Deamidation methods for proteins

Abstract

The present invention addresses the problem of promoting a deamidation reaction with a protein deamidation enzyme.　The present invention aims to promote the deamidation reaction by reacting a protein deamidation enzyme with a protein in the presence of a salt or a polysaccharide. According to the present invention, the reaction efficiency can be improved. The present invention is especially useful for the deamidation of a vegetable protein such as a pea protein. A deamidated protein (especially a deamidated vegetable protein) produced by employing the present invention can be used in various foods or beverages.

Description

Technical Field

[0001] The present invention relates to the deamidation of proteins. Specifically, it relates to a method for deamidating proteins and its uses, a protein improver used in the method for deamidating proteins, and the like.

Background Art

[0002] Backgrounded by the increasing health consciousness in recent years, the demand for plant proteins and the like has significantly increased. On the other hand, deamidation of proteins by deamidating enzymes is expected to be used in various applications including foods and beverages in order to improve the solubility of proteins and impart functionality as an emulsifier or a foaming agent (see, for example, Patent Document 1).

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] Since the deamidation reaction by a protein deamidating enzyme is an enzymatic reaction, it is affected by conditions such as the substrate and the reaction temperature, and the reaction may become insufficient. In other words, because it is an enzymatic reaction, there are cases where the desired effects (such as improvement in the functionality of proteins by deamidation) cannot be obtained. Therefore, an object of the present invention is to provide a method for promoting the deamidation reaction by a protein deamidating enzyme.

Means for Solving the Problems

[0005] As a result of intensive studies to solve the above problems, the present inventors have found that when a protein deamidating enzyme is allowed to act in the presence of salts and / or polysaccharides, the cross-linking reaction of proteins is promoted, and the present invention shown below has been completed. [1] A method for deamidating a protein, characterized by acting a protein deamidase on a protein in the presence of salts and / or polysaccharides. [2] The protein deamide method according to [1], wherein the salts are at least one selected from the group consisting of carbonates, phosphates, hydrogen phosphates, polyphosphates, and citrates. [3] The protein deamide method according to [1] or [2], wherein the polysaccharide is gellan gum. [4] The protein deamide method according to any one of [1] to [3], wherein the protein deamide enzyme is protein glutaminase. [5] The protein deamide method according to any one of [1] to [4], wherein the protein is a plant protein and / or an animal protein. [6] A protein modifier containing salts and / or polysaccharides, and a protein deamidase. [7] The protein modifier according to [6], wherein the salt is a phosphate, polyphosphate, or citrate. [8] The protein modifier according to [6] or [7], wherein the polysaccharide is gellan gum. [9] A protein modifier according to any one of items [6] to [8], wherein the protein deamidase is protein glutaminase.

[10] A method for producing a deamide protein, comprising the following steps: (1) A step of preparing a solution containing protein, salts and / or polysaccharides, (2) A step in which the prepared solution is treated with a protein deamidation enzyme.

[11] Methods for producing food or pharmaceuticals, comprising the following steps: (1) A step of preparing a food ingredient or pharmaceutical ingredient containing protein, and a solution containing salts and / or polysaccharides. (2) A step in which the prepared solution is treated with a protein deamidation enzyme.

[12] The method for producing salts according to

[10] or

[11] , wherein the salts are at least one selected from the group consisting of carbonates, phosphates, hydrogen phosphates, polyphosphates, and citrates.

[13] A method for producing a product according to any one of items

[10] to

[12] , wherein the polysaccharide is gellan gum.

[14] The method for producing a protein according to any one of

[10] to

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

[15] The method for producing a protein according to any one of

[10] to

[14] , wherein the protein is a plant protein and / or an animal protein. [Brief explanation of the drawing]

[0006] [Figure 1] This figure shows the results of measuring the reaction efficiency of deamidation when Z-Gln-Gly reacted with protein glutaminase using a solvent prepared by supplementing a solution containing disodium hydrogen phosphate with either sodium dihydrogen phosphate or sodium carbonate. [Figure 2] This figure shows the results of measuring the deamidation reaction efficacy when various substrate proteins were reacted with protein glutaminase using a solvent prepared by supplementing a solution containing disodium hydrogen phosphate with sodium carbonate. [Figure 3] This figure shows the results of measuring the thermal stability of protein glutaminase in a solvent prepared by supplementing a solution containing disodium hydrogen phosphate with sodium carbonate. [Modes for carrying out the invention]

[0007] 1. Methods for deamidating proteins The first aspect of the present invention relates to a method for deamidating proteins (protein deamidation method). In this invention, the protein deamidation reaction is promoted by treating the protein raw material with a protein deamidation enzyme in the presence of salts and / or polysaccharides.

