CN112251429B

CN112251429B - Preparation method and application of ZIF-8@FDH nanometer hybrid material

Info

Publication number: CN112251429B
Application number: CN202011068097.1A
Authority: CN
Inventors: 王生杰; 修阳; 李�杰; 尹三立; 杨宏琳; 贾超群; 曹美文; 夏永清
Original assignee: China University of Petroleum East China
Current assignee: China University of Petroleum East China
Priority date: 2020-10-08
Filing date: 2020-10-08
Publication date: 2023-06-02
Anticipated expiration: 2040-10-08
Also published as: CN112251429A

Abstract

The invention provides a preparation method and application of a ZIF-8@FDH nanometer hybrid material. Belongs to the field of nano materials, and can solve the problems of complex system, poor dispersibility, damage to enzyme in the reaction process and the like of the existing enzyme immobilization method. The technical scheme includes that (1) zinc acetate dihydrate is dissolved in water to obtain solution A; (2) Dissolving 2-methylimidazole in water, adding FDH powder, and uniformly mixing to obtain a solution B; (3) Dropwise adding the solution A into the solution B gradually under stirring, and reacting to obtain ZIF-8@FDH suspension; (4) And (3) washing and centrifuging the suspension, and vacuum drying the precipitate for 12 hours to obtain the ZIF-8@FDH nano hybrid material. The ZIF-8@FDH nanometer hybrid material obtained by the method can be used for catalyzing the specific conversion of carbon dioxide to formic acid.

Description

Preparation method and application of ZIF-8@FDH nanometer hybrid material

Technical Field

The invention belongs to the technical field of nano materials, and particularly relates to a method for fixing Formate Dehydrogenase (FDH) by using ZIF-8 and application thereof.

Technical Field

As a biocatalyst, formate dehydrogenase can efficiently catalyze the reduction of formic acid to carbon dioxide and reduced nicotinamide adenine dinucleotide NADH at normal temperature and pressure, or catalyze the reduction of carbon dioxide to formic acid in the presence of a suitable electron donor. However, enzymes are expensive and have poor stability. Is easy to be deactivated in high temperature, acid-base environment or organic solution. And the enzyme in the product is difficult to separate, which is unfavorable for recycling the enzyme and can cause pollution of the product. Therefore, immobilization of enzymes is one of the important measures to improve the efficiency of use of FDH and to reduce costs.

In recent years, enzyme immobilization techniques have been widely studied, and immobilization carriers and immobilization methods have been reported. Wherein the metal-organic framework (MOF) is a porous material consisting of metal-containing nodes and organic ligands linked by coordination bonds. Because of the abundance of geometry and connectivity of metal nodes and ligands, its topology can be designed and tailored for specific functions. Because of their excellent controllable framework structure and simple preparation, they have become an important method for immobilized enzymes (Lian X, fang Y, joseph E, et al enzyme-MOF (metal-organic framework) compositions [ J ]. Chemical Society Reviews,2017,46 (11): 3386-3401.). And the MOF has extremely high specific surface area and pore volume, and the pore diameter is easy to adjust. The synthesis mode is mild, and the enzyme is not damaged.

Methods utilizing MOF immobilized enzymes can be divided into four types, surface attachment, covalent bonding, pore encapsulation, and co-precipitation. Wherein, the coprecipitation method mainly forms a zeolite imidazolium salt framework (ZIF-8) by coordination of zinc ions and 2-methylimidazole (HmIm), can fix zymogen sites in the synthesis process, and allows guest molecules with larger pore diameters to be encapsulated in the ZIF structure. Not only is the structure of ZIF-8 important for enzyme immobilization and enzyme activity maintenance, the presence of enzyme molecules counteracts ZIF-8, affecting its framework structure and pore size, and thus affecting the activity of the immobilized enzyme. Accordingly, there is a need to design ZIF-8 framework structures suitable for FDH fixation. In addition, the presently reported methods have severe ZIF-8 agglomeration and are disadvantageous for dispersion in solution to increase the contact of the enzyme with the substrate, resulting in poor catalytic efficiency. Therefore, a new preparation method is urgently needed to improve the dispersibility of ZIF-8 in an aqueous solution.

