CN111534505A - Immobilized laccase and preparation method and application thereof - Google Patents
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Abstract
The invention relates to an immobilized laccase and a preparation method and application thereof. The technical scheme is as follows: mixing metal organic framework material MIL-125 with laccase solution with the pH value of 3.0-8.0, adsorbing for 0.5-3 h at the rotation speed of 150-200 r/min and the temperature of 20-80 ℃, centrifuging, washing, and drying to obtain the immobilized laccase. The immobilized enzyme prepared by the invention has the advantages of high catalytic efficiency, high stability and the like, and can be recycled.
Description
Technical Field
The invention belongs to the field of enzyme engineering, and particularly relates to immobilized laccase as well as a preparation method and application thereof.
Background
Chlorophenols are a kind of refractory compounds, and have been widely used in the production of wood preservation, pesticides and herbicides for a long time, wherein 2, 4-dichlorophenol (2,4-DCP) is a toxic and refractory pollutant and widely exists in production wastewater of pesticides, fuels, plasticizers and the like. Countries such as the united states and china have included "black lists" of harmful or priority pollutants in water. Therefore, how to eliminate the pollution of chlorophenol compounds in the environment is a subject of great importance.
With the enhancement of the public green environmental protection consciousness and the development of biotechnology in society, the enzyme is increasingly applied to industries such as biological industry, pharmaceutical industry, paper industry and the like as a green, environment-friendly and efficient biological agent. However, how to solve the problems of poor stability, poor repeated availability, low local reaction rate, etc. of free enzyme has become a hot spot of research. Laccase is polyphenol oxidase capable of green catalyzing phenols, and has potential application value in the aspects of sewage treatment, lignin degradation and the like.
The immobilization of the enzyme is a technique of immobilizing the enzyme on a carrier matrix while maintaining the activity and properties of the enzyme, so that the enzyme and its derivatives can be recycled. At present, the immobilized laccase has four main preparation methods: adsorption, cross-linking, entrapment and covalent bonding. Because the recovery rate of the immobilized enzyme prepared by the traditional method is relatively low, the enzyme activity of the immobilized enzyme is obviously reduced after the immobilized enzyme is used for a certain time, and the application performance of the enzyme is greatly reduced, a proper carrier is necessarily sought, so that the immobilized enzyme has the characteristics of high stability, strong operability, simple and convenient process and the like, and is expected to be applied to industrial production.
In the last decades, mesoporous materials have been developed very slowly compared to the efforts to control the synthesis of mesoporous materials. Strictly speaking, at present, no breakthrough progress is made in the application aspect of the mesoporous material. The mesoporous material has the advantages of continuously adjustable aperture, large specific surface area, easy adjustment of pore chemical composition, good biocompatibility and stability and the like. Compared with the traditional inorganic nano material, the molecular structure of the metal organic framework Materials (MOFs) contains a large number of organic ligands (functional groups), and the metal organic framework materials can form specific intermolecular hydrogen bonds, covalent bonds or hydrophobic intermolecular interaction with biological protein molecules, and are expected to have wide application prospects in the field of enzyme immobilization.
Disclosure of Invention
The invention aims to provide a method for immobilizing laccase by using a metal-organic framework material MIL-125 as a carrier, and the method is applied to oxidation reaction of phenols and arylamine compounds to obtain the immobilized laccase which has high enzyme activity and stability and can be recycled.
In order to achieve the purpose, the invention adopts the following technical scheme: an immobilized laccase is prepared by adsorbing laccase with metal organic framework material MIL-125 as carrier.
The preparation method of the immobilized laccase comprises the following steps: mixing metal organic framework material MIL-125 with laccase solution with the pH value of 3.0-8.0, adsorbing for 0.5-3 h at the rotation speed of 150-200 r/min and the temperature of 20-80 ℃, centrifuging, washing, and drying to obtain the immobilized laccase.
Further, the preparation method of the metal-organic framework material MIL-125 comprises the following steps: adding terephthalic acid and tetrabutyl titanate into a mixed solution of N, N-Dimethylformamide (DMF) and methanol by adopting a hot melting method, stirring at room temperature, transferring the mixed solution into a reaction kettle, reacting at 140-160 ℃ for 16-20h, cooling to room temperature, centrifuging, washing the obtained precipitate with DMF and methanol, and drying to obtain the metal-organic framework material MIL-125.
Further, the preparation method of the laccase solution with the pH value of 3.0-8.0 comprises the following steps: the laccase is dissolved in citric acid-sodium dihydrogen phosphate buffer solution with the pH value of 3.0-8.0 to prepare the laccase-modified enzyme preparation.
