Immobilized enzyme and method for producing the same

Abstract

The main objective of the present invention is to provide an immobilized enzyme having heat resistance and high activity, which can be used in the food, pharmaceutical, and chemical fields, and a method for producing the same. [Solution] An immobilized enzyme comprising an enzyme and a hydrogen-bonding organic structure, characterized in that the relative enzyme activity R represented by the following formula 1 is 1.5 or more and 20 or less. [Math 1] JPEG2026165777000007.jpg13132 (X represents the enzyme activity value of the immobilized enzyme. Y represents the enzyme activity value of the immobilized enzyme synthesized under temperature conditions of 20°C to 30°C. The enzyme activity value means the amount of enzyme exhibiting enzyme activity divided by the total amount of supported enzyme.)

Description

Technical Field

[0001] The present invention relates to immobilized enzymes used as labeling substances in fields such as analytical chemistry, immobilized enzymes used as catalysts in the synthesis of food additives and pharmaceuticals, and immobilized enzymes used in the treatment of industrial wastewater.

[0002] Enzymes, which are a type of biocatalyst, have extremely high selectivity for specific substrates and the property of efficiently promoting chemical reactions. For this reason, enzymes are utilized in a wide range of industrial fields including pharmaceuticals, food processing, and environmental conservation.

[0003] In the industrial utilization of enzymes, materials that take into account stability, reusability, and economic efficiency are required. As one of the techniques for imparting stability, reusability, and economic efficiency, enzyme immobilization techniques are known. The enzyme immobilization technique refers to a technique of immobilizing an enzyme within a certain space to obtain an enzyme that enables continuous reaction, recovery after the reaction, and reuse. Hereinafter, the enzyme obtained by the above immobilization technique is referred to as an immobilized enzyme.

[0004] As one of the methods for immobilizing an enzyme within a certain space, a method of immobilizing an enzyme on an insoluble carrier is known. As the insoluble carrier, porous materials are mainly utilized, and activated carbon, polymers, silica, zeolites, metal-organic frameworks (MOFs), covalent organic frameworks (COFs), hydrogen-bonded organic frameworks (HOFs) and the like are known.

[0005] Methods for producing immobilized enzymes can be broadly classified into two categories: the carrier binding method and the entrapment method. The carrier binding method refers to a method of immobilizing an enzyme in the pores of a porous material such as activated carbon, polymer beads, silica, or zeolite by physical adsorption or chemical adsorption. The entrapment method refers to a method for synthesizing an immobilized enzyme, in which an enzyme is added simultaneously with the synthesis of a porous material including an organic structure such as a crosslinked polymer, MOF, COF, or HOF, to composite the porous material and the enzyme.

[0006] When immobilized enzymes are synthesized using MOF, COF, or HOF as a support material via an inclusive method, the three-dimensional structure of the enzyme is maintained by the fine and regular crystalline structure of the MOF, COF, or HOF. Therefore, the enzyme contained in the immobilized enzyme can obtain excellent heat resistance.

[0007] In particular, HOF is a structure that utilizes the rigidity of hydrogen bonds, which are reversible bonds, and has the advantage of allowing the enzyme to be reused after the immobilized enzyme that was synthesized has been broken down.

[0008] Patent Document 1 discloses a method for producing immobilized enzymes using HOF as a support by an inclusion method.

[0009] Examples of enzymes that can be immobilized include oxidoreductases, transferases, hydrolases, addition-release enzymes, isomerases, synthesizers, and transport enzymes.

[0010] Peroxidases, a type of oxidoreductase, are used as labeling agents in the field of analytical chemistry. Furthermore, they can be used in the field of environmental chemistry, such as in the treatment of industrial wastewater, and are attracting attention as enzymes with industrial utility. [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] International Publication No. 2020 / 252536 [Overview of the project] [Problems that the invention aims to solve]

[0012] For HOF-supported immobilized enzymes, there is a market demand to improve the reaction efficiency of reactions using immobilized enzymes in order to expand their usable applications. If it is possible to increase the amount of enzyme that can be effectively used and to create an immobilized enzyme that is also heat-resistant, the application of immobilized enzymes could rapidly advance.

