A method for removing organic pollutants by immobilizing laccase using magnetic polyethyleneimine

Abstract

The present invention provides a method for removing organic pollutants by immobilizing laccase using magnetic polyethyleneimine nanomaterials. First, magnetic Fe3O4 nanoparticles are synthesized by a solvent thermal method, and the magnetic Fe3O4 nanoparticles are surface-modified with a carboxyl-functionalized ionic liquid. Then, polyethyleneimine is grafted by covalent bonds, and finally, the laccase is immobilized by copper ion chelation. The enzyme loading and activity recovery rate of the immobilized laccase prepared by this method are as high as 52.3 mg / g and 91.7%, respectively, and its stability, pollutant removal efficiency and reusability are significantly improved. The immobilized laccase has good oxidative removal ability for common organic pollutants such as 2,4-DCP, phenol, bisphenol A, 4-CP, indole, anthracene and phenanthrene, has good application value in removing organic pollutants, and is conducive to the further application of immobilized laccase in organic wastewater treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of water pollution treatment, and particularly relates to a method for removing organic pollutants by immobilizing laccase with magnetic polyethyleneimine. Background Art

[0002] Organic compounds are widely used in a variety of industries, including organic synthesis, pharmaceuticals, and pesticides. However, this also leads to the presence of large amounts of organic pollutants in the wastewater generated by these industries. These organic pollutants pose serious risks to nature and human health. Effectively removing pollutants from wastewater has become a key concern in the environmental field.

[0003] The traditional treatment methods for organic pollutants mainly include physical adsorption and chemical catalysis, but they usually have disadvantages such as low efficiency and easy to cause secondary pollution. The enzymatic method is considered to be an effective method for degrading organic pollutants because of its high specificity, environmental friendliness, and high catalytic efficiency. Among them, laccase (EC1.10.3.2) is a type of oxidoreductase with copper as the catalytic center. It has a strong redox ability and acts on a wide range of substrates. The only by-product produced is water, which is very friendly to the environment. However, free laccase faces a very complex environment when treating actual industrial wastewater, which can easily cause the activity and stability of laccase to decrease. Seyed Mehdi (Bioresour Technol. 2020, 306, 123169) et al. used aminated magnetic iron oxide to immobilize laccase by glutaraldehyde cross-linking. Although the stability was improved, the activity recovery rate was only 27%. Chen Chao et al. (Chem Eng J. 2018, 350, 949-959) modified the surface of magnetic nanoparticles with polydopamine and immobilized laccase using dialdehyde starch as a crosslinker. Although stability and activity retention were good, the removal efficiency of 2,4-dichlorophenol was only 91%, and after eight reuses, only 77% of 2,4-dichlorophenol was removed. To address these issues, developing a suitable immobilized enzyme carrier is of great significance to improving the catalytic efficiency, stability, and reusability of laccase. Summary of the Invention

[0004] The present invention provides a magnetic polyethyleneimine-immobilized laccase composite biocatalyst. Using magnetic polyethyleneimine nanoparticles as a carrier, the laccase is immobilized on the carrier through copper ion chelation and applied to the removal of organic pollutants in water. The immobilized laccase prepared by this method achieves enzyme loading and activity recovery rates as high as 52.3 mg / g and 91.7%, respectively. Furthermore, its stability, pollutant removal efficiency, and reusability are significantly improved.

[0005] The technical solution adopted by the present invention is as follows: a method for removing organic pollutants by immobilizing laccase with magnetic polyethyleneimine, comprising the following steps:

[0006] (1) Synthesis of magnetic Fe3O4 nanoparticles by coprecipitation method;

[0007] (2) Preparation of carboxyl-functionalized ionic liquid-modified magnetic Fe3O4 nanoparticles;

[0008] (3) grafting polyethyleneimine onto carboxyl-functionalized ionic liquid-modified magnetic Fe3O4 nanoparticles;

[0009] (4) performing copper ion chelation modification on the magnetic polyethyleneimine nanoparticles obtained in step (3);

[0010] (5) Preparation of magnetic polyethyleneimine-immobilized laccase: adding the copper ion chelated modified magnetic polyethyleneimine nanoparticles obtained in step (4) to a laccase solution (the laccase was purchased from Ningxia Xiashengzyme Bioengineering Co., Ltd. and extracted by fermentation with Aspergillus oryzae) with a concentration of 0.3-0.7 mL laccase solution / mL, a temperature of 20-50° C., a pH of 3.0-5.0, and shaking for 4-8 hours to carry out a laccase immobilization reaction;

[0011] (6) The immobilized laccase prepared in step (5) is applied to the removal of various organic pollutants.