[0008] In the present invention, the protein subjected to enzymatic treatment (substrate protein) is not particularly limited as long as it is subject to the action of the above-mentioned enzyme, and there are no particular restrictions on its origin, properties, etc. It may be any of the following: plant protein, animal protein, fungal protein, algae protein, etc.

[0009] Examples of vegetable proteins include proteins derived from beans such as soy beans, green peas, lentils, chickpeas, black beans, and fava beans; proteins derived from cereals such as wheat, barley, oats, rye, 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.

[0010] The animal protein may be derived from mammals or insects. Examples of mammalian-derived 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. Examples of insect-derived proteins include proteins derived from crickets.

[0011] Examples of fungal-derived proteins include yeast-derived proteins, filamentous fungus-derived proteins, and fungal-derived proteins (such as mycoprotein derived from mushrooms).

[0012] Among these substrate proteins, vegetable proteins and animal proteins are preferably used as the targets for deamidation, and vegetable proteins are more preferably used.

[0013] The substrate protein may also be a chemically partially decomposed protein by an acid, an alkali, etc., an enzymatically partially decomposed protein by a protease, etc., a chemically modified protein by various reagents, or a synthetic peptide, etc.

[0014] The substrate protein may be used alone or in combination of two or more.

[0015] The substrate protein as described above is used in the reaction in the form of a solution, slurry or paste, and its concentration is not particularly limited, and the concentration may be determined according to the desired properties and state of the target deamidated protein. Further, not limited to an aqueous solution, a solution, slurry or paste in the form of an emulsion with an oil or fat may be used in the reaction. Furthermore, salts, sugars, proteins, flavors, humectants, colorants, etc. may be added to the solution, slurry or paste of the substrate protein as necessary.

[0016] The salts used in the present invention are preferably water-soluble salts. The types of water-soluble salts are not particularly limited, and examples include inorganic salts such as sulfates, hydrochlorides, nitrates, dioxide salts, phosphates, hydrogen phosphates, polyphosphates, thiocyanates, thiosulfates, carbonates, hydrogen carbonates; and organic acid salts such as acetates, tartrates, citrates, sorbates. Also, the types of metal atoms constituting the water-soluble salts are not particularly limited, and examples include alkali metals such as sodium and potassium; alkaline earth metals such as magnesium and calcium; and other metals such as manganese, copper, and zinc. Among these, alkali metals are preferred. [[ID=于5]]

[0017] Among the salts, the polyphosphate may be linear, branched or cyclic, but is preferably cyclic. Also, the degree of polymerization of phosphoric acid in the polyphosphate is not particularly limited, and examples include 2 to 1,000, preferably 3 to 100, more preferably 4 to 10, and even more preferably 5 to 7. Among the polyphosphates, hexametaphosphate is preferably mentioned.

[0018] The salts used in the present invention are preferably phosphates, hydrogen phosphates, polyphosphates, citrates, sorbates, and carbonates; more preferably, potassium phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, trisodium phosphate, sodium polyphosphate, trisodium citrate, and sodium carbonate. In particular, when carbonates are used as salts, deamination of substrate proteins can be carried out much more efficiently, and the thermal stability of the protein deamidase can also be improved.

[0019] These salts may be used individually or in combination of two or more. An example of a combination of two salts is a combination of hydrogen phosphate and carbonate. When using hydrogen phosphate and carbonate in combination, the ratio is not particularly limited, but for example, 4 to 46 parts by weight, preferably 15 to 43 parts by weight, and more preferably 20 to 41 parts by weight of carbonate per 1 part by weight of hydrogen phosphate.

[0020] The polysaccharides used in the present invention are preferably water-soluble polysaccharides. The types of water-soluble polysaccharides are not particularly limited, and examples include starch, dextrin, dextran, cellulose, xylan, tamarind seed gum, guar gum, tara gum, locust bean gum, gum arabic, carrageenan, gellan gum, xanthan gum, karaya gum, pectin, polygalacturonic acid, and alginic acid. These polysaccharides may also be in the form of salts. Examples of polysaccharide salts include alkali metal salts such as sodium salts and potassium salts.