[0013] Furthermore, if peroxidases with quality stability as oxidoreductases can be immobilized on HOF, it could lead to technological advancements in fields such as food, pharmaceuticals, and chemistry.

[0014] The main objective of the present invention is to provide an immobilized enzyme having heat resistance and high activity, which can be used in the food, pharmaceutical, and chemical fields, and a method for producing the same. [Means for solving the problem]

[0015] The inventors of this invention have diligently researched methods for synthesizing immobilized enzymes using HOF as a support, and as a result have discovered that by optimizing the temperature conditions in the comprehensive method, it is possible to produce immobilized enzymes that possess both heat resistance and highly efficient enzyme activity, leading to the present invention.

[0016] The immobilized enzyme and method for producing the same according to the present invention have the following configurations [1] to

[10] . [1] Enzymes and, An immobilized enzyme comprising a hydrogen-bonding organic structure, An immobilized enzyme characterized in that its relative enzyme activity R, represented by the following formula 1, is between 1.5 and 20.

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[10] In the step (a), among the ligands, at least one type of ligand is an amidine group, and among the ligands, at least one type of ligand is a carbonyl group, according to the method for producing the immobilized enzyme of any one of [7] to [9]. [Effects of the Invention]

[0017] According to the present invention, it is possible to provide an immobilized enzyme that has heat resistance and high enzyme activity, and can be used in fields such as the food field, the pharmaceutical field, and the chemical field, as well as a production method thereof. [Brief Description of the Drawings]

[0018] ​ [Figure 1] This is a schematic diagram illustrating the crystal structure of HOF. [Figure 2] These are the thermogravimetric curves (TGA curves) of the HOF (HoF without enzyme) used in the synthesis of the immobilized enzyme, the enzyme itself, and the synthesized immobilized enzyme. [Figure 3] This is a graph of the calibration curve used to calculate the amount of enzyme exhibiting enzyme activity in the examples and comparative examples. [Modes for carrying out the invention]

[0019] The present invention will be described below. However, the embodiments described below are merely illustrative. The present invention is not limited in any way to the embodiments described below.

[0020] 1. Enzymes Examples of enzymes according to the present invention include oxidoreductases, transferases, hydrolases, addition-desorption enzymes, isomerases, synthases, and transport enzymes, based on classification according to enzyme commission numbers (EC numbers). As the enzyme included in the immobilized enzyme, one enzyme may be selected from the examples above, or two or more enzymes may be selected in combination.

[0021] Examples of oxidoreductases include peroxidase, dehydrogenase, reductase, oxidase, oxygenase, transhydrogenase, and catalase.

[0022] Peroxidases are used in a wide range of fields, including analysis, clinical testing, immunochemistry, histochemistry, and cell chemistry. In recent years, their demand has expanded beyond analytical chemistry to include environmental chemistry due to their usefulness in environmental remediation, such as industrial wastewater treatment.

[0023] Examples of peroxidases include horseradish peroxidase (HRP), myeloperoxidase, lactoperoxidase, guaiacolperoxidase, and ascorbic acid peroxidase. In particular, HRP is preferred because it can oxidize a wide variety of organic compounds with high catalytic efficiency, making it suitable for use in a wide range of fields.

[0024] 2. Hydrogen bonding organic framework (HOF) HOF is a porous material in which organic ligands self-assemble through hydrogen bonding. The organic ligands have functional groups capable of forming hydrogen bonds.

[0025] Examples of functional groups capable of forming hydrogen bonds include carbonyl groups (C=O), ether groups (ROR), nitrile groups (-C≡N), amidine groups (-C(=NH)-NH2), guanidine groups (-C(=NH)-NH-NH2), hydrazine groups (-NH-NH2), hydrazone groups (-C(=NNH2)-), pyridine rings (-C5H5N), imidazole groups (=N in the ring), urea groups (-C(=O)(NH2)2), sulfonyl groups (-SO2-), imine groups (-C=NH), isocyanide groups (-N≡C), hydroxyl groups (-OH), thiol groups (-SH), amino groups (-NH2), and carbamoyl groups (-C(=O)-NH2). Furthermore, these functional groups contained in the ligand may also be ionized salts.

