CN111484990B - Cobaltose peroxidase-loaded nanoreactor modified by polydopamine and prepared from cobalt hierarchical porous material and application of nanoreactor - Google Patents
Cobaltose peroxidase-loaded nanoreactor modified by polydopamine and prepared from cobalt hierarchical porous material and application of nanoreactor Download PDFInfo
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Abstract
The invention discloses a polydopamine-modified cobalt hierarchical pore material loaded horseradish peroxidase nanoreactor and application thereof. The enzyme reactor is Co obtained by calcining Co-MOF in air or nitrogen 3 O 4 Or the Co/C hierarchical porous material is prepared by modifying polydopamine and then serving as a carrier to load Horse Radish Peroxidase (HRP). The carrier simultaneously has the characteristics of micropores and mesopores, the pore diameter of the mesopores is kept between 8 and 12nm through the accurate regulation and control of the pore diameter, the single-molecule array trapping of HRP can be realized, and a certain space is reserved for the twisting and overturning of enzyme molecules in the biological catalysis process; micropores densely distributed around the mesopores can enrich the substrate to reduce diffusion resistance; the polydopamine does not influence the performance of hierarchical pores, and can improve the biocompatibility of the material and the capacity of combining with enzyme molecules. The enzyme nano reactor has high catalysisThe catalyst has the advantages of chemical activity, thermal stability and reusability, is used for phenol degradation, can keep 66.1 percent of catalytic activity after being reused for 25 times, and has the degradation rate of nearly 100 percent within 15 min.
Description
Technical Field
The invention belongs to the technical field of enzyme immobilization, and particularly relates to a hierarchical pore Co functionally modified by the surface of a polydopamine biomimetic membrane 3 O 4 Or Co/C is used as a carrier, and the nano-reactor is used for loading horseradish peroxidase and application thereof.
Background
Free enzyme is used as an efficient and green biocatalyst, and is widely applied to the fields of synthetic chemistry, food processing, pharmacy, wastewater treatment and the like due to the advantages of good specificity, high catalytic efficiency, mild reaction conditions and the like. However, since the free enzyme has the disadvantages of poor operation stability, easy inactivation under extreme conditions, inability of reuse and recovery, etc. in practical applications, it is considered that the construction of a supported bio-enzyme nanoreactor by loading the free enzyme into a solid phase carrier is one of the most effective ways to improve the stability of the enzyme.
Polydopamine (PDA) is an adhesive polymer with a unique surface modification function, can be used for surface modification of almost any chemical material, and has good hydrothermal stability and biocompatibility.
Horse Radish Peroxidase (HRP) is a glycoprotein complex enzyme containing iron porphyrin prosthetic group, and is the most widely studied peroxidase at present. In the presence of hydrogen peroxide, horseradish peroxidase can catalyze the oxidation of various compounds, especially substances containing large pi conjugated systems, such as phenols, aromatics, anilines, indoles and other compounds. It has high specific activity, stability, small molecular weight, easy preparation of pure enzyme, wide distribution in plant kingdom, and high content of horseradish.
Metal-Organic Frameworks (MOFs for short) are crystalline porous materials with periodic network structures formed by self-assembly of inorganic Metal centers (Metal ions or clusters) and Organic ligands. As the MOFs material used as the carrier has the excellent characteristics of large specific surface area, uniform and stable pore size distribution and the like, the MOFs material loaded enzyme has wide application prospects in the aspects of biomedicine, biocatalysis, new material preparation, chemical production and the like as a novel solid catalyst. The existing preparation method of MOFs material loaded enzyme mainly comprises four methods, namely an adsorption method, an embedding method, a covalent immobilization method and a coprecipitation method. The method for preparing the porous nano material based on MOF precursor calcination is simple, the conversion rate is high, the prepared porous material not only has the advantages of MOF, but also has the excellent characteristics of adjustable pore structure, good thermal stability, chemical stability and the like, and can become an ideal enzyme-loaded carrier, but the large-scale application of the MOFs material as a template to prepare the porous material is greatly limited due to the complex and difficult control of the operation process of thermally decomposing the MOFs to prepare the porous material and adopting the porous material to load the enzyme.
