Cross-linking method of proteins

Abstract

The present application has an object to provide a novel protein cross-linking method. By allowing both an oxidoreductase such as laccase and a protein deamidase such as protein glutaminase to act on a substrate protein, a cross-linking reaction is promoted.

Description

Technical Field

[0001] This invention relates to a novel cross-linking method for proteins using enzymes. More specifically, it relates to a cross-linking method for proteins using both oxidoreductases and protein deamidases. Background Technology

[0002] Enzymes that have the potential to polymerize proteins through cross-linking reactions have been known in the past, including transglutaminase, lysyl oxidase, protein disulfide isomerase, protein disulfide reductase, thiol oxidase, lipid oxygenase, polyphenol oxidase (tyrosinase), peroxidase, etc. (see, for example, Non-Patent Literature 1).

[0003] For transglutaminase, one of the enzymes mentioned above, the protein cross-linking method is well known. It is well known that transglutaminase derived from microorganisms is inexpensive and does not require calcium in the reaction, thus it is widely used, primarily in the food processing field (see Patent Document 1, Non-Patent Document 2).

[0004] However, protein cross-linking reactions based on transglutaminase have the following problems. Transglutaminase is an enzyme that forms cross-linked structures within or between protein molecules through an acyl transfer reaction between the γ-carboxyl group of a glutamine residue and the ε-amino group of a lysine residue. Therefore, depending on the protein type, some proteins may be difficult to use as substrates due to a lack of glutamine or lysine residues. For example, albumin-like proteins cannot serve as substrates for transglutaminase in their native state.

[0005] Thus, in the past, as an enzyme-based protein cross-linking method, the possibility of using a variety of enzymes was pointed out, but there were almost no practical methods that met the requirements of supply, cost, and ease of purification. Even in the only method that could be called based on transglutaminase derived from microorganisms, the cross-linking reaction could not occur depending on the type of protein, thus limiting its application.

[0006] In response, a cross-linking method based on proteins containing multi-copper oxidases such as laccase, bilirubin oxidase, ascorbic acid oxidase, and cellulase was proposed, which is completely different from the reaction mechanism of transglutaminase. This expands the range of proteins that can be targeted in the transglutaminase-based protein cross-linking method. In addition, it makes it possible to prepare protein gels with new physical properties and characteristics (see Patent Document 2).

[0007] Existing technical documents

[0008] Patent documents

[0009] Patent Document 1: Japanese Patent Publication No. 6-65280

[0010] Patent Document 2: Japanese Patent Application Publication No. 11-276162

[0011] Non-patent literature

[0012] Non-Patent Literature 1: Matheis and Whitaker, J. Food Biochemistry 11, 309-327, 1987

[0013] Non-patent literature 2: Journal of the Japanese Society for Agricultural Chemistry, Vol. 69, No. 10, pp. 1301-1308 Summary of the Invention

[0014] The technical problem that the invention aims to solve

[0015] However, in the protein cross-linking method based on polycopper oxidase as described above, there are problems such as the need for a large amount of enzyme and a long reaction time due to the low reactivity of polycopper oxidase to proteins.

[0016] Means for solving technical problems

[0017] In order to solve the above-mentioned technical problems, the inventors conducted in-depth research and discovered a new method for deamidating proteins, which significantly improved the cross-linking reaction of proteins based on oxidoreductases such as polycopper oxidases, thereby completing the present invention as shown below.

[0018] [1] A method for cross-linking proteins, characterized in that oxidoreductase and protein deamidase act on the protein.

[0019] [2] According to the protein cross-linking method described in [1], the oxidoreductase is a polycopper oxidase.

[0020] [3] According to the protein cross-linking method described in [2], wherein the polycopper oxidase is laccase and / or bilirubin oxidase.

[0021] [4] According to the protein cross-linking method described in [2], the copper oxidase is laccase.

[0022] [5] The protein crosslinking method according to any one of [1] to [4], wherein the protein deamidase is an enzyme that acts on glutamine residues in the protein.

[0023] [6] According to the protein cross-linking method described in [5], the protein deamidase is protein glutaminase.

[0024] [7] A protein modifier containing an oxidoreductase and a protein deamidase.

[0025] [8] The protein modifier according to [7], wherein the oxidoreductase is a polycopper oxidase.

[0026] [9] The protein modifier according to [8], wherein the polycopper oxidase is laccase and / or bilirubin oxidase.

[0027]

[10] According to the protein modifier described in [8], wherein the polycopper oxidase is laccase.

[0028]

[11] The protein modifier according to any one of [7] to

[10] , wherein the protein deamidase is an enzyme that acts on glutamine residues in the protein.

[0029]

[12] According to the protein modifier described in

[11] , wherein the protein deamidase is protein glutaminase.

[0030]

[13] A method for preparing a cross-linked protein, comprising the following steps:

[0031] (1) The process of treating proteins with protein deamidase; and

[0032] (2) The process of treating proteins that have undergone protein deamidation with oxidoreductase.

[0033]

[14] A method for preparing a cross-linked protein, comprising the following steps:

[0034] (1) The process of preparing proteins treated with protein deamidase; and

[0035] (2) The process of treating the prepared protein with oxidoreductase.

[0036]

[15] A method for preparing a cross-linked protein, comprising the step of simultaneously treating the protein with an oxidoreductase and a protein deamidase.

[0037]

[16] A method for preparing food or medicine, comprising the following steps:

[0038] (1) A process for preparing protein-containing food or pharmaceutical raw materials treated with protein deamidase; and

[0039] (2) The process of treating prepared food or pharmaceutical raw materials with oxidoreductase.

