Protein cross-linking method

Abstract

The present invention addresses the problem of providing a novel protein crosslinking method. In the present invention, a crosslinking reaction is accelerated by causing both an oxidoreductase such as a laccase and a protein deamidase such as a protein glutaminase to act on a substrate protein.

Description

[Technical Field]

[0001] The present invention relates to a novel method for crosslinking proteins using an enzyme, and more particularly to a method for crosslinking proteins using a combination of an oxidoreductase and a protein deamidase. [Background technology]

[0002] Enzymes that have the potential to polymerize proteins through cross-linking reactions include transglutaminase, lysyl oxidase, protein disulfide isomerase, protein disulfide reductase, sulfhydryl oxidase, lipoxygenase, polyphenol oxidase (tyrosinase), and peroxidase (see, for example, Non-Patent Document 1).

[0003] Among the enzymes mentioned above, transglutaminase is well known for its use in protein cross-linking. As is well known, the discovery of inexpensive microbial transglutaminase that does not require the presence of calcium for reaction has led to its widespread use, particularly in the food processing industry (see Patent Document 1 and Non-Patent Document 2).

[0004] However, transglutaminase-mediated protein cross-linking reactions have the following problems: Transglutaminase is an enzyme that forms cross-links within or between protein molecules as a result of an acyl transfer reaction between the γ-carboxyl group of glutamine residues and the ε-amino group of lysine residues in proteins, so some proteins are difficult to use as substrates due to a lack of glutamine or lysine residues. For example, albumin proteins cannot serve as substrates for transglutaminase in their native state.

[0005] As described above, the possibility of using several enzymes for enzymatic protein cross-linking has been pointed out in the past, but there are almost no practical methods that satisfy the requirements in terms of supply amount, cost, ease of purification, etc., and even the only method that uses transglutaminase derived from microorganisms has limited applications because the cross-linking reaction does not occur depending on the type of protein.

[0006] In response to this, a method for cross-linking proteins using multicopper oxidases, which include laccase, bilirubin oxidase, ascorbic acid oxidase, and ceruloplasmin, which have a completely different reaction mechanism from transglutaminase, has been proposed. This method expands the range of proteins that can be cross-linked using transglutaminase, which was previously limited, and also makes it possible to produce protein gels with new physical properties and characteristics (see Patent Document 2). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Special Publication No. 6-65280 [Patent Document 2] Japanese Patent Application Publication No. 11-276162 [Non-patent literature]

[0008] [Non-Patent Document 1] MatheisandWhitaker,J.FoodBiochemistry11,309-327,1987 [Non-patent document 2] Japanese Society of Agricultural Chemistry, Vol. 69, No. 10, pp. 1301-1308 Summary of the Invention [Problem to be solved by the invention]

[0009] However, the above-mentioned protein crosslinking method using multicopper oxidase has problems such as the need for a large amount of enzyme and a long reaction time due to the low reactivity of multicopper oxidase with proteins. [Means for solving the problem]

[0010] As a result of extensive research aimed at solving the above-mentioned problems, the present inventors have newly discovered that the protein cross-linking reaction catalyzed by an oxidoreductase such as multicopper oxidase can be significantly improved by using an enzyme that deamidates proteins in combination, and have thus completed the present invention described below. [1] A protein cross-linking method, characterized by allowing an oxidoreductase and a protein deamidating enzyme to act on a protein. [2] The protein cross-linking method according to [1], wherein the oxidoreductase is a multicopper oxidase. [3] The protein cross-linking method according to [2], wherein the multicopper oxidase is laccase and / or bilirubin oxidase. [4] The protein cross-linking method according to [2], wherein the multicopper oxidase is laccase. [5] The protein cross-linking method according to any one of [1] to [4], wherein the protein deamidase is an enzyme that acts on glutamine residues in a protein. [6] The protein cross-linking method according to [5], wherein the protein deamidase is protein glutaminase. [7] A protein improver containing an oxidoreductase and a protein deamidating enzyme. [8] The protein improving agent according to [7], wherein the oxidoreductase is a multicopper oxidase. [9] The protein improving agent according to [8], wherein the multicopper oxidase is laccase and / or bilirubin oxidase.

[10] The protein improver according to [8], wherein the multicopper oxidase is laccase.

[11] The protein improving agent according to any one of [7] to

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

[12] The protein improving agent according to

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

[13] A method for producing a cross-linked protein, comprising the steps of: (1) treating a protein with a protein deamidating enzyme; (2) A step of treating the deamidated protein with an oxidoreductase.

[14] A method for producing a cross-linked protein, comprising the steps of: (1) preparing a protein treated with protein deamidase; (2) A step of treating the prepared protein with an oxidoreductase.

[15] A method for producing a crosslinked protein, comprising the step of simultaneously treating a protein with an oxidoreductase and a protein deamidating enzyme.

[16] A method for producing a food or drug, comprising the following steps: (1) preparing a protein-containing food material or pharmaceutical material treated with protein deamidase; (2) A step of treating the prepared food or pharmaceutical raw materials with an oxidoreductase.

