CN116409885A - Hydrogen bond organic framework enzyme biological composite material and preparation method and application thereof - Google Patents
Hydrogen bond organic framework enzyme biological composite material and preparation method and application thereof Download PDFInfo
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- CN116409885A CN116409885A CN202211501650.5A CN202211501650A CN116409885A CN 116409885 A CN116409885 A CN 116409885A CN 202211501650 A CN202211501650 A CN 202211501650A CN 116409885 A CN116409885 A CN 116409885A
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/308—Dyes; Colorants; Fluorescent agents
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
- C02F2103/30—Nature of the water, waste water, sewage or sludge to be treated from the textile industry
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/20—Air quality improvement or preservation, e.g. vehicle emission control or emission reduction by using catalytic converters
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- Life Sciences & Earth Sciences (AREA)
- Microbiology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Hydrology & Water Resources (AREA)
- Engineering & Computer Science (AREA)
- Environmental & Geological Engineering (AREA)
- Water Supply & Treatment (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Fire-Extinguishing Compositions (AREA)
Abstract
The invention discloses a hydrogen bond organic framework enzyme biological composite material, a preparation method and application thereof. The hydrogen bond organic frame enzyme biological composite material takes an organic building unit as a frame main body, and enzyme is wrapped in the frame main body; the organic building unit is a carboxylic acid hydrogen bond organic framework material; the enzyme is laccase. The raw materials used in the synthesis method are easy to obtain, the synthesis method is mild and simple, and the enzyme activity can be reserved to the maximum extent. The Lac@HOF is applied to the catalytic degradation of organic pollutants in water, not only shows excellent catalytic performance, but also obviously improves the resistance to environmental factors, and has extremely high value in practical industrial application.
Description
Technical Field
The invention belongs to the technical field of hydrogen bond organic frame materials, and particularly relates to a hydrogen bond organic frame enzyme biological composite material, a preparation method and application thereof.
Background
Wastewater produced by the textile industry is one of the main sources of pollution for surface and ground water. The synthetic dyes used in dyeing are the main components of wastewater pollutants, because the dyes adhered to fabrics in actual production are very small and most of the dyes remain in the wastewater. About 3X 10 per year worldwide 8 The kg of dye is discharged into the waste water. In addition, due to their good solubility in water, the discharge of wastewater containing these dyes directly into rivers and natural streams can lead to serious environmental problems. The complex aromatic structure of dye molecules and the chemical composition thereof; making them difficult to decompose by oxygen, heat, light and certain oxidizing agents. Thus, the removal or degradation of these dyes is one of the difficulties faced in wastewater treatment. Malachite green is a common triphenylmethane dye and is widely applied to the printing and dyeing industry. Numerous studies have reported that they can produce mutagenic, carcinogenic and teratogenic effects in humans.
At present, the wastewater treatment methods mainly comprise a physicochemical method and a biological method, and the physicochemical methods such as chemical oxidation, membrane separation technology, adsorption and the like are used for wastewater treatment. Although these methods have a certain treatment effect, they are very expensive and may cause secondary pollution. Compared with other methods, the biological method uses plants, microorganisms and enzymes to treat the wastewater, and has the characteristics of easy operation, lower cost, environmental friendliness, safety and the like. Unfortunately, plants and microorganisms are very sensitive to some toxic contaminants in wastewater, which greatly limits their range of applications.
In contrast, enzymes are an effective biocatalyst that can function rapidly and selectively in harsh environments. They have specific active sites that bind to specific substrates and reduce the activation energy of the reaction during enzymatic processes to promote degradation of toxic environmental pollutants. Therefore, has high reaction kinetics and specificity. In addition, enzymatic biodegradation has the advantages of more flexible operating conditions, no need of nutrient supply, less formation of byproducts, fast mass transfer rate and the like. However, the enzyme is easily dissolved in water, is difficult to separate after use, has high environmental requirements, is easy to inactivate, and has expensive purification technology, thus preventing the enzyme from being applied to actual production. Immobilized enzymes are a method developed to overcome a series of problems encountered in enzyme use, and are mainly classified into the following categories: adsorption, entrapment, covalent bonding, and the like.
