CN113832135B - Method for immobilizing laccase by modified polypropylene microporous membrane - Google Patents
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Abstract
The invention discloses a method for immobilizing laccase by a modified polypropylene microporous membrane. In the method, the polypropylene microporous membrane is soaked in n-butyl alcohol to initially improve the surface hydrophilicity; soaking the membrane by using a mixed solvent, so that the hydrophilicity of the membrane is improved again, and the surface smoothness of the membrane is improved at the same time; then coating with aluminum hydroxide colloid to ensure that the aluminum hydroxide colloid is firmly and uniformly coated on the surface of the membrane; and finally, the high-efficiency deposition of dopamine is realized through the close bonding of dopamine and colloid, and the effect of immobilizing laccase is achieved. The modified polypropylene microporous membrane prepared by the method has small contact angle and good hydrophilicity; the immobilized laccase obtained by taking the modified polypropylene microporous membrane as the carrier has good stability, high enzyme curing amount, good degradation and adsorption effects on the phenol-containing wastewater and good application prospect.
Description
Technical Field
The invention belongs to the technical field of laccase immobilization, and particularly relates to a method for immobilizing laccase by using a modified polypropylene microporous membrane.
Background
Laccase belongs to blue oxidase, and has been widely applied to the technical fields of biochemical ring materials and the like since the discovery of the 19 th century. Because the free laccase has the defects of no acid and alkali resistance, no high temperature resistance and the like in the using process, the laccase immobilization technology is widely developed and applied.
Through long-term development, the immobilization methods of laccase at present are various and comprise a chemical method, a physical method, an embedding method, a self-immobilization method and the like. In various methods, the use of an immobilization support is generally not isolated; due to the structural and functional adjustability, membranes have been studied as carriers for laccase immobilization. For example, patent document CN101671665A reports a method for immobilizing laccase by using an electrodischarge fiber membrane, which comprises two steps of preparation of an enzyme-loaded electrospun fiber membrane and membrane-immobilized cross-linked laccase, the whole process is complex, and the enzyme activity recovery rate fluctuates greatly and is only 82.8% at most; patent document CCN107034207A prepares an inorganic hybrid film for fixing laccase, and the decolorization of Congo red dye can reach more than 90%, but does not consider the treatment effect of phenolic wastewater with more complex components; patent document CN109012219A reports a preparation method of an in-situ immobilized laccase ultrafiltration membrane, the relative enzyme activity of the immobilized laccase is only 64.7% at 20-60 ℃, and the removal rate of phenol-containing wastewater is only 78.4-81.5%. Therefore, it is necessary to develop a laccase membrane carrier with simple preparation method, good immobilized enzyme stability and high activity.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method for immobilizing laccase by using a modified polypropylene microporous membrane, which is used for improving the hydrophilicity and the smoothness of the surface of the membrane by modifying the microporous membrane, efficiently grafting dopamine, realizing efficient immobilization of the laccase, and having good stability and good degradation and adsorption capacity on phenol-containing wastewater.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for immobilizing laccase by using a modified polypropylene microporous membrane comprises the following steps:
(1) Preparing an immobilized laccase carrier;
(2) Immobilizing laccase;
the preparation method of the immobilized laccase carrier comprises the following steps:
s1, pretreatment of a polypropylene microporous membrane: soaking a polypropylene microporous membrane in a n-butanol solvent, taking out, washing with deionized water for multiple times, and naturally drying for later use;
s2, solvent modification of a polypropylene microporous membrane: placing the polypropylene microporous membrane obtained by the pretreatment in the step S1 into a mixed solvent for soaking, taking out after soaking, washing with deionized water for multiple times, and naturally drying for later use;
s3, secondary modification of the polypropylene microporous membrane: placing the polypropylene microporous membrane subjected to solvent modification in the step S2 into a container, and coating by using aluminum hydroxide colloid;
s4, depositing the dopamine on the surface: and (4) placing the polypropylene microporous membrane coated in the step (S3) in a buffer solution containing dopamine, carrying out oscillation reaction, and washing and drying after the reaction is finished to obtain the immobilized laccase carrier.
Preferably, the soaking time in the step S1 is 3 to 10min.
Preferably, the mixed solvent in the step S2 is prepared by mixing the following components in a mass ratio of 100:5 deionized water and N, N-dimethylacetamide.
