CN108396023B

CN108396023B - Preparation of magnetic MOF materials by milling and use for enzyme immobilization

Info

Publication number: CN108396023B
Application number: CN201810133548.1A
Authority: CN
Inventors: 张海霞; 邹玉琳
Original assignee: Zhongwei High-Tech Research Institute Lanzhou University; Lanzhou University
Current assignee: Zhongwei High-Tech Research Institute Lanzhou University; Lanzhou University
Priority date: 2018-02-09
Filing date: 2018-02-09
Publication date: 2021-02-02
Anticipated expiration: 2038-02-09
Also published as: CN108396023A

Abstract

The invention relates to a method for preparing magnetic MOF materials by grinding and used for enzyme immobilization. With zinc oxide and 2-methylimidazole and Fe₃O₄One-step method for simply and rapidly synthesizing magnetic Fe by taking magnetic nanoparticles as raw materials₃O₄@ ZIF-8 material. The novel preparation method has the advantages that the novel preparation method is safe, quick and pollution-free, the material is successfully used for immobilizing the lipase, the enzyme activity is kept and the stability is greatly improved after the lipase is immobilized, and the material has magnetism, is easy to separate and realizes repeated use. And the material is also suitable for fixation of trypsin, which indicates Fe₃O₄The @ ZIF-8 material has universality for enzyme immobilization. The free enzyme becomes a relatively ideal catalyst after immobilization, and has a good application prospect for modern industry.

Description

Preparation of magnetic MOF materials by milling and use for enzyme immobilization

Technical Field

The invention relates to a method for preparing magnetic MOF material (Fe) by using a grinding method₃O₄@ ZIF-8) and is used for enzyme immobilization, belonging to the technical field of functional nano material preparation.

Background

Free enzymes are not an ideal catalyst for modern industry. The temperature and pH value range of the survival of the free enzyme are narrow, and the enzyme is easy to denature and inactivate. The catalyst is unstable to heat, acid, alkali and organic solvents, and has the defects difficult to overcome in the aspect of catalytic reaction: (1) it is susceptible to microbial degradation or aggregation and inactivation. (2) In a homogeneous catalytic system, the enzyme is difficult to separate from the product, resulting in impure product and affecting the quality of the product. (3) Continuous operation cannot be achieved. (4) Free enzymes are not easily recovered, are difficult to reuse, and are expensive. In order to overcome the disadvantages of free enzyme, people explore to fix water-soluble enzyme, so that the catalytic activity of the enzyme is kept, the stability of the enzyme is improved, the fixed enzyme is easy to separate solid from liquid, and the enzyme can be reused. The MOF (metal organic framework material) is a microporous reticular framework formed by transition metal ions or metal clusters and organic ligands through complexation, the internal arrangement of the framework is regular, and the framework has a relatively large specific surface area, adjustable pores, good mechanical properties and good stability in a solvent. Many researchers have demonstrated that MOF materials are a very good enzyme immobilization material, but most MOF materials are prepared by high temperature and high pressure. The mode of high temperature and high pressure has many disadvantages, such as energy consumption, non-conformity with the concept of environmental protection, need the participation of large-scale instruments, high cost, danger, easy occurrence of major safety accidents, etc.

Disclosure of Invention

In view of the above, it is an object of the present invention to provide a process for the preparation of magnetic MOF materials (Fe) by grinding₃O₄@ZIF-8）。

It is another object of the present invention that the magnetic MOF material is used for the immobilization of enzymes.

The purpose of the invention is realized by the following technical scheme

Magnetic MOF materials (Fe)₃O₄@ ZIF-8), which comprises the following steps:

adding Fe into a mortar₃O₄ZnO and 2-methylimidazole (HMeIM), ground in a mortar for 10min, and collected solid particles (Fe)₃O₄@ ZIF-8), washing the solid particles with ultrapure water for 5-6 times, approaching the solid particles with a magnet to realize magnetic recovery, and vacuum drying the solid particles at 60 ℃ for 12 h.

The magnetic MOF material (Fe)₃O₄@ ZIF-8) for the immobilization of enzymes, the steps of which are:

weighing enzyme, dissolving in water, centrifuging at 12000 rpm at 4 deg.C for 15 min, collecting supernatant, lyophilizing, and standing at 4 deg.C. Weighing 100mgThe lyophilized enzyme of (2) was dissolved in a 100m phosphate buffer solution of pH =7.4 and 10 mM, and 5mg of Fe was added to 5ml of the buffer solution₃O₄@ ZIF-8 material, solid particles and solution were mixed well using a vortex machine and left overnight in a shaker at 25 ℃. The loading amount of the enzyme was measured by Beford method as being capable of immobilizing 28 μ g of the enzyme per mg of solid particles, magnetically recovering the immobilized enzyme using a magnet, and washing the material 3 to 4 times with a buffer of pH =7.4,10 mM phosphoric acid to remove the enzyme physically adsorbed on the material and the free enzyme not immobilized in the solution.