[0040]

[17] A method for preparing a food or pharmaceutical product, comprising the step of simultaneously treating a food or pharmaceutical raw material containing a protein with an oxidoreductase and a protein deamidase. Attached Figure Description

[0041] Figure 1This is a diagram showing the electrophoretic pattern of SDS-polyacrylamide gel electrophoresis in Experiment Example 1.

[0042] Figure 2 This is a diagram showing the electrophoretic pattern of SDS-polyacrylamide gel electrophoresis in Experiment Example 2.

[0043] Figure 3 This is a diagram showing the electrophoretic pattern of SDS-polyacrylamide gel electrophoresis in Experiment Example 3.

[0044] Figure 4 This is a graph showing the electrophoretic pattern of SDS-polyacrylamide gel electrophoresis in Experiment Example 5.

[0045] Figure 5 This is a diagram showing the electrophoretic pattern of SDS-polyacrylamide gel electrophoresis in Experiment Example 6.

[0046] Figure 6 This is a diagram showing the electrophoretic pattern of SDS-polyacrylamide gel electrophoresis in Experiment Example 7.

[0047] Figure 7 This is a graph showing the electrophoretic pattern of SDS-polyacrylamide gel electrophoresis in Experiment Example 8.

[0048] Figure 8 This is a graph representing the viscosity change in Experiment Example 9. Detailed Implementation

[0049] 1. Methods for cross-linking proteins

[0050] The cross-linking method of the present invention is characterized by the action of oxidoreductases and protein deamidases on the protein. The oxidoreductases referred to in this invention are enzymes that cross-link proteins through redox reactions, and are not particularly limited; examples include the following enzymes.

[0051] (1) Enzymes that crosslink proteins by oxidizing the ε-amino group of lysine in proteins to produce highly reactive aldehydes and forming Schiff bases with the amino groups of other protein molecules (e.g., lysyl oxidase).

[0052] (2) Enzymes that cross-link proteins by oxidizing the sulfhydryl groups of cysteine ​​in proteins and forming disulfide bonds with other protein molecules (e.g., thiol oxidases).

[0053] (3) Enzymes that crosslink proteins (e.g., tyrosinases) by oxidizing the hydroxyl groups of tyrosine in proteins to produce highly reactive orthoquinones and reacting with quinones, amino groups or thiol groups of other protein molecules.

[0054] (4) It has broad substrate specificity and mainly acts on the hydroxyl group of tyrosine, the sulfhydryl group of cysteine ​​and the ε-amino group of lysine in proteins, and crosslinks proteins through any of the mechanisms in (1) to (3) above (e.g., polycopper oxidases such as laccase).

[0055] (5) Although it catalyzes the same reaction as (4), the enzyme (e.g., peroxidase) requires hydrogen peroxide as an oxygen donor in the oxidation reaction.

[0056] Here, polycopper oxidases refer to a group of enzymes containing multiple copper atoms within their molecules that utilize molecular oxygen to oxidize polyphenols, methoxyphenols, diamines, bilirubin, ascorbic acid, etc. Currently, the known number of copper atoms is typically 2 to 8, but this number can vary depending on the state of the enzyme preparation and the analytical method used, and therefore is not strictly limited to this. Examples of enzymes classified as polycopper oxidases include laccase, bilirubin oxidase, ascorbic acid oxidase, and cellulase.

[0057] Laccase ([EC1.10.3.2]) is a type of copper protein that is an enzyme with low substrate specificity that acts on quinol, hydroquinone, or often aminophenol or phenylenediamine. The resulting semiquinone further undergoes enzymatic or non-enzymatic reactions. Examples of such laccases include those derived from plants such as lacquer or microorganisms such as bacteria or fungi. Examples of microbial laccases include those derived from the genera *Aspergillus*, *Neurospora*, *Podospora*, *Botrytis*, *Collybia*, *Fomes*, *Lentinus*, *Pleurotus*, *Pycnoporus*, *Pyricularia*, *Trametes*, *Rhizoctonia*, *Rigidoporus*, *Coprinus*, *Psatyrella*, *Myceliophtera*, *Schtalidium*, *Polyporus*, *Phlebia*, and *Coriolus*.

[0058] Bilirubin oxidase (EC1.3.3.5) is a type of copper protein that is an enzyme that primarily acts on bilirubin. Examples of such bilirubin oxidases include enzymes derived from the genera Penicillium, Myrothecium, and Trachyderma.

[0059] Ascorbic acid oxidase (EC1.10.3.3) is a type of copper protein that is an enzyme that mainly acts on L-ascorbic acid. It is derived from plants such as cucumber, pumpkin, and zucchini, as well as microorganisms such as bacteria or fungi.

[0060] Cellulase (EC1.16.3.1) is a type of copper protein that is a multifunctional protein that maintains copper homeostasis in organisms, and has oxidase activity and amine oxidase activity. It is found in the serum of animals and birds.

