CN114921450A

CN114921450A - Method for preparing immobilized carbonic anhydrase and application thereof

Info

Publication number: CN114921450A
Application number: CN202210568663.8A
Authority: CN
Inventors: 崔建东; 许家宝
Original assignee: Tianjin University of Science and Technology
Current assignee: Tianjin University of Science and Technology
Priority date: 2022-05-24
Filing date: 2022-05-24
Publication date: 2022-08-19

Abstract

The invention provides a method for preparing immobilized carbonic anhydrase and application thereof. The method has the advantages of simple operation, low cost, good repeatability, and good stability, reusability and CO content of the obtained immobilized enzyme ₂ Good trapping and conversion effect, and can be well applied to industrialized trapping and conversion of CO ₂ 。

Description

Method for preparing immobilized carbonic anhydrase and application thereof

Technical Field

The invention belongs to the field of enzyme catalysis, and particularly relates to carbonic anhydrase immobilization with acid-resistant MOF as a carrier, and a preparation method and application thereof.

Background

The climate change meeting of 25 th united nations who held in madrid in 2019 urges governments around the world to comply with the 'paris agreement' recommendation, and the global average temperature rise is kept below 2 ℃ relative to the level before industrialization. Achieving this goal requires powerful emission reduction measures in all sectors that result in carbon dioxide emissions. Currently, Carbon Capture and Sequestration (CCS) technology is being developed in various countries around the world to reduce CO emissions from thermal power plants, building material plants, and the like ₂ 。

Carbonic Anhydrase (CA) is an ancient metalloenzyme ubiquitous in nature and available as CO ₂ As substrate, efficiently catalyze CO ₂ Acceleration of HCO ₃ ^- And further CaCO is formed by means of mineralization ₃ To solidify CO ₂ . With physicochemical CO capture ₂ Compared with the technology, the enzyme method has the advantages of high efficiency and specificity, mild reaction condition and environmental protection. Thus, CO capture conversion with CA ₂ Has good application prospect.

In the absorption of CO ₂ In the process of (3), CA is dissolved in the solvent. Thus, in the subsequent CO ₂ During thermal desorption, the temperature required in the stripper can denature CA. On the other hand, CO ₂ Recovered as carbonate/bicarbonate crystals, and high ionic strength and pH after feeding the saturated solvent to the crystallization unit also leads to CA deactivation. Therefore, CA needs to be immobilized in industrial applications. The immobilization can improve the recovery rate and the recycling property of the enzyme, reduce the use cost of the enzyme and improve partial performance of the enzyme.

Metal-organic frameworks (MOFs) are a promising class of materials consisting of metal centers or clusters and organic ligands. It is a porous crystal structure material, and has become a commonly used carrier material for immobilized enzymes due to its flexible structure, adjustable pore size and large specific surface area.

Most of the carriers used for immobilized CA do not adsorb or trap CO by themselves ₂ Significantly affects the conversion of immobilized CA to CO ₂ The ability of the cell to perform.

Disclosure of Invention

The invention provides carbonic anhydrase immobilization with acid-resistant MOF as a carrier, and a preparation method and application thereof. The method has the advantages of simple operation, low cost, good repeatability, and good stability, reusability and CO of the obtained immobilized enzyme ₂ Good trapping and conversion effect, and can be well applied to industrialized trapping and conversion of CO ₂ 。

The invention provides a method for preparing immobilized carbonic anhydrase, which comprises the steps of taking a Metal Organic Framework (MOF) as a carrier, adding a modifier into the metal organic framework to obtain a modified metal organic framework, simultaneously adding a cross-linking agent and Carbonic Anhydrase (CA) into the modified metal organic framework, and activating and crosslinking to obtain the immobilized enzyme.