Disclosure of Invention
The invention aims to provide a polydopamine biomimetic membrane surface functionalized modified cobalt hierarchical pore material loaded horseradish peroxidase nano-reactor with high catalytic activity, good thermal stability and reusability, and provides a new application for the enzyme nano-reactor.
Aiming at the aim, the enzyme nano-reactor adopted by the invention is Co with mesoporous aperture of 8-12 nm, which is subjected to surface functional modification by a poly-dopamine biomimetic membrane 3 O 4 The material is obtained by taking a hierarchical pore material or a Co/C hierarchical pore material with a mesoporous aperture of 8-12 nm, which is subjected to surface functionalization modification by a polydopamine biomimetic membrane, as a carrier and loading horseradish peroxidase through electrostatic interaction and covalent interaction.
In the enzyme nano-reactor, the mass ratio of the carrier to the horseradish peroxidase is 1.1-0.2.
The mesoporous aperture of the Co is 8-12 nm 3 O 4 The hierarchical porous material is obtained by roasting Co-MOF for 2h at 500 ℃ in an air atmosphere.
The Co/C hierarchical porous material with the mesoporous aperture of 8-12 nm is obtained by roasting Co-MOF for 2 hours at 600 ℃ in a nitrogen atmosphere.
The carrier is Co with the mesoporous aperture of 8-12 nm 3 O 4 The material is prepared by dispersing a hierarchical pore material or a Co/C hierarchical pore material with a mesoporous aperture of 8-12 nm and dopamine hydrochloride in ultrapure water and then adding hexamethylenetetramine.
The polydopamine modified cobalt hierarchical pore material loaded horseradish peroxidase nano-reactor is applied to degrading organic pesticide phenol.
Compared with the prior art, the invention has the following beneficial effects:
1. according to the invention, co-MOF prepared by a hydrothermal method is taken as a precursor, the precursor is respectively calcined in an air atmosphere and a nitrogen atmosphere, and Co is obtained by controlling the calcination time and temperature 3 O 4 And the aperture of the Co/C hierarchical pore material is accurately regulated, so that the material has the characteristics of micropores and mesopores, and the pore diameter of the mesopores is matched with the size of enzyme molecules. Then, the surface of the poly-dopamine biomimetic membrane is functionally modified and then is used as a carrier to fix horse radish peroxidase on the multi-level hole Co through electrostatic interaction and covalent interaction 3 O 4 And mesoporous channels of Co/C. Co 3 O 4 And the mesoporous aperture of the Co/C hierarchical porous material is 8-12 nm, so that the single-molecule array loading of horseradish peroxidase can be realized, a certain space is reserved for the twisting and overturning of enzyme molecules in the biological catalysis process of horseradish peroxidase, and micropores densely distributed around the mesopores play a role of enriching substrates, can reduce the diffusion resistance of the substrate molecules, and is very favorable for the enzyme catalysis process. Co coating by using poly-dopamine biomimetic membrane 3 O 4 And the Co/C hierarchical pore material can not block the pore channel of the material, can not influence the performance of the hierarchical pore, and further improves the biocompatibility of the material and the capability of combining with enzyme molecules.
2. Compared with free enzyme, the enzyme nano-reactor disclosed by the invention can be used for synergistically improving the catalytic activity and the thermal stability of horseradish peroxidase, overcoming the defect of enzyme molecule leakage in the repeated use process and increasing the repeated use times.
3. The enzyme nanoreactor disclosed by the invention is used for degrading the organic pesticide phenol in the simulated wastewater, and the result shows that a very good degradation effect can be achieved in a short time in an actual reaction environment with non-optimal reaction conditions.
Drawings
FIG. 1 is a MHCo prepared in example 1 3 O 4 Transmission electron microscopy.
FIG. 2 is FITC-HRP @ PDA @ MHCo prepared in example 1 3 O 4 Confocal laser microscopy.
FIG. 3 is a field emission transmission electron micrograph of Co/C prepared in example 2.
FIG. 4 is a confocal laser micrograph of FITC-HRP @ PDA @ Co/C prepared in example 2.
Fig. 5 is an isothermal titration calorimetry curve: (a) HRP @ PDA @ MHCo 3 O 4 -500；(b)HRP@PDA@MHCo 3 O 4 -600；(c)HRP@PDA@Co/C-500；(d)HRP@PDA@Co/C-600；(e)HRP@PDA@Co/C-800。
FIG. 6 is free HRP, HRP @ PDA @ MHCo prepared in example 1 3 O 4 And the ultraviolet spectrogram of the ABTS peroxidation catalyzed by HRP @ PDA @ Co/C prepared in example 2.
