Phosphotriesterase-crosslinked magnetic hybrid nanoflower as well as preparation method and application thereof

Abstract

The invention discloses a phosphotriesterase-cross-linked magnetic hybrid nanoflower as well as a preparation method and application of the phosphotriesterase-cross-linked magnetic hybrid nanoflower. The preparation method of the phosphotriesterase-crosslinked magnetic hybrid nanoflower comprises the following steps: adding phosphotriesterase into a phosphate buffer solution, then adding a CoCl2 solution and synthesized magnetic composite microspheres Fe3O4-PDA (at) PEI, placing the mixture at a certain temperature for incubation, and then adding glutaraldehyde for reaction to obtain the phosphotriesterase-crosslinked magnetic hybrid nanoflower. The phosphotriesterase-crosslinked magnetic hybrid nanoflower prepared by the invention is relatively high in activity, has excellent degradation performance on parathion-methyl, fenitrothion, chlorpyrifos and ethion, is convenient to recycle, has good acid-base stability, temperature stability and storage stability, and still has relatively high activity after being repeatedly used. The phosphotriesterase-crosslinked magnetic hybrid nanoflower prepared by the preparation method disclosed by the invention is high in catalytic activity and good in mechanical strength, and has efficient recoverability.

Description

Technical Field

[0001] The invention belongs to the technical field of immobilized enzymes, and specifically relates to a preparation method and application of phosphotriesterase-cross-linked magnetic hybrid nanoflowers. Background Art

[0002] In recent decades, organophosphorus pesticides have been widely used in agriculture, forestry and animal husbandry due to their excellent insecticidal efficacy. The excessive and unreasonable use of organophosphorus compounds has caused great harm to the environment and the food chain. The development of efficient and environmentally friendly purification solutions is a long-term challenge. Methods for treating organophosphorus pollutants include alkaline hydrolysis, incineration and landfill disposal. However, these methods are inefficient and produce toxic byproducts. The bioenzymatic method is safe, efficient, green and environmentally friendly, and has mild operating conditions, showing potential application prospects. At present, enzymatic degradation and removal of organophosphorus compounds in the environment has attracted more and more attention. However, due to the high production cost, low stability and poor reusability of free enzymes, practical applications are limited. Immobilized enzymes can improve the stability and reusability of enzymes. At present, a variety of support materials have been used to immobilize organophosphorus hydrolases, such as nylon, cellulose, carbon nanotubes, metal organic frameworks, and graphene oxide. However, although these immobilized enzymes improve the stability of the enzymes, they reduce the catalytic activity of the enzymes. In 2012, Ge Jun's team accidentally discovered that Cu 2+ As an inorganic component, the enzyme is an organic component, which forms a flower-like nanocomposite material through self-assembly. When an enzyme is used as an organic component of a hybrid nanoflower, it exhibits higher enzyme activity and stability than free enzymes. Since then, a variety of enzymes including laccase, lipase, horseradish peroxidase, glucose oxidase, etc. have been reported to be used to prepare hybrid nanoflowers and show good catalytic ability. The large specific surface area, reduced mass transfer limitations and synergistic effect of nanozyme molecules of hybrid nanoflowers are considered to be the reasons for their good performance. At present, various enzymes, as well as DNA, proteins and plant extracts are used as organic components to synthesize hybrid nanoflower catalysts. However, the synthesis of traditional hybrid nanoflowers has the disadvantages of low mechanical strength, long preparation time, low number of effective reuses, and cumbersome separation process, which need to be solved in practical applications. In general, the poor reusability of enzyme-inorganic hybrid nanoflowers is related to their weak mechanical strength, and the petal structure of hybrid nanoflowers is easily broken during use or centrifugal recovery.

[0003] Therefore, it is urgent to develop an effective synthesis method to improve the robustness of the hybrid nanoflower system while maintaining high catalytic activity and efficient recycling. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a preparation method and application of phosphotriesterase-cross-linked magnetic hybrid nanoflowers. The phosphotriesterase-cross-linked magnetic hybrid nanoflowers prepared by the present invention have high catalytic activity, good mechanical strength, and high efficiency in recycling.

[0005] The technical solution of the present invention is as follows:

[0006] A method for preparing phosphotriesterase-cross-linked magnetic hybrid nanoflowers, the preparation method comprising the following steps:

[0007] (1) Preparation of magnetic composite microspheres: Fe3O4 was dispersed in Tris-HCl buffer to obtain a magnetic powder suspension, dopamine hydrochloride and polyethyleneimine were added, and after ultrasonic and oscillation incubation, magnetic composite microspheres Fe3O4-PDA@PEI were obtained;

[0008] (2) Preparation of phosphotriesterase-cross-linked magnetic hybrid nanoflowers: Add phosphotriesterase enzyme solution into phosphate buffer solution, then add CoCl2 solution and magnetic composite microspheres Fe3O4-PDA@PEI for incubation to complete self-assembly, and then add glutaraldehyde solution for reaction to obtain phosphotriesterase-cross-linked magnetic hybrid nanoflowers.