[0061] The protein deamidase referred to in this invention is an enzyme that catalyzes the reaction of free ammonia from proteins. Specifically, examples include: enzymes that convert glutamine residues in proteins to glutamate residues (i.e., protein glutaminase), enzymes that convert asparagine residues to aspartic acid residues (i.e., protein asparaginase), and protein deamidases that convert arginine residues in proteins to citrulline residues. Enzymes that deamidate glutamine residues in proteins are known, for example, protein glutaminases derived from Chryseobacterium proteolyticum (Eur J Biochem, 268(5), 1410, 2001, Protein-glutaminase From Chryseobacterium Proteolyticum, an Enzyme That Deamidates Glutaminyl Residuesin Proteins. Purification, Characterization and Gene Cloning, SYamaguchi1, DJ Jeenes, DB Archer or Front Microbiol, 9, 1975, 2018, Complete Genome Sequence and Characterization of a Protein-Glutaminase Producing Strain, Chryseobacterium proteolyticum QSH1265, RuidanQu, XiaoyuZhu, MinTian, ​​YingjieLiu, WenjuanYan, JianYe, HongliangGao, JingHuang), but are not limited thereto. Examples of enzymes that deamidate asparagine residues in proteins include, for example, protein asparaginases disclosed in WO2015 / 133590, but are not limited thereto. Examples of enzymes that deamidate arginine residues in proteins include, for example, arginine deiminoases derived from Fusarium graminearum.

[0062] Generally, deamidation of glutamine and / or asparagine residues in proteins to generate carboxyl groups increases the protein's negative charge, and / or neutralizes the protein by deamidation of strongly basic arginine residues. This results in a decrease in the isoelectric point and an increase in hydration. Furthermore, the increased electrostatic repulsion leads to reduced interactions between proteins, i.e., decreased association. These changes significantly increase the protein's solubility and water dispersibility. Additionally, the increased negative charge causes the protein to unfold, altering its higher-order structure and exposing hydrophobic regions buried within the molecule to the molecular surface. Therefore, deamidated proteins are amphiphilic and become ideal surfactants, significantly improving their emulsifying power, emulsion stability, foaming ability, and foam stability. Thus, deamidation of proteins improves various functional properties, significantly increasing their applicability (e.g., Molecular Approaches to Improving Food Quality and Safety, D. Chatnagar and TEClevel and, eds., Van Nostr and Reinhold, New York, 1992, p. 37). Furthermore, deamidation of arginine residues in proteins also increases their hydrophobicity and alters their higher-order structure.

[0063] Therefore, not adhering to theory, the inventors have newly discovered "the phenomenon of promoting cross-linking reactions of redox enzyme-based proteins through the action of protein deamidases" (see the examples described later). Due to the action of protein deamidases, proteins unfold and their higher-order structures change. This result demonstrates that by exposing amino acid residues such as cysteine ​​and lysine, which are targets of redox enzymes and are embedded inside the protein molecule, to the surface of the protein molecule, it becomes more susceptible to the action of redox enzymes.

[0064] Next, the protein cross-linking method of the present invention will be described in more detail. There are no particular limitations on the types or sources of oxidoreductases and protein deamidases that can be used in the present invention. Regarding the source, it can be derived from animals, plants, or microorganisms. Furthermore, in the case of enzymes derived from microorganisms, the enzyme can be an enzyme accumulated either inside or outside the cell of the microorganism. Furthermore, not only naturally occurring enzymes can be used, but also enzymes produced through genetic engineering or cell engineering methods. Additionally, enzyme proteins modified by protein engineering methods can also be used. Furthermore, while it is preferable to use purified, high-purity oxidoreductases (e.g., polycopper oxidases) and protein deamidases (e.g., protein glutaminases), their purity is not limited as long as the desired reaction can be carried out. Alternatively, enzyme preparations can be used as oxidoreductases and protein deamidases; in this case, various salts, sugars, proteins, lipids, surfactants, etc., can be added to the enzyme preparation as enzyme stabilizers.

[0065] The cross-linking method of this invention is applicable to a variety of proteins for which cross-linking is desired. There are no particular limitations on the origin or properties of the proteins that serve as substrates for oxidoreductases and protein deamidases. For example, if the protein is plant-based, examples include proteins derived from legumes such as soybeans, greenpeas, lentils, chickpeas, and black beans; proteins derived from cereals such as wheat, barley, oats, and rice; proteins derived from nuts such as almonds and peanuts; and proteins derived from seeds such as hemp seeds, chia seeds, quinoa, and amaranth. Additionally, proteins derived from insects such as crickets, yeast, filamentous fungi, fungi known as mushrooms, and algae such as spirulina can also be used. If the protein is animal protein, examples include: casein, β-lactoglobulin and other milk proteins; ovalbumin and other ovalbumin; myosin, actin and other meat proteins; serum albumin and other blood proteins; gelatin, collagen and other tendon proteins. Additionally, substrate proteins can be obtained by using chemicals based on acids or bases to partially break down proteins, proteins partially hydrolyzed by proteases, proteins chemically modified with various reagents, or synthetic peptides.

[0066] While the substrate proteins described above are provided for reaction in a fluid composition such as a solution, slurry, or paste, the concentration of the substrate protein in the fluid composition is not particularly limited. The concentration is determined based on the desired properties and state of the target protein crosslink. Generally, a low concentration yields a solution or precipitate with increased viscosity, while a high concentration yields a gel. However, a concentration of 1% by weight or more of the substrate protein allows for adequate gelation. Furthermore, the fluid composition containing the substrate protein is not limited to aqueous solutions, aqueous dispersions, or aqueous pastes of the protein; fluid compositions in the form of emulsions of these proteins and oils can also be provided for reaction. Additionally, salts, sugars, proteins, fragrances, humectants, colorants, etc., can be added to the fluid composition containing the substrate protein as needed.

