JPH0328195B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing adenosine-5'-triphosphate (hereinafter referred to as "ATP"). ATP is not only directly involved in energy metabolism in the body, but also has an important effect on the metabolism of proteins, carbohydrates, and lipids through coenzymes, and is used as a pharmaceutical.
It is also important as a biochemical research reagent. The method for producing ATP is to biochemically phosphorylate 5'-adenylic acid using yeast (Japanese Patent Publication No.
19119), a method for phosphorylating adenosine using yeast cells treated with a surfactant (Japanese Patent Publication No. 1973-
20759), a method of phosphorylating 5'-adenylic acid using yeast cells that have been subjected to a freeze-thaw treatment (Japanese Patent Publication No. 1983-45197), yeast cells capable of assimilating hydrocarbons, or A method of phosphorylating adenosine or 5'-adenylic acid using the treated product (Tokuko Sho
50-9873), yeast that has the ability to phosphorylate adenosine, 5'-adenylic acid, or adenosine-5'-diphosphate, and that has not been subjected to any treatment that would substantially damage the cell membrane of its bacterial cells. Methods using bacterial cells (Japanese Unexamined Patent Publication No. 55-34036) are known, but all of these methods require expensive raw materials such as adenosine, 5'-adenylic acid, and adenosine-5'-diphosphoric acid. There is a drawback. An industrially cheaper method using adenine as a raw material is a method of culturing microorganisms belonging to Previbacterium ammoniagenes in a medium containing an extremely low concentration of manganese ions (Japanese Patent Publication No. 1973-
26912), by adding metal complex-forming agents such as aliphatic amines, aromatic amines, condensed phosphoric acid, nitrocarboxylic acid, salicylaldehyde and its derivatives, and β-diketones to a medium containing excessive manganese ions. Therefore, a method for cultivating bacteria capable of producing nucleotides by reducing available manganese ions in the medium (Japanese Patent Publication No. 13114/1983),
A method for culturing microorganisms capable of producing ATP in a medium containing a surfactant (Japanese Patent Publication No. 49-28996), a culture solution of yeast or bacteria capable of producing ATP from adenine in the presence of a surfactant Method using body or its processed material
No.) etc. are known. However, none of these methods produce a sufficient amount of ATP, and it is hoped that an industrially cheaper ATP production method will be developed. As a result of studies aimed at improving the conventional ATP production method, the present inventors found that a surfactant was used to generate ATP through the action of bacteria that have the ability to generate ATP from adenine, a phosphate donor, and an energy donor. It has been found that the amount of ATP produced is significantly increased by adding phytic acid to an aqueous medium in the presence of an organic solvent. Furthermore, it has been found that when the above method is carried out in the presence of magnesium ions, the amount of ATP produced is further improved. The present invention will be explained in detail below. In the present invention, energy donors refer to ATP precursors (AMP,
A compound that provides the energy necessary to biosynthesize ATP from ADP). The microorganisms used in the present invention include adenine, phosphate donors, and energy donors.
Any microorganism that has the ability to produce ATP can be used. Furthermore, mutant strains obtained by mutating these microorganisms can also be used as long as they have the above-mentioned ATP-producing ability. Preferred strains belong to the genus Corynebacterium or Brevibacterium, and a specific example of a strain is Brevibacterium ammoniagenes.
ATCC21170 and Corynebacterium glutamicum ATCC21171. The medium used for culturing the microorganism may be either a natural medium or an artificial medium as long as it contains a suitable amount of carbon source, nitrogen source, and organic and inorganic nutrient sources. Carbon sources used for culture include carbohydrates such as glucose, fructose, sucrose, molasses, and starch hydrolysates, hydrocarbons such as normal paraffin and kerosene, alcohols such as methanol, ethanol, glycerin, and sorbitol, pyruvic acid, lactic acid, Organic acids such as acetic acid, amino acids such as glycine, alanine, glutamic acid, aspartic acid, etc. are used, and the concentration used is 5 to 30%.
% is preferred. The carbon source referred to here is something that microorganisms require for growth, and is distinguished from the energy donor required for the reaction that produces ATP. However, if there is an excess carbon source in the medium and the carbon source is brought into the ATP production reaction,
The carbon source is used as an energy donor. Nitrogen sources used for culture include various inorganic and organic ammonium salts such as ammonia, ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium sulfate, and urea, nitrogen-containing substances such as peptone, meat extract, protein hydrolyzate, and fish meal. , usually used at a concentration of 0.1-10%. Inorganic substances used in the culture medium include phosphates such as monopotassium phosphate, dipotassium phosphate, monosodium phosphate, disodium phosphate, and ammonium phosphate, sulfates, hydrochlorides, and nitrates of magnesium, manganese, calcium, iron, zinc, etc. is used in appropriate amounts. Furthermore, if the microorganism is an auxotrophic strain, compounds required for growth must be added to the culture solution. The culture conditions for microorganisms include a temperature of 20 to 40°C, under aerobic conditions such as shaking and aeration, and adjusting the pH to around neutrality using ammonia, aqueous ammonia, urea, caustic soda, caustic potash, etc. While, 1~
Cultivate for 8 days. The microorganism culture thus obtained can be used for the reaction as it is, or the culture may be subjected to various treatments and the treated product obtained may be used for contact reaction with adenine and a phosphate donor. . Processed products include concentrated or dried culture products, liquid or bacterial cells obtained by processing cultures with a centrifuge, dried bacterial cells, acetone-treated products, surfactant-treated products, and sonicated products. Examples include lytic enzyme-treated products, immobilized bacterial cells, and enzyme preparations extracted from bacterial cells. The contact reaction can be carried out in any aqueous medium, but it is economically advantageous to add an adenine phosphate donor, an energy donor, a surfactant, or an organic solvent, phytic acid, or magnesium ion to the medium during microbial culture. Examples of methods include accumulating the target substance in the culture solution by allowing the target substance to exist in the culture medium, or carrying out a contact reaction after the completion of culture. In the former case, necessary components such as adenine may be present in the medium at the time of inoculation or during culture, but the surfactant and/or organic solvent may be added afterwards. In the latter case, adenine, a phosphate donor, an energy donor, a surfactant and/or an organic solvent are added to the culture, bacterial cells, or their processed material after cultivation, and other substances are added if necessary. In addition, under aerobic conditions at 20-50℃
The target product can be accumulated by reacting for 48 hours. At this time, it is desirable to adjust the pH to 6-8. In either case, the concentration of adenine is 1 to 100 g/, preferably 1 to 40 g/. Energy donors include carbohydrates such as glucose, fructose, sucrose, molasses, and starch hydrolysates, pyruvic acid, lactic acid, acetic acid, and α-
Organic acids such as ketoglutaric acid, amino acids such as glycine-alanine, aspartic acid, and glutamic acid are used. These are used in concentrations of approximately 5 to 150 g/. As the phosphate donor, any of the phosphates used in the culture medium for the aforementioned bacteria can be used. These are 1~
Used at a concentration of 100g/. The surfactants used include cationic surfactants such as polyoxyethylene stearylamine (e.g. Nymeen S-215, NOF K.
anionic surfactants such as sodium lauryl sulfate, sodium oleylamide sulfate, nonionic surfactants such as polyoxyethylene sorbitan monostearate (e.g. Nonion ST221, NOF
amphoteric surfactants such as lauryl betaine (e.g. Anon BF, manufactured by NOF KK);
These are usually used at a concentration of 0.1 to 50 g/, preferably 1 to 20 g/. As the organic solvent, toluene, xylene, aliphatic alcohol, benzene, ethyl acetate, etc. are used, and the concentration thereof is 0.1 to 50 ml, preferably 1
~20ml/ is good. The phytic acid used may be the free acid or its salts such as sodium, potassium, and magnesium salts, and natural products containing phytic acid (for example, corn, steep, etc.) may be used.
Liquor, etc.) may be used. In the case of a method in which adenine is added during the cultivation of microorganisms, phytic acid may be added to the aqueous medium at any time from the time of inoculation to 12 hours after addition of the surfactant and/or organic solvent. In addition, in the case of a method in which the contact reaction is carried out after completion of the culture, it may be carried out at any time from the time of addition of the surfactant and/or organic solvent to 12 hours. In addition, the amount of phytic acid added is 1 to 1.
50g/, preferably 3-20g/. As the magnesium ion, any of salts of inorganic acids such as sulfates, nitrates, and hydrochlorides, salts of organic acids such as magnesium citrate, and other natural products with a high content of magnesium ions (such as phytin) can be used. Magnesium ions may be added to the aqueous medium at any time, as long as they are added at the same time as phytic acid, or after that, or they may be added several times at different times. The amount added is usually 0.01~
It is 0.20 mole. ATP accumulated in the aqueous medium can be collected by using activated carbon, ion exchange resin, etc., as exemplified in the Examples. Examples of the present invention are shown below. Example 1 Glucose 15%, urea 0.5% (separate sterilization)
KH ₂ PO ₄ 1.0%, K ₂ HPO ₄ 1.0%, MgSO ₄・
_7H2O1.0 %, _CaCl2・_2H2O0.01 %, biotin 30μ
A fermentation medium consisting of 0.5% yeast extract, 0.5% meat extract (adjusted to pH 7.2 with caustic soda), 20ml each in a 250ml Erlenmeyer flask, was heated and sterilized, and Billevibacterium ammoniagenes was added to it. ATCC21170 was inoculated and cultured with shaking at 32°C for 48 hours. After culturing, the bacterial cells were separated by centrifugation.
Obtained bacterial cells (wet bacterial weight 30%), glucose
50g/, adenine 7g/, NaHPO ₄ 15g/
, phytic acid 3.5g/, xylene 10ml/,
20ml of solution containing 2156g of Naimeen S-250
ml Erlenmeyer flask and adjust the pH with ammonia water.
At 32°C, adjust the temperature to around 7.4 and add as needed to keep the concentration of phosphoric acid in the reaction supernatant around 10 g/anhydrous disodium phosphate.
The reaction was carried out under shaking conditions for 24 hours. Table 1 shows the results of comparing the amount of ATP produced when phytic acid and magnesium ions were added alone, when both were added at the same time, and when no addition was made.

