CN111411102B - Preparation method of ZIF-8/enzyme composite material - Google Patents
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Abstract
The invention relates to a preparation method of a ZIF-8/enzyme composite material, which comprises the steps of enabling ZIF-8 crystals to grow on the surface of an enzyme in a lamellar structure by adjusting the concentration of a precursor, mixing a metal salt aqueous solution with a ligand aqueous solution and a mixed solution of the enzyme, stirring for a period of time at room temperature, and separating a solid product to obtain the ZIF-8/enzyme composite material. Different from the composite material obtained by the traditional method, the substrate molecule is diffused without depending on the pore channel of ZIF-8, and the large-size substrate contacts the active center of the enzyme through the gap between the ZIF-8 lamellar structures, so that the catalytic reaction is completed. The ZIF-8 nanoflower growing on the surface of the enzyme can well protect the enzyme from reacting under severe conditions, and meanwhile, the ZIF-8/enzyme nanoflower composite material also has good cyclability and storage capacity.
Description
Technical Field
The invention relates to the technical field of composite material preparation, in particular to a preparation method of a ZIF-8/enzyme composite material.
Background
The enzyme is a very important biological macromolecule and has far higher catalytic efficiency than an artificial catalyst in the aspect of catalyzing biological reaction. Therefore, the enzyme has great value and prospect in industrial production. However, the enzyme is difficult to be industrially applied because of its low thermostability, narrow applicable pH range, poor resistance to organic solvents and metal ions, and difficulty in separation after the reaction. To address this problem, immobilization of enzymes on Metal Organic Frameworks (MOFs) is a viable strategy. The strategy can effectively improve the stability of enzyme storage and reaction, and simultaneously, the enzyme can be recycled.
The MOF is a hybrid material formed by connecting metal nodes and organic ligands through coordination bonds. They have the advantages of high specific surface area, easy regulation and modification, high chemical and thermal stability, mild synthesis conditions and the like, and are good carriers for immobilizing enzymes. In particular, the zeolitic imidazolate framework-8 (ZIF-8) has very mild synthesis conditions, high chemical and thermal stability and excellent biocompatibility, and thus it is possible to encapsulate enzymes in ZIF-8 in situ by a co-precipitation method under aqueous solution conditions through ZIF-8 precursor assembly. The strategy can simply, conveniently and rapidly prepare the ZIF-8/enzyme composite material without being influenced by the size of the enzyme. Meanwhile, the ZIF-8 shell of the composite material can protect the enzyme, reduce the leaching of the enzyme and improve the circulating capacity of the enzyme.
However, this method has great limitations. Since the pore diameter of ZIF-8 is small (about)
) The diffusion of large-size substrates is severely limited, so that only a small amount of small-substrate enzymes (such as catalase, cytochrome c, glucose oxidase and the like) are suitable for the strategy at present, and the application of the strategy in the industry is greatly hindered.
Disclosure of Invention
The present invention has been made to solve the above problems, and an object of the present invention is to provide a novel method for preparing a ZIF-8/enzyme composite material.
The purpose of the invention is realized by the following technical scheme:
a preparation method of a ZIF-8/enzyme composite material comprises the steps of enabling ZIF-8 crystals to grow on the surface of enzyme in a lamellar structure by adjusting the concentration of a precursor, and synthesizing the ZIF-8/enzyme nanoflower composite material by a one-pot method. Different from the composite material obtained by the traditional method, the substrate molecule is diffused without depending on the pore channel of ZIF-8, and the large-size substrate contacts the active center of the enzyme through the gap between the ZIF-8 lamellar structures, so that the catalytic reaction is completed. The ZIF-8 nanoflower growing on the surface of the enzyme can well protect the enzyme from reacting under severe conditions (including high temperature, organic solvent and trypsin solution), and meanwhile, the ZIF-8/enzyme nanoflower composite material also has good cyclability and storage capacity.
