JPS5951799A - Preparation of adenosine-5'-triphosphate - Google Patents

Abstract

PURPOSE:To prepare adenosine-5'-triphosphate efficiently, by bringing a mold of a bacterium into contact with adenine, etc. in the presence of phytic acid in an aqueous medium containing a surface active agent and/or an organic solvent. CONSTITUTION:A culture of a bacterium capable of producing adenosine-5'- triphosphate from adenine, a donor of phosphoric acid, and a donor of energy, preferably a bacterium belonging to the genus Brevibacterium or Corynebacterium, its mold or a treated material thereof, is brought into contact with adenine, the donor of phosphoric acid, and the donor of energy in an aqueous medium containing a surface active agent and/or an organic solvent such as toluene, xylene, ethyl acetate, etc. in the presence of 1-50g/l, preferably 3-20g/l phytic acid or its salt, and, if necessary, 0.01-0.20mol magnesium ion.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing adenosine-51-triphosphate (hereinafter referred to as "ATPJ"). It exerts an important effect on metabolism through coenzymes and is important as a medicine and as a reagent for biochemical research.

ATP is produced by biochemically phosphorylating 5'-adenylic acid using yeast (Japanese Patent Publication No. 40-19119).
(Japanese Patent Publication No. 46-20759), a method of phosphorylating adenosine using yeast cells treated with a surfactant (Japanese Patent Publication No. 46-20759),
- Method for phosphorylating adenylic acid (Japanese Patent Publication No. 55-4519
7), a method for phosphorylating adenosine or 51-adenylic acid using yeast cells capable of assimilating hydrocarbons or a processed product thereof (Japanese Patent Publication No. 50-9873), adenosine, 51-adenylic acid or adenosine-51-
A method using yeast cells that have the ability to phosphorylate diphosphoric acid and that has not been subjected to any treatment that would substantially damage the cell membrane of the cells (Japanese Patent Laid-Open No. 55-34096) is known. However, these methods all use adenosine and 51-adenylic acid as raw materials.

A disadvantage is that adenosine-5'-diphosphate is expensive.

A method using adenine, which is technically cheaper as a raw material, is a method of culturing microorganisms belonging to Brevibacterium ammoniagenes in a medium containing an extremely low concentration of manganese ions (Japanese Patent Publication No. 44-261912). , aliphatic amines, aromatic amines, condensed phosphoric acids, and nitrocarboxylic acids in a medium containing excessive manganese ions.

A method for culturing bacteria capable of producing nucleotides by reducing the availability of manganese ions in the medium by adding salicylaldehyde, its fatty conductor, β-diketone, and other metal complex-forming agents (Japanese Patent Publication No. 13114/1983) ), a method for culturing microorganisms capable of producing ATP in a surfactant-containing medium (Japanese Patent Publication No. 49-28996),
A method using a culture solution or bacterial cells of yeast having the ability to generate ATP from adenine in the presence of a surfactant, or a processed product thereof, (%Kaimei No. 53-136591)
etc. are known. However, in all of these methods, the amount of ATP produced is insufficient, and industrially cheaper AT is used.
The development of a method for producing P is expected.

The present inventors investigated to improve the conventional ATP production method, and as a result, ATP was produced from adenine, a phosphate donor, and a saccharide.
9ATP is produced by the action of bacteria capable of producing phytic acid and magnesium ions in an aqueous medium in the presence of a surfactant and/or an organic solvent. It was found that the amount of ATP produced was significantly increased.

The present invention will be explained in detail below.

The microorganisms used in the present invention include adenine,
Any microorganism capable of producing ATP from a phosphate donor and sugars can be used. Furthermore, mutant strains obtained by mutagenic treatment of these microorganisms also have the above-mentioned AT.
It is used only if it has the ability to generate P.

Preferred strains belong to the genus Corynebacterium or Brevibacterium; specific examples of strains include Brevibacterium , Ammoniagenes AT (1''c21170) and Corynebacterium glutamicum ATCIC2117.
It is 1.