Further, according to the preparation method, the laccase, MIL-125, is (2-18) and 1 in mass ratio.
Furthermore, according to the preparation method, the laccase: MIL-125 is 14:1 in mass ratio.
Further, the preparation method comprises the steps of mixing the metal organic framework material MIL-125 with a laccase solution with the pH value of 6.0, adsorbing for 1-3 hours at the rotation speed of 170r/min and the temperature of 50 ℃, centrifuging, washing and drying to obtain the immobilized laccase.
The application of the immobilized laccase in catalyzing the oxidation reaction of phenols or arylamine compounds. The method comprises the following steps: mixing the immobilized laccase with a solution of a phenol or arylamine compound, and carrying out an enzyme catalytic reaction at a pH of 3.0-8.0, a rotation speed of 150-200 r/min and a temperature of 20-70 ℃.
Further, the phenolic compound is a chlorophenol compound.
Further, the chlorophenol compound is 2, 4-dichlorophenol (2, 4-DCP).
Further, according to the mass ratio, 2,4-DCP and immobilized laccase (50-500) are 1.
Furthermore, the mass ratio of the 2,4-DCP to the immobilized laccase is 200: 1.
The invention has the following beneficial effects:
1. at present, the metal ions used in metal-organic framework materials are mainly divalent or trivalent ions including 3d transition metals, 3p metals or lanthanides. Among them, titanium is widely used in metal organic framework materials because of its low toxicity, good redox activity and photocatalytic performance. MIL-125 is a crystalline titanium dicarboxylate consisting of octahedral titanium units sharing corners or edges, connected to 12 other cyclic octamers via 1, 4-phthalate (BDC) linkers to form a porous three-dimensional structure. Compared with other metal organic framework materials, the MIL-125 has larger surface area, good porosity and strong thermal stability. The invention takes MIL-125 as a carrier to adsorb laccase to prepare the immobilized laccase, has the characteristics of low toxicity, stability and the like, and can be applied to the industries of food, medicine and the like.
2. The immobilized laccase provided by the invention is simple in preparation process, saves resources, and is suitable for industrial mass production. Has wide application in various fields, such as pharmacy, food industry, agriculture and the like, and therefore has wide commercial prospect. The special catalytic property of laccase enables the laccase to be widely applied to the aspects of lignin degradation, paper pulp wastewater treatment, paper industry wastewater detoxification and the like, and also to be widely applied to the fields of biosensors and biofuel cells.
3. The immobilized laccase prepared by the invention can be recycled, and the tolerance of the laccase to the conditions of temperature, pH and the like is improved.
Drawings
FIG. 1 is an XRD spectrum of MIL-125 prepared in accordance with the present invention.
FIG. 2 is an SEM scanning electron micrograph of MIL-125 prepared according to the present invention.
FIG. 3 is the effect of pH on MIL-125 adsorption of laccase.
FIG. 4 is the effect of temperature on MIL-125 adsorption of laccase.
FIG. 5 shows the effect of immobilized laccase prepared according to the invention on the catalytic performance of 2,4-DCP at different pH.
FIG. 6 shows the effect of the immobilized laccase prepared according to the invention on the catalytic performance of 2,4-DCP at different temperatures.
FIG. 7 shows the relative enzyme activities of the immobilized laccase prepared by the present invention after repeating the procedure 5 times.
Detailed Description
The present invention will be described in further detail with reference to the following examples and accompanying drawings. The following specific examples are not to be construed as limiting the invention but merely as illustrative thereof.
Laccase meal from Denmark Novoxil reagent in the following examples; the various reagents and procedures used in the examples are conventional in the art, unless otherwise indicated.
1. The calculation method of the laccase adsorption rate refers to a formula (1);
wherein: a (%) is laccase adsorption rate;
C0(mg/L) is the initial concentration of enzyme protein;
Ce(mg/L) is the concentration of enzyme protein in the supernatant obtained by centrifugation after adsorption
2. The calculation method of the removal rate of 2,4-DCP refers to the formula (2)
Wherein: d (%) is the removal rate of 2, 4-DCP;
C1(mg/L) is the initial concentration of 2, 4-DCP;
C2(mg/L) is the final concentration of 2,4-DCP after enzymolysis
3. The calculation method of the enzyme activity of the immobilized laccase refers to the formula (3)
Wherein: q (U/mL. min) is the enzyme activity;
t (min) is the time of the enzymolysis reaction
Example 1
The preparation method of the immobilized laccase comprises the following steps:
1. preparation of metal organic framework material MIL-125
Adding terephthalic acid and tetrabutyl titanate into a mixed solution of N, N-dimethylformamide and methanol, stirring at room temperature, transferring the mixed solution into a reaction kettle, reacting at 140-160 ℃ for 16-20h, cooling to room temperature, centrifuging, washing the obtained precipitate with N, N-dimethylformamide and methanol, and drying to obtain the metal organic framework material MIL-125.