Preferably, the specific steps are as follows: and (3) mixing the metal salt aqueous solution with the mixed solution of the ligand aqueous solution and the enzyme, stirring for a period of time at room temperature, and separating a solid product to obtain the ZIF-8/enzyme composite material.
Preferably, the metal salt is zinc salt, and further preferably zinc nitrate hexahydrate or anhydrous zinc acetate, and the concentration of the aqueous solution of the metal salt is 0.5-24 g/L.
Preferably, the ligand is 2-methylimidazole, and the concentration of the ligand aqueous solution is 1.6-7 g/L.
Preferably, the molar ratio of the metal salt to the ligand is 1: 4.
preferably, the enzyme comprises a large-size substrate enzyme, and further preferably, the enzyme is a human S-adenosylmethionine synthetase or alcohol dehydrogenase.
Preferably, the solid product is separated by centrifugation at 8000-.
Preferably, the solid product is washed with tris (hydroxymethyl) aminomethane buffer at a concentration of 0.1mol/L and a pH of 8.0.
The ZIF-8/enzyme nanoflower composite material prepared by the method can be used for catalyzing enzyme reaction, and can be recycled after the reaction.
The invention has the beneficial effects that: the method of the invention has simple operation, low price and high efficiency. The ZIF-8 nanoflower shell basically does not affect the activity of the enzyme, and meanwhile, the enzyme can be protected from the influence of high temperature, organic solvents, trypsin and other environments, and the circulating capacity and the storage capacity of the enzyme are improved. More importantly, the ZIF-8/enzyme nanoflower composite allows large size substrates to enter the ZIF-8 shell, so the method is applicable to most enzymes. Experiments show that the ZIF-8/enzyme nanoflower composite material prepared by the method has excellent catalytic capability, reaction stability, circulation capability and storage capability, and can allow large-size substrates to contact the active center of the enzyme through gaps between ZIF-8 lamellar structures, so that the application range of the strategy of fixing the enzyme on a metal organic framework is greatly widened.
Drawings
FIG. 1 is a scanning electron micrograph of HSAMS @ ZIF-8.
FIG. 2 is an X-ray diffraction pattern of ZIF-8 and HSAMS @ ZIF-8.
FIG. 3 is a Fourier transform infrared spectrum of ZIF-8 and HSAMS @ ZIF-8.
FIG. 4 is a simultaneous thermogram of ZIF-8 and HSAMS @ ZIF-8.
FIG. 5 is a graph comparing the activity of free HSAMS and HSAMS @ ZIF-8.
FIG. 6 is a graph comparing the reaction stability of free HSAMS and HSAMS @ ZIF-8.
FIG. 7 is a graph demonstrating the cycling capability of HSAMS @ ZIF-8.
FIG. 8 is a graph demonstrating the storage capability of HSAMS @ ZIF-8.
FIG. 9 is a scanning electron micrograph of AlcDH @ ZIF-8.
FIG. 10 is an X-ray diffraction pattern of ZIF-8 and AlcDH @ ZIF-8.
FIG. 11 is a Fourier transform infrared spectrum of ZIF-8 and AlcDH @ ZIF-8.
FIG. 12 is a simultaneous thermogram of ZIF-8 and AlcDH @ ZIF-8.
FIG. 13 is a graph comparing the activity of free AlcDH and AlcDH @ ZIF-8.
FIG. 14 is a graph comparing the reaction stability of free AlcDH and AlcDH @ ZIF-8.
FIG. 15 is a graph showing the results of demonstrating the circulating ability of AlcDH @ ZIF-8.
FIG. 16 is a graph showing the results of demonstrating the storage capacity of AlcDH @ ZIF-8.
Detailed Description
The invention is further elucidated with reference to the figures and embodiments.