[Table] Heat-treat reaction solution 1 in Table 1 at 85°C for 20 minutes, remove solids by centrifugation, and obtain a supernatant. This supernatant liquid was adjusted to pH 3.5 with sulfuric acid, 45 g of activated carbon was added to this to adsorb ATP, and after washing with water, the pH was adjusted to 3.5.
% ethanol-ammonia mixture [28% ammonia aqueous solution and ethanol (99%) 1:1 (v/
v)] and concentrate the eluate to around PH8. Next, the concentrated solution was passed through a strongly basic anion exchange resin Dowex-1 x 4 prepared in advance as a Cl type, and after washing with water, a mixture of hydrochloric acid and common salt [0.02NHCl solution (PH1.7)] was added.
0.2M NaCl solution by dissolving NaCl in
Elute with and collect the ATP fraction. After neutralizing this eluate with caustic soda, it was passed through a column packed with activated carbon and eluted with 15% aqueous ammonia.
Concentrate the eluate to remove ammonia. By adding methanol to the concentrate
16.4 g of crystals of the sodium salt of ATP are obtained. Example 2 The same strain as in Example 1 was used, except that the amount of magnesium ions added during the reaction was 10 g/in the form of magnesium sulfate heptahydrate, and the amount of phytic acid was as shown in Table 2. A method similar to Example 1 was carried out to obtain the results shown in Table 2.