2. preparation of laccase solution with pH value of 3.0-8.0
The laccase is dissolved in citric acid-sodium dihydrogen phosphate buffer solution with the pH value of 3.0-8.0 to prepare the laccase-modified enzyme preparation.
3. Preparation of immobilized laccase
Mixing metal organic framework material MIL-125 with laccase solution with the pH value of 3.0-8.0, adsorbing for 0.5-3 h at the rotation speed of 150-200 r/min and the temperature of 20-80 ℃, centrifuging, washing, and drying to obtain the immobilized laccase.
(I) influence of pH on the adsorption of laccase to MIL-125
1. Preparation of metal organic framework material MIL-125
Adding 2.20g of terephthalic acid and 2.4mL of tetrabutyl titanate into 40mL of mixed solution of N, N-Dimethylformamide (DMF) and methanol (9:1, v/v), stirring at room temperature for 20min, transferring the mixed solution into a reaction kettle, reacting at 150 ℃ for 16h, cooling to room temperature, centrifuging, washing obtained precipitates twice with DMF and methanol respectively, and drying in a vacuum drying oven at 70 ℃ for 12h to obtain the metal-organic framework material MIL-125.
FIG. 1 is an XRD spectrum of MIL-125 prepared. As can be seen from fig. 1, the peak position is located at 2 θ of 6.7 °, 9.7 °, 11.6 °, etc., and is a characteristic peak of MIL-125.
FIG. 2 is an SEM scanning electron micrograph of the MIL-125 prepared. As can be seen from FIG. 2, the MIL-125 prepared had a disk shape and a particle size of about 2 μm.
2. Preparation of laccase solutions
Weighing 3.00g laccase respectively, dissolving in 500mL citric acid-sodium dihydrogen phosphate buffer solution with pH of 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0, centrifuging to obtain supernatant, obtaining laccase solutions with different pH concentrations of 6mg/mL, and determining enzyme protein content (C)0) And stored at 4 ℃ for later use.
3. Preparation of immobilized laccase
And respectively taking 9mL of laccase solution with the pH value of 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0 and the concentration of 6mg/mL, adding 6mg MIL-125, uniformly mixing, placing in a constant temperature oscillator with the rotation speed of 170r/min, adsorbing at 50 ℃ for 40min, respectively centrifuging the mixed solution, washing the obtained precipitate with deionized water, and freeze-drying to respectively obtain the immobilized laccase prepared under different pH values. The supernatant obtained by centrifugation was subjected to separate measurement of the enzyme protein content (C)e)。
And (3) calculating the laccase adsorption rate of the immobilized laccase prepared under different pH values by referring to the formula (1). The results are shown in fig. 3, and it can be seen from fig. 3 that the adsorption rate of MIL-125 to laccase increases significantly with increasing pH, and reaches a plateau until pH 6.0, so that pH 6.0 can be selected as the optimal pH for MIL-125 to adsorb laccase.
(II) Effect of temperature on MIL-125 laccase adsorption
1. Preparation of metal organic framework material MIL-125
Adding 2.20g of terephthalic acid and 2.4mL of tetrabutyl titanate into 40mL of mixed solution of N, N-Dimethylformamide (DMF) and methanol (9:1, v/v), stirring at room temperature for 20min, transferring the mixed solution into a reaction kettle, reacting at 150 ℃ for 16h, cooling to room temperature, centrifuging, washing obtained precipitates twice with DMF and methanol respectively, and drying in a vacuum drying oven at 70 ℃ for 12h to obtain the metal-organic framework material MIL-125.
2. Preparation of laccase solutions
Weighing 3.00g of laccase, dissolving in 500mL of citric acid-sodium dihydrogen phosphate buffer solution with pH of 6.0, centrifuging, collecting supernatant to obtain laccase solution with pH of 6.0 and concentration of 6mg/mL, and determining enzyme protein content (C)0) And stored at 4 ℃ for later use.
