JPS6248396A - Production of glutathione - Google Patents

Abstract

PURPOSE:To produce glutathione easily and inexpensively in high yield, by cultivating a filamentous, theremostable mold of the genus Phormidium of blue-green algae in a specific culture solution and collecting formed and accumulated glutathione. CONSTITUTION:A live cell of a filamentous, thermostable mold of the genus Phormidium of blue-green algae is used as a biological material and cultivated in any of various artificial medium such as modified Fitzgerald medium, etc., or a culture solution of natural hot spring water (e.g., hot spring water of MATSUE) or a phosphoric acid buffer solution as a fundamental medium preferably under a light condition by feeding L-glutamic acid, L-cysteine and glycine. Glutathione is formed and accumulated in the culture solution and the cell is collected. In order to promote biosynthesis and accumulation of glutathione, preferably about 15-100mM borate is added to the culture solution. The borate can be formed in the solution by adding boric acid, depending upon a basic substance contained in the culture solution or base added to adjust pH.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a method for producing glutathione, particularly to a method for producing glutathione by biosynthesis using filamentous thermophilic cyanobacteria.

Glutathione is a biochemically important tripeptide that is involved in protecting sulfhydryl groups (SH groups) of biomolecules such as proteins, maintaining normal redox potential, and active transport of amino acids in living organisms. Furthermore, recently it has been reported that glutathione is involved in the control of cell division, and it is also attracting attention from the aspect of anti-cancer properties. fact,
Glutathione is currently used in pharmaceuticals as a liver tonic, partly because it has clinically strong detoxifying effects.

(Prior art) Chemical synthesis methods, biosynthesis methods, etc. have been industrialized to date to produce highly useful glutathione, but they have many drawbacks in terms of reaction steps and economic efficiency. have. An enzymatic synthesis method has also been developed, but since it requires expensive ATP, the question is how to increase the efficiency of ATP use in the production of glutathione, and how to efficiently regenerate and reuse ATP. The big question is how to get it. Published by Kagaku Dojin Co., Ltd., Kagaku Special Edition No. 103 (June 20, 1984) No. 123-13
On page 6, Mokumitsu et al. have developed an Escherichia coli strain with enhanced glutathione-producing ability through genetic engineering technology, and report on its use and usefulness. According to their report, the maximum yield of glutathione obtained by culturing under optimal conditions is around 2% per liter of culture solution. Although culturing E. coli is easy to manage operationally, it requires aseptic operation, and it is necessary to add various types of carbohydrates, amino acids, vitamins, etc. as nutrients to the culture medium. In this example, an external supply of ATP is required to increase the production of glutathione.

(Problems to be Solved by the Invention) In view of the many problems associated with the conventional production methods of various glutathione as mentioned above, the present inventors have conducted intensive research and discovered that a specific filamentous thermophilic cyanobacterium Phormidium (Phormid
By utilizing the glutathione biosynthesis ability of bacteria of the genus M.ium, we have succeeded in solving all of these existing problems, and have arrived at the present invention.

That is, the object of the present invention is to produce glutathione easily, inexpensively, and with high yield using a biosynthetic method without requiring the addition of expensive external energy, complicated sterilization operations, or strict sterile control. There is a way to get it.

(Means for Solving the Problems) The above object is to cultivate a filamentous thermophilic cyanobacterium Phormidium in a culture solution to which monoglutamic acid, L-cysteine and glycine are added as raw material substrates, Biosynthesis/
This is achieved by a method for producing glutathione, which is characterized by collecting accumulated glutathione.

More specifically, the present invention uses living cells of a filamentous thermophilic cyanobacterium of the genus Phormidium as a biological material, various artificial media such as Fitzgerald's modified medium, natural hot spring water culture solution, or Glutathione is biosynthesized using a phosphate buffer or the like as a basic medium, supplying L-glutamic acid, L-cysteine, and glycine under light or dark conditions, preferably light conditions, preferably in the presence of borate. This is a method for producing glutathione, in which glutathione is produced and accumulated in the culture medium and cells, and then collected.

Generally, cyanobacteria are prokaryotic organisms like E. coli, but
It has photosynthetic ability and can grow and multiply using only light energy as external energy. That is, aerobic
Under light conditions, it grows by fixing carbon dioxide gas, so the necessary factors for growth are light energy, carbon dioxide gas,
Water and inorganic salts are sufficient.

As a result of various studies on such cyanobacteria, the present inventors have found that filamentous thermophilic cyanobacteria Phormidium spp., collected and isolated from the Matsue Onsen hot spring source area, are photosynthetic under aerobic light and dark conditions. It was confirmed that Phormidium PM has the ability to efficiently produce and accumulate glutathione, and it was named Phormidium-PM (hereinafter abbreviated as this bacterium).