[0021] Among these polysaccharides, acidic polysaccharides (polysaccharides having a carboxyl group in at least one of their constituent monosaccharides); more preferably, carrageenan, gellan gum, xanthan gum, karaya gum, pectin, polygalacturonic acid, alginic acid; and even more preferably, gellan gum.

[0022] The protein deamidases referred to in this invention are enzymes that deamidate glutamine residues and asparagine residues in proteins, and examples include the following. (1) An enzyme that deamidates glutamine residues in proteins and converts them to glutamic acid (e.g., protein glutaminase). (2) An enzyme that deamidates asparagine residues in proteins and converts them to aspartic acid (e.g., protein asparaginase). (3) Enzymes that deimide arginine residues in proteins and convert them to citrulline (e.g., arginine deiminase, protein arginine deiminase, peptidyl arginine deiminase).

[0023] Generally, deamidating glutamine and asparagine residues in a protein to generate carboxyl groups increases the negative charge of the protein, resulting in a decrease in the isoelectric point and an increase in hydration power. Furthermore, the increased electrostatic repulsion leads to a decrease in interprotein interactions, i.e., a decrease in association. These changes significantly increase the solubility and water dispersibility of the protein. The increase in the negative charge of the protein also unfolds the protein, altering its higher-order structure and exposing hydrophobic regions that were previously embedded within the molecule to the molecular surface. Therefore, deamidated proteins become amphiphilic and ideal surfactants, greatly improving the emulsifying power, emulsification stability, foaming ability, and foam stability of the protein. Thus, deamidation of proteins leads to improvements in various functional properties of the protein, dramatically increasing its applications. Similarly, deiminoating arginine residues in a protein also increases the hydrophobicity of the protein and alters its higher-order structure.

[0024] Examples of protein deamide enzymes include those derived from the genera Chryseobacterium, Flavobacterium, Empedobacter, Sphingobacterium, Aureobacterium, or Myroides. These protein deamide enzymes are disclosed in Japanese Patent Publication No. 2000-50887, Japanese Patent Publication No. 2001-218590, WO2006 / 075772, etc.

[0025] Furthermore, a specific example of an enzyme that deamides the side chains of glutamine residues in proteins is protein glutaminase derived from the genus Chryseobacterium.

[0026] Furthermore, specific examples of enzymes that deamide asparagine residues in proteins include protein deamide enzymes derived from the genera Luteimicrobium, Agromyces, Microbacterium, or Leifsonia. These protein deamide enzymes are disclosed, for example, in WO2015 / 133590.

[0027] Furthermore, an example of an enzyme that deiminates arginine residues in proteins is arginine deiminase derived from the genus Fusarium. Such a protein deamide enzyme is disclosed, for example, in WO2008 / 000714.

[0028] Preferably, these protein deamide enzymes include protein glutaminase, more preferably protein glutaminase derived from the genus Chryseobacterium, and even more preferably protein glutaminase derived from Chryseobacterium proteoricum. Protein glutaminase derived from Chryseobacterium proteoricum is commercially available, for example, as Protein Glutaminase "Amano" 500 (manufactured by Amano Enzyme Co., Ltd.), and this commercially available product can be used.

[0029] Furthermore, the protein deamidation enzyme may be an enzyme modified by protein engineering techniques.

[0030] The protein deamide enzyme can be prepared from a culture medium of a microorganism that produces the protein deamide enzyme. The microorganism used to prepare the protein deamide enzyme is not particularly limited, but any microorganism that produces the enzyme, such as those belonging to the genera Chryseobacterium, Flavobacterium, Empedobacter, Sphingobacterium, Aureobacterium, or Myroides, can be used. Alternatively, microorganisms into which the protein deamide enzyme gene has been introduced by genetic engineering may be used. A specific example of a microorganism suitable for preparing the protein deamide enzyme is Chryseobacterium sp. No. 9670, which belongs to the genus Chryseobacterium.

