KR20160116940A - Carbonic anhydrase immobilized silk hydrogel and conversion or fixation of carbon dioxide using the same - Google Patents

Abstract

The present invention relates to carbonic anhydrase-immobilized silk hydrogel and the conversion or the fixation of carbon dioxide using the same. According to the present invention, the carbonic anhydrase-immobilized silk hydrogel or a composition comprising the same is dual crosslinked by the photocrosslinking and alcohol treatment, thereby being eco-friendly and economic, having excellent thermal and storage stability, and being used in a repeated manner. The carbonic anhydrase-immobilized silk hydrogel or the composition comprising the same has significantly excellent effects of enzymatic activities, thereby removing, converting, or fixing carbon dioxide.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a carbonic anhydrase-immobilized silk hydrogel,

The present invention relates to a silk hydrogel in which a carbonic anhydrase is immobilized and to the conversion or immobilization of carbon dioxide using the silk hydrogel.

Global warming, which is becoming a global environmental problem today, is known to be caused mainly by carbon dioxide (CO ₂ ) and various greenhouse gases generated by the combustion process of fossil fuels that have been used for the past 100 years. Specifically, it has been reported that the accumulation of carbon dioxide and various greenhouse gases in the atmosphere absorbs the infrared rays emitted from the earth, thereby raising the temperature of the earth, which causes melting of polar glaciers and rising sea level . Techniques for treating carbon dioxide that have been developed so far include physical methods such as adsorption and membrane separation methods, and methods of storing carbon dioxide in lipids using chemical means and converting them into useful materials. Particularly, methods of capturing, storing, and converting carbon dioxide using chemical means have been extensively studied, but this method requires a lot of energy to raise the hydration rate of carbon dioxide, which is uneconomical . Therefore, it is very important to find a way to increase the rate of CO2 conversion in an efficient and environmentally friendly manner.

Carbonic anhydrase (CA) is the fastest-growing enzyme on the planet and hydrates carbon dioxide molecules. Hydrated carbon dioxide molecules are present in the form of bicarbonate, and abundant amounts of bicarbonate can be converted into useful minerals. In general, immobilization of an enzyme means that the enzyme can be easily separated from a reaction mixture in a biochemical process, or an enzyme is immobilized on a specific support in order to enhance the stability of the enzyme. Thus, enzyme treatment is convenient through enzyme immobilization, The enzyme can be easily separated from the obtained product, and an expensive enzyme can be easily recovered and reused, and the enzyme can be utilized in a continuous process without a large change in activity in a harsh environment. Therefore, much research has been conducted to immobilize the CA enzyme on various supports such as porous SBA-15, porous glass, or nanoparticles. However, this method requires a complicated multistage reaction process and use of toxic chemicals, secondary contamination occurs, and enzyme activity is lost.

Silk fibroin is a protein polymer composed of a large hydrophobic region and a small hydrophilic region. Silk fibroin has been studied as a biological support for covalent or non-covalent enzyme fixation and is known to enhance stability due to intermolecular interactions between silk and enzymes. Such silk fibroin may be produced in a wide variety of formulations such as fibers, films, sponges, powders and gels, but hydrogel-based enzyme immobilization among these formulations can protect the enzyme against harsh conditions and maintain enzyme activity Has been widely used.

KR 10-2013-0096424

The inventors of the present invention have found that a silk hydrogel in which carbonic anhydrase-immobilized double-crosslinked by photo-crosslinking and alcohol treatment is an environmentally friendly, economical, excellent thermal and storage stability , It can be used repeatedly, and the enzyme activity is excellent, so that the carbon dioxide can be effectively removed, converted or fixed, and the present invention has been completed.

Accordingly, the present invention provides a composition for converting or fixing carbon dioxide, which comprises a silk hydrogel in which a carbonic anhydrase is immobilized.

In addition, the present invention provides a silk hydrogel in which the carbonic anhydrase is immobilized, and a method for producing the silk hydrogel.

The present invention also provides a method for converting or fixing carbon dioxide, comprising the step of reacting the composition for converting or fixing carbon dioxide with a saturated solution of carbon dioxide.

The present invention also provides a device for converting or fixing carbon dioxide, comprising a composition for converting or fixing the carbon dioxide.

In order to achieve the above object,

The present invention provides a composition for the conversion or fixing of carbon dioxide, which comprises a silk hydrogel fixed with carbonic anhydrase.

In addition,

(1) preparing a mixed solution of a carbonic anhydrase and a silk protein;

(2) photo-crosslinking the mixed solution to produce a silk hydrogel; And

(3) subjecting the silk hydrogel to an alcohol treatment, wherein the silk hydrogel is immobilized with a carbonic anhydrase.

In addition, the present invention provides a silk hydrogel in which a carbonic anhydrase is immobilized, which is produced according to the above-described production method.

The present invention also provides a method for converting or fixing carbon dioxide, comprising the step of reacting the composition for converting or fixing carbon dioxide with a saturated solution of carbon dioxide.