[0067] The amount of enzyme used, reaction time, temperature, and pH of the reaction solution are not particularly limited. Generally, the enzyme amount relative to 1g of protein is 1–1,000,000 U for oxidoreductases, preferably 10–500,000 U, more preferably 100–200,000 U; and 0.01–100,000 U for protein deamidases, preferably 0.1–50,000 U, more preferably 1–10,000 U. The reaction temperature is 5–80°C, preferably 20–60°C. The pH of the reaction solution is 2–10, preferably 4–8. The reaction time is 10 seconds–48 hours, preferably 10 minutes–24 hours. Under these reaction conditions, a gel-like substance of a protein polymerized cross-linked product or a flowable composition can be obtained. These reaction conditions can be appropriately selected based on the physical properties and moisture content of the target protein cross-linked product or flowable composition gel-like substance. Furthermore, the optimal reaction conditions can be determined through preliminary experiments.

[0068] When using copper oxidases as redox enzymes, various polyphenols can be added as mediators to promote the reaction, such as hydroquinone, catechol, guaiacol, ferulic acid, vanillic acid, p-coumaric acid, syringaldehyde, and p-phenylenediamine.

[0069] In the protein cross-linking method of the present invention, the substrate protein is treated with protein cross-linking enzymes, namely oxidoreductases and protein deamidases. The order of enzyme action (i.e., the order of oxidoreductase treatment and protein deamidase treatment) is not particularly limited, but simultaneous treatment with both enzymes is preferred, or treatment with protein deamidase followed by oxidoreductase treatment. For the purpose of improving work efficiency, simultaneous treatment is more preferred. In the case of treatment with protein deamidase followed by oxidoreductase treatment, a step of inactivating the protein deamidase can be added after the protein deamidase treatment. By adding the inactivation step, the amount of protein deamidation can be adjusted.

[0070] 2. Methods for preparing cross-linked proteins, or food or pharmaceuticals containing cross-linked proteins.

[0071] By using the cross-linking method of the present invention, cross-linked proteins or food or pharmaceutical products containing them can be prepared. One method for preparing cross-linked proteins includes the following steps (1) and (2). Alternatively, a protein deamidase inactivation step can be added after step (1).

[0072] (1) The process of treating proteins with protein deamidase

[0073] (2) The process of treating proteins that have undergone protein deamidation with oxidoreductase.

[0074] In another approach, the following steps (1) and (2) are performed.

[0075] (1) The process of preparing proteins that have been treated with protein deamidase.

[0076] (2) The process of treating the prepared protein with oxidoreductase.

[0077] In another approach, the cross-linked protein is prepared by the following step (i).

[0078] (i) A process of simultaneously treating proteins with oxidoreductases and protein deamidases.

[0079] On the other hand, one method of preparing food or medicine includes the following (1) and (2).

[0080] (1) The process of preparing food or pharmaceutical raw materials containing protein that have been treated with protein deamidase.

[0081] (2) Process of treating prepared food or pharmaceutical raw materials with oxidoreductase.

[0082] In another manner, food or medicine is prepared through the following process (i).

[0083] (i) A process of simultaneously treating food or pharmaceutical ingredients containing protein with oxidoreductases and protein deamidases.

[0084] 3. Protein modifiers

[0085] This invention also provides a protein modifier capable of being used for the cross-linking of proteins. The protein modifier of this invention is typically used in the cross-linking method or preparation method of this invention. The protein modifier of this invention contains, as an active ingredient, an oxidoreductase and a protein deamidase, which are enzymes used for protein cross-linking. The protein modifier of this invention can be used as a protein cross-linking agent, preferably as a thickener in a flowable composition containing proteins, and more preferably as a gelling agent in a flowable composition containing proteins. Details of the oxidoreductase and protein deamidase are as described above (section 1. Methods for Cross-linking Proteins), therefore their description is omitted.

[0086] The present invention will be further illustrated below with reference to examples.

[0087] Example

[0088] In the following examples, unless otherwise specified, the enzyme activity assay of laccase was performed using 2,2'-adiazono-bis-[3-ethylbenzothiazoline sulfonate (6)] (ABTS, manufactured by Boehringer Ingelheim) as the substrate, and according to the method described below.

[0089] <Activity Assay>

[0090] ABTS was dissolved at a concentration of 1.0 mg / mL in 25 mM citrate buffer (pH 3.2) to prepare the substrate solution. 3.0 mL of this substrate solution was placed in a test tube, preheated at 25°C, and then 0.1 mL of enzyme solution was added. The mixture was stirred and incubated at 25°C. The absorbance at 405 nm was measured after 1 minute and 3 minutes. One unit of enzyme was defined as the amount that increased the absorbance at 405 nm by 1.0 OD within 1 minute under these conditions.

[0091] On the other hand, unless otherwise specified, the enzyme activity assay of protein glutaminase shall be performed using Z-Gln-Gly as the substrate, and in accordance with the method described below.

[0092] <Activity Assay>

[0093] Add 10 μL of enzyme solution to 100 μL of 176 mmol / L phosphate buffer (pH 6.5) containing 10 mmol / L Z-Gln-Gly, incubate at 37 °C for 60 min, then add 100 μL of 12% trichloroacetic acid solution to stop the reaction. After centrifugation (15000 rpm, 4 °C, 5 min), analyze the supernatant using F-kitammonia (manufactured by Boehringer Ingelheim) as described below (A1). Alternatively, use water instead of enzyme solution and perform the assay in the same manner (A2). Add 10 μL of supernatant and 190 μL of water to 100 μL of F-kitammonia reagent 2, incubate at room temperature for 5 min, and then measure the absorbance at 340 nm using 100 μL (E1). Add 1.0 μL of reagent 3 (glutamate dehydrogenase) to the remaining 200 μL, and then let it stand at room temperature for 20 minutes. Measure the absorbance (E2) of the remaining 200 μL at 340 nm. Calculate the enzyme concentration by taking the amount of enzyme that releases 1 μmol of ammonia per minute under the above conditions as one unit, using the following formula.