[0012] Preferably, the concentration of the laccase solution in step (5) is 0.6 mL laccase solution / mL, the temperature is 35° C., the pH is 3.5, and the reaction time is 5 hours.

[0013] Preferably, the preparation method of the magnetic Fe3O4 nanoparticles in step (1) is as follows: 5.11g of ferric chloride hexahydrate and 1.99g of ferrous chloride tetrahydrate are dissolved in 100ml of deionized water and heated to 70°C under a nitrogen atmosphere; then the pH value of the system is adjusted to 10 with ammonia water (volume fraction 28%), and the temperature is adjusted to 80°C, and the reaction is carried out for 1.5h; after the solution is cooled, the Fe3O4 particles are separated by a magnet, washed with deionized water until neutral, and placed in a vacuum drying oven for drying.

[0014] Preferably, the preparation method of the carboxyl-functionalized ionic liquid-modified magnetic Fe3O4 nanoparticles in step (2) is as follows: 3.0 g of Fe3O4 nanoparticles are dispersed in toluene, 15 mmol of 3-chloropropyltrimethylsilane (CPTMO) is added dropwise, and the mixture is reacted at 95°C for 7 h under nitrogen protection, and then the solid is separated with a magnet, washed with ethanol several times and dried; the obtained solid is then dispersed in chloroform together with imidazole (15 mmol), reacted at 35°C for 24 h under nitrogen protection, separated with a magnet, washed with ethanol several times and dried; the dried solid is then alkylated with excess chloroacetic acid (15-30 mmol) in acetonitrile at 50°C for 12 h, separated with a magnet, washed with ethanol several times and dried; finally, the intermediate product is subjected to anion exchange with sodium tetrafluoroborate (NaBF4) in acetone for a certain time, separated with a magnet, washed with ethanol several times, and dried; the final product is named MCIL.

[0015] Preferably, the preparation method of the carboxyl-functionalized ionic liquid-modified magnetic Fe3O4 nanoparticles grafted with polyethyleneimine in step (3) is as follows: 0.1 mol N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), 0.1 mol N-hydroxysuccinimide (NHS) and 1.0 g MCIL are dispersed in 50 mL of citrate buffer solution pH 7.0 and stirred at room temperature for 2 hours; then 30 mL of polyethyleneimine (PEI) solution 1.0%-3.0% w / v is added, reacted at room temperature for 12 hours, separated by a magnet, washed several times with water and ethanol and dried, and the product is named MCIL-PEI.

[0016] Preferably, the preparation method of the copper ion chelated magnetic polyethyleneimine nanoparticles described in step (4) is as follows: 1.0 g of MCIL-PEI is dispersed in 25 mL of acetonitrile solution with different concentrations of CuCl2, and reacted for a certain time under N2 protection; the solid is separated with a magnet and washed with ethanol; the product is named MCIL-PEI-Cu.

[0017] Preferably, the laccase immobilization process in step (5) is as follows: adding the magnetic polyethyleneimine nanoparticles obtained in step (4) to the laccase solution to carry out a laccase immobilization reaction; then separating by applying an external magnetic field, washing, and freeze-drying to obtain magnetic polyethyleneimine immobilized laccase.

[0018] Preferably, the immobilized laccase is applied to the removal of various organic pollutants such as 2,4-dichlorophenol, 4-chlorophenol, phenol, bisphenol A, indole, anthracene or phenanthrene.

[0019] Using Fe3O4 modified with carboxyl functionalized ionic liquid as the matrix, polyethyleneimine was grafted via N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and laccase was then immobilized using the chelation effect of copper ions. The specific steps are as follows:

[0020] 1) Fe3O4 nanoparticles were synthesized by coprecipitation. 5.11 g of FeCl3·6H2O and 1.99 g of FeCl2·4H2O were dissolved in 100 ml of deionized water and heated to 70°C under a nitrogen atmosphere. The pH of the solution was then adjusted to 10 with 28% ammonia water, and the temperature was adjusted to 80°C for 1.5 hours. After the solution cooled, the Fe3O4 particles were separated using a magnet, washed with deionized water until neutral, and dried in a vacuum oven to obtain magnetic Fe3O4 nanoparticles.