Therefore, finding a frame carrier with excellent performance and combining it with a biological enzyme by a suitable immobilization method to obtain a biological composite material with excellent catalytic activity is an important problem to be solved.
Disclosure of Invention
In view of the above-mentioned prior art problems, a first object of the present invention is to provide a hydrogen bond organic framework enzyme biocomposite which not only effectively retains the activity of biological enzymes and has excellent removal ability for organic pollutants in sewage, but also has significantly improved resistance to environmental factors, excellent thermal stability, storage stability and bioavailability, and is resistant to strong acids, strong bases and metal ions, and has extremely high value in practical industrial applications.
The second object of the invention is to provide a preparation method of the hydrogen bond organic framework enzyme biological composite material.
A third object of the present invention is to provide the use of the hydrogen bonding organic framework enzyme biocomposite described above for removing organic contaminants.
In order to achieve the above object, the present invention is realized by the following technical scheme:
a hydrogen bond organic frame enzyme biological composite material takes an organic building unit as a frame main body, and enzyme is wrapped in the frame main body; the organic building unit is a carboxylic acid hydrogen bond organic framework material; the enzyme is laccase.
Hydrogen bond organic frameworks (HOFs) are a class of porous materials with periodic structures formed by self-assembly of organic building blocks through hydrogen bonding and pi-pi stacking and van der waals interactions. HOFs do not contain metal nodes, have mild synthesis conditions, have lower densities and larger theoretical pore volumes than MOFs composed of inorganic metal and organic unit linkages, which is highly advantageous for certain biological applications. Laccase (laccase) is a polyphenol oxidase containing multiple copper and has strong catalytic ability, so that the laccase has a wide range of substrates, and can catalyze more than 250 substrates according to statistics. However, laccase has poor stability, catalytic capability is easily influenced by external environment such as extreme pH, temperature and the like, and free laccase has strong water solubility, is not easy to separate from a system after the reaction is finished, cannot be reused, increases cost, and greatly limits the application of the laccase in the industrial field.
The inventor discovers through a large number of screening and experiments that when the hydrogen bond organic framework is used as an immobilization carrier, preferably laccase is used as an immobilization object, the prepared hydrogen bond organic framework enzyme biological composite material has excellent performance. The inventor finds that the laccase can be better dispersed in the aqueous solution of the organic ligand of the hydrogen bond organic framework by adopting a biomimetic mineralization method and controlling the pH value in a specific range in the preparation process, and the laccase can be better encapsulated in the hydrogen bond organic framework taking the organic building block as the framework body by adopting a one-pot method, and the activity of the laccase in the hydrogen bond organic framework can be effectively reserved. The prepared hydrogen bond organic framework enzyme biological composite material has excellent removing capability for organic pollutants, and can quickly remove the pollutants without adding additional reaction medium during reaction. In addition, the hydrogen bond organic framework enzyme biological composite material not only has excellent removal capability on organic pollutants in sewage, but also has remarkably improved resistance capability on environmental factors, has excellent heat stability, storage stability and repeated availability, and can resist strong acid, strong alkali and metal ions. The hydrogen bond organic frame enzyme biological composite material has the advantages that for malachite green solution with the concentration of 50mg/L, the adding amount of the material is only 10mg/L, and under the same condition, the adding amount of the material is far lower than that of other reported materials, and the hydrogen bond organic frame enzyme biological composite material has extremely high value in practical industrial application.
Preferably, the organic building block is 1,3,6, 8-tetrakis (4-carboxylbenzenepyrene).
Preferably, in the hydrogen bond organic framework enzyme biological composite material, the mass fraction of laccase is 8-10%.
Further preferably, in the hydrogen bond organic framework enzyme biocomposite material, the mass fraction of the laccase is 10%.
In addition, the invention also provides a preparation method of the hydrogen bond organic framework enzyme biological composite material, which comprises the following preparation steps:
(1) Dissolving the organic building unit in a solvent to obtain a solution A;
(2) Laccase is dissolved in a buffer solution with the pH value of 3.5-4.5 to obtain a solution B;
(3) And mixing and stirring the solution A and the solution B, standing, and centrifuging to obtain the hydrogen bond organic framework enzyme biological composite material.