Disclosure of Invention

Aiming at the problems existing in the existing MOF biological enzyme immobilization method, the invention provides a ZIF-8 FDH immobilization method and application. The preparation method is simple, the reaction condition is mild, and the enzyme is not damaged. When the obtained immobilized material is used as a catalyst for enzymatic reaction, the immobilized material can keep higher enzyme activity, has better tolerance to environment, is easy to separate, can be recycled and the like. The specific invention comprises the following steps:

the invention provides a preparation method of a ZIF-8@FDH nanometer hybrid material, which is characterized by comprising the following steps:

s1: zinc acetate dihydrate is dissolved in water to obtain a solution A;

s2: dissolving 2-methylimidazole in water, adding FDH powder, and uniformly mixing to obtain a solution B;

s3: dropwise adding the solution A into the solution B gradually under stirring, and reacting to obtain ZIF-8@FDH suspension;

s4: and (3) after the suspension is centrifugally washed, carrying out vacuum drying on the precipitate for 12 hours to obtain the ZIF-8@FDH nanometer hybrid material.

Preferably, the zinc acetate dihydrate concentration is 10-40mM and the 2-methylimidazole concentration is 50-160mM.

Preferably, the volume ratio of the solution A to the solution B is 0.5-4.

Preferably, the FDH is present in a concentration of 0.05-0.5mg/mL. .

Preferably, the reaction is carried out at room temperature for a reaction time of 12 to 48 hours.

Preferably, the centrifugation speed of the ZIF-8@FDH suspension is 5000-12000rpm, and the centrifugation time is 10-30min.

The invention provides a ZIF-8@FDH nanometer hybrid material prepared by the preparation method.

The invention provides an application of the ZIF-8@FDH nanometer hybrid material prepared by the preparation method in catalyzing the specific conversion of carbon dioxide to formic acid.

Compared with free enzyme, the activity of FDH in the ZIF-8@FDH nanometer hybrid material prepared by the invention is maintained at more than 80%, and the tolerance to the environment is improved.

Compared with the prior art, the invention has the advantages and positive effects that:

1. the invention utilizes the zeolite imidazole salt framework formed by coordination of zinc ions and 2-methylimidazole to fix the FDH in situ in the aqueous solution, and the ZIF-8@FDH nanometer hybrid material has better dispersibility in the aqueous phase. The stability of the FDH is improved, and the activity of the immobilized enzyme is better maintained, so that the FDH can be recycled.

2. The preparation method provided by the invention is simple, the water-based environment is friendly to enzyme, the price is low, and the further industrialized generation and commercial popularization are facilitated.

Drawings

FIG. 1 is a Transmission Electron Microscope (TEM) photograph of ZIF-8@FDH nanohybrid material prepared in example 3 of the present invention;

FIG. 2 is an optical photograph of a ZIF-8@FDH nanohybrid material prepared in example 3 of the present invention dispersed in an aqueous solution;

FIG. 3 is a Fourier transform infrared spectrum (FTIR) of a ZIF-8@FDH nanohybrid material prepared in example 3 of the present invention with pure ZIF-8;

FIG. 4A is a graph showing the standard curve of absorbance in the UV-visible absorption spectrum corresponding to aqueous NADH solutions of different concentrations;

FIG. 4B shows the catalytic action of NAD when the ZIF-8@FDH nanohybrid material prepared in example 3 of the present invention and free FDH are used as catalysts ⁺ In the reduction process, the generated product NADH is related to the reaction time;

FIG. 5 shows the enzymatic activities of ZIF-8@FDH and free FDH prepared in example 3 of the present invention at different temperatures;

FIG. 6 is a graph showing the relationship between NADH and reaction time of the ZIF-8@FDH nano hybrid material prepared in example 3 of the present invention during cyclic catalysis;

FIG. 7 is a graph showing the conversion rate versus time of the ZIF-8@FDH nanohybrid material prepared in example 3 of the present invention catalyzing the conversion of CO2 to formic acid.