In the following examples, Zeolite Imidazolate framework-8 is labeled ZIF-8, human S-adenosylmethionine synthetase is labeled HSAMS, ethanol dehydrogenase is labeled AlcDH, ZIF-8/human S-adenosylmethionine synthetase is labeled HSAMS @ ZIF-8, ZIF-8/ethanol dehydrogenase is labeled AlcDH @ ZIF-8, and Tris (hydroxymethyl) aminomethane is labeled Tris-HCl.
Example 1
Screening and optimizing the conditions for preparing HSAMS @ ZIF-8. The method comprises the following steps:
(1) respectively dissolving 0.65mg, 1.3mg, 1.9mg, 2.5mg, 5mg and 10mg of zinc nitrate hexahydrate in 420 mu L of water to obtain zinc nitrate solutions 1, 2, 3, 4, 5 and 6;
(2) dissolving 0.7mg, 1.4mg, 2.1mg, 2.8mg, 5.5mg and 11mg of 2-methylimidazole in 420 μ L of water, adding 20 μ L of HSAMS (concentration of 6mg/mL), stirring for 5min to obtain 2-methylimidazole/ enzyme solutions 1, 2, 3, 4, 5 and 6;
(3) mixing 2-methylimidazole/ enzyme solutions 1, 2, 3, 4, 5 and 6 with corresponding zinc nitrate solutions, stirring at room temperature for 4 hours, centrifuging (10000rpm, 5min), and washing the solid with Tris-HCl buffer solution with the concentration of 0.1mol/L and the pH of 8.0 for 3 times to obtain HSAMS @ ZIF-8 samples 1, 2, 3, 4, 5 and 6;
(4) a mixed solution of the HSAMS reaction substrates is prepared, and the serial number of the mixed solution is solution a. The preparation method comprises the following steps: 37.3mg of potassium chloride (KCl), 52.9mg of magnesium chloride hexahydrate (MgCl)2·6H2O), 78.7mg of disodium adenosine triphosphate trihydrate (ATP), 14.9mg of methionine (Met) are dissolved in 1mL of Tris-HCl buffer solution at a concentration of 0.1mol/L and a pH of 8.0;
(5) to each of the HSAMS @ ZIF-8 samples 1, 2, 3, 4, 5 and 6 was added 20 μ L of solution a, 180 μ L of Tris-HCl buffer at a concentration of 0.1mol/L and a pH of 8.0, and the mixture was stirred at room temperature for 4 hours. After the reaction was completed, the reaction was quenched with 5. mu.L of trifluoroacetic acid (TFA), and the product S-adenosylmethionine (SAM) was detected by a liquid phase mass spectrometer (LC-MS), and the preparation conditions of HSAMS @ ZIF-8 sample 2 were determined to be optimal.
FIG. 1 is a scanning electron micrograph of the prepared HSAMS @ ZIF-8, which is known to be lamellar.
Example 2
The HSAMS @ ZIF-8 is proved to have good catalytic capability, and the reaction activities of the HSAMS @ ZIF-8 and free HSAMS are compared. The method comprises the following steps:
(1) dissolving 1.3mg of zinc nitrate hexahydrate in 420 mu L of water to obtain a zinc nitrate solution;
(2) dissolving 1.4mg of 2-methylimidazole in 420 μ L of water, adding 20 μ L of HSAMS (concentration of 6mg/mL) respectively, and stirring for 5min to obtain 2-methylimidazole/enzyme solution;
(3) mixing the 2-methylimidazole/enzyme solution with a zinc nitrate solution, stirring at room temperature for 4 hours, centrifuging (10000rpm, 5min), and washing the solid for 3 times by using Tris-HCl buffer solution with the concentration of 0.1mol/L and the pH value of 8.0 to obtain HSAMS @ ZIF-8;
(4) to HSAMS @ ZIF-8 was added 20 μ L of solution a, 180 μ L of Tris-HCl buffer at 0.1mol/L pH 8.0; to 20. mu.L of HSAMS (6 mg/mL) was added 20. mu.L of solution a, 160. mu.L of Tris-HCl buffer at a concentration of 0.1mol/L and pH 8.0. Both were stirred at room temperature. In reactions 1, 2, 3, 6, 17, 24, 30, 41 and 48h, 10. mu.L of each reaction solution was aspirated, 190. mu.L of 5% TFA was added, and the product S-adenosylmethionine (SAM) was detected by liquid chromatography mass spectrometry (LC-MS).