Preferably, the metal organic framework is an acid-resistant metal organic framework. Adding a modifier into the acid-resistant MOF for modification to obtain the modified acid-resistant MOF, simultaneously adding a cross-linking agent and CA into the acid-resistant MOF, and performing activation cross-linking on a rotary table to obtain the immobilized enzyme. The acid-resistant MOF is preferably at least one of UIO-66, MIL-125, MIL-101, MOF-808 and NMOFs. In CO ₂ When the reaction system is changed into acid, the acid-resistant MOF is suitable for fixing CA, and is beneficial to the capture of CO by CA ₂ The stability and activity of the enzyme are maintained.

In any of the above, preferably, the modifier is at least one of dopamine, polyethyleneimine, APTES, and ethylenediamine. The modifier is preferably APTES, and the APTES can react with hydroxyl on the MOF surface so as to be grafted to the MOF surface; the modifier is preferably ethylenediamine, which contains two amino groups, one of which can serve as a Lewis base to bind the metal sites of the MOF and the other of which can serve as CO ₂ The adsorption site of (a); the modifying agent is preferably PEI or dopamine, and PEI or dopamine introduces ammonia in the solidification process, so that the PEI or dopamine not only generates covalent fixation with CA, but also can adsorb CO ₂ The function of (1).

In any one of the above, preferably, the crosslinking agent is at least one of glutaraldehyde, hexamethylenediamine, genipin, maleic anhydride, bisazo benzene, and an isocyanic acid derivative.

In any one of the above, it is preferable that the mass fraction of the crosslinking agent is 0 to 35%. The mass fraction of the crosslinking agent is preferably 0, 12, 23, 34, 35%. The role of the cross-linker is to activate the modified MOF.

Preferably in any of the above, the cross-linking time is 1-6 h. The crosslinking time is preferably 1, 2, 3, 4, 5, 6 h.

Preferably in any of the above, the carbonic anhydrase concentration is 0.1-0.5 mg/mL. The carbonic anhydrase concentration is preferably 0.1, 0.2, 0.3, 0.4, 0.5 mg/mL.

In any of the above, the amount of the modified metal organic framework added is preferably 1 to 15 mg. The amount of the modified metal organic framework added is preferably 1, 2.5, 3.5, 4.5, 7.5, 9.5, 12.5, 13.5, 14, 15 mg.

The invention also provides a metal-organic framework solidified carbonic anhydrase prepared by any one of the methods.

The invention also provides a method for preparing immobilized carbonic anhydrase or immobilized carbonic anhydrase immobilized on metal-organic framework in CO, which is described in any one of the above ₂ Trapping applications in conversion.

The invention provides a modified acid-resistant MOF immobilized CA prepared by the method.

The invention provides the method for immobilizing CA in CO by using the modified acid-resistant MOF ₂ Trapping the application in the aspect of conversion.

The invention provides a method for preparing immobilized CA, which comprises the following steps:

adding a modifier into the MOF for modification to obtain the modified MOF, simultaneously adding a cross-linking agent and CA into the MOF, and performing activation crosslinking on a rotary table to obtain the immobilized enzyme. The MOF is preferably acid-resistant MOF, and the modified acid-resistant MOF is obtained after treatment of a modifying agent.

In the invention, the carrier of the acid-resistant MOF is adopted, so that CO can be solved ₂ The damage of the immobilized carrier caused by the acid environment of the aqueous solution is a problem. By adding the modifier, a large amount of amino is introduced, and CO is solved by the carrier while cross-linking sites are provided for enzyme ₂ The problem of incompatibility.

I. Preparation of acid-resistant MOF

Preferably, the acid-resistant MOF is at least one of UIO-66, MIL-125, MIL-101, MOF-808, and NMOFs.

More preferably, the acid resistant MOF is MOF-808.

Preferably, zirconium chloride and trimesic acid are used as raw materials, glacial acetic acid is used for regulating crystal form, and metal and organic ligand are combined into MOF after heating.