[0026] The hydrogen bonds that form the crystal structure of HOF may be between identical functional groups or between different functional groups. When different functional groups form hydrogen bonds with each other, a charge difference may arise depending on the combination, making it possible to form charge-supported hydrogen bonds that share charge. Charge-supported hydrogen bonds have stronger bonding strength than ordinary hydrogen bonds and do not easily break apart. From the viewpoint of improving durability during the production of immobilized enzymes, it is preferable to synthesize HOF using ligands having amidine and carbonyl groups.

[0027] The HOF crystal structure preferably contains ligands that include at least eight nitrogen atoms. Including at least eight nitrogen atoms makes it easier to immobilize the enzyme and allows for the production of highly heat-resistant immobilized enzymes.

[0028] 3. Immobilized enzymes An immobilized enzyme consists of a support and the enzyme, with the enzyme immobilized on the support. Examples of immobilization structures include structures in which the support and enzyme are physically or chemically bonded, or structures in which the enzyme is incorporated into the support. When immobilizing an enzyme using HOF as the support, a structure in which the enzyme is incorporated into HOF with excellent crystallinity is preferred from the viewpoint of heat resistance and freedom in enzyme selection for the support.

[0029] Structures in which the enzyme is incorporated into HOF include, for example, structures in which the enzyme is completely encapsulated within the HOF crystal structure, structures in which the enzyme is partially immobilized on the HOF crystal structure, and structures in which the enzyme is bound to the surface of HOF. From the viewpoint of achieving both heat resistance and excellent enzyme activity, a structure in which the enzyme is partially immobilized on the HOF crystal structure is preferred.

[0030] The immobilized enzyme preferably has a relative enzyme activity R represented by the following formula 1 that is between 1.5 and 20, more preferably between 1.5 and 15, and even more preferably between 1.5 and 10.

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[0031] By setting the relative enzyme activity R to 1.5 or higher, the enzyme is not completely contained within the HOF crystal structure, resulting in a more highly active immobilized enzyme. Furthermore, by setting R to 20 or lower, the enzyme is partially protected by the highly crystalline HOF, improving the enzyme's heat resistance.

[0032] 4. Method for producing immobilized enzymes Firstly, ligand solutions are prepared by dissolving ligands in a solvent to form the HOF. If there are multiple types of ligands that make up the HOF, ligand solutions containing each ligand are prepared individually. The ligand solution containing an amidine group (hereinafter referred to as ligand solution A) and the ligand solution containing a carbonyl group (hereinafter referred to as ligand solution B) mentioned above will be used as examples and explained in detail below.

[0033] The ligand solution concentration in ligand solution A is preferably 0.5 mg / ml to 20 mg / ml, more preferably 1.0 mg / ml to 15 mg / ml, and even more preferably 1.0 mg / ml to 10 mg / ml. The ligand solution concentration in ligand solution B is preferably 0.5 mg / ml to 20 mg / ml, more preferably 1.0 mg / ml to 15 mg / ml, and even more preferably 1.0 mg / ml to 10 mg / ml. By setting the ligand solution concentrations in ligand solution A and ligand solution B within the above ranges, the solubility of the ligand can be increased while synthesizing the immobilized enzyme in a short time.

[0034] Next, the enzyme is prepared. While there are no particular limitations on the enzyme to be immobilized, the method for producing the immobilized enzyme will be described in detail below, using the aforementioned HRP as an example.

[0035] Next, prepare the solvent (synthesis solvent) to be used during the synthesis of the immobilized enzyme. Examples of solvents include water, methanol, ethanol, propyl alcohol, acetone, ethyl acetate, dimethylformamide, dimethylacetamide, dimethyl sulfoxide, acetonitrile, tetrahydrofuran, N-methyl-2-pyrrolidone, and 1,4-dioxane. From the viewpoint of synthesizing the immobilized enzyme while maintaining the enzyme in a more highly active state, it is preferable to use water as the synthesis solvent.