[0031] For example, protein deamidase enzymes can be obtained from the culture medium or cells of the above-mentioned microorganisms. That is, secreted proteins can be recovered from the culture medium, and other proteins can be recovered from the cells. Methods for preparing protein deamidase enzymes from the culture medium include known protein separation and purification methods (centrifugation, UF concentration, salting out, various chromatography methods using ion exchange resins, etc.). For example, the culture medium can be centrifuged to remove the cells, and then the target enzyme can be obtained by combining salting out, chromatography, etc. When recovering the enzyme from the cells, for example, the cells can be crushed by pressurization, sonication, etc., and then the target enzyme can be obtained by separation and purification in the same manner as above. Alternatively, the cells can be recovered from the culture medium beforehand by filtration, centrifugation, etc., before performing the above series of steps (crushing, separation, and purification of cells). The enzyme may be powdered by drying methods such as freeze-drying or vacuum drying, and appropriate excipients and drying aids may be used at that time.

[0032] While it is preferable to use highly purified protein deamidation enzymes in the present invention, any purity is acceptable as long as it catalyzes the desired reaction. Furthermore, these enzyme preparations may contain various salts, sugars, proteins, lipids, or surfactants as enzyme stabilizers.

[0033] The enzymatic activity of protein glutaminase, one of the protein deamidation enzymes, is measured using Z-Gln-Gly (Benzyloxycarbonyl-L-glutaminylglycine) as the substrate, unless otherwise specified, by the method described below. <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, incubate at 37°C for 60 minutes, then add 100 μl of 12% trichloroacetic acid solution to stop the reaction. After centrifugation (15000 rpm, 4°C, 5 minutes), measure the supernatant using the Ammonia Assay Kit (Sigma-Aldrich) as follows (A1). Alternatively, measure similarly using water instead of enzyme solution (A2). Add 10 μl of supernatant and 190 μl of water to 100 μl of F-kit ammonia reagent 2, let stand at room temperature for 5 minutes, and then measure the absorbance at 340 nm (E1) using 100 μl. After adding 1.0 μl of reagent 3 (glutamate dehydrogenase) to the remaining 200 μl, and leaving it at room temperature for another 20 minutes, the absorbance (E2) of the remaining 200 μl at 340 nm is measured. Under the above conditions, the amount of enzyme that releases 1 μmol of ammonia per minute is defined as 1 unit, and is calculated according to the following formula. U / ml=1.76×[A1(E1-E2)-A2(E1-E2)]

[0034] Protein deamidation is carried out by incubating a reaction mixture containing a protein, salts and / or polysaccharides, and a protein deamidase enzyme.

[0035] As long as the effect of the present invention, namely the deamidation reaction of proteins, is promoted, the conditions for treatment with protein deamidation enzymes are not particularly limited. The optimal reaction conditions can be set according to the enzyme used by adjusting the protein concentration, reaction temperature, reaction pH, reaction time, amount of salts added (if salts are added), amount of polysaccharides added (if polysaccharides are added), and amount of enzyme added (enzyme concentration) through preliminary experiments.

[0036] While not limited to this example, the amount of protein in the reaction solution is 0.1 to 30% by weight, preferably 0.5 to 20% by weight, and more preferably 0.9 to 11% by weight. The amount of enzyme is 0.01 to 1000 U of deamide enzyme per 1 g of protein, preferably 0.1 to 100 U, more preferably 0.5 to 20 U, and even more preferably 1 to 10 U. The amount of salts in the reaction solution is 0.01 to 20% (W / V%), preferably 0.05 to 15% (W / V%), and more preferably 0.1 to 11% (W / V%). The amount of polysaccharides in the reaction solution is 0.001 to 10% (W / V%), preferably 0.005 to 5% (W / V%), and more preferably 0.01 to 1% (W / V%). The reaction temperature is 5 to 80°C, preferably 20 to 70°C, and more preferably 30 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, more preferably 30 minutes to 12 hours, and even more preferably 30 minutes to 4 hours. Deamidated proteins can be obtained under these reaction conditions. These reaction conditions are appropriately selected depending on the properties of the protein, the salts, polysaccharides, and protein deamidation enzymes used. The optimal reaction conditions can be determined through preliminary experiments.