[0009] Furthermore, in step (1), the concentration of the Tris-HCl buffer is 10-50 mmol / L, and the pH is 7.0-10.0; the amount of the Tris-HCl buffer is to ensure that the concentration of Fe3O4 is 40-60 g / L.

[0010] Furthermore, in step (1), the mass ratio of dopamine hydrochloride to Fe3O4 is 1:5; the mass ratio of polyethyleneimine to Fe3O4 is 1:5.

[0011] Furthermore, in step (1), the power of the ultrasound is 50-200 W, and the time is 3-9 min; the temperature of the oscillating incubation is 20-35° C., the rotation speed is 150-250 rpm, and the time is 4-8 h.

[0012] Furthermore, in step (2), the amino acid sequence of the phosphotriesterase is as shown in SEQ ID NO.1; the specific enzyme activity of the phosphotriesterase is 6.45 U / mg; the concentration of the phosphate buffer solution is 10-40 mmol / L, and the pH is 7.0-10.0; the concentration of the phosphotriesterase in the phosphate buffer is 0.1-0.15 mg / mL; the concentration of the CoCl2 in the phosphate buffer is 2-5 mmol / L; and the concentration of the magnetic composite microspheres Fe3O4-PDA@PEI in the phosphate buffer is 0.5-2 g / L.

[0013] Furthermore, in step (2), the temperature of the ultrasonic incubation is 35-40° C., and the time is 2-12 h.

[0014] Furthermore, in step (2), the concentration of glutaraldehyde in phosphate buffer solution is 0.1-0.15%; and the reaction time is 0.5-2.5h.

[0015] A phosphotriesterase-cross-linked magnetic hybrid nanoflower prepared by the preparation method.

[0016] An application of the phosphotriesterase-cross-linked magnetic hybrid nanoflower, wherein the phosphotriesterase-cross-linked magnetic hybrid nanoflower is used for degrading organophosphorus pesticides.

[0017] Furthermore, the organophosphorus pesticides include methyl parathion, fenitrothion, chlorpyrifos, and ethion.

[0018] The beneficial technical effects of the present invention are:

[0019] The present invention provides a method for preparing phosphotriesterase-crosslinked magnetic hybrid nanoflowers, wherein magnetic nanoparticles modified with dopamine and polyethyleneimine are combined with phosphotriesterase hybrid nanoflowers, and then crosslinked by glutaraldehyde. The obtained phosphotriesterase-crosslinked magnetic hybrid nanoflowers can be quickly separated from the reaction solution, better reused, and have better pH stability and temperature stability. The high specific surface area of ​​the phosphotriesterase-crosslinked magnetic hybrid nanoflowers and the activation effect of cobalt ions significantly improve the enzyme activity.

[0020] The present invention further optimizes the preparation conditions of phosphotriesterase-cross-linked magnetic hybrid nanoflowers, and obtains the optimal preparation conditions of phosphotriesterase-cross-linked magnetic hybrid nanoflowers as follows: phosphotriesterase concentration is 0.1 mg / mL, Co 2+ When the concentration is 2mmol / L, the mass of the magnetic composite microspheres is 0.5mg, the incubation temperature is 37℃, the glutaraldehyde concentration is 0.1% and the immobilization time is 6h, the enzyme activity of the phosphotriesterase-cross-linked magnetic hybrid nanoflowers prepared under the optimal conditions can reach 298% of the free enzyme.

[0021] The stability of the phosphotriesterase-crosslinked magnetic hybrid nanoflowers prepared by the present invention in temperature, pH and organic solvent is significantly higher than that of the free enzyme. After storage at room temperature for 30 days, the activity of the free enzyme basically disappears, while the relative activity of the phosphotriesterase-crosslinked magnetic hybrid nanoflowers is 84.8%. In addition, the phosphotriesterase-crosslinked magnetic hybrid nanoflowers can still maintain a relative activity of 90.32% after being reused five times.

[0022] The present invention applies phosphotriesterase-cross-linked magnetic hybrid nanoflowers to the degradation experiment of methyl parathion, cypermethrin, chlorpyrifos and ethion. When the concentration of organophosphorus pesticide is 100 mg / L, the enzyme concentration is 0.02 mg / ml, the temperature is 40°C, and the reaction time is 1 hour. The degradation rates of the four organophosphorus pesticides by phosphotriesterase-cross-linked magnetic hybrid nanoflowers are 99%, 96%, 92% and 85%, respectively, which are significantly higher than free enzymes. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Graph showing the effect of preparation conditions on the activity of phosphotriesterase-cross-linked magnetic hybrid nanoparticles.