The bacterial deposit number at Honkan's Institute of Microbial Technology, Agency of Industrial Science and Technology is Microtechnology Research Institute No. 8332 (deposited on July 6, 1985)
It has the following mycological properties.

■ Growth status in each medium 1. Liquid culture (1) Hot spring water enriched medium Temperature: 47°C; Air aeration rate: 1 liter of culture solution/1.5 liters per minute; Light intensity: Culture conditions of 2.000 lux , a steady state is reached in 10 to 14 days. This medium shows the best growth among the media used so far. The orchid mass has a film-like or mantle-like shape, and is attached to a substrate or floats in water, but it can be cultured more or less uniformly by proper stirring.

(2) Figgeerard modified medium The growth rate is as good as the hot spring water enriched medium.

Other diamonds (Dyer) and gaffor (Gaffor)
Although it is possible to grow on the improved medium d) and the BG-11 medium, the growth situation is slightly inferior compared to cases (1) and (2) above.

2. Agar flat culture (1) Hot spring water enriched medium After applying living cyanobacterial cells (inoculum) to an agar flat surface in a petri dish, growth can be visually confirmed in 4 to 5 days. Further, good growth is shown after 10 to 14 days. The light intensity at this time is 1. Same as (1), it is 2,000 lux.

■Various physiological and ecological properties 1, optimal growth conditions Optimum growth pH 7.8-8.8 Optimum temperature 45-50℃ Optimum light intensity 2.000-3.000 lux Optimum C
D□ concentration: about 5%2, conditions for growth: pH 5-10, temperature: 10-55°C, light intensity: 500-30,000 lux3, notable characteristics: Only carbon dioxide gas (CD□) is used as a carbon source.

Although any of the NO3, N02 and NH4 nitrogen sources can be used as a nitrogen source, NO+ gives the best results for growth.

2.6 in the state of living cells. Photoreduction of redox dyes such as dichlorophenol indophenol or methyl viologen is possible.

As for chlorophyll, it has only chlorophyll a and does not contain chlorophyll b.

■, Morphological properties Regarding living cyanobacterial cells grown in hot spring water enriched medium,
Observation using ordinary microscopic specimens revealed that the algal bodies consist of filamentous bodies called trichomes, do not branch, do not produce exospores or endospores, and do not have heterocysts. The trichomes are straight or slightly bent, with a width of about 1.5 μm, and no constrictions are observed at the junctions between cells. The length of the cells varies from 3 to 6 μm depending on the growth stage, and the tips of filamentous cell groups do not become thin and are generally round. The cell content is homogeneous with few granules.

Based on the above properties, this bacterium is Phormidium rapideum (P
hormidium lapideum).

■Medium composition Both are used after being adjusted to pH 8.0.

1. Hot spring water enriched medium KNO31g K2HPO40.02g Mg5L ・7H200.25g Fe50. o, 005g hot spring water
1 liter2. Fitzgerald's modified medium 1 cup per knot, KNO31g K21 (Pct4Q, 03g!, IgS04 7H200, 25gcac
+2・2L0 0.036gNaHCO30,8
40g Na2S+03.9H200゜058g iron citrate
0.006g Citric acid 0.006g εDTA 0.001g A3 solution
1,0m1 3, Dia and Gafoud improved medium (inorganic medium for thermophilic cyanobacteria) MgSL 0.15g KH, Po, 0.25g K2+1PO40,25g NaN03o, 5g KNO30,5g CaCL 0.06g83B
O30,034g εDT8・Na
0.03g (NH4) 6Mo7024 ・Old 1
20 0.022gFeC1:+ ・6H2
0θ, 016g1,1nc12・41-120
0.004gCu5O<・5820
0.0019g2nSO4・7820
0.00066gCo(NO3)2.6H201,5
xlO-5g Distilled water 1 liter 4,8G-11 NaNO+ 1.5gK2HP0
．． 0.04g! J g S O4・
7H200,075gCaCL ・'2H2Q
O, (136g citric acid 0.
006g Iron ammonium citrate 0.006g EDT
A -Mg 0.001gNa2C0
3o, 02g A, solution 1mβ distilled water 1 liter This bacteria is 47~
It is adapted to temperatures around 50°C, and therefore, when culturing this bacterium at this temperature, strict sterilization and aseptic control are not required as with E. coli, and ATP is not photophosphorized. It can self-produce and regenerate through oxidation reactions.

In the method of the present invention, living cells of the present invention are placed in an enriched medium such as an artificial culture medium such as the above-mentioned Fitzgierard, a natural culture medium such as Matsue hot spring water, or a buffer solution such as a phosphate buffer at a bacterial cell concentration of approximately Suspend to a concentration of 200 to 500 mg/ml and culture aerobically, preferably around pH 7.0 to 8.0.

