Method for producing protein-immobilized carbon material

Abstract

To provide a method for efficiently immobilizing proteins on a carbon material.SOLUTION: Provided is a method for producing a protein-immobilized carbon material, comprising: step A of preparing a carbon material having a carboxyl group; step B of preparing a substance having a biotin moiety and an amino group at its terminus; step C of obtaining a carbon material having a biotin moiety by amide bonding in an aprotic organic solvent between the carboxyl group of the carbon material and the amino group of the substance having a biotin moiety and an amino group at its terminus; step D of preparing a protein having an avidin moiety; and step E of obtaining a protein-bound carbon material by binding the biotin moiety of the carbon material having a biotin moiety obtained in step C with the avidin moiety of the protein having an avidin moiety prepared in step D.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing a protein-immobilized carbon material. [Background technology]

[0002] Enzymes and other proteins have been used in a wide range of fields, including food, biomedical, and cosmetics, but enzymes are difficult to separate and recover because they are water-soluble and have a low sedimentation coefficient. Therefore, proteins have been immobilized on glass beads, magnetic beads, or the surface of yeast cells.

[0003] Patent Document 1 lists, as examples of carriers for immobilizing proteins, plastics represented by polyethylene, polypropylene, polystyrene, polymethacrylate, and polyvinyl alcohol; natural materials represented by hydrogel, agarose, dextran, cellulose, and chitosan; inorganic materials represented by silica, glass, and ceramics; and metal materials represented by gold, alumina, and silver. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2011-132145 A Summary of the Invention [Problem to be solved by the invention]

[0005] However, general-purpose immobilization materials are generally expensive. In particular, conductive immobilization materials, such as metal-based carriers such as Pt, are even more expensive. Meanwhile, no method has been established for immobilizing proteins on inexpensive and conductive carbon materials.

[0006] The present invention has been made in view of the above-mentioned problems, and an object of the present invention is to provide a method for efficiently immobilizing proteins on carbon materials. [Means for solving the problem]

[0007] The present inventors have conducted extensive research into protein-immobilized carbon materials, and as a result have found that a protein-immobilized carbon material can be efficiently obtained by employing the following production method, thereby completing the present invention.

[0008] That is, the present invention provides the following. [1] Step A of preparing a carbon material having a carboxyl group; Step B of preparing a substance having a biotin moiety and an amino group at its terminus; a step C of forming an amide bond between the carboxyl group of the carbon material and the amino group of a substance having a biotin moiety and an amino group at its terminal in an aprotic organic solvent to obtain a carbon material having a biotin moiety; A step D of providing a protein having an avidin moiety; and A step E of obtaining a carbon material having a protein bound thereto by binding the biotin moiety of the carbon material having the biotin moiety obtained in the step C to the avidin moiety of the protein having the avidin moiety prepared in the step D. A method for producing a protein-immobilized carbon material, comprising:

[0009] According to the configuration [1], a carbon material having a biotin moiety is obtained by amide bonding between a carboxyl group of a carbon material and an amino group of a substance having a biotin moiety and an amino group at its terminal (step C), and a carbon material having a protein bonded thereto is obtained by bonding the biotin moiety of the carbon material having a biotin moiety obtained in step C with the avidin moiety of a protein having an avidin moiety (obtaining step). That is, according to the configuration [1] above, a protein can be immobilized on a carbon material via a substance having a biotin moiety and an amino group at its terminal. In particular, in the above [1], a carboxyl group of a carbon material and an amino group of a substance having a biotin moiety and an amino group at the terminal are amide-bonded in an aprotic organic solvent. Since an amide bond is formed by dehydration condensation, the use of a non-aqueous solvent allows the amide bond to be formed efficiently. As a result, the protein can be efficiently immobilized on the carbon material.

[0010] Furthermore, the present invention provides the following: [2] The method for producing a protein-immobilized carbon material according to [1] above, wherein the aprotic organic solvent is dimethyl sulfoxide.

[0011] Dimethyl sulfoxide is an aprotic and polar solvent. Amide bonds are formed by dehydration condensation, so non-aqueous solvents are preferred. Furthermore, the use of dimethyl sulfoxide, an aprotic and polar solvent, for activation of carboxylic acids (active esters) allows the amide bonds to be formed more efficiently.

[0012] Furthermore, the present invention provides the following: [3] The method for producing a protein-immobilized carbon material according to [1] or [2] above, wherein the substance having a biotin moiety and an amino group at its terminal is biotin hydrazide.