[0024] Figure: A, the effect of the amount of magnetic composite microspheres Fe3O4-PDA@PEI on the activity of phosphotriesterase-cross-linked magnetic hybrid nanoflowers; B, Co 2+ A, is the effect of phosphotriesterase concentration on the activity of phosphotriesterase-cross-linked magnetic hybrid nanoflowers; C, is the effect of phosphotriesterase concentration on the activity of phosphotriesterase-cross-linked magnetic hybrid nanoflowers; D, is the effect of self-assembly temperature on the activity of phosphotriesterase-cross-linked magnetic hybrid nanoflowers; E, is the effect of glutaraldehyde concentration on the activity of phosphotriesterase-cross-linked magnetic hybrid nanoflowers; F, is the effect of self-assembly incubation time on the activity of phosphotriesterase-cross-linked magnetic hybrid nanoflowers.

[0025] Figure 2 Transmission electron micrographs of Fe3O4, Fe3O4-PDA@PEI and phosphotriesterase-cross-linked magnetic hybrid nanoflowers prepared in Example 2 of the present invention.

[0026] In the figure: A, is a transmission electron microscopy image of Fe3O4; B, is a transmission electron microscopy image of Fe3O4-PDA@PE; C, is a transmission electron microscopy image of phosphotriesterase-cross-linked magnetic hybrid nanoflowers.

[0027] Figure 3 Scanning electron microscope images of Fe3O4, Fe3O4-PDA@PEI and phosphotriesterase-cross-linked magnetic hybrid nanoflowers prepared in Example 2 of the present invention.

[0028] In the figure: A, is a scanning electron micrograph of Fe3O4; B, is a scanning electron micrograph of Fe3O4-PDA@PE; C, is a scanning electron micrograph of phosphotriesterase-cross-linked magnetic hybrid nanoflowers.

[0029] Figure 4 The temperature tolerance and acid-base tolerance graphs of the free enzyme prepared in Example 1 and the phosphotriesterase-cross-linked magnetic hybrid nanoflowers prepared in Example 2 of the present invention.

[0030] In the figure: A is the temperature tolerance diagram; B is the acid-base tolerance diagram.

[0031] Figure 5 The stability diagram of the free enzyme prepared in Example 1 and the phosphotriesterase-cross-linked magnetic hybrid nanoflower prepared in Example 2 of the present invention.

[0032] In the figure: A is the high temperature stability diagram; B is the organic solvent stability diagram; C is the storage stability diagram; D is the repeated use performance diagram.

[0033] Figure 6 This is a graph showing the degradation performance of organophosphorus pesticides by the free enzyme prepared in Example 1 and the phosphotriesterase-cross-linked magnetic hybrid nanoflowers prepared in Example 2 of the present invention.

[0034] In the figure: A, is the degradation performance of methyl parathion; B, is the degradation performance of fenitrothion; C, is the degradation performance of chlorpyrifos; D, is the degradation performance of ethion. DETAILED DESCRIPTION

[0035] The present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0036] The phosphotriesterase described in the present invention is derived from the strain Brevundimonas diminuta, and the amino acid sequence is shown in SEQ ID NO.1.

[0037] SEQ ID NO.1:

[0038] DRINTVRGPITISEAAGFTLTHEHICGSSAGFLRAWPEFFGSRKALAEKAVRGLRRARAAGVRTIVDVSTFDIGRDVSLLAEVSRAADVHIVAATGLWFDPPLSMRLRSVEELTQFFLREIQYGIEDTGIRAGIIKVATTGKATPFQELVLKAAARASLATGVPVT THTAASQRDGEQQAAIFESEGLSPSRVCIGHSDDTDDLSYLTALAARGYLIGLDHIPYSAIGLEDNASASALLGIRSWQTRALLIKALIDQGYMKQILVSNDWTFGFSSYVTNIMDVMDRVNPDGMAFIPLRVIPFLREKGVPQETLAGITVTNPARFLSPTLRA

[0039] Example 1

[0040] The preparation of phosphotriesterase comprises the following steps:

[0041] (1) Amplification of target gene

[0042] According to the amino acid sequence of phosphotriesterase SEQ ID NO.1, the target gene was synthesized according to the codon preference of Escherichia coli, and then the target gene was connected to the pMD19-T Simple Vector vector, and PCR amplification was performed with the primers involved. The PCR product was subjected to agarose gel electrophoresis. After verifying that the band size was correct, the target band was cut and placed in a 1.5 mL centrifuge tube, recovered using a product purification kit, and the nucleic acid concentration was determined using Nanodrop.

[0043] (2) Construction of recombinant plasmid

[0044] The pTIG-trx expression plasmid was cut with EcoRⅠ and Hind III to make it linearized, and the target gene obtained by PCR was connected with the linearized pTIG-trx expression plasmid by DNA ligase to obtain a recombinant plasmid.