The present invention also provides a device for converting or fixing carbon dioxide, comprising a composition for converting or fixing the carbon dioxide.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for the conversion or fixing of carbon dioxide, which comprises a silk hydrogel fixed with carbonic anhydrase.

In the present invention, "carbonic anhydrase" is an enzyme that catalyzes the conversion of carbon dioxide and water into hydrogen carbonate ion and hydrogen ion. The reaction by the carbonic anhydrase is as follows.

[Reaction] CO ₂ + H ₂ O <---> HCO ₃ ^- + H ⁺

This reaction occurs mainly in tissues with a high concentration of carbon dioxide and the rate is known to be the fastest among the reactions caused by the enzyme. The reverse reaction of this reaction occurs in the lungs and kidney nephron or plant cells, which are fast but require no catalyst and low carbon dioxide concentrations. Such carbonic anhydrase is also called carbonic anhydrase. In the natural world, there are several different kinds of carbonic anhydrases such as α, β, γ, δ and ε types, and α-type carbonic anhydrase , Β-type carbonic anhydrase in plants, and δ-type carbonic anhydrase with cadmium instead of zinc in diatoms.

In the present invention, preferably, alpha -form carbonic anhydrase (ngCA) derived from gonococcus Neisseria gonorrhoeae is used.

The carbonic anhydrase of the present invention includes not only a carbonic anhydrase having the amino acid sequence of SEQ ID NO: 1, but also mutants thereof or homologs thereof. Specifically, any carbonic anhydrase can be used as long as the tyrosine residue does not affect the active site.

The carbonic anhydrase is immobilized on a silk protein support.

The "silk protein" of the present invention is not limited to any one including a tyrosine residue. The silk protein may be a natural protein, that is, a natural sequence, or a synthetic protein, that is, a synthetic gene based protein whose primer sequence corresponds broadly to the natural sequence. Specifically a protein of natural or recombinant origin, derived from Arachnida or Insecta, and preferably a protein such as Bombyx mori, green lacewings (Chrysoperla carnea), araneus (Araneus diadematus, or silkworm spider (Nephila clavipes). More preferably, the silk protein may be silk fibroin extracted from silkworm. The silk fibroin can participate in the covalent bond with the tyrosine residue of the carbonic anhydrase that is immobilized by including ~ 5% of the tyrosine residue.

In order to immobilize the carbonic anhydrase on the silk hydrogel, a covalent bond-based photo-crosslinking and non-covalent bond-based alcohol treatment method can be used.

The alcohol may be an alcohol aqueous solution having a ratio of alcohol to water of 1: 0.1 to 1: 10 by volume (v / v), and methanol or ethanol may be used as the alcohol.

The carbon dioxide can be converted or fixed to a bicarbonate or a carbonate compound by reacting the carbonic anhydrase-immobilized silk hydrogel, or a composition containing the same, with a saturated solution of carbon dioxide.

The bicarbonate compound is selected from the group consisting of sodium bicarbonate (Na ₂ HCO ₃ ), potassium bicarbonate (KHCO ₃ ), magnesium bicarbonate (Mg (HCO ₃ ) ₂ ), calcium bicarbonate (Ca (HCO ₃ ) ₂ ), ammonium bicarbonate ((NH ₄₎ HCO _3), and the like.

The carbonate compound may be at least one selected from the group consisting of carbonic acid (H ₂ CO ₃ ), lithium carbonate (Li ₂ CO ₃ ), sodium carbonate (Na ₂ CO ₃ ), potassium carbonate (K ₂ CO ₃ ), rubidium carbonate (Rb ₂ CO ₃ ) carbonate (Cs ₂ CO _3), beryllium carbonate (BeCO _3), magnesium carbonate (MgCO _3), calcium carbonate (CaCO _3), strontium carbonate (SrCO _3), barium carbonate (BaCO _3), manganese carbonate (MnCO _3), ferrous carbonate (FeCO _3), cobalt carbonate (CoCO _3), nickel carbonate (NiCO _3), copper carbonate (CuCO _3), silver carbonate (Ag ₂ CO _3), zinc carbonate (ZnCO _3), cadmium carbonate (CdCO ₃₎ , Aluminum carbonate (Al ₂ (CO ₃ ) ₃ ), thallium carbonate (Tl ₂ CO ₃ ), lead carbonate (PbCO ₃ ), lanthanum carbonate (La ₂ (CO ₃ ) ₃ ) and the like.

(1) preparing a mixed solution of a carbonic anhydrase and a silk protein; (2) photo-crosslinking the mixed solution to produce a silk hydrogel; And (3) subjecting the silk hydrogel to an alcohol treatment. The present invention also provides a method for producing a silk hydrogel fixed with carbonic anhydrase.

The step (1) is a step of preparing a mixed solution of a carbonic anhydrase and a silk protein.