Preferably, in the step (1), the organic building unit (1, 3,6, 8-tetrakis (4-carboxylbenzenepyrene) is dissolved in a solvent by ultrasonic treatment.
Preferably, in the step (1), the solvent is N, N-dimethylformamide.
Preferably, the mass ratio of the 1,3,6, 8-tetra (4-carboxybenzene) pyrene to the laccase is 1.5-3: 1.
preferably, in the step (2), the buffer solution is a phosphoric acid/phosphate buffer solution.
In the step (2), the pH of the buffer solution is 4.
Preferably, the time of the standing treatment is 10 to 30 minutes.
Further preferably, the time of the standing treatment is 20 minutes.
In addition, the invention also protects the application of the hydrogen bond organic framework enzyme biological composite material in removing organic pollutants.
Preferably, the organic contaminant is one or more of malachite green, methylene blue, congo red, bisphenol a.
Further preferably, the organic contaminant is malachite green.
Preferably, the method comprises the steps of, the pH of the organic pollutant is 3-11.
Further preferably, the pH of the organic contaminant is 3 to 9.
It is further preferred that the organic contaminants are removed under light conditions.
Preferably, the reaction time of removing the organic pollutants by the hydrogen bond organic framework enzyme biological composite material is more than or equal to 0.5h.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention provides a hydrogen bond organic frame enzyme biological composite material, which not only effectively reserves the activity of laccase, has excellent removal capability on organic pollutants in sewage, but also remarkably improves the resistance capability on environmental factors, has excellent thermal stability, storage stability and repeated availability, can resist strong acid, strong alkali and metal ions, and has extremely high value in practical industrial application.
(2) The hydrogen bond organic framework enzyme biological composite material provided by the invention has the advantages that the adding amount of the material is only 10mg/L for malachite green solution with the concentration of 50mg/L. Under the same conditions, the addition amount of the material is far lower than that of other reported materials, and the material has extremely high value in practical industrial application.
(3) According to the invention, the pH value of the hydrogen bond organic frame enzyme biological composite material is optimized during preparation, and the hydrogen bond organic frame enzyme biological composite material is aged by increasing the standing condition, so that the synthesis of the hydrogen bond organic frame enzyme biological composite material is promoted.
Drawings
FIG. 1 is an infrared spectrum of a hydrogen bond organic framework enzyme biocomposite in example 1.
FIG. 2 is a CLSM graph of the hydrogen bond organoframed enzyme biocomposites of example 1.
FIG. 3 shows the removal rate of malachite green from hydrogen bond organic framework enzyme biocomposites of example 1, lac+HOF prepared in comparative example 1, and control HOF at various times.
FIG. 4 shows the removal rate of hydrogen bond organic framework enzyme biocomposites, laccase, HOF for malachite green in example 1 at different reaction times.
FIG. 5 is a graph showing the effect of sodium chloride and its mass fraction on the removal of malachite green by hydrogen bond organic framing enzyme biocomposites in example 1.
FIG. 6 is a graph showing the effect of natural organic matter and its concentration on removal of malachite green by hydrogen bond organic framing enzyme biocomposites in example 1.
FIG. 7 is a graph showing the effect of different temperatures on the removal of malachite green by hydrogen bond organic framework enzyme biocomposites in example 1.
FIG. 8 is a graph showing the effect of different reaction pH on the removal of malachite green by hydrogen bonding organic framework enzyme biocomposites in example 1.
FIG. 9 is a graph showing the stability of the hydrogen bond organic framework enzyme biocomposites at different pH values in example 1.
FIG. 10 is a graph showing the effect of different metal ions on the removal of malachite green by hydrogen bonding organic framework enzyme biocomposites in example 1.
FIG. 11 shows the removal of malachite green from hydrogen bond organic framing enzyme biocomposites of example 1 at different storage times.