[0045] (3) Introduce the recombinant plasmid into competent cells and induce expression

[0046] The recombinant plasmid was transformed into competent cells of E.coli BL21 (DE3), and single colonies were verified by colony PCR. The single colonies that successfully produced phosphotriesterase were picked and inoculated into TB liquid culture medium containing 100 mg / L ampicillin, and cultured overnight at 37°C and 200 rpm to obtain seed solution. Subsequently, 1 mL of seed solution was added to 50 mL of TB liquid culture medium containing 100 mg / L ampicillin, and cultured at 37°C and 200 rpm for 2 h; then IPTG (final concentration 0.1 mmol / L) was added, and expression was induced at 16°C and 200 rpm for 24 h. The bacteria were collected by centrifugation, washed twice with PBS, and the supernatant was collected by centrifugation after ultrasonic disruption. The protein was purified by His Trap FF affinity chromatography column, and the target protein eluate was dialyzed with a PBS solution containing 20 mmol / L and pH 8.0 to remove high-concentration imidazole. The pure enzyme solution of phosphotriesterase was used for subsequent phosphotriesterase-cross-linked magnetic hybrid nanoflower experiments. The specific enzyme activity of the purified phosphotriesterase was measured to be 6.45 U / mg.

[0047] Example 2

[0048] A method for preparing phosphotriesterase-cross-linked magnetic hybrid nanoflowers comprises the following steps:

[0049] (1) Preparation of magnetic composite microspheres: 200 mg of Fe3O4 was dispersed in 4 mL of Tris-HCl buffer (25 mmol / L, pH 8.5) to obtain a magnetic powder suspension, and then 40 mg of dopamine hydrochloride and 40 mg of polyethyleneimine were added. The suspension was ultrasonically treated at 100 W for 5 min, and then incubated at 25 ° C and 200 rpm for 6 h. The incubation product was separated from the solution by a magnet and washed with ultrapure water for 3 times until the washing solution was colorless to obtain magnetic composite microspheres Fe3O4-PDA@PEI;

[0050] (2) Preparation of phosphotriesterase-cross-linked magnetic hybrid nanoflowers: 0.4 mL of 0.25 mg / mL phosphotriesterase enzyme solution was added to 1 mL of phosphate buffer solution (20 mmol / L, pH 8.0), and then 16.7 μL of 120 mmol / L CoCl2 solution (Co 2+ The concentration of glutaraldehyde in phosphate buffer solution was 2 mmol / L) and 0.5 g of magnetic composite microspheres Fe3O4-PDA@PEI were incubated at 37°C for 6 h to complete self-assembly, and then 5 μL of 20% glutaraldehyde solution (the concentration of glutaraldehyde in phosphate buffer solution was 0.1%) was added to react for 1 h to obtain phosphotriesterase-cross-linked magnetic hybrid nanoflowers.

[0051] Embodiment 3-4

[0052] The preparation method of Example 3 is basically the same as that of Example 2, except that the amount of magnetic composite microspheres Fe3O4-PDA@PEI in step (2) of Example 3-4 is adjusted to 1 g and 2 g respectively, and the rest of the contents remain unchanged.

[0053] Embodiment 5-7

[0054] The preparation method of Example 5-7 is basically the same as that of Example 2, except that the amount of the 120 mmol / L CoCl2 solution in step (2) of Example 5-7 is adjusted to 25 μL (Co 2+ The concentration in phosphate buffer solution is 3mmol / L), 33.3μL (Co 2+ The concentration in phosphate buffer solution is 4mmol / L), 41.7μL (Co 2+ The concentration in phosphate buffer solution is 5mmol / L), and the rest of the contents remain unchanged.

[0055] Example 8

[0056] The preparation method of Example 8 is basically the same as that of Example 2, except that the amount of 0.25 mg / mL phosphotriesterase solution in step (2) of this example is adjusted to 0.6 mL, and the rest of the contents remain unchanged.

[0057] Example 9

[0058] The preparation method of Example 9 is basically the same as that of Example 2, except that the amount of 20% glutaraldehyde solution in step (2) of this example is adjusted to 7.5 μL, and the rest of the contents remain unchanged.

[0059] Comparative Examples 1-3

[0060] The preparation method of Comparative Examples 1-3 is basically the same as that of Example 2, except that the amount of magnetic composite microspheres Fe3O4-PDA@PEI in step (2) of Comparative Examples 1-3 is adjusted to 0.2g, 3g, and 4g respectively, and the rest of the contents remain unchanged.

[0061] Comparative Examples 4-5

[0062] The preparation method of Comparative Example 4-5 is basically the same as that of Example 2, except that the amount of the 120 mmol / L CoCl2 solution in step (2) of Comparative Example 4-5 is adjusted to 0 μL (Co 2+ The concentration in phosphate buffer solution is 0mmol / L), 8.3μL (Co 2+ The concentration in phosphate buffer solution is 1mmol / L), and the rest of the contents remain unchanged.