[17] A method for producing a food or pharmaceutical product, comprising the step of simultaneously treating a protein-containing food or pharmaceutical raw material with an oxidoreductase and a protein deamidase. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the electrophoretic pattern of SDS-polyacrylamide gel electrophoresis in Test Example 1. [Figure 2] FIG. 1 shows the electrophoretic pattern of SDS-polyacrylamide gel electrophoresis in Test Example 2. [Figure 3] FIG. 1 shows the electrophoretic pattern of SDS-polyacrylamide gel electrophoresis in Test Example 3. [Figure 4] FIG. 1 shows the electrophoretic pattern of SDS-polyacrylamide gel electrophoresis in Test Example 5. [Figure 5] FIG. 1 shows the electrophoretic pattern of SDS-polyacrylamide gel electrophoresis in Test Example 6. [Figure 6]FIG. 10 shows the electrophoretic pattern of SDS-polyacrylamide gel electrophoresis in Test Example 7. [Figure 7] FIG. 10 shows the electrophoretic pattern of SDS-polyacrylamide gel electrophoresis in Test Example 8. [Figure 8] 10 is a graph showing viscosity changes in Test Example 9. DETAILED DESCRIPTION OF THE INVENTION

[0012] 1. Protein cross-linking method The cross-linking method of the present invention is characterized by allowing a protein to undergo the action of an oxidoreductase and a protein deamidase. The oxidoreductase referred to in the present invention is not particularly limited as long as it is an enzyme that cross-links proteins by an oxidation-reduction reaction, and examples thereof include the following: (1) An enzyme (e.g., lysyl oxidase) that crosslinks proteins by oxidizing the ε-amino group of lysine in proteins to produce a highly reactive aldehyde, which then combines with the amino group of another protein molecule to form a Schiff base. (2) An enzyme (e.g., sulfhydryl oxidase) that crosslinks proteins by oxidizing the sulfhydryl groups of cysteines in proteins and forming disulfhydryl bonds with other protein molecules. (3) An enzyme (e.g., tyrosinase) that crosslinks proteins by oxidizing the hydroxyl group of tyrosine in proteins to produce a highly reactive o-quinone, which then reacts with the quinone, amino group, or sulfhydryl group of another protein molecule. (4) Enzymes with broad substrate specificity, acting primarily on the hydroxyl groups of tyrosine, sulfhydryl groups of cysteine, and ε-amino groups of lysine in proteins, cross-linking proteins by any of the mechanisms (1) to (3) above (e.g., multicopper oxidases such as laccase). (5) Enzymes that catalyze the same reaction as (4) but require hydrogen peroxide as the oxygen donor in the oxidation reaction (e.g., peroxidases).

[0013] Here, multicopper oxidase refers to a group of enzymes that contain multiple copper atoms in their molecules and oxidize polyphenols, methoxyphenols, diamines, bilirubin, ascorbic acid, etc., with molecular oxygen. The number of copper atoms contained in previously known enzymes is typically 2 to 8, but this number is not particularly limited, as it varies depending on the state of the enzyme preparation at the time of analysis and the analytical method. Enzymes classified as multicopper oxidases include, for example, laccase, bilirubin oxidase, ascorbic acid oxidase, and ceruloplasmin.

[0014] Laccase (EC 1.10.3.2) is a type of multicopper protein with low substrate specificity, acting on O-quinol, p-quinol, and sometimes also on aminophenols and phenylenediamines. The resulting semiquinones undergo further enzymatic or non-enzymatic reactions. Examples of such laccases include those derived from plants such as lacquer and from microorganisms such as bacteria and fungi. Examples of laccases derived from microorganisms include those from the genera Aspergillus, Neurospora, Podospora, Botrytis, Collybia, Fomes, Lentinus, Pleurotus, Pycnoporus, Pyricularia, Trametes, Rhizoctonia, Rigidoporus, Coprinus, Psatyrella, Myceliophtera, Schtalidium, Polyporus, Phlebia, and Coriolus.

[0015] Bilirubin oxidase (EC1.3.3.5) is a type of multi-copper protein, an enzyme that acts mainly on bilirubin. Examples of such bilirubin oxidase include enzymes derived from the genera Penicillium, Myrothecium, and Trachyderma.

[0016] Ascorbic acid oxidase (EC1.10.3.3) is a type of multi-copper protein, an enzyme that acts primarily on L-ascorbic acid, and is found in plants such as cucumber, pumpkin, and zucchini, as well as in microorganisms such as bacteria and fungi.

[0017] Ceruloplasmin (EC1.16.3.1) is a type of multicopper protein, a multifunctional protein that maintains copper homeostasis in the body and has ferroxidase and amine oxidase activities, and is present in the serum of animals and birds.

[0018] The protein deamidating enzyme referred to in the present invention refers to an enzyme that catalyzes a reaction that liberates ammonia from a protein, and specifically includes an enzyme that converts glutamine residues in a protein to glutamic acid residues (i.e., protein glutaminase), an enzyme that converts asparagine residues to aspartic acid residues (i.e., protein asparaginase), and a protein deiminase that converts arginine residues in a protein to citrulline residues. An example of an enzyme that deamidates glutamine residues in a protein is protein glutaminase derived from Chryseobacterium proteolyticum (EurJ Biochem, 268(5), 1410, 2001, Protein-glutaminase From Chryseobacterium Proteolyticum, An Enzyme That Deamidates Glutaminyl Residues in Proteins. Purification, Characterization and Gene Cloning, S Yamaguchi, DJ Jeanes, DB Archer, or Front Microbiol, 9, 1975, 2018, Complete Genome Sequence and Characterization of a Protein-Glutaminase Producing Strain, Chryseobacterium proteolyticum QSH1265, Ruidan Qu, Xiaoyu Zhu, Min Tian, ​​Yingjie Liu, Wenjuan Yan, Jian Ye, Hongliang Gao, Jing Huang) are well known, but are not limited to these. Enzymes that deamidate asparagine residues in proteins include, but are not limited to, the protein asparaginase disclosed in WO 2015 / 133590. Known enzymes that deiminate arginine residues in proteins include, for example, arginine deiminase derived from Fusarium graminearum.

