How to Generate Cysteine ​​from Glutathione

Abstract

The present invention addresses the problem of providing a practical means for producing cysteine from glutathione, said means being suited for use in the field of foodstuffs. The present invention produces cysteine from glutathione using a first step in which a γ-glutamyl peptide hydrolase derived from a microorganism acts on reduced glutathione to produce cysteinylglycine, and a second step in which an acid protease derived from a microorganism acts on the cysteinylglycine to produce cysteine.

Description

[Technical field]

[0001] The present invention relates to a method for producing cysteine ​​from glutathione and its use. This application claims priority based on Japanese Patent Application No. 2019-124052 filed on July 2, 2019, the entire contents of which are incorporated by reference. [Background technology]

[0002] Amino acids and peptides in food are important not only as nutrients, but also in the taste and flavor of food. For example, glutamic acid and aspartic acid have umami and sour tastes, glycine, alanine, threonine, etc. have sweet tastes, and tryptophan, isoleucine, valine, etc. have bitter tastes. Some amino acids also react with other components (sugars, lipids, etc.) to produce unique flavors. A specific example is cysteine, whose Maillard reaction product imparts a meaty flavor. For example, heat treatment of foods or ingredients containing cysteine ​​can enhance the umami flavor. Adding cysteine ​​to foods and other foods and carrying out the same treatment can be expected to impart or enhance the umami flavor. However, cysteine ​​is mainly extracted from animal-derived raw materials (hair and feathers), and the use of cysteine ​​as an additive in foods and other foods is not preferred.

[0003] Incidentally, some foods contain relatively large amounts of glutathione, a cysteine ​​compound. Therefore, if cysteine ​​could be produced from glutathione, the above problems associated with the addition of cysteine ​​could be resolved.

[0004] In vivo, glutathione is decomposed stepwise by enzymes (see, for example, Non-Patent Documents 1 and 2). Specifically, first, γ-glutamyltranspeptidase cleaves the γ-glutamyl bond of glutathione to generate glutamic acid (Glu) and the dipeptide cysteinylglycine (CysGly). Next, CysGly is decomposed by dipeptidase to generate cysteine ​​(Cys) and glycine (Gly). If such two-step decomposition in vivo can be utilized in foods, etc., it would be possible to generate cysteine ​​from a component (glutathione) originally contained in foods, etc., and to impart or enhance flavor. Here, as a technology related to the first-step reaction, it has been proposed to generate CysGly from glutathione using microbial glutaminase to enhance the flavor of foods, etc. (Patent Document 1). On the other hand, regarding the second-step reaction, there have been reports of the decomposition of CysGly using enzymes derived from animals (e.g., rats, rabbits, pigs, and horses) (Non-Patent Documents 1 and 2). However, it is difficult to use animal-derived enzymes in food, etc. There have been no successful examples of decomposition of CysGly using enzymes suitable for use in the food industry. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 4453057 Public Relations [Non-patent literature]

[0006] [Non-Patent Document 1] J. Biol. Chem. 1950, 186:731-735. [Non-Patent Document 2] J. Biol. Chem. 1937, 120:209-217. Summary of the Invention [Problem to be solved by the invention]

[0007] Although the production of cysteine ​​from glutathione that is originally present in foods etc. is a promising means for imparting or enhancing the flavor of foods etc., it has not yet been realized. In view of this situation, an object of the present invention is to provide a practical means for producing cysteine ​​from glutathione that is also suitable for use in the food industry (food applications). [Means for solving the problem]

