CN110129308B - Surface charge-controlled functionalized dendritic mesoporous SiO 2 Immobilized chloroperoxidase reactor and application thereof - Google Patents

Surface charge-controlled functionalized dendritic mesoporous SiO 2 Immobilized chloroperoxidase reactor and application thereof Download PDF

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CN110129308B
CN110129308B CN201910393910.3A CN201910393910A CN110129308B CN 110129308 B CN110129308 B CN 110129308B CN 201910393910 A CN201910393910 A CN 201910393910A CN 110129308 B CN110129308 B CN 110129308B
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蒋育澄
王兰兰
宋艺超
胡满成
李淑妮
翟全国
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Abstract

The invention discloses a surface charge-controlled functionalized dendritic mesoporous SiO 2 An immobilized chloroperoxidase reactor for preparing the mesoporous SiO with surface amino group modified by dendritic medium-pore structure and its application 2 The nano particles are crosslinked with hyaluronic acid or amino protonated chitosan to be used as a carrier, and the immobilized chloroperoxidase is obtained through electrostatic interaction and hydrogen bond interaction. The carrier pore channels used by the enzyme reactor are loaded with rich negative charges or positive charges and are rich in functional groups such as hydroxyl groups, so that the binding force between the carrier and enzyme molecules is greatly enhanced, and the loading capacity of the enzyme is improved. The enzyme reactor has good catalytic activity, thermal stability and reusability, has good tolerance in organic solvents such as DMF, methanol, acetonitrile and the like, is used for degrading drugs such as levofloxacin, rifaximin and the like in wastewater, and has high degradation speed and high degradation rate.

Description

Surface charge-controlled functionalized dendritic mesoporous SiO 2 Immobilized chloroperoxidase reactor and application thereof
Technical Field
The invention belongs to the technical field of enzyme immobilization, and particularly relates to dendritic mesoporous SiO with rich charges and rich hydroxyl functional groups and the like loaded in a pore 2 An immobilized chloroperoxidase reactor and application thereof.
Background
The biological enzyme is an organic matter which is generated by living cells and has a catalytic action, most of the biological enzyme is protein, and the biological enzyme is a non-toxic and environment-friendly biological catalyst. The biological enzyme has high catalytic efficiency and high specificity, one enzyme can only catalyze one or one type of chemical reaction, and the reaction strip is mild. However, free enzymes are easy to inactivate in environments of high temperature, strong acid, strong base, organic solvent and the like, and the resistance of the enzymes to temperature, acid, alkali and organic solvent can be effectively improved by adopting an immobilized enzyme mode. And the immobilized enzyme has wide application prospect in the aspect of sewage treatment due to the characteristics of green, recoverability and the like.
Chloroperoxidase (CPO) is a heme peroxidase (42 kDa) isolated from the marine fungus Caldariomyces fumago, combines the catalytic characteristics of various enzymes such as heme peroxidase, catalase and cytochrome P-450, and is currently considered to be the most widely used enzyme in the peroxidase family. CPO can catalyze a variety of organic reactions, such as: halogenation reaction, peroxidation reaction, hydroxylation reaction, epoxidation reaction and sulfonation oxidation reaction, so that the CPO has great application potential.
Mesoporous silica refers to a silica material with a pore size of 2-50 nm, has a high specific surface area, a large pore volume and an ordered pore network, and has strong chemical stability, thermal stability and operational stability. The aperture of the three-dimensional center radiation dendritic mesoporous silica is adjustable, and the three-dimensional center radiation dendritic mesoporous silica has a vertical pore structure and has wide application prospect in the aspect of enzyme immobilization. However, since the silica itself has a weak electric property, the electrostatic interaction between the silica and the CPO is relatively weak, and the enzyme molecules are easily leaked during repeated use.
Disclosure of Invention
The invention aims to provide the functionalized dendritic mesoporous SiO with high catalytic activity, good thermal stability, reusability and organic solvent tolerance and adjustable surface charge 2 An immobilized chloroperoxidase reactor and provides new applications for the enzyme reactor.