[0037] 2. Method for producing deamide proteins, or foods or pharmaceuticals containing deamide proteins. The protein deamide method of the present invention can be used to produce deamided proteins, or foods and pharmaceuticals containing them. Therefore, the present invention also provides a method for producing deamided proteins and a method for producing foods or pharmaceuticals containing deamided proteins. A typical embodiment of the method for producing deamided proteins includes the following steps (1) and (2). A step of inactivating the protein deamide enzyme may be added after step (2). The specific conditions for the following steps (1) and (2) are as described in the "1.1. Protein Deamide Method" section above. (1) A step of preparing a solution containing protein, salts and / or polysaccharides, (2) A step in which the prepared solution is treated with a protein deamidation enzyme.

[0038] On the other hand, a typical embodiment of a method for producing food or pharmaceuticals includes the following steps (1) and (2). A step of deactivating the protein deamidation enzyme may be added after step (2). The specific conditions for the following steps (1) and (2) are as described in the section "1.1. Method for Deamidating Proteins" above. (1) A step of preparing a food ingredient or pharmaceutical ingredient containing protein, and a solution containing salts and / or polysaccharides. (2) A step in which the prepared solution is treated with a protein deamidation enzyme.

[0039] An example of a solution prepared in step (1) above is a plant-based milk prepared using plant protein to which salts and / or polysaccharides have been added.

[0040] The deamide proteins produced by the method of the present invention have commercial value in themselves. On the other hand, they are also useful as ingredients or components in various foods and beverages. Therefore, this application also provides foods or beverages containing deamide proteins obtained by the production method of the present invention. The foods and beverages referred to here are not particularly limited. Examples of food and beverages include processed seafood products (chikuwa, kamaboko, hanpen, shikiika, dried fish, salted seafood, fish sausage, tsukudani, canned goods, etc.), processed meat products (ham, bacon, sausage, jerky, corned beef, processed meat, etc.), processed vegetables (canned and bottled vegetables, processed tomatoes, processed mushrooms, pickled vegetables, dried vegetables, tsukudani vegetables, etc.), noodles and bread (various types of noodles, sliced ​​bread, sweet bread, etc.), processed grain products (cereals, oatmeal, muesli, processed rice products, fu (wheat gluten), barley tea, etc.), dairy products (milk, processed milk, milk beverages, concentrated milk, powdered milk, condensed milk, fermented milk, lactic acid bacteria beverages, yogurt, butter, cheese, ice cream, etc.), and processed fruits (canned and bottled fruits, jam, marmalade, dried fruit, etc.). ), confectionery and desserts (biscuits, baked goods, rice crackers, fried sweets, Japanese fresh confectionery, Western fresh confectionery, semi-fresh confectionery, Japanese dried confectionery, candies, chocolates, chewing gum, snack foods, jellies, frozen desserts, etc.), beverages (soft drinks, carbonated drinks, fruit juices, coffee drinks, vegetable juices, tea-based beverages, non-alcoholic beverages, alcoholic beverages, etc.), seasonings (sauces, dipping sauces, dressings, etc.), soups, tofu, noodles, bread, plant-based alternative meat and dairy products (alternative cheese, alternative fermented dairy products), roux (curry roux, stew roux, etc.), nutritional supplements and beverages (protein powder, protein drinks, supplements, energy drinks, etc.), pet food, and pet nutritional supplements. Furthermore, as a novel protein-derived material, it is used in a wide range of industries, including cosmetics, medical supplies, microcapsule materials, and carriers for immobilized enzymes.

[0041] 3. Protein modifiers The present invention further provides a protein modifier that can be used for protein deamidation. The protein modifier of the present invention is typically used in the protein deamidation method, the method for producing deamidated proteins, or the method for producing food or pharmaceuticals. The protein modifier of the present invention contains salts and / or polysaccharides as active ingredients in addition to a protein deamidation enzyme. Details of the protein deamidation enzyme, salts and polysaccharides are as described above and will not be explained further. Although not limited to this example, the protein deamidation enzyme content is 1 to 2000 U, preferably 10 to 1500 U, more preferably 50 to 1000 U per gram of formulation. The salt content is 0.1 to 10 g, preferably 0.2 to 9 g, more preferably 0.5 to 8 g per 1 U of protein deamidation enzyme. The polysaccharide content is 0.01 to 10 mg, preferably 0.05 to 5 mg, more preferably 0.1 to 1 mg per 1 U of protein deamidation enzyme. Furthermore, the final form of the protein modifier may be liquid or solid (including powder). Protein modifiers may contain, in addition to the active ingredient, excipients, buffers, suspending agents, stabilizers, preservatives, antiseptics, physiological saline solution, etc. [Examples]