The above-mentioned carbonic anhydrase may be any type as long as the tyrosine residue does not affect the active site, and it is preferably an alpha form derived from Neisseria gonorrhoeae . The carbonic anhydrase may be a carbonic anhydrase having the amino acid sequence of SEQ. ID. NO. 1 as well as its variants or homologous thereto. The silk protein is not limited to any one including a tyrosine residue, and silk fibroin extracted from silkworm can be used. The solvent used to prepare the mixed solution is preferably Tris HCl buffer having a pH of 7 to 9.

In addition, the mixed solution may contain a photoinitiator. The photoinitiator may be a visible light or UV initiator, or both, preferably a visible light initiator. The components may be present in varying amounts depending on the combination of components and the desired composition. (Bipyridine) ruthenium (II) chloride, camphorquinone peroxyester, 9-fluorenecarboxylic acid peroxyester, dl-camphorquinone, dicumyl peroxide, Agar Cure 784DC, and the like. In the present invention, tris (bipyridine) ruthenium is preferably used.

It is preferable that the photoinitiator is included in an amount of 0.001 to 0.1 part by weight based on 1 part by weight of the silk protein in consideration of the content of the silk protein contained in the mixed solution.

The step (2) is a step of photo-crosslinking a mixed solution of a carbonic anhydrase and a silk protein to produce a silk hydrogel.

The silk protein may have a photoinduced covalent bond with the tyrosine residue of the carbonic anhydrase that is immobilized by containing ~ 5% of the tyrosine residue. In addition, the silk protein and the carbonic anhydrase may form a silk hydrogel in which the carbonic anhydrase is immobilized through intramolecular or intermolecular interactions between the tyrosine residues.

The photo-crosslinking is preferably performed by visible light, and may be performed for 1 to 100 seconds (s), preferably for 1 to 30 seconds.

In the step (3), the photo-crosslinked silk hydrogel is subjected to alcohol treatment to form? -Sheet crosslinking.

When the photo-crosslinked silk hydrogel is subjected to alcohol treatment, the irregular coil structure is changed to a regular [beta] -sheet structure, so that the mechanical properties and stability can be improved. The alcohol may be methanol or ethanol or may be an aqueous methanol solution or an aqueous ethanol solution in which the ratio of alcohol to water is 1: 0.1 to 1: 10, preferably 1: 0.1 to 1: 5 (v / v) . The alcohol may be treated for 0.1 to 2 hours, preferably 0.5 to 1.5 hours. By treating the alcohol at a high concentration for a short period of time, the activity of the enzyme can be improved to improve the mechanical mechanical properties.

In addition, the present invention provides a silk hydrogel in which a carbonic anhydrase is immobilized, which is produced according to the above-described production method.

The present invention also provides a method for converting or fixing carbon dioxide, comprising the step of reacting the composition for converting or fixing carbon dioxide with a saturated solution of carbon dioxide.

The present invention also provides a device for converting or fixing carbon dioxide, comprising a composition for converting or fixing the carbon dioxide.

The apparatus for converting or fixing carbon dioxide of the present invention may be a conventional apparatus for extracting carbon dioxide from air and converting it into bicarbonate and fixing it. For example, the conversion or fixation device for carbon dioxide includes upright towers, a chamber, and a source of water, the upright tower having at least one air inlet at the bottom And at least one air outlet at the top of the tower, the chamber being located between at least one air inlet and at least one air outlet and for collecting carbon dioxide And a collector for wetting the resin to release carbon dioxide. The collector for capturing the carbon dioxide may include a composition for converting or fixing the carbon dioxide of the present invention.

The carbonic anhydrase immobilized silk hydrogel according to the present invention is excellent in thermal and storage stability and can be repeatedly used. In addition, the silk hydrogel has an advantage that it can effectively remove, convert, or fix carbon dioxide have.

The carbonic anhydrase-immobilized silk hydrogel according to the present invention or a composition containing the same is double-crosslinked by photo-crosslinking and alcohol treatment, thereby being environmentally friendly, economical, excellent in thermal and storage stability, But the enzyme activity is remarkably excellent, so that there is an effect that the carbon dioxide can be removed, converted or fixed.