Aiming at the aim, the enzyme reactor adopted by the invention is dendritic mesoporous SiO with the surface amino modified 2 The nano particles are crosslinked with hyaluronic acid or amino protonated chitosan to be used as a carrier, and the immobilized chloroperoxidase is obtained through electrostatic interaction and hydrogen bond interaction.
In the enzyme reactor, the compound adopted for surface amino modification is any one of 3-aminopropyltriethoxysilane, tetraethylenepentamine and N-aminoethyl-3-aminopropylmethyldimethoxysilane.
In the enzyme reactor, the hyaluronic acid reacts with the surface amino-modified dendritic mesoporous SiO by the action of N-hydroxysuccinimide sulfonic acid sodium salt and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride under the condition that the pH value is 8-10 2 Crosslinking the nano particles; the surface amino modified dendritic mesoporous SiO 2 Crosslinking hyaluronic acid with nano particles, and standing at pH 3-4The chloroperoxidase is immobilized by electrical and hydrogen bonding.
In the enzyme reactor, the amino protonated chitosan is dendritic mesoporous SiO modified by glutaraldehyde and surface amino 2 Crosslinking the nano particles; the surface amino modified dendritic mesoporous SiO 2 After chitosan is crosslinked by the nano particles, under the condition that the pH value is 5-6, the chloroperoxidase is fixed through electrostatic action and hydrogen bond action.
The invention relates to surface charge-controlled functionalized dendritic mesoporous SiO 2 The immobilized chloroperoxidase reactor can be used for degrading levofloxacin and rifaximin.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention leads the dendritic mesoporous SiO 2 The nano particles are modified by amino and then are further crosslinked with hyaluronic acid or amino protonated chitosan, so that the dendritic mesoporous SiO 2 The pore channels are loaded with rich negative charges or positive charges and simultaneously are rich in functional groups such as hydroxyl groups and the like, and then the porous material is taken as a carrier, and the pH value of the solution is adjusted to ensure that the chloroperoxidase is fixed on the dendritic mesoporous SiO in a manner of electrostatic interaction and hydrogen bond combination 2 In the pore canal, the binding force between the carrier and the enzyme molecules is greatly enhanced, and the immobilized amount of the enzyme is improved.
2. The enzyme reactor can maintain the catalytic activity of the chloroperoxidase, overcome the defects of free enzyme, improve the stability of the molecular structure of the enzyme and the operation stability, overcome the defect of enzyme molecule leakage in the process of repeated use, improve the times of repeated use, and has good tolerance in organic solvents such as DMF, methanol, acetonitrile and the like.
3. The enzyme reactor is used for degrading levofloxacin and rifaximin in wastewater, and can achieve a better degradation effect in a shorter time.
Drawings
FIG. 1 is a transmission electron micrograph of DSP-HA prepared in example 1.
FIG. 2 is a confocal laser micrograph of CPO @ DSP-HA prepared in example 1.
FIG. 3 is a transmission electron micrograph of the DSP-CHIT prepared in example 2 by field emission.
FIG. 4 is a confocal micrograph of CPO @ DSP-CHIT laser prepared in example 2.
FIG. 5 is a graph showing the effect of temperature on the catalytic activities of CPO @ DSP-HA and CPO @ DSP-CHIT.
FIG. 6 is a plot of the reusability of CPO @ DSP-HA in buffered solutions.
FIG. 7 is a plot of the reusability of CPO @ DSP-CHIT in buffered solutions.
FIG. 8 is a graph showing the effect of methanol on the catalytic activities of CPO @ DSP-HA and CPO @ DSP-CHIT.
FIG. 9 is a graph showing the effect of acetonitrile on the catalytic activities of CPO @ DSP-HA and CPO @ DSP-CHIT.
FIG. 10 is a graph showing the effect of DMF on the catalytic activity of CPO @ DSP-HA and CPO @ DSP-CHIT.
FIG. 11 is a graph showing the effect of various concentrations of levofloxacin degraded by CPO @ DSP-HA.