Further preferably, zirconium chloride (116.5mg, 0.5mmol), trimesic acid (35mg, 0.168mmol) and glacial acetic acid (2.8mL, 49mmol) are mixed, dissolved in 5mL DMF (N, N-Dimethylformamide) and then placed in a preheated 135 ℃ oven for reaction for 24 h. The precipitate obtained by centrifugation was washed with DMF and methanol, respectively. The solid obtained after washing was dried under vacuum at 60 ℃ overnight.

Preparation of amino-modified MOF-808

Preferably, the modifier is Dopamine (DA) and Polyethyleneimine (PEI). DA and PEI can be subjected to oxidative polymerization under an alkaline condition and deposited on the MOF surface, and PEI contains more amino groups and is resistant to CO ₂ Has high affinity. The preferred mass ratio of the modifier is: PEI: DA ═ 1: 2,1.5: 2,1: 1,2.5: 2,3: 2,3.5: 2 or 2: 1

More preferably, the mass ratio of the modifier is 1: 1.

in some embodiments, the modification steps for PEI and DA are as follows:

0.1g of MOF-808 was dissolved in 19mL of Tris-hydrochloric acid buffer (pH 8.5, 50mM) and sonicated for 10 min. Preferably, the mass ratio of PEI to DA is 1: 2-2: 1; preferably, PEI and DA (10 mg: 20mg, 20: 20mg, 20: 10mg) are weighed into a 5mL tube, and 1mL Tris-hydrochloric acid buffer (pH 8.5, 50mM) is added to mix them uniformly, at which time the DA continues to undergo oxidative autopolymerization to form PDA (polydopamine).

1mL of PEI/PDA solution was added to the MOF-808 suspension in a 28 ℃ water bath with magnetic stirring and reacted for 30 min. The resulting suspension was centrifuged (12000rpm,10min) and the pellet was washed twice with deionized water. And then drying the mixture overnight in vacuum at 60 ℃ to obtain solid powder, namely PEI/PDA-MOF-808.

Immobilization of enzymes

Preferably, 3-11mg PEI/PDA-MOF-808 is dispersed in PBS buffer (

pH

8, 10 mM);

preferably, a quantity of CA enzyme solution (1mg/mL, Milli-Q) is added to a final concentration of 0.2-0.4 mg/mL.

Preferably, the cross-linking agent is glutaraldehyde.

More preferably, 10. mu.L of an aqueous glutaraldehyde solution (0-25% wt) is added.

Preferably, the mixture is placed on a multi-purpose rotary shaker at a speed set between 20 and 60 r/min.

Preferably, the reaction is carried out for 1 to 4 hours.

The solid was collected by centrifugation (10000rpm, 10min), and washed twice with PBS buffer to obtain the immobilized enzyme.

A second object of the invention is to use the PEI/PDA-MOF-808 to immobilize CA on CO ₂ Trapping the application in the aspect of conversion.

Compared with the prior art, the invention has the following advantages:

according to the invention, acid-resistant MOF-808 is used as a carrier, PEI and DA are used for carrying out amination modification on the acid-resistant MOF-808 and are used for fixing carbonic anhydrase, and compared with carrier immobilized enzymes only modified by DA, the enzyme activity recovery rates are respectively 43.9% and 20.5%. Compared with other MOF immobilized carbonic anhydrases, the acid resistance and the reusability of the PEI/PDA-MOF-808 immobilized carbonic anhydrase are obviously improved, and 53.6% of initial enzyme activity is kept after 1h of treatment under the condition that the pH value is 3. After 8 times of repeated use, 56.0 percent of the initial enzyme activity is still kept. By investigating catalytic CO ₂ The hydration cyclability found that the calcium carbonate production remained consistent with the initial value after 8 cycles.

1. The invention provides a method for preparing immobilized CA by taking acid-resistant MOF as a carrier for the first time. CO 2 ₂ The acidic condition caused by the hydration reaction of (2) often causes damage to the immobilized carrier. The metal ion zirconium and the ligand trimesic acid required for synthesis of MOF-808 are both acid resistant. Can ensure the integrity of the immobilized enzyme in the catalysis process.