To promote the biosynthesis and accumulation of glutathione, approximately 1
5+nM to about Loom! , l, preferably about 30mM
It is preferable to add about J degree of borate to the culture solution.

A boric acid salt can be formed in a culture solution by adding boric acid to the culture solution, using a basic substance contained in the culture solution, or a base added to adjust pH and JI. Examples of borates include sodium borate (sodium tetraborate), potassium borate (potassium tetraborate), ammonium borate, and the like.

To the above culture solution, monoglutamic acid, L-instine, and glycine, which are amino acid units constituting glutathione, are added as raw material substrates. The appropriate amount to add is 5~
The amount corresponds to a concentration of 20mM.

Although the bacterial cells suspended in the culture medium thus prepared produce a considerable amount of glutathione even under dark conditions, their biosynthetic ability increases markedly under light conditions. Therefore, in the method of the present invention, it is best to expose the culture solution to intermittent or continuous light irradiation during cell culture, such as a visible light source, such as a daylight fluorescent light, a fluorescent light for plant cultivation, a tungsten incandescent light bulb, or sunlight. preferable. The light intensity of the irradiating visible light source is 1.0
About 00 to 10,000 lux, preferably 2,000
A range of ~3,000 lux is good. In addition, in order to obtain good results, the temperature of the culture solution during light irradiation should be kept at room temperature to about 5
It is important to maintain the temperature within a medium to high temperature range of 5°C.

When aerobic culture, such as shaking, aeration, stirring culture, etc., is carried out under the above-mentioned light conditions, the bacterial cells actively produce glutathione and accumulate it within the bacterial cells and in the culture solution.

In the method of the present invention, there is no need to supply ATP as external energy because water locks have the ability to self-produce ATP, but by adding about 2 m) of ATP, the yield of glutathione, especially in the dark, can be improved. The yield under the conditions can be improved.

70% of the total amount of glutathione synthesized in this way is
Since glutathione is secreted and accumulated in the culture solution, glutathione can be easily harvested by gently fractionating the living cells used. Furthermore, glutathione accumulated in cells can be easily and efficiently harvested by hot water extraction from bacterial cells. Furthermore, when hot water extraction is not performed, the living cells can be returned to the reaction tank and used again to produce glutathione under the same conditions as above.

(Function) In carrying out the method of the present invention, as described above, an appropriate amount of hydrophilic cells is suspended in the above culture solution, the pH is adjusted to a suitable range using boric acid, caustic alkali, aqueous ammonia, etc., and Add predetermined amounts of raw substrates, namely L-glutamic acid, L-cystine and glycine. This liquid is subjected to aerobic culture by shaking culture or a tank culture method involving aeration, stirring, etc. while continuously or intermittently irradiating the solution with moderate intensity light from a visible light source as described above. During cultivation, the temperature is maintained at the appropriate temperature, and the pH is periodically adjusted three times to maintain the pH at an appropriate value.

During the above operation, this bacterium uses the inorganic salts in the culture solution as a nutrient source and uses light energy to fix carbon dioxide gas, using its photosynthetic ability to grow and multiply. Utilizing self-produced and self-regenerated ATP through oxidation reactions, it biosynthesizes amino acids as raw material substrates, produces and accumulates glutathione within cells, and at the same time secretes and accumulates it in the culture medium. I am going.

The above biosynthetic reaction can be carried out to some extent under dark conditions, but the yield of glutathione under dark conditions is, for example, 2.5
45-55% of the yield under light conditions under 00 lux irradiation
Therefore, it can be seen that the effect of the above-mentioned light irradiation on the reaction mechanism is large.

In addition, although the above-mentioned glutathione production reaction proceeds even without the addition of borate, borate has a strong reaction promoting effect, and for example, the addition of about 30mM boric acid reduces the glutathione yield without the addition of boric acid. It has been confirmed that this increases to 170-180% of that in the case of

Furthermore, as mentioned above, since water locks have the ability to self-produce and self-regenerate ATP, unlike conventional methods, addition of ATP is not particularly required, but it has been found that adding it contributes to increased yield. are doing. For example, 2 m! , 1 A
It was experienced that the addition of TP increased the glutathione yield to 107-108% of that without the addition under 2.500 lux light irradiation, while it increased to about 155% under dark conditions.

In this way, glutathione can be produced photosynthetically at a high yield using the orchid ring, or water lock, as a biological material. Approximately 3 milligrams per gram. The substrate conversion rate in this case exceeded 10%, and was about 6% based on the dry weight of cyanobacteria, which is about three times the yield in the conventional yeast method.

(Example) The invention will be further described with reference to the following examples.