The medium used for culturing the microorganism may be any natural medium 9 artificial medium as long as it contains a suitable amount of carbon source, nitrogen source, and organic and inorganic nutrient sources.

Glucose is used as a carbon source for culture.

Fructose Book Sucrose Molasses, l#! & carbohydrates such as hydrolysates, normal paraffins.

Hydrocarbons such as kerosene, alcohols such as methanol, ethanol, glycerin, and sorbitol, biruvic acid,
Organic acids such as lactic acid and acetic acid, amino acids such as glycine, t-alanine, 9-glutamic acid and aspartic acid are used, and the concentration thereof is preferably 5 to 30%.

Ammonia is used as a nitrogen source for culture.

Various inorganic and organic ammonium salts such as ammonium chloride-ammonium nitrate, ammonium phosphate t4af & ammonium phosphate, urine R, peptone, meat extract, protein hydrolyzate, fish meal, etc. are used, and nitrogen-containing substances such as o, i~ It is used at a concentration of 10%.

The inorganic substance used in the medium is phosphoric acid-potassium.

Phosphates such as dipotassium phosphate, sodium phosphate, disodium phosphate and ammonium phosphate, sulfates, hydrochlorides and nitrates of magnesium, manganese, calcium, iron, zinc and the like are used in appropriate amounts. Furthermore, if the microorganism is an auxotrophic strain, chemical substances required for growth must be added to the culture solution.

The culture conditions for microorganisms include a temperature of 20 to 40'C;
Shake 9 Under aerobic conditions such as aeration and stirring, ammonia, aqueous ammonia, urea, and caustic soda.

While adjusting the pH to around neutral using caustic potash etc.
The microbial culture obtained in this manner by culturing for ~8 days can be used as it is for the reaction, or it can be treated with adenine and phosphate donors using the treated products obtained by various treatments of the culture. may be subjected to a contact reaction. Processed products include concentrated or dried culture products, p liquid or bacterial cells obtained by treating the culture with a centrifuge, dried bacterial cells, acetone-treated products, surfactant-treated products, and sonication. thing.

Examples include lytic enzyme-treated products, immobilized bacterial cells, and enzyme preparations extracted from bacterial cells.

The catalytic reaction can be carried out in any aqueous medium, and is most economically viable if an adenine phosphate donor, sugar, surfactant or organic solvent, phytic acid, or magnesium ion is present in the medium during the cultivation of the microorganism. Examples of methods include allowing the target substance to accumulate in the culture solution at least, or conducting 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 and phosphate donors are added to the culture, bacterial cells, or processed products thereof after culturing.

Sugars, surfactants and/or organic solvents are added, and other substances are added as necessary, and the desired product is accumulated by reacting under aerobic conditions at 20 to 50°C for 3 to 48 hours. be able to. At this time, adjust the pH to 6.
It is desirable to adjust it to ~8. In any case, the concentration of adenine is between 1 and 100 f'//, preferably between 1 and 4
0 t/11 is good.

Examples of the surfactant used include cationic surfactants such as polyoxyethylene stearylamine (for example, Nymeen S-215, manufactured by NOF Corporation), cetyltrimethylammonium bromide, cetylpyridium chloride, and the like.

Anionic surfactant 1, such as sodium lauryl sulfate, sodium oleylamide sulfate, nonionic surfactant, such as polyoxyethylene sorbitan monostearate (f[Banonion 5T221, manufactured by NOF KX), amphoteric surfactant, For example, lauryl betaine (for example, Anon BF, manufactured by Nippon Oil & Fats Corporation) is used, and these are usually used at a concentration of 0.1 to 501/l, preferably 1 to 201/l.

As the organic solvent, toluene, xylene, aliphatic alcohol nabenzene, ethyl acetate, etc. are used, and the concentration thereof is 0.1 to 50 m4/l, preferably 1 to 20 m4/l.
l/l is good.

The phytic acid used may be the free acid or its salts such as sodium salt, potassium salt, magnesium salt, etc., and natural products containing phytic acid (
For example, corn, steep 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, any time from the time of addition of the surfactant and/or organic solvent to the 12th hour may be used. Further, the amount of phytic acid added is 1 to 50μ, preferably 3 to 50μ.
20 f/l is good.