FIG. 7 is the reaction product of free HRP and HRP @ PDA @ MHCo 3 O 4 Thermal stability profile of (a).
FIG. 8 is a graph of the thermal stability of free HRP versus HRP @ PDA @ Co/C.
FIG. 9 is HRP @ PDA @ MHCo 3 O 4 The reuse number of (c) figure.
FIG. 10 is a graph of the number of reuses of HRP @ PDA @ Co/C.
FIG. 11 is HRP @ PDA @ MHCo 3 O 4 Degradation pattern for phenol.
FIG. 12 is a graph showing the degradation of phenol by HRP @ PDA @ Co/C.
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.
The Co-MOF used in the examples below is based onThe preparation method comprises the following steps: 0.1455g of Co (NO) 3 ) 2 ·6H 2 Placing O, 0.1051g of trimesic acid, 1.31g of polyvinylpyrrolidone, 0.0721g of pyrazine and 30mL of DMF in a 50mL polytetrafluoroethylene hydrothermal reaction kettle, carrying out ultrasonic treatment for 30min, sealing, heating to 150 ℃ within 5h, keeping the temperature for 24h, slowly cooling to room temperature, washing the product with absolute ethyl alcohol for multiple times, and placing in a vacuum drying oven at 60 ℃ for drying for 6h to obtain the spherical Co-MOF with regular morphology.
Example 1
Placing 100mg Co-MOF in a quartz porcelain boat, placing in a resistance furnace, heating to 500 deg.C at a rate of 1 deg.C/min, maintaining at 500 deg.C for 2 hr, naturally cooling to room temperature, and converting the sample from original purple color to black glossy crystal to obtain Co 3 O 4 Hierarchical porous material (noted as MHCo) 3 O 4 ) As shown in fig. 1.
0.241g of MHCo 3 O 4 0.0095g of dopamine hydrochloride is dispersed in 50mL of ultrapure water, 0.102g of hexamethylenetetramine is added, after 30s of sealed vortex, the mixture is incubated for 3h at 90 ℃, naturally cooled to room temperature, centrifuged for 3min at 10000r/min, supernatant is removed, the mixture is washed for a plurality of times by water and absolute ethyl alcohol and dried in a vacuum drying oven at 60 ℃ for 12h to obtain the Co modified by the surface functionalization of the poly-dopamine biomimetic membrane 3 O 4 Hierarchical porous material (marked as PDA @ MHCo 3 O 4 )。
Mixing 5mg PDA @ MHCo 3 O 4 Adding the mixture into 1.5mL of 0.1mol/L phosphate buffer solution with pH =3, then adding 100 mu L of 0.25mmol/L HRP aqueous solution, placing the mixture into a freezing water bath constant temperature oscillator for oscillating for 2h, centrifuging the mixture at 6000r/min for 3min, separating supernatant from solid, washing the solid with phosphate buffer solution for 2-3 times, centrifuging the solid under the same condition after each washing to remove the HRP not loaded on the surface of the carrier, and finally drying the solid in vacuum at 30 ℃ for 12h to obtain the polydopamine modified Co 3 O 4 Hierarchical porous material loaded horse radish peroxidase nano-reactor (marked as HRP @ PDA @ MHCo) 3 O 4 )。
Horseradish peroxidase labeled with Fluorescein Isothiocyanate (FITC) (labeled as FITC-HRP) instead of the HRP described above, FITC-HRP @ PDA @ MHCo was prepared 3 O 4 For confocal laser microscopy, the results are shown in FIG. 2. A number of uniformly distributed green fluorescent spots are visible in FIG. 2, MHCo due to the support PDA @ 3 O 4 It is not a fluorescent material by itself, so the observed fluorescent signal is derived from FITC-HRP incorporated into the carrier. The above experimental results confirm that HRP was successfully loaded to MHCo 3 O 4 In (1).