2. The invention firstly proposes that PEI and DA are used for jointly modifying MOF, and the introduction of a large amount of amino can improve the effect of an immobilized carrier on substrate CO ₂ Of (c) is determined.

3. According to the application, through adding PEI, the number of amino groups on the surface of the carrier can be increased, the crowding state is reduced, and the recovery rate of enzyme activity is improved.

4. In the invention, the amination condition is mild, the time is short, and the process operation is simple.

5. The acid resistance of the immobilized CA obtained by the invention is goodThe enzyme has good acid resistance and reusability, and 53.6 percent of initial enzyme activity is reserved after the treatment for 1 hour under the condition that the pH value is 3; by investigating the catalytic CO ₂ The hydration cyclability found that the calcium carbonate production remained consistent with the initial value after 8 cycles.

Drawings

FIG. 1 is a fluorescent microscope image of modified acid-resistant MOF immobilized CA prepared in example 1 of the present invention.

FIG. 2 is a graph showing the effect of the crosslinking time on the immobilized enzyme in Experimental example 1 of the present invention;

FIG. 3 is a graph showing the effect of CA concentration on immobilized enzyme in Experimental example 2 of the present invention;

FIG. 4 is a graph showing the effect of the amount of added carrier on immobilized enzyme in Experimental example 3 of the present invention;

FIG. 5 shows the effect of the rotational speed of the rocking bed on the immobilized enzyme in Experimental example 4 of the present invention;

FIG. 6 shows the pH tolerance of immobilized CA of Experimental example 5 of the present invention;

FIG. 7 shows the cyclic applicability of immobilized CA in Experimental example 5 of the present invention;

FIG. 8 shows the use of empty carrier and immobilized CA for CO in Experimental example 5 of the present invention ₂ Comparison of the transformation.

FIG. 9 shows the results of experiment example 5 of the present invention in which CA is immobilized on CO ₂ Recyclability in the conversion.

FIG. 10 is a flow chart of the preparation of the preferred embodiments 1 to 5 of the present invention.

Detailed Description

The present invention will be more clearly and completely described in the following embodiments, but the described embodiments are only a part of the embodiments of the present invention, and not all of them. The examples are provided to aid understanding of the present invention and should not be construed to limit the scope of the present invention.

The preferred preparation scheme for examples 1-5 is shown in FIG. 10.

Example 1 preparation of modified MOF-808 immobilized CA

The modified acid-resistant MOF-808 immobilized CA was synthesized as follows:

5mg PEI/PDA-MOF-808 was dispersed in 800. mu.L PBS buffer (pH 8, 10mM), followed by 200. mu.L CA enzyme solution (1mg/mL, Milli-Q) and finally 10. mu.L glutaraldehyde aqueous solution (20% wt). The mixture was placed on a multi-purpose rotary shaker and allowed to react for 3h each. Then, the solid was collected by centrifugation (10000rpm, 10min), and washed twice with PBS buffer to obtain the immobilized enzyme.

The enzyme activity determination method comprises the following steps: to a 1.5mL EP tube, 600. mu.L of PBS (10mM, pH 7.4), 100. mu.L of a Milli-Q-resuspended immobilized enzyme solution and 300. mu. L p-NPA (3mM) were added, and after reaction for 3min at room temperature, the filtrate was filtered through a disposable nylon filter, and the absorbance value at 348nm of the filtrate was measured; and subtracting the light absorption value of the control group added with the empty carrier to obtain the light absorption value variation caused by enzyme catalysis. And finally calculating the enzyme activity according to the formula.

The enzyme activity calculation formula is as follows:

the enzyme activity recovery rate calculation formula is as follows:

the experimental result shows that the enzyme activity recovery rate of the modified MOF-808 immobilized CA prepared by adding 5mg of the carrier is 41.9%.