[0021] 2) 3.0 g of Fe₃O₄ nanoparticles were dispersed in toluene, and 15 mmol of 3-chloropropyltrimethylsilane (CPTMO) was added dropwise. The reaction was carried out at 95°C under nitrogen for 7 h. The solid was then separated with a magnet, washed several times with ethanol, and dried. The resulting solid was then dispersed with imidazole (15 mmol) in chloroform and reacted at 35°C under nitrogen for 24 h. The solid was then separated with a magnet, washed several times with ethanol, and dried. The intermediate product was then alkylated with excess chloroacetic acid in acetonitrile at 50°C for 12 h, separated with a magnet, washed several times with ethanol, and dried. Finally, the intermediate product was anion exchanged with NaBF₄ in acetone for a specified time. After separation with a magnet, it was washed several times with ethanol and dried to obtain carboxylated ionic liquid-modified magnetic Fe₃O₄ nanoparticles, designated MCIL.

[0022] The excess chloroacetic acid is 15-30 mmol, preferably 20 mmol.

[0023] The anion exchange time is 24-36 hours, preferably 48 hours.

[0024] 3) Disperse 0.1 mol EDC, 0.1 mol NHS, and 1.0 g MCIL in 50 mL of citrate buffer (pH 7.0) and stir at room temperature for 2 hours. Then, add 30 mL of PEI solution of varying concentrations and react at room temperature for 12 hours. After magnetic separation, wash the mixture several times with water and ethanol, and dry it to obtain magnetic polyethyleneimine nanoparticles, designated MCIL-PEI.

[0025] The concentration of the PEI solution is 1.0%-3.0% w / v, preferably 2.5% w / v.

[0026] 4) To verify the effect of ionic liquid addition on the immobilization capacity of the support, a comparative support was designed. 3.0 g of Fe₃O₄ nanoparticles were dispersed in toluene, and 15 mmol of 3-chloropropyltrimethylsilane (CPTMO) was added dropwise. The reaction was carried out at 95°C under nitrogen for 7 h. The solid was then separated with a magnet, washed several times with ethanol, and dried. The intermediate product was then reacted with 30 mL of PEI solution (2.5% w / v) at room temperature for 12 h, separated with a magnet, washed several times with water and ethanol, and dried. The product was named MPEI.

[0027] 5) 1.0 g of MCIL-PEI was dispersed into 25 mL of CuCl2 acetonitrile solution at different concentrations and reacted for a certain period of time under N2 protection. The solid was separated with a magnet, washed with ethanol, and dried, and named MCIL-PEI-Cu. MPEI, MCIL-PEI, and MCIL-PEI-Cu (all 10 mg) were then added to laccase solutions at different concentrations and shaken in a water bath shaker for a certain period of time under different temperature and pH conditions to perform laccase immobilization reaction. The immobilized laccases were then separated by an external magnetic field, washed, and freeze-dried to obtain immobilized laccases. The three immobilized laccases were named MPEI-lac, MCIL-PEI-lac, and MCIL-PEI-Cu-lac, respectively.

[0028] The concentration of the CuCl2 acetonitrile solution is 0.5-1.0 mol / L, preferably 0.6 mol / L.

[0029] The reaction time under N2 protection is 36-72 hours, preferably 48 hours.

[0030] The concentration of the laccase solution in the immobilization reaction is 0.3-0.7 mL laccase solution / mL, preferably 0.6 mL laccase solution / mL.

[0031] The immobilization reaction time is 4-8 hours, preferably 5 hours.

[0032] The temperature of the immobilization reaction is 20-50°C, preferably 35°C.

[0033] The pH of the immobilization reaction is 3.0-5.0, preferably 3.5.

[0034] The oscillation speed of the immobilization reaction water bath shaker is 130-200 r / min, preferably 150 r / min.

[0035] The laccase activity assay in the method is as follows: a 1 mmol / L ABTS solution is prepared using a citric acid buffer solution (pH 3.0), an appropriate amount of immobilized laccase is added to 5 mL of the ABTS solution, and the reaction is carried out at 30°C for 5 minutes. The absorbance of the supernatant at 420 nm is then measured using a UV-visible spectrophotometer. The enzyme activity unit (1U) is defined as the amount of laccase required to catalyze the oxidation of 1 μmol of ABTS in one minute under certain conditions. The average value of the measurements is obtained by performing three replicates. The activity is calculated using the following formula:

[0036] Apparent activity (U / g biocatalyst) = A × 10 6 ×V t / (36000×t×m1)

[0037] Specific activity (U / g protein) = A × 10 6 ×V t / (36000×t×m2)

[0038] A–ABTS ·+ absorbance at 420 nm;

[0039] V t –Total volume of reaction solution (L);

[0040] 36000–ABTS ·+ The molar extinction coefficient (M -1 cm -1 );

[0041] t – reaction time (min);

[0042] m1 – mass of immobilized enzyme (g);

[0043] m2 – the amount of protein contained in the immobilized enzyme (g).

