WO2015057004A1

WO2015057004A1 - Method for stabilizing metalloprotein by cross-linking

Info

Publication number: WO2015057004A1
Application number: PCT/KR2014/009767
Authority: WO
Inventors: 김중배; 전승현
Original assignee: 고려대학교 산학협력단
Priority date: 2013-10-18
Filing date: 2014-10-17
Publication date: 2015-04-23
Also published as: KR20150045896A; KR101651235B1

Abstract

The present invention relates to: a metalloprotein-insoluble carrier for adsorbing a metalloprotein in an insoluble carrier to stabilize the metalloprotein and maintaining the catalytic activity of a metalloprotein for a long time by cross-linking the metalloproteins so as to prevent a metal ion from escaping out of the protein; and a preparation method therefor. The metalloprotein-insoluble carrier conjugate of the present invention and the preparation method therefor effectively form a metalloprotein aggregate, thereby allowing aggregates of an equal weight of insoluble carriers and a large amount of metalloproteins to be accumulated, and are stable toward external environment changes. As one embodiment of the present invention, the efficiency of carbonic anhydrase in a carbon dioxide conversion process can be maximized. In addition, carbon dioxide can be converted so as to be stored in a desired place, and calcium carbonate generated by using the carbon dioxide is a main material of cement, and can be applied to various high value-added industries such as various neutralizing agents.

Description

Method of stabilizing metal proteins by crosslinking

The present invention relates to a method of stabilizing a metal protein by crosslinking, and more particularly, to a metal protein capable of maintaining the catalytic activity of the metal protein for a long time by preventing crosslinking between the metal proteins to prevent ions from being released from the metal protein. It relates to a stabilization method.

Metalloprotein (metalloprotein) is a protein containing a metal ion in the protein, exists in vivo to perform a variety of functions. For example, iron in heme can perform both oxidation and reduction (Fe ²⁺ -Fe ³⁺ ) to perform a variety of biological functions, consisting of heme protein (hemeprotein), metalloenzyme (metalloenzyme) have. Changes or loss of protein function degrade enzyme function. Loss of enzyme function makes life a lot difficult. Heavy metals that have penetrated the body penetrate (bind) the protein and interfere with its own function. Keratin, which makes up hair and nails, hemoglobin that carries oxygen, and collagen, which attaches calcium to bone and makes up bone, are all proteins. Hemoglobin contains iron in the globin protein. However, heavy metals, mercury, can't transport oxygen if they drive iron out of its place. In addition, mercury adheres to collagen, which weakens bones and fractures easily. When heavy metals bind to enzyme proteins, workers lose their inherent function. Therefore, if the metal contained in the metal protein is released, it can be a big problem because it loses the function of the metal protein.

Carbonic anhydrase (CA), known as a representative metal protein, is known as the optimal metal protein that can be used in biological carbon dioxide abatement techniques. This metal protein contains zinc in its interior, and carbon dioxide interacts with it to convert bicarbonate ions (HCO ^{3 −} ) very quickly. Carbonic anhydrase converts 10 ⁶ carbon dioxide molecules per second, which means that a sufficient supply of carbon dioxide can theoretically convert more than 4,600 tons of carbon dioxide per hour using 1 kg of metal protein. The converted bicarbonate ions can be used for various purposes, and their potential value is very high. The stabilization of carbonic anhydrase allows development of a process to convert carbon dioxide to calcium carbonate. When bicarbonate ions are continuously supplied by stabilized carbonic anhydrase, it can react with calcium chloride to form calcium carbonate. Calcium carbonate is the main raw material of cement and is used in various neutralizing agents such as these.

Metallic proteins are relatively expensive catalysts and are therefore usually immobilized on a carrier for reuse after use in a process. The immobilized metal protein increases the activity of the catalyst and can be prolonged while maintaining the activity. Thus, the immobilization method is an essential element in the use of metal proteins as industrial biocatalysts. As a general metal protein immobilization method using nanostructures, a simple adsorption method, which is an enzyme immobilization method, or a covalent attachment method using covalent bonds, has been used. The high surface area provided by the nanostructures may increase the amount of metal protein supported and also increase the activity of metal protein per unit weight. However, there are still many problems with stability and persistence.