Example 3
The HSAMS @ ZIF-8 is proved to have better reaction stability than free HSAMS, and the reaction activities of the HSAMS @ ZIF-8 and the free HSAMS are compared under the conditions of high temperature, organic solvent and trypsin solution. The method comprises the following steps:
(1) 5 parts HSAMS @ ZIF-8 were prepared, numbered a1, a2, a3, a4, a5, respectively. The experimental procedure was as in steps (1) (2) (3) of example 2. Taking 5 parts of 20 mu L HSAMS (the concentration is 6mg/mL), and numbering b1, b2, b3, b4 and b5 respectively;
(2) to a1, 20 μ L of solution a, 80 μ L of Tris-HCl buffer at 0.1mol/L pH 8.0, 100 μ L of methanol; to a2 was added 20 μ L of solution a, 30 μ L of Tris-HCl buffer at 0.1mol/L pH 8.0, 150 μ L of Dimethylsulfoxide (DMSO); to a3 was added 20. mu.L of solution a, 180. mu.L of Tris-HCl buffer at 0.1mol/L pH 8.0, 0.01mg trypsin (trypsin); adding 20. mu.L of solution a, 180. mu.L of Tris-HCl buffer with the concentration of 0.1mol/L and the pH value of 8.0 into a4 and a5 respectively; to b1 was added 20 μ L of solution a, 60 μ L of Tris-HCl buffer at 0.1mol/L pH 8.0, 100 μ L of methanol; to b2, 20 μ L of solution a, 10 μ L of Tris-HCl buffer at a concentration of 0.1mol/L and pH 8.0, 150 μ L of Dimethylsulfoxide (DMSO) were added; to b3, 20 μ L of solution a, 160 μ L of Tris-HCl buffer at a concentration of 0.1mol/L and pH 8.0, 0.01mg trypsin (trypsin) was added; to b4 and b5, 20. mu.L of solution a and 160. mu.L of Tris-HCl buffer at a concentration of 0.1mol/L and pH 8.0 were added, respectively. a1, a2, a3, a5, b1, b2, b3 and b5 react for 4 hours at room temperature; a4, b4 reacted at 50 ℃ for 4 h. After the reaction was completed, the reaction was quenched with 5. mu.L of trifluoroacetic acid (TFA), and the product S-adenosylmethionine (SAM) was detected by a liquid phase mass spectrometer (LC-MS), and the results are shown in FIG. 6.
Example 4
The HSAMS @ ZIF-8 is proved to have excellent circulating capacity, and a circulating reaction experiment is carried out on the HSAMS @ ZIF-8. The method comprises the following steps:
(1) preparation of HSAMS @ ZIF-8. Experimental procedure reference was made to steps (1) (2) (3) of example 2;
(2) to HSAMS @ ZIF-8 was added 20. mu.L of solution a, 180. mu.L of Tris-HCl buffer at a concentration of 0.1mol/L and pH 8.0, stirred at room temperature for 4h, and centrifuged (10000rpm, 5min) after completion of the reaction. The supernatant was quenched with 5. mu.L of trifluoroacetic acid (TFA) and the product, S-adenosylmethionine (SAM), was detected by liquid mass spectrometry (LC-MS). The solid was washed 2 times with Tris-HCl buffer at a concentration of 0.1mol/L at pH 8.0;
(3) repeating the step (2)4 times.
The results are shown in FIG. 7.
Example 5
The HSAMS @ ZIF-8 is proved to have excellent storage capacity, and a storage experiment is carried out on the HSAMS @ ZIF-8.