FIG. 12 is a graph showing the effect of various concentrations of levofloxacin degraded by CPO @ DSP-CHIT.
Figure 13 is a graph of the effect of different concentrations of rifaximin on degradation by cpo @ dsp-HA.
Figure 14 is a graph of the effect of different concentrations of rifaximin on degradation by cpo @ dsp-CHIT.
Detailed Description
The invention will be further described in detail with reference to the following figures and examples, but the scope of the invention is not limited to these examples.
Lower dendritic mesoporous SiO 2 Nanoparticles (DSP) according to the document "Shen D K, yang J P, li X M, et al, phase formation Approach to Three-Dimensional Dentistic Biodegradable Silicone Nanospheres [ J]Nano Letters,2014,14 (2): 923 ".
Example 1
Dispersing 1g of DSP nanoparticles in 30mL of anhydrous toluene, adding 0.5mL of 97 vol% aqueous 3-aminopropyltriethoxysilane solution dropwise thereto, refluxing at 110 deg.C for 15h, centrifuging the resulting mixture, washing with acetone three times, and vacuum drying 1 at room temperature2h, obtaining the amino modified dendritic mesoporous SiO 2 (DSP-NH 2 )。
100mg of DSP-NH 2 Uniformly dispersing in 100mL of deionized water to form a solution a; adding 0.37g N-hydroxysuccinimide sulfonic acid sodium salt and 0.20g 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride to 20mL of deionized water to dissolve the solid, adding 60mL of an aqueous solution containing 0.113g of Hyaluronic Acid (HA) to the solution, and mixing the solution sufficiently to form a solution b; uniformly mixing the solution a and the solution b, adjusting the pH of the mixed solution to 9 by triethylamine, stirring for 12h at 38 ℃, performing centrifugal separation, washing the product with deionized water for three times, and performing vacuum drying for 12h at room temperature to obtain the dendritic mesoporous SiO of the cross-linked hyaluronic acid 2 (DSP-HA), FIG. 1 is a transmission electron micrograph of the obtained DSP-HA.
To 10mg of DSP-HA were added 1400. Mu.L of pH 3 PBS buffer and 100. Mu.L of CPO solution (2.0X 10) - 5 mmol·L -1 pH = 4.0), sonicated for 5min to disperse uniformly, and then shaken at 20 ℃ for 24h to allow the CPO to be sufficiently fixed. After the reaction is finished, centrifugally separating the product, centrifugally cleaning the product for three times by using PBS (phosphate buffer solution) with the pH value of 3, and drying the product in vacuum at room temperature to obtain the functionalized dendritic mesoporous SiO of the surface cross-linked hyaluronic acid 2 Immobilized chloroperoxidase reactor (CPO @ DSP-HA), FIG. 2 is a confocal laser micrograph of CPO @ DSP-HA. The test shows that the solid loading of the chloroperoxidase in the obtained enzyme reactor is 30.68 mg.g -1 。
Example 2
Preparation of DSP-NH according to example 1 2 . 100mg of DSP-NH 2 Uniformly dispersed in 2.5mL0.1mol.L - 1 PBS buffer solution with pH 7.5 and 100 μ L of 50% glutaraldehyde aqueous solution, standing at room temperature for 1h, centrifuging the mixed solution, and adding 0.1 mol. L -1 Centrifuging and washing the solution for three times by using PBS buffer solution with the pH of 7.5, removing supernatant fluid to obtain DSP-NH of the cross-linked glutaraldehyde 2 And (3) dispersing the mixture. 50mg of Chitosan (CHIT) was added to 2.5mL of 0.1mol. L -1 Completely dissolving chitosan solid in HCl aqueous solution; then the above-mentioned DSP-NH of cross-linked glutaraldehyde 2 Dispersion liquidMixing with chitosan solution, stirring at room temperature for 12h, centrifuging, washing the product with PBS buffer solution with pH of 8 for three times, vacuum drying at room temperature to obtain crosslinked chitosan dendritic mesoporous SiO 2 (DSP-CHIT), and FIG. 3 is a transmission electron micrograph of the obtained DSP-CHIT.