[0008] Under the above-mentioned circumstances, the present inventors have conducted extensive research aimed at efficiently producing cysteine ​​from glutathione using microbial enzymes, while considering applications in the food industry. As a result, they have succeeded in efficiently producing cysteine ​​from glutathione by using glutaminase, a type of γ-glutamyl peptide hydrolase, in combination with acid protease. They have also found particularly effective reaction conditions and information useful for practical application. Based on these results, the following inventions are provided. [1] In the first step, microbial gamma-glutamyl peptide hydrolase acts on reduced glutathione to produce cysteinylglycine; a second step in which a microbial acid protease acts on the cysteinylglycine to produce cysteine; 13. A method for producing cysteine ​​from glutathione, comprising: [2] The method according to [1], wherein a microbial γ-glutamyl peptide hydrolase and a microbial acidic protease are added to a glutathione-containing composition or a solution thereof, or a glutathione solution, and then the first step is carried out by reacting at a pH of 3 to 9 and a temperature of 15°C to 70°C, and then the reaction solution is adjusted to a pH of 2 to 7 and reacted at a temperature of 20°C to 70°C, thereby carrying out the second step. [3] The method according to [1], wherein the first step is carried out by adding a microbial γ-glutamyl peptide hydrolase to a glutathione-containing composition or a solution thereof, or a glutathione solution, and then reacting at a pH of 3 to 9 and a temperature of 15°C to 70°C, and then adding a microbial acidic protease to the reaction solution, and reacting at a pH of 2 to 7 and a temperature of 20°C to 70°C, thereby carrying out the second step. [4] The method according to claim 1, wherein the first step and the second step are carried out by adding a microbial γ-glutamyl peptide hydrolase and a microbial acidic protease to a glutathione-containing composition or a solution thereof, or a glutathione solution, and then reacting them at a pH of 3 to 6 and at a temperature of 15°C to 70°C. [5] The method according to [2] or [3], wherein the reaction conditions for the first step are pH 4 to 8 and a temperature of 30°C to 60°C, and the reaction conditions for the second step are pH 3 to 6 and a temperature of 20°C to 60°C. [6] The method according to [2] or [3], wherein the reaction conditions for the first step are pH 5 to 7 and a temperature of 30°C to 50°C, and the reaction conditions for the second step are pH 3 to 5 and a temperature of 30°C to 50°C. [7] The method according to any one of [1] to [6], wherein the microbial γ-glutamyl peptide hydrolase is glutaminase, γ-glutarate transferase or γ-glutamyl cyclotransferase. [8] The method according to any one of [1] to [6], wherein the microbial γ-glutamyl peptide hydrolase is a glutaminase derived from a microorganism of the genus Bacillus. [9] The method according to any one of [1] to [6], wherein the microbial γ-glutamyl peptide hydrolase is glutaminase derived from Bacillus amyloliquefaciens.

[10] The method according to any one of [1] to [9], wherein the microbial acid protease is an Aspergillus microbial acid protease.

[11] The method according to any one of [1] to [9], wherein the microbial acid protease is an Aspergillus oryzae-derived acid protease.

[12] The method according to any one of [1] to

[11] , wherein the glutathione-containing composition is meat, a processed meat product, seafood, a processed seafood product, a vegetable, a processed vegetable product, yeast extract or a meat extract. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present invention relates to a method for producing cysteine ​​from glutathione (hereinafter, also referred to as the "cysteine ​​production method of the present invention"). The cysteine ​​production method of the present invention is highly versatile and can be used for the purpose of producing cysteine ​​from glutathione present in food or ingredients, obtaining cysteine ​​from purified or crudely purified glutathione, etc. As described below, in the present invention, foods or ingredients containing glutathione, or glutathione solutions, etc. are treated (processed materials), and it is sufficient that at least a part of the glutathione in the processed material is in a form (e.g., reduced form) that can be decomposed by the enzyme (microorganism-derived γ-glutamyl peptide hydrolase) used in the present invention.

[0010] The cysteine ​​production method of the present invention uses roughly two types of enzymes to produce cysteine ​​from glutathione through a two-step cleavage or decomposition reaction. Specifically, cysteine ​​is produced from glutathione through a step (first step) in which a microbial γ-glutamyl peptide hydrolase acts on reduced glutathione to produce cysteinylglycine, and a step (second step) in which a microbial acid protease acts on the produced cysteinylglycine to produce cysteine.

[0011] The first step is a reaction in which the γ-glutamyl bond of reduced glutathione is cleaved by the action of a microbial γ-glutamyl peptide hydrolase to produce cysteinylglycine (CysGly) (glutamic acid is also produced as a by-product). As long as the cleavage of the γ-glutamyl bond of reduced glutathione can be achieved, the microbial γ-glutamyl peptide hydrolase is not particularly limited. Examples of microbial γ-glutamyl peptide hydrolase include glutaminase, γ-glutamyltransferase, and γ-glutamylcyclotransferase derived from Bacillus microorganisms. An example of glutaminase derived from Bacillus microorganisms is glutaminase derived from Bacillus amyloliquefaciens (e.g., glutaminase SD-C100S provided by Amano Enzyme Inc.). The microbial γ-glutamyl peptide hydrolase does not need to be a purified product, and for example, a culture solution, a disruption solution / extract, or a partially purified product thereof of a microorganism producing the γ-glutamyl peptide hydrolase may be used. Two or more types of microbial γ-glutamyl peptide hydrolases may be used in combination. Some microbial γ-glutamyl peptide hydrolases are commercially available (for example, the above-mentioned glutaminase SD-C100S) and are easily available and usable.