FIG. 12 shows the removal of malachite green by hydrogen bond organic framework enzyme biocomposites of different reuse times.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Example 1
(1) 10mg of organic linker 1,3,6, 8-tetra (4-carboxybenzene) pyrene was dissolved in 1mL of N, N-Dimethylformamide (DMF) by ultrasonic treatment to obtain solution A;
(2) Dissolving 5mg laccase (Lac) in phosphate buffer solution with pH of 4 to obtain solution B;
(3) And (3) mixing and stirring the solution A in the step (1) and the solution B in the step (2) for 5min, standing for 20min, and centrifugally collecting to obtain the hydrogen bond organic framework enzyme biological composite material (Lac@HOF).
(4) Post-treatment: washing the hydrogen bond organic frame enzyme biological composite material with deionized water for 2 times and ethanol for 1 time in sequence, removing redundant solvents and impurities, directly dispersing the hydrogen bond organic frame enzyme biological composite material in the deionized water, and storing at 4 ℃.
The content of copper ions in Lac@HOF is detected by an inductively coupled plasma emission spectrometer (ICP), and the mass fraction of laccase in the hydrogen bond organic framework enzyme biological composite material is determined to be 10%.
FIG. 1 is an infrared spectrum of the hydrogen bond organic framework enzyme biocomposite prepared in example 1. As can be seen from FIG. 1, the absorption peaks of the hydrogen bond organic framework (HOF) and the hydrogen bond organic framework enzyme biocomposite (Lac@HOF) are substantially the same, indicating that the addition of laccase (Lac) did not change the structure of HOF, and in addition, the absorption peak of Lac also appears in Lac@HOF, indicating that laccase is successfully encapsulated.
To further demonstrate the presence of laccase in lac@hof, we observed lac@hof samples using a laser scanning confocal microscope (CLSM). FIG. 2 is a CLSM graph of the hydrogen bond organic framework enzyme biocomposite material prepared in example 1, and as can be seen from FIG. 2, blue fluorescence emission generated by laccase can be clearly observed in fluorescence field, which confirms successful doping of laccase.
Example 2
This embodiment differs from embodiment 1 in that: in the step (2), the addition amount of laccase is 5.5mg; the pH was 3.5; in the step (3), the standing treatment time is 30min. The mass fraction of laccase in the hydrogen bond organic framework enzyme biological composite material is 9%.
Example 3
This embodiment differs from embodiment 1 in that: in the step (2), the addition amount of laccase is 4.5mg; the pH was 4.5; in the step (3), the standing treatment time is 10min. The mass fraction of laccase in the hydrogen bond organic framework enzyme biological composite material is 8%.
Comparative example 1
The difference between this comparative example and example 1 is that:
(1) Obtaining HOF material: 10mg of the organic linker 1,3,6, 8-tetrakis (4-carboxylbenzenepyrene) (H4 TBAPy) was dissolved in 1mL of N, N-Dimethylformamide (DMF) by ultrasonic treatment, followed by standing for 20 minutes. The HOF-101 material obtained was collected by centrifugation, washed sequentially with deionized water 2 times and ethanol 1 time. HOF-101 was dispersed directly in deionized water and stored at 4 ℃.
(2) The same amount of HOF-101 material synthesized in step (1) of this example and laccase as in example 1 was weighed, then HOF-101 material was added to 5mL of purified water in which laccase was dissolved, and shaking was performed for 30 minutes to obtain Lac+HOF, which was then used for removal of malachite green.
FIG. 3 shows the organic contaminant removal rates of Lac@HOF prepared in example 1, lac+HOF prepared in comparative example 1 and HOF of the control group, respectively, added to sewage at different times, wherein C 0 The absorbance after the material addition was calculated, and C was the absorbance after only malachite green addition. The experimental result is shown in fig. 3, the removal rate of Lac+HOF prepared by physical mixing in comparative example 1 is basically the same as that of HOF, which shows that laccase and HOF do not generate good synergistic effect in the process, which proves that in the preparation process of hydrogen bond organic framework enzyme biological composite material, a specific preparation method (biomimetic mineralization method) is needed to be adopted and specific technological conditions (such as pH, standing treatment and the like) are controlled to synthesize Lac@HOF, and the synthesized Lac@HOF has unique advantages, and the catalytic activity of the material can be obviously improved.