[0042] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0043] <Example 1> To investigate whether the deamidation reaction of protein glutaminase is promoted by the presence of salts, experiments were conducted using pea protein (product name: NOW® Sports Organic Pea Protein) as a substrate. 10% by weight (final concentration) of pea protein, protein glutaminase (product name: Protein Glutaminase "Amano" 500, manufactured by Amano Enzyme Co., Ltd.), and 0.5% (W / V%) of sodium citrate (final concentration) were mixed in purified water. The pH was adjusted to 7.5 using hydrochloric acid or sodium hydroxide, and the mixture was incubated at 52.5°C for 1.5 hours to allow the reaction to proceed. After the reaction was complete, the ammonia liberated by protein glutaminase was quantified as follows: The reaction solution was stopped by treating it at 85°C for 10 minutes. 1 mL of the reaction solution was centrifuged (13,000 rpm, 10 min), and the supernatant obtained was diluted 100-fold with ultrapure water. Ammonia was quantified using the Ammonia Assay Kit (Sigma-Aldrich). For the enzyme concentration, 7.5 U of protein glutaminase (final concentration) was used per gram of substrate protein. The deamidation rate was calculated by dividing the amount of ammonia released per gram of pea protein by protein glutaminase by the amount of ammonia released by acid hydrolysis of 1 gram of pea protein (reacting in 6N hydrochloric acid (final concentration), 110°C, for 24 hours). For comparison, the deamidation rate was also measured by performing the enzymatic reaction under the same conditions as above, except that sodium citrate was not added.

[0044] The results are shown in Table 1. Reacting protein glutaminase in the presence of sodium citrate significantly improved the deamidation rate of pea protein compared to the absence of salts. [Table 1]

[0045] <Example 2> We investigated whether the deamidation reaction of protein glutaminase is promoted by the presence of inorganic salts. The reaction and evaluation were carried out in the same manner as in Example 1, except that 1.0% (W / V%) of dipotassium hydrogen phosphate (DKP) (final concentration) was used instead of 0.5% (W / V%) of sodium citrate (final concentration). For comparison, the enzymatic reaction was carried out under the same conditions as above, except that dipotassium hydrogen phosphate was not added, and the deamidation rate was measured.

[0046] The results are shown in Table 2. Reacting protein glutaminase in the presence of DKP significantly improved the deamidation rate of pea protein compared to the absence of inorganic salts. [Table 2]

[0047] <Example 3> The effects of treatment pH and inorganic salt concentration on promoting the deamidation reaction by inorganic salts were investigated. The reaction and evaluation were carried out in the same manner as in Example 2, except that the pea protein was changed (in this example, product name: PURIS Pea Protein 870, manufactured by PURIS Foods) and the treatment pH and dipotassium hydrogen phosphate (DKP) concentration were changed. The pH was adjusted using hydrochloric acid or sodium hydroxide.

[0048] The results are shown in Table 3. Under all pH conditions (pH 6.5 to 8.5), regardless of DKP concentration, the deamidation rate of pea protein by protein glutaminase improved in the presence of DKP compared to the absence of DKP. [Table 3]

[0049] <Example 4> We investigated whether the deamidation reaction of protein glutaminase is promoted by the presence of polysaccharides. The reaction and evaluation were carried out in the same manner as in Example 1, except that the pea protein was changed (in this example, product name: PURIS Pea Protein 870, manufactured by PURIS Foods) and gellan gum 0.02% (W / V%) (final concentration) was used instead of sodium citrate 0.5% (W / V%) (final concentration). For comparison, the deamidation rate was measured by performing the enzymatic reaction under the same conditions as above, except that gellan gum was not added.

[0050] The results are shown in Table 4. Reacting with protein glutaminase in the presence of gellan gum significantly improved the deamidation rate of pea protein compared to the absence of gellan gum. [Table 4]

[0051] <Example 5> We investigated whether the deamidation reaction of protein glutaminase is promoted by the presence of polysaccharides and inorganic salts. The reaction and evaluation were carried out in the same manner as in Example 4, except that 1.0% (W / V%) (final concentration) of dipotassium hydrogen phosphate (DKP) was used in addition to 0.02% (W / V%) (final concentration) of gellan gum. For comparison, the enzymatic reaction was carried out under the same conditions as above, except that neither gellan gum nor DKP was added, and the deamidation rate was measured.