[0012] The second step is a reaction in which CysGly produced in the first step is decomposed by the action of a microbial acid protease to produce cysteine ​​(glycine is also produced as a by-product). As long as the decomposition of CysGly can be achieved, the microbial acid protease is not particularly limited. An example of the microbial acid protease is an acid protease derived from a microorganism of the genus Aspergillus. An example of the acid protease derived from a microorganism of the genus Aspergillus is an acid protease derived from Aspergillus oryzae (for example, Protease M "Amano" SD and Protease HF "Amano" 150SD provided by Amano Enzyme Co., Ltd.). The microbial acid protease does not need to be a purified product, and for example, a culture solution, a disruption solution / extract, or a partially purified product thereof of a microorganism producing an acid protease may be used. Two or more types of microbial acid proteases may be used in combination. In addition, several acid proteases derived from microorganisms are commercially available (for example, the above-mentioned Protease M "Amano" SD and Protease HF "Amano" 150S) and are easily available and usable.

[0013] In the present invention, it is sufficient that the first step is followed by the second step in the entire process. Thus, in one aspect (first aspect) of the present invention, a microbial γ-glutamyl peptide hydrolase and a microbial acid protease are added to a glutathione-containing composition or a solution thereof, or a glutathione solution, and then the first step is carried out by reacting under conditions in which the microbial γ-glutamyl peptide hydrolase acts (referred to as "first conditions"), and then the conditions are changed to conditions in which the microbial acid protease acts (referred to as "second conditions"), and the second step is carried out by reacting. According to this aspect, it is not necessary to add an enzyme (microbial acid protease) midway, improving workability.

[0014] A glutathione-containing composition or a solution thereof is used as one of the objects to be treated. The glutathione-containing composition is not particularly limited, and may be, for example, a food or food ingredient containing glutathione. The term "food ingredient" is interpreted in a broad sense, and includes natural food materials such as meat, fish, and vegetables, as well as processed products, extracts, and seasonings thereof. Specific examples of food and food ingredients include meat, processed meat products, seafood, processed seafood products, vegetables, processed vegetable products, yeast extract, and meat extract.

[0015] In addition to the glutathione-containing composition or its solution, the glutathione solution can also be the subject of the present invention. Usually, glutathione is dissolved in a suitable solvent (e.g., water) to prepare a glutathione solution. Glutathione is produced by extraction from yeast or organic synthesis, and is provided (commercially available) as a reagent, medicine, pharmaceutical raw material, etc. As described above, at least a part of the glutathione in the subject must be in reduced form, and usually, reduced glutathione is used to prepare the glutathione solution.

[0016] The conditions under which the microbial γ-glutamyl peptide hydrolase acts, i.e., the first conditions, are, for example, pH 3 to 9 and reaction temperature 15°C to 70°C. The pH is preferably 4 to 8, more preferably 5 to 7. The reaction temperature is preferably 30°C to 60°C, more preferably 30°C to 50°C. The reaction time and the amount of enzyme are not particularly limited as long as the expected action is exhibited. An example of the reaction time is 5 minutes to 48 hours. The amount of enzyme is such that the concentration of the microbial γ-glutamyl peptide hydrolase in the reaction solution is, for example, 0.001% (W / W) to 10% (W / W), preferably 0.01% (W / W) to 1% (W / W). When glutaminase derived from Bacillus amyloliquefaciens is used as the microbial γ-glutamyl peptide hydrolase, particularly preferred reaction conditions are pH 5 to 7 and reaction temperature 35°C to 45°C.

[0017] The conditions under which the microbial acid protease acts, i.e., the second conditions, are, for example, pH 2 to 7 and reaction temperature 20°C to 70°C. The pH is preferably 3 to 6, more preferably 3 to 5. The reaction temperature is preferably 20°C to 60°C, more preferably 30°C to 50°C. The reaction time and the amount of enzyme are not particularly limited as long as the expected action is exhibited. An example of the reaction time is 5 minutes to 12 hours. If the reaction time is long, there is a concern that the generated cysteine ​​may be oxidized, so it is preferable to increase the reaction efficiency by increasing the amount of enzyme, optimizing the reaction conditions (especially pH and temperature), etc. The amount of enzyme is such that the concentration of the microbial acid protease in the reaction solution is, for example, 0.001% (W / W) to 10% (W / W), preferably 0.01% (W / W) to 2% (W / W). When the acid protease derived from Aspergillus oryzae is used as the microbial acid protease, particularly preferred reaction conditions are a pH of 3 to 5 and a reaction temperature of 30°C to 50°C.