Detailed Description

The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The embodiment of the invention provides a preparation method of a ZIF-8@FDH nanometer hybrid material, which is characterized by comprising the following steps:

s1: zinc acetate dihydrate is dissolved in water to obtain a solution A;

The principle of the method disclosed in the above examples is the zeolite imidazolium framework formed by coordination of zinc ions with 2-methylimidazole. In this process, the biomacromolecule can adjust the size, morphology and crystallinity of the framework material by participating in the framework construction process, and simultaneously encapsulate itself in the porous framework material, and then create new cavities which closely surround the biomacromolecule and interact with the framework thereof, thereby fixing the enzyme molecule in situ in the solution, and finally forming a polyhedral structure with a specific morphology, as shown in fig. 1. The advantage of this reaction is that: the reaction is carried out in aqueous solution, the condition is mild, the operation is simple, and the activity of enzyme is not damaged in the preparation process.

In a preferred embodiment, the zinc acetate dihydrate concentration is 10-40mM and the 2-methylimidazole concentration is 50-160mM. It should be noted that each enzyme molecule surface will bind a fixed amount of zinc ions and HmIm ligand, and increasing the local concentration of metal cations and organic ligands will promote the pre-nucleation of ZIF-8 around the biomacromolecule, thereby allowing the formation of a controlled structure of crystals. Thus, the concentration of zinc ions and HmIm is critical and can have an impact on the structure and size of the complex formed by the enzyme and ions. And ultimately affect the activity of the immobilized product, thus its concentration needs to be fixed. Too high or too low a concentration affects the structure and size of the complex formed by the enzyme and the ions. And ultimately affect product activity. In this example, zinc acetate dihydrate concentration is preferably 10-40mM, and 2-methylimidazole concentration is preferably 50-160mM, and a regular framework structure can be formed.

In a preferred embodiment, the volume ratio of solution A to solution B is 0.5-4. It is understood that by controlling the ratio of zinc ions to HmIm, crystals can be produced only in the presence of enzymes, reducing the effect of pure ZIF-8 crystals on the product. When the HmIm concentration is too high, pure ZIF-8 is rapidly formed without adding an enzyme, and removal is difficult, so that the overall activity of the final product is affected.

In a preferred embodiment, the FDH is present at a concentration of 0.05-0.5mg/mL. As described above, each enzyme molecule surface will bind a fixed amount of zinc ions and HmIm ligand, and the excess enzyme molecule will not bind the ligand ions. And may be adsorbed on the crystal surface of ZIF-8 to increase the particle size of the product and form a precipitate. The substrate is not easy to fully diffuse to contact with the catalyst, so that the catalytic efficiency is affected. In this example, FDH concentration of 0.05-0.5mg/mL was selected to meet the requirements of good dispersibility and less agglomeration (as shown in FIG. 2). And the characteristic peaks of N-H and amide bands of the FDH are shown in the reaction product besides the characteristic of the ZIF-8 group through the characteristic of an infrared spectrogram (shown in figure 3), so that the successful synthesis of the ZIF-8@FDH nano hybrid material is shown.

In a preferred embodiment, the reaction is carried out at room temperature for a period of time ranging from 12 to 48 hours. The reaction is carried out at room temperature in order to better maintain the activity of the biological enzyme. The reaction time is controlled between 12 and 48 hours at room temperature, the reaction is more complete, and the ZIF-8@FDH nanometer hybrid material with a regular structure and good catalytic property can be obtained.

In a preferred embodiment, the ZIF-8@FDH suspension is centrifuged at 5000-12000rpm for 10-30min. The purpose of centrifugation is to separate the immobilized ZIF-8@FDH from the solution, thereby removing unreacted reactants and the non-immobilized FDH molecules, to avoid contaminating the product in subsequent enzyme catalysis experiments, and to increase the difficulty of purification. The rotation speed and time of centrifugation are mainly determined by the size of the separated product, and the difference of the sizes of ZIF-8@FDH nanometer hybrid materials can be caused by different reactant ratios and FDH concentrations, so that the proper rotation speed is selected during centrifugation. In this example, the centrifugation speed is selected to be 5000-12000rpm, and the centrifugation time is preferably selected to be 10-30min.