[0013] When the substance having a biotin moiety and an amino group at its terminal is biotin hydrazide, it is easily available and has an excellent cost. Furthermore, among the substances having a biotin moiety and an amino group at its terminal, biotin hydrazide is particularly preferred because it has a simple structure and is less likely to cause secondary reactions.

[0014] Furthermore, the present invention provides the following: [4] The method for producing a protein-immobilized carbon material according to any one of [1] to [3] above, wherein the step A includes a step A-1 of oxidizing a carbon material with nitric acid to provide a carboxyl group.

[0015] The method of oxidizing a carbon material with nitric acid to obtain a carbon material having a carboxyl group is advantageous in that a carbon material having a carboxyl group can be obtained efficiently. Effect of the Invention

[0016] According to the present invention, a method for efficiently immobilizing a protein on a carbon material can be provided. [Brief description of the drawings]

[0017] [Figure 1] FIG. 2 is a schematic diagram for explaining a method for producing a protein-immobilized carbon material according to the present embodiment. [Diagram 2] 1 is a graph showing activity retention rates in Examples and Comparative Examples.

[0018] Hereinafter, an embodiment of the present invention will be described.

[0019] A method for producing a protein-immobilized carbon material according to the present embodiment includes the steps of: A step A of preparing a carbon material having a carboxyl group; Step B of preparing a substance having a biotin moiety and an amino group at its terminus; a step C of forming an amide bond between the carboxyl group of the carbon material and the amino group of a substance having a biotin moiety and an amino group at its terminal in an aprotic organic solvent to obtain a carbon material having a biotin moiety; A step D of providing a protein having an avidin moiety; and The method further includes a step E of obtaining a carbon material having a protein bound thereto by binding the biotin moiety of the carbon material having the biotin moiety obtained in the step C to the avidin moiety of the protein having the avidin moiety prepared in the step D.

[0020] FIG. 1 is a schematic diagram for explaining a method for producing a protein-immobilized carbon material according to this embodiment.

[0021] <Process A> In the method for producing a protein-immobilized carbon material according to this embodiment, first, a carbon material having a carboxyl group is prepared (see the right side of FIG. 1). A carbon material having a carboxyl group can be obtained by carboxylating a carbon material that does not have a carboxyl group. In other words, it can be obtained by introducing a carboxyl group (-COOH) into the hydrocarbon skeleton of the carbon material.

[0022] The method of oxidizing a carbon material to form a carboxyl group is not particularly limited, but examples thereof include a method of oxidizing a carbon material with nitric acid, a method of oxidizing a carbon material with sulfuric acid, a method of oxidizing a carbon material by heating in air, a method of oxidizing a carbon material with ozone, a method of oxidizing a carbon material with hydrogen peroxide, and a method of oxidizing a carbon material with potassium permanganate. In other words, it is preferable that the step A includes a step A-0 of oxidizing a carbon material to provide a carboxyl group. In particular, the step A-0 is preferably a step A-1 of oxidizing a carbon material with nitric acid, since a carbon material having a carboxyl group can be efficiently obtained. The concentration of the aqueous nitric acid solution used in oxidizing the carbon material with nitric acid is preferably from 1% by mass to 50% by mass, and more preferably from 5% by mass to 20% by mass. The concentration of the slurry (concentration of the carbon material in the aqueous nitric acid solution) when the carbon material is oxidized with nitric acid is preferably from 1 mass % to 30 mass %, more preferably from 5 mass % to 15 mass %. The temperature during the oxidation of the carbon material with nitric acid is preferably 25°C or higher and 100°C or lower, and more preferably 60°C or higher and 100°C or lower. The time for oxidizing the carbon material with nitric acid is preferably 10 minutes or more, and more preferably 30 minutes or more. By setting the nitric acid oxidation conditions as described above, the carbon material can be suitably carboxylated.

[0023] In addition, as the carbon material having a carboxyl group, a commercially available material may be used.

[0024] The carbon material is not particularly limited, and examples thereof include carbon black, activated carbon, hard carbon, soft carbon, mesoporous carbon, graphite, carbon nanotubes, etc. Among them, carbon black is preferred from the viewpoint of forming mesopores (pores with a diameter of 2-50n) during granulation. That is, when the carbon material is carbon black, the enzyme can enter the pores (mesopores) and the enzyme can be fixed to the carbon black surface in a highly dispersed state.