To 10mg of DSP-CHIT were added 1400. Mu.L of pH 5 PBS buffer and 100. Mu.L of CPO solution (2.0X 10) -5 mmol·L -1 pH = 4.0), sonicated for 5min to disperse uniformly, and then shaken at 20 ℃ for 24h to sufficiently fix CPO by electrostatic interaction and hydrogen bonding. After the reaction is finished, centrifugally separating the product, centrifugally cleaning the product for three times by using PBS (phosphate buffer solution) with the pH value of 5, and drying the product in vacuum at room temperature to obtain the surface chitosan functionalized dendritic mesoporous SiO 2 Immobilized chloroperoxidase reactor (CPO @ DSP-CHIT), FIG. 4 is a confocal laser micrograph of CPO @ DSP-CHIT. Tests show that the solid loading of the chloroperoxidase in the obtained enzyme reactor is 31.12mg g -1 。
The inventor carries out performance tests on CPO @ DSP-HA prepared in example 1 and CPO @ DSP-CHIT prepared in example 2, and the specific tests are as follows:
1. experiment of catalytic Activity
The catalytic activity of the enzyme reactor is inspected by taking catalytic 2-chloro-5,5-dimethyl-1,3-cyclohexanedione (MCD) as a model reaction, and the specific steps are as follows: 5 μ L of free CPO solution (2.0X 10) -5 mmol·L -1 pH = 4.0), 5mg CPO @ DSP-HA and 5mg CPO @ DSP-CHIT were added to 1420. Mu.L of 0.1 mol. L -1 PBS buffer solution pH =2.75 and 50 μ L2.5 mmol · L -1 Adding 30 mu L0.1 mol.L into the mixed solution of MCD aqueous solution -1 H 2 O 2 Placing the water solution in shaking table, oscillating for 15min, taking out, centrifuging, measuring absorbance value of supernatant at 278nm with ultraviolet spectrophotometer, and recording as A t . Equal amounts of PBS buffer, aqueous MCD and H were added 2 O 2 The aqueous solution was mixed well and the absorbance at 278nm was measured and recorded as A 0 . The conversion rate of MCD is calculated by the following formula:
Figure BDA0002057536450000051
in the formula A t : the absorbance value of the supernatant at the time t; a. The 0 : absorbance value of MCD at 0min of isoconcentration reaction. The results showed that CPO @ DSP-HA and CPO @ DSP-CHIT retained high catalytic activity, about 95.34% and 96.72%, based on 100% catalytic activity of free CPO.
2. Thermal stability test
Respectively mixing CPO @ DSP-HA and CPO @ DSP-CHIT with equal enzyme amount of free CPO (2 μ L,2.0 × 10) -5 mmol·L -1 ) The thermal stability of CPO @ DSP-HA and CPO @ DSP-CHIT was expressed by taking out after 1h incubation at various temperatures (50-90 deg.C.), cooling to room temperature, determining the catalytic activity of the MCD by using a model reaction catalyzing MCD, regarding the highest conversion rate to MCD as 100%, and plotting the catalytic activity at other temperatures with its relative activity against time, and the results are shown in FIG. 5.
As can be seen in fig. 5, the residual activity of both enzyme reactors to catalyze the MCD chlorination reaction decreases with increasing temperature, and at each temperature the retained activity of the enzyme reactor is higher than the free CPO. Wherein, the CPO @ DSP-HA and the CPO @ DSP-CHIT can respectively maintain 73.40 percent of catalytic activity and 58.28 percent of catalytic activity after being placed for 1h at 70 ℃; after being placed at 80 ℃ for 1h, the CPO @ DSP-HA and the CPO @ DSP-CHIT still can respectively maintain 47.65 percent of catalytic activity and 35.25 percent of catalytic activity, and have good thermal stability at high temperature compared with free CPO.