[0019] Generally, when glutamine and / or asparagine residues in a protein are deamidated to generate carboxyl groups, increasing the protein's negative charge and / or when highly basic arginine residues in the protein are deiminated and neutralized, the isoelectric point decreases and the hydration potential increases. Furthermore, increased electrostatic repulsion leads to decreased protein-protein interactions, i.e., decreased association. These changes significantly increase the protein's solubility and water dispersibility. Furthermore, the increased negative charge of the protein unfolds the protein, changing its higher-order structure and exposing hydrophobic regions previously buried within the molecule to the molecular surface. Therefore, deamidated proteins possess amphiphilic properties, making them ideal surfactants, significantly improving the emulsifying power, emulsion stability, foaming ability, and foam stability of proteins. Thus, protein deamidation improves various functional properties of proteins, dramatically expanding the uses of the proteins (e.g., Molecular Approaches to Improving Food Quality and Safety, D. Chatnagar and T. E. Cleveland, eds., Van Nostrand Reinhold, New York, 1992, p. 37). Deimination of arginine residues in proteins also increases the hydrophobicity of the proteins, changing their higher-order structure.

[0020] Therefore, without being bound by theory, the present inventors' newly discovered phenomenon that "the cross-linking reaction of proteins catalyzed by oxidoreductases is promoted by the action of protein deamidase" (see Examples below) can be explained as follows: the action of protein deamidase causes the folding of proteins to unfold, resulting in a change in the higher-order structure, and as a result, amino acid residues that are targets of oxidoreductases, such as tyrosine as well as cysteine ​​and lysine, which were buried inside the protein molecule, are exposed on the surface of the protein molecule, making them more susceptible to the action of oxidoreductases.

[0021] Next, the protein cross-linking method of the present invention will be described in more detail. The type and origin of the oxidoreductase and protein deamidase that can be used in the present invention are not particularly limited. The origin may be, for example, animal, plant, or microbial. Furthermore, in the case of microbial enzymes, they may be accumulated either intracellularly or extracellularly within the microorganism. Furthermore, they may be naturally occurring enzymes or enzymes produced by genetic engineering or cell engineering techniques. They may also be enzyme proteins modified by protein engineering techniques. Furthermore, it is desirable to use highly purified oxidoreductases (e.g., multicopper oxidases) and protein deamidases (e.g., protein glutaminase), but their purity is not important as long as the desired reaction is possible. Furthermore, enzyme preparations may be used as the oxidoreductase and protein deamidase. In such cases, the enzyme preparations may contain various salts, sugars, proteins, lipids, surfactants, and the like as enzyme stabilizers.

[0022] The cross-linking method of the present invention can be applied to various 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 oxidoreductase and protein deamidase. Examples of plant proteins include proteins derived from legumes such as soybeans, green peas, lentils, chickpeas, and black beans; proteins derived from grains such as wheat, barley, oats, and rice; proteins derived from nuts such as almonds and peanuts; and proteins derived from seeds such as hemp, chia, quinoa, and amaranth. Other proteins that can be used include proteins derived from insects such as crickets, fungi such as mycoproteins from yeast, filamentous fungi, and mushrooms, and algae such as spirulina. Examples of animal proteins include milk proteins such as casein and β-lactoglobulin, egg proteins such as ovalbumin, meat proteins such as myosin and actin, blood proteins such as serum albumin, and tendon proteins such as gelatin and collagen. Also usable as substrate proteins are proteins partially hydrolyzed chemically with acid or alkali, proteins partially hydrolyzed enzymatically with protease, proteins chemically modified with various reagents, and synthetic peptides.

[0023] The substrate protein described above is subjected to the reaction in a state contained in a flowable composition such as a solution, slurry, or paste. However, the concentration of the substrate protein in the flowable composition is not particularly limited, and the concentration may be determined depending on the desired properties and state of the target protein cross-linked product. Generally, a low concentration results in a solution or precipitate with increased viscosity, while a high concentration results in a gel-like product. However, a substrate protein concentration of 1% by weight or higher is sufficient to produce a gelled product. Furthermore, the flowable composition containing the substrate protein is not limited to a flowable composition in the form of an aqueous solution, aqueous dispersion, or aqueous dispersion paste of the protein. A flowable composition in the form of an emulsion of these with fats or oils may also be subjected to the reaction. Furthermore, salts, sugars, proteins, flavors, humectants, colorants, and the like may be added to the flowable composition containing the substrate protein as needed.

[0024] The amount of enzyme used, reaction time, temperature, and pH of the reaction solution are not particularly limited. Typically, the amount of enzyme is 1 to 1,000,000 U, preferably 10 to 500,000 U, and more preferably 100 to 200,000 U, per gram of protein, for oxidoreductase, and 0.01 to 100,000 U, preferably 0.1 to 50,000 U, and more preferably 1 to 10,000 U, for protein deamidating enzyme. The reaction temperature is 5 to 80°C, preferably 20 to 60°C. The pH of the reaction solution is 2 to 10, preferably 4 to 8. The reaction time is 10 seconds to 48 hours, preferably 10 minutes to 24 hours. Under the above reaction conditions, a crosslinked product of polymerized protein or a gel-like product of a flowable composition can be obtained. These reaction conditions are appropriately selected depending on the physical properties and water content of the desired protein crosslinked product or gel-like product of a flowable composition. The optimal reaction conditions can be determined through preliminary experiments.