[0025] The carbon material may be in the form of a powder or a plate. When the carbon material is a powder, the particle diameter D50 is preferably 1 μm or more and 2000 μm or less, and more preferably 10 μm or more and 500 μm or less. When the particle diameter D50 is within the above numerical range, proteins can be suitably immobilized. When the carbon material is in the form of a plate, the size may be, for example, a plate having an area of ​​about 0.5 to 5 cm square or a disk having a diameter of about 0.5 to 5 cm. The carbon material may also be a molded body obtained by solidifying a powdered carbon material using a binder (e.g., resin, pitch). The size of the molded body is not particularly limited, but may be, for example, a sphere having a diameter of 10 μm to 5 mm or a cylinder having a diameter of 1 to 10 mm.

[0026] <Process B> In the method for producing a protein-immobilized carbon material according to the present embodiment, a substance having a biotin moiety and an amino group at its terminal is prepared. In Fig. 1, biotin hydrazide is shown as an example of the substance having a biotin moiety and an amino group at its terminal.

[0027] The substance having a biotin moiety and an amino group at its end is not particularly limited as long as it has a biotin moiety and an amino group, and examples thereof include biotin hydrazide, biotin-PEG4-amine, N-(6-hydrazinyl-6-oxohexyl)biotinamide, 5-(biotinamide)pentylamine, and neutravidin. Among these, biotin hydrazide is particularly preferred because it has a simple structure and is less likely to cause secondary reactions. Also, biotin hydrazide is preferred from the viewpoint of material cost.

[0028] <Process C> Next, the carboxyl group of the carbon material and the amino group of the substance having the biotin moiety and an amino group at its terminal are amide-bonded in an aprotic organic solvent (amidation reaction) to obtain a carbon material having a biotin moiety. Figure 1 shows the condensation reaction between the carbon material having a carboxyl group and the amino group of biotin hydrazide to form an amide bond.

[0029] The aprotic organic solvent is not particularly limited, but examples thereof include dimethyl sulfoxide, acetone, dichloromethane, diethyl ether, acetonitrile, dimethylformamide, tetrahydrofuran, etc. Among them, dimethyl sulfoxide, which is an aprotic and polar solvent, is preferable. Since the amide bond is formed by dehydration condensation, a non-aqueous solvent is preferable, and further, by using dimethyl sulfoxide, which is an aprotic and polar solvent, for activation of carboxylic acid (active ester), the amide bond is formed more efficiently.

[0030] The concentration of the substance having a biotin moiety and an amino group at its terminus during the amidation reaction is preferably 0.5 mM (mmol / L) to 5 mM, more preferably 1 mM to 2 mM, per 1000 μL of the aprotic organic solvent.

[0031] The concentration of the carbon material during the amidation reaction is preferably from 1 mg to 100 mg, more preferably from 5 mg to 20 mg, per 1000 μL of the aprotic organic solvent.

[0032] The temperature for the amidation reaction is preferably 10°C or higher and 50°C or lower, and more preferably 20°C or higher and 30°C or lower.

[0033] The time for the amidation reaction is preferably from 1 hour to 6 hours, and more preferably from 1.5 hours to 3 hours.

[0034] During the amidation reaction, rotational stirring may be performed using a rotator etc. The rotation speed during rotational stirring is, for example, preferably 5 rpm or more and 50 rpm or less, more preferably 10 rpm or more and 20 rpm or less.

[0035] After the amidation reaction, the carbon material is recovered by centrifugation, etc. At this time, washing with phosphate-buffered saline, etc. may be carried out as appropriate.

[0036] <Process D> In the method for producing a protein-immobilized carbon material according to the present embodiment, a protein having an avidin site is prepared (see the left side of FIG. 1). In FIG. 1, streptavidin is shown as an example of a protein having an avidin site.

[0037] The protein having an avidin site is not particularly limited as long as it has an avidin site, and examples thereof include streptavidin, neutravidin, and the like.

[0038] The protein having an avidin site may be prepared by introducing an avidin site into a protein that does not have an avidin site using a known technique. In this case, the protein that does not have an avidin site is not particularly limited.

[0039] <Process E> Next, the biotin moiety of the carbon material having the biotin moiety obtained in step C is bound to the avidin moiety of the protein having the avidin moiety prepared in step D, thereby obtaining a carbon material having a protein bound thereto (step E).

[0040] Examples of the solvent used in step E include phosphate-buffered saline.

[0041] When performing step E, when phosphate buffered saline is used, the concentration of the protein having an avidin site is preferably 10 to 10,000 times diluted.

[0042] When carrying out step E, when using phosphate buffered saline, the concentration of the carbon material having a biotin moiety is preferably 0.1% by mass or more and 50% by mass or less, and more preferably 0.5% by mass or more and 2% by mass or less.

[0043] The temperature when step E is carried out is preferably 10°C or higher and 50°C or lower, and more preferably 20°C or higher and 30°C or lower.