Preferably, the soaking temperature in the step S2 is room temperature, and the soaking time is 0.5 to 2d.
Preferably, the specific operation of coating in step S3 includes the steps of: and (3) placing the polypropylene microporous membrane subjected to solvent modification in a container, enabling the flow direction of the polypropylene microporous membrane to be parallel to the flow direction of the aluminum hydroxide colloid, and then coating the polypropylene microporous membrane by using the prepared aluminum hydroxide colloid.
Preferably, the pH value of the aluminum hydroxide colloid in the step S3 is 7.0, and the concentration of aluminum ions is 0.035mol/L.
Preferably, the coating temperature in step S3 is 25 ℃ and the coating time is 3 to 10min.
Preferably, the buffer in step S4 is Tris buffer at pH =8.0, and the concentration of dopamine is 2mg/mL.
Preferably, in the step S4, the oscillation reaction temperature is room temperature, and the oscillation reaction time is 8 to 24h.
Preferably, the laccase immobilization step (2) comprises the steps of: placing 0.5g of the prepared immobilized laccase carrier in a reaction bottle, then adding 1.5mL of laccase-containing acetic acid-sodium acetate buffer solution with the pH value of 5.5, oscillating for 4h at room temperature, washing with the acetic acid-sodium acetate buffer solution after oscillation is finished, and finally washing with water and storing for later use;
wherein the concentration of the laccase is 0.6mg/mL.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention provides a method for immobilizing laccase by a modified polypropylene microporous membrane, which comprises the steps of soaking a polypropylene microporous membrane in n-butyl alcohol to preliminarily improve the surface hydrophilicity; soaking the membrane by using a mixed solvent, so that the hydrophilicity of the membrane is improved again, and the surface smoothness of the membrane is improved at the same time; then coating with aluminum hydroxide colloid to ensure that the aluminum hydroxide colloid is firmly and uniformly coated on the surface of the membrane; and finally, the dopamine is tightly bonded with the colloid, so that the high-efficiency deposition of the dopamine is realized, and the effect of immobilizing the laccase is achieved.
(2) In the invention, the n-butyl alcohol is adopted for soaking, so that on one hand, the effect of removing impurities on the surface of the polypropylene microporous membrane can be achieved, and meanwhile, the hydrophilicity of the surface of the polypropylene microporous membrane can be preliminarily improved; the mixed solvent of deionized water and N, N-dimethylacetamide is used for modification, so that chain segments on the surface of the polypropylene microporous membrane are recombined, the hydrophilicity of the membrane is further improved, and the surface energy of the membrane is reduced; the modified polypropylene microporous membrane is coated with aluminum hydroxide colloid, so that the coating effect of the aluminum hydroxide can be improved, and the coating of the aluminum hydroxide can improve the internal and external hydrophilicity of the polypropylene microporous membrane and provide a binding activity point for the subsequent deposition of dopamine; dopamine can be firmly fixed on the surface of the polypropylene microporous membrane through active point positions and aluminum hydroxide in a bonding mode, so that the hydrophilicity of the surface of the membrane is improved again on one hand, and the surface of the dopamine has rich functional groups to realize the efficient fixation of laccase on the other hand.
(3) The modified polypropylene microporous membrane prepared by the method has small contact angle and good hydrophilicity; the immobilized laccase obtained by taking the modified polypropylene microporous membrane as a carrier has good stability and high enzyme curing amount, and the curing amount exceeds 160mg/g; the degradation and adsorption effect on the phenol-containing wastewater is good, and the degradation and adsorption rate exceeds 96%.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further explained in detail with reference to the embodiments.
It is particularly emphasized that laccases are commercially available from Aldrich; polypropylene microporous membranes were purchased from Membrana, germany, with specifications: the average pore diameter was 0.2 μm, and the porosity was 80%. Other materials or reagents in the present invention are also commercially available unless otherwise specified.
Example 1
A method for immobilizing laccase by a modified polypropylene microporous membrane comprises the following steps:
(1) Preparing an immobilized laccase carrier;
(2) And (3) laccase immobilization: placing 0.5g of the prepared immobilized laccase carrier in a reaction bottle, then adding 1.5mL of laccase-containing acetic acid-sodium acetate buffer solution with the pH value of 5.5, oscillating for 4h at room temperature, washing with the acetic acid-sodium acetate buffer solution after oscillation is finished, and finally washing with water and storing for later use; wherein the concentration of laccase is 0.6mg/mL.