1 is an optical image of a methanol-treated m-ngCA-fixed silk hydrogel and a non-methanol-treated ngCA-fixed silk hydrogel according to Example 1. Fig.
Fig. 2 shows a schematic view of the structural form of the m-ngCA-fixed silk hydrogel according to Example 1 and thereby the conversion of carbon dioxide to calcium carbonate. Fig.
Figure 3 shows the amount of carbonic anhydrase that can be immobilized in a 10% (w / v) silk hydrogel.
Fig. 4 shows the enzymatic activity of m-ngCA-fixed silk hydrogels and ngCA-fixed silk hydrogels according to Example 1. Fig.
5 is a scanning electron microscope (SEM) image of m-ngCA-fixed silk hydrogels and ngCA-fixed silk hydrogels according to Example 1. Fig.
Figure 6 is a stress-strain curve of (a) m-ngCA-fixed silk hydrogel and (b) ngCA-fixed silk hydrogel according to Example 1, (c) (d) m-ngCA-fixed silk hydrogel and (e) loading-unloading cycle curve of ngCA-fixed silk hydrogel and ngCA-fixed silk hydrogel, and (f) Lt; RTI ID = 0.0 &gt; m-ngCA-fixed &lt; / RTI &gt; silk hydrogels and ngCA-fixed silk hydrogels as a function of compression cycle.
7 is a graph showing the activity upon re-use of the m-ngCA-fixed silk hydrogel and the ngCA-fixed silk hydrogel according to Example 1. Fig.
8 is a diagram showing storage stability of (a) m-ngCA-fixed silk hydrogel according to Example 1 and (b) free ngCA.
9 is an optical image of m-ngCA-fixed silk hydrogel and ngCA-fixed silk hydrogel according to Example 1 after storage for 7 days at 30 ° C.
10 is a graph showing the thermal stability of (a) m-ngCA-fixed silk hydrogel and (b) ngCA-fixed silk hydrogel according to Example 1;
11 is a SEM image of calcium carbonate (CaCO ₃ ) precipitated by m-ng CA-fixed silk hydrogel.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example  1. Preparation of Silk Hydrogel Having Carbonic Anhydrase (ngCA) Immobilized

1-1. Preparation and purification of carbonic anhydrase (ngCA)

The? -Type CA (ngCA) protein from Neisseria gonorrhoeae was expressed and purified according to known methods (see Chemospere 2012, 87, 1091). First, the recombinant E. coli containing the ngCA-expression vector was cultured in LB (Luria-Bertani) medium (0.5% yeast extract, 1% tryptophan) containing 50 μg / mL ampicillin at 37 ° C. , And 1% NaCl) until the absorbance was 0.6-0.8 at 600 nm. Then, 1 mM isopropyl-beta-D-thiogalactopyranoside and 0.1 mM ZnSO ₄ were added to the culture medium to induce the expression of ngCA protein. The recombinant cells were further grown at 37 DEG C for 10 hours and then centrifuged at 4000 rpm for 10 minutes to recover the cells. The recovered cells were resuspended in a solution for cell destruction (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 10 mM imidazole, pH 8.0) and then disrupted for 10 minutes at 50% power using an ultrasonic mill. The shredded cells were centrifuged at 10,000 rpm for 30 minutes at 4 &lt; 0 &gt; C to recover the aqueous fraction.

Thereafter, the aqueous fraction was applied to a nickel-NTA column for purification, and the elution of the protein from the column was then carried out using elution buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 250 mM imidazole, pH 8.0) Respectively. The eluate was dialyzed against 20 mM Tris-SO ₄ (pH 8.3). The concentration of protein was determined by Bradford assay using bovine serum albumin (BSA) as a standard.

1-2. Preparation of silk hydrogel with fixed carbonic anhydrase (ngCA)

Carbonic anhydrase (ngCA) - fixed silk hydrogel was prepared by photochemical crosslinking and methanol treatment. First, silk fibroin was mixed with ngCA solution (20 mM Tris-HCl (pH 8.3)) and dissolved with stirring for 1 hour. Thereafter, tris (bipyridine) ruthenic acid (II) chloride ([Ru (bpy) ₃ ] ²⁺ ) and sodium persulfate were added to the mixed solution, and until the solution reached a uniform phase And mixed while stirring.

In addition, for gelation, the ngCA-fixed silk hydrogel was prepared by exposing the mixed solution to visible light (~ 450 nm) for 15 seconds and the ngCA-fixed silk hydrogel was exposed to 90% ( v / v) methanol to prepare an m-ngCA-fixed silk hydrogel.

An optical image of the m-ngCA-fixed silk hydrogel and ngCA-fixed silk hydrogel according to Example 1 is shown in Fig.

Further, a schematic view of the structural form of the m-ngCA-fixed silk hydrogel according to Example 1 and the conversion of carbon dioxide into calcium carbonate thereby is shown in Fig.

Experimental Example  1. Analysis of silk hydrogel activity by immobilization of ngCA enzyme and methanol treatment

1.1 Analysis Method

The ability of the silk hydrogel according to Example 1 to immobilize the ngCA enzyme and its effect on the methanol treatment was evaluated using an esterase activity assay involving the hydrolysis of para-nitrophenylacetate (PNA). Production of para-nitrophenyl was measured at an absorbance of 348 nm (? _Max ) using a UV-vis spectrometer. A 30 mM PNA solution was prepared by dissolving an appropriate amount of PNA in 100% (v / v) acetonitrile, and the analysis was carried out using 900 μL of 20 mM potassium phosphate (pH 7.0), 100 μL of 30 mM PNA (final concentration 3 mM) Gt; 25 C &lt; / RTI &gt; in a total reaction volume of 1 mL consisting of a fixed silk hydrogel. The absorbance was also measured every 2 minutes for 12 minutes at 348 nm.

The analytical method was also used to evaluate the reusability, thermal stability and storage stability of the silk hydrogels.