[0044] The method for removing organic pollutants by using magnetic polyethyleneimine immobilized laccase is as follows: adding immobilized laccase to an aqueous solution of various organic pollutants to react for a certain time, and measuring the removal efficiency of various pollutants by high performance liquid chromatography after the reaction is completed.

[0045] RE(%)=(A0-A t ) / A0×100

[0046] Among them, A0 and A t Represent the peak areas of pollutants detected by HPLC before and after removal, respectively.

[0047] The beneficial effects of the present invention are:

[0048] (1) The magnetic polyethyleneimine-immobilized laccase of the present invention exhibited a high loading capacity and activity recovery rate of 52.3 mg / g and 91.7%, respectively. Furthermore, its thermal stability was significantly improved, with 64.6% of the enzyme activity remaining after 6 h at 60°C. This demonstrates a significant competitive advantage over other immobilized laccases.

[0049] (2) The modification of carboxyl functionalized ionic liquid can enhance the dispersibility of the carrier and the activity of immobilized laccase. The activity recovery rate of MCIL-PEI-lac is increased by 10.2% compared with MPEI.

[0050] (3) The use of copper ion chelation to immobilize laccase can effectively enhance the catalytic activity of immobilized laccase, which helps to further improve the removal efficiency of immobilized laccase for organic pollutants.

[0051] (4) The magnetic polyethyleneimine-immobilized laccase of the present invention not only has a removal efficiency of more than 90% for common phenolic organic pollutants, but also has a removal efficiency of more than 80% for organic pollutants such as indole and anthracene that are more difficult to remove. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The present invention will be further described below with reference to the accompanying drawings.

[0053] Figure 1 1 is the XRD spectrum of the products Fe3O4, MCIL, MCIL-PEI and MCIL-PEI-Cu in the synthesis process of magnetic polyethyleneimine nanoparticles prepared in Example 1.

[0054] Figure 2 It is the hysteresis loop spectrum of the ferroferric oxide nanoparticles Fe3O4 and the magnetic polyethyleneimine nanoparticles MCIL-PEI-Cu prepared in Example 2.

[0055] Figure 3 These are scanning electron micrographs of ferroferric oxide nanoparticles Fe3O4 (a, c) and magnetic polyethyleneimine nanoparticles MCIL-PEI-Cu (b, d) prepared in Example 2. DETAILED DESCRIPTION

[0056] The present invention will be further described below with reference to the following examples, which are provided for illustration only and to illustrate that the spirit and scope of the present invention are not limited to the details and modifications herein.

[0057] Example 1:

[0058] (1) Ferroferric oxide nanoparticles were synthesized by coprecipitation. 5.11 g of FeCl₃·6H₂O and 1.99 g of FeCl₂·4H₂O were dissolved in 100 ml of deionized water and heated to 70°C under a nitrogen atmosphere. The pH of the system was then adjusted to 10 with aqueous ammonia (28% by volume), and the temperature was adjusted to 80°C for 1.5 h. After the solution cooled, the Fe₃O₄ particles were separated using a magnet, washed with deionized water until neutral, and dried in a vacuum oven to obtain magnetic ferroferric oxide nanoparticles.

[0059] (2) 3.0 g of Fe3O4 nanoparticles were dispersed in toluene, and 15 mmol of 3-chloropropyltrimethylsilane (CPTMO) was added dropwise. The mixture was reacted at 95°C for 7 h under nitrogen protection. The solid was then separated with a magnet, washed several times with ethanol, and dried. The resulting solid was then dispersed in chloroform with imidazole (15 mmol). The mixture was reacted at 35°C for 24 h under nitrogen protection. The solid was then separated with a magnet, washed several times with ethanol, and dried. The intermediate product was then alkylated with 20 mmol of chloroacetic acid in acetonitrile at 50°C for 12 h, separated with a magnet, washed several times with ethanol, and dried. Finally, the intermediate product was anion exchanged with NaBF4 in acetone for 48 h. After separation with a magnet, the solid was washed several times with ethanol, and dried to obtain carboxylated ionic liquid-modified magnetic Fe3O4 nanoparticles, named MCIL.

[0060] (3) 0.1 mol EDC, 0.1 mol NHS, and 1.0 g of the carboxylated ionic liquid-modified magnetic Fe3O4 nanoparticles obtained in step (2) were dispersed in 50 mL of citrate buffer solution (pH 7.0) and stirred at room temperature for 2 h. 30 mL of PEI solution (2.5% w / v) was then added and the mixture was reacted at room temperature for 12 h. After separation with a magnet, the mixture was washed several times with water and ethanol and dried to obtain magnetic polyethyleneimine nanoparticles, designated MCIL-PEI.