The preparation method of the immobilized laccase carrier comprises the following steps:
s1, pretreatment of a polypropylene microporous membrane: placing the polypropylene microporous membrane in n-butanol solvent for soaking for 4min, taking out, washing with deionized water for multiple times, and naturally drying for later use;
s2, solvent modification of a polypropylene microporous membrane: placing the polypropylene microporous membrane obtained by the pretreatment in the step S1 in 30mL of mixed solvent, soaking for 1d at room temperature, taking out after soaking, washing with deionized water for multiple times, and naturally drying for later use; wherein the mixed solvent is prepared from the following components in percentage by mass of 100:5, mixing the deionized water and the N, N-dimethylacetamide;
s3, secondary modification of the polypropylene microporous membrane: placing the polypropylene microporous membrane subjected to solvent modification in the step S2 into a container, and then coating the polypropylene microporous membrane with aluminum hydroxide colloid with the pH =7 and the concentration of 0.035mol/L, wherein the placing mode of the polypropylene microporous membrane is parallel to the flow direction of the aluminum hydroxide colloid; the coating temperature is 25 ℃, and the coating time is 5min;
s4, surface deposition of dopamine: and (4) placing the polypropylene microporous membrane coated in the step (S3) in 25mL of a dopamine (concentration is 2 mg/mL) containing Tris buffer solution with the pH value of 8.0, carrying out oscillation reaction at room temperature for 8h, and washing and drying after the reaction is finished to obtain the immobilized laccase carrier.
Example 2
A method for immobilizing laccase by a modified polypropylene microporous membrane comprises the following steps:
(1) Preparing an immobilized laccase carrier;
(2) And (3) laccase immobilization: placing 0.5g of the prepared immobilized laccase carrier in a reaction bottle, then adding 1.5mL of laccase-containing acetic acid-sodium acetate buffer solution with the pH value of 5.5, oscillating for 4h at room temperature, washing with the acetic acid-sodium acetate buffer solution after oscillation is finished, and finally washing with water and storing for later use; wherein the concentration of the laccase is 0.6mg/mL.
The preparation method of the immobilized laccase carrier comprises the following steps:
s1, pretreatment of a polypropylene microporous membrane: placing the polypropylene microporous membrane in n-butanol solvent for soaking for 5min, taking out, washing with deionized water for multiple times, and naturally drying for later use;
s2, solvent modification of a polypropylene microporous membrane: placing the polypropylene microporous membrane obtained by the pretreatment in the step S1 in 30mL of mixed solvent, soaking at room temperature for 1.5 days, taking out after soaking, then washing with deionized water for multiple times, and naturally drying for later use; wherein the mixed solvent is prepared from the following components in percentage by mass of 100:5, mixing the deionized water and the N, N-dimethylacetamide;
s3, secondary modification of the polypropylene microporous membrane: placing the polypropylene microporous membrane subjected to solvent modification in the step S2 into a container, and then coating the polypropylene microporous membrane with aluminum hydroxide colloid with the pH =7 and the concentration of 0.035mol/L, wherein the placing mode of the polypropylene microporous membrane is parallel to the flow direction of the aluminum hydroxide colloid; the coating temperature is 25 ℃, and the coating time is 6min;
s4, surface deposition of dopamine: and (4) placing the polypropylene microporous membrane coated in the step (S3) in 25mL of a dopamine (concentration is 2 mg/mL) containing Tris buffer solution with the pH value of 8.0, carrying out oscillation reaction at room temperature for 12h, and washing and drying after the reaction is finished to obtain the immobilized laccase carrier.
Example 3
A method for immobilizing laccase by a modified polypropylene microporous membrane comprises the following steps:
(1) Preparing an immobilized laccase carrier;
(2) And (3) laccase immobilization: placing 0.5g of the prepared immobilized laccase carrier in a reaction bottle, then adding 1.5mL of laccase-containing acetic acid-sodium acetate buffer solution with the pH value of 5.5, oscillating for 4h at room temperature, washing with the acetic acid-sodium acetate buffer solution after oscillation is finished, and finally washing with water and storing for later use; wherein the concentration of laccase is 0.6mg/mL.