3. Reusability test
Adding 5mg of CPO @ DSP-HA and CPO @ DSP-CHIT to 1420 μ L of 0.1 mol. L -1 PBS buffer solution at pH 2.75 and 50. Mu.L of 2.5 mol. L -1 To the mixture of MCD aqueous solution, 30. Mu.L 0.1mmol. L was added -1 H 2 O 2 And (3) placing the aqueous solution in a shaking table, oscillating for 15min, taking out, centrifuging, and measuring the absorbance value of the supernatant at 278nm by using an ultraviolet spectrophotometer. After each reaction, the reaction solution was centrifuged, and after the supernatant was aspirated, the same amounts of PBS buffer, MCD aqueous solution and H were added 2 O 2 The aqueous solution to start the next reaction. Will be used for the first timeThe catalytic activity was regarded as 100%, and the catalytic activity of each subsequent time was compared with that of the first time and expressed as the remaining activity. The results are shown in FIGS. 6 and 7.
As can be seen from the figure, after the CPO @ DSP-HA is repeatedly used for 12 times, the catalytic activity of 56.11 percent can still be kept; after the CPO @ DSP-CHIT is repeatedly used for 16 times, 62.48 percent of catalytic activity can still be kept, which indicates that the reusability is good.
4. Organic solvent resistance test
(1) Tolerance to methanol
Mixing CPO @ DSP-HA and CPO @ DSP-CHIT with equal amount of free CPO solution (2 μ L,2.0 × 10) -5 mmol·L -1 ) Adding 1.5mL of methanol aqueous solution with different volume concentrations (volume fractions are 0%, 5%, 10%, 15%, 20%, 25% and 30%) respectively, and standing at room temperature for 1h for catalyzing MCD chlorination reaction. The conversion rates of CPO @ DSP-HA, CPO @ DSP-CHIT and free CPO solution to MCD without adding methanol aqueous solution were respectively regarded as 100%, and the relative activities of CPO @ DSP-HA, CPO @ DSP-CHIT and free CPO solution at different methanol volume concentrations were plotted against the methanol volume concentration to express the methanol tolerance of CPO @ DSP-HA, CPO @ DSP-CHIT and free CPO, and the results are shown in FIG. 8.
As can be seen from FIG. 8, the residual activity of free CPO was less than 20% after treatment with 30% by volume of aqueous methanol, while the residual activities of CPO @ DSP-HA and CPO @ DSP-CHIT both reached 88.56% and 85.32%.
(2) Tolerance to acetonitrile
Mixing CPO @ DSP-HA and CPO @ DSP-CHIT with equal amount of free CPO solution (2 μ L,2.0 × 10) -5 mmol·L -1 ) The catalyst was added to 1.5mL of acetonitrile aqueous solution (volume fraction of 0%, 5%, 10%, 15%, 20%, 25%, 30%) at different volume concentrations and left at room temperature for 1h for catalyzing the MCD chlorination reaction. The conversion rates of CPO @ DSP-HA, CPO @ DSP-CHIT and free CPO solution to MCD without adding acetonitrile aqueous solution are respectively regarded as 100%, and the acetonitrile volume concentration is plotted by the relative activities of CPO @ DSP-HA, CPO @ DSP-CHIT and free CPO solution under different acetonitrile volume concentrations to represent the acetonitrile tolerance of CPO @ DSP-HA, CPO @ DSP-CHIT and free CPO, and the result is shown in figure 9.
As can be seen from FIG. 9, the free CPO was almost completely inactivated by the treatment with 20% by volume aqueous acetonitrile, whereas CPO @ DSP-HA and CPO @ DSP-CHIT retained about 88.46% and 83.93% of the activity.
(3) Tolerance to N, N-Dimethylformamide (DMF)
Mixing CPO @ DSP-HA and CPO @ DSP-CHIT with equal amount of free CPO solution (2 μ L,2.0 × 10) -5 mmol·L -1 ) Respectively adding 1.5mL of DMF aqueous solution with different volume concentrations (volume fractions are 0%, 5%, 10%, 15%, 20%, 25% and 30%) and standing at room temperature for 1h for catalyzing MCD chlorination reaction. The conversion rates of CPO @ DSP-HA, CPO @ DSP-CHIT and free CPO solution to MCD without adding DMF aqueous solution were respectively regarded as 100%, and the DMF tolerance of CPO @ DSP-HA, CPO @ DSP-CHIT and free CPO was represented by plotting the relative activities of CPO @ DSP-HA, CPO @ DSP-CHIT and free CPO solution at different DMF volume concentrations against the DMF volume concentration, and the results are shown in FIG. 10.