[0061] (4) To verify the effect of ionic liquid addition on the immobilization ability of the support, a comparative support was designed. 3.0 g of Fe3O4 nanoparticles were dispersed in toluene, 15 mmol of 3-chloropropyltrimethylsilane (CPTMO) was added dropwise, and the mixture was reacted at 95°C for 7 h under nitrogen protection. The solid was then separated with a magnet, washed several times with ethanol, and dried. The intermediate product was then reacted with 30 mL of PEI solution (2.5% w / v) at room temperature for 12 h, separated with a magnet, washed several times with water and ethanol, and dried. The product was named MPEI.

[0062] (5) 1.0 g of the magnetic polyethyleneimine nanoparticles obtained in step (3) was dispersed into an acetonitrile solution (25 mL) containing CuCl2 (0.6 mol / L) and reacted under N2 protection for 48 h. The solid was separated by a magnet, washed with ethanol and dried. The product was named MCIL-PEI-Cu. Then, MPEI, MCIL-PEI, and MCIL-PEI-Cu (all 10 mg) were added to a laccase solution (0.6 mL laccase solution / mL). The laccase was purchased from Ningxia Xiasheng Enzyme Bioengineering Co., Ltd. and extracted by fermentation of Aspergillus oryzae. The laccase was immobilized in a water bath shaker at 150 r / min for 5 hours at 35°C and pH 3.5. After separation by an external magnetic field, washing, and freeze-drying, the magnetic polyethyleneimine immobilized laccase was obtained. The three immobilized laccases were named MPEI-lac, MCIL-PEI-lac, and MCIL-PEI-Cu-lac, respectively. Their enzymatic properties are shown in Table 1.

[0063] Table 1 Laccase immobilization results a

[0064]

[0065] The protein content of the laccase solution was 282.9 mg / L, and the activity of the free laccase was 2967.84 U g -1 .

[0066] (6) To measure the thermal stability of the three immobilized laccases, free laccase and immobilized laccase were placed in a 60°C water bath shaker for 6 h, and their residual enzyme activities were measured every hour. The results showed that free laccase was quickly inactivated at 60°C, with only 12.3% of the enzyme activity remaining after 6 h, while MPEI-lac, MCIL-PEI-lac, and MCIL-PEI-Cu-lac retained 46.2%, 57.4%, and 64.6% of the enzyme activities, respectively.

[0067] (7) To measure the storage stability of the three immobilized laccases, free and immobilized laccases were stored at 3°C for one month, and their residual enzyme activities were measured every five days. The results showed that free laccase only retained 40.21% of its activity after 30 days of storage, while MPEI-lac, MCIL-PEI-lac, and MCIL-PEI-Cu-lac retained 70.6%, 79.2%, and 88.3% of their activities, respectively.

[0068] (8) Circular dichroism spectroscopy was performed on free laccase and three immobilized laccases, and the results are shown in Table 2. The β-sheet structure is very important for maintaining the conformation of the active center of laccase. It can be found that when laccase is immobilized on MPEI or MCIL-PEI, the molecular structure of laccase is severely damaged, and its β-sheet ratio decreases significantly, which may be the main reason for the decrease in activity of MPEI-lac or MCIL-PEI-lac. MCIL-PEI-Cu-lac maintains its secondary structure to the greatest extent, and therefore, the activity is well preserved.

[0069] Table 2 Secondary structural components of various laccases

[0070]

[0071] Example 2:

[0072] (1) Ferroferric oxide nanoparticles were synthesized by coprecipitation. 5.11 g of FeCl₃·6H₂O and 1.99 g of FeCl₂·4H₂O were dissolved in 100 ml of deionized water and heated to 70°C under a nitrogen atmosphere. The pH of the system was then adjusted to 10 with 28% ammonia water, and the temperature was adjusted to 80°C for 1.5 h. After the solution cooled, the Fe₃O₄ particles were separated using a magnet, washed with deionized water until neutral, and dried in a vacuum oven to obtain magnetic ferroferric oxide nanoparticles.

[0073] (2) 3.0 g of Fe3O4 nanoparticles were dispersed in toluene, and 15 mmol of 3-chloropropyltrimethylsilane (CPTMO) was added dropwise. The mixture was reacted at 95°C for 7 h under nitrogen protection. The solid was then separated with a magnet, washed several times with ethanol, and dried. The resulting solid was then dispersed in chloroform with imidazole (15 mmol). The mixture was reacted at 35°C for 24 h under nitrogen protection. The solid was then separated with a magnet, washed several times with ethanol, and dried. The intermediate product was then alkylated with 15 mmol of chloroacetic acid in acetonitrile at 50°C for 12 h, separated with a magnet, washed several times with ethanol, and dried. Finally, the intermediate product was anion exchanged with NaBF4 in acetone for 36 h. After separation with a magnet, the solid was washed several times with ethanol, and dried to obtain carboxylated ionic liquid-modified magnetic Fe3O4 nanoparticles, named MCIL.