The preparation method of the immobilized laccase carrier comprises the following steps:
s1, pretreatment of a polypropylene microporous membrane: placing the polypropylene microporous membrane in n-butanol solvent for soaking for 6min, taking out, washing with deionized water for multiple times, and naturally drying for later use;
s2, solvent modification of a polypropylene microporous membrane: placing the polypropylene microporous membrane obtained by the pretreatment in the step S1 in 35mL of mixed solvent, soaking for 2d at room temperature, taking out after soaking, washing with deionized water for multiple times, and naturally drying for later use; wherein the mixed solvent is prepared from the following components in percentage by mass of 100:5, mixing the deionized water and the N, N-dimethylacetamide;
s3, secondary modification of the polypropylene microporous membrane: placing the polypropylene microporous membrane subjected to solvent modification in the step S2 in a container, and then coating the polypropylene microporous membrane with aluminum hydroxide colloid with the pH =7 and the concentration of 0.035mol/L, wherein the placing mode of the polypropylene microporous membrane is parallel to the flow direction of the aluminum hydroxide colloid; the coating temperature is 25 ℃, and the coating time is 7min;
s4, surface deposition of dopamine: and (4) placing the polypropylene microporous membrane coated in the step (S3) in 30mL of a dopamine (with the concentration of 2 mg/mL) containing Tris buffer solution with the pH value of 8.0, carrying out oscillation reaction at room temperature for 16h, and washing and drying after the reaction is finished to obtain the immobilized laccase carrier.
Comparative example 1
A method for immobilizing laccase by a modified polypropylene microporous membrane comprises the following steps:
(1) Preparing an immobilized laccase carrier;
(2) And (3) laccase immobilization: placing 0.5g of the prepared immobilized laccase carrier in a reaction bottle, then adding 1.5mL of laccase-containing acetic acid-sodium acetate buffer solution with the pH value of 5.5, oscillating for 4h at room temperature, washing with the acetic acid-sodium acetate buffer solution after oscillation is finished, and finally washing with water and storing for later use; wherein the concentration of the laccase is 0.6mg/mL.
The preparation method of the immobilized laccase carrier comprises the following steps:
s1, pretreatment of a polypropylene microporous membrane: placing the polypropylene microporous membrane in n-butanol solvent for soaking for 4min, taking out, washing with deionized water for multiple times, and naturally drying for later use;
s2, secondary modification of a polypropylene microporous membrane: placing the polypropylene microporous membrane pretreated in the step S1 in a container, and then coating the polypropylene microporous membrane with aluminum hydroxide colloid with the pH =7 and the concentration of 0.035mol/L, wherein the placing mode of the polypropylene microporous membrane is parallel to the flow direction of the aluminum hydroxide colloid; the coating temperature is 25 ℃, and the coating time is 5min;
s3, surface deposition of dopamine: and (3) placing the polypropylene microporous membrane coated in the step (S2) in 25mL of a dopamine (concentration is 2 mg/mL) containing Tris buffer solution with the pH value of 8.0, carrying out oscillation reaction at room temperature for 8h, and washing and drying after the reaction is finished to obtain the immobilized laccase carrier.
Comparative example 2
A method for immobilizing laccase by a modified polypropylene microporous membrane comprises the following steps:
(1) Preparing an immobilized laccase carrier;
(2) And (3) laccase immobilization: placing 0.5g of the prepared immobilized laccase carrier in a reaction bottle, then adding 1.5mL of laccase-containing acetic acid-sodium acetate buffer solution with the pH value of 5.5, oscillating for 4h at room temperature, washing with the acetic acid-sodium acetate buffer solution after oscillation is finished, and finally washing with water and storing for later use; wherein the concentration of the laccase is 0.6mg/mL.