The embodiment of the invention provides a ZIF-8@FDH nano hybrid material prepared by the preparation method according to any one of the above embodiments, and the dispersion result of the ZIF-8@FDH nano hybrid material in water is shown in figure 2. The ZIF-8@FDH nanometer hybrid material provided by the invention improves the stability of the enzyme on one hand, so that the enzyme can withstand different environments (shown in figure 5); on the other hand, the enzyme is conveniently separated from the product and can be recycled (as shown in FIG. 6). This is important to reduce contamination of the product by the enzyme and to reduce waste of the enzyme.

The embodiment of the invention provides an application of the ZIF-8@FDH nano hybrid material prepared by the preparation method in catalyzing the specific conversion of carbon dioxide to formic acid, and CO2 can be specifically converted into formic acid (shown in figure 7).

In a preferred embodiment, the FDH in the ZIF-8@FDH nano hybrid material prepared by the method has the activity kept above 80% compared with the free enzyme (as shown in FIG. 4), and the environmental tolerance is improved.

In order to more clearly and in detail introduce the preparation method and application of the ZIF-8@FDH nanometer hybrid material provided by the embodiment of the invention, the preparation method and application are described below with reference to specific embodiments.

Example 1

Zinc acetate dihydrate was prepared as an aqueous solution to give solution a, wherein the zinc acetate dihydrate concentration was 10mM.

Preparing 2-methylimidazole into aqueous solution. Formate dehydrogenase was dissolved in 2-methylimidazole solution to give solution B, wherein the concentration of 2-methylimidazole was 50mM and the concentration of FDH was 0.05mg/mL.

Adding the solution A into the solution B under stirring, and reacting for 12 hours at room temperature to obtain ZIF-8@FDH suspension.

And centrifuging the suspension, cleaning the precipitate, and vacuum drying for 12 hours at room temperature to obtain the ZIF-8@FDH nanometer hybrid material.

Example 2

Zinc acetate dihydrate was prepared as an aqueous solution to give solution a, in which the zinc acetate dihydrate concentration was 40mM.

Preparing 2-methylimidazole into aqueous solution. Formate dehydrogenase was dissolved in 2-methylimidazole solution to give solution B, wherein the concentration of 2-methylimidazole was 160mM and the concentration of FDH was 0.5mg/mL.

Adding the solution A into the solution B under stirring, and reacting for 48 hours at room temperature to obtain ZIF-8@FDH suspension.

Example 3

Zinc acetate dihydrate was prepared as an aqueous solution to give solution a, wherein the zinc acetate dihydrate concentration was 25mM.

Preparing 2-methylimidazole into aqueous solution. Formate dehydrogenase was dissolved in 2-methylimidazole solution to give solution B, wherein the concentration of 2-methylimidazole was 100mM and the concentration of FDH was 0.3mg/mL.

Adding the solution A into the solution B under stirring, and reacting for 36h at room temperature to obtain ZIF-8@FDH suspension.

Example 4

Fourier infrared spectrometer is adopted, model: the following test was performed at an experimental temperature of 25℃with Nicolet6700, and the materials prepared in the above examples were exemplified.

And measuring the transmittance of the sample in the infrared region by combining a Fourier infrared spectrometer, specifically, mixing and grinding the sample and potassium bromide, and tabletting for measurement, wherein the measurement range is 400-4000nm.

The test results are shown in FIG. 2, wherein the ZIF-8 sample with FDH encapsulated therein was at 3300cm for pure ZIF-8 ^-1 And 1650cm ^-1 Two new peaks were generated corresponding to the N-H bond and the amide bond of the protein, respectively. The composition of the prepared ZIF-8@FDH nanometer hybrid material is proved.

Example 5

An ultraviolet-visible spectrophotometer is adopted, and the model is as follows: UV-1700PharmaSpec, the following test was performed at an experimental temperature of 25℃and the materials obtained in the above examples are exemplified.

NADH has an absorbance at 340nm in the UV-visible spectrum, the intensity of which is related to its concentration (as shown in FIG. 4A), this example combines with the UV-visible spectrum to measure the NADH content in the system.