3. Preparation of immobilized laccase
And respectively taking 9mL of laccase solution with the pH value of 6.0 and the concentration of 6mg/mL, adding 6mg of MIL-125, uniformly mixing, placing in a constant temperature oscillator with the rotating speed of 170r/min, respectively adsorbing at 20, 30, 40, 50, 60, 70 and 80 ℃ for 40min, respectively centrifuging the mixed solution, washing the obtained precipitate with deionized water, and freeze-drying to respectively obtain the immobilized laccase prepared at different temperatures. The supernatant obtained by centrifugation was subjected to separate measurement of the enzyme protein content (C)e)。
The laccase adsorption rate is calculated by referring to the formula (1), and as a result, as shown in FIG. 4, the adsorption rate of laccase gradually increases with the increase of temperature, and in order to prevent the inactivation of laccase during the immobilization process, 50 ℃ is selected as the optimal temperature for the MIL-125 immobilized laccase.
Example 2
1. Preparation of metal organic framework material MIL-125
Adding 2.20g of terephthalic acid and 2.4mL of tetrabutyl titanate into 40mL of mixed solution of N, N-Dimethylformamide (DMF) and methanol (9:1, v/v), stirring at room temperature for 20min, transferring the mixed solution into a reaction kettle, reacting at 150 ℃ for 16h, cooling to room temperature, centrifuging, washing obtained precipitates twice with DMF and methanol respectively, and drying in a vacuum drying oven at 70 ℃ for 12h to obtain the metal-organic framework material MIL-125.
2. Preparation of laccase solutions
Weighing 3.00g of laccase, dissolving in 500mL of citric acid-sodium dihydrogen phosphate buffer solution with pH of 6.0, centrifuging, collecting supernatant to obtain laccase solution with pH of 6.0 and concentration of 6mg/mL, and determining enzyme protein content (C)0) And stored at 4 ℃ for later use.
3. Preparation of immobilized laccase
And (2) adding 6mg MIL-125 into 9mL of laccase solution with the pH value of 6.0 and the concentration of 6mg/mL, uniformly mixing, placing in a constant-temperature oscillator with the rotation speed of 170r/min, adsorbing at 50 ℃ for 2h, centrifuging the mixed solution, washing the obtained precipitate with deionized water, and freeze-drying to obtain the immobilized laccase. The supernatant obtained by centrifugation was subjected to measurement of the enzyme protein content (C)e). Referring to equation (1), the laccase adsorption rate was calculated to be 72.6%.
Example 3 catalysis of 2,4-DCP Oxidation by immobilized laccase
The following experiment was performed using the immobilized laccase prepared in example 2.
The method comprises the following steps: mixing the immobilized laccase with a solution of a phenol or arylamine compound, and carrying out an enzyme catalytic reaction at a pH of 3.0-8.0, a rotation speed of 150-200 r/min and a temperature of 20-70 ℃.
(one) influence of immobilized laccase on catalytic performance of 2,4-DCP at different pH values
The method comprises the following steps: adding 20mg of immobilized laccase into 10mL of 2,4-DCP solution with the concentration of 2.5mmol/L, uniformly mixing, respectively adjusting the pH of the mixed solution to 3.0, 4.0, 5.0, 6.0, 7.0 and 8.0, and respectively carrying out enzymolysis for 10min at the rotation speed of 170r/min and the temperature of 30 ℃.
Centrifuging to obtain supernatant, measuring absorbance at 595nm with ultraviolet spectrophotometer, calculating the concentration of 2,4-DCP in the supernatant, and calculating the removal rate of 2,4-DCP with reference to formula (2), the result is shown in FIG. 5. As can be seen in FIG. 5, the removal rate of 2,4-DCP increased gradually with increasing pH, reaching a maximum at pH 6.0, and subsequently decreased with increasing pH of the solution, so that the pH optimum for enzymatic digestion of the immobilized laccase was 6.0.
(II) influence of immobilized laccase on catalytic performance of 2,4-DCP at different temperatures
The method comprises the following steps: adding 20mg of immobilized laccase into 10mL of 2,4-DCP solution with the concentration of 2.5mmol/L, uniformly mixing, adjusting the pH of the mixed solution to 6.0, and performing enzymolysis for 10min at the rotation speed of 170r/min and the temperatures of 20, 30, 40, 50, 60 and 70 ℃.
Centrifuging to obtain supernatant, measuring absorbance at 595nm with ultraviolet spectrophotometer, calculating the concentration of 2,4-DCP in the supernatant, and calculating the removal rate of 2,4-DCP with reference to formula (2), the result is shown in FIG. 6. As can be seen from FIG. 6, the removal rate of 2,4-DCP gradually increased with increasing temperature, reaching the highest at 30 ℃ and then decreased with increasing temperature, so that the optimum temperature for the enzymatic hydrolysis of the immobilized laccase was 30 ℃.
