JPH043959B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing glutathione, and more specifically, it has the ability to produce glutathione from L-glutamic acid, L-cysteine and/or L-cytetin, and glycine in the presence of adenosine triphosphate (hereinafter referred to as ATP). Using bacteria and bacteria that have the ability to convert adenosine diphosphate (hereinafter referred to as ADP) to ATP, L-glutamic acid, glycine, L-cysteine and/or L-
The present invention relates to a method for producing glutathione from cystine. Conventionally known methods for producing glutathione include an extraction method from yeast cells and a method for producing glutathione from the specific amino acid by enzymatic reaction using the enzymatic activity of microorganisms. In this enzymatic reaction, the presence of ATP is required, but ATP is expensive. A method for producing glutathione using yeast having the ability to regenerate ATP is known (Japanese Patent Application Laid-Open No. 53-94090). As a result of studying methods for producing glutathione using bacteria capable of regenerating ATP, we found that when producing glutathione from bacteria that can produce glutathione from L-glutamic acid, glycine, L-cysteine and/or L-cystine and ATP,
It has been found that by using bacteria capable of producing ATP from ADP as an ATP source, it is possible to produce a significant amount of glutathione. According to the present invention, the following A and B can be replaced by C, D, H,
Glutathione can be obtained in good yield by contacting in an aqueous medium containing H and G. B: Bacterial cultures, cells or processed products thereof that have the ability to produce glutathione from L-glutamic acid, L-cysteine and/or L-cystine, and glycine B: L-glutamic acid, L-cysteine and/or L-cystine and glycine: Energy donor (E) D: Phosphate ion (P) E: Magnesium ion (Mg) F: Surfactant and/or organic solvent G: From ADP in the presence of E, P, and Mg Bacterial cultures, bacterial cells, or processed products thereof that have the ability to produce ATP. As the glutathione-producing bacteria used in the present invention, any bacteria that produces glutathione from the above-mentioned specific amino acids in the presence of ATP can be used. Specific examples include bacteria of the genus Escherichia, genus Alcaligenes, and genus Azotobacter, such as Escherichia coli ATCC 11303, Escherichia coli FERM BP48, Alcaligenes viscolactis ATCC 9036, and Azotobacter vinlandii ATCC 12518. These bacteria may be cultured in the same manner as normal bacterial culture. That is, either a synthetic medium or an organic medium can be used as long as the medium contains appropriate amounts of carbon sources, nitrogen sources, inorganic substances, and other nutrients. As the carbon source, various carbohydrate raw materials such as gucose, glycerol, fructose, sucrose, maltose, mannose, mannitol, xylose, galactose, starch, starch hydrolyzate, and molasses can be used. Furthermore, various organic acids such as pyruvic acid, acetic acid, and lactic acid, and various amino acids such as aspartic acid and alanine can also be used. Nitrogen sources include ammonia or various inorganic and organic ammonium salts such as ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium nitrate, ammonium carbonate, ammonium acetate, or urea and other nitrogen-containing compounds, as well as peptone, NZ amine, meat extract, yeast extract. ,
Various nitrogenous organic substances such as corn steep liquor, casein hydrolyzate, fish meal or its digested product, defatted soybean meal or its digested product, pupa hydrolyzate, aspartic acid, glutamic acid, threonine, etc. can be used. Furthermore, as inorganic substances, potassium dihydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium carbonate, etc. are used. Fermentation is carried out under aerobic conditions such as shaking culture or submerged culture with aeration and stirring. The culture temperature is 20-40°C.
It is desirable to adjust the pH of the medium during culture to around neutrality. As the neutralizing agent, aqueous ammonia, sodium hydroxide, ammonium carbonate, calcium carbonate, etc. can be used. The culture solution can be used as it is, or the bacterial cells can be separated from it. As a treatment for bacterial cells, washing,
Treated bacterial cells that have been subjected to treatments such as drying, freezing, lyophilization, acetone drying, contact with organic solvents or surfactants, lysozyme treatment, ultrasonication, mechanical grinding, etc. can be used. Furthermore, enzyme protein fractions having enzyme activity for producing glutathione from L-glutamic acid, L-cysteine, and glycine obtained from these bacterial cells or processed bacterial cells can also be used. Furthermore, immobilized products of bacterial cells, insolubilized products of treated bacterial cells, etc. can also be used. Energy donors include carbohydrates such as glucose, fructose, sucrose, molasses, precipitated hydrolysates, pyruvic acid, lactic acid, acetic acid, α
- Organic acids such as ketoglutaric acid, amino acids such as glycine, alanine, and aspartic acid are used. These are usually used in concentrations of approximately 1 to 200 g/g. The concentration of phosphate ions and magnesium ions in the aqueous solution is preferably in the range of 0.1 to 70 g/in, and if they are insufficient, they are replenished so that the concentration falls within this range. As the phosphate ion, either sodium salt, potassium salt, etc. can be used. As the magnesium ion, either an inorganic salt or an organic acid salt can be used. Bacterial cells having the ability to regenerate ATP include any bacteria that has the ability to generate ATP from ADP in an aqueous solution containing an energy donor, phosphate ions, magnesium ions, and a surfactant and/or an organic solvent. The following are examples of typical bacteria that can be used: Brevibacterium ammoniagenes ATCC21170 Corynebacterium glutamicum ATCC21171 The bacterial cells are used at a wet bacterial cell concentration of 1 to 500 g/preferably 40 to 300 g/. The ATP-regenerating bacteria may be cultured in the same manner as described above using the medium used for culturing the glutathione-producing bacteria. The surfactant used is polyoxyethylene stearylamine (for example, Nymeen S).
-215, manufactured by NOF KK), cationic surfactants such as alkyldimethylbenzylammonium chloride, alkylisoquinolium bromide, anionic surfactants such as dioctyl sodium sulfosuccinate, alkyl amines, alkyldimethylamines Amine-based surfactants such as
It is used at a concentration of 0.1 to 50 g/, preferably 1 to 20 g/. The organic solvent used is xylene, toluene, benzene, etc., and its concentration is 0.1 to 50 ml/, preferably 1 to 20 ml/. The reaction for producing glutathione from L-glutamic acid, glycine, and L-cysteine and/or L-cystine is carried out in an aqueous solution with appropriate stirring for 1 to 48 hours, usually 2 to 24 hours. More preferable results can be obtained by carrying out the reaction while controlling the temperature and pH within the ranges of 10 to 70°C and PH of 4 to 10, respectively. If necessary, raw material amino acids may be added as necessary during the reaction. By carrying out the reaction under the above conditions, glutathione is produced and accumulated in the reaction solution. As a method for simple purification of glutathione from a reaction solution, a conventional resin method, copper salt method, etc. can be applied. Aspects of the present invention will be explained below with reference to Examples. Example 1 1 Culturing of glutathione-producing bacteria Escherichia coli FERM BP48 is used.
glucose 10g/, yeast extract 10g/, peptone 10g/, meat extract 5g/,
300 ml of a medium containing 1 g of MgSO ₄ .7H ₂ O and 5 g of KH ₂ PO ₄ and adjusted to pH 7.0 is placed in a 2-fold Erlenmeyer flask and sterilized by heating. The bacteria, which had been precultured in a yeast bouillon medium, was inoculated into this, and cultured with shaking at 30°C overnight. The resulting culture solution was centrifuged to collect the bacterial cells and incubated at -20°C.
Freeze in a refrigerator. 2 Cultivation of ATP regenerating bacterial cells Brevibacterium ammoniagenes
Use ATCC21170. Glucose 15%, urea
0.5% (separate sterilization), KH ₂ PO ₄ , K ₂ HPO ₄ ,
_MgSO4・_7H2O , each 1.0%, _CaCl2・_2H2O0.01
%, biotin 30 μg/, yeast extract 0.5%, meat extract 0.5%, and adjusted to pH 7.2.
-Pour 300ml into a folded Erlenmeyer flask and sterilize by heating. This is inoculated with bacteria pre-cultured in yeast bouillon medium and shaken overnight at 30°C. Bacterial cells are collected from the obtained culture solution by centrifugation and stored frozen in a -20°C freezer. 3. Glutathione production reaction 20 ml of the reaction solution having the composition shown in Table 1 is placed in a 200 ml beaker, stirred at 950 rpm with a magnetic stirrer while maintaining the temperature at 37° C. and pH 7.4, and the reaction is carried out for 6 hours. As a result, 9.8 m
M glutathione is produced. The amount of glutathione produced when Brevibacterium cells were removed from the above reaction solution was 1.6 mM.