1.2 Results of analysis

The activity of m-ngCA-fixed silk hydrogel as a function of the amount of carbonic anhydrase (ngCA) is shown in Fig.

As shown in FIG. 3, as the amount of ngCA enzyme increased from 100 to 1400 μg, the activity linearly increased and a high coefficient of crystallinity (R ² ) of 0.99 or more was obtained. At 1400 ug or more, the activity reached almost saturation and up to ~ 1800 ug of ngCA enzyme could be fixed in 20 mg of silk hydrogel.

In addition, the activity of m-ngCA-fixed silk hydrogels and ngCA-fixed silk hydrogels depending on treatment with water-soluble methanol is shown in Fig.

As shown in Fig. 4, the alcohol generally acts as a denaturant and adversely affects the enzyme activity because it interferes with the hydrophobic interaction of the enzyme, whereas the methanol-treated m-ngCA-fixed silk hydrogel has a high 92% Activity. These results indicate that the ngCA enzyme immobilized in the silk fibroin due to the covalent and non-covalent bonding between ngCA and silk fibroin retains the initial activity even after treatment with water-soluble methanol since the ngCA enzyme has a very high stiffness compared to the free enzyme I think.

Experimental Example 2. Evaluation of Swelling Characteristics of m-ngCA-Fixed Silk Hydrogel

2.1 Analysis Method

The dried m-ngCA-fixed silk hydrogel according to Example 1 was immersed in a solution of 20 mM Tris-HCl (pH 8.3)) at 4 占 폚 for 1 day. After the excess water was removed, the weight of the swollen silk high road gel was measured. Three separate tests were performed on each sample, and the degree of swelling was defined as follows.

Degree of swelling _{= [(W t - W d} ) / W d], the fluid recovery _{(%) = [(W t} - W d) / ((W g - W d) × 100]

Where W _t is the weight of the hydrogel swelled at time t and W _d is the weight of the dried silk hydrogel.

2.2 Results of analysis

The swelling characteristics of the m-ngCA-fixed silk hydrogel and the ngCA-fixed silk hydrogel are shown in Table 1.

m-ngCA-fixed silk hydrogel ngCA-fixed silk hydrogel Swelling degree 1.17 ± 0.05 8.39 ± 0.27 Fluid recovery (%) 24.65 + - 0.76 86.01 + - 0.91

As shown in Table 1, the degree of swelling of the m-ngCA-fixed silk hydrogel was 1.17 and the recovery of fluid (%) was 24.65, while the degree of swelling of the ngCA-fixed silk hydrogel was 8.39 and the recovery of fluid was 86.01 Respectively. Thus, the low swelling of m-ngCA-fixed silk hydrogel compared to ngCA-fixed silk hydrogel may be due to the high content of β-sheet due to methanol treatment.

An SEM image of m-ngCA-fixed silk hydrogel and ngCA-fixed silk hydrogel is also shown in Fig.

As shown in FIG. 5, the ngCA-fixed silk hydrogel exhibits a high porous honeycomb structure, while the m-ngCA-fixed silk hydrogel exhibits a compact structure with relatively low porosity . These results support the swelling results of the m-ngCA-fixed silk hydrogel.

Experimental Example 3. Evaluation of mechanical properties of m-ngCA-fixed silk hydrogel

3.1 Analysis Method

An unconfined compression test on the m-ngCA-fixed silk hydrogel (8 mm diameter, 2 mm height) and ngCA-fixed silk hydrogel (10 mm diameter, 3 mm height) according to Example 1 was performed on a 2 kN load cell RTI ID = 0.0 &gt; Instron &lt; / RTI &gt; 3344 material test system. Each sample was compressed at a rate of 1 mm / min until a maximum strain of ~ 50% was reached (n = 3).

After obtaining a stress-strain curve, the compressive tangent modulus was calculated at 20% and 40% stress. In addition, the hydrogel was subjected to 20 loading-unloading cycles at 20% stress. The 20 cycles were recorded and the energy dispersion was calculated as follows;

Energy dispersion =

3.2 Results of analysis

(a) m-ngCA-fixed silk hydrogel, and (b) a stress-strain curve of ngCA-fixed silk hydrogel, (c) (d) m-ngCA-fixed silk hydrogel, and (e) the loading-unloading cycle curve of the ngCA-fixed silk hydrogel, and (f) The energy dispersions of -ngCA-stationary silk hydrogels and ngCA-stationary silk hydrogels are shown in Fig.

As shown in Figures 6 (a) - (c), m-ngCA-fixed silk hydrogels (~ 1300 kPa and ~ 11000 kPa for 20% and 40% stress, respectively) 50 times higher stiffness compared to ~ 30 kPa and ~ 200 kPa for 20% and 40% stress). This high stiffness of the m-ngCA-fixed silk hydrogel may be attributed to the fact that the high content of? -Sheet due to methanol treatment induced physical crosslinking and improved the compression coefficient.