[0025] When multicopper oxidase is used as the oxidoreductase, various polyphenols such as hydroquinone, catechol, guaiacol, ferulic acid, vanillic acid, p-coumaric acid, syringaldehyde, and p-phenylenediamine may be added as mediators to promote the reaction.

[0026] In the protein cross-linking method of the present invention, a substrate protein is treated with protein cross-linking enzymes, i.e., oxidoreductase and protein deamidase. The order in which the enzymes are acted upon (i.e., the order of oxidoreductase treatment and protein deamidase treatment) is not particularly limited, but it is preferable to treat with both enzymes simultaneously, or to treat with protein deamidase followed by oxidoreductase. For purposes such as improving work efficiency, simultaneous treatment is more preferable. When treatment with protein deamidase is performed followed by oxidoreductase, a step of inactivating protein deamidase may be added after protein deamidase treatment. The amount of protein deamidation can be adjusted by adding the inactivation step.

[0027] 2. Method for producing cross-linked proteins or foods and medicines containing cross-linked proteins By using the crosslinking method of the present invention, it is possible to produce a crosslinked protein, or a food or pharmaceutical containing the same. One embodiment of the method for producing a crosslinked protein comprises the following steps (1) and (2). Note that a step of inactivating protein deamidase may be added after step (1). (1) A step of treating proteins with protein deamidating enzymes (2) A step of treating the deamidated protein with an oxidoreductase

[0028] In another embodiment, the following steps (1) and (2) are carried out: (1) preparing a protein treated with protein deamidating enzyme (2) A step of treating the prepared protein with an oxidoreductase

[0029] In yet another embodiment, the cross-linked protein is produced by the following step (i): (i) a step of simultaneously treating a protein with an oxidoreductase and a protein deamidase

[0030] On the other hand, one embodiment of the method for producing foods and medicines includes the following (1) and (2). (1) A step of preparing a food material or pharmaceutical material containing protein that has been treated with protein deamidating enzyme. (2) A step of treating prepared food or pharmaceutical raw materials with oxidoreductases.

[0031] In another embodiment, a food or pharmaceutical product is produced by the following step (i): (i) a step of simultaneously treating a food material or pharmaceutical material containing a protein with an oxidoreductase and a protein deamidating enzyme;

[0032] 3.Protein improver The present invention also provides a protein improving agent that can be used for protein cross-linking. The protein improving agent of the present invention is typically used in the cross-linking method or production method of the present invention. The protein improving agent of the present invention contains, as active ingredients, protein cross-linking enzymes, oxidoreductase and protein deamidase. The protein improving agent of the present invention can be used as a protein cross-linking agent, preferably as a thickener for a protein-containing fluid composition, and more preferably as a gelling agent for a protein-containing fluid composition. Details of the oxidoreductase and protein deamidase are as described above (section 1. Protein cross-linking method), and therefore will not be described here.

[0033] The present invention will now be further described with reference to examples. [Example]

[0034] In the following examples, unless otherwise specified, the enzymatic activity of laccase was measured using 2,2'-Azino-di-[3-ethylbenzthiazolinesulfonate(6)] (ABTS, manufactured by Boehringer Mannheim) as a substrate by the method described below.

[0035] <Activity measurement method> ABTS was dissolved in 25 mM citrate buffer (pH 3.2) at a concentration of 1.0 mg / ml to prepare the substrate solution. 3.0 ml of this substrate solution was placed in a cuvette and preheated to 25°C. 0.1 ml of the enzyme solution was added, stirred, and incubated at 25°C. The absorbance at 405 nm was measured after 1 and 3 minutes. The amount of enzyme that increases the absorbance at 405 nm by 1.0 OD per minute under these conditions is defined as 1 unit.

[0036] On the other hand, the enzyme activity of protein glutaminase was measured by the method described below using Z-Gln-Gly as a substrate unless otherwise specified. <Activity measurement method> 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 and incubate at 37°C for 60 minutes. Then, add 100 μl of 12% trichloroacetic acid solution to stop the reaction. After centrifugation (15,000 rpm, 4°C, 5 minutes), measure the supernatant using F-kitammonia (Boehringer Mannheim) as follows (A1). Separately, measure in the same manner using water instead of enzyme solution (A2). Add 10 μl of the supernatant and 190 μl of water to 100 μl of F-kitammonia Reagent 2 and incubate at room temperature for 5 minutes. Measure the absorbance at 340 nm (E1) using 100 μl of the supernatant. To the remaining 200 μl, add 1.0 μl of Reagent 3 (glutamate dehydrogenase), leave at room temperature for an additional 20 minutes, and then measure the absorbance (E2) at 340 nm of the remaining 200 μl. The amount of enzyme that liberates 1 μmol of ammonia per minute under the above conditions is defined as 1 unit, and is calculated according to the following formula: u / ml=1.76×[A1(E1-E2)-A2(E1-E2)] <Test Example 1> The effect of laccase (LC) and protein glutaminase (PG) on protein cross-linking was investigated using egg albumin (Fujifilm Wako Pure Chemical Industries, Ltd.). Egg albumin was mixed at a final concentration of 5% by weight with 50 mM potassium-sodium phosphate buffer (pH 7.0), laccase (product name: Laccase Y120, Amano Enzyme Inc.), and protein glutaminase (product name: Protein Glutaminase "Amano" 500, Amano Enzyme Inc.) at 40°C for 24 hours with shaking at 160 rpm. After the reaction, a portion of the reaction mixture was subjected to 2-25% polyacrylamide gel electrophoresis to observe the increase in molecular weight of the substrate protein. Cross-linking and the cross-linking promotion effect compared to laccase alone were assessed. The amounts of the enzymes added were 100 U of laccase (final concentration) and 500 mU of protein glutaminase (final concentration) per 1 mg of substrate protein.

