JPH0424994B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing glutathione. Glutathione is an important peptide that is used medicinally as a treatment for liver diseases or as an antidote. Methods for producing glutathione include organic synthesis,
A fermentation method for intracellular accumulation and a microbial or enzymatic conversion method are known. As an organic synthesis method, L-glutamic acid and L-
When condensing cysteine and glycine, a step of introducing and removing a protecting group is necessary, making it a complicated method. As a method for accumulation in bacteria by fermentation, a method using yeast and adding L-glutamic acid, L-cysteine, or glycine (Japanese Unexamined Patent Publication No. 48
-92579, 53-94089), but since the enzyme system involved in this glutathione production system is a synthetic enzyme that requires ATP, it is necessary to perform glutathione production at the same time as culturing to produce ATP. is essential, and since the produced glutathione is accumulated in small amounts within the bacterial body, it is difficult to collect glutathione. As a microbial or enzymatic conversion method, a method in which microorganisms belonging to the genus Pseudomonas, Proteus, Bacillus, Enterobacter, and Erwinia are allowed to act on L-glutamic acid, L-cysteine, and glycine in an aqueous medium (Japanese Patent Application Laid-open No. 57−
2698) or a method in which microorganisms belonging to the genus Escherichia are reacted in the same manner as above (JP-A-58-
(20188), but since the enzyme system involved in the glutathione production system requires ATP, the addition of expensive ATP was essential for the reaction. Against this background, the present inventors
As a result of intensive research to find an inexpensive method for producing glutathione that does not require the addition of ATP and does not require the production of glutathione while culturing bacterial strains, we have found that microorganisms are incubated with L-glutamic acid in an aqueous medium that does not contain ATP. The present inventors have discovered that glutathione can be efficiently produced simply by acting on L-cysteine and glycine, leading to the completion of the present invention. That is, the present invention is directed to Achromobacter, Serratia, Flavobacterium, Agrobacterium, Escherichia, Citrobacter, Corynebacterium, Zarcina, Nocardia, Cytelomyces, Paxoren, and Trigonopsis. , Schizoblastosporion spp., Debaryomyces spp., Trichosporon spp., Candeida spp., Rhodotorula spp., Satucharomyces spp., Pychia spp., Cryprococcus spp., Geotrichum spp., Torulopsis spp., Hansenula spp., Alcaligenes spp.
Belongs to the genus Brevibacterium, Arthrobacter, Micrococcus, and Kluyveromyces L
- Glutathione, which is characterized in that a microorganism capable of condensing glutamic acid, L-cysteine, and glycine to produce glutathione is made to act on L-glutamic acid, L-cysteine, and glycine in an aqueous medium to produce glutathione. This is the manufacturing method. A method for converting L-glutamic acid, L-cysteine, and glycine into glutathione by allowing a microorganism capable of producing glutathione to act on L-glutamic acid, L-cysteine, and glycine is to prepare L-glutamic acid, L-cysteine, glycine, and the bacterial cells of the above microorganism in an aqueous medium. It may be brought into contact with a culture solution or a treated bacterial cell material. L-glutamic acid and L- used in the present invention
Examples of microorganisms that have the ability to condense cysteine and glycine into glutathione include:

[Table] To produce glutathione by allowing the cells of these microorganisms to react, it is sufficient to use a normal medium and react L-glutamic acid, L-cysteine, and glycine using the cells after culturing. . The medium used in the method using the cultured bacterial cells is a normal medium containing L-glutamic acid, L-cysteine, and glycine, as well as normal carbon sources, nitrogen sources, and inorganic ions. Additionally, desirable results can often be obtained by adding organic micronutrients such as vitamins and amino acids. As the carbon source, carbohydrates such as glucose and sucrose, organic acids such as acetic acid, alcohols, and others are used as appropriate. As the nitrogen source, ammonia gas, aqueous ammonia, ammonium salt, and others are used. As the inorganic ions, magnesium ions, phosphate ions, potassium ions, iron ions, and others are used as appropriate. Cultivation is carried out under aerobic conditions, pH 4 to 9, temperature 25
1 to 10 while controlling the temperature within an appropriate range of 40℃ to 40℃.
The desired results can be obtained by culturing for 1 day. As bacterial cells, the culture solution as it is after cultivation is completed,
Both bacterial cells isolated from the culture solution and washed bacterial cells can be used. The bacterial cell treatments include freeze-dried bacterial cells, acetone-dried bacterial cells, bacterial cells that have been brought into contact with toluene, surfactants, etc., bacterial cells that have been treated with lysozyme, bacterial cells that have been exposed to ultrasound, and mechanically ground bacterial cells. In addition to bacterial cells, enzyme protein fractions having enzyme activity that converts L-glutamic acid, L-cysteine, and glycine obtained from these processed bacterial cells into glutathione, as well as immobilized products of these bacterial cells, bacterial cells, etc. Insolubilized products of treated products and others can also be used. As the water-soluble medium, those containing water, buffers, and organic solvents such as ethanol can be used. Furthermore, as necessary, nutrients necessary for the growth of microorganisms,
Antioxidants, surfactants, coenzymes, hydroxylamine, metal ions, etc. can also be added to the aqueous medium. The method of bringing the culture solution, cultured cells, or treated bacterial cells of the above microorganisms into contact with L-glutamic acid, L-cysteine, and glycine to cause them to act includes the following:
Adjust an aqueous medium in which glutamic acid, L-cysteine, glycine, culture solution, cultured bacterial cells, or treated bacterial cells are dissolved or suspended to an appropriate temperature of 10°C to 70°C, and maintain the pH between 4 and 9. It may be left standing for a while or stirred. Thus, after 5 to 100 hours, a large amount of glutathione is produced and accumulated in the aqueous medium. The generated glutathione can be separated and purified by a known separation method. The generated glutathione was measured using an amino acid analyzer. Example 1 Glucose 2.0g/dl, (NH ₄ ) ₂ SO ₄ 0.5g/
dl, KH ₂ PO ₄ 0.1g/dl, K ₂ HPO ₄ 0.1g/dl,
MgSO ₄・7H ₂ O 0.05g/dl, FeSO ₄・7H ₂ O 1
mg/dl, MnSO ₄・4H ₂ O 1mg/dl, yeast extract
Medium containing 1.0g/dll, peptone 1.0g/dll, calcium carbonate 4.0g/dll, (separately sterilized) (PH7.0)
50 ml of the mixture was placed in a 500 ml flask and sterilized at 120°C for 15 minutes. One platinum loopful of the microorganisms listed in Table 2 that had been cultured on a bouillon agar medium at 30°C for 24 hours was inoculated into this, and the mixture was incubated at 30°C for 20 hours.
Cultured for hours. Bacterial cells were collected from this culture solution by centrifugation, washed once with physiological saline in the same amount as the culture solution, and collected. These bacterial cells were added to reaction solution A shown in Table 1 at 5 g/dl.
(final pH 6.0, 5 ml) and heated to 37℃.
The reaction was maintained for 16 hours. The glutathione produced at this time was converted to the tetrathionate method (Fahey, RC; J. Bacteriol. 121: 144-
151, 1975) and are shown in Table 2.

【table】

[Table] Conspicyura

[Table] S. fragilis

Claims (1)

[Claims] 1 Achromobacter sp., Serratia sp., Flavobacterium sp., Agrobacterium sp., Escherichia sp., Citrobacter sp., Corynebacterium sp., Zarutina sp., Nocardia sp., Sitelomyces sp., Baxoren sp., Trigonopsis sp., Schizoblastospo Lyon, Debaryomyces, Trichosporon, Candeida, Rhodotorula,
It belongs to the genera Satucharomyces, Vichia, Cryptococcus, Geotrichum, Torulopsis, Hansenula, Alcaligenes, Brevibacterium, Arthrobacter, Micrococcus, and Kluyveromyces, and in the absence of ATP. Microorganisms that have the ability to produce glutathione from L-glutamic acid, L-cysteine, and glycine
By acting on L-glutamic acid, L-cysteine, and glycine in an aqueous medium that does not contain ATP,
A method for producing glutathione, characterized by producing glutathione.