In addition, such a metal protein is sensitive to the environment, there is a problem that the metal ion easily escapes even if a small change does not perform its function.

Accordingly, the present inventors have stabilized the metal protein and thus have a high catalytic activity and create a method for storing metal ions in the protein.

The first problem to be solved by the present invention is to provide a method for stabilizing the metal protein by preventing metal ions in the metal protein from being released through crosslinking.

The second problem to be solved by the present invention is to provide a metal protein complex that exhibits high catalytic activity and is easy to separate and reuse even after use.

The present invention has been made to solve the above problems,

A first aspect of the present invention provides a method for preparing an insoluble carrier comprising (a) immobilizing a metal protein on an insoluble carrier; And (b) adding a crosslinking agent to form a crosslink between the immobilized metal protein.

According to one embodiment of the present invention, after the step (a), it may further comprise the step of adding a precipitation agent to the insoluble carrier to precipitate the metal protein immobilized on the insoluble carrier.

According to another embodiment of the present invention, after the step (b), it may further comprise the step of removing the added cross-linking agent and / or precipitation agent.

In addition, a second aspect of the present invention provides a metal protein stabilized by the above method.

In addition, a third aspect of the invention provides an insoluble carrier; And a metal protein immobilized on the insoluble carrier, and a crosslink is formed between the metal proteins to prevent desorption of metal ions.

The insoluble carrier may be any one or more selected from the group consisting of polymer nanofibers, carbon nanofibers, ceramic membranes, activated carbon, silica carriers, alumina carriers, celite carriers and zeolites.

The polymer nanofibers may be poly (styrene-co-maleic anhydride), polyacrylonitrile, nylon, polyester, polyurethane, polyvinyl chloride, polystyrene, cellulose, chitosan, polylactic acid, polylactic-co- It may be any one or more selected from the group consisting of glycolic acid, polyglycolic acid polycaprolactone, collagen, polypyrrole, polyaniline and polyvinyl alcohol.

The metal protein is carbonated anhydrous metal protein, heme protein, transferrin, metallothionein, formic acid dehydrogenase, formaldehyde dehydrogenase, alcohol dehydrogenase, glycerol dehydrogenase, nitrogen fixase, calmodulin, troponin It may be one or more selected from the group consisting of pulp albumin and calpine.

The precipitation agent is methanol, ethanol, 1-propanol, 2-propanol, butyl alcohol, acetone, polyethylene glycol, ammonium sulfate, ammonium sulfide, sodium chloride, sodium sulfate, sodium phosphate, potassium chloride, potassium sulfate, potassium phosphate and their The aqueous solution may be single or mixed.

The crosslinking agent is glutaraldehyde, diisocyanate, dianhydride, diepoxide, dialdehyde, diimide, 1-ethyl-3-dimethyl aminopropylcarbodiimide, bis (imido ester), bis (succinimidyl Esters) and diacid chlorides.

In the method of stabilizing metal proteins through cross-linking of the present invention, metal proteins are efficiently formed in aggregates, so that metal ions do not flow out of the protein, and a large amount of metal protein aggregates can be accumulated in a carrier of the same weight. As it is stable to environmental changes, it is possible to maximize the catalytic activity efficiency and maintain stability for a long time.

1 is a schematic diagram of a method for preparing a carbonic anhydrase-polymer nanofiber complex according to an embodiment of the present invention.

Figure 2 is a graph showing the initial activity and stability of the immobilized carbonic anhydrase according to an embodiment of the present invention.

Figure 3 is a graph showing the stability according to the leakage prevention of metal ions in the immobilized carbonic anhydrase according to an embodiment of the present invention.

Figure 4 is a graph showing the stability in various environments of the immobilized carbonic anhydrase according to an embodiment of the present invention. 4A is a graph showing the stability of immobilized metalloproteins in agitation conditions in a seawater environment. 4B is a graph showing the stability of the immobilized metal protein under carbon dioxide injection conditions in a seawater environment. Figure 4c is a graph showing the stability of the immobilized metal protein according to various similar environments distilled water, tap water, pH.

Hereinafter, the present invention will be described in more detail.