[0037] The results are shown in Figure 1 and Table 1. [Table 1]

[0038] As shown in Figure 1, when laccase was used alone, no cross-linking or polymerization of the substrate protein occurred. In contrast, when laccase was used in combination with protein glutaminase, a band representing a protein that had become so polymerized that it could not pass through the mesh of the polyacrylamide gel was observed at the top of lane 3, confirming the cross-linking or polymerization of the substrate protein. In other words, the combined use of laccase and protein glutaminase was found to have the effect of promoting the cross-linking of egg albumin protein.

[0039] <Test Example 2> The effect of combining laccase and protein glutaminase on promoting protein cross-linking was investigated using LYZAMINE-S (Pea Protein, Rocket Japan). Lyzamine-S (5 wt. % final concentration) was mixed with 50 mM potassium-sodium phosphate buffer (pH 7.0), laccase (product name: Laccase Y120, Amano Enzyme Inc.), and protein glutaminase (product name: Protein Glutaminase "Amano" 500, Amano Enzyme Inc.) and incubated at 40°C for 24 hours with shaking at 160 rpm. After completion of the reaction, a portion of the reaction mixture was taken and subjected to 2-25% polyacrylamide gel electrophoresis to observe the increase in molecular weight of the substrate protein. Cross-linking and the cross-linking promotion effect were assessed relative to the results obtained when laccase was used alone. The amounts of the enzymes added were 100 U of laccase (final concentration) and 500 mU of protein glutaminase (final concentration) per 1 mg of substrate protein.

[0040] The results are shown in Figure 2 and Table 2. [Table 2]

[0041] As shown in Figure 2, when laccase was used alone, no cross-linking or polymerization of the substrate protein occurred. In contrast, when laccase was used in combination with protein glutaminase, the band of the protein that had polymerized to the point that it could not pass through the mesh of the polyacrylamide gel (the band visible at the top of lane 3) became darker, confirming the cross-linking or polymerization of the substrate protein. In other words, the combined use of laccase and protein glutaminase was found to have the effect of promoting the cross-linking of pea protein.

[0042] <Test Example 3> The effect of combining laccase and protein glutaminase on protein cross-linking was investigated using soybean protein powder (Fujifilm Wako Pure Chemical Industries, Ltd.). Soybean protein powder (5% by weight, final concentration) was mixed with 50 mM potassium-sodium phosphate buffer (pH 7.0), laccase (product name: Laccase Y120, Amano Enzyme Inc.), and protein glutaminase (product name: Protein Glutaminase "Amano" 500, Amano Enzyme Inc.) and incubated at 40°C for 24 hours with shaking at 160 rpm. After completion of the reaction, a portion of the reaction mixture was subjected to 2-25% polyacrylamide gel electrophoresis to observe the increase in molecular weight of the substrate protein. The degree of cross-linking and the cross-linking promotion effect were assessed relative to the level achieved by using laccase alone at the same concentration. The amounts of enzymes added were 100 U of laccase (final concentration) and 500 mU of protein glutaminase (final concentration) per 1 mg of substrate protein. Similar reactions were also performed under conditions in which the amounts of enzymes (laccase and protein glutaminase) added were reduced to 1 / 10 and 1 / 100.

[0043] The results are shown in Figure 3 and Table 3. [Table 3]

[0044] As shown in Figure 3, the combined use of laccase and protein glutaminase improved the efficiency of cross-linking and polymerizing the substrate protein compared to when laccase was used alone, and at both enzyme concentrations, the bands of proteins that had polymerized so much that they could not pass through the mesh of the polyacrylamide gel (the bands that can be seen at the top of each lane (lanes 5 to 7)) were observed to be darker, confirming cross-linking and polymerizing. In other words, the combined use of laccase and protein glutaminase was found to have the effect of promoting the cross-linking of soybean-derived proteins.

[0045] <Test Example 4> The effect of combining laccase and protein glutaminase on protein cross-linking was investigated using egg albumin (Fujifilm Wako Pure Chemical Industries, Ltd.). Egg albumin (5% by weight, final concentration) was mixed with 50 mM potassium-sodium phosphate buffer (pH 7.0) and protein glutaminase (product name: Protein Glutaminase "Amano" 500, Amano Enzyme Inc.) at 40°C for 4 hours with shaking at 160 rpm. Laccase (product name: Laccase Y120, Amano Enzyme Inc.) was then added and the reaction was continued at 40°C for 20 hours with shaking at 160 rpm. After the reaction was complete, a portion of the reaction mixture was taken and subjected to 2-25% polyacrylamide gel electrophoresis to observe the increase in molecular weight of the substrate protein and assess cross-linking. The amounts of the enzymes added were 100 U of laccase (final concentration) and 500 mU of protein glutaminase (final concentration) per 1 mg of substrate protein.