[0036] Furthermore, from the viewpoint of synthesizing immobilized enzymes while maintaining the enzymes in a more highly active state, it is preferable that the pH range of the solvent be 4 ≤ pH < ≤ 10.

[0037] Next, ligand solution A and HRP are mixed to obtain a mixed solution in which the ligand and enzyme are dissolved in the solvent. Then, ligand solution B is added dropwise to the mixed solution and mixed. Upon mixing of the mixed solution and ligand solution B, charge-assisted hydrogen bonds are formed between the amidine group and the carbonyl group, and the crystalline structure of HOF is formed. At this time, because HRP is present as an enzyme, HRP is immobilized on HOF during the formation of the crystalline structure, and a solid immobilized enzyme is synthesized. (See Figure 1)

[0038] In the above example, a method is described in which a mixed solution of ligand solution A and HRP is prepared as a preliminary step to synthesizing the immobilized enzyme, and then the resulting mixed solution is mixed with ligand solution B to synthesize the immobilized enzyme. However, it is also acceptable to prepare a mixed solution of ligand solution B and HRP, and then mix the resulting mixed solution with ligand solution A.

[0039] The temperature conditions from the preparation of the above mixed solution to the synthesis of the immobilized enzyme are preferably 0°C to 10°C or lower, more preferably 1°C to 10°C, and even more preferably 3°C to 8°C. By setting the temperature conditions within this range, the enzyme is not completely contained within the crystalline structure of HOF, resulting in a more highly active immobilized enzyme. Furthermore, the enzyme is partially protected by HOF, which has excellent crystallinity, thus providing heat resistance to the enzyme.

[0040] The resulting solid immobilized enzyme can be isolated from the solvent by centrifugation or filtration.

[0041] The proportion of the total enzyme amount in the immobilized enzyme is preferably 0.1% by mass or more and 20% by mass or less, more preferably 1% by mass or more and 15% by mass or less, and even more preferably 5% by mass or more and 15% by mass or less. By setting it within the above range, the decrease in enzyme activity due to aggregation of enzymes is suppressed, and the amount of enzyme that can be effectively used can be increased. [Examples]

[0042] The present invention will be described in more detail below based on specific examples, but the present invention is not limited in any way to the following examples and can be implemented with appropriate modifications without changing the gist of the invention.

[0043] [Measurement and evaluation methods]

[0044] (Measurement of total enzyme content in immobilized enzymes) The total amount of enzyme in an immobilized enzyme can be calculated by thermogravimetric analysis (TGA). First, TGA was performed on HOF (HoF without enzyme) used in the synthesis of the immobilized enzyme, the enzyme itself, and the synthesized immobilized enzyme. In the obtained TGA curve, assuming 150°C as the pre-combustion temperature and 890°C as the post-combustion temperature, a graph was output with the weight factor on the vertical axis and temperature (°C) on the horizontal axis, with the post-combustion temperature set to 1. (See Figure 2) Based on the difference in weight factor at 150°C, the total amount of enzyme immobilized in the immobilized enzyme was calculated based on the proportion of enzyme in the immobilized enzyme. Since moisture from the air is adsorbed into the pores of HOF, the weight % before combustion was compared at 150°C, which is above the boiling point of water. In this invention, the above weight % was used as mass % to calculate the total amount of enzyme.

[0045] [Conditions for thermogravimetric analysis] Analytical instrument (TGAQ50, manufactured by T.A. Instruments Japan) Analysis environment: Under N2 nitrogen Heating rate: 10℃ / min Temperature range: 30℃~900℃ Sample size: 10 mg Analysis container: Platinum pan