Test examples
(1) Influence of different reaction time on removal of malachite green by hydrogen bond organic framework enzyme biological composite material
To 5mL of malachite green solution having a concentration of 50mg/L, 0.05mg of the hydrogen bond organic framework enzyme biocomposite prepared in example 1, 0.01mg of laccase, and 0.05mg of the hydrogen bond organic framework (HOF) synthesized in comparative example 1, respectively, were added, and then the reaction solution was shaken in the dark for 30 minutes to reach adsorption equilibrium. Then, the laccase reaction system is placed under a dark condition, and the hydrogen bond organic framework enzyme biological composite material reaction system is placed under an illumination condition (40 w wavelength is 365nm ultraviolet lamp). And taking out the sample at different reaction times, and then measuring the absorbance of the malachite green solution at the maximum absorption wavelength of 617nm by using an ultraviolet-visible spectrophotometer, so as to calculate the malachite green removal rate.
As shown in fig. 4, fig. 4 shows the removal rate of organic pollutants from hydrogen bond organic framework enzyme biocomposites, laccase and HOF at different reaction times. From the graph, the removal rate of laccase to malachite green is extremely low, and after 2 hours of reaction, the removal rate is only 3.4%. The removal rate of HOF is only 40.8%, however, the hydrogen bond organic framework enzyme biological composite material has good removal effect on malachite green, 85% of malachite green can be removed after 30 minutes of reaction, and the removal rate of malachite green is up to 96% after 2 hours, which proves that the removal performance of malachite green can be greatly improved after laccase is compounded with HOF.
(2) Influence of sodium chloride and mass fraction thereof on removal of malachite green by hydrogen bond organic framework enzyme biological composite material
And respectively dissolving a proper amount of sodium chloride into 5mL of malachite green solution with the concentration of 50mg/L to obtain reaction solutions with the mass fractions of 0%, 3.5%, 10% and 20% of sodium chloride, respectively adding 0.05mg of the hydrogen bond organic framework enzyme biological composite material prepared in the example 1 and 0.04mg of laccase, and vibrating the reaction solutions in the dark for 30 minutes to reach adsorption balance. Then, the laccase reaction system reacts for 24 hours under the dark condition, and the hydrogen bond organic framework enzyme biological composite material reaction system reacts for 2 hours under the illumination condition (40 w wavelength is 365nm ultraviolet lamp). After the reaction is completed, the absorbance of the malachite green solution is measured by an ultraviolet-visible spectrophotometer at the maximum absorption wavelength of 617nm, and the malachite green removal rate is calculated.
As shown in FIG. 5, laccase is very sensitive to sodium chloride, and its removal rate rapidly decreases with increasing mass fraction of sodium chloride, and even at 3.5% by mass, the enzyme removal rate rapidly decreases to 2.2%. The tolerance of the hydrogen bond organic framework enzyme biological composite material to high-concentration sodium chloride is obviously improved, and the removal rate is always kept to be more than 96%, which fully proves that the hydrogen bond organic framework provides a relatively mild microenvironment for laccase, so that the laccase is not easy to undergo severe conformational change, and the stability of the laccase is greatly improved. This has high value for the treatment of actual dye wastewater.
(3) Influence of natural organic matters and concentration thereof on removal of malachite green by hydrogen bond organic framework enzyme biological composite material
A proper amount of natural organic matters are respectively dissolved in 5mL of malachite green solution with the concentration of 50mg/L, so that the concentrations of the natural organic matters in the solution are 0, 10, 50 and 100mg/L respectively, then 0.05mg of the hydrogen bond organic framework enzyme biological composite material prepared in the example 1 and 0.04mg of laccase are added, and the reaction solution is vibrated for 30 minutes in the dark to reach adsorption balance. Then, the laccase reaction system reacts for 24 hours under the dark condition, and the hydrogen bond organic framework enzyme biological composite material reaction system reacts for 2 hours under the illumination condition (40 w wavelength is 365nm ultraviolet lamp). After the reaction is completed, the absorbance of the malachite green solution is measured by an ultraviolet-visible spectrophotometer at the maximum absorption wavelength of 617nm, and the malachite green removal rate is calculated.