[0094] u / mL=1.76×[A1(E1-E2)-A2(E1-E2)]

[0095] <Experimental Example 1>

[0096] This study investigated the promoting effect of laccase (LC) and protein glutaminase (PG) on protein cross-linking using egg-derived albumin (manufactured by Fujifilm and Kojun Pharmaceutical Co., Ltd.). 5% (final) w / w of egg-derived albumin, 50 mM (final) potassium sodium phosphate buffer (pH 7.0), laccase (product name: laccase Y120, manufactured by Amano Enzyme Co., Ltd.), and protein glutaminase (product name: protein glutaminase "Amano" 500, manufactured by Amano Enzyme Co., Ltd.) were mixed and reacted at 40°C with shaking at 160 rpm for 24 hours. After the reaction, a portion of the reaction solution was aliquoted and subjected to 2–25% polyacrylamide electrophoresis. The increase in molecular weight of the substrate protein was observed to determine the cross-linking and the cross-linking promoting effect compared to the use of laccase alone. The enzyme addition amount is 100U of laccase (final concentration) and 500mU of protein glutaminase per 1mg of substrate protein.

[0097] The results are shown in Figure 1 See Table 1.

[0098] [Table 1]

[0099]

[0100] like Figure 1As shown, no cross-linking and polymerization of the substrate protein occurred when laccase was used alone. In contrast, when laccase and protein glutaminase were used together, bands of protein polymerized to the point that they could not pass through the mesh of the polyacrylamide gel were observed at the top of lane 3, confirming the cross-linking and polymerization of the substrate protein. That is, by using protein glutaminase in conjunction with laccase, the promoting effect of cross-linking of albumin proteins derived from eggs was confirmed.

[0101] <Experimental Example 2>

[0102] The promoting effect of laccase and protein glutaminase on protein cross-linking was investigated using the LYZAMINE-S method (pea protein, manufactured by ROCKET JAPAN). 5% (final) wt% LYZAMINE-S, 50 mM (final) potassium sodium phosphate buffer (pH 7.0), laccase (product name: laccase Y120, manufactured by Amano Enzyme Co., Ltd.), and protein glutaminase (product name: protein glutaminase "Amano" 500, manufactured by Amano Enzyme Co., Ltd.) were mixed and reacted at 40°C with shaking at 160 rpm for 24 hours. After the reaction, a portion of the reaction solution was aliquoted and subjected to 2–25% polyacrylamide gel electrophoresis. The increase in molecular weight of the substrate protein was observed to determine the cross-linking and the cross-linking promoting effect compared to laccase alone. The enzyme addition was 100 U of laccase (final concentration) and 500 mU of protein glutaminase (final concentration) per 1 mg of substrate protein.

[0103] The results are shown in Figure 2 And Table 2.

[0104] [Table 2]

[0105]

[0106] like Figure 2 As shown, no cross-linking and polymerization of the substrate protein occurred when laccase was used alone. In contrast, when laccase and protein glutaminase were used together, a denser band of protein polymerized to the point of being unable to pass through the mesh of the polyacrylamide gel (the band that could be observed at the top of lane 3) was confirmed, indicating cross-linking and polymerization of the substrate protein. That is, by using protein glutaminase in conjunction with laccase, a promoting effect on the cross-linking of pea protein was confirmed.

[0107] <Experimental Example 3>

[0108] The promoting effect of laccase and protein glutaminase on protein cross-linking was studied using soybean-derived protein powder (manufactured by Fujifilm and Kojun Pharmaceutical Co., Ltd.). 5% by weight (final concentration) of soybean-derived protein powder, 50 mM (final concentration) potassium sodium phosphate buffer (pH 7.0), laccase (product name: laccase Y120, manufactured by Amano Enzyme Co., Ltd.), and protein glutaminase (product name: protein glutaminase "Amano" 500, manufactured by Amano Enzyme Co., Ltd.) were mixed and reacted at 40°C with shaking at 160 rpm for 24 hours. After the reaction, a portion of the reaction solution was aliquoted and subjected to 2–25% polyacrylamide electrophoresis. The increase in molecular weight of the substrate protein was observed to determine the cross-linking and the cross-linking promoting effect compared to the use of laccase alone at the same concentration. The enzyme addition was 100 U of laccase (final concentration) and 500 mU of protein glutaminase (final concentration) per 1 mg of substrate protein. In addition, the same reaction was carried out under the condition that the amount of enzyme added (laccase and protein glutaminase) was reduced to 1 / 10 and 1 / 100, respectively.

[0109] The results are shown in Figure 3 And Table 3.

[0110] [Table 3]

[0111]

[0112] like Figure 3 As shown, compared with the use of laccase alone, the combined use of laccase and protein glutaminase improved the efficiency of cross-linking and polymerization of substrate proteins. Regardless of the enzyme concentration, it was confirmed that the protein bands polymerized to the point of being unable to pass through the mesh of the polyacrylamide gel (bands that could be identified at the top of each lane (lanes 5-7)) were more concentrated, confirming cross-linking and polymerization. That is, by combining laccase with protein glutaminase, the cross-linking-promoting effect of soybean-derived proteins was confirmed.