[0046] (Measurement of enzyme levels indicating enzyme activity) In immobilized enzymes, the amount of enzyme exhibiting enzyme activity can be calculated by activity measurement. The method of activity measurement varies depending on the enzyme, but for example, when the enzyme is HRP, a method utilizing the pyrogallol color reaction shown in Equation 2 below can be used. 10 μl of HRP aqueous solution (3.47 μg / ml), 1.5 ml of purified water, 0.2 ml of phosphate buffer (0.1 M), and 0.1 ml of hydrogen peroxide solution (0.15 M) were added to a sample vial and preheated at 25°C for 5 minutes. 0.2 ml of pyrogallol aqueous solution (5 w / v%) was added and reacted for 5 minutes. After 5 minutes, the absorbance at 420 nm was measured. [ka] Next, the above procedure was repeated twice, varying the amount of HRP aqueous solution to 15 μl and 20 μl, to obtain a calibration curve between the amount of HRP indicating enzyme activity and the absorbance. (See Figure 3) Then, the above HRP was replaced with an immobilized enzyme whose total enzyme amount was known, and the same measurement was performed. From the relationship between the absorbance when replaced with the immobilized enzyme and the calibration curve, the amount of HRP that exhibits enzyme activity in the immobilized enzyme was calculated.

[0047] [Conditions for absorbance analysis] Analytical instrument (Hitachi High-Tech Science Co., Ltd., UH3900D) Wavelength: 420nm

[0048] (Method for calculating relative enzyme activity R) The specific enzyme activity R was calculated using the following formula 1 based on the total enzyme amount and HRP amount obtained from the immobilized enzymes measured above.

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[0049] In the examples and comparative examples of this application, the enzyme activity value of Comparative Example 2 was denoted as Y, and the enzyme activity values ​​of the other examples and comparative examples were denoted as X, and the value of the relative enzyme activity R was calculated from Equation 1.

[0050] (Example 1) Ligand solution A was prepared by dissolving 40 mg of 4,4',4'',4'''-methanetetrayltetrabenzimidamide tetrahydrochloride (CAS: 220586-69-0) in 4.4 ml of purified water, and then dissolving 8.5 mg of horseradish peroxidase (HRP) to prepare a mixed solution. Ligand solution B (7.67 mg / ml) was prepared by adding 30 mg of 4,4',4'',4'''-methanetetrayltetrabenzoic acid (CAS: 160248-28-2) to 4.1 ml of purified water, and then adding 0.5 ml of sodium hydroxide aqueous solution (0.48 mol / L) to dissolve 35.3 mg of the sodium salt of 4,4',4'',4'''-methanetetrayltetrabenzoic acid in 4.6 ml of purified water. A mixed solution containing ligand solution A and HRP was prepared at 5.8°C. Ligand solution B at room temperature was then slowly added dropwise at a rate of 41.8 ml / min, and the mixture was stirred for 30 minutes while maintaining the temperature at 5.8°C after the addition was complete. The precipitated solid was settled by centrifugation at 3000 rpm for 15 minutes, and the supernatant was removed and collected. The collected solid was then suspended in purified water and washed again by centrifugation. The washing process was performed twice. After washing, the water was removed by vacuum drying for 20 hours to prepare an immobilized enzyme, which was a white solid. 8.3 mg of the obtained immobilized enzyme was weighed, and the total enzyme amount was calculated by the thermogravimetric analysis described above. In addition, 1.8 mg was weighed into a sample bottle, and the amount of HRP, which indicates enzyme activity in the immobilized enzyme, was calculated by the absorbance analysis described above.

[0051] (Example 2) Ligand solution A was prepared by dissolving 40 mg of 4,4',4'',4'''-methanetetrayltetrabenzimidamide tetrahydrochloride in 26.0 ml of purified water, and then dissolving 17.3 mg of horseradish peroxidase (HRP) to prepare a mixed solution. Ligand solution B (4.58 mg / ml) was prepared by adding 30 mg of 4,4',4'',4'''-methanetetrayltetrabenzoic acid to 7.2 ml of purified water, then adding 0.5 ml of sodium hydroxide aqueous solution (0.48 mol / L), and dissolving 35.3 mg of the sodium salt of 4,4',4'',4'''-methanetetrayltetrabenzoic acid in 7.7 ml of purified water. A mixed solution containing ligand solution A and HRP was prepared at 9.0°C. Ligand solution B at room temperature was then slowly added dropwise at a rate of 18.1 ml / min, and the mixture was stirred for 30 minutes after the addition was complete. After collection, the same procedure as in Example 1 was performed.