As can be seen from FIG. 10, the residual activity of free CPO was almost zero after treatment with 15% by volume of DMF aqueous solution, while the residual activities of CPO @ DSP-HA and CPO @ DSP-CHIT could still reach 82.15% and 73.13%, respectively.
5. Electrostatic driving force of binding between CPO and support
DSP, DSP-NH 2 DSP-HA and DSP-CHIT were ultrasonically dispersed into PBS buffer solution of pH =3 and pH =5, respectively, and their Zeta potentials at different pH were determined with a laser particle sizer, as in table 1.
TABLE 1
Figure BDA0002057536450000071
As can be seen from Table 1, in PBS buffer solutions with pH 3 and pH 5, both DSPs were negatively charged and modified with amino groups to give DSP-NH 2 The electric properties in both buffer solutions became positive. Due to the existence of free carboxyl in hyaluronic acid, the DSP-HA is electronegative, when the pH of the PBS buffer solution is 3, the electric property of the CPO is positive, and the CPO and the DSP-HA carrier are mainly combined through electrostatic interaction; due to amino groups in chitosan chainsUnder acidic conditions protonation to-NH occurs 3 + The DSP-CHIT is strong positive, when the pH of the PBS buffer solution is 5, the electric property of the CPO is negative, and the CPO and the carrier DSP-HA are combined mainly through electrostatic interaction.
Example 2
CPO @ DSP-HA and CPO @ DSP-CHIT degradation drug
1. Degradation of levofloxacin
Adding 5mg CPO @ DSP-HA or CPO @ DSP-CHIT, 2480 mu L0.1mol.L into 10mL centrifuge tube -1 PBS buffer solution with pH of 2.75, 500. Mu.L of standard solution of levofloxacin with different concentrations (making the initial concentration of levofloxacin in the final reaction system 10. Mu.g/mL, 20. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL), and finally 20. Mu.L of 0.1 mol. L -1 H 2 O 2 The aqueous solution starts the reaction. The reaction was carried out for 25min at room temperature under magnetic stirring, and after the reaction was completed, it was extracted with ethyl acetate 3 times. And finally, completely evaporating and removing the extract liquor by using a rotary evaporator, and dissolving a sample by using chromatographic pure acetonitrile to obtain a crude sample. Filtering the crude sample by a 0.22 mu m organic phase filter membrane, and analyzing and determining by using a high performance liquid chromatography (HPLC-15C) under the following determination conditions: under the mode of equal gradient, acetonitrile-KH is adopted 2 PO 4 An aqueous solution (V/V = 20).
The degradation rate (η) is calculated according to the formula: eta = (C) 0 -C t )/C 0 X 100%, wherein C t Showing the concentration of levofloxacin at time t after the addition of enzyme, C 0 The levofloxacin concentration in the reaction system without the addition of enzyme is shown.
The experimental result shows that when the initial concentration of the levofloxacin is 10 mu g/mL, the degradation rates of the CPO @ DSP-HA and the CPO @ DSP-CHIT on the levofloxacin are 97.52% and 96.93% respectively; when the concentration of the levofloxacin is 20 mug/mL, the degradation rates of CPO @ DSP-HA and CPO @ DSP-CHIT on the levofloxacin are 96.21% and 96.52% respectively; when the initial concentration of the levofloxacin is 50 mug/mL, the degradation rates of CPO @ DSP-HA and CPO @ DSP-CHIT on the levofloxacin are 95.49% and 95.34% respectively; when the concentration of the levofloxacin is 100 mu g/mL, the degradation rates of the CPO @ DSP-HA and the CPO @ DSP-CHIT on the levofloxacin are 87.17 percent and 87.04 percent respectively.