As can be seen from FIG. 1, the appearance of the fluorescence microscope for preparing modified MOF-808 immobilized CA in example 1 of the present invention shows that a large amount of fluorescent material is carried on the surface of the carrier, indicating that the fluorescently labeled enzyme is successfully bound to the carrier.

Example 2 preparation of modified MOF-808 immobilized CA

The modified acid-resistant MOF-808 immobilized CA was synthesized as follows:

5mg PEI/PDA-MOF-808 was dispersed in 800. mu.L Milli-Q, followed by 200. mu.L CA enzyme solution (1mg/mL, Milli-Q) and finally 10. mu.L glutaraldehyde aqueous solution (20% wt). The mixture was placed on a multi-purpose rotary shaker and reacted for 3h each. Then, the solid was collected by centrifugation (10000rpm, 10min), and washed twice with PBS buffer to obtain the immobilized enzyme.

The enzyme activity was calculated according to the method of example 1.

As a result of the experiment, the enzyme activity recovery rate of the modified MOF-808 immobilized CA prepared in Milli-Q is 39.7%.

Example 3 preparation of modified MOF-808 immobilized CA

The modified acid-resistant MOF-808 immobilized CA was synthesized as follows:

10mg PEI/PDA-MOF-808 was dispersed in 1600. mu.L PBS buffer (pH 8, 10mM), followed by addition of 400. mu.L CA enzyme solution (1mg/mL, Milli-Q) and finally 20. mu.L glutaraldehyde aqueous solution (20% wt). The mixture was placed on a multi-purpose rotary shaker and reacted for 3h each. Then, the solid was collected by centrifugation (10000rpm, 10min), and washed twice with PBS buffer to obtain the immobilized enzyme.

The enzyme activity was calculated according to the method of example 1.

The experimental result shows that the recovery rate of the enzyme activity of the prepared modified MOF-808 immobilized CA is 40.3% when the addition amount of the carrier is 10 mg.

Example 4 preparation of modified MOF-808 immobilized CA

The modified acid-resistant MOF-808 immobilized CA was synthesized as follows:

30mg of PEI/PDA-MOF-808 was dispersed in 4800. mu.L of PBS buffer (pH 8, 10mM), followed by 1200. mu.L of CA enzyme solution (1mg/mL, Milli-Q) and finally 60. mu.L of glutaraldehyde aqueous solution (20% wt). The mixture was placed on a multi-purpose rotary shaker and allowed to react for 3h each. Then, the solid was collected by centrifugation (10000rpm, 10min), and washed twice with PBS buffer to obtain the immobilized enzyme.

The enzyme activity was calculated according to the method of example 1.

The experimental result shows that the recovery rate of the enzyme activity of the prepared modified MOF-808 immobilized CA is 42.0% when the addition amount of the carrier is 30 mg.

Example 5 preparation of modified MOF-808 immobilized CA

The modified acid-resistant MOF-808 immobilized CA was synthesized as follows:

5mg PEI/PDA-MOF-808 was dispersed in 800. mu.L PBS buffer (pH 8, 10mM), followed by 200. mu.L CA enzyme solution (1mg/mL, Milli-Q) and finally 20. mu.L glutaraldehyde aqueous solution (20% wt). The mixture was placed on a multi-purpose rotary shaker and reacted for 3h each. The solid was then collected by centrifugation (10000rpm, 10min) and washed twice with Milli-Q to obtain the immobilized enzyme.

The enzyme activity was calculated according to the method of example 1.

As a result of the experiment, the recovery rate of enzyme activity of modified MOF-808 immobilized CA prepared when Milli-Q was used to wash the carrier was 41.5%.