The method comprises the following steps:
(1) 4 parts of HSAMS @ ZIF-8 are prepared. The experimental procedure was as in steps (1) (2) (3) of example 2. Storing 4 parts of HSAMS @ ZIF-8 at room temperature for 1 week, 2 weeks, 3 weeks, and 6 weeks, respectively;
(2) referring to step (5) of example 1, 20. mu.L of solution a, 180. mu.L of Tris-HCl buffer at a concentration of 0.1mol/L and pH 8.0 was added to 4 parts of HSAMS @ ZIF-8, respectively, and stirred at room temperature for 4 hours. After the reaction was completed, the reaction was quenched with 5. mu.L of trifluoroacetic acid (TFA), and the product, S-adenosylmethionine (SAM), was detected by liquid phase mass spectrometer (LC-MS). The results are shown in FIG. 8.
Example 6
Screening and optimizing the conditions for preparing AlcDH @ ZIF-8. The method comprises the following steps:
(1) dissolving 0.385mg, 0.5775mg, 0.77mg, 1.155mg and 1.54mg of anhydrous zinc acetate in 420 mu L of water respectively to obtain zinc acetate solutions 1, 2, 3, 4 and 5;
(2) dissolving 0.7mg, 1.05mg, 1.4mg, 2.1mg and 2.8mg of 2-methylimidazole in 420 mu L of water, adding 20 mu L of AlcDH (the concentration is 6mg/mL) respectively, and stirring for 5min to obtain 2-methylimidazole/ enzyme solutions 1, 2, 3, 4 and 5;
(3) mixing 2-methylimidazole/ enzyme solutions 1, 2, 3, 4 and 5 with corresponding zinc acetate solutions, stirring at room temperature for 4-5h, centrifuging (10000rpm, 5min), and washing the solid with Tris-HCl buffer solution with the concentration of 0.1mol/L and the pH of 8.0 for 3 times to obtain AlcDH @ ZIF-8 samples 1, 2, 3, 4 and 5;
(4) and preparing an AlcDH reaction substrate mixed solution, wherein the number of the AlcDH reaction substrate mixed solution is solution b. The preparation method comprises the following steps: 66.343mg of oxidized coenzyme I (NAD)+) 5.84. mu.L of ethanol was dissolved in 1mL of Tris-HCl buffer (pH 8.0) at a concentration of 0.1 mol/L;
(5) to each of the HSAMS @ ZIF-8 samples 1, 2, 3, 4 and 5 was added 20 μ L of solution b, 180 μ L of Tris-HCl buffer at a concentration of 0.1mol/L and pH 8.0, and the mixture was stirred at room temperature for 4 hours. After the reaction is finished, quenching the reaction by using 1 mu L of trifluoroacetic acid (TFA), detecting the product reductive coenzyme I (NADH) by using a liquid phase mass spectrometer (LC-MS), and determining the preparation condition of the AlcDH @ ZIF-8 sample 2 as the optimal condition.
Example 7
The AlcDH @ ZIF-8 is proved to have good catalytic capability, and the reaction activities of the AlcDH @ ZIF-8 and free AlcDH are compared. The method comprises the following steps:
(1) 0.5775mg of anhydrous zinc acetate is dissolved in 420 mu L of water to obtain a zinc acetate solution;
(2) dissolving 1.05mg of 2-methylimidazole in 420 mu L of water, respectively adding 20 mu L of AlcDH (the concentration is 6mg/mL), and stirring for 5min to obtain a 2-methylimidazole/enzyme solution;
(3) mixing the 2-methylimidazole/enzyme solution with the zinc acetate solution, stirring at room temperature for 5h, centrifuging (10000rpm, 5min), and washing the solid for 3 times by using Tris-HCl buffer solution with the concentration of 0.1mol/L and the pH value of 8.0 to obtain AlcDH @ ZIF-8;
(4) to AlcDH @ ZIF-8 were added 20. mu.L of solution b, 20. mu.L of methylene blue solution at a concentration of 1mmol/L, 160. mu.L of Tris-HCl buffer at a concentration of 0.1mol/L and pH 8.0; to 20. mu.L of AlcDH (6 mg/mL) were added 20. mu.L of solution b, 20. mu.L of methylene blue solution at a concentration of 1mmol/L, and 140. mu.L of Tris-HCl buffer at a concentration of 0.1mol/L and pH 8.0. Both were stirred at room temperature. After 10, 30, 60 and 120min of reaction, 10. mu.L of the reaction solution was aspirated, 1. mu.L of 5% TFA was added, and after centrifugation, the absorbance corresponding to the maximum absorption peak was measured at 664nm with an ultraviolet spectrophotometer, respectively.