[0113] <Experimental Example 4>

[0114] The promoting effect of protein cross-linking based on the combined use of laccase and protein glutaminase was studied using egg-derived albumin (manufactured by Fujifilm and Kojun Pharmaceutical Co., Ltd.). 5% (final) w / w of egg-derived albumin, 50 mM (final) potassium sodium phosphate buffer (pH 7.0), and protein glutaminase (product name: Amano 500, manufactured by Amano Enzyme Co., Ltd.) were mixed and shaken at 160 rpm for 24 hours at 40°C. Then, laccase (product name: Laccase Y120, manufactured by Amano Enzyme Co., Ltd.) was added, and the reaction was allowed to proceed at 160 rpm for 24 hours at 40°C. After the reaction, a portion of the reaction solution was aliquoted and subjected to 2–25% polyacrylamide electrophoresis. The increase in molecular weight of the substrate protein was observed to determine the cross-linking and the cross-linking promoting effect compared to the use of laccase alone. The enzyme addition amount is 100U of laccase (final concentration) and 500mU of protein glutaminase per 1mg of substrate protein.

[0115] The results are shown in Table 4.

[0116] [Table 4]

[0117] swimming lane Add enzymes Crosslinking 1 No enzymes added none 2 PG→LC have

[0118] Furthermore, in the electrophoresis images, even after laccase treatment following protein glutaminase treatment, bands of proteins polymerized to the point of being unable to pass through the mesh of the polyacrylamide gel could be observed at the top of lane 2, confirming the cross-linking and polymerization of the substrate proteins. A comparison between treatment with laccase alone (Example 1) and treatment with laccase following protein glutaminase treatment in this example demonstrates that by combining protein glutaminase with laccase, regardless of the order of enzyme addition, a promoting effect on the cross-linking of albumin proteins from eggs was confirmed.

[0119] <Experimental Example 5>

[0120] This study investigated the promoting effect of laccase and protein glutaminase on protein cross-linking using almond-derived protein powder. 5% (final concentration) of almond-derived protein powder, 50 mM (final concentration) potassium sodium phosphate buffer (pH 7.0), laccase (product name: laccase Y120, manufactured by Amano Enzyme Co., Ltd.), and protein glutaminase (product name: protein glutaminase "Amano" 500, manufactured by Amano Enzyme Co., Ltd.) were mixed and reacted at 40°C with shaking at 160 rpm for 24 hours. After the reaction, a portion of the reaction solution was aliquoted and subjected to 2–25% polyacrylamide gel electrophoresis. The increase in molecular weight of the substrate protein was observed to determine cross-linking and the cross-linking promoting effect compared to laccase alone. The enzyme addition was 100 U of laccase (final concentration) and 500 mU of protein glutaminase (final concentration) per 1 mg of substrate protein. In addition, the same reaction was carried out under the condition of co-dilution of enzyme addition (laccase and protein glutaminase).

[0121] The results are shown in Figure 4 See Table 5.

[0122] [Table 5]

[0123]

[0124]

[0125] like Figure 4 As shown, compared with the use of laccase alone, the combined use of laccase and protein glutaminase improved the efficiency of cross-linking and polymerization of substrate proteins. Regardless of the enzyme concentration, the presence of proteins polymerized to approximately 200 kDa or higher was newly confirmed, confirming cross-linking and polymerization (lanes 5-7). In other words, by combining laccase with protein glutaminase, the cross-linking-promoting effect of almond-derived proteins was confirmed.

[0126] <Experimental Example 6>

[0127] This study investigated the promoting effect of laccase and protein glutaminase on protein cross-linking using chickpea protein powder. 5% (final concentration) of chickpea protein powder, 50 mM (final concentration) potassium sodium phosphate buffer (pH 7.0), laccase (product name: laccase Y120, manufactured by Amano Enzyme Co., Ltd.), and protein glutaminase (product name: protein glutaminase "Amano" 500, manufactured by Amano Enzyme Co., Ltd.) were mixed and reacted at 40°C with shaking at 160 rpm for 24 hours. After the reaction, a portion of the reaction solution was aliquoted and subjected to 2–25% polyacrylamide gel electrophoresis. The increase in molecular weight of the substrate protein was observed to determine cross-linking and the cross-linking promoting effect compared to laccase alone. The enzyme addition was 100 U of laccase (final concentration) and 500 mU of protein glutaminase (final concentration) per 1 mg of substrate protein. In addition, the same reaction was carried out under the condition of co-dilution of enzyme addition (laccase and protein glutaminase).

[0128] The results are shown in Figure 5 See Table 6.

[0129] [Table 6]

[0130]

[0131] like Figure 5 As shown, compared with the use of laccase alone, the combined use of laccase and protein glutaminase improved the efficiency of cross-linking and polymerization of substrate proteins. Regardless of the enzyme concentration, it was confirmed that the protein bands polymerized to the point of being unable to pass through the mesh of the polyacrylamide gel (bands that could be identified at the top of each lane (lanes 5-7)) were more concentrated, confirming cross-linking and polymerization. That is, by combining laccase with protein glutaminase, the promoting effect of cross-linking of chickpea-derived proteins was confirmed.

[0132] <Experimental Example 7>

[0133] The promoting effect of bilirubin oxidase (BO) and protein glutaminase on protein cross-linking was investigated using the Merck Millipore method. 5% (final) protein, 50 mM (final) potassium sodium phosphate buffer (pH 7.0), bilirubin oxidase (product name: BO "Amano" 3, manufactured by Amano Enzyme Co., Ltd.), and protein glutaminase (product name: protein glutaminase "Amano" 500, manufactured by Amano Enzyme Co., Ltd.) were mixed and reacted at 40°C with shaking at 160 rpm for 24 hours. After the reaction, a portion of the reaction solution was aliquoted and subjected to 2–25% polyacrylamide gel electrophoresis. The increase in molecular weight of the substrate protein was observed to determine the cross-linking and the cross-linking promoting effect compared to the same concentration of bilirubin oxidase used alone. The enzyme addition was 100 U of bilirubin oxidase (final concentration) and 500 mU of protein glutaminase (final concentration) per 1 mg of substrate protein. In addition, the same reaction was carried out under the condition of co-diluting enzyme addition (bilirubin oxidase and protein glutaminase).