[0074] (3) 0.1 mol EDC, 0.1 mol NHS, and 1.0 g of the carboxylated ionic liquid-modified magnetic Fe3O4 nanoparticles obtained in step (2) were dispersed in 50 mL of citrate buffer solution (pH 7.0) and stirred at room temperature for 2 h. 30 mL of PEI solution (2.5% w / v) was then added and the mixture was reacted at room temperature for 12 h. After separation with a magnet, the mixture was washed several times with water and ethanol and dried to obtain magnetic polyethyleneimine nanoparticles, designated MCIL-PEI.

[0075] (4) To verify the effect of ionic liquid addition on the immobilization ability of the support, a comparative support was designed. 3.0 g of Fe3O4 nanoparticles were dispersed in toluene, 15 mmol of 3-chloropropyltrimethylsilane (CPTMO) was added dropwise, and the mixture was reacted at 95°C for 7 h under nitrogen protection. The solid was then separated with a magnet, washed several times with ethanol, and dried. The intermediate product was then reacted with 30 mL of PEI solution (2.5% w / v) at room temperature for 12 h, separated with a magnet, washed several times with water and ethanol, and dried. The product was named MPEI.

[0076] (5) 1.0 g of the magnetic polyethyleneimine nanoparticles obtained in step (3) was dispersed in an acetonitrile solution (25 mL) containing CuCl2 (0.5 mol / L) and reacted under N2 protection for 48 h. The solid was separated by a magnet, washed with ethanol and dried. The product was named MCIL-PEI-Cu. Then, MPEI, MCIL-PEI, and MCIL-PEI-Cu (all 10 mg) were added to the laccase solution and shaken in a water bath shaker at 150 r / min for 5 h at 35°C and pH 3.5 to carry out the laccase immobilization reaction. After separation by an external magnetic field, washing, and freeze-drying, the magnetic polyethyleneimine immobilized laccase was obtained. The three immobilized laccases were named MPEI-lac, MCIL-PEI-lac, and MCIL-PEI-Cu-lac, respectively.

[0077] When the amount of chloroacetic acid and the anion exchange time were reduced in step (2), the loading amount of the immobilized laccase MCIL-PEI-lac was 32.4 mg / g, which was lower than the loading amount of MCIL-PEI-lac obtained in Example 1. When the concentration of the CuCl2 solution was reduced in step (5), the loading amount and activity recovery rate of the immobilized laccase MCIL-PEI-Cu-lac were 46.5 mg / g and 80.3%, respectively, which were significantly lower than those of the MCIL-PEI-Cu-lac obtained in Example 1.

[0078] Example 3:

[0079] The synthesis process of immobilized laccase refers to Example 1. The difference is that the amount of chloroacetic acid in step (2) is 30 mmol, and the concentration of CuCl2 solution in step (5) is 0.8 mol / L. The loading amount and activity of the obtained immobilized laccase MCIL-PEI-lac are 33.5 mg / g and 51.3%, respectively. The loading amount changes less than that of the MCIL-PEI-lac obtained in Example 1, but the activity recovery rate decreases significantly. The loading amount and activity recovery rate of the obtained immobilized laccase MCIL-PEI-Cu-lac are 55.6 mg / g and 89.3%, respectively. The performance change is not obvious compared with the MCIL-PEI-Cu-lac obtained in Example 1. Considering the economic benefit, a 0.6 mol / L CuCl2 solution is selected.

[0080] Example 4:

[0081] The synthesis process of immobilized laccase was similar to that of Example 1. The difference was that the concentration of the PEI solution in steps (3) and (4) was 1.0% w / v. The resulting immobilized laccase MCIL-PEI-lac had a loading capacity of 30.2 mg / g, which was significantly lower than that of the MCIL-PEI-lac obtained in Example 1. This may be due to the lower concentration of the PEI solution, which resulted in too little PEI grafted onto the support, leading to a lower loading capacity.

[0082] Example 5:

[0083] The synthesis process of immobilized laccase was similar to that of Example 1. The difference was that the concentration of the PEI solution in steps (3) and (4) was 3.0% w / v. The resulting immobilized laccase MCIL-PEI-lac had a loading of 33.2 mg / g, which was not significantly different from that of the MCIL-PEI-lac obtained in Example 1. This may be because the grafted PEI had already reached saturation, and further increasing the concentration of the PEI solution had little effect.