[0063] Comparative Examples 6-8

[0064] The preparation method of Comparative Example 6-8 is basically the same as that of Example 2, except that the amount of 0.25 mg / mL phosphotriesterase solution in step (2) of Comparative Example 6-8 is adjusted to 0.2 mL, 0.8 mL, and 1.0 mL, respectively, and the rest of the contents remain unchanged.

[0065] Comparative Examples 9-10

[0066] The preparation method of Comparative Examples 9-10 is basically the same as that of Example 2, except that the temperature of the self-contained device in step (2) of Comparative Examples 6-8 is adjusted to 4° C. and 25° C. respectively, and the rest of the contents remain unchanged.

[0067] Comparative Examples 11-14

[0068] The preparation method of Comparative Examples 11-14 is basically the same as that of Example 2, except that the amount of 20% glutaraldehyde solution in step (2) of Comparative Examples 11-14 is adjusted to 2.5 μL, 10 μL, 12.5 μL, and 15 μL, respectively, and the rest of the contents remain unchanged.

[0069] Examples 10-12

[0070] The preparation methods of Examples 10-12 are substantially the same as those of Example 2, except that the incubation time for self-assembly in step (2) of this Example is adjusted to 8 h, 10 h, and 12 h, respectively, and the rest of the contents remain unchanged.

[0071] Comparative Examples 15-16

[0072] The preparation method of Comparative Examples 15-16 is basically the same as that of Example 2, except that the incubation time of self-assembly in step (2) of this example is adjusted to 2 h and 4 h respectively, and the rest of the contents remain unchanged.

[0073] Test Case

[0074] (1) Effect of the dosage of magnetic composite microspheres Fe3O4-PDA@PEI on the activity of phosphotriesterase-cross-linked magnetic hybrid nanoflowers

[0075] The phosphotriesterase-cross-linked magnetic hybrid nanoflowers prepared in Examples 2-4 of the present invention and Comparative Examples 1-3 were taken, and methyl parathion was used as the reaction substrate to measure the relative enzyme activity (the enzyme activity of free phosphotriesterase was defined as 100%). The results are as follows: Figure 1 As shown in A. Figure 1 The results in Figure A show that the amount of magnetic composite microspheres Fe3O4-PDA@PEI affects the relative activity of phosphotriesterase-cross-linked magnetic hybrid nanoflowers. When the amount of Fe3O4-PDA@PEI added to the enzyme solution is 0.5-2 g / L, the relative enzyme activity of the prepared phosphotriesterase-cross-linked magnetic hybrid nanoflowers is 291.35%-308.32%; and when the amount of Fe3O4-PDA@PEI added to the enzyme solution is 3-4 g / L, the relative enzyme activity decreases significantly, down to 239.17%-25. 5.99%; in addition, when the addition amount of Fe3O4-PDA@PEI relative to the enzyme solution is 0.2g / L, the relative enzyme activity of the prepared phosphotriesterase-cross-linked magnetic hybrid nanoflowers is higher, but considering that too low an addition amount of magnetic composite microspheres will reduce the magnetic separation ability of phosphotriesterase-cross-linked magnetic hybrid nanoflowers, therefore, considering the effect of magnetic composite microspheres on magnetic separation ability and relative activity, the addition amount of magnetic composite microspheres Fe3O4-PDA@PEI in the present invention is preferably 0.5-2g / L, among which the addition amount of 0.5g / L is optimal.

[0076] (2)Co 2+ Effect of concentration on the activity of phosphotriesterase-cross-linked magnetic hybrid nanoflowers

[0077] The phosphotriesterase-cross-linked magnetic hybrid nanoflowers prepared in Examples 2, 5-7 and Comparative Examples 4-5 of the present invention were taken, and methyl parathion was used as the reaction substrate to measure the relative enzyme activity (the enzyme activity of free phosphotriesterase was defined as 100%). The results are as follows: Figure 1 As shown in B. Figure 1 As shown in Figure B, the activity of phosphotriesterase-cross-linked magnetic hybrid nanoflowers increases with the increase of Co 2+ The concentration increases first and then decreases. 2+ The relative activity is better when the concentration in phosphate buffer solution is 2-5mmol / L, among which Co 2+ The relative activity is optimal when the concentration is 2 mmol / L in phosphate buffer solution.

[0078] (3) Effect of phosphotriesterase concentration on the activity of phosphotriesterase-cross-linked magnetic hybrid nanoflowers

[0079] The phosphotriesterase-cross-linked magnetic hybrid nanoflowers prepared in Examples 2 and 8 of the present invention and Comparative Examples 6-8 were taken, and methyl parathion was used as the reaction substrate to measure the relative enzyme activity (the enzyme activity of free phosphotriesterase was defined as 100%). The results are as follows: Figure 1 C. From Figure 1 From the results in Figure C, it can be seen that the activity of phosphotriesterase-cross-linked magnetic hybrid nanoflowers shows a trend of first increasing and then decreasing with the increase of phosphotriesterase concentration. When the concentration of phosphotriesterase in phosphate buffer solution is 0.1-0.15 mg / mL, the relative activity is better. Among them, the relative activity is optimal when the concentration of phosphotriesterase in phosphate buffer solution is 0.1 mg / mL.