Further, as shown in Figs. 6 (d) to 6 (f), in the case of the m-ngCA-fixed silk hydrogel, the area of the hysteresis loop rapidly decreased during the initial five consecutive cycles, And finally reached a steady state with an energy dispersion of ~ 5 kJ / m ₃ . These results indicate that the hydrogel is not ruptured under these conditions, and the initial sharp reduction is thought to be due to the loss of water present in the gel. Because m-ngCA-fixed silk hydrogels have a lower water content than ngCA-fixed silk hydrogels, water can be affected by the water being pushed out of the gel during initial loading. On the other hand, in the case of ngCA-fixed silk hydrogels, a specific stress-strain curve was maintained for 20 consecutive cycles. From the above results, it can be seen that the m-ngCA-fixed silk hydrogel has high resistance and elasticity against external impact.

Experimental Example 4. Evaluation of reusability of m-ngCA-fixed silk hydrogel

4.1 Analysis Method

The reusability of the m-ngCA-fixed silk hydrogel according to Example 1 was evaluated using the above-described esterase assay. For further use, the product in the hydrogel was removed by dialysis against 20 mM Tris-SO ₄ buffer (pH 8.3) and the hydrogel was stored at 4 ° C prior to use.

4.2 Analysis Results

The activity upon re-use of the m-ngCA-fixed silk hydrogel and the ngCA-fixed silk hydrogel according to Example 1 is shown in Fig.

As shown in Figure 7, the m-ngCA-fixed silk hydrogel retains more than 90% of the initial activity after 9 re-cycles, while the ngCA-fixed silk hydrogel retains 84% of the initial activity appear. The high reusability of such m-ngCA-fixed silk hydrogels is believed to be due to the hydrolysis of silk hydrogels. That is, the water-soluble methanol treatment can prevent the hydrogel from being hydrolyzed by forming a? -Sheet crosslink. The high re-usability of the m-ngCA-fixed silk hydrogel was significantly improved with chitosan beads (~ 43% active after reuse 5), amine-based silica (~57 re-use after ~ 87% activity) and R5 peptide- (After 10 re-use, > 90% active), silver nanoparticle-bound porous SBA-15 (10 times more reusable) than the reusability of the amine-functionalized SBA- , &Gt; 90% active), and magnetic particles (> 85% active after 10 times reuse).

Experimental Example 5. Evaluation of storage and thermal stability of m-ngCA-fixed silk hydrogel

5.1 Analysis Method

The storage and thermal stability of the m-ngCA-fixed silk hydrogel according to Example 1 was evaluated using the above esterase assay. To measure storage stability, m-ngCA-stationary silk hydrogel was stored at 30 ° C and tested at intervals of ~7 days.

To measure thermal stability, m-ngCA-fixed silk hydrogel was incubated at 50 캜 for 1 hour and 12 hours, and then stored at 4 캜 until activity was measured.

5.2 Results of analysis

The storage stability of (a) m-ngCA-fixed silk hydrogel according to Example 1 and (b) free ngCA according to Example 1 is shown in Fig. An optical image of the m-ngCA-fixed silk hydrogel and ngCA-fixed silk hydrogel according to Example 1 after storage at 30 DEG C for 7 days is also shown in Fig.

As shown in Fig. 8, m-ngCA-fixed silk hydrogel showed no activity decrease during storage for 30 days but decreased to ~ 11% of initial activity after 10 days storage for free ngCA.

Also, as shown in Fig. 9, the ngCA-fixed silk hydrogel appeared to be fully hydrolyzed within 7 days. The high storage stability of the m-ngCA-fixed silk hydrogel is believed to be due to the structural stability of the silk hydrogel due to the methanol treatment, and the ngCA trapped within the silk hydrogel can be stable for long periods of time maintaining the initial activity. From the above results, it can be seen that the methanol-treated m-ngCA-fixed silk hydrogel exhibits excellent activity even when stored for a long period of time.

The thermal stability of (a) m-ngCA-fixed silk hydrogel according to Example 1 and (b) ngCA-fixed silk hydrogel according to Example 1 is shown in FIG. 10 compared to free ngCA.

As shown in Fig. 10, the m-ngCA-fixed silk hydrogel exhibited ~ 91% and ~ 41% activity of the initial activity after 1 hour and 12 hours of culture at 50 ° C, respectively, And decreased to ~ 4% active level within 1 hour. This result may be due to the limited structure of the ngCA enzyme trapped within the silk hydrogel. However, m-ngCA-fixed silk hydrogels exhibited slightly lower thermal stability values than the ngCA-fixed silk hydrogels exhibiting ~ 96% and ~ 60% activity of the initial activity, respectively. The reason seems to arise from the different opportunities of enzymes for refolding / denaturation. CA enzymes experience two transitions separated by a stable molten globule intermediate and have the opportunity to change from an unfolding transition state to a stable intermediate state or an initial state, unless completely denatured. Thus, while stored at 4 ° C prior to testing, the m-ngCA-fixed silk hydrogel may have resulted in a lower refolding due to the limited mobility of internally captured ngCA enzyme relative to the ngCA-fixed silk hydrogel .