As mentioned above, immobilization of metal proteins is an essential element in the use of metal proteins as industrial biocatalysts. As a general metal protein immobilization method, simple adsorption or covalent attachment, which is an enzyme immobilization method, is used, but there are still many problems such as deterioration of the catalytic reaction due to mass transfer resistance and inactivation of the action site. have.

In the present invention, (a) fixing the metal protein to the insoluble carrier; And (b) adding a crosslinking agent to crosslink the immobilized metal protein. As a result, a large amount of metal protein per unit area is immobilized on the surface of the carrier and metal ions can be escaped from the protein as compared to the conventionally immobilized metal protein, thereby maintaining the function of the metal protein for a long time.

Metallic proteins used in the present invention are carbonic anhydrase, heme protein, transferrin, metallothionein, formic acid dehydrogenase, formaldehyde dehydrogenase, alcohol dehydrogenase, glycerol dehydrogenase, nitrogen fixase, calmodulin It may be one or more selected from the group consisting of, troponin, pulp albumin and calpine.

The carrier is an insoluble carrier, and may be any one or more selected from the group consisting of polymer nanofibers, carbon nanofibers, ceramic membranes, activated carbon, silica carriers, alumina carriers, celite carriers and zeolites. It doesn't work.

As the carrier, the polymer nanofibers may be poly (styrene-co-maleic anhydride), polyacrylonitrile, nylon, polyester, polyurethane, polyvinyl chloride, polystyrene, cellulose, chitosan, polylactic acid, polylactic It may be any one or more selected from the group consisting of -co-glycolic acid, polyglycolic acid polycaprolactone, collagen, polypyrrole, polyaniline and polyvinyl alcohol.

According to an embodiment of the present invention, the method may further include adding a precipitation agent to the insoluble carrier to precipitate the attached metal protein. The precipitation agents include methanol, ethanol, 1-propanol, 2-propanol, butyl alcohol, acetone, polyethylene glycol, ammonium sulfate, ammonium sulfide, sodium chloride, sodium sulfate, sodium phosphate, potassium chloride, potassium sulfate, potassium phosphate and these The aqueous solution of may be single or mixed.

1 is a schematic diagram showing a method for preparing a metal protein-polymer nanofiber composite according to an embodiment of the present invention. Referring to Figure 1, by adding a carbonic anhydride enzyme to the polymer nanofibers attached to the surface of the polymer nanofibers, the enzyme is present in a nearly single layer on the surface of the polymer nanofibers. The attached enzyme is thus precipitated to form an aggregate consisting of tens to hundreds of enzymes. These aggregates are not monolayers, but enzymes form dozens or hundreds of layers through crosslinking, so that much more enzymes can be immobilized per polymer nanofiber surface area than in the prior art, as well as more tightly fixed, Do not allow the metal ions in the enzyme to escape even after passing.

In addition, after the step (b), preferably washing the stabilized metal protein may further comprise the step of removing the added cross-linking agent and / or precipitation agent.

The present invention provides a metal protein stabilized by the above method. The metal protein may be reused while maintaining the nature and function of the metal protein, and may be applied to a carbon dioxide capture technology, a biomedical field, etc. because of excellent storage and stability.

In addition, the present invention is an insoluble carrier; And a metal protein immobilized on the insoluble carrier, and cross-linking is formed between the metal proteins to prevent desorption of metal ions. The metal protein is immobilized on an insoluble carrier through adsorption or covalent bonds, and crosslinks are formed between the metal proteins to prevent the metal ions inside the metal protein from escaping from the outside of the metal protein, thereby maintaining the function and activity of the metal protein for a long time. Do it. Therefore, the metal protein complex of the present invention not only facilitates storage and reacquisition, but also maintains the activity of the metal ions so that the catalytic activity is not reduced, so that the metal protein complex has the same efficiency as that of the unfixed metal protein.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1

Preparation of the ingredients

(1) metal protein

As a metal protein for immobilization, carbonic anhydrase derived from bovine was used. Carbonic anhydrase was purchased from Sigma-Aldrich (St. Louis, MO, USA).