[0046] The results are shown in Table 4. [Table 4]

[0047] Furthermore, in the electrophoresis image, even when laccase treatment was performed after protein glutaminase treatment, a band of protein that had polymerized to the point that it could not pass through the meshes of the polyacrylamide gel was confirmed at the top of lane 2, confirming cross-linking and polymerization of the substrate protein. As is clear from a comparison between the case in Test Example 1 in which laccase was used alone and the case in this Test Example in which laccase treatment was performed after protein glutaminase treatment, the combined use of laccase and protein glutaminase was found to have the effect of promoting cross-linking of egg-derived albumin protein, regardless of the order in which these enzymes were added.

[0048] <Test Example 5> The effect of combining laccase and protein glutaminase on protein cross-linking was investigated using almond-derived protein powder. Almond-derived protein powder (5% by weight, final concentration) was mixed with 50 mM potassium-sodium phosphate buffer (pH 7.0), laccase (product name: Laccase Y120, Amano Enzyme Inc.), and protein glutaminase (product name: Protein Glutaminase "Amano" 500, Amano Enzyme Inc.) and incubated at 40°C for 24 hours with shaking at 160 rpm. After completion of the reaction, a portion of the reaction mixture was subjected to 2-25% polyacrylamide gel electrophoresis to observe the increase in molecular weight of the substrate protein. Cross-linking and the cross-linking promotion effect were assessed relative to the results obtained when laccase was used alone at the same concentration. The amounts of enzymes added were 100 U (final concentration) of laccase and 500 mU (final concentration) of protein glutaminase per 1 mg of substrate protein. The same reaction was also carried out under conditions in which the enzymes (laccase and protein glutaminase) were both diluted.

[0049] The results are shown in Figure 4 and Table 5. [Table 5]

[0050] As shown in Figure 4, the combined use of laccase and protein glutaminase improved the efficiency of cross-linking and polymerizing the substrate protein compared to when laccase was used alone, and at both enzyme concentrations, the presence of proteins polymerized to approximately 200 kDa or more was newly confirmed, confirming cross-linking and polymerizing (lanes 5-7). In other words, the combined use of laccase and protein glutaminase was found to have an effect of promoting the cross-linking of almond-derived proteins.

[0051] <Test Example 6> The effect of combining laccase and protein glutaminase on protein cross-linking was investigated using chickpea-derived protein powder. Chickpea-derived protein powder (5 wt. % final concentration) was mixed with 50 mM potassium-sodium phosphate buffer (pH 7.0), laccase (product name: Laccase Y120, Amano Enzyme Inc.), and protein glutaminase (product name: Protein Glutaminase "Amano" 500, Amano Enzyme Inc.) and incubated at 40°C for 24 hours with shaking at 160 rpm. After completion of the reaction, a portion of the reaction mixture was subjected to 2-25% polyacrylamide gel electrophoresis to observe the increase in molecular weight of the substrate protein. Cross-linking and the cross-linking promotion effect were assessed relative to the results obtained when laccase was used alone at the same concentration. The amounts of enzymes added were 100 U (final concentration) of laccase and 500 mU (final concentration) of protein glutaminase per 1 mg of substrate protein. The same reaction was also carried out under conditions in which the enzymes (laccase and protein glutaminase) were both diluted.

[0052] The results are shown in Figure 5 and Table 6. [Table 6]

[0053] As shown in Figure 5, the combined use of laccase and protein glutaminase improved the efficiency of cross-linking and polymerizing the substrate protein compared to when laccase was used alone, and at both enzyme concentrations, the bands of proteins that had polymerized to the point that they could not pass through the mesh of the polyacrylamide gel (the bands that can be seen at the top of each lane (lanes 5 to 7)) were observed to be darker, confirming cross-linking and polymerizing. In other words, the combined use of laccase and protein glutaminase was found to have the effect of promoting the cross-linking of chickpea-derived proteins.

[0054] <Test Example 7> The effect of bilirubin oxidase (BO) and protein glutaminase on protein cross-linking was investigated using casein (Merck Millipore). Protein was mixed at a final concentration of 5% by weight in 50 mM potassium-sodium phosphate buffer (pH 7.0), bilirubin oxidase (BO "Amano" 3, Amano Enzyme Inc.), and protein glutaminase (Protein Glutaminase "Amano" 500, Amano Enzyme Inc.) at 40°C for 24 hours with shaking at 160 rpm. After the reaction, a portion of the reaction mixture was subjected to 2-25% polyacrylamide gel electrophoresis to observe the increase in molecular weight of the substrate protein. Cross-linking and the cross-linking promotion effect were assessed relative to the results obtained when bilirubin oxidase was used alone at the same concentration. The amounts of enzymes added were 100 U of bilirubin oxidase (final concentration) and 500 mU of protein glutaminase (final concentration) per 1 mg of substrate protein. The same reaction was also performed under conditions in which the amounts of enzymes added (bilirubin oxidase and protein glutaminase) were both diluted.