Experimental Example 6. Evaluation of Carbon Dioxide Conversion of m-ngCA-Fixed Silk Hydrogel

6.1 Analysis Method

The conversion of carbon dioxide to calcium carbonate for the m-ngCA-fixed silk hydrogel according to Example 1 was performed using the calcium carbonate precipitation method.

First, a CO ₂ -saturated aqueous solution was prepared by bubbling carbon dioxide (CO ₂ ) in water at 18 ° C in an open system. Then 5 mL of CO ₂ -saturated aqueous solution was added to 5 mL of reaction buffer (1M Tris-SO ₄ , 20 mM CaCl 2) containing a sample (free ngCA enzyme, ngCA-fixed silk hydrogel and m-ngCA-fixed silk hydrogel) ₂ , pH 9.5) and the reaction was carried out at ambient temperature for 150 seconds in the closed system. The final product was filtered through a 0.1 μm-pore-membrane and dried overnight at 85 ° C. The precipitated calcium carbonate (CaCO ₃ ) was weighed and its morphology was observed by SEM.

6.2 Results of analysis

The conversion rates of carbon dioxide to calcium carbonate by the free ngCA enzyme, ngCA-fixed silk hydrogel and m-ngCA-fixed silk hydrogel according to Example 1 are shown in Table 2 and measured by m-ngCA-fixed silk hydrogel An SEM image of the precipitated CaCO ₃ is shown in FIG.

Weight of precipitate (mg) Removed CO ₂ (mg) Yield (%) Non-catalyst 2.7 ± 0.7 1.2 ± 0.3 13.2 ± 3.6 Free ngCA 9.5 ± 0.6 4.2 ± 0.3 46.4 ± 2.8 m-ngCA-fixed silk hydrogel 5.4 ± 0.3 2.4 ± 0.1 26.2 ± 2.0 ngCA-fixed silk hydrogel 5.1 ± 0.1 2.2 ± 0.1 24.9 ± 0.7

As shown in Table 2, the m-ngCA-fixed silk hydrogel exhibited about 2-fold increase in CO ₂ hydration activity compared to the non-catalytic reaction, and a ~ 60% level of CO ₂ hydration activity. The reduced activity of the m-ngCA-fixed silk hydrogel relative to the free ngCA enzyme will be due to diffusive transport of the substrate and / or product from the hydrogel. In addition, the m-ngCA-fixed silk hydrogel exhibited CO ₂ hydration activity similar to the ngCA-fixed silk hydrogel. From the above results, it can be seen that the m-ngCA-fixed silk hydrogel can be used as an effective biocatalyst for environmentally converting carbon dioxide to calcium carbonate.

<110> POSTECH ACADEMY-INDUSTRY FOUNDATION <120> Carbonic anhydrase immobilized silk hydrogel and conversion or          fixation of carbon dioxide using the same <130> DPP20102958KR <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 236 <212> PRT <213> Artificial Sequence <220> <223> CA-1 <400> 1 Met Gly His Gly Asn His Thr His Trp Gly Tyr Thr Gly His Asp Ser   1 5 10 15 Pro Glu Ser Trp Gly Asn Leu Ser Glu Glu Phe Arg Leu Cys Ser Thr              20 25 30 Gly Lys Asn Gln Ser Pro Val Asn Ile Thr Glu Thr Val Ser Gly Lys          35 40 45 Leu Pro Ala Ile Lys Val Asn Tyr Lys Pro Ser Met Val Asp Val Glu      50 55 60 Asn Asn Gly His Thr Ile Gln Val Asn Tyr Pro Glu Gly Gly Asn Thr  65 70 75 80 Leu Thr Val Asn Gly Arg Thr Tyr Thr Leu Lys Gln Phe His Phe His                  85 90 95 Val Pro Ser Glu Asn Gln Ile Lys Gly Arg Thr Phe Pro Met Glu Ala             100 105 110 His Phe Val His Leu Asp Glu Asn Lys Gln Pro Leu Val Leu Ala Val         115 120 125 Leu Tyr Glu Ala Gly Lys Thr Asn Gly Arg Leu Ser Ser Ile Trp Asn     130 135 140 Val Met Pro Met Thr Ala Gly Lys Val Lys Leu Asn Gln Pro Phe Asp 145 150 155 160 Ala Ser Thr Leu Leu Pro Lys Arg Leu Lys Tyr Tyr Arg Phe Ala Gly                 165 170 175 Ser Leu Thr Thr Pro Pro Cys Thr Glu Gly Val Ser Trp Leu Val Leu             180 185 190 Lys Thr Tyr Asp His Ile Asp Gln Ala Gln Ala Glu Lys Phe Thr Arg         195 200 205 Ala Val Gly Ser Glu Asn Asn Arg Pro Val Gln Pro Leu Asn Ala Arg     210 215 220 Val Val Ile Glu Gly Gly His His His His His 225 230 235

Claims (23)