[0080] (4) Effect of self-assembly temperature on the activity of phosphotriesterase-cross-linked magnetic hybrid nanoflowers

[0081] The phosphotriesterase-cross-linked magnetic hybrid nanoflowers prepared in Example 2 of the present invention and Comparative Examples 9-10 were taken, and methyl parathion was used as the reaction substrate to measure the relative enzyme activity (the enzyme activity of free phosphotriesterase was defined as 100%). The results are as follows: Figure 1 D. From Figure 1 As can be seen from the results in Figure D, the activity of the prepared phosphotriesterase-cross-linked magnetic hybrid nanoflowers is low when self-assembly is carried out at low temperature and room temperature, while the relative activity is significantly improved when self-assembly is carried out at 37°C.

[0082] (5) Effect of glutaraldehyde concentration on the activity of phosphotriesterase-cross-linked magnetic hybrid nanoflowers

[0083] The phosphotriesterase-cross-linked magnetic hybrid nanoflowers prepared in Examples 2 and 9 of the present invention and Comparative Examples 11-14 were taken, and methyl parathion was used as the reaction substrate to measure the relative enzyme activity (the enzyme activity of free phosphotriesterase was defined as 100%). The results are as follows: Figure 1 E. From Figure 1From the results in Figure E, it can be seen that the activity of phosphotriesterase-cross-linked magnetic hybrid nanoflowers shows a trend of first increasing and then decreasing with the increase of the concentration of glutaraldehyde in the phosphate buffer solution. When the concentration of glutaraldehyde in the phosphate buffer solution is 0.05%, the activity is low, and when the concentration of glutaraldehyde in the phosphate buffer solution is increased to 0.2-0.3%, the enzyme activity also gradually decreases; when the concentration of glutaraldehyde in the phosphate buffer solution is 0.1-0.15%, the relative activity is better, among which the relative activity is optimal when the concentration of glutaraldehyde in the phosphate buffer solution is 0.1%.

[0084] (6) Effect of self-assembly incubation time on the activity of phosphotriesterase-cross-linked magnetic hybrid nanoflowers

[0085] The phosphotriesterase-cross-linked magnetic hybrid nanoflowers prepared in Examples 2, 10-12 and Comparative Examples 15-16 of the present invention were taken, and methyl parathion was used as the reaction substrate to measure the relative enzyme activity (the enzyme activity of free phosphotriesterase was defined as 100%). The results are as follows: Figure 1 F. From Figure 1 As can be seen from the results in Figure F, the activity of phosphotriesterase-cross-linked magnetic hybrid nanoflowers gradually increases with the increase of self-assembly incubation time. When the incubation time is 6-12h, the enzyme activity reaches stability, and the relative activity is lower than 6h.

[0086] (7) Structural characterization of phosphotriesterase-cross-linked magnetic hybrid nanoflowers

[0087] The morphology and structure of Fe3O4, the magnetic composite microspheres Fe3O4-PDA@PEI prepared in step (1) of Example 2 of the present invention, and the phosphotriesterase-cross-linked magnetic hybrid nanoflowers prepared in Example 2 of the present invention were observed by a transmission electron microscope of FEI Talos F200x, USA. The results are as follows: Figure 2 As shown. Figure 2 As can be seen from Figure A, the shape and size of the Fe3O4 magnetic microspheres are uniform, with an average diameter of about 200nm. Figure 2 As can be seen from Figure B, after PDA and PEI co-deposition modification, the Fe3O4-PDA@PEI magnetic nanospheres have an obvious dopamine layer on the outer layer and a clear core-shell structure. Figure 2 As can be seen from Figure C, the phosphotriesterase-cross-linked magnetic hybrid nanoflowers are composed of many lamellar crystal structures, and there are magnetic Fe3O4 particles in the crystals, which indicates the successful preparation of the phosphotriesterase-cross-linked magnetic hybrid nanoflowers.

[0088] The morphology of Fe3O4, the magnetic composite microspheres Fe3O4-PDA@PEI prepared in step (1) of Example 2 of the present invention, and the phosphotriesterase-cross-linked magnetic hybrid nanoflowers prepared in Example 2 of the present invention were observed by a German ZEISS Sigma 300 scanning electron microscope. The results are as follows: Figure 3 As shown. Figure 3 As can be seen from Figures A and B, Fe3O4 and Fe3O4-PDA@PEI have similar morphologies, both consisting of many small nanocrystalline particles. Figure 3 As can be seen from Figure C, the phosphotriesterase-cross-linked magnetic hybrid nanoflower has a complete flower-like structure, Fe3O4-PDA@PEI nanoparticles are attached to the petals of the nanoflower, and the size of the nanoflower is about 12μm.