The advantages and the beneficial effects of the invention are as follows:

the invention has the advantages that (1) the method is simple and rapid, and the magnetic Fe can be obtained only by grinding in a mortar for 10min₃O₄@ ZIF-8 material; (2) the method is green and environment-friendly, and the solid-phase reaction does not need the participation of an organic solvent, does not pollute the environment and does not harm the health of human beings; (3) devices such as heating, centrifugation and the like are not needed in the material preparation process and the material using process, so that the energy consumption and the cost are low; the method has the advantages that (4) loss is avoided, a large amount of non-magnetic MOF materials can be synthesized in the preparation process of the traditional magnetic MOF materials, so that the cost is increased, the magnetic MOF materials are ground in a mortar, and magnetic nano particles are introduced, so that the materials can be quickly subjected to magnetic separation from a solution, and the existing centrifugal process is avoided; after the enzyme is fixed, the enzyme can be recycled, so that the cost of the catalyst is greatly reduced. Fe prepared by the invention₃O₄The @ ZIF-8 material is successfully used for immobilizing lipase and trypsin, and the immobilized enzyme has good enzymatic activity, stability and reusability.

Drawings

FIG. 1 is a schematic diagram of a magnetic MOF material synthesized by the invention and used for Lipase immobilization, wherein HMeIM is 2-methylimidazole, and Lipase is Lipase.

FIG. 2 shows Fe in the present invention₃O₄@ZIF-8、Lipase@Fe₃O₄The infrared spectra of @ ZIF-8 and Lipase.

FIG. 3 shows pure Fe synthesized by the present invention₃O₄ZnO, ZIF-8 and Fe₃O₄SEM spectrogram of @ ZIF-8, wherein (a) is Fe₃O₄(b) ZnO, (c) ZIF-8, and (d) Fe₃O₄@ZIF-8。

FIG. 4 shows Fe in the present invention₃O₄、Fe₃O₄@ZIF-8、Lipase@Fe₃O₄@ ZIF-8, in which (a) is Fe₃O_4，(b) Is Fe₃O₄@ ZIF-8, (c) is Lipase @ Fe₃O₄@ZIF-8。

FIG. 5 shows kinetic curves and kinetic constants of Free enzyme (Free lipase) and Immobilized enzyme (Immobilized lipase) according to the present invention.

FIG. 6 shows the relative enzyme activities of Free enzyme (Free lipase) and Immobilized enzyme (Immobilized lipase) at different pH values in the present invention.

FIG. 7 shows relative enzyme activities of Free enzyme (Free lipase) and Immobilized enzyme (Immobilized lipase) in the present invention incubated at 55 ℃ for various periods of time.

FIG. 8 shows relative enzyme activities of Free enzyme (Free lipase) and Immobilized enzyme (Immobilized lipase) in the present invention at room temperature for different periods of time.

FIG. 9 shows reusability of the immobilized enzyme in the present invention.

Detailed Description

The enzyme referred to in this example is lipase. In recent years, lipases have attracted much attention due to their unique catalytic properties, and they can catalyze various organic reactions such as ester hydrolysis, ester exchange, esterification, etc. In addition, lipases can achieve high enantioselectivity and high regioselectivity for substrates under very mild conditions.

The technical scheme of the invention is further explained by combining the drawings, the embodiments and the experimental examples as follows:

example 1

Magnetic MOF materials (Fe)₃O₄@ ZIF-8) preparation and enzyme immobilization

60mg of Fe was added to the mortar₃O₄120mg ofZnO and 242mg of 2-methylimidazole (HMeIM) were ground in a mortar for 10min, and solid particles (Fe) were collected₃O₄@ ZIF-8), washing the solid particles with ultrapure water for 5-6 times, using a magnet to approach the solid particles to realize magnetic recovery, and drying the solid particles at 60 ℃ for 12 hours in vacuum. Weighing enzyme, dissolving in water, centrifuging at 12000 rpm at 4 deg.C for 15 min, collecting supernatant, lyophilizing, and standing at 4 deg.C. 100mg of the lyophilized enzyme was weighed out and dissolved in 100ml of a 10 mM phosphate buffer with pH =7.4, 5ml of the buffer was taken, and 5mg of Fe was added₃O₄@ ZIF-8 material, solid particles and solution were mixed well using a vortex machine and left overnight in a shaker at 25 ℃. The loading of the enzyme was determined by Beford method (Bradford method: Coomassie brilliant blue G250, a compound which forms a blue color after binding to the protein, has a maximum absorption peak at 595nm, and the shade of blue is proportional to the protein concentration, which is a rapid, accurate, and reproducible method for protein quantification) as Fe per mg₃O₄The @ ZIF-8 material was able to immobilize 28 micrograms of enzyme, magnetically recover the immobilized enzyme using a magnet, and wash the material 3-4 times with a buffer of 10 mM phosphoric acid at pH =7.4 to remove the enzyme physically adsorbed on the material and the free enzyme not immobilized in the solution.