The preparation method of the immobilized laccase carrier comprises the following steps:
s1, pretreatment of a polypropylene microporous membrane: placing the polypropylene microporous membrane in n-butanol solvent for soaking for 4min, taking out, washing with deionized water for multiple times, and naturally drying for later use;
s2, solvent modification of a polypropylene microporous membrane: placing the polypropylene microporous membrane obtained by the pretreatment in the step S1 in 30mL of mixed solvent, soaking for 1d at room temperature, taking out after soaking, then washing with deionized water for multiple times, and naturally airing for later use; wherein the mixed solvent is prepared from the following components in a mass ratio of 100:5, deionized water and N, N-dimethylacetamide;
s3, surface deposition of dopamine: and (3) placing the polypropylene microporous membrane subjected to solvent modification in the step (S2) in 25mL of a Tris buffer solution containing dopamine (with the concentration of 2 mg/mL) and having the pH value of 8.0, carrying out oscillation reaction at room temperature for 8h, and washing and drying after the reaction is finished to obtain the immobilized laccase carrier.
Carrying out water contact angle tests on the modified polypropylene microporous membranes prepared in the examples 1 to 3 and the comparative examples 1 to 2; the immobilized laccases prepared in the examples 1 to 3 and the comparative examples 1 to 2 are subjected to enzyme activity test, immobilized laccase quantity test, degradation adsorption performance test and thermal stability test. The specific test method is as follows:
(1) Water contact Angle test
The test was carried out with reference to GB/T30447-2013.
Table 1 water contact angle test results of modified polypropylene microporous membranes
Contact angle/° | |
Example 1 | 25 |
Example 2 | 27 |
Example 3 | 29 |
Comparative example 1 | 34 |
Comparative example 2 | 38 |
(2) Enzyme Activity assay
Adding 2mL of 1mg/mL free laccase or 2mg of immobilized laccase obtained into 1mmol/L ABTS solution, carrying out oscillation reaction for 15min at 25 ℃, placing in an ice bath to terminate the reaction, carrying out centrifugal separation, and measuring the absorbance change of the supernatant at 420nm by using an ultraviolet-visible spectrophotometer to calculate the corresponding enzyme activity.
In this example, the model of the uv-vis spectrophotometer is new century T6.
(3) Immobilized laccase quantity test
a) Drawing a protein standard curve: respectively adding 1000 mug/mL of standard protein solution and distilled water according to different amounts, then respectively and sequentially adding 5mL of Coomassie brilliant blue G-250 reagent, measuring the absorbance after dyeing for 5min, and drawing a protein standard curve by taking the protein content as a horizontal coordinate and the absorbance as a vertical coordinate.
b) Determination of the amount of immobilized laccase: testing the laccase amount by adopting a Coomassie brilliant blue method; wherein, the enzyme amount calculation formula is as follows:
Q(%)=(Co-C)V/W*100;
in the above formula, Q represents the enzyme adsorption amount, CoThe concentration of laccase in the solution before immobilization; c is the concentration of laccase in the immobilized solution, V is the total volume of the immobilized solution, and W is the mass of the carrier. In the present case, Co=0.6 g/L,V=1.5mL=1.5*10-3L, W =0.5g. The specific test and calculation results are shown in table 2.
TABLE 2 immobilized laccase quantity test results
Amount of immobilized laccase/(mg/g) | |
Example 1 | 163.5 |
Example 2 | 164.1 |
Example 3 | 162.9 |
Comparative example 1 | 132.4 |
Comparative example 2 | 129.3 |
As can be seen from Table 2, the immobilization amount of laccase in each example of the invention exceeds 160mg/g, and the immobilization efficiency is high.
(4) Degradation adsorption of 2, 4-dichlorophenol
Mixing 0.2g of immobilized laccase with 15mL of 10 mg/L2, 4-dichlorophenol, reacting for 5h at room temperature, centrifuging, diluting 10mL of supernatant, and sequentially adding 1.25mL of 0.5mol/L NH3·H2O,0.5mL of 2% 4-aminoantipyrine and 0.5mL of 80g/L potassium ferricyanide solution are stirred for reaction for 15min, and the absorption change of the immobilized laccase to the 2, 4-dichlorophenol can be calculated by measuring the absorbance change at the wavelength of 510nm through an ultraviolet spectrophotometer.
In this example, HPLC is Shimadzu LC-20A.
TABLE 3 results of the 2, 4-dichlorophenol degradation adsorption test
Degradation adsorption rate/% | |
Example 1 | 97.23 |
Example 2 | 96.89 |
Example 3 | 97.01 |
Comparative example 1 | 83.46 |
Comparative example 2 | 80.35 |
From the above table, it can be seen that the immobilized laccase prepared in the examples of the present application has a better adsorption degradation rate for 2, 4-dichlorophenol.