[0052] The results are shown in Table 5. Reacting protein glutaminase in the presence of gellan gum and DKP significantly improved the deamidation rate of pea protein compared to the absence of polysaccharides and DKP. [Table 5]

[0053] <Example 6> We investigated whether the deamidation reaction of protein glutaminase is promoted by the presence of polysaccharides and polyphosphates. The reaction and evaluation were carried out in the same manner as in Example 5, except that sodium polyphosphate (sodium hexametaphosphate) (SPP) 0.5% (W / V%) (final concentration) was used instead of dipotassium hydrogen phosphate (DKP) 1.0% (W / V%) (final concentration). For comparison, the deamidation rate was measured by performing the enzymatic reaction under the same conditions as above, except that neither gellan gum nor SPP was added.

[0054] The results are shown in Table 6. Reacting protein glutaminase in the presence of gellan gum and SPP significantly improved the deamidation rate of pea protein compared to the absence of polysaccharides and SPP. [Table 6]

[0055] <Example 7> The reaction between Z-Gln-Gly (Benzyloxycarbonyl-L-glutaminylglycine) and protein glutaminase was carried out using a solvent prepared by supplementing a solution containing disodium hydrogen phosphate with sodium dihydrogen phosphate or sodium carbonate, and the effect of supplementing with sodium dihydrogen phosphate or sodium carbonate on the reaction efficiency of deamidation was evaluated. Specifically, a solution was prepared by adding Z-Gln-Gly (final concentration 60 mM) to McIlbine buffer (pH 7.0, containing 16.5 mM disodium hydrogen phosphate and 1.8 mM citrate), and then supplementing with sodium dihydrogen phosphate or sodium carbonate (final concentration of the added amount 0-1.0 M) to adjust the pH to 7.0. To this solution, 0.1 ml of enzyme solution containing 0.13 U / ml of protein glutaminase (product name: Protein Glutaminase "Amano" 500, manufactured by Amano Enzyme Co., Ltd.) was added, and the reaction was carried out at 37°C for 20 minutes. Subsequently, 1.0 ml of 0.4 M trichloroacetic acid solution was added to stop the reaction. Next, the amount of ammonia liberated by protein glutaminase was measured using an ammonia measurement kit (product name: Ammonia Test Wako, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). The amount of liberated ammonia under conditions without supplementation of sodium dihydrogen phosphate or sodium carbonate (i.e., containing only 16.5 mM disodium hydrogen phosphate as a salt) was set as 100%, and the relative amount of liberated ammonia when sodium dihydrogen phosphate or sodium carbonate was supplemented was determined.

[0056] The results are shown in Figure 1. The results showed that when sodium dihydrogen phosphate or sodium carbonate was added to a solution containing disodium hydrogen phosphate, the deamination reaction efficiency improved in a concentration-dependent manner with respect to the sodium dihydrogen phosphate or sodium carbonate. In particular, when sodium carbonate was added to a solution containing sodium hydrogen phosphate, the deamination reaction efficiency improved dramatically, indicating that the deamination reaction efficiency can be improved even when sodium carbonate is used alone as a salt.

[0057] <Example 8> The reaction efficiency of deamidation was evaluated by using a solvent prepared by supplementing a solution containing disodium hydrogen phosphate with sodium carbonate to carry out reactions with various substrate proteins and protein glutaminase. Specifically, a solution was prepared by adding egg albumin, milk casein, chickpea protein, pea protein, soy protein, or wheat gluten (final concentration 1.0 W / V%) to McIlbein buffer (pH 7.0, containing 16.5 mM disodium hydrogen phosphate and 1.8 mM citrate), and then supplementing with sodium carbonate (final concentration 0.5 M) to adjust the pH to 7.0. To this solution, 0.1 ml of enzyme solution containing 0.13 U / ml of protein glutaminase (product name: Protein Glutaminase "Amano" 500, manufactured by Amano Enzyme Co., Ltd.) was added and the reaction was carried out at 37°C for 20 minutes. Subsequently, 1.0 ml of 0.4 M trichloroacetic acid solution was added to stop the reaction. Next, the amount of ammonia released by protein glutaminase was measured using an ammonia measurement kit (product name: Ammonia Test Wako, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.). The same measurement was performed under conditions without the addition of sodium carbonate (i.e., containing only 16.5 mM disodium hydrogen phosphate as a salt), and the relative value of the amount of ammonia released when sodium carbonate was added was determined, with the amount of ammonia released under these conditions set to 100.