[Table] * Dry bacterial weight 4 Isolation and purification of glutathione The centrifuged supernatant of reaction solution 1 (containing 10mM glutathione) obtained by the same reaction as above was concentrated under reduced pressure and added to Amberlite IR-120 (H ⁺ type) column to adsorb glutathione, wash with water and elute with 0.1NH ₂ SO ₄ , concentrate the eluate and add ethanol to obtain crystals. Further, recrystallization is performed using ethanol to obtain 2.0 g of purified glutathione. Example 2 The cells are cultured in the same manner as in Example 1, except that the bacteria shown in Table 2 are used as glutathione-producing strains, and the resulting cells are frozen. Using this bacterial cell, a reaction was carried out in the same manner as in Example 1, and the results are shown in Table 2.

[Table] Shows the amount of glutathione produced when removed.
Example 3 The same glutathione-producing bacterial cells as in Example 1 and
The ATP-regenerating bacterial strain was cultured in the same manner as in Example 1, and the glutathione-producing bacteria were collected, a part of which was frozen.
In addition, the remainder is used as is for each reaction,
ATP regenerating bacteria use the broth as is for the reaction. Add Escherichia coli cells, glucose, phosphoric acid, magnesium sulfate, K ₂ SO ₄ , Nymeen, and xylene to 14 ml of culture solution of ATP-regenerating bacteria [300 g (wet bacterial weight) 1] to a final concentration of Table 1. The reaction was carried out in the same manner as in Example 1, with the liquid volume adjusted to 20 ml and the results are shown in Table 3.

[Table] Example 4 The same procedure as in Example was carried out except that Corynebacterium glutamicum ATCC21171 was used as the ATP regenerating bacterium, yielding 3.52 mM glutathione. Note that when Corynebacterium was not added, the amount of glutathione produced was 1.47 mM.

Claims (1)

[Scope of Claims] 1. A method for producing glutathione, which comprises bringing the following A and B into contact in an aqueous medium containing C, D, E, H, and G. B: Bacterial cultures, cells or processed products thereof that have the ability to produce glutathione from L-glutamic acid, L-cysteine and/or L-cystine, and glycine B: L-glutamic acid, L-cysteine and/or L-cystine and glycine: Energy donor (E) D: Phosphate ion (P) E: Magnesium ion (Mg) F: Surfactant and/or organic solvent G: Adenosine and Bacterial cultures, bacterial cells, or processed products of bacteria that have the ability to produce adenosine/triphosphate from diphosphate