(5) Stability test
The immobilized laccase prepared in the examples 1 to 3 and the free enzyme are stored for 1.5h at different temperatures, and the residual activity of the enzyme is calculated according to the enzyme activity test method.
Test results show that the residual activity of the immobilized enzyme obtained in example 1 is still kept over 75% after the immobilized enzyme is stored for 2 hours at 60 ℃; the remaining activity of the enzyme in comparative example 1 was 69%; the remaining activity of the enzyme in comparative example 2 was 65%; while the residual activity of the free laccase is less than 60%.
The above description describes a preferred embodiment of the present invention and should not be taken as limiting the scope of the invention as claimed. Any modification, equivalent replacement and improvement without departing from the principle and spirit of the present invention should be considered as being within the protection scope of the present claims.
Claims (8)
1. A method for immobilizing laccase by a modified polypropylene microporous membrane is characterized by comprising the following steps:
(1) Preparing an immobilized laccase carrier;
(2) Immobilizing laccase;
the preparation method of the immobilized laccase carrier comprises the following steps: s1, pretreatment of a polypropylene microporous membrane: soaking the polypropylene microporous membrane in n-butanol solvent, taking out, washing with deionized water for multiple times, and naturally drying for later use;
s2, solvent modification of a polypropylene microporous membrane: placing the polypropylene microporous membrane obtained by the pretreatment in the step S1 into a mixed solvent for soaking, taking out after soaking, washing with deionized water for multiple times, and naturally drying for later use;
s3, secondary modification of a polypropylene microporous membrane: placing the polypropylene microporous membrane subjected to solvent modification in the step S2 into a container, and coating by using aluminum hydroxide colloid;
s4, surface deposition of dopamine: placing the polypropylene microporous membrane coated in the step S3 in a buffer solution containing dopamine, carrying out oscillation reaction, washing and drying after the reaction is finished, and thus obtaining the immobilized laccase carrier;
wherein, the mixed solvent in the step S2 is prepared by mixing the following components in a mass ratio of 100:5, mixing deionized water and N, N-dimethylacetamide;
wherein, the laccase immobilization in the step (2) comprises the following steps: placing 0.5g of the prepared immobilized laccase carrier in a reaction bottle, then adding 1.5ml of laccase-containing sodium acetate buffer solution with the pH value of 5.5, oscillating for 4 hours at room temperature, washing with the sodium acetate buffer solution after oscillation is finished, and finally washing with water and storing for later use; wherein the concentration of the laccase is 0.6mg/mL.
2. The method for immobilizing laccase by using the modified polypropylene microporous membrane as claimed in claim 1, wherein the soaking time in step S1 is 3-10 min.
3. The method for immobilizing laccase by using a modified microporous polypropylene membrane as claimed in claim 1, wherein the soaking temperature in step S2 is room temperature, and the soaking time is 0.5-2 days.
4. The method for immobilizing laccase by using the modified polypropylene microporous membrane as claimed in claim 1, wherein the step S3 comprises the following steps: and (3) placing the polypropylene microporous membrane subjected to solvent modification in a container, enabling the flow direction of the polypropylene microporous membrane to be parallel to the flow direction of the aluminum hydroxide colloid, and then coating the polypropylene microporous membrane by using the prepared aluminum hydroxide colloid.
5. The method for immobilizing laccase by using modified microporous polypropylene membrane as claimed in claim 4, wherein the pH of aluminum hydroxide colloid in step S3 is 7.0, and the concentration of aluminum ions is 0.035mol/L.
6. The method for immobilizing laccase by using modified polypropylene microporous membrane as claimed in claim 5, wherein the coating temperature in step S3 is 25 ℃ and the coating time is 3-10 min.
7. The method for immobilizing laccase on a modified polypropylene microporous membrane according to claim 1, wherein the buffer in step S4 is Tris buffer with pH =8.0, and the concentration of dopamine is 2mg/mL.
8. The method for immobilizing laccase by using a modified microporous polypropylene membrane as claimed in claim 7, wherein the oscillation reaction temperature in step S4 is room temperature, and the oscillation reaction time is 8-24 h.
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