In FIG. 3, (a) is Fe₃O₄(b) ZnO, (c) ZIF-8, and (d) Fe₃O₄Comparison of the @ ZIF-8 with the four materials shows that (a) Fe₃O₄The magnetic nanoparticles are relatively uniform in size, the particle size is approximately 20nm, and the particle size of the (c) pure MOF material prepared by grinding is approximately 200 nm. Adding Fe₃O₄After the magnetic nano particles are obtained, the obtained (d) magnetic MOF material has more uneven surface than the pure (c) MOF material, and Fe can be determined₃O₄The magnetic nanoparticles are successfully embedded on the surface of the MOF material, which indicates that the material synthesis is successful. As can be seen from the IR spectrum of FIG. 4, the immobilized enzyme (Lipase @ Fe)₃O₄@ ZIF-8) with not only material (Fe)₃O₄@ ZIF-8) and an absorption peak specific to the enzyme (Lipase), indicating that the enzyme is successfully immobilized to Fe₃O₄@ ZIF-8 material surface.As can be seen from the hysteresis curve of FIG. 5, Fe is represented by (a)₃O_4，(b) Is Fe₃O₄@ ZIF-8, (c) is Lipase @ Fe₃O₄@ ZIF-8, (c) magnetic properties of the enzyme-immobilized magnetic MOF material and (a) pure Fe₃O₄The magnetic properties of the magnetic nanoparticles are comparatively reduced, but magnetic separation is still possible.

Kinetic equation study of immobilized enzymes

The michaelis equation parameters for immobilized and free enzymes, including the michaelis constant Km and the maximum reaction rate Vmax of the immobilized and free enzymes, were calculated by measuring the reaction rates at different substrate concentrations of p-nitrophenylpalmitate (pNPP) at 25 ℃, pH = 7.4. Wherein the Michaelis constant Km represents the affinity of the enzyme with the substrate, the smaller Km, the stronger the affinity, Vmax represents the maximum reaction rate of the enzyme-catalyzed reaction, and the larger Vmax, the stronger the activity of the enzyme. As shown in FIG. 5, the Km of Free enzyme (Free lipase) was 29.2, and Vmax was 3.3; the Km of the Immobilized enzyme (Immobilized lipase) was 83.7, and Vmax was 8.7. After immobilization of the enzyme, Km is nearly tripled, which is consistent with many literature reports. This is because the water-soluble enzyme is immobilized on the solid material to increase the steric hindrance of the enzyme to the substrate, so that the affinity of the enzyme to the substrate is decreased. The Vmax of the immobilized enzyme was also increased as compared with that of the free enzyme, and it was found that Fe₃O₄@ ZIF-8 has certain enhancement on the activity of lipase.

Stability study of immobilized enzymes

Stability of pH

The immobilized enzyme and the free enzyme were placed in buffer solutions of different pH (pH = 4-9), respectively, and after 2h at room temperature, the activities of the free enzyme and the immobilized enzyme at different pH were determined by measuring the content of p-nitrophenol (pNP), which is a hydrolysate of p-nitrophenylpalmitate (pNPP). pNP has an absorption maximum at an ultraviolet wavelength of 405 nm. As can be seen from FIG. 6, the Immobilized enzyme (Immobilized lipase) still retained the property of Free enzyme, and both had an optimum pH of about 7 for the Free enzyme (Free lipase), but after immobilizing the enzyme on the MOF material, it was more resistant to alkaline strips than the Free enzymeAnd (3) a component. Thus, the free enzyme passes through Fe₃O₄The pH stability is improved to a certain extent after the @ ZIF-8 material is fixed.