[0058] The results are shown in Figure 2. These results indicate that adding 0.5 M sodium carbonate to a solution containing disodium hydrogen phosphate promotes the deamination of substrate proteins by protein glutaminase. Furthermore, in the reaction system with added salts, the deamination reaction by protein glutaminase was promoted for both plant and animal proteins, and the effect of promoting the deamination reaction was particularly high for plant proteins.

[0059] <Example 9> The thermal stability of protein glutaminase was evaluated using a solvent prepared by supplementing a solution containing disodium hydrogen phosphate with sodium carbonate. Specifically, a solution was prepared by supplementing a McIlbine buffer (pH 7.0, containing 41.2 mM disodium hydrogen phosphate and 4.4 mM citrate) with sodium carbonate (final concentration 1.0 M) to adjust the pH to 7.0. Protein glutaminase (product name: Protein Glutaminase "Amano" 500, manufactured by Amano Enzyme Co., Ltd.) was added to this solution at a concentration of 0.13 U / ml and incubated at 50°C for 120 minutes or at 60°C for 60 minutes. The activity of protein glutaminase was measured over time. The activity of protein glutaminase was determined using the method described above. The residual activity of protein glutaminase was calculated by setting the activity value of protein glutaminase before incubation as 100%. For comparison, the test was conducted under the same conditions as above, except that 1.0 M sodium carbonate was not added, and the activity of protein glutaminase was measured over time.

[0060] The results are shown in Figure 3. These results confirm that the thermal stability of protein glutaminase is improved in the presence of sodium carbonate. [Industrial applicability]

[0061] The protein deamide method of the present invention promotes the deamide reaction and improves reaction efficiency. The deamided proteins obtained by the present invention have commercial value in themselves and can also be used as materials or ingredients in various foods, beverages, or pharmaceuticals. In other words, the present invention is particularly expected to have applications in the food and beverage and pharmaceutical fields.

[0062] This invention is not limited in any way to the descriptions of embodiments and examples of the invention described above. Various modifications are also included in this invention, provided that they do not depart from the scope of the claims and are easily conceivable by those skilled in the art. The contents of papers, published patent gazettes, and other documents explicitly mentioned herein shall be cited in their entirety.

Claims

1. A method for deamidering a protein, comprising acting a protein glutaminase on a protein in the presence of gellan gum.

2. The protein deamide method according to claim 1, wherein protein glutaminase is applied to a protein in the presence of gellan gum and salts.

3. The protein deamide method according to claim 2, wherein the salt is at least one selected from the group consisting of carbonates, phosphates, hydrogen phosphates, polyphosphates, and citrates.

4. The protein deamide method according to any one of claims 1 to 3, wherein the protein is a plant protein and / or an animal protein.

5. A protein modifier containing gellan gum and protein glutaminase.

6. Furthermore, the protein modifier according to claim 5, further containing salts.

7. The protein modifier according to claim 6, wherein the salts are carbonates, phosphates, hydrogen phosphates, polyphosphates, or citrates.

8. A method for producing a deamidated protein, comprising the following steps: (1) A step of preparing a solution containing protein and gellan gum, (2) The process of treating the prepared solution with protein glutaminase.

9. A method for producing food or pharmaceuticals, including the following steps: (1) A step of preparing a food ingredient or pharmaceutical ingredient containing protein and a solution containing gellan gum, (2) The process of treating the prepared solution with protein glutaminase.

10. The manufacturing method according to claim 8 or 9, wherein the solution prepared in step (1) further contains salts.

11. The manufacturing method according to claim 10, wherein the salt is at least one selected from the group consisting of carbonates, phosphates, hydrogen phosphates, polyphosphates, and citrates.

12. The method for producing a protein according to any one of claims 8 to 11, wherein the protein is a plant protein and / or an animal protein.

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