[0052] (Example 3) Ligand solution A was prepared by dissolving 40 mg of 4,4',4'',4'''-methanetetrayltetrabenzimidamide tetrahydrochloride in 10.2 ml of purified water, and then dissolving 19.8 mg of horseradish peroxidase (HRP) to prepare a mixed solution. 30 mg of 4,4',4'',4'''-methanetetrayltetrabenzoic acid was added to 4.7 ml of purified water, and then 0.5 ml of sodium hydroxide aqueous solution (0.48 mol / L) was added to prepare ligand solution B (6.79 mg / ml) by dissolving the sodium salt of 4,4',4'',4'''-methanetetrayltetrabenzoic acid, equivalent to 35.3 mg, in 5.2 ml of purified water. After preparing the mixed solution at 9.0°C, ligand solution B was slowly added dropwise at a rate of 4.6 ml / min, and the mixture was stirred for 30 minutes after the completion of the dropwise addition. After collection, the same procedure as in Example 1 was performed.

[0053] (Comparative Example 1) Ligand solution A was prepared by dissolving 40 mg of 4,4',4'',4'''-methanetetrayltetrabenzimidamide tetrahydrochloride in 4.4 ml of purified water, and then dissolving 2.0 mg of horseradish peroxidase (HRP) to prepare a mixed solution. 30 mg of 4,4',4'',4'''-methanetetrayltetrabenzoic acid was added to 13.2 ml of purified water, and then 0.5 ml of sodium hydroxide aqueous solution (0.48 mol / L) was added to prepare ligand solution B (2.58 mg / ml) by dissolving the sodium salt of 4,4',4'',4'''-methanetetrayltetrabenzoic acid, equivalent to 35.3 mg, in 13.7 ml of purified water. After preparing the mixed solution at 11.2°C, ligand solution B was slowly added dropwise at a rate of 14.6 ml / min, and the mixture was stirred for 30 minutes after the completion of the dropwise addition. After collection, the same procedure as in Example 1 was performed.

[0054] (Comparative Example 2) Ligand solution A was prepared by dissolving 40 mg of 4,4',4'',4'''-methanetetrayltetrabenzimidamide tetrahydrochloride in 8.9 ml of purified water, and then dissolving 7.3 mg of horseradish peroxidase (HRP) to prepare a mixed solution. 30 mg of 4,4',4'',4'''-methanetetrayltetrabenzoic acid was added to 12.9 ml of purified water, and then 0.5 ml of sodium hydroxide aqueous solution (0.48 mol / L) was added to prepare ligand solution B (2.63 mg / ml) by dissolving the sodium salt of 4,4',4'',4'''-methanetetrayltetrabenzoic acid, equivalent to 35.3 mg, in 13.4 ml of purified water. After preparing the mixed solution at 26.0°C, ligand solution B was slowly added dropwise at a rate of 39.9 ml / min, and the mixture was stirred for 30 minutes after the completion of the dropwise addition. After collection, the same procedure as in Example 1 was performed. (Comparative Example 3) Ligand solution A was prepared by dissolving 40 mg of 4,4',4'',4'''-methanetetrayltetrabenzimidamide tetrahydrochloride in 4.4 ml of purified water, and then dissolving 17.9 mg of horseradish peroxidase (HRP) to prepare a mixed solution. 30 mg of 4,4',4'',4'''-methanetetrayltetrabenzoic acid was added to 18.4 ml of purified water, and then 0.5 ml of sodium hydroxide aqueous solution (0.48 mol / L) was added to prepare ligand solution B (1.87 mg / ml) by dissolving the sodium salt of 4,4',4'',4'''-methanetetrayltetrabenzoic acid, equivalent to 35.3 mg, in 18.9 ml of purified water. After preparing the mixed solution at 32.5°C, ligand solution B was slowly added dropwise at a rate of 63.4 ml / min, and the mixture was stirred for 30 minutes after the completion of the dropwise addition. After collection, the same procedure as in Example 1 was performed.