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 can 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 can be added several times at different times.

The amount added is usually 0.01 to 0.20 mol.

ATP accumulated in the aqueous medium can be collected by using activated carbon, ion exchange resin, etc., as exemplified in Examples.

Wear.

Examples of the present invention are shown below.

Example 1 Glucose 15%, urea 0.5% (separate sterilization).

KH2PO41.0%, K2HP0.1.0%, M
PS○4・7H201,0%, C'aC12-2H2
00,01% l piotine 30μ? /l.

A fermentation medium consisting of 0.5% yeast extract, 0.5% meat extract (adjusted to pH 7.2 with caustic soda) was
Pour 20ml each into a 0d Erlenmeyer flask and sterilize by heating, then add Brevibacterium ammoniagenes ATCC2.
1170 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 -11ii30 cm), glucose 50 t/l
, adenine 7 f/l, Na) (Po, 15
f/l, phytic acid 3.5t/l, xylem/
10 seagulls 4. Solution 20α containing Naimeen 8-2156ψ
Processed into a 250111 volume Erlenmeyer flask, adjusted the pH to around 7.4 with aqueous ammonia, and added as appropriate midway through to maintain the concentration of phosphoric acid in the reaction liquid supernatant at around 10971 as anhydrous disodium phosphate. 24 at 32℃ while
The reaction was carried out under shaking conditions for hours. Table 1 shows the results of comparing the amounts 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 1 Additives (r/l) ATP production amount phytic acid
M? 804.7H20 ($1/waio
0 12. On (157,0 Ill 3.5 0
15.8■3.5 5 20.
5. Heat-treat reaction solution 11 in Table 1 (2) 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, activated carbon 459 was added to it to adsorb ATP, and after washing with water, it was mixed with a mixture of 50% ethanol and ammonia [28% ammonia aqueous solution and ethanol (99%) in 1 part: 1 (mixing at v/v)] and adjust the eluate to pH 8.

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, N
aCJ was dissolved to make a 0.2 M NaCl solution] and the ATP fraction was collected.

After neutralizing this eluate with caustic soda, it is passed through a column filled with activated carbon, eluted with 15% aqueous ammonia, and the eluate is concentrated to scatter the ammonia. By adding methanol to the concentrate, ATP sodium salt crystal 16.4F is obtained.

Example 2 Using the same strain as in Example 1, the amount of magnesium ions added during the reaction was 1 in the form of magnesium sulfate heptahydrate.
0 f/l and using the amount of phytic acid shown in Table 2, the same method as in Example 1 was carried out to obtain the results shown in Table 2.

Table 2 Phytic acid added bamboo ATP production amount (y/l) (y,/ll) 05.1 2, 10.94
19.26
26.98 2
6.510 26.0 Example 3 Using the same bacterial cells as in Example 1, the culture solution was cultured under the same conditions as glucose 50 t/II, adenine 7 y/l,
Na2HPO41s, y/l, ywso4-rH
2o 1 oyA.

Phytic acid 8 V'l*xylene 1 o rut/l,
After adding Naimeen S-21541 and continuing shaking at 32° C. for an additional 10 hours, %24.7 f/l of ATP was produced. ATP production without adding phytic acid and magnesium sulfate is 7.1 f/II
It is.

Example 4 The same strain as in Example 3 was used, the amount of adenine was set to 10 y/l, and in addition to adding WSO4.7H20 at 10'IA when adding nymeen and xylene, 2, 5.7.
9. At the 11th hour, each 62/1 was added at a total of 40 y/l,
In addition, the amount of phytic acid added is t sy/l and 8 f/
The same method as in Example 3 was carried out except that the second stage of "IT" was used, and the results are shown in Table 3.

Table 3 1.5 8.0 0 0 02
7.0 7.0 5 16.0 16.
07 19.1 23
．． 19 21.4 2
7.111 22.9 28.9 13 22.6 30.3 24 22.4 37
．． 0 Corynebacterium glutamicum ATCC211
Using 71, release F';): Starch hydrolyzate (glucose conversion '1:15 split), urea 2V pan-NH,C
l13□1 KH2P0.10□, 2HPO410φ
.