Example 2
Uniformly spreading 100mg of Co-MOF in a quartz porcelain boat, placing in a tube furnace, heating to 600 ℃ at the speed of 1 ℃/min under the nitrogen atmosphere, preserving the heat at 600 ℃ for 2h under the nitrogen atmosphere, naturally cooling to room temperature, and keeping the sample in the nitrogen atmosphere during the temperature reduction process. The sample turned from the original purple color to black, shiny crystals with a yield of about 35%. The obtained black crystals were fully ground to obtain a Co/C hierarchical porous material, as shown in FIG. 3.
Dispersing 0.200g of Co/C hierarchical porous material and 0.0095g of dopamine hydrochloride in 50mL of ultrapure water, adding 0.102g of hexamethylenetetramine, sealing vortex for 30s, incubating at 90 ℃ for 3h, naturally cooling to room temperature, centrifuging at 10000r/min for 3min, removing supernatant, washing with water and absolute ethyl alcohol for several times, and drying in a vacuum drying oven at 60 ℃ for 12h to obtain the Co/C hierarchical porous material (marked as PDA @ Co/C) with the surface of the polydopamine biomimetic membrane subjected to functional modification.
Adding 5mg PDA @ Co/C into 1.5mL0.1mol/L phosphate buffer with pH =3, then adding 100 mu L0.25mmol/L HRP aqueous solution, placing the mixture in a freezing water bath constant temperature oscillator for oscillation for 2h, centrifuging at the rotating speed of 6000r/min for 3min, separating supernatant and solid, washing the solid with phosphate buffer for 2-3 times, centrifuging under the same condition after each washing to remove the HRP not loaded on the surface of the carrier, and finally drying in vacuum at 30 ℃ for 12h to obtain the polydopamine modified Co/C hierarchical porous material loaded horseradish peroxidase nano-reactor (marked as HRP @ PDA @ Co/C).
FITC-HRP @ PDA @ Co/C was prepared using fluorescein isothiocyanate labeled horseradish peroxidase (denoted FITC-HRP) in place of the HRP described above and used for confocal laser microscopy testing, and the results are shown in FIG. 4. A number of uniformly distributed spots of green fluorescence are visible in FIG. 4, since the support PDA @ Co/C is not a fluorescent material by itself, and the observed fluorescence signal is derived from FITC-HRP incorporated into the support. The above experimental results confirm that HRP was successfully loaded in the Co/C hierarchical porous material.
To determine the preparation conditions of the hierarchical porous materials of examples 1 and 2, the inventors prepared two MHCo materials by heating Co-MOF as a template to 500 ℃ and 600 ℃ respectively in an air atmosphere 3 O 4 Hierarchical porous material, labelled MHCo 3 O 4 500 and MHCo 3 O 4 -600; three Co/C hierarchical porous materials are prepared by respectively heating to 500 ℃, 600 ℃ and 800 ℃ in a nitrogen atmosphere, and are marked as Co/C-500, co/C-600 and Co/C-800. Then, a series of screening tests are carried out on the two prepared materials, and the carrier is evaluated through enzyme loading capacity and enzyme catalytic activity, wherein the specific test steps are as follows: the two types of materials with different pore sizes are sequentially selected as carriers, a physical adsorption method is utilized to load HRP respectively, and the enzyme loading amount is calculated according to a formula (1):
in formula (1), Δ a: the change of the absorbance value of HRP at the ultraviolet wavelength of 403 nm; v: final volume of experimental system (mL); epsilon: molar absorptivity of HRP (102000L. Mol) -1 ·cm -1 ) (ii) a l: width (cm) of cuvette used in experiment; m: the mass of the carrier (g) added. The results are shown in Table 1.
And the enzyme catalytic activity of the supported enzyme was tested as follows: the HRP catalytic activity is measured by taking stable blue-green ABTS · + free radical generated by HRP catalytic oxidation of 2,2' -biazoyl-bis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) as a model reaction, and the specific method comprises the following steps: adding 30 μ L of 0.01mol/L ABTS aqueous solution and free HRP or equivalent amount of free enzyme-loaded enzyme into pH 3 phosphate buffer, and finally adding 30 μ L of 0.1mol/L H 2 O 2 Shaking the aqueous solution, and shaking in a shaking tableCentrifuging for 2min after 20min, measuring the absorbance value of the reaction solution at 415nm by using an ultraviolet spectrophotometer, and calculating the conversion rate of ABTS according to the formula (2):
in the formula, A: actually measuring an absorbance value by an ultraviolet spectrophotometer; epsilon 415nm : ABTS. + molar absorption coefficient at 415 nm; b: width (cm) of cuvette used in measurement; c ABTS : ABTS substrate concentration (mol/L) before reaction. That is, ABTS is catalyzed and oxidized by using free enzyme and supported enzyme with the same amount, and the catalytic activity of the free enzyme and the catalytic activity of the supported enzyme are compared, and the result is shown in Table 1.