Three sewage water sources (artificial lake water, black river water and domestic sewage) around schools are selected as experimental water samples to be applied to a reaction system for catalyzing and degrading levofloxacin by an enzyme reactor. Adding three water samples and levofloxacin solutions with different concentrations (prepared by water sample) into 5mg CPO @ DSP-HA or CPO @ DSP-CHIT, and adding 10 μ L of 0.1 mol. L -1 H of (A) to (B) 2 O 2 The final volume of the aqueous solution was 1500. Mu.L, and the reaction was carried out for 25min under magnetic stirring to complete the reaction. After the reaction was completed, it was extracted 3 times with ethyl acetate. And finally, completely evaporating and removing the extract liquor by using a rotary evaporator, and dissolving a sample by using isometric chromatographic pure acetonitrile to obtain a crude sample. The crude sample was filtered through a 0.22 μm organic phase filtration membrane and then subjected to high performance liquid chromatography (HPLC-15C) analysis, and the results are shown in FIGS. 11 and 12.
As can be seen from the figure, when the initial concentration of the levofloxacin in the water sample is 20 mug/mL, the degradation rates of the CPO @ DSP-HA and the CPO @ DSP-CHIT on the levofloxacin in the three water sources within 25min can reach more than 80%; when the initial concentration of the levofloxacin reaches 100 mu g/mL, the degradation rates of CPO @ DSP-HA on the levofloxacin in the black river water and the artificial lake water can respectively reach 80.17 percent and 85.45 percent, the degradation rate on the levofloxacin in the domestic sewage can reach 60.32 percent, the degradation rates of CPO @ DSP-CHIT on the levofloxacin in the black river water and the artificial lake water can respectively reach 78.62 percent and 82.18 percent, and the degradation rate on the levofloxacin in the domestic sewage can reach 71.36 percent. Therefore, CPO @ DSP-HA and CPO @ DSP-CHIT can achieve better degradation effect on levofloxacin in different water qualities.
2. Degrading rifaximin
Preparing rifaximin standard solutions with different concentrations by using a mixed solution of methanol and acetonitrile, 0.075mol/L potassium dihydrogen phosphate aqueous solution and 1.0mol/L citric acid aqueous solution with the volume ratio of 30; adding 500 μ L rifaximin standard solution and 2400 μ L PBS buffer with pH =2.75 into five 10mL centrifuge tubesDissolving, 5mg CPO @ DSP-HA or CPO @ DSP-CHIT, 100 μ L0.1 mol/L H 2 O 2 Aqueous solution, room temperature reaction for 25min. After the reaction is finished, ethyl acetate is used for extraction for 3 times, all extraction liquid is evaporated and removed by a rotary evaporator, and then a sample is dissolved by chromatographic pure acetonitrile to obtain a crude sample. The crude sample was filtered through a 0.22 μm organic phase filtration membrane and then subjected to analysis and determination by high performance liquid chromatography (HPLC-15C). The high performance liquid chromatography determination conditions are as follows: under an isocratic mode, a mixed solution of methanol and acetonitrile, 0.075mol/L potassium dihydrogen phosphate aqueous solution and 1.0mol/L citric acid aqueous solution in a volume ratio of 30; the detection conditions are as follows: flow rate 1.0mL/min -1 The detection wavelength is 254nm, the column temperature is room temperature, and the sample injection amount is 20 mu L.
The experimental result shows that when the initial concentration of the rifaximin is 10 mug/mL, the rifaximin degradation rates of CPO @ DSP-HA and CPO @ DSP-CHIT are 95.60% and 95.52% respectively; when the rifaximin concentration is 20 mug/mL, the rifaximin degradation rates of CPO @ DSP-HA and CPO @ DSP-CHIT are 93.25% and 94.21%, respectively; when the rifaximin concentration is 50 mu g/mL, the rifaximin degradation rates of CPO @ DSP-HA and CPO @ DSP-CHIT are 90.33% and 91.49%, respectively; when the initial concentration of the rifaximin is 100 mu g/mL, the degradation rates of the rifaximin by CPO @ DSP-HA and CPO @ DSP-CHIT are 86.12% and 88.10%, respectively.