Example 1 Approximately 300 mg of living cells of this bacterium were suspended in phosphate buffer (final concentration 50 mM, pH 7, 5, 10 ml), and 10 mM Coconi L-glutamic acid 5 L-cysteine 5 m
Add N (10mM glycine, 30mM borate (all final concentrations), and use a shaking culture apparatus [Taiyo Kagaku Kogyo Co., Ltd.].
Co., Ltd., trade name, Taiyomonocin II'] was cultured for 24 hours at 48° C. and 2.500 lux (National Nano Behomolux Fluorescent Light) with gentle shaking.

After the cultivation was completed, the algal cells were centrifuged and the supernatant liquid was quantified, and it was found that 2.0% of the algal cells per gram (wet weight) of the algal cells.
Accumulated production of glutathione of 5 μIJ grams was observed. In addition, when the algal cells used were subjected to hot water extraction treatment,
An additional 1 milligram of glutathione was obtained.

Example 2 The same water lock as in Example 1 above was suspended in a Matsue hot spring water medium. This hot spring water medium contains 0.02 g of potassium monophosphate, 1 g of potassium nitrate, 0.25 g of magnesium sulfate, and 0.005 g of ferrous sulfate per liter of natural spring water obtained from the Matsue hot spring source. The pH was adjusted to 8.0. In the same manner as in Example 1, borate, L-glutamic acid, L-cystine, and glycine were added to prepare Quiyomonodine [A.

Light was irradiated at 48° C. with 10.000 lux (tungsten incandescent bulb) in an L-shaped test tube. During the reaction process, the pH was adjusted at times to remain around 8. After 12 hours, the reaction supernatant was obtained by centrifugation, and when examined, it was found that 2 milligrams of glutathione was obtained per gram (wet weight) of algal cells. Furthermore, when the algal cells were extracted with hot water, 1 milligram of glutathione was obtained.

However, at this time, without treating the algal cells with hot water, they were returned to the same medium containing amino acids and boric acid as in the beginning and the glutathione production reaction proceeded under light irradiation of 2,500 lux.
After 8 hours of re-reaction, 2 milligrams (per wet weight of 1 gram of blue-green algae cells) of glutathione was harvested from the reaction solution.

(Effects of the Invention) With the advent of the method of the present invention, glutathione, which is a biochemically important tripeptide and is useful as a pharmaceutical and is attracting attention especially from the viewpoint of anticancer properties, can be produced industrially with great ease and cost. It has become possible to manufacture it with great advantage.

That is, the water lock applied to the method of the present invention grows and multiplies by its inherent photosynthetic ability using only inexpensive light energy, carbon dioxide gas, water, and inorganic salts as necessary growth factors, and uses self-produced and self-regenerated ATP. Since it efficiently biosynthesizes and accumulates raw material substrate amino acids, it can be used as a medium for external energy such as various nutrient sources, which has been a major drawback in conventional enzymatic methods and biochemical methods such as the use of modified Escherichia coli through genetic manipulation. does not require the addition of AT, which is particularly expensive.
The fact that the method of the present invention enables efficient production of glutathione at a significantly high yield without requiring the addition of P is that the method of the present invention provides an easy process free from the trouble of special sterilization operations and aseptic control. Together with this, it greatly contributes to improving productivity, that is, reducing production costs.

Claims (1)

[Claims] 1. A filamentous thermophilic cyanobacteria of the genus Phormidium is cultivated in a culture medium to which L-glutamic acid, L-cysteine and glycine are added as raw material substrates, and biosynthesized and accumulated glutathione is collected. A method for producing glutathione, characterized by: 2. The method for producing glutathione according to claim 1, wherein the culturing is performed under irradiation with a visible light source. 3. The light intensity of the visible light source is 1,000 to 10,000.
The method for producing glutathione according to claim 2, which is lux. 4. The method for producing glutathione according to any one of claims 1 to 3, wherein the culture solution is a Fitzgerald artificial medium. 5. The method for producing glutathione according to any one of claims 1 to 3, wherein the culture solution is a natural medium enriched with Matsue hot spring water. 6. The method for producing glutathione according to any one of claims 1 to 3, wherein the culture solution is a phosphate buffer. 7. The method for producing glutathione according to any one of the claims, wherein each of the raw material substrates is added in an amount of 5 to 20 mM. 8. The above culture is maintained at pH 7.0 to 8.0 and at room temperature to 5.
A method for producing glutathione according to any one of the preceding claims, which is carried out at a temperature of 5°C. 9. The method for producing glutathione according to any one of the claims, wherein the culture solution contains borate. 10. The method for producing glutathione according to claim 9, wherein borate is contained in a range of about 15mM to about 100mM. 11. The method for producing glutathione according to any one of the claims, wherein ATP is added to the culture solution. 12. Glutathione according to any of the preceding claims, wherein glutathione accumulated in the culture solution is collected by fractionation of living cells, and glutathione accumulated in bacterial cells is collected by hot water extraction. manufacturing method.