As shown in FIG. 6, the addition of natural organic matters has a great influence on removal of malachite green by laccase, the removal rate of laccase is rapidly reduced with the increase of the concentration of the natural organic matters, and when the concentration of the natural organic matters is more than 50mg/L, the laccase is basically deactivated, because the spatial structure of the laccase is severely changed by the natural organic matters, so that the laccase is deactivated. In contrast, even when the concentration of natural organic matters reaches 100mg/L, the removal rate of malachite green of the hydrogen bond organic framework enzyme biological composite material is still up to 96%, and the reasons are that laccase is not directly contacted with natural organic matters due to the wrapping of the hydrogen bond organic framework, so that denaturation caused by excessive water loss is avoided. In addition, the spatial structure of laccase is limited by the carrier, so that the structure of laccase is not easy to change, and the activity of the material is maintained.
(4) Enzyme organisms with hydrogen bond organic frameworks at different temperatures influence of composite material on removal of malachite green
0.05mg of the hydrogen bond organic framework enzyme biological composite material prepared in example 1 and 0.04mg of laccase are dissolved in 5mL of ultrapure water respectively, and incubated (20, 30, 40, 50, 60, 80 ℃) in a water bath for 24 hours at different temperatures, and malachite green dye is added after incubation is finished, so that the dye concentration is 50mg/L. The reaction solution was then shaken in the dark for 30 minutes to reach adsorption equilibrium. Then, the laccase reaction system reacts for 24 hours under the dark condition, and the hydrogen bond organic framework enzyme biological composite material reaction system reacts for 2 hours under the illumination condition (40 w wavelength is 365nm ultraviolet lamp). After the reaction is completed, the absorbance of the malachite green solution is measured by an ultraviolet-visible spectrophotometer at the maximum absorption wavelength of 617nm, and the malachite green removal rate is calculated.
As shown in FIG. 7, when the incubation temperature was below 40℃the removal rate of malachite green by laccase increased with increasing temperature and showed optimal stability at 40℃with removal rate up to 45.3%, however, with further increasing temperature the activity decreased rapidly and the laccase had substantially lost activity at 80 ℃. In contrast, hydrogen bond organic framework enzyme biocomposites show extremely strong thermal stability due to the protection of the carrier, and the removal rate of malachite green is more than 93% in the range of 20-80 ℃, probably because the hydrogen bond organic framework limits the spatial structure of the enzyme and prevents the enzyme from folding at high temperature.
(5) Influence of reaction pH on removal of malachite green by hydrogen bond organic framework enzyme biological composite material
Buffer solutions with pH of 3, 5, 6, 7, 8, 9 and 11 are prepared by using phosphoric acid and phosphate buffer solutions respectively for standby. 5mL of malachite green solution at a concentration of 50mg/L was prepared using buffers of different pH. Then, the hydrogen bond organic framework enzyme biocomposite material and laccase prepared in example 1 were added separately, and the reaction solution was shaken for 30 minutes in the dark to reach adsorption equilibrium. Then, the laccase reaction system reacts for 24 hours under the dark condition, and the hydrogen bond organic framework enzyme biological composite material reaction system reacts for 2 hours under the illumination condition. After the reaction is completed, the absorbance of the malachite green solution is measured by an ultraviolet-visible spectrophotometer at the maximum absorption wavelength of 617nm, and the malachite green removal rate is calculated.
As shown in FIG. 8, the pH change has a large effect on laccase, and when the pH is less than 4 or greater than 9, the laccase efficiency of removal of organic contaminants is significantly reduced. The hydrogen bond organic framework enzyme biological composite material has no obvious change in the removal of organic pollutants when the pH value is 3-9, and the removal rate of the organic pollutants is obviously reduced when the pH value is more than 9.
(6) Influence of stability of hydrogen bond organic framework enzyme biological composite material under different pH values
The hydrogen bond organic framework enzyme biological composite material and laccase prepared in the example 1 are respectively dissolved in 5mL buffer solutions with different pH values, malachite green dye is added after incubation for 24 hours, the dye concentration is 50mg/L, and the reaction solution is oscillated for 30 minutes in the dark to reach adsorption equilibrium. Then, the laccase reaction system reacts for 24 hours under the dark condition, and the laccase @ HOF reaction system reacts for 2 hours under the light condition. After the reaction is completed, the absorbance of the malachite green solution is measured by an ultraviolet-visible spectrophotometer at the maximum absorption wavelength of 617nm, and the malachite green removal rate is calculated.