Example 8
The reaction stability of AlcDH @ ZIF-8 is proved to be better than that of free AlcDH, and the reaction activities of AlcDH @ ZIF-8 and free AlcDH are compared under the conditions of high temperature, organic solvent and trypsin solution. The method comprises the following steps:
(1) 5 parts of AlcDH @ ZIF-8 are prepared, numbered c1, c2, c3, c4, c5, respectively. The experimental procedure was as in steps (1) (2) (3) of example 7. Taking 5 parts of 20 mu L AlcDH (the concentration is 6mg/mL) with the numbers of d1, d2, d3, d4 and d5 respectively;
(2) to c1 was added 20 μ L of solution b, 80 μ L of Tris-HCl buffer at 0.1mol/L pH 8.0, 100 μ L of methanol; to c2 was added 20 μ L of solution b, 30 μ L of Tris-HCl buffer at 0.1mol/L pH 8.0, 150 μ L of dimethyl sulfoxide (DMSO); to c3 was added 20 μ L of solution b, 180 μ L of Tris-HCl buffer at 0.1mol/L pH 8.0, 0.01mg trypsin (trypsin); to c4 and c5 were added 20 μ L of solution b, 180 μ L of Tris-HCl buffer at 0.1mol/L pH 8.0, respectively; to d1 was added 20 μ L of solution b, 60 μ L of Tris-HCl buffer at 0.1mol/L pH 8.0, 100 μ L of methanol; to d2 was added 20 μ L of solution b, 10 μ L of Tris-HCl buffer at 0.1mol/L pH 8.0, 150 μ L of dimethyl sulfoxide (DMSO); to d3 was added 20 μ L of solution b, 160 μ L of Tris-HCl buffer at 0.1mol/L, pH 8.0, 0.01mg trypsin (trypsin); to d4 and d5 were added 20. mu.L of solution b, 160. mu.L of Tris-HCl buffer at a concentration of 0.1mol/L and pH 8.0, respectively. c1, c2, c3, c5, d1, d2, d3 and d5 react for 4 hours at room temperature; c4, d4 at 50 ℃ for 4 h. After the reaction was completed, the reaction was quenched with 1. mu.L of trifluoroacetic acid (TFA), and the product of reduced coenzyme I (NADH) was detected by liquid phase mass spectrometer (LC-MS), and the results are shown in FIG. 14.
Example 9
The AlcDH @ ZIF-8 is proved to have excellent circulating capacity, and a circulating reaction experiment is carried out on the AlcDH @ ZIF-8. The method comprises the following steps:
(1) preparation of AlcDH @ ZIF-8. Experimental procedure reference was made to steps (1) (2) (3) of example 7;
(2) to AlcDH @ ZIF-8 was added 20. mu.L of solution b, 180. mu.L of Tris-HCl buffer (0.1 mol/L) at pH 8.0, and the mixture was stirred at room temperature for 4 hours, followed by centrifugation (10000rpm, 5min) after completion of the reaction. The supernatant was quenched with 1. mu.L of trifluoroacetic acid (TFA) and the product reduced coenzyme I (NADH) was detected by liquid phase mass spectrometry (LC-MS). The solid was washed 2 times with Tris-HCl buffer at a concentration of 0.1mol/L at pH 8.0;
(3) repeating the step (2)4 times.