[0044] The time for carrying out step E is preferably from 1 minute to 60 minutes, and more preferably from 5 minutes to 30 minutes.

[0045] In step E, rotational stirring may be performed using a rotator or the like. The rotation speed during rotational stirring is, for example, preferably 5 rpm or more and 50 rpm or less, more preferably 10 rpm or more and 20 rpm or less.

[0046] After step E, washing may be carried out appropriately with phosphate buffered saline or the like.

[0047] In this manner, a carbon material having a protein immobilized thereon is obtained.

[0048] When a carbon material has electrical conductivity, the carbon material on which a protein is immobilized will also have electrical conductivity. Therefore, if an enzyme is immobilized on a carbon material, it can be used as a sensor in which the carbon captures electrons generated by the oxidation-reduction reaction of the enzyme. Another possible use is to apply a voltage to promote the enzymatic reaction of the immobilized enzyme.

[0049] The method for producing a protein-immobilized carbon material according to this embodiment has been described above. EXAMPLES

[0050] The present invention will be described in detail below using examples, but the present invention is not limited to the following examples as long as it does not depart from the gist of the present invention.

[0051] <Preparation of protein-immobilized carbon materials> Example 1 1. Preparation of Carbon Materials Polyvinyl alcohol (PVA500, manufactured by Kanto Chemical Co., Ltd.) was dissolved in ion-exchanged water to obtain a 0.85% by mass PVA aqueous solution (polyvinyl alcohol aqueous solution). 50 g of carbon black (MOGUL, manufactured by CABOT Co., Ltd.) was dispersed in 588 g of the PVA aqueous solution to obtain a CB-dispersed PVA aqueous solution, which was then spray-dried using a spray dryer. The resulting dried product was heated in a cylindrical furnace with N 2 A spheroidized CB product was obtained by heat treatment at 800°C for 2 hours in a stream. The CB spheroidized product was added to a 5% by mass aqueous solution of nitric acid so that the slurry concentration was 10% by mass, and boiled for 60 minutes. After that, it was suction filtered, and the powder was washed with warm water at 60°C. The washed powder was added to ion-exchanged water so that the slurry concentration was 10% by mass, and boiled for 60 minutes. After that, it was suction filtered, and the powder was washed with warm water at 60°C, and then dried overnight (24 hours) in a dryer at 115°C to obtain a carbon material having a carboxyl group.

[0052] 2. Amide bond formation between the carboxyl group of the carbon material and the amino group of biotin hydrazide Biotin hydrazide (Tokyo Chemical Industry Co., Ltd., product name: Biotin Hydrazide (100 mg)) was prepared. A DMSO (dimethyl sulfoxide) solution in which the concentration of biotin hydrazide was adjusted to 1.3 mM (mmol / L) was prepared. 1000 μL of the prepared DMSO solution and 10 mg of the carbon material having a carboxyl group obtained above were added to a reaction tube. The reaction tube was placed in a Microtube Rotator MTR-103 (ASONE, Osaka, Japan) and reacted at 20 rpm for 2 hours at 23°C. After the reaction, the precipitate was collected by centrifugation (15,000 x g, 1 minute, 23°C) and the supernatant was discarded. The collected material was washed twice with 1 mL of PBS (phosphate buffer solution). As a result, the carboxyl group of the carbon material and the amino group of the biotin hydrazide were amide-bonded to obtain a carbon material having a biotin moiety.

[0053] 3. Immobilization of horseradish peroxidase (enzyme) The entire carbon material obtained in 2 above (carbon material having a biotin moiety) was immersed in 1 mL of PBS (phosphate buffer solution) containing horseradish peroxidase (HRP)-labeled streptavidin (Proteintech Group Inc., Illinois, USA) at a dilution ratio of 1:1000. Then, the material was treated at 20 rpm and 23° C. for 5 minutes on a Microtube Rotator MTR-103. The treated carbon material was washed twice with 1 mL of PBS (phosphate buffer solution). As a result, the carbon material with immobilized protein according to Example 1 was obtained.

[0054] Comparative Example 1 1. Preparation of Carbon Materials A carbon material having a carboxyl group was obtained in the same manner as in "1. Preparation of carbon material" of Example 1.