[0018] When changing from the first conditions to the second conditions, the pH of the reaction solution can usually be adjusted using an acid (e.g., hydrochloric acid, sulfuric acid, nitric acid, acetic acid) or an alkali (e.g., sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, calcium hydroxide, magnesium hydroxide).

[0019] The produced cysteine ​​is recovered or purified as necessary. For example, when a glutathione solution is used as the substance to be treated, recovery / purification is performed to obtain highly pure cysteine. For example, column chromatography can be used for the recovery / purification.

[0020] In another aspect (second aspect) of the present invention, a microorganism-derived γ-glutamyl peptide hydrolase is added to a glutathione-containing composition or its solution, or a glutathione solution, and then the first step is carried out by reacting under conditions in which the microorganism-derived γ-glutamyl peptide hydrolase acts (referred to as "first conditions"). Thereafter, a microorganism-derived acidic protease is added to the reaction solution, and the reaction is carried out under conditions in which the microorganism-derived acidic protease acts (referred to as "second conditions") to carry out the second step. According to this aspect, the microorganism-derived acidic protease is not present in the reaction solution during the first step (cleavage of the γ-glutamyl bond by the microorganism-derived γ-glutamyl peptide hydrolase), and there is no risk of the microorganism-derived γ-glutamyl peptide hydrolase being decomposed (digested) by the microorganism-derived acidic protease. Therefore, good or efficient cleavage of the γ-glutamyl bond by the microorganism-derived γ-glutamyl peptide hydrolase can be expected. The first and second conditions of the second aspect are respectively equivalent to the first and second conditions of the above-mentioned first aspect. The reaction time and the amount of enzyme are similar.

[0021] In another embodiment (third embodiment) of the present invention, the microbial γ-glutamyl peptide hydrolase and the microbial acid protease are added to the glutathione-containing composition or its solution, or the glutathione solution, and then the reaction is carried out under conditions in which both the microbial γ-glutamyl peptide hydrolase and the microbial acid protease can act. That is, the first step and the second step are carried out simultaneously. According to this embodiment, there is no need to add the enzyme during the reaction, and there is no need to change the reaction conditions, improving workability. Conditions in which the microbial γ-glutamyl peptide hydrolase and the microbial acid protease can act simultaneously include, for example, pH 3 to 6 and a reaction temperature of 20°C to 70°C. The pH is preferably 4 to 6. The reaction temperature is preferably 20°C to 60°C, more preferably 30°C to 50°C. The reaction time and the amount of enzyme are not particularly limited as long as the expected action is exhibited. An example of the reaction time is 5 minutes to 24 hours. If the reaction time is long, there is a concern that the generated cysteine ​​may be oxidized, and therefore it is preferable to increase the reaction efficiency by increasing the amount of enzyme, optimizing the reaction conditions (particularly pH and temperature), and the like. EXAMPLES

[0022] The following experiments were conducted with the aim of establishing a method for efficiently producing cysteine ​​from glutathione, with the aim of application in the food industry.

[0023] 1. Cysteine ​​production by single enzyme treatment (1) Method 0.020mL of 5% (w / v) enzyme solution was added to 1.0mL of 50mmol / L reduced glutathione, and the mixture was left to stand at 37℃ for 16 hours, after which it was analyzed by HPLC. The amount of cysteine ​​produced was evaluated based on the ratio of the L-cysteine ​​peak area to the total peak area. The enzymes used are shown in the following table (Table 1). [Table 1]

[0024] (2) Results Almost no L-cysteine ​​was produced by single enzyme treatment (Table 2). [Table 2]

[0025] 2. Cysteine ​​production by the combined use of glutaminase and various enzymes (1) Method 0.02 mL of 5% (w / v) enzyme solution was added to 1.0 mL of 50 mmol / L reduced glutathione (pH 4, 5, 6, or 7), and the mixture was incubated at 37°C for 12 hours, after which it was analyzed by HPLC. The amount of cysteine ​​produced was evaluated as the ratio of the L-cysteine ​​peak area to the total peak area.