(2) polymer nanofibers

In the present invention, a polymer nanofiber is used as a carrier. The polymers used to make the polymer nanofibers are PS (Polystyrene, molecular weight (MW) = 950,400) and Poly (styrene-co-maleic anhydride) (Poly (styrene-co-maleic anhydride), PSMA, Mw = 224,000). Tetrahydrofuran (THF) and acetone were used as the organic solvent for dissolving the polymer. These materials were purchased from Sigma-Aldrich (St. Louis, MO, USA).

(3) buffers and chemicals

As a buffer, phosphate buffer (PB) 10, 100 mM, pH 7.6 was used, and glutaraldehyde was used for the carbonic anhydride coating. Tris buffer (50mM, pH 7.6) was used to prevent maleic anhydride groups in the polymer nanofibers after carbonic anhydrase coating. Para-nitrophenyl acetate (NPA) was used as a reactor to measure the activity and physical properties of carbonic anhydrase.

Example 2

Immobilization of Carbonic Anhydrase in an Insoluble Carrier

(1) Preparation of PS + PSMA Nanofibers by Electrospinning Method and Alcohol Treatment of Polymeric Nanofibers

Nanofibers for immobilization of carbonic anhydrase are made of polystyrene (PS) and poly (styrene-co-maleic anhydride) (PSMA). (maleic anhydride) is immobilized covalently with the amine group of the carbonic anhydrase. The production method of PS + PSMA nanofiber is as follows. PS and PSMA were mixed at a weight ratio of 2: 1 at room temperature, dissolved in tetrahydrofuran (THF), and then mixed for about 3 hours using a magnetic stirrer. Thereafter, the acetone solution was mixed to lower the viscosity of the polymer solution, and then the polymer solution was placed in a 5 mL syringe with a 30 gauge stainless steel needle. The operating condition of the voltage was 7 kV, and the flow rate was 0.1 ml / hr using a syringe pump. The nanofibers from the electrospinning were collected in clean aluminum foil.

The alcohol treatment process for making dispersed nanofibers is as follows. Nanofibers were placed in a vial containing 50% v / v alcohol solution (ethanol), followed by shaking for 10 minutes at 200 rpm. Once the nanofibers were completely dispersed, they were washed without drying until alcohol was completely removed from the solution. The dispersed nanofibers were stored in buffer solution until carbonic anhydrase immobilization.

(2) Covalently Enzyme Immobilization Using Insoluble Carriers

Immobilization of metal protein (carbonic anhydrase) using an insoluble carrier (polymer nanofibers) was carried out using the following two types of covalent enzyme immobilization method using alcohol-treated nanofibers (CA-CA / EtOH-NF). ), Enzyme precipitation coating method using alcohol-treated nanofibers (EPC-CA / EtOH-NF)].

The dispersed nanofibers prepared above were incubated in a carbonic anhydride solution (10 mg / ml, 50 mM sodium phosphate buffer, pH 7.6) for carbonic anhydrase immobilization. The vial containing the carbonic anhydrase solution and the nanofibers was shaken at 200 rpm for 30 minutes, and then incubated at 4 ° C. for 2 hours to immobilize the carbonic anhydrase with the covalent bond to the nanofibers. (CA-CA / EtOH-NF).

For the synthesis of enzyme precipitate coating of carbonic anhydrase (EPC-CA), 45% ammonium sulfide was added to biocatalytic nanofibers (CA-CA / EtOH-NF) for 1 hour, and then A glutaraldehyde solution (0.5% w / v) as a crosslinking agent was added thereto, and then incubated overnight at 4 ° C (EPC-CA / EtOH-NF).

The biocatalyst nanofibers (CA-CA / EtOH-NF, EPC-CA / EtOH-NF) incubated overnight were washed with 100 mM sodium phosphate buffer (pH 7.6) and the reaction remaining on the nanofibers. Shaking was performed for 30 minutes using 100 mM sodium phosphate buffer (pH 7.6) to prevent aldehyde groups. The finished biocatalyst nanofibers were completely washed with 50 mM sodium phosphate buffer (pH 7.6) and stored at 4 ° C.