[0055] 2. Amide bond formation between the carboxyl group of the carbon material and the amino group of biotin hydrazide Biotin hydrazide (Tokyo Chemical Industry Co., Ltd., product name: Biotin Hydrazide (100 mg)) was prepared. An aqueous acetate buffer solution in which the concentration of biotin hydrazide was adjusted to 1.3 mM (mmol / L) was prepared. To a reaction tube, 1000 μL of the prepared acetate buffer solution and 10 mg of the carbon material having a carboxyl group obtained above were added. The reaction tube was placed in a Microtube Rotator MTR-103 (ASONE, Osaka, Japan) and reacted at 20 rpm for 2 hours at 23°C. After the reaction, the precipitate was collected by centrifugation (15,000 x g, 1 minute, 23°C) and the supernatant was discarded. The collected material was washed twice with 1 mL of PBS (phosphate buffer solution). As a result, the carboxyl group of the carbon material and the amino group of the biotin hydrazide were amide-bonded to obtain a carbon material having a biotin moiety.

[0056] 3. Immobilization of horseradish peroxidase (enzyme) A carbon material having a protein immobilized thereon according to Comparative Example 1 was obtained in the same manner as in "3. Immobilization of horseradish peroxidase (enzyme)" in Example 1, except that the carbon material having a biotin moiety obtained in "2. Amide bond formation between the carboxyl group of the carbon material and the amino group of biotin hydrazide" in Comparative Example 1 above was used.

[0057] Comparative Example 2 1. Preparation of Carbon Materials A carbon material having a carboxyl group was obtained in the same manner as in "1. Preparation of carbon material" of Example 1.

[0058] 2. Immobilization of horseradish peroxidase (enzyme) The entire amount of the carbon material having a carboxyl group (carbon material having no biotin moiety) obtained in 1 above was immersed in 1 mL of PBS (phosphate buffer solution) containing horseradish peroxidase (HRP)-labeled streptavidin (Proteintech Group Inc., Illinois, USA) at a dilution ratio of 1:1000. Then, the material was treated at 20 rpm and 23°C for 5 minutes using a Microtube Rotator MTR-103. The treated carbon material was washed twice with 1 mL of PBS (phosphate buffer solution). As a result, a carbon material having a protein immobilized thereon according to Comparative Example 2 was obtained.

[0059] [Verification of protein immobilization] The carbon material on which the protein was immobilized was introduced into 1 mL of PBS (phosphate buffer solution) containing 3,3',5,5'-tetramethylbenzidene (TMB) (Funakoshi, Tokyo, Japan) as a substrate at a mass ratio of (carbon material on which the protein was immobilized):(PBS containing TMB) = 1:10, and the activity of HRP (peroxidase) on the carbon material on which the protein was immobilized was confirmed. Specifically, solid and supernatant components were separated by natural settling for about 30 seconds at room temperature, atmospheric pressure, and in the air gas phase. The supernatant obtained by settling was measured for absorbance at 650 nm, which is within the absorption band of oxidized TMB, using an ASUV-1100 UV-Visible Spectrophotometer. The measurement conditions for the UV-Visible Spectrophotometer were room temperature, atmospheric pressure, in the air gas phase, with a wavelength of 650 nm and an optical path length of 1 cm. When using the carbon material repeatedly, new TMB-containing PBS was added to the carbon material that had been washed twice with 1 mL of PBS. The activity retention rate at the third use was calculated as the ratio of the absorbance value at 650 nm at the third use to the absorbance value at 650 nm at the first use. The activity retention rate was calculated as a ratio by dividing the absorbance value at 650 nm for each measurement by the absorbance value at 650 nm for the first measurement. The results are shown in Figure 2. As shown in Figure 2, it can be seen that the activity retention rate is high when biotin hydrazide is bound to a carbon material in DMSO.

[0060]

Table 1

Claims

1. A step A of preparing a carbon material having a carboxyl group; Step B of preparing a substance having a biotin moiety and an amino group at its terminal end; a step C of forming an amide bond between the carboxyl group of the carbon material and the amino group of a substance having the biotin moiety and an amino group at its terminal in an aprotic organic solvent to obtain a carbon material having a biotin moiety; A step D of providing a protein having an avidin site; and A step E of obtaining a carbon material having a protein bound thereto by binding the biotin moiety of the carbon material having the biotin moiety obtained in the step C to the avidin moiety of the protein having the avidin moiety prepared in the step D. A method for producing a protein-immobilized carbon material, comprising:

2. 2. The method for producing a protein-immobilized carbon material according to claim 1, wherein the aprotic organic solvent is dimethyl sulfoxide.

3. 3. The method for producing a protein-immobilized carbon material according to claim 1, wherein the substance having a biotin moiety and an amino group at its terminal is biotin hydrazide.

4. 3. The method for producing a protein-immobilized carbon material according to claim 1, wherein the step A includes a step A-1 of oxidizing the carbon material with nitric acid to provide a carboxyl group.

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