b. Thermal stability

The immobilized enzyme and free enzyme were dispersed in a buffer of phosphoric acid at pH =7.4, and then incubated at a high temperature of 55 ℃. The immobilized enzyme and the free enzyme are taken out every 1h for enzyme activity measurement, and the thermal stability of the immobilized enzyme and the free enzyme at 55 ℃ is examined. As shown in fig. 7, the activity of Free enzyme (Free lipase) decreased to 45% after 5h in an environment of pH =7.4 and temperature of 55 ℃. While the activity of Immobilized enzyme (Immobilized lipase) is still kept above 90%, and the activity of Free enzyme (Free lipase) is gradually lost with the increase of incubation time. Therefore, the thermal stability of the enzyme after the enzyme is immobilized is greatly improved, which undoubtedly expands the use temperature range of the enzyme.

c. Stability over time

After the enzyme was immobilized, the stability of the immobilized enzyme at room temperature over time was examined. First, the immobilized enzyme and the free enzyme were dispersed in a phosphate buffer solution with pH =7.4, and the immobilized enzyme and the free enzyme were taken out at intervals of 2 days at room temperature, respectively, and the enzyme activity was measured. As shown in FIG. 8, the activity of Immobilized enzyme (Immobilized lipase) was maintained at 80% or more and the activity of Free enzyme (Free lipase) was reduced to 20% even after standing at room temperature for 8 days. As can be seen from the above, the free enzyme passes through Fe₃O₄After the @ ZIF-8 material is fixed, the time stability is improved, and the immobilized enzyme can well solve the problem of harsh enzyme storage conditions.

Reusability study of immobilized enzymes

Compared with free enzyme, the immobilized enzyme has the greatest advantage that the immobilized enzyme can be quickly subjected to magnetic separation from a solution, and the free enzyme is dissolved in water, cannot be recycled and cannot be reused, so that the reuse of the immobilized enzyme is realized, and the economic cost is reduced. The reusability of the immobilized enzyme was examined. As can be seen from FIG. 9, the activity of the immobilized enzyme was maintained at about 70% after 6 times of repeated use. Therefore, the immobilized enzyme has good stability and reusability. If the catalyst is applied to the industrial field, the cost of the catalyst can be greatly reduced.

Example 2

Fe₃O₄Universality study of @ ZIF-8 material

Trypsin is one of proteolytic enzymes that can efficiently hydrolyze denatured proteins, and thus is widely used in sample pretreatment of complex biological samples and in the medical field. To explore the synthesized Fe₃O₄The invention uses the magnetic MOF material for the immobilization of trypsin. The preparation of the magnetic MOF material is consistent with that of a material for fixing lipase, and the preparation method is completely the same. The material is determined to have the capability of fixing trypsin, and the loading rate of the material reaches per milligram of Fe₃O₄The @ ZIF-8 material was able to immobilize 40 micrograms of trypsin, which retained 82% of the activity of the free enzyme. Thus, the milled synthetic magnetic MOF material (Fe)₃O₄@ ZIF-8) can be used as an enzyme immobilization material and applied to immobilization of different enzymes.

Claims

1. A method for preparing magnetic MOF material and for the immobilization of enzymes by grinding, comprising the steps of:

magnetic MOF material Fe₃O₄Preparation of @ ZIF-8

60mg of Fe was added to the mortar₃O₄120mg of ZnO and 242mg of 2-methylimidazole HMeIM, grinding in a mortar for 10min, and collecting solid particles Fe₃O₄@ ZIF-8, washing the solid particles with ultrapure water for 5-6 times, enabling the washed solid particles to be close to the magnet, realizing magnetic recovery, and drying the magnetic solid particles at 60 ℃ for 12 hours in vacuum;

immobilization of enzymes

Weighing enzyme, dissolving in water, centrifuging at 12000 rpm at 4 deg.C for 15 min, collecting supernatant, lyophilizing, and placing at 4 deg.C, weighing 100mg lyophilized enzyme, dissolving in 100ml 10 mM phosphoric acid buffer solution with pH =7.4, collecting buffer solution 5ml, adding 5mg Fe₃O₄@ ZIF-8 material, using a vortex machine to mix solid particles and solution uniformly, and in a shakerOvernight at medium 25 ℃; the loading amount of the enzyme was measured by the Beford method as being capable of immobilizing 28. mu.g of the enzyme per mg of the solid particles, magnetically recovering the immobilized enzyme using a magnet, and washing the material 3 to 4 times with a buffer solution of pH =7.4,10 mM phosphoric acid to remove Fe₃O₄@ ZIF-8 material physically adsorbed enzymes and free enzymes not immobilized in solution.

2. A process for the preparation of magnetic MOF materials and for the immobilization of enzymes by grinding according to claim 1, wherein: the enzymes used above were: lipase or trypsin.