[0134] The results are shown in Figure 6 See Table 7.

[0135] [Table 7]

[0136]

[0137] like Figure 6 As shown, compared with the use of bilirubin oxidase alone, the combined use of bilirubin oxidase and protein glutaminase improved the efficiency of cross-linking and polymerization of substrate proteins. Regardless of the enzyme concentration, it was confirmed that the polymerized protein bands were denser in the upper part of each lane (lanes 5-7), confirming cross-linking and polymerization. In other words, the promoting effect of combining bilirubin oxidase with protein glutaminase was confirmed.

[0138] <Experimental Example 8>

[0139] The promoting effect of the combined use of tyrosinase (TyrA) and protein glutaminase on protein cross-linking was investigated using the Merck Millipore method. 5% (final) protein, 50 mM (final) potassium sodium phosphate buffer (pH 7.0), tyrosinase (TyrA) (manufactured by Merck, product name: tyrosinase from mushroom), and protein glutaminase (manufactured by Amano Enzyme Co., Ltd.) were mixed and reacted at 40°C with shaking at 160 rpm for 24 hours. After the reaction, a portion of the reaction solution was aliquoted and subjected to 2–25% polyacrylamide gel electrophoresis. The increase in molecular weight of the substrate protein was observed to determine the cross-linking and the cross-linking promoting effect compared to the use of tyrosinase alone at the same concentration. The enzyme addition was 100 U of tyrosinase (final concentration) and 500 mU of protein glutaminase (final concentration) per 1 mg of substrate protein. In addition, the same reaction was carried out under the condition of co-dilution of enzyme addition (tyrosinase and protein glutaminase).

[0140] The results are shown in Figure 7 See Table 8.

[0141] [Table 8]

[0142]

[0143] like Figure 7 As shown, compared with the use of tyrosinase alone, the combined use of tyrosinase and protein glutaminase improved the efficiency of substrate protein cross-linking and polymerization. Regardless of the enzyme concentration, it was confirmed that the polymerized protein bands were denser in the upper part of each lane (lanes 5-6), and / or new polymerized protein bands were newly confirmed in the uppermost part of each lane (lanes 6-7), confirming cross-linking and polymerization. That is, by combining tyrosinase with protein glutaminase, the promoting effect of casein protein cross-linking was confirmed.

[0144] <Experimental Example 9>

[0145] (Confirmation of the viscosity-increasing effect of casein solution and wheat gluten solution)

[0146] A mixture comprising 5% (w / v) casein solution or wheat gluten solution, 100 mU / mL protein glutaminase (product name: Protein Glutaminase "Amano" 500, manufactured by Amano Enzyme Co., Ltd.), 50 U / mL laccase (product name: Laccase Y120, manufactured by Amano Enzyme Co., Ltd.), and 100 mM phosphate buffer (pH 7.0) was treated at 40°C, and the viscosity at various time points was measured using an EMS-1000 (Kyoto Electronics Manufacturing Co., Ltd., Tokyo, Japan). The shear rate during measurement was 200 s⁻¹, and a 30-second pre-measurement was performed to maintain the flow state. Casein solution without added enzyme, casein solution treated with laccase (50 U / mL), and casein solution treated with protein glutaminase (100 mU / mL) were also measured as controls.

[0147] The results are shown in Figure 8 The viscosity of casein (or gluten) solutions without added enzymes, casein (or gluten) solutions with laccase alone, and casein (or gluten) solutions with protein glutaminase alone did not change during the assay. On the other hand, in casein solutions using both laccase and protein glutaminase, the concentration increased with reaction time.

[0148] Furthermore, in the electrophoresis images, the efficiency of cross-linking and polymerization of substrate proteins was improved when laccase and protein glutaminase were used in combination compared to when laccase was used alone. Regardless of the enzyme concentration, the presence of proteins polymerized to approximately 200 kDa or higher was newly confirmed, confirming cross-linking and polymerization.

[0149] That is, by using laccase in combination with protein glutaminase, the promoting effect of cross-linking between casein protein and wheat gluten protein was confirmed.

[0150] (Gelization study of casein solution)

[0151] <Experimental Example 10>

[0152] A mixture comprising 5% (w / v) casein solution, 100 mU / mL protein glutaminase (product name: Protein Glutaminase "Amano" 500, manufactured by Amano Enzyme Co., Ltd.), 50 U / mL laccase (product name: Laccase Y120, manufactured by Amano Enzyme Co., Ltd.), and 100 mM phosphate buffer (pH 7.0) was added to a test tube. After treatment at 40°C for 24 hours, the test tube was tilted to confirm the presence or absence of gelation and the cross-linking promotion effect compared to when laccase was used alone.

[0153] The results are shown in Table 9.

[0154] [Table 9]

[0155]

[0156] As shown in Table 9, the properties of casein solutions without added enzyme, casein solutions with laccase alone, and casein solutions with protein glutaminase alone remained unchanged. On the other hand, the casein solution using only laccase and protein glutaminase gelled. That is, by using protein glutaminase in conjunction with laccase, the promoting effect of casein cross-linking was confirmed.