As shown in FIG. 9, incubation at different pH's has a large influence on the effect of laccase, and when the incubation pH is less than 4 or greater than 10, the stability of laccase is greatly affected, and the removal rate of organic pollutants is further affected. The hydrogen bond organic framework enzyme biological composite material gradually reduces the removal rate of organic pollutants along with the gradual increase of incubation pH, but also always maintains higher removal rate (> 52%).
(7) Influence of metal ions on removal of malachite green from hydrogen bond organic framework enzyme biological composite material
Respectively adding a certain amount of FeSO 4 、CuSO 4 、MgSO 4 、CaCl 2 KCl was dissolved in 5mL of ultrapure water at a concentration of 2mmol/L for each metal ion, and malachite green dye was then added to give a dye concentration of 50mg/L. Then, 0.05mg of the hydrogen bond organic framework enzyme biocomposite prepared in example 1 and 0.04mg of laccase were added to the reaction solution, respectively, and they were shaken in the dark for 30 minutes to reach adsorption equilibrium. Finally, the laccase reaction system is reacted for 24 hours under the dark condition, and the hydrogen bond organic framework enzyme biological composite material reaction system is reacted for 50 minutes under the illumination condition (the wavelength of 40w is 365nm ultraviolet lamp). After the reaction is completed, the absorbance of the malachite green solution is measured by an ultraviolet-visible spectrophotometer at the maximum absorption wavelength of 617nm, and the malachite green removal rate is calculated.
As shown in FIG. 10, almost all metal ions have an inhibition effect on removing malachite green from laccase, laccase is strongly inhibited in copper ion and calcium ion solutions, the removal rate is reduced to 22% and 10%, and particularly in ferrous ion solutions, the laccase has almost no removal effect on malachite green. However, the hydrogen bond organic framework enzyme biological composite material can keep high removal rate in different metal ion solutions, and only slightly reduces (66%) in copper ion solutions, which indicates that the hydrogen bond organic framework can obviously enhance the rigidity of laccase, so that functional groups in the structure are not easy to combine with external metal ions, and the stability of the laccase biological composite material is well maintained.
(8) Storage stability study of hydrogen bond organic framework enzyme biological composite material
The hydrogen bond organic frame enzyme biological composite material and laccase prepared in example 1 were stored at room temperature of about 20 ℃ in an external environment without light-shielding treatment, and 0.05mg of the hydrogen bond organic frame enzyme biological composite material and 0.04mg of laccase were dissolved in 5mL of malachite green and were shaken in the dark for 30 minutes to reach adsorption equilibrium at storage times of 3, 7, 14, 21 and 35 days, respectively. And then reacting the laccase reaction system for 24 hours under the dark condition, and reacting the hydrogen bond organic framework enzyme biological composite material reaction system for 2 hours under the illumination condition (40 w wavelength is 365nm ultraviolet lamp). After the reaction is completed, the absorbance of the malachite green solution is measured by an ultraviolet-visible spectrophotometer at the maximum absorption wavelength of 617nm, and the malachite green removal rate is calculated.
As shown in FIG. 11, the activity of the free laccase decreased rapidly with time, and the removal rate of malachite green decreased from 31% to 6.9% only for 7 days, and the laccase had substantially lost activity after 21 days. In contrast, hydrogen bond organoframed enzyme biocomposites have excellent storage stability, maintaining extremely high removal rates (96%) for malachite green even after 5 weeks of standing at 20 ℃. The improved stability of the immobilized enzyme may be associated with a reduced structural mobility of the protein. Because laccase is embedded in a carrier, the rigidity of laccase is improved, and the capability of immobilizing laccase to maintain stable protein conformation is enhanced, so that enzyme denaturation can be prevented.