[0084] Example 6:

[0085] The synthesis process of immobilized laccase was similar to that of Example 1. The difference was that the reaction time under N2 protection in the copper ion chelation step in step (5) was 24 hours. The loading amount of the immobilized laccase MCIL-PEI-Cu-lac was 47.2 mg / g, which was significantly lower than that of the MCIL-PEI-Cu-lac obtained in Example 1. The possible reason was that the Cu ions were reduced after the reaction time was shortened. 2+ The ligand was not fully coordinated to the support, resulting in a low loading capacity.

[0086] Example 7:

[0087] The synthesis process of immobilized laccase is as described in Example 1. The difference is that the laccase immobilization reaction conditions in step (5) are different. The loading amount and activity recovery rate of immobilized laccase MCIL-PEI-Cu-lac under different reaction conditions are shown in Table 3:

[0088] Table 3 Effects of different immobilization conditions on MCIL-PEI-Cu-lac immobilization results

[0089]

[0090]

[0091] Example 8:

[0092] The synthesis process of immobilized laccases is described in Example 1. Three immobilized laccases (all 1U) were added to 10mL of a 10mg / L 2,4-DCP aqueous solution and reacted with stirring at 25°C. The liquid phase was sampled every hour to monitor the removal effect. After 10 hours, the 2,4-DCP removal efficiencies of MPEI-lac, MCIL-PEI-lac, and MCIL-PEI-Cu-lac were 81%, 90%, and 100%, respectively. The immobilized laccases were collected in an external magnetic field. After washing with pure water to remove reaction byproducts, the next batch of the above reaction was continued. After six consecutive reactions, the removal efficiency of MCIL-PEI-Cu-lac for the 10mg / L 2,4-DCP aqueous solution could still reach 92.3%, while the removal efficiencies of MPEI-lac and MCIL-PEI-lac could only reach 43.2% and 48.9%, respectively.

[0093] Example 9:

[0094] The synthesis process of immobilized laccases is described in Example 1. Three immobilized laccases (all at 1 U) were added to 10 mL of a 10 mg / L bisphenol A aqueous solution and reacted with stirring at 25°C. Liquid samples were taken every hour to monitor the removal efficiency. After 24 hours, the removal efficiencies of MPEI-lac, MCIL-PEI-lac, and MCIL-PEI-Cu-lac on 10 mg / L bisphenol A reached maximum values of 61.4%, 69.3%, and 98.6%, respectively. The immobilized laccases were collected in an external magnetic field. After washing with pure water and removing reaction byproducts, the immobilized laccases were subsequently added to the next batch of the above reaction. After six consecutive additions, MCIL-PEI-Cu-lac still achieved a removal efficiency of 83.1% on the 10 mg / L bisphenol A aqueous solution, while MPEI-lac and MCIL-PEI-lac only achieved 28.2% and 35.1%, respectively.

[0095] Example 10:

[0096] The synthesis of immobilized laccase was performed as described in Example 1. 1 U of MCIL-PEI-Cu-lac (previously described in Example 1) was added to 10 mL of a 10 mg / L aqueous 4-CP solution. The reaction was stirred at 25°C, and the liquid phase was sampled every hour to monitor the removal efficiency. After 24 hours, the 4-CP removal efficiency reached a maximum of 95.9%.

[0097] Example 11:

[0098] The synthesis of immobilized laccase was performed as described in Example 1. 1 U of MCIL-PEI-Cu-lac (previously described in Example 1) was added to 10 mL of a 10 mg / L aqueous phenol solution. The reaction was stirred at 25°C, and liquid samples were taken every hour to monitor the removal efficiency. After 24 hours, the phenol removal efficiency reached a maximum of 93.6%.

[0099] Example 12:

[0100] The synthesis of immobilized laccase was performed as described in Example 1. 1 U of MCIL-PEI-Cu-lac (previously described in Example 1) was added to 10 mL of an anthracene aqueous solution (10 mg / L). The reaction was stirred at 25°C, and liquid samples were taken every hour to monitor the removal efficiency. After 24 hours, the anthracene removal efficiency reached a maximum of 84.6%.

[0101] Example 13:

[0102] The synthesis of immobilized laccase was performed as described in Example 1. 1 U of MCIL-PEI-Cu-lac (previously described in Example 1) was added to 10 mL of an aqueous solution of indole (10 mg / L). The reaction was stirred at 25°C, and the liquid phase was sampled every hour to monitor the removal efficiency. After 24 hours, the indole removal efficiency reached a maximum of 80.9%.