Firstly, preparing NADH solutions with different concentrations, measuring ultraviolet absorption at 340nm, taking the absorption peak intensity of the NADH aqueous solution with different concentrations at 340nm as an abscissa, taking the corresponding concentration as an ordinate, and making a standard curve of NADH, wherein as shown in FIG. 4A, the data points have a better linear relation, and the linear equation is: y=0.00814+0.18833x, and by measuring the ultraviolet visible absorption intensity at 340nm in different reaction systems, the concentration of NADH in the system can be calculated according to the relation, so that the conversion rate of NADH can be further calculated.

Example 6

Comparing ZIF-8@FDH nano hybrid material provided by the embodiment of the invention with free FDH vs NAD ⁺ The catalytic effect is specifically tested as follows:

the reaction substrate consisted of 13.4mg NAD ⁺ 48mg of formic acid and phosphate buffer (pH 7) and the total volume of the solution was 4mL. The ZIF-8@FDH nanohybrid material prepared in example 3 and the same amount of FDH were added to the substrate, respectively. Samples were taken every three minutes and the concentration of NADH was determined by measuring the absorbance of the solution at 340nm with an ultraviolet-visible spectrophotometer.

FIG. 4B shows the UV-visible absorption spectrum of a sample after 20 minutes of catalytic reaction using the ZIF-8@FDH nanohybrid material provided in example 3 of the present invention and the same amount of free FDH as catalysts, respectively. From the graph, when the ZIF-8@FDH nano hybrid material provided by the embodiment 3 of the invention is used for catalysis, the absorption peak intensity of a system at 340nm is not greatly different from that of the free FDH serving as a catalyst, so that the prepared ZIF-8@FDH nano hybrid material well maintains the catalytic activity of the FDH.

FIG. 4B shows the NADH formation curve when the ZIF-8@FDH nanohybrid material provided in example 3 of the present invention and the same amount of free FDH were used as catalysts, respectively. More than 80% of the catalytic activity of the free FDH was retained over the time period observed. It is well shown that the FDH immobilization method provided by the invention does not cause damage to the enzyme activity.

FIG. 5 shows a comparison of the catalytic NADH conversion at different temperatures using the ZIF-8@FDH nanohybrid material provided in example 3 of the present invention and the same amount of free FDH as catalysts, respectively. The ZIF-8@FDH nanometer hybrid material has a good protection effect on the FDH when the temperature is raised, and shows good environmental tolerance.

FIG. 6 shows the relationship between NADH and reaction time when the ZIF-8@FDH nano catalytic material provided in example 3 of the present invention is recovered for re-catalysis after catalytic reaction. The figure shows that the recovered ZIF-8@FDH nano catalytic material can still catalyze the conversion reaction of NADH, so that the ZIF-8@FDH nano hybrid material provided by the embodiment of the invention can be recycled, and the loss of enzyme and pollution caused by the enzyme in a product are reduced.

FIG. 7 shows that the ZIF-8@FDH nano catalytic material prepared in the embodiment 3 of the invention is used as a catalyst, NADH is used as a coenzyme, and carbon dioxide is successfully converted into formic acid, so that the nano material provided by the invention can be used as a catalyst for specifically converting carbon dioxide into formic acid.

Claims

1. The preparation method of the ZIF-8@FDH nanometer hybrid material is characterized by comprising the following steps of:

dissolving zinc acetate dihydrate with the concentration of 10-40mM in water to obtain a solution A;

dissolving 50-160mM 2-methylimidazole in water, adding FDH powder, and mixing to obtain solution B;

gradually dripping the solution A into the solution B under stirring, and reacting for 12-48 hours at room temperature to obtain ZIF-8@FDH suspension, wherein the volume ratio of the solution A to the solution B is 0.5-4;

and (3) after the suspension is centrifugally washed, the precipitate is dried in vacuum for 12 hours, and the ZIF-8@FDH nanometer hybrid material is obtained, wherein the concentration of the FDH is 0.05-0.5mg/mL.

2. The method of claim 1, wherein the ZIF-8@fdh suspension is centrifuged at 5000-12000rpm for 10-30min.

3. The ZIF-8@FDH nanometer hybrid material prepared by the preparation method according to claim 1 or 2.

4. Use of the ZIF-8@fdh nanohybrid material according to claim 3 for catalyzing the specific conversion of carbon dioxide to formic acid.