JPH0147154B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing glutathione, and more specifically, a method for producing glutathione by an extraction method from the cells of a microorganism belonging to the genus Escherichia in which the control mechanism of the glutathione synthase system has been released. Regarding. Glutathione is a tripeptide consisting of L-glutamic acid, L-cysteine and glucine,
It is a substance useful as a treatment for liver diseases, an antidote, etc. It is also a substance useful as a biochemical reagent. Conventionally, methods for producing glutathione using microorganisms include a method of extracting glutathione from yeast cells, and a method of contacting dry yeast with good membrane permeability with a substrate solution containing L-glutamic acid, L-cysteine, and glycine. There are known methods for producing glutathione, and methods for conjugating an ATP regeneration system when producing glutathione by contacting yeast or Escherichia coli cells with a substrate solution. However, when viewed as an industrial production method, these methods do not necessarily produce a satisfactory amount of glutathione. In view of this situation, the present inventors have conducted various studies to find a method for industrially advantageous production of glutathione using microorganisms, and have found that glutathione belongs to the genus Escherichia. The present inventors have discovered that microorganisms that have the activity and whose γ-glutamylcysteine enzyme is no longer inhibited by glutathione accumulate a significant amount of glutathione within their cells, leading to the completion of the present invention. That is, the present invention is a method for producing glutathione, which comprises culturing the above-mentioned microorganism and extracting glutathione from the microorganisms thus obtained. The microorganism used in the present invention belongs to the genus Escherichia and has γ-glutamylcysteine synthase (E.
C.6.3.2.2., hereinafter referred to as GSH-) activity and glutathione synthase (EC6.3.2.3., hereinafter referred to as GSH-) activity, and has glutathione-mediated GSH- Any microorganism that has been released from inhibition can be used, and a typical example of such a microorganism is GSH-
Suitable examples include Escherichia coli which has GSH-activity and GSH-activity and in which the inhibition of GSH- by glutathione has been released. The above bacterial strain can be obtained, for example, as follows. First, GSH-activity and a wild strain of E. coli with GSH-activity (e.g.
A cysteine-requiring strain and a methylglyoxal-resistant strain are obtained by inducing mutations in Escherichia coli B355). Mutations can be induced by conventional mutagenesis treatments, such as N-methyl-
This can be carried out by treatment with a mutagenic agent such as N'-nitro-N-nitrosoguanidine. To obtain a cysteine auxotroph, the resulting strain is subjected to mutagenesis in a minimal medium containing 2×10 ^-5 M cysteine (e.g., K ₂ HPO ₄ 0.7%, KH ₂ PO ₄ 0.3%,
It can be obtained by culturing in a medium containing 0.1% (NH ₄ ) ₂ SO ₄ and 0.5% glucose (hereinafter referred to as DM medium) and separating the resulting small colonies. On the other hand, a methylglyoxal-resistant strain can be obtained by culturing the strain obtained by the above mutagenesis treatment in a DM medium containing methylglyoxal, and separating the resulting large colonies by fishing. Next, the above-mentioned methylglyoxal-resistant strain is cultured in a minimal medium containing the cysteine-requiring strain obtained above, and colonies that do not form a halo around the colony are isolated to obtain a GSH-deficient strain (e.g., Escherichia coli C912). get. Next, mutations were induced in this GSH-deficient strain using the same treatment methods as described above, followed by culturing it in a DM medium containing 8-hydroxyquinoline, and the resulting colonies were isolated by fishing to induce GSH-deficient strains.
- activity and GSH- activity, and in which the inhibition of GSH- by glutathione has been released. Examples of bacterial strains obtained in this way include, for example, Escherichia coli.
An example is RC912 (Feikoken Jokyo No. 47). The medium used for culturing the microorganism of the present invention obtained as described above includes a carbon source,
Either a synthetic medium or a natural medium can be used as long as it contains a suitable amount of nitrogen sources, inorganic substances, etc. As the carbon source, various hydrocarbons such as glucose, sucrose, fructose, starch, starch hydrolyzate, and molasses can be used, and the amount used is preferably about 0.5 to 5.0%. Examples of nitrogen sources include various inorganic and organic ammoniums such as ammonium sulfate, ammonium phosphate, ammonium carbonate, and ammonium acetate;
Alternatively, nitrogenous organic substances such as peptone, yeast extract, corn steep liquor, and casein hydrolyzate can be used, and the amount used is preferably about 0.5 to 2.0%. Further, as the inorganic substance, for example, potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, manganese sulfate, etc. can be used, and the amount used is preferably about 0.005 to 0.5%. The culture is preferably carried out under aerobic conditions such as shaking culture or aeration agitation culture. The suitable culture temperature is 25-37℃, and the culture period is usually 16-40℃.
About an hour is enough. Thus, a significant amount of glutathione accumulates within the bacterial cells. After culturing, glutathione inside the bacterial cells is extracted. Extraction of glutathione can be easily carried out by heating extraction with water. Glutathione can be isolated from the extract by treating the extract with a known method such as ion exchange resin treatment. Examples of the present invention will be shown below. Example 1 The strains shown in Table 1 below were mixed with 0.5% glucose, 0.3% potassium hydrogen phosphate, 0.7% potassium dihydrogen phosphate, and 0.01% magnesium sulfate heptahydrate.
%, ammonium sulfate 0.1% (PH
7.0) and cultured with shaking at 37°C for 16 hours.
After culturing, collect the bacterial cells by centrifugation and reduce to 0.85%.
After washing with physiological saline, glutathione in the bacterial cells was extracted by heating with water. The amount of glutathione in the extract was quantified, and the amount of glutathione accumulated (μmole) per 1 g (wet weight) of bacterial cells was calculated. The results are shown below.
It was as shown in the table. 【table】

Claims (1)

[Scope of Claims] 1. A microorganism belonging to the genus Escherichia that has γ-glutamylcysteine synthetase activity and glutathione synthetase activity and in which inhibition of γ-glutamylcysteine synthetase by glutathione has been released is cultured, A method for producing glutathione, which comprises extracting glutathione from collected bacterial cells. 2. The production according to claim 1, wherein the microorganism is Escherichia coli which has γ-glutamylcysteine synthetase activity and glutathione synthetase activity, and in which inhibition of γ-glutamylcysteine synthetase by glutathione has been released. Law.