Meanwhile, carriers are screened by means of an isothermal titration micro calorimetry (ITC) technology, the change of heat flow of a supported enzyme catalytic reaction prepared by the carrier material with the surface functionalized modification along with time is measured, an isothermal titration calorimetry curve (shown in figure 5) is obtained, a nonlinear least square method is adopted for fitting to obtain thermodynamic parameters, and the result is shown in table 2.
TABLE 1 results of Carrier screening test and pore characteristics
TABLE 2 thermodynamic parameters of the Supported enzymes
As can be seen from Table 1, MHCo 3 O 4 500 and MHCo 3 O 4 The mesoporous aperture ranges of the-600 two materials are respectively 8-12 nm and 3-19 nm, wherein the mesoporous aperture is MHCo with the mesoporous aperture of 8-12 nm 3 O 4 The material has relatively high enzyme loading capacity and enzyme catalytic activity; the mesoporous aperture ranges of the Co/C-500, co/C-600 and Co/C-800 materials are respectively 6-9 nm, 8-12 nm and 3-5 nm, wherein the Co/C material with the mesoporous aperture range of 8-12 nm has the advantages thatHigh enzyme loading and enzyme catalytic activity. As is apparent from Table 2 and FIG. 5, MHCo was obtained in an air atmosphere 3 O 4 500 and MHCo 3 O 4 The catalytic reaction of the supported enzyme prepared from the-600 two materials belongs to endothermic reaction, and HRP @ PDA @ MHCo 3 O 4 Binding constant value (Ka) of-500 greater than HRP @ PDA @ MHCo 3 O 4 Binding constant value (Ka) of-600, indicating HRP @ PDA @ MHCo 3 O 4 Binding affinity of-500 to substrate vs. HRP @ PDA @ MHCo 3 O 4 -600 large; the supported enzyme catalysis reactions prepared from the Co/C-500, co/C-600 and Co/C-800 materials prepared in the nitrogen atmosphere belong to endothermic reactions, and the binding affinity of HRP @ PDA @ Co/C-600 to a substrate is greater than that of HRP @ PDA @ Co/C-500 and HRP @ PDA @ Co/C-800. Therefore, the invention selects MHCo with the mesoporous aperture of 8-12 nm 3 O 4 Hierarchical porous materials and Co/C hierarchical porous materials, i.e. MHCo 3 O 4 And-500 and Co/C-600 are carriers to prepare an enzyme nano reactor loaded with HRP. As can be seen from Table 2 and FIG. 6, HRP @ PDA @ MHCo prepared in example 1 3 O 4 And HRP @ PDA @ Co/C prepared in example 2 were loaded with horseradish peroxidase at 109.5mg/g, 137.4mg/g, respectively, and the results showed that it retained catalytic activities of 96.8% and 93.7%, respectively, compared with the free enzyme.
HRP @ PDA @ MHCo prepared in example 1 above 3 O 4 The heat stability and reusability tests were carried out with HRP @ PDA @ Co/C prepared in example 2, as follows:
1. thermal stability test
5mg of HRP @ PDA @ MHCo prepared in example 1 3 O 4 Or the HRP @ PDA @ Co/C prepared in the example 2 and the equivalent amount of free HRP are respectively incubated for 1h at different temperatures (30-100 ℃), the temperature is naturally cooled and then the temperature is used for catalyzing ABTS to generate oxidation reaction, the conversion rate when the temperature is 0min is regarded as 100%, and the temperature is plotted by the relative activity of the oxidation activity at other times relative to 0min, so that the free HRP, the HRP @ PDA @ MHCo and the like are represented 3 O 4 Thermal stability of HRP @ PDA @ Co/C, results are shown in FIGS. 7 and 8.