Example 10
The AlcDH @ ZIF-8 is proved to have excellent storage capacity, and a storage experiment is carried out on the AlcDH @ ZIF-8.
The method comprises the following steps:
(1) 4 parts of AlcDH @ ZIF-8 are prepared. The experimental procedure was as in steps (1) (2) (3) of example 7. Storing 4 parts of AlcDH @ ZIF-8 at room temperature for 1 week, 2 weeks, 3 weeks and 6 weeks respectively;
(2) referring to step (5) of example 6, 20. mu.L of solution b, 180. mu.L of Tris-HCl buffer at a concentration of 0.1mol/L and pH 8.0 was added to 4 parts of AlcDH @ ZIF-8, respectively, and the mixture was stirred at room temperature for 4 hours. After the reaction was completed, the reaction was quenched with 1. mu.L of trifluoroacetic acid (TFA), and the product reduced coenzyme I (NADH) was detected by liquid phase mass spectrometer (LC-MS). The results are shown in FIG. 16.
In summary, the invention provides a preparation method of a novel ZIF-8/enzyme composite material, which is characterized in that a ZIF-8 crystal grows on the surface of an enzyme in a lamellar structure by screening and optimizing a proper precursor concentration, and finally the ZIF-8/enzyme nanoflower composite material is formed. ZIF-8 has excellent biocompatibility and mild synthesis conditions, so that the activity of the enzyme is not influenced basically, and meanwhile, ZIF-8 can protect the enzyme to react under severe conditions of high temperature, organic, protease and the like, so that the circulation and storage capacity of the enzyme are improved. The greatest advantage of the present invention compared to the traditional co-precipitation strategy is that it allows large size substrates to pass through the gaps between the ZIF-8 lamellar structures to contact the active center of the enzyme, thus greatly broadening the scope of application of the strategy.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (5)
1. A preparation method of a ZIF-8/enzyme composite material is characterized in that ZIF-8 crystals grow on the surface of enzyme in a lamellar structure by adjusting the concentration of a precursor to form the ZIF-8/enzyme nanoflower composite material;
the method comprises the following specific steps: mixing the metal salt aqueous solution with the mixed solution of the ligand aqueous solution and the enzyme, stirring for a period of time at room temperature, and separating out a solid product to obtain the ZIF-8/enzyme composite material;
the metal salt is zinc nitrate hexahydrate or anhydrous zinc acetate, and the concentration of the metal salt aqueous solution is 0.5-24 g/L; the ligand is 2-methylimidazole, and the concentration of the ligand aqueous solution is 1.6-7 g/L; the molar ratio of the metal salt to the ligand is 1: 4.
2. the method of making a ZIF-8/enzyme composite as claimed in claim 1, wherein the enzyme comprises a large-sized substrate enzyme.
3. The method of preparing ZIF-8/enzyme composite according to claim 1, wherein the enzyme is a human S-adenosylmethionine synthetase or an alcohol dehydrogenase.
4. The method of claim 1, wherein the solid product is separated by centrifugation at 12000rpm, 8000 and 3-6 min.
5. The method of manufacturing ZIF-8/enzyme composite according to claim 1, wherein the solid product is washed with tris (hydroxymethyl) aminomethane buffer at a concentration of 0.1mol/L and pH = 8.0.
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| CN112175931A (en) * | 2020-10-16 | 2021-01-05 | 安徽师范大学 | Immobilized carboxylesterase, and preparation method and application thereof |
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| CN115418363A (en) * | 2022-08-30 | 2022-12-02 | 华侨大学 | Method for encapsulating yeast by using ZIF-8 nano material based on biomimetic mineralization and composite material and application thereof |
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