environment can only achieve 25%degradation efficiency,OPH@mZIF-8 enzyme preparation is still up to 70%.