[Test Example 1]

Determination of Activity and Stability of Stabilized Metal Proteins

Carbonic anhydrase activity was measured by hydrolysis of para-nitrophenyl acetate (NPA), which is a reactive substance of carbonic anhydrase in aqueous buffer. NPA solution for activity measurement was dissolved in NPA using acetonitrile (10.9 mg / ml in acetonitrile) as a solvent, and then the solution was diluted to 1/100 using 50 mM sodium phosphate buffer (pH 7.6). Diluted to use. In the case of enzymes immobilized on the nanostructure, it is impossible to measure in real time, so that the biocatalytic nanofibers were added to 20 mL of NPA solution at 200 rpm, and then 1 mL of sample was taken every hour. Then, after diluting to 1/10 with 50 mM sodium phosphate buffer (pH 7.6), the absorbance of the product was measured with a spectrophotometer at 348 nm. The stability of the enzyme was measured by measuring the activity after the immobilized sample was incubated at 200 rpm stirring conditions, the same sample was reused over time to measure the activity in the same manner as compared to the initial activity.

As a result of initial activity measurement, the initial activities of covalently bound carbonic anhydrase (CA-CA / EtOH-NF) and enzyme-coated carbonic anhydrase (EPC-CA / EtOH-NF) were 0.23 and 10.2 uM / min, respectively. It was measured as (FIG. 2). In other words, when immobilized using the same amount of nanofibers, it means that the carbonic anhydrase immobilization method by enzyme precipitation coating is about 45 times higher activity than the acid anhydrase immobilization method by covalent bonding. This means that a myriad of enzyme aggregates are immobilized on the nanofibers.

Next, as a result of the stability measurement, the covalently bonded carbonic anhydrase loses its activity within a few days, whereas the enzyme-coated carbonic anhydrase does not lose the activity of the enzyme for 790 days and is 80% of the initial activity. It can be seen that it is maintained (FIG. 2). This proves that the enzyme aggregate coating through enzyme precipitation maintains the activity of the enzyme without losing the activity of the external environment.

[Test Example 2]

Identification of resistance to leakage of metal ions in stabilized metal proteins

Ethylenediaminetetraacetic acid (EDTA) is an organic compound, and the chemical formula is C10H16N2O8. It can act as a six-digit ligand and combine with metal ions to form a chiral chelate compound. It can act as a six-digit ligand and combine with metal ions to form a chiral chelate compound. EDTA can co-ordinate six vertices of an octahedron centered on metal ions, resulting in the central metal being surrounded by a ligand. EDTA thus has a strong affinity for certain metal ions. In biochemistry and molecular biology, EDTA is used to remove metal ions needed by enzymes to prevent DNA from damaging the sample.

When metal ions escape from metal enzymes, EDTA binds to metal ions and metal ions in metal enzymes are removed, thereby preventing the activity of metal enzymes. Therefore, in this experiment, ethylenediaminetetraacetic acid was added at various concentrations to prevent the desorbed metal ions from entering the enzyme, and then cultured under stirring conditions. It was reused to determine the stability of the enzyme. As a result of stability measurement, covalently bonded carbonic anhydrase loses most of its activity within 24 hours because metal ions in the enzyme are easily desorbed and the desorbed metal ions bind to ethylenediaminetetraacetic acid and cannot enter the enzyme again. On the other hand, in the case of cross-linked carbonic anhydrase, the activity of some enzymes is reduced but the stability can be seen (FIG. 2). This demonstrates that the crosslinked metal protein-polymer nanofiber complex prevents the desorption of metals in the enzyme, thereby maintaining the activity of the metal protein even when the external environment changes.

[Test Example 3]

Identification of stability in stabilized metal proteins in various environments

Seawater is abundant in nature and contains high concentrations of salts, which are expected to be economical and have sufficient buffering effects. Therefore, in the present invention, the stability of the enzyme was confirmed using a seawater solution having a seawater composition. As a result, it was confirmed that the stability of the enzyme was maintained when the stability under the 200 rpm stirring condition and the performance stability were measured while directly injecting carbon dioxide (FIG. 4). In addition, in the case of a buffer solution used in a general biochemical process, it can significantly impair the economics of the process in terms of cost, it is necessary to secure the stability of the enzyme in a variety of aqueous solution to solve this problem. Therefore, distilled water (DI water), tap water (tap water), the enzyme stability according to the pH was confirmed. As a result of the experiment, in the case of the carbonic anhydrase immobilized by the enzyme precipitation coating method, it was confirmed that it showed stability even under various conditions (FIG. 4).