Three sewage sources (artificial lake water, black river water and domestic sewage) around schools are selected as experimental water samples to be applied to a reaction system for catalytic degradation of rifaximin by an enzyme reactor. Adding three water samples and rifaximin solutions with different concentrations into 5mg CPO @ DSP-HA or CPO @ DSP-CHIT respectively, and finally adding 10 mu L0.1mol.L -1 H of (A) to (B) 2 O 2 The final volume was kept at 1500. Mu.L, and the reaction was carried out for 25min under magnetic stirring to complete the reaction. After the reaction is finished, extracting for 3 times by using ethyl acetate, completely evaporating and removing extraction liquid by using a rotary evaporator, and dissolving a sample by using chromatographic pure acetonitrile to obtain a crude sample. The crude sample was filtered through a 0.22 μm organic phase filtration membrane and then subjected to high performance liquid chromatography (HPLC-15C) analysis, and the results are shown in FIGS. 13 and 14.
As can be seen from the figure, when the initial concentration of rifaximin in a water sample is 20 mug/mL, the rifaximin degradation rates of CPO @ DSP-HA and CPO @ DSP-CHIT can reach more than 90% within 25min, and the degradation effect is good; when the initial concentration of rifaximin in a water sample reaches 100 mug/mL, the degradation rates of CPO @ DSP-HA to rifaximin in black river water and artificial lake water can reach 82.32% and 88.55% respectively, and the degradation rate to rifaximin in domestic sewage can reach 76.32%; the degradation rates of CPO @ DSP-CHIT to rifaximin in black river water and artificial lake water can respectively reach 83.36% and 88.62%, and the degradation rate to rifaximin in domestic sewage can reach 78.25%.

Claims (6)

1. Surface charge-controlled functionalized dendritic mesoporous SiO 2 An immobilized chloroperoxidase reactor, characterized in that: the enzyme reactor is dendritic mesoporous SiO with surface amino modified 2 The nano particles are crosslinked with hyaluronic acid or amino protonated chitosan to be used as a carrier, and are obtained by fixing chloroperoxidase through electrostatic interaction and hydrogen bond interaction;
the surface amino modified dendritic mesoporous SiO 2 After the nano particles are crosslinked with hyaluronic acid, under the condition that the pH value is 3-4, fixing chloroperoxidase through electrostatic interaction and hydrogen bond interaction;
the surface amino modified dendritic mesoporous SiO 2 After the nano particles are crosslinked with amino protonated chitosan, under the condition that the pH value is 5-6, the chloroperoxidase is fixed through electrostatic action and hydrogen bond action.
2. The surface charge-controlled functionalized dendritic mesoporous SiO of claim 1 2 An immobilized chloroperoxidase reactor, characterized by: the compound adopted for surface amino modification is any one of 3-aminopropyltriethoxysilane, tetraethylenepentamine and N-aminoethyl-3-aminopropylmethyldimethoxysilane.
3. The surface charge-controlled functionalized dendritic mesoporous SiO of claim 1 2 An immobilized chloroperoxidase reactor, characterized in that: the hyaluronic acid is under the condition that the pH value is 8-10Reacting with N-hydroxysuccinimide sulfonic acid sodium salt and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride to modify dendritic mesoporous SiO with surface amino 2 And (4) crosslinking the nanoparticles.
4. The surface charge-controlled functionalized dendritic mesoporous SiO of claim 1 2 An immobilized chloroperoxidase reactor, characterized in that: the amino protonated chitosan is prepared by modifying glutaraldehyde and surface amino with dendritic mesoporous SiO 2 And (4) crosslinking the nanoparticles.
5. The surface charge-controlled functionalized dendritic mesoporous SiO of claim 1 2 Application of an immobilized chloroperoxidase reactor in degradation of levofloxacin.
6. The surface charge-controlled functionalized dendritic mesoporous SiO of claim 1 2 Use of an immobilized chloroperoxidase reactor for the degradation of rifaximin.
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