As can be seen from the figure, free HRP and HRP@PDA@MHCo 3 O 4 The catalytic activity of HRP @ PDA @ Co/C for catalyzing ABTS oxidation reaction is reduced along with the increase of temperature. Free HRP and HRP @ PDA @ MHCo 3 O 4 The heat stability of HRP @ PDA @ Co/C is not greatly different between 30 ℃ and 50 ℃, the optimal reaction temperature is 40 ℃, but when the temperature exceeds 60 ℃, the catalytic activity of free HRP is obviously reduced, the catalytic activity can only be kept at 18.1% after the HRP @ PDA @ Co/C is incubated for 1h at 70 ℃, and under the same condition, the HRP @ MHCo/C can only be kept at 18.1% after the HRP @ PDA @ Co/C is incubated for 1h 3 O 4 And HRP @ PDA @ Co/C can still maintain 84.8% and 70.8% of catalytic activity, and can still maintain 82.6% and 55.6% of catalytic activity even after being incubated for 1h at 90 ℃, while HRP @ MHCo 3 O 4 (non-polydopamine modified MHCo) 3 O 4 Prepared by loading HRP on a carrier) and HRP @ Co/C (prepared by loading HRP on a carrier without polydopamine modification) can only maintain 65.7 percent and 57.3 percent of catalytic activity, and can only maintain 45.2 percent and 42.8 percent of catalytic activity even when the carrier is incubated for 1h at 90 ℃. The above experimental results show that HRP @ PDA @ MHCo 3 O 4 And the thermal stability of HRP @ PDA @ Co/C is obviously improved compared with that of free HRP, and further proves that the enzyme nano-reactor prepared after the polydopamine is functionally modified on the surface of the cobalt hierarchical porous material has good thermal stability.
2. Reusability test
HRP @ PDA @ MHCo 3 O 4 Or HRP @ PDA @ Co/C is used for ABTS peroxidation, after reaction for 30min, the absorbance value of the upper layer reaction liquid at 415nm is centrifugally determined, and the lower layer HRP @ PDA @ MHCo is used 3 O 4 Or HRP @ PDA @ Co/C is used for catalyzing next ABTS peroxidation reaction, the first ABTS conversion rate is 100%, the conversion rate of each time is compared with that of the first time, and the residual activity is used for representing HRP @ PDA @ MHCo 3 O 4 And reusability of HRP @ PDA @ Co/C, the results are shown in FIG. 9 and FIG. 10.
As can be seen from FIGS. 9 and 10, it is shown that the HRP @ MHCo 3 O 4 Compared with HRP @ Co/C, HRP @ PDA @ MHCo 3 O 4 And HRP @ PDA @ Co/C, the HRP @ PDA @ MHCo 3 O 4 Can still maintain 92.4 percent of catalytic activity after being repeatedly used for 10 timesThe result shows that the catalyst can maintain 66.1% of catalytic activity even after being repeatedly used for 25 times, and the catalyst can maintain 85.4% of catalytic activity even after being repeatedly used for 10 times by HRP @ PDA @ Co/C, and can maintain 58.7% of catalytic activity even after being repeatedly used for 20 times, so that the catalyst has good reusability.
Example 3
Example 1 preparation of HRP @ PDA @ MHCo 3 O 4 And example 2 preparation of HRP @ PDA @ Co/C degradation simulation wastewater in phenol application
Glucose (6 g/L), ammonium nitrate (1.0 g/L), potassium dihydrogen phosphate (0.5 g/L), dipotassium hydrogen phosphate (1.5 g/L), sodium chloride (0.5 g/L), potassium chloride (0.5 g/L) and xylene, toluene and benzene all at a concentration of 50mg/L were added to tap water, and then 2mL of CuSO containing 0.1g/L was added 4 And 0.2g/L of ZnSO 4 The water solution is used for preparing artificial simulated organic wastewater containing 15.67mg/L of tap water. Adding artificial simulated wastewater into 10mL centrifuge tube wrapped by aluminum foil, and adding 2mg HRP @ PDA @ MHCo 3 O 4 Or HRP @ PDA @ Co/C and phenol solution, and then adding a certain volume of 10mmol/L H into the reaction system 2 O 2 Aqueous solution, maintaining a final volume of 3mL. After 15min of dark reaction at room temperature, centrifugation was carried out, 1mL of supernatant was transferred to a 10mL centrifuge tube using a micropipette, and an equal volume of ethyl acetate was added for extraction. Transferring the extracted upper layer liquid into a 100mL flask for rotary evaporation, and adding 1mL of methanol into the flask for dissolution after the solvent is evaporated to dryness to obtain a sample. After the sample was filtered through an organic phase filtration membrane of 0.22 μm, the sample was analyzed by high performance liquid chromatography, and the degradation rate of the substrate was calculated according to the formula (3).