[Table] Example 3 Using the same bacterial cells as in Example 1, 50 g of glucose/7 g/adenine was added to the culture solution cultured under the same conditions.
, NaHPO ₄ 15g/, MgSO ₄・7H ₂ O 10g/
, 8 g of phytic acid, 10 ml of xylene, and 154 g of Naimeen S-2 were added, and the shaking and stirring at 32°C was continued for another 10 hours. As a result, 24.7 g of
Generated by ATP. ATP production without adding phytic acid and magnesium sulfate is 7.1g/
It is. Example 4 Using the same strain as in Example 3, the amount of adenine was reduced to 10
In addition to adding 10 g of MgSO ₄ 7H ₂ O at the time of addition of Nymeen and xylene, 6 g each at 2, 5, 7, 9, and 11 hours thereafter/total 40
g/added, and the amount of phytic acid added was 1.5
The same method as in Example 3 was carried out except that the two stages of g/ and 8 g/ were used, and the results are shown in Table 3.

[Table] Example 5 Corynebacterium glutamicum
Using ATCC21171, starch hydrolysis solution (15% glucose equivalent), urea 2g/,
NH ₄ Cl3g/, KH ₂ PO ₄ 10g/, K ₂ HPO ₄ 10
g/, MgSO ₄・7H ₂ O10g/, MnCl ₂・
4H ₂ O 5mg/, corn stave liquor 10
The cells are cultured under the same conditions as in Example 1 using peptone 2 g/(PH7.6). After shaking culture at 32°C for 72 hours, the bacterial cells were separated by centrifugation, and the obtained bacterial cells (dry bacterial weight 10%), glucose 50 g/,
A reaction was carried out under the same conditions as in Example 1 using a solution containing 7 g of adenine, 15 g of Na ₂ HPO ₄ /, 10 g of phytic acid, and 6 g of Naimeen S-215, and as a result, 17 g of ATP was produced. . The amount of ATP produced when phytic acid and magnesium sulfate are not added is 4.7 g/.

Claims (1)

[Scope of Claims] 1 Belonging to the genus Corynebacterium or Brevibacterium, adenosine-5'-triphosphate (hereinafter referred to as
Cultures, cells, or processed products of microorganisms that have the ability to produce ATP) and adenine, phosphate donors, and energy donors are mixed in an aqueous medium containing a surfactant and/or an organic solvent. A method for producing ATP characterized by contacting it in the presence of an acid. 2. The manufacturing method according to claim 1, wherein the contact is performed during culturing of the microorganism.