A composition for the conversion or fixing of carbon dioxide, comprising a silk hydrogel fixed with carbonic anhydrase. The method according to claim 1,
Wherein the carbonic anhydrase is an? -Type derived from Neisseria gonorrhoeae . The method according to claim 1,
Wherein the carbonic anhydrase has the amino acid sequence of SEQ ID NO: 1. The method according to claim 1,
Wherein the carbonic anhydrase is characterized in that the tyrosine residue does not affect the active site. The method according to claim 1,
Wherein the silk is a silk protein derived from a silkworm (Bombyx mori), green lacewings (Chrysoperla carnea), araneus (Araneus diadematus), or Nephila clavipes. &Lt; / RTI &gt; 6. The method of claim 5,
Wherein the silk protein is silk fibroin extracted from silkworm. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt; The method according to claim 1,
Wherein the carbonic anhydrase is immobilized on a silk hydrogel by photo-crosslinking and alcohol treatment. 8. The method of claim 7,
Wherein the alcohol is an alcohol aqueous solution in which the ratio of alcohol to water is 1: 0.1 to 1: 10 by volume (v / v). 8. The method of claim 7,
Wherein the alcohol is methanol or ethanol. The method according to claim 1,
Wherein the carbon dioxide is converted into a bicarbonate or a carbonate compound. 11. The method of claim 10,
The bicarbonate compound may be selected from the group consisting of sodium bicarbonate (Na ₂ HCO ₃ ), potassium bicarbonate (KHCO ₃ ), magnesium bicarbonate (Mg (HCO ₃ ) ₂ ), calcium bicarbonate (Ca (HCO ₃ ) ₂ ) and ammonium bicarbonate that is, conversion or fixing composition for carbon dioxide, characterized in that at least one member selected from the group consisting of _{((NH 4) HCO 3)} . 11. The method of claim 10,
The carbonate compound may be at least one selected from the group consisting of carbonic acid (H ₂ CO ₃ ), lithium carbonate (Li ₂ CO ₃ ), sodium carbonate (Na ₂ CO ₃ ), potassium carbonate (K ₂ CO ₃ ), rubidium carbonate (Rb ₂ CO ₃ ) carbonate (Cs ₂ CO _3), beryllium carbonate (BeCO _3), magnesium carbonate (MgCO _3), calcium carbonate (CaCO _3), strontium carbonate (SrCO _3), barium carbonate (BaCO _3), manganese carbonate (MnCO _3), ferrous carbonate (FeCO _3), cobalt carbonate (CoCO _3), nickel carbonate (NiCO _3), copper carbonate (CuCO _3), silver carbonate (Ag ₂ CO _3), zinc carbonate (ZnCO _3), cadmium carbonate (CdCO ₃₎ , aluminum carbonate _{(Al 2 (CO 3) 3} ), thallium carbonate (Tl ₂ CO _3), lead carbonate (PbCO ₃₎ and lanthanum carbonate, characterized in that at least one member selected from the group consisting of _{(La 2 (CO 3) 3} ) By weight based on the total weight of the composition. (1) preparing a mixed solution of a carbonic anhydrase and a silk protein;
(2) photo-crosslinking the mixed solution to produce a silk hydrogel; And
(3) subjecting the silk hydrogel to an alcohol treatment; and (3) subjecting the silk hydrogel to an alcohol treatment. 14. The method of claim 13,
The method for producing a silk hydrogel as set forth in (1), wherein the solvent for preparing the solution is Tris HCl buffer having a pH of 7 to 9. 14. The method of claim 13,
The method for producing a silk hydrogel as set forth in (1) above, wherein the mixed solution comprises a photoinitiator. 16. The method of claim 15,
Wherein the photoinitiator is selected from the group consisting of tris (bipyridine) ruthenium (II) chloride, camphorquinone peroxyester, 9-fluorenecarboxylic acid peroxyester, dl-camphorquinone, and Agar Cure 784DC Wherein the carbonic anhydrase is immobilized on at least one surface of the silk hydrogel. 14. The method of claim 13,
Wherein the photo-crosslinking is performed for 1 to 100 seconds (s) by visible light in the step (2). 14. The method of claim 13,
In the step (3), the alcohol is an alcohol aqueous solution having a ratio of alcohol to water of 1: 0.1 to 1: 10 by volume (v / v), wherein the hydrolyzate of carbonic anhydrase- Gt; 14. The method of claim 13,
Wherein the alcohol is methanol or ethanol in the step (3), wherein the carbonic anhydrase is immobilized. 14. The method of claim 13,
Wherein the alcohol is treated for 0.1 to 2 hours in the step (3). &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; 20. A silk hydrogel in which a carbonic anhydrase is immobilized, which is produced according to the method of any one of claims 13 to 20. A method for converting or fixing carbon dioxide, comprising the step of reacting a composition for converting or fixing carbon dioxide according to any one of claims 1 to 12 with a saturated solution of carbon dioxide. A device for converting or fixing carbon dioxide, comprising a composition for the conversion or fixing of carbon dioxide according to any one of claims 1 to 12.

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