Experimental example 1 preparation method of modified MOF-808 immobilized CA under different cross-linking time conditions

To illustrate the effect on the recovery of modified MOF-808 immobilized CA at different cross-linking times, experiments were divided into four groups, where each group was prepared as follows:

the immobilized enzyme is synthesized according to the following steps:

5mg PEI/PDA-MOF-808 was dispersed in 800. mu.L PBS buffer (pH 8, 10mM), followed by 200. mu.L CA enzyme solution (1mg/mL, Milli-Q) and finally 10. mu.L glutaraldehyde aqueous solution (20% wt). The mixture was placed on a multi-purpose rotary shaker and reacted for 1, 2, 3, 4h, respectively. Then, the solid was collected by centrifugation (10000rpm, 10min), washed twice with PBS buffer solution, and the enzyme activity and the recovery rate of the immobilized enzyme were calculated according to the method for measuring enzyme activity in example 1.

The experimental results are as follows: referring to fig. 2, the recovery rates of the enzyme activities of the immobilized CA of the synthetic modified MOF-808 were 29.4%, 32.4%, 35.4%, and 30.4% under the conditions of carrying different cross-linking times.

Experimental example 2 preparation method of modified MOF-808 immobilized CA under different CA concentration conditions

To illustrate the effect of different CA concentrations on the recovery of modified MOF-808 immobilized CA, the experiments were divided into five groups, where each group was prepared as follows:

the immobilized enzyme is synthesized according to the following steps:

5mg PEI/PDA-MOF-808 was dispersed in PBS buffer (pH 8, 10mM), followed by 200, 250, 300, 350, 400. mu.L CA enzyme solution (1mg/mL, Milli-Q) with a retention system of 1mL, and finally 10. mu.L glutaraldehyde aqueous solution (20% wt). The mixture was placed on a multi-purpose rotary shaker for 3 h. Then, the solid was collected by centrifugation (10000rpm, 10min), washed twice with PBS buffer, and the enzyme activity and the recovery rate of the immobilized enzyme were calculated according to the method for measuring enzyme activity in example 1.

The experimental results are as follows: referring to fig. 3, the recovery rates of the enzymes of the immobilized CA of the synthetically modified MOF-808 were 36.3%, 37.45%, 40.75%, 38.25%, 32.55% under the conditions of carrying different cross-linking times.

Experimental example 3 preparation method of modified MOF-808 immobilized CA under different addition amounts of carriers

To illustrate the effect of different carrier addition amounts on the recovery of immobilized MOF-808 CA, experiments were divided into five groups, wherein each group was prepared as follows:

the immobilized enzyme is synthesized according to the following steps:

3, 5, 7, 9, 11mg PEI/PDA-MOF-808, respectively, were dispersed in 800. mu.L PBS buffer (pH 8, 10mM), followed by 200. mu.L CA enzyme solution (1mg/mL, Milli-Q), and finally 10. mu.L glutaraldehyde aqueous solution (20% wt). The mixture was placed on a multi-purpose rotary shaker and allowed to react for 3 h. Then, the solid was collected by centrifugation (10000rpm, 10min), washed twice with PBS buffer solution, and the enzyme activity and the recovery rate of the immobilized enzyme were calculated according to the method for measuring enzyme activity in example 1.

The experimental results are as follows: referring to fig. 4, the recovery rates of the enzyme activities of the immobilized CA of the synthetic modified MOF-808 were 21.1%, 36.2%, 37%, 38.1%, and 39.2% under the condition of loading different amounts of the carriers.

Experimental example 4 preparation method of modified MOF-808 immobilized CA under different table rotation speeds

To illustrate the effect of varying table rotation speed on the recovery of immobilized MOF-808 CA, experiments were divided into three groups, wherein each group was prepared as follows:

synthesizing immobilized enzyme according to the following steps:

5mg PEI/PDA-MOF-808 was dispersed in 800. mu.L PBS buffer (pH 8, 10mM), followed by 200. mu.L CA enzyme solution (1mg/mL, Milli-Q) and finally 10. mu.L glutaraldehyde aqueous solution (20% wt). The mixture was placed on a multi-purpose rotary shaker and reacted for 3h at 20, 40, 60rpm, respectively. Then, the solid was collected by centrifugation (10000rpm, 10min), washed twice with PBS buffer, and the enzyme activity and the recovery rate of the immobilized enzyme were calculated according to the method for measuring enzyme activity in example 1.