[0055] (Comparative Example 4) Ligand solution A was prepared by dissolving 40 mg of 4,4',4'',4'''-methanetetrayltetrabenzimidamide tetrahydrochloride in 5.6 ml of purified water, and then dissolving 0.3 mg of horseradish peroxidase (HRP) to prepare a mixed solution. 30 mg of 4,4',4'',4'''-methanetetrayltetrabenzoic acid was added to 22.6 ml of purified water, and then 0.5 ml of sodium hydroxide aqueous solution (0.48 mol / L) was added to prepare ligand solution B (1.53 mg / ml) by dissolving the sodium salt of 4,4',4'',4'''-methanetetrayltetrabenzoic acid, equivalent to 35.3 mg, in 23.1 ml of purified water. After adjusting the mixed solution to 39.2°C, ligand solution B was slowly added dropwise at a rate of 2.4 ml / min, and the mixture was stirred for 30 minutes after the completion of the dropwise addition. After collection, the same procedure as in Example 1 was performed.

[0056] [Table 1]

[0057] In Examples 1-3, where the synthesis temperature of the immobilized enzyme was 10°C or lower, the value of the specific enzyme activity R was significantly higher than that of Comparative Examples 1-4. This is because, by synthesizing at a temperature of 10°C or lower, the enzyme is not completely contained within the crystalline structure of HOF, resulting in a more highly active immobilized enzyme. Furthermore, since the immobilized enzyme of the present invention is supported on HOF, which has excellent crystallinity, it is considered to have heat resistance. [Industrial applicability]

[0058] The present invention can be suitably used in immobilized enzymes used as labeling substances in fields such as analytical chemistry, immobilized enzymes used as catalysts in the synthesis of food additives and pharmaceuticals, and immobilized enzymes used in the treatment of industrial wastewater.

Claims

1. Enzymes and, An immobilized enzyme comprising a hydrogen-bonding organic structure, An immobilized enzyme characterized in that its relative enzyme activity R, represented by the following formula 1, is between 1.5 and 20. Equation 1 (X represents the enzyme activity value of the immobilized enzyme. Y represents the enzyme activity value of the immobilized enzyme synthesized under temperature conditions of 20°C to 30°C. The enzyme activity value means the amount of enzyme exhibiting enzyme activity divided by the total amount of supported enzyme.)

2. The immobilized enzyme according to claim 1, wherein the enzyme is an oxidoreductase.

3. The immobilized enzyme according to claim 1 or 2, wherein the enzyme is a peroxidase.

4. The immobilized enzyme according to claim 1 or 2, wherein the enzyme is horseradish peroxidase.

5. The immobilized enzyme according to claim 1 or 2, wherein the hydrogen-bonding organic structure comprises a ligand containing at least eight nitrogen atoms.

6. The hydrogen-bonding organic structure comprises at least two ligands, Of the aforementioned ligands, at least one ligand is an amidine group. The immobilized enzyme according to claim 1 or 2, wherein at least one of the ligands is a carbonyl group.

7. A method for producing immobilized enzymes, (a) A step of preparing at least two ligands by separately adding at least two ligands to the same solvent, (b) A step of adding an enzyme to at least one of the ligand solutions, (c) A step of obtaining an immobilized enzyme by contacting all of the ligand solutions, (d) A step of separating the immobilized enzyme from the solvent, Equipped with, A method for producing immobilized enzymes, characterized in that the step in (c) is carried out at a temperature of 10°C or lower.

8. The method for producing an immobilized enzyme according to claim 7, wherein the solvent in step (a) is water.

9. The method for producing an immobilized enzyme according to claim 7 or 8, wherein in step (a), the pH of the solvent is 4 or more and 10 or less.

10. In step (a) above, Of the aforementioned ligands, at least one ligand is an amidine group. The method for producing an immobilized enzyme according to claim 7 or 8, wherein at least one of the ligands is a carbonyl group.

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