[0103] Example 14:

[0104] The synthesis of immobilized laccase was performed as described in Example 1. 1 U of MCIL-PEI-Cu-lac (previously described in Example 1) was added to 10 mL of a 10 mg / L phenanthrene aqueous solution. The reaction was stirred at 25°C, and liquid samples were taken every hour to monitor the removal efficiency. After 24 hours, the phenanthrene removal efficiency reached a maximum of 77.3%.

Claims

1. A method for removing organic pollutants by using magnetic polyethyleneimine-immobilized laccase, characterized in that: The following steps are involved: (1) Synthesis of magnetic Fe3O4 nanoparticles by coprecipitation method; (2) Preparation of carboxyl-functionalized ionic liquid-modified magnetic Fe3O4 nanoparticles; (3) Grafting polyethyleneimine onto carboxyl-functionalized ionic liquid-modified magnetic Fe3O4 nanoparticles; (4) performing copper ion chelation modification on the magnetic polyethyleneimine nanoparticles obtained in step (3); (5) Preparation of magnetic polyethyleneimine-immobilized laccase: The copper ion chelated modified magnetic polyethyleneimine nanoparticles obtained in step (4) were added to a laccase solution having a concentration of 0.6 mL laccase solution / mL, a temperature of 35°C, a pH of 3.5, and shaken for 5 hours to carry out a laccase immobilization reaction; (6) applying the immobilized laccase prepared in step (5) to the removal of organic pollutants; The preparation method of the carboxyl functionalized ionic liquid modified magnetic Fe3O4 nanoparticles in step (2) is as follows: 3.0 g of Fe3O4 nanoparticles are dispersed in toluene, 15 mmol of 3-chloropropyltrimethylsilane CPTMO is added dropwise, and the mixture is reacted at 95°C for 7 h under nitrogen protection, and then the solid is separated by a magnet, washed with ethanol several times and dried; the obtained solid is then dispersed in chloroform together with 15 mmol of imidazole, reacted at 35°C for 24 h under nitrogen protection, and then the solid is separated by a magnet, washed with ethanol several times and dried; the dried solid is then alkylated with 20 mmol of chloroacetic acid in acetonitrile at 50°C for 12 h, separated by a magnet, washed with ethanol several times and dried; finally, the intermediate product is subjected to anion exchange with sodium tetrafluoroborate NaBF4 in acetone for 48 h, separated by a magnet, washed with ethanol several times and dried; the final product is named MCIL; Step (3) Preparation of magnetic Fe3O4 nanoparticles modified with carboxyl functionalized ionic liquid grafted with polyethyleneimine is as follows: 0.1 mol N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), 0.1 mol N-hydroxysuccinimide (NHS) and 1.0 g MCIL are dispersed in 50 mL of citrate buffer solution (pH 7.0) and stirred at room temperature for 2 h; then 30 mL of 2.5% w / v polyethyleneimine (PEI) solution is added and reacted at room temperature for 12 h, separated by magnet, washed several times with water and ethanol and dried. The product is named MCIL-PEI; Step (4) Preparation of copper ion chelated magnetic polyethyleneimine nanoparticles: 1.0 g MCIL-PEI was dispersed in 25 mL of 0.6 mol / L CuCl2 acetonitrile solution and reacted under N2 protection for 48 h; the solid was separated with a magnet and washed with ethanol; the product was named MCIL-PEI-Cu; The immobilized laccase process in step (5) is as follows: adding the MCIL-PEI-Cu obtained in step (4) to the laccase solution to carry out a laccase immobilization reaction; then separating by applying an external magnetic field, washing, and freeze-drying to obtain magnetic polyethyleneimine-immobilized laccase; The immobilized laccase is applied to the removal of organic pollutants such as 2,4-dichlorophenol, 4-chlorophenol, phenol, bisphenol A, indole, anthracene or phenanthrene.

2. The method for removing organic pollutants by using magnetic polyethyleneimine-immobilized laccase according to claim 1, characterized in that: The preparation method of the magnetic Fe3O4 nanoparticles in step (1) is as follows: 5.11 g of ferric chloride hexahydrate and 1.99 g of ferrous chloride tetrahydrate are dissolved in 100 ml of deionized water and heated to 70°C under a nitrogen atmosphere; the pH value of the system is then adjusted to 10 with 28% by volume ammonia water, and the temperature is adjusted to 80°C, and the reaction is carried out for 1.5 hours; after the solution is cooled, the Fe3O4 particles are separated by a magnet, washed with deionized water until neutral, and dried in a vacuum drying oven.

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