(9) Enzyme biological compound of hydrogen bond organic framework study of the Material's bioavailability
To 10mL of malachite green solution having a concentration of 50mg/L, 0.1mg of the hydrogen bond organic framework enzyme biocomposite prepared in example 1 was added, and then the reaction solution was shaken in the dark for 30 minutes to reach adsorption equilibrium. Then, the hydrogen bond organic framework enzyme biological composite material reaction system reacts for 2 hours under the illumination condition (50 w blue light with the wavelength of 420 nm). After the reaction was completed, 0.25mL of the reacted solution was removed and diluted with 2.75mL of ultrapure water for use in the subsequent detection. Then, malachite green was added again to the reaction system, and the mixture was again irradiated with light. The operation steps are the same as before, and the method is repeatedly used for 10 times. After each reaction, the addition amount of malachite green is proportionally reduced so as to control the concentration of pollutants in the reaction system to be unchanged. And all diluted samples to be tested are subjected to ultraviolet-visible spectrophotometry to measure the absorbance of the malachite green solution at the maximum absorption wavelength of 617nm, so that the malachite green removal rate is calculated.
As shown in fig. 12, the hydrogen bond organoframed enzyme biocomposite still has a high removal rate (> 92%) of malachite green even after 10 repeated uses, which may benefit from the material having a large number of active sites, thus enabling rapid removal of contaminants, avoiding a large accumulation of contaminants on the surface of the material. In addition, the protection effect of the hydrogen bond organic framework on the enzyme ensures that the functional group on the surface of the enzyme is not easy to combine with the organic micromolecule in the solution, reduces the damage of active substances generated by the reaction on laccase activity, and has excellent reusability, thereby reducing the cost of practical application.
The foregoing examples are illustrative only and serve to explain some features of the method of the invention. The claims that follow are intended to claim the broadest possible scope as conceivable and the embodiments presented herein are demonstrated for the applicant's true test results. It is, therefore, not the intention of the applicant that the appended claims be limited by the choice of examples illustrating the features of the invention. Some numerical ranges used in the claims also include sub-ranges within which variations in these ranges should also be construed as being covered by the appended claims where possible.
Claims (10)
1. The hydrogen bond organic frame enzyme biological composite material is characterized in that the composite material takes an organic building unit as a frame main body, and enzyme is wrapped in the frame main body; the organic building unit is a carboxylic acid hydrogen bond organic framework material; the enzyme is laccase.
2. The hydrogen bonding organic framework enzyme biocomposite according to claim 1, wherein the organic building block is 1,3,6, 8-tetrakis (4-carboxylbenzenepyrene).
3. The hydrogen bond organic framework enzyme biocomposite according to claim 1, wherein the mass fraction of laccase in the hydrogen bond organic framework enzyme biocomposite is 8-10%.
4. A method for preparing the hydrogen bond organic framework enzyme biological composite material according to any one of claims 1 to 3, which is characterized by comprising the following preparation steps:
(1) Dissolving the organic building unit in a solvent to obtain a solution A;
(2) Laccase is dissolved in a buffer solution with the pH value of 3.5-4.5 to obtain a solution B;
(3) And mixing and stirring the solution A and the solution B, standing, and centrifuging to obtain the hydrogen bond organic framework enzyme biological composite material.
5. The method according to claim 4, wherein in the step (1), the solvent is N, N-dimethylformamide.
6. The preparation method according to claim 4, wherein the mass ratio of the organic building block to laccase is 1.5-3: 1.
7. the method according to claim 4, wherein in the step (2), the buffer solution is a phosphoric acid/phosphate buffer solution.
8. The method according to claim 4, wherein in the step (2), the buffer solution has a pH of 4.
9. The use of the hydrogen bond organic framework enzyme biocomposite material prepared by the preparation method of any one of claims 1 to 3 or any one of claims 4 to 8 in removing organic pollutants.
10. The use according to claim 9, wherein the organic contaminant is one or more of malachite green, methylene blue, congo red, bisphenol a.
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| CN114621924B (en) * | 2022-04-03 | 2024-05-17 | 中国科学院长春应用化学研究所 | Porous carbon sphere nano enzyme doped hydrogen bond organic framework shell and preparation method thereof |
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