In the formula (3), C 0 : an initial concentration of the substrate; c t : concentration of substrate at time t. The results are shown in Table 3, FIG. 11 and FIG. 12.
TABLE 3
As can be seen from FIGS. 11 and 12, the HRP @ MHCo 3 O 4 HRP @ PDA @ MHCo/C, as compared with HRP @ Co/C 3 O 4 And HRP @ PDA @ Co/C show higher catalytic efficiency in the process of degrading phenol in simulated wastewater, and the two enzyme nano-reactors can realize the complete degradation of phenol with the concentration of 3mmol/L within 15 min. In addition, the two enzyme nano-reactors can also process high-concentration phenol, when the concentration of phenol reaches 9mmol/L, the degradation rate of HRP @ PDA @ Co/C can still reach 96% within 15min, while the degradation rate of HRP @ PDA @ MHCo can still reach 96% within 15min 3 O 4 The degradation rate of the supported enzyme is even close to 100%, and as shown in table 3, the catalytic efficiency of the free enzyme to the substrate is reduced compared with that of the enzyme nano reactor under the same conditions, namely the catalytic activity of the supported enzyme is improved compared with that of the free enzyme. The above results indicate HRP @ PDA @ MHCo 3 O 4 And HRP @ PDA @ Co/C has better degradation effect on the phenol which is an organic poison in the simulated wastewater.
Claims (4)
1. A polydopamine modified cobalt hierarchical pore material load horse radish peroxidase nanoreactor is characterized in that: the enzyme nano-reactor is Co with mesoporous aperture of 8-12 nm, which is functionally modified on the surface of a polydopamine biomimetic membrane 3 O 4 The material is obtained by taking a hierarchical pore material or a Co/C hierarchical pore material with mesoporous aperture of 8-12 nm, which is subjected to surface functionalization modification by a polydopamine biomimetic membrane, as a carrier and loading horseradish peroxidase through electrostatic interaction and covalent interaction;
the mesoporous aperture is 8-12 nm of Co 3 O 4 The hierarchical porous material is obtained by roasting Co-MOF for 2 hours at 500 ℃ in an air atmosphere;
the Co/C hierarchical porous material with the mesoporous aperture of 8-12 nm is obtained by roasting Co-MOF for 2 hours at 600 ℃ in a nitrogen atmosphere;
the Co-MOF is prepared according to the following method: 0.1455g Co (NO) 3 ) 2 ·6H 2 O, 0.1051g trimesic acid, 1.31g polyvinylpyrrolidone, 0.0721g pyrazine and 30mL DMF in 50mL polytetrafluoro-tetrafluoroethaneAnd (3) performing ultrasonic treatment for 30min in an ethylene hydrothermal reaction kettle, sealing, heating to 150 ℃ within 5h, keeping the temperature for 24h, then slowly cooling to room temperature, washing the product with absolute ethyl alcohol for multiple times, and drying in a vacuum drying oven at 60 ℃ for 6h to obtain the spherical Co-MOF with regular morphology.
2. The polydopamine-modified cobalt hierarchical pore material-loaded horseradish peroxidase nanoreactor according to claim 1, characterized in that: in the enzyme nano-reactor, the mass ratio of the carrier to the horseradish peroxidase is 1.
3. The polydopamine-modified cobalt hierarchical pore material-loaded horseradish peroxidase nanoreactor according to claim 1, characterized in that: the carrier is Co with the mesoporous aperture of 8-12 nm 3 O 4 The material is prepared by dispersing a hierarchical pore material or a Co/C hierarchical pore material with a mesoporous aperture of 8-12 nm and dopamine hydrochloride in ultrapure water and then adding hexamethylenetetramine.
4. The application of the polydopamine modified cobalt hierarchical pore material loaded horseradish peroxidase nanoreactor in degrading organic pesticide phenol, according to claim 1.
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