[0055] The results are shown in Figure 6 and Table 7. [Table 7]

[0056] As shown in Figure 6, the combined use of bilirubin oxidase and protein glutaminase improved the efficiency of cross-linking and polymerizing the substrate protein compared to when bilirubin oxidase was used alone, and at all enzyme concentrations, the bands of the polymerized proteins were observed as darker bands at the top of each lane (lanes 5 to 7), confirming cross-linking and polymerizing. In other words, the combined use of bilirubin oxidase and protein glutaminase was found to have the effect of promoting the cross-linking of casein protein.

[0057] <Test Example 8> The effect of tyrosinase (TyrA) and protein glutaminase on promoting protein cross-linking was investigated using casein (Merck Millipore). Protein was mixed at a final concentration of 5% by weight in 50 mM potassium-sodium phosphate buffer (pH 7.0), tyrosinase (product name: Tyrosinase from mushroom, Merck) and protein glutaminase (product name: Protein Glutaminase "Amano" 500, Amano Enzyme Inc.) and incubated at 40°C for 24 hours with shaking at 160 rpm. After completion of the reaction, a portion of the reaction mixture was subjected to 2-25% polyacrylamide gel electrophoresis to observe the increase in molecular weight of the substrate protein. Cross-linking and the cross-linking promotion effect were assessed relative to the results obtained when tyrosinase was used alone at the same concentration. The amounts of enzymes added were 100 U of tyrosinase (final concentration) and 500 mU of protein glutaminase (final concentration) per 1 mg of substrate protein. Similar reactions were also performed under conditions in which the amounts of enzymes added (tyrosinase and protein glutaminase) were both diluted.

[0058] The results are shown in Figure 7 and Table 8. [Table 8]

[0059] 7, compared to the use of tyrosinase alone, the combined use of tyrosinase and protein glutaminase improved the efficiency of cross-linking and polymerizing the substrate protein, and at either enzyme concentration, the polymerized protein bands were observed as darker bands at the top of each lane (lanes 5 and 6) and / or new polymerized protein bands were observed at the top of each lane (lanes 6 and 7), confirming cross-linking and polymerizing. In other words, the combined use of tyrosinase and protein glutaminase was found to have the effect of promoting the cross-linking of casein protein.

[0060] <Test Example 9> (Confirmation of the viscosity-improving effect of casein solution and wheat gluten solution) A mixture containing 5% (w / v) casein or wheat gluten solution, 100 mU / mL of protein glutaminase (product name: Protein Glutaminase "Amano" 500, Amano Enzyme Inc.), 50 U / mL of laccase (product name: Laccase Y120, Amano Enzyme Inc.), and 100 mM phosphate buffer (pH 7.0) was treated at 40°C, and the viscosity was measured at various times using an EMS-1000 (Kyoto Electronics Manufacturing Co., Ltd., Tokyo, Japan). The shear rate during measurement was 200 s-1, and a preliminary measurement was performed for 30 s to maintain a fluid state. During the measurement, casein solutions without enzymes, treated with laccase (50 U / mL), and treated with protein glutaminase (100 mU / mL) were also measured as controls.

[0061] The results are shown in Figure 8. The viscosity of the casein (or gluten) solution without added enzyme, the casein (or gluten) solution with laccase only, and the casein (or gluten) solution with protein glutaminase only did not fluctuate during the measurement. On the other hand, the concentration of the casein solution with both laccase and protein glutaminase increased in a reaction time-dependent manner.

[0062] Furthermore, in the electrophoretic images, the combined use of laccase and protein glutaminase improved the efficiency of cross-linking and polymerizing the substrate protein compared to the use of laccase alone, and at both enzyme concentrations, the presence of proteins polymerized to 200 kDa or more was newly confirmed, confirming cross-linking and polymerizing.

[0063] In other words, the combined use of laccase and protein glutaminase was found to have the effect of promoting the cross-linking of casein protein and wheat gluten protein.

[0064] (Gelling study of casein solution) <Test Example 10> A mixture containing 5% (w / v) casein solution, 100 mU / mL of protein glutaminase (product name: Protein Glutaminase "Amano" 500, manufactured by Amano Enzyme Inc.), 50 U / mL of laccase (product name: Laccase Y120, manufactured by Amano Enzyme Inc.), and 100 mM phosphate buffer (pH 7.0) was added to a test tube and treated at 40°C for 24 hours. After this, the test tube was tilted to check for the presence or absence of gelation and the cross-linking promotion effect compared to when laccase was used alone.

[0065] The results are shown in Table 9. [Table 9]

[0066] As shown in Table 9, the properties of casein solutions with no enzymes added, with laccase alone added, and with protein glutaminase alone added remained unchanged. However, only the casein solution containing both laccase and protein glutaminase gelled. In other words, the combined use of laccase and protein glutaminase was found to promote the cross-linking of casein protein.

[0067] (Study on milk gelation) <Test Example 11> A mixture of commercially available milk and 100mU / mL of protein glutaminase (product name: Protein Glutaminase "Amano" 500, manufactured by Amano Enzyme Inc.), 50U / mL of laccase (product name: Laccase Y120, manufactured by Amano Enzyme Inc.), and 100mM phosphate buffer (pH 7.0) was added to a test tube and treated at 40°C for 24 hours. The test tube was then tilted to check for gelation and the cross-linking promotion effect compared to when laccase was used alone.

[0068] The results are shown in Table 10. [Table 10]

[0069] As shown in Table 10, the properties of milk without added enzymes, milk with laccase alone, and milk with protein glutaminase alone did not change. On the other hand, only milk treated with both laccase and protein glutaminase gelled. In other words, the combined use of laccase and protein glutaminase promoted the cross-linking of milk proteins.