[0026] (2) Results At pH 4 to 6, the combination of glutaminase and acid protease efficiently produced cysteine ​​(Table 3). [Table 3]

[0027] 3. Consideration of processing conditions (1) Method 0.8mL of 5% (w / v) glutaminase solution was added to 40mL of 50mmol / L reduced glutathione (pH 6.0), and the mixture was left to stand at 37℃ for 12 hours. The pH was then adjusted to 4.0 with hydrochloric acid, and the mixture was diluted to 50mL. Protease HF "Amano" 150SD was added to a final concentration of 0.5% (w / v) or 2.0% (w / v), and the mixture was left to stand at 37℃ or 50℃ for 4 to 24 hours, after which it was analyzed by HPLC. The amount of cysteine ​​produced was evaluated as the ratio of the peak area of ​​L-cysteine ​​to the total peak area.

[0028] (2) Results The more protease used, the faster the cysteine ​​was produced. Also, the amount of cysteine ​​produced was greater at 50°C than at 37°C (Table 4). [Table 4]

[0029] 4. Consideration of yield improvement (1) Method 100 mg of glutaminase SD-C100S and 400 mg of protease HF "Amano" 150SD were added to 9 mL of 0.153 g (w / v) reduced glutathione (pH 6.0) (final concentration 50 mM), and the mixture was left to react at 37°C for 3 hours, after which the pH was adjusted to 4.0 and the mixture was left to react at 37°C or 50°C for another 3 hours. The reaction solution was analyzed by HPLC, and the concentration of L-cysteine ​​produced was calculated.

[0030] (2) Results Approximately 40 mM cysteine ​​was produced from 50 mM reduced glutathione (Table 5). Even when the reaction temperature was set to 50°C after adjusting the pH to 4.0, a sufficient amount of cysteine ​​was produced, but the amount produced was slightly less than that at 37°C. It is presumed that the high temperature reaction caused the oxidation of SH groups, resulting in a decrease in concentration. [Table 5] [Industrial Applicability]

[0031] The present invention is useful, for example, for increasing the amount of cysteine ​​in food or food ingredients. In other words, according to the present invention, food or food ingredients with increased cysteine ​​content can be produced. Therefore, the present invention can also be understood as a method for producing a composition (e.g., food, food ingredients) with increased cysteine ​​content. In addition to increasing the amount of cysteine ​​in food or food ingredients, the present invention can also be used for the purpose of obtaining cysteine ​​from purified or crudely purified glutathione. Thus, the present invention is highly versatile and is expected to be used in a variety of fields.

[0032] The present invention is not limited to the above-mentioned embodiment 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 publications, patent publications, etc. specified in this specification are all incorporated by reference.

Claims

1. a first step in which a microbial gamma-glutamyl peptide hydrolase acts on reduced glutathione to produce cysteinylglycine; A second step of producing cysteine ​​by reacting the cysteinylglycine with a microbial acid protease, the first step and the second step are carried out by adding a microbial γ-glutamyl peptide hydrolase and a microbial acid protease to a glutathione-containing composition or a solution thereof, or a glutathione solution, and then reacting them at a pH of 4 to 6; A method for producing cysteine ​​from glutathione.

2. The method according to claim 1, wherein the microbial γ-glutamyl peptide hydrolase is glutaminase, γ-glutarate transferase or γ-glutamyl cyclotransferase.

3. The method according to claim 1 or 2, wherein the microbial γ-glutamyl peptide hydrolase is a glutaminase derived from a microorganism of the genus Bacillus.

4. The method according to any one of claims 1 to 3, wherein the microbial γ-glutamyl peptide hydrolase is glutaminase derived from Bacillus amyloliquefaciens.

5. The method according to any one of claims 1 to 4, wherein the microbial acid protease is an Aspergillus microbial acid protease.

6. The method according to any one of claims 1 to 5, wherein the microbial acid protease is an Aspergillus oryzae-derived acid protease.

7. The microbial gamma-glutamyl peptide hydrolase is a glutaminase derived from a microorganism of the genus Bacillus, The method according to any one of claims 1 to 6, wherein the microbial acid protease is an Aspergillus microbial acid protease.

8. The method of claim 7, wherein the reaction is carried out at 30 to 50°C.

9. The microbial gamma-glutamyl peptide hydrolase is glutaminase derived from Bacillus amyloliquefaciens, The method according to any one of claims 1 to 6, wherein the microbial acid protease is an Aspergillus oryzae-derived acid protease.

10. The method of claim 9, wherein the reaction is carried out at 30 to 50°C.

11. The method according to any one of claims 1 to 10, wherein the glutathione-containing composition is meat, a processed meat product, seafood, a processed seafood product, a vegetable, a processed vegetable product, yeast extract or a meat extract.

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