[0089] (8) Temperature and acid-base tolerance of phosphotriesterase-cross-linked magnetic hybrid nanoflowers

[0090] The phosphotriesterase-crosslinked magnetic hybrid nanoflower (immobilized enzyme) prepared in Example 2 of the present invention and the free enzyme prepared in Example 1 were respectively placed at 30-80°C to react with methyl parathion for 10 min (the pH of the reaction was controlled to be 10), and then the relative enzyme activity was determined (the maximum activity of the free phosphotriesterase was defined as 100%). The results are as follows: Figure 4 As shown in A. Figure 4 As shown in Figure A, the optimum temperatures for free enzyme and phosphotriesterase-cross-linked magnetic hybrid nanoflowers are 40°C and 50°C, respectively. In addition, phosphotriesterase-cross-linked magnetic hybrid nanoflowers have a wider temperature application range than free enzymes.

[0091] The phosphotriesterase-crosslinked magnetic hybrid nanoflower (immobilized enzyme) prepared in Example 2 of the present invention and the free enzyme prepared in Example 1 were respectively placed at a pH value of 6-11 and reacted with methyl parathion for 10 minutes (the reaction temperature was controlled at 40°C), and then the relative enzyme activity was determined (the maximum activity of the free phosphotriesterase was defined as 100%). The results are as follows: Figure 4 As shown in B. Figure 4 As can be seen from the results of Figure B, the optimum pH values ​​of free enzyme and phosphotriesterase-cross-linked magnetic hybrid nanoflowers are both 10. In addition, the enzyme activity of phosphotriesterase-cross-linked magnetic hybrid nanoflowers at each pH value is much higher than that of free enzyme, indicating that the phosphotriesterase-cross-linked magnetic hybrid nanoflowers prepared by the present invention improve the temperature tolerance and acid-base tolerance of phosphotriesterase.

[0092] (9) Stability of phosphotriesterase-cross-linked magnetic hybrid nanoflowers

[0093] The phosphotriesterase-crosslinked magnetic hybrid nanoflower (immobilized enzyme) prepared in Example 2 of the present invention and the free enzyme prepared in Example 1 were placed in a phosphate buffer solution (20 mmol / L, pH 8) and incubated at 60°C for 4 h. During this period, equal amounts of protein free enzyme and phosphotriesterase-crosslinked magnetic hybrid nanoflower were taken every 1 h to react with the substrate at 40°C for 10 min to determine the enzyme activity. The initial activity was defined as 100%. The results are as follows Figure 5 As shown in A. Figure 5 As can be seen from Figure A, the free enzyme activity basically disappeared after 4 hours, while the cross-linked magnetic hybrid nanoflowers can still maintain 71.23% of the initial activity.

[0094] The phosphotriesterase-crosslinked magnetic hybrid nanoflower (immobilized enzyme) prepared in Example 2 of the present invention and the free enzyme prepared in Example 1 were placed in methanol, ethanol, ethyl acetate, and dimethyl sulfoxide (all organic solvents were 50% in concentration, v / v), respectively, and incubated at 40°C for 4 hours. Then, equal amounts of protein free enzyme and phosphotriesterase-crosslinked magnetic hybrid nanoflower were reacted with methyl parathion at 40°C for 10 minutes to determine the enzyme activity, and the initial activity was defined as 100%. The results are as follows Figure 5 As shown in B. Figure 5 As can be seen from Figure B, the free enzyme maintained 59.75% of its initial activity after incubation in 50% methanol for 4 hours, while the phosphotriesterase-cross-linked magnetic hybrid nanoflowers could still maintain up to 93.67% of their initial activity. In addition, the relative activity of the phosphotriesterase-cross-linked magnetic hybrid nanoflowers after incubation in ethanol, ethyl acetate and dimethyl sulfoxide for 4 hours was much higher than that of the free enzyme.

[0095] The phosphotriesterase-crosslinked magnetic hybrid nanoflowers (immobilized enzyme) prepared in Example 2 of the present invention and the free enzyme prepared in Example 1 were stored at room temperature for 30 days. During this period, equal amounts of protein free enzyme and phosphotriesterase-crosslinked magnetic hybrid nanoflowers were taken every 6 days to react with methyl parathion at 40°C for 10 minutes to determine the enzyme activity. The initial activity was defined as 100%. The results are as follows Figure 5 C. From Figure 5 As can be seen from Figure C, the activity of the free enzyme was only 5.51% after 30 days of storage, while the phosphotriesterase-cross-linked magnetic hybrid nanoflowers retained 84.8% of the initial activity.