The method for stabilizing metal proteins by crosslinking of the present invention can be used in various industries by effectively stabilizing metal proteins used in various fields. In particular, carbonic anhydrase, one of the representative metal proteins, may be used in an enzyme-based carbon dioxide conversion and utilization process. In the representative carbon dioxide capture technology, reducing energy consumption of the desorption process is one of the most important issues. As a solution to this problem, by using carbonic anhydrase, a metal protein of which the lifespan of the present invention is dramatically improved, it is possible to simply convert carbon dioxide to bicarbonate at room temperature without adsorption and desorption, and to convert bicarbonate to various carbon dioxide. By using it, it can be used industrially by developing an enzyme-based carbon dioxide conversion and utilization process that can replace the existing carbon dioxide capture and storage technology in the long term.

Claims

(a) immobilizing a metal protein on an insoluble carrier; And

(b) adding a crosslinking agent to form a crosslink between the immobilized metal proteins

Metal protein stabilization method comprising a.
According to claim 1, After the step (a),

And adding a precipitation agent to the insoluble carrier to precipitate the immobilized metal protein.
The method of claim 1, wherein after step (b),

The method of stabilizing the metal protein further comprising the step of removing the crosslinking agent.
The method of claim 2, wherein after step (b),

The method of stabilizing the metal protein further comprising the step of removing the precipitation agent and crosslinking agent.
The method of claim 1,

The insoluble carrier is a method of stabilizing a metal protein of any one or more selected from the group consisting of polymer nanofibers, carbon nanofibers, ceramic membranes, activated carbon, silica carriers, alumina carriers, celite carriers and zeolites.
The method of claim 5,

The polymer nanofibers may be poly (styrene-co-maleic anhydride), polyacrylonitrile, nylon, polyester, polyurethane, polyvinyl chloride, polystyrene, cellulose, chitosan, polylactic acid, polylactic-co- Method for stabilizing a metal protein of at least one or more selected from the group consisting of glycolic acid, polyglycolic acid polycaprolactone, collagen, polypyrrole, polyaniline and polyvinyl alcohol.
The method of claim 1,

The metal protein is carbonic anhydrase, heme protein, transferrin, metallothionein, formic acid dehydrogenase, formaldehyde dehydrogenase, alcohol dehydrogenase, glycerol dehydrogenase, nitrogen fixase, calmodulin, troponin, A method of stabilizing a metal protein, which is at least one or more metal proteins selected from the group consisting of pulp albumin and calpine.
The method of claim 1,

The crosslinking agent is glutaraldehyde, diisocyanate, dianhydride, diepoxide, dialdehyde, diimide, 1-ethyl-3-dimethyl aminopropylcarbodiimide, bis (imido ester), bis (succinimidyl Ester) and diacid chloride, wherein the method of stabilizing a metal protein is at least one or more compounds selected from the group consisting of.
The method of claim 2,

The precipitation agent is methanol, ethanol, 1-propanol, 2-propanol, butyl alcohol, acetone, polyethylene glycol, ammonium sulfate, ammonium sulfide, sodium chloride, sodium sulfate, sodium phosphate, potassium chloride, potassium sulfate, potassium phosphate and their A method for stabilizing a metal protein, in which an aqueous solution is used alone or in combination.
A metal protein stabilized according to the method of any one of claims 1 to 9.
Insoluble carriers; And a metal protein immobilized on the insoluble carrier, wherein a crosslink is formed between the metal proteins to prevent desorption of metal ions.
The method of claim 11,

The insoluble carrier is at least one selected from the group consisting of polymer nanofibers, carbon nanofibers, ceramic membranes, activated carbon, silica carriers, alumina carriers, celite carriers and zeolites.
The method of claim 11,

The metal protein is carbonic anhydrase, heme protein, transferrin, metallothionein, formic acid dehydrogenase, formaldehyde dehydrogenase, alcohol dehydrogenase, glycerol dehydrogenase, nitrogen fixase, calmodulin, troponin, A crosslinked metal protein complex of any one or more selected from the group consisting of pulp albumin and calpine.