The experimental results are as follows: referring to fig. 5, the recovery rates of the enzymes of the immobilized CA of the synthetic modified MOF-808 were 37.6%, 37.6% and 32.7% under the conditions of different table rotation speeds.

Experimental example 5 Performance test of immobilized enzyme

(I) pH tolerance

The experimental method comprises the following steps: 0.1mg/mL of free CA and immobilized CA having the same enzymatic activity as free CA are respectively placed in 10mM PBS buffer solution with pH of 5.5, 6.5, 7.5, 8.5, 9.5, 10 or 10.5, after being placed for 1h, the enzymatic activity is calculated according to the method for measuring the enzymatic activity in example 1, and the reaction system without the CA sample is used as a blank control group. The highest enzyme activity is defined as 100%, and the highest enzyme activity in the ratio of the rest enzyme activities is the tolerance under different pH values. The enzyme activity determination result refers to the attached figure 6, after the enzyme is processed for 1 hour under the condition that the pH value is 3, the free enzyme almost loses the enzyme activity, and 53.6 percent of the initial enzyme activity of the immobilized enzyme is kept, which shows that the acid resistance of the immobilized enzyme is obviously improved.

Table 1 pH tolerance of the immobilized enzymes

(II) repeated use property

The experimental method comprises the following steps: 1000 mul of immobilized enzyme liquid (containing 5mg of immobilized enzyme), 2200 mul of PBS buffer solution and 560mL of 4-NPA solution are added into a 5mL test tube, reaction is carried out for 3min at room temperature, then centrifugation is carried out for 2min at 10000rpm, and the enzyme activity is calculated by the enzyme activity measuring method in example 1. The enzyme activity determination result refers to figure 7, the immobilized enzyme has good reusability, and 56.0% of initial enzyme activity can be retained after 8 times of reusability. The loss of enzyme activity may be due to frequent centrifugal washing (20 times) in the experiment, resulting in loss of immobilized enzyme.

TABLE 2 reusability of immobilized enzymes

(III) immobilized enzyme for CO ₂ Trapping transformation

The experimental method comprises the following steps: a certain amount of immobilized CA, free CA having the same enzymatic activity, or modified carrier having the same mass as the immobilized CA (no enzyme) was added to each flask containing 50mL of glycine-sodium hydroxide buffer (0.05M, pH 10). Introducing CO into the mixture ₂ Gas for about 5min, continuously detecting pH change of buffer solution with pH meter until pH value is 7.0, stopping introducing CO ₂ A gas. And (3) centrifuging the reaction liquid at 10000rpm for 10min, and separating to obtain the immobilized enzyme and a supernatant. Mixing the separated supernatants, adding 10mL CaCl ₂ The solution (5% wt, 0.5M, Tris-HCl buffer pH 9.5) was allowed to stand for 60min, and the resulting white precipitate was filtered. Drying the filter paper with the precipitate in an oven at 60 ℃, weighing every 1h until the weight is not changed, and recording data.

The experimental results are as follows: referring to FIG. 8, the amounts of calcium carbonate produced in the blank group, free CA, modified empty vector and immobilized enzyme were 321.65, 322.75, 333.7 and 356.7mg, respectively. Since pH 10 is not the optimum catalytic pH for CA, the 5min bubbling time is short and CA does not perform well with hydrated CO ₂ The function of (1); the PEI/PDA-MOF-808 carrier can adsorb CO more because of containing PEI ₂ CaCO obtained ₃ The yield was 333.7mg, but after one conversion, no adsorption capacity was obtained, the yield was 319.9mg, which is consistent with the blank, since the amino group was replaced by CO ₂ And (4) occupied. In contrast, CA on the immobilized enzyme can adsorb CO due to the synergistic effect of CA and PEI ₂ Efficient hydration, thus producing CaCO ₃ The highest yield is 356.7 mg.