MrSO,・7H2010? /1. λinC12・4H
205FQ/13 * Corn Steep Liquor 10
1. Pefton 21(1)H7,6) was used and cultured under the same conditions as in Example 1. After shaking culture at 32°C for 72 hours, the bacterial cells were collected by centrifugation, and the obtained bacterial cells (dried bacterial cells M
10 g), glucose 50 f/11, adeni:y
7 fi'/l, N, a2HP0.15? /l
As a result of carrying out the reaction under the same conditions as in Example 1-IV using a solution containing phytic acid 1°74, and Naimeen S-21,560,000, 17f/no ATP was produced.

The amount of A'l'P produced when phytic acid and magnesium sulfate are not added is 4.7? /11.

Patent Applicant (102), Kyowa Hakko Kogyo Co., Ltd. Representative Kinoshita Yoshibe Procedural Amendment Letter October 72, 1980 1st Office Commissioner 1, Indication of Case 1987') ¥ Permit No. 76/'1871 No. 2, name of the invention t1 of adenosine-5'-triphosphate! Construction method 3, relationship with 41 amendments tp ke Applicant postal code 100 Address 6-1 Otemachi-chome, Chiyoda-ku, Tokyo Name
(102) Kyowa M Hakkounara Co., Ltd. (Location: 03-201-
7211 Extension 2751) Representative: Ikki Kinoshita Section 4, [Detailed Description of the Invention-1 and "Claims" of the specification to be amended, Part 5, contents of the amendment as shown in the appendix (2) Clear δ(l Correct “saccharide” in the next part of 〒′) to “energy donor”.

■Page 4, lines 9 and 17 ■ Page 7, line 12 ■ Page 8, 3rd illustration (3) Specification page 4, line 12 Delete "and magnesilano, ion."

(4) Ming? (Add the following sentence between lines 14 and 15 on page 4 of Book I.

"Furthermore, it has been found that when the above method is carried out in the presence of magnesium ions, the production of ATP is further improved." (5) Specification ψ1: page 8,
Add the following sentence between lines 10 and 11.

[Energy donors include carbohydrates such as glucose, fructose, sucrose, molasses, and starch hydrolysates, organic acids such as pyruvic acid, lactic acid, acetic acid, and α-ketoglutaric acid, and amino acids such as glycinealanine, aspartic acid, and glutamic acid. used. These are approximately 5~
Used at f★ degrees of 150 f/l.

As a phosphate donor, it is used in the cultivation medium of the above-mentioned bacteria:
Any lXj acid salt can be used. These are 1 to 100
``Used in the v-view i of μ'' Claim + 1) Adenine, Tsu 2 Me? Acid donor and energy ■ (from one adenosine-51-3:', Qt-C
f, q, biological culture materials, cup bodies, or their places that have the ability to generate ATP)■11! In the presence of phytic acid in an aqueous mash containing a surfactant and a solvent, adenine, a qM acid donor, and an energy 4L donor are mixed with )'4f
'l・lt, +47j, A with /I''# sign
T p //): Q construction method.

+2) If the microorganism is a member of the genus Corynebacterium or Brevibacterium spp.
Manufacturing method described in section.

(3) The method according to claim 1 or 2, wherein the contact is performed during culturing of the microorganism.

-5n

Claims (3)

[Claims] (1) Cultures, microbial cells, or processed products thereof of microorganisms that have the ability to produce adenosine-51-triphosphate (hereinafter referred to as ATP) from adenine, a phosphate donor, and saccharides, and adenine, phosphate donors, and saccharides. 1. A method for producing ATP, which comprises contacting with 1) a surfactant and/or an organic solvent, and 2) an aqueous medium containing phytic acid and magnesium ions. (2) The production method according to claim 1, wherein the microorganism belongs to the genus Corynebacterium or Brevibacterium. (3) The method according to claim 1 or 2, wherein the contact is performed during culturing of the microorganism.