[0096] The phosphotriesterase-cross-linked magnetic hybrid nanoflower prepared in Example 2 of the present invention was used to test its reusability. After each catalytic reaction of methyl parathion, it was recovered by a magnet for the next catalytic reaction. The results are as follows: Figure 5 D. From Figure 5As can be seen from Figure D, the phosphotriesterase-cross-linked magnetic hybrid nanoflowers still have 90.32% of the initial enzyme activity after the fifth use, indicating that the phosphotriesterase-cross-linked magnetic hybrid nanoflowers locked by the present invention have good reusability.

[0097] (10) Degradation performance of phosphotriesterase-cross-linked magnetic hybrid nanoflowers on organophosphorus pesticides

[0098] 0.02 mg / mL of the phosphotriesterase-cross-linked magnetic hybrid nanoflower (immobilized enzyme) prepared in Example 2 of the present invention and the free enzyme prepared in Example 1 were placed in four organophosphorus pesticides (methyl parathion, fenitrothion, chlorpyrifos and ethion) with a concentration of 100 mg / L, reacted at 40°C for 1 hour, and the degradation performance was tested. The results are as follows: Figure 6 As shown. Figure 6 The results show that the degradation rates of free enzyme and phosphotriesterase-cross-linked magnetic hybrid nanoflowers for four organophosphorus pesticides increase slowly with time. The degradation rates of cross-linked magnetic hybrid nanoflowers for methyl parathion, cypermethrin, chlorpyrifos and ethion within 1 hour were 99%, 96%, 92% and 85%, respectively, while those of free enzyme were 97%, 93%, 82% and 77%, respectively, indicating that phosphotriesterase-cross-linked magnetic hybrid nanoflowers have higher degradation efficiency for a variety of organophosphorus pesticides than free enzymes.

[0099] The above is only a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and changes directly derived or associated by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included in the protection scope of the present invention.

Claims

1. A method for preparing phosphotriesterase-cross-linked magnetic hybrid nanoflowers, characterized in that: The preparation method comprises the following steps: (1) Preparation of magnetic composite microspheres: Fe3O4 was dispersed in Tris-HCl buffer to obtain a magnetic powder suspension, dopamine hydrochloride and polyethyleneimine were added, and after ultrasonic and oscillation incubation, magnetic composite microspheres Fe3O4-PDA@PEI were obtained; (2) Preparation of phosphotriesterase-cross-linked magnetic hybrid nanoflowers: Add phosphotriesterase enzyme solution into phosphate buffer solution, then add CoCl2 solution and magnetic composite microspheres Fe3O4-PDA@PEI for incubation to complete self-assembly, and then add glutaraldehyde solution for reaction to obtain phosphotriesterase-cross-linked magnetic hybrid nanoflowers.

2. The preparation method according to claim 1, characterized in that: In step (1), the concentration of the Tris-HCl buffer is 10-50 mmol / L, and the pH is 7.0-10.0; the amount of the Tris-HCl buffer is to ensure that the concentration of Fe3O4 is 40-60 g / L.

3. The preparation method according to claim 1, characterized in that: In step (1), the mass ratio of dopamine hydrochloride to Fe3O4 is 1:5; the mass ratio of polyethyleneimine to Fe3O4 is 1:

5.

4. The preparation method according to claim 1, characterized in that: In step (1), the power of the ultrasound is 50-200 W, and the time is 3-9 min; the temperature of the oscillating incubation is 20-35° C., the rotation speed is 150-250 rpm, and the time is 4-8 h.

5. The preparation method according to claim 1, characterized in that: In step (2), the amino acid sequence of the phosphotriesterase is as shown in SEQ ID NO.1; the specific enzyme activity of the phosphotriesterase is 6.45U / mg; the concentration of the phosphate buffer solution is 10-40mmol / L, and the pH is 7.0-10.0; the concentration of the phosphotriesterase in the phosphate buffer is 0.1-0.15mg / mL; the concentration of the CoCl2 in the phosphate buffer is 2-5mmol / L; the concentration of the magnetic composite microspheres Fe3O4-PDA@PEI in the phosphate buffer is 0.5-2g / L.

6. The preparation method according to claim 1, characterized in that: In step (2), the temperature of the ultrasonic incubation is 35-40° C. and the time is 2-12 h.

7. The preparation method according to claim 1, characterized in that: In step (2), the concentration of glutaraldehyde in phosphate buffer solution is 0.1-0.15%; and the reaction time is 0.5-2.5h.

8. A phosphotriesterase-cross-linked magnetic hybrid nanoflower prepared by the preparation method according to any one of claims 1 to 7.

9. An application of the phosphotriesterase-cross-linked magnetic hybrid nanoflower according to claim 8, characterized in that: The phosphotriesterase-cross-linked magnetic hybrid nanoflowers are used to degrade organophosphorus pesticides.

10. The use according to claim 9, characterized in that: The organophosphorus pesticides include methyl parathion, fenitrothion, chlorpyrifos and ethion.

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