(III) repeatability test
The method comprises the following steps: adding 20mg of immobilized laccase into 10mL of 2,4-DCP solution with the concentration of 2.5mmol/L, uniformly mixing, adjusting the pH of the mixed solution to 6.0, and performing enzymolysis for 10min at the rotation speed of 170r/min and the temperature of 30 ℃ respectively. Centrifuging, measuring absorbance of the supernatant at 595nm with an ultraviolet spectrophotometer, and calculating the concentration of 2,4-DCP in the supernatant. The precipitate was immobilized laccase, washed and dried, and the experiment was repeated.
The enzyme activity was calculated with reference to the formula (3), and the results are shown in FIG. 7. As can be seen in FIG. 7, after 5 times of repetition, the immobilized laccase still maintained more than 60% of the enzyme activity, which indicates that MIL-125 is a good carrier for the immobilized laccase.
Claims (10)
1. An immobilized laccase is characterized in that the immobilized laccase is prepared by adsorbing laccase by using a metal organic framework material MIL-125 as a carrier.
2. The method for preparing the immobilized laccase of claim 1, which comprises the steps of: mixing metal organic framework material MIL-125 with laccase solution with the pH value of 3.0-8.0, adsorbing for 0.5-3 h at the rotation speed of 150-200 r/min and the temperature of 20-80 ℃, centrifuging, washing, and drying to obtain the immobilized laccase.
3. The method according to claim 2, wherein the metal-organic framework material MIL-125 is prepared by a method comprising the steps of: adding terephthalic acid and tetrabutyl titanate into a mixed solution of N, N-dimethylformamide and methanol, stirring at room temperature, transferring the mixed solution into a reaction kettle, reacting at 140-160 ℃ for 16-20h, cooling to room temperature, centrifuging, washing the obtained precipitate with N, N-dimethylformamide and methanol, and drying to obtain the metal organic framework material MIL-125.
4. The preparation method according to claim 2, wherein the preparation method of the laccase solution with the pH of 3.0-8.0 comprises the following steps: the laccase is dissolved in citric acid-sodium dihydrogen phosphate buffer solution with the pH value of 3.0-8.0 to prepare the laccase-modified enzyme preparation.
5. The preparation method according to claim 2, wherein laccase is MIL-125 ═ 1 (2-18) by mass ratio.
6. The preparation method according to claim 5, wherein laccase is MIL-125 ═ 14:1 by mass ratio.
7. The preparation method according to claim 2, characterized in that the metal-organic framework material MIL-125 is mixed with laccase solution with pH of 6.0, and the mixture is adsorbed for 1-3 h at 50 ℃ at the rotation speed of 170r/min, centrifuged, washed and dried to obtain the immobilized laccase.
8. The use of the immobilized laccase of claim 1 for catalyzing the oxidation of phenolic or aromatic amines.
9. Use according to claim 8, characterized in that the method is as follows: mixing the immobilized laccase of claim 1 with a solution of a phenol or arylamine compound, and carrying out an enzyme catalytic reaction at a pH of 3.0-8.0, a rotation speed of 150-200 r/min, and a temperature of 20-70 ℃.
10. Use according to claim 8 or 9, characterized in that the phenolic compound is a chlorophenol compound.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110129290A (en) * | 2019-04-03 | 2019-08-16 | 江苏大学 | Metal-organic framework material immobilization laccase and its preparation method and application |
| CN113000069A (en) * | 2021-02-25 | 2021-06-22 | 广西大学 | Preparation method and application of bionic laccase functionalized imine covalent organic framework nanoenzyme |
| CN115404225A (en) * | 2022-10-14 | 2022-11-29 | 兰州大学 | Nitrogen-doped titanium dioxide and titanium carbide co-modified PVDF (polyvinylidene fluoride) film immobilized laccase as well as method and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110129290A (en) * | 2019-04-03 | 2019-08-16 | 江苏大学 | Metal-organic framework material immobilization laccase and its preparation method and application |
| CN113000069A (en) * | 2021-02-25 | 2021-06-22 | 广西大学 | Preparation method and application of bionic laccase functionalized imine covalent organic framework nanoenzyme |
| CN113000069B (en) * | 2021-02-25 | 2022-10-11 | 广西大学 | Preparation method and application of bionic laccase functionalized imine covalent organic framework nanoenzyme |
| CN115404225A (en) * | 2022-10-14 | 2022-11-29 | 兰州大学 | Nitrogen-doped titanium dioxide and titanium carbide co-modified PVDF (polyvinylidene fluoride) film immobilized laccase as well as method and application thereof |
| CN115404225B (en) * | 2022-10-14 | 2024-03-15 | 兰州大学 | Nitrogen-doped titanium dioxide and titanium carbide co-modified PVDF membrane immobilized laccase and method and application thereof |
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