[0070] (Wheat gluten gelation study) <Test Example 12> A mixture of 5% (w / v) wheat gluten solution, 100mU / mL of protein glutaminase (product name: Protein Glutaminase "Amano" 500, manufactured by Amano Enzyme Inc.), 50U / mL of laccase (product name: Laccase Y120, manufactured by Amano Enzyme Inc.), and 100mM phosphate buffer (pH 7.0) was added to a test tube and treated at 40°C for 24 hours. The test tube was then tilted to check for gelation and the cross-linking promotion effect compared to when laccase was used alone.

[0071] The results are shown in Table 11. [Table 11]

[0072] As shown in Table 11, the properties of the wheat gluten solution without added enzymes, the wheat gluten solution with laccase alone, and the wheat gluten solution with protein glutaminase alone did not change. On the other hand, only the wheat gluten solution containing both laccase and protein glutaminase gelled. In other words, the combined use of laccase and protein glutaminase promoted the cross-linking of wheat gluten proteins. [Industrial Applicability]

[0073] The enzymatic protein cross-linking method of the present invention significantly accelerates the protein cross-linking reaction catalyzed by oxidoreductases, which has previously been difficult to put into practical use due to its low reactivity. The cross-linked, polymerized, or gelled protein materials produced by the present invention can be used in food processing applications, such as fish paste, kamaboko (fish paste), fish and meat sausages, tofu, noodles, confectionery and bread, food adhesives, meat sheet foods, yogurt, jelly, cheese, and plant-based meat and dairy alternatives (e.g., cheese alternatives and fermented milk alternatives). Furthermore, as novel protein-derived materials, they are expected to be used in a wide range of industries, including cosmetics, medical supplies, microcapsule materials, and carriers for immobilized enzymes. Because the reaction mechanism of cross-linking by oxidoreductases is thought to be different from that of transglutaminase, which is frequently used for protein cross-linking, the present invention is also expected to be used and applied to the production of protein polymers and gels with new qualities.

[0074] The present invention is not limited to the above-described embodiments and examples. Various modifications within the scope of the claims and within the scope that can be easily conceived by a person skilled in the art are also included in the present invention. The contents of papers, published patent applications, patent publications, and other publications explicitly stated in this specification are incorporated herein by reference in their entirety.

Claims

1. The method comprises allowing an oxidoreductase and a protein deamidase to act on a protein, A method for cross-linking a protein, wherein the oxidoreductase is laccase and / or bilirubin oxidase, or tyrosinase, and the protein deamidase is protein glutaminase and / or protein asparaginase.

2. 2. The method for cross-linking proteins according to claim 1, wherein the oxidoreductase is a multicopper oxidase.

3. 3. The method for cross-linking proteins according to claim 2, wherein the multicopper oxidase is laccase and / or bilirubin oxidase.

4. 3. The method for cross-linking proteins according to claim 2, wherein the multicopper oxidase is laccase.

5. 2. The protein cross-linking method according to claim 1, wherein the protein deamidase is protein glutaminase.

6. Contains oxidoreductase and protein deamidase, A protein improving agent, wherein the oxidoreductase is laccase and / or bilirubin oxidase, or tyrosinase, and the protein deamidase is protein glutaminase and / or protein asparaginase.

7. 7. The protein improving agent according to claim 6, wherein the oxidoreductase is a multicopper oxidase.

8. 8. The protein improving agent according to claim 7, wherein the multicopper oxidase is laccase and / or bilirubin oxidase.

9. 8. The protein improving agent according to claim 7, wherein the multicopper oxidase is laccase.

10. 7. The protein improving agent according to claim 6, wherein the protein deamidase is protein glutaminase.

11. The following steps: (1) treating a protein with a protein deamidase; (2) treating the deamidated protein with an oxidoreductase; A method for producing a crosslinked protein, wherein the oxidoreductase is laccase and / or bilirubin oxidase, or tyrosinase, and the protein deamidase is protein glutaminase and / or protein asparaginase.

12. The following steps: (1) preparing a protein treated with protein deamidase; (2) treating the prepared protein with an oxidoreductase; A method for producing a crosslinked protein, wherein the oxidoreductase is laccase and / or bilirubin oxidase, or tyrosinase, and the protein deamidase is protein glutaminase and / or protein asparaginase.

13. comprising a step of simultaneously treating a protein with an oxidoreductase and a protein deamidase, A method for producing a crosslinked protein, wherein the oxidoreductase is laccase and / or bilirubin oxidase, or tyrosinase, and the protein deamidase is protein glutaminase and / or protein asparaginase.

14. The following steps: (1) preparing a food material or pharmaceutical material containing a protein that has been treated with protein deamidase; (2) treating the prepared food or pharmaceutical raw material with an oxidoreductase; A method for producing a food or pharmaceutical product, wherein the oxidoreductase is laccase and / or bilirubin oxidase, or tyrosinase, and the protein deamidase is protein glutaminase and / or protein asparaginase.

15. The method comprises the step of simultaneously treating a food material or pharmaceutical material containing a protein with an oxidoreductase and a protein deamidase, A method for producing a food or pharmaceutical product, wherein the oxidoreductase is laccase and / or bilirubin oxidase, or tyrosinase, and the protein deamidase is protein glutaminase and / or protein asparaginase.

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