(IV) immobilized enzyme for CO ₂ Capturing converted reuse performance

The experimental method comprises the following steps: respectively taking a certain amount of immobilized CA or free CA with the same enzyme activity as the immobilized CA or modified carrier (without enzyme) with the same mass as the immobilized CAThe mixture was added to a flask containing 50mL of glycine-sodium hydroxide buffer (0.05M, pH 10). Introducing CO into the mixture ₂ The gas is kept for about 5min, and the pH change of the buffer solution is continuously detected by a pH meter until the pH value is 7.0, and the CO introduction is stopped ₂ A gas. And (3) centrifuging the reaction liquid at 10000rpm for 10min, and separating to obtain the immobilized enzyme and a supernatant. The immobilized enzyme was washed twice with glycine-sodium hydroxide buffer (0.05M, pH 10), and 50mL of glycine-sodium hydroxide buffer (0.05M, pH 10) was added again while CO was passed through ₂ The experimental procedure described above was repeated. Mixing the supernatant obtained from each separation, adding 10mL CaCl ₂ The solution (5% wt, 0.5M, Tris-HCl buffer pH 9.5) was allowed to stand for 60min, and the resulting white precipitate was filtered. The filter paper with the precipitate was dried in an oven at 60 ℃ and weighed every 1h until no further change in weight occurred, and the data was recorded.

Referring to the figure 9, the cycle performance detection result of the immobilized enzyme is that the modified MOF-808 immobilized CA prepared by the invention can realize good cycle effect, and the initial yield consistent with the initial value can be kept after 8 times of use.

TABLE 3 reusability of immobilized enzymes to produce calcium carbonate

Example 6

The preparation of modified MOFs in example 6 was similar to examples 1 to 5, except that,

the cross-linking agent adopts hexamethylene diamine, genipin, maleic anhydride, bisazo benzene and isocyanic acid derivatives.

The modifier adopts APTES and ethylenediamine.

Preparation of immobilized enzyme by obtained modified MOF, activity and stability of enzyme and CO capture ₂ The ability of (2) was similar to those of Experimental examples 1 to 5.

The above examples merely express several embodiments of the present invention, and the description thereof is more specific and detailed, but the technical scope thereof is not limited to the above embodiments. It will be apparent to those skilled in the art that various modifications and embodiments can be made without departing from the spirit of the invention, and these are within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A method for preparing immobilized carbonic anhydrase uses a metal organic framework as a carrier, and is characterized in that a modifier is added into the metal organic framework to obtain a modified metal organic framework, a cross-linking agent and carbonic anhydrase are simultaneously added into the modified metal organic framework, and the immobilized enzyme is obtained by activation and cross-linking.

2. The method of claim 1, wherein the metal organic framework is an acid resistant metal organic framework.

3. The method of claim 1, wherein the modifying agent is at least one of dopamine, polyethyleneimine, APTES, ethylenediamine.

4. The method of claim 1, wherein the cross-linking agent is at least one of glutaraldehyde, hexamethylenediamine, genipin, maleic anhydride, and bisazo benzenes, isocyanic acid derivatives.

5. The method of claim 4, wherein the cross-linking agent is present in an amount of 0 to 35% by mass.

6. The method of claim 5, wherein the crosslinking time is 1 to 6 hours.

7. The method of claim 1, wherein the carbonic anhydrase concentration is from 0.1 to 0.5 mg/mL.

8. The method of claim 1, wherein the modified metal organic framework is added in an amount of 1-15 mg.

9. A metal-organic framework immobilized carbonic anhydrase prepared by the process of any one of claims 1-8.

10. The process for preparing immobilized carbonic anhydrase of any one of claims 1 to 8 or the metal-organic framework immobilized carbonic anhydrase of claim 9 in CO ₂ Application in capture conversion.