[0157] (Study on the gelation of milk)

[0158] <Experimental Example 11>

[0159] A mixture containing commercially available milk, 100 mU / mL protein glutaminase (product name: Protein Glutaminase "Amano" 500, manufactured by Amano Enzyme Co., Ltd.), 50 U / mL laccase (product name: Laccase Y120, manufactured by Amano Enzyme Co., Ltd.), and 100 mM phosphate buffer (pH 7.0) was added to a test tube. After treatment at 40°C for 24 hours, the test tube was tilted to confirm the presence or absence of gelation and the cross-linking promotion effect compared to when laccase was used alone.

[0160] The results are shown in Table 10.

[0161] [Table 10]

[0162]

[0163] As shown in Table 10, the properties of milk without added enzymes, milk with added laccase alone, and milk with added protein glutaminase alone remained unchanged. On the other hand, milk using only laccase and protein glutaminase gelled. That is, by using protein glutaminase in conjunction with laccase, the promoting effect of cross-linking of milk proteins was confirmed.

[0164] (Gelation study of wheat gluten)

[0165] <Experimental Example 12>

[0166] A mixture comprising 5% (w / v) wheat gluten solution, 100 mU / mL protein glutaminase (product name: Protein Glutaminase "Amano" 500, manufactured by Amano Enzyme Co., Ltd.), 50 U / mL laccase (product name: Laccase Y120, manufactured by Amano Enzyme Co., Ltd.), and 100 mM phosphate buffer (pH 7.0) was added to a test tube. After treatment at 40°C for 24 hours, the test tube was tilted to confirm the presence or absence of gelation and the cross-linking promotion effect compared to when laccase was used alone.

[0167] The results are shown in Table 11.

[0168] [Table 11]

[0169]

[0170] As shown in Table 11, the properties of wheat gluten solutions without added enzymes, wheat gluten solutions with laccase alone, and wheat gluten solutions with protein glutaminase alone remained unchanged. On the other hand, the wheat gluten solution using only laccase and protein glutaminase gelled. That is, by using protein glutaminase in conjunction with laccase, the promoting effect of protein cross-linking in wheat gluten solutions was confirmed.

[0171] Industrial availability

[0172] The enzyme-based protein cross-linking method of the present invention can significantly promote the cross-linking reaction of redox enzyme-based proteins, which has been difficult to apply to practical applications to date due to its low reactivity. The cross-linked, polymerized, or gelled protein materials manufactured according to the present invention can be applied in the processing of foods such as fish paste, fish cakes, fish / meat sausages, tofu, pasta, pasta / bread, food binders, meat flakes, yogurt, jelly, cheese, plant-based meat substitutes, and dairy substitutes (cheese substitutes, fermented dairy substitutes). Furthermore, as a novel protein-derived material, it is anticipated to have applications in a wide range of industries, including cosmetics, medical products, microcapsule materials, and carriers for immobilized enzymes. Since the reaction mechanism of redox enzyme-based cross-linking is considered different from that of transglutaminase, which is frequently used for protein cross-linking, the use and application of the present invention in the manufacture of protein polymers or gels with novel qualities are also expected.

[0173] This invention is not limited to the description of the embodiments and examples described above. This invention also includes various modifications that are readily apparent to those skilled in the art without departing from the scope of the claims. All contents of papers, published patent gazettes, and patent gazettes explicitly stated in this specification are incorporated herein by reference.

Claims

1. A protein cross-linking method, characterized in that, This allows copper oxidase and protein glutaminase to act on proteins. Specifically, the dosage of polycopper oxidase is 100–200,000 U relative to 1 g of protein, and the dosage of protein glutaminase is 1–10,000 U. The copper oxidase is laccase and / or bilirubin oxidase.

2. The protein cross-linking method according to claim 1, wherein, The copper oxidase is laccase.

3. A method for preparing a cross-linked protein, comprising the following steps: (1) The process of deamidating proteins using protein glutaminase; and (2) The process of treating proteins that have undergone protein deamidation with polycopper oxidase. in, The dosage of polycopper oxidase relative to 1g of protein is 100–200,000 U, and the dosage of protein glutaminase is 1–10,000 U. The copper oxidase is laccase and / or bilirubin oxidase.

4. A method for preparing a cross-linked protein, comprising the following steps: (1) The process of preparing proteins that have undergone deamidation treatment with protein glutaminase; and (2) The process of treating the prepared protein with polycopper oxidase. in, The dosage of polycopper oxidase relative to 1g of protein is 100–200,000 U, and the dosage of protein glutaminase is 1–10,000 U. The copper oxidase is laccase and / or bilirubin oxidase.

5. A method for preparing a cross-linked protein, comprising the step of simultaneously treating the protein with a polycopper oxidase and a protein glutaminase. in, The dosage of polycopper oxidase relative to 1g of protein is 100–200,000 U, and the dosage of protein glutaminase is 1–10,000 U. The copper oxidase is laccase and / or bilirubin oxidase.

6. A method for preparing a food or pharmaceutical product, comprising the following steps: (1) The process of preparing food or pharmaceutical raw materials containing protein that have been treated with protein glutaminase; and (2) The process of treating prepared food or pharmaceutical raw materials with copper oxidase. in, The dosage of polycopper oxidase relative to 1g of protein is 100–200,000 U, and the dosage of protein glutaminase is 1–10,000 U. The copper oxidase is laccase and / or bilirubin oxidase.

7. A method for preparing a food or pharmaceutical product, comprising the step of simultaneously treating a protein-containing food or pharmaceutical raw material with a polycopper oxidase and a protein glutaminase. in, The dosage of polycopper oxidase relative to 1g of protein is 100–200,000 U, and the dosage of protein glutaminase is 1–10,000 U. The copper oxidase is laccase and / or bilirubin oxidase.

Images