JPS6359680B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing adenosine-5'-triphosphate (hereinafter abbreviated as ATP) by a fermentation method. ATP plays an important role in energy metabolism as a high-energy phosphoric acid compound in living organisms, and is considered important as a pharmaceutical and biochemical reagent. In addition, in recent years, in order to use ATP efficiently in bioreactors that use ATP,
The use of pyruvate kinase, an enzyme that regenerates adenosine-5'-diphosphate into ATP, is being considered in the ATP regeneration system. A cheap supply of ATP is desired for production. Conventional methods for producing ATP include direct isolation and purification from animal muscle, organic chemical synthesis, and 5'-
Examples include a method of enzymatically phosphorylating adenylic acid and a fermentation method. Among these, fermentation methods include, for example, a method in which bacteria belonging to Brevibacterium ammoniagenes are cultivated in a medium containing adenine or its derivatives to produce and accumulate ATP (Special Publication No. 17634-1971), and a method in which ATP production capacity is Culture microorganisms with a surfactant in a medium containing surfactants, and
A method of collecting ATP (Japanese Patent Publication No. 49-28996) and a method of adding a compound having a phenolic hydroxyl group when the production of ATP in the medium reaches the maximum (Japanese Patent Publication No. 53-6490) have been proposed. ing. However, in all of these conventional fermentation methods, fermentation is carried out in the presence of adenine or its derivatives, such as adenosine, in the medium, and ATP is produced by phosphorylating this adenine or its derivatives as a substrate. It is something that accumulates. Therefore, since the conventional method uses expensive adenine or its derivatives as a raw material, it cannot be said to be an industrially advantageous method. As a result of studying a method for industrially producing ATP at low cost by fermentation, the present inventor found that instead of adenine or its derivatives, methanol or a chemical substance that shows the same metabolic pathway as methanol, and a specific range of inorganic phosphoric acid. The present inventors have discovered that ATP accumulates in a high concentration in a medium by culturing ATP-producing bacteria capable of assimilating methanol in a medium containing ATP as a substrate, leading to the present invention. Therefore, an object of the present invention is to provide a method that can industrially advantageously produce ATP. The present invention will be explained in detail below. As described above, the present invention uses a medium containing methanol or a chemical substance that exhibits the same metabolic pathway as methanol as a substrate, so the ATP-producing bacteria used in the present invention must be capable of assimilating methanol. No. Examples of ATP-producing bacteria capable of assimilating methanol include the following. Methylomonas probus FERM-P3193, Methylomonas methylophora ATCC21369, belonging to the genus Methylomonas; Pseudomonas methylotropha NCIB10508, Pseudomonas rosea NCIB10597, Pseudomonas insueta ATCC21276, Seudomonas belonging to the genus Pseudomonas Monas methanolica ATCC21704, Pseudomonas metanica
ATCC21439; Protaminobacter ruber belonging to the genus Protaminobacter ATCC8457;
Candida ATCC21372; Achromobacter belonging to the genus Achromobacter
Methanolophylla ATCC21275; Corynebacterium sp. ATCC21232, which belongs to the genus Corynebacterium; and Bacillus cereus, which belongs to the genus Bacillus.
ATCC14579, Bacillus subtilis var.
NB1001FERM−P1373. As a technology for producing useful substances using the above-mentioned bacteria, conventional methods have been to grow bacterial cells as a protein source by culturing them in a medium with methanol as a substrate (Japanese Unexamined Patent Publication No.
52-28988), production of flavin adenine dinucleotide [J. Ferment. Technol. 55, 630 (1977)],
Production of vitamin B ₁₂ [Appl.Microbiol.30, 477
(1975)], production of lipids and polysaccharides, collection of lecture abstracts from the 1975 Japanese Society of Agricultural Chemistry Conference, Symposium C ₁ , Utilization of Microorganisms 551-556), and production of amino acids (collection of lecture abstracts mentioned above). There is, but ATP
There are no reports yet on the use of the above bacteria for production purposes. The present invention has the above-mentioned methanol assimilation ability.
ATP-producing bacteria are used as substrates for methanol or a chemical substance that shows the same metabolic pathway as methanol, and inorganic phosphate as a phosphate radical (PO ₄ ) at 4 to 35 g/
It is characterized by being cultured in a medium containing. Here, "chemical substances exhibiting the same metabolic pathway as methanol" means substances that can be utilized in the same metabolic pathway as methanol, such as methane, methylamine, formaldehyde, and formic acid. Methanol and the above substances are used as carbon sources for the culture medium,
It should be noted that if their concentration in the medium is too high, bacterial growth will be inhibited. Generally, the concentration is 0.2 to 4 for methanol and methylamine.
Volume %, 0.01 to 0.1 for formic acid and formaldehyde
Volume % is appropriate, and methane is preferably used at a concentration corresponding to its solubility. In addition, these carbon sources may be added in their entirety to the medium at the beginning of culture, but it is recommended to keep the concentration of carbon sources in the medium low at the beginning and supplement them as the carbon source is consumed as the culture progresses. , more favorable results in ATP production can be obtained. In addition to the carbon sources mentioned above, the present invention requires the presence of inorganic phosphate as PO ₄ in the medium of 4 to 35 g/medium, but the concentration of inorganic phosphate in such a medium is usually This corresponds to several times to several tens of times that of the bacterial culture medium. Note that if the concentration of inorganic phosphoric acid (as PO ₄ ) in the medium is lower than 4g/, no increase in ATP production will be observed, while if it is higher than 35g/, the growth of ATP-producing bacteria will be suppressed and the amount of ATP produced will decrease. This will reduce the Incidentally, inorganic phosphoric acid acts to prevent ATP produced during culture from being secreted outside the bacterial cell and being degraded by phosphatase. The inorganic phosphate used here is one used in ordinary fermentation media, and examples thereof include (NH ₄ ) ₂ HPO ₄ , KH ₂ PO ₄ , and K ₂ HPO ₄ . In the present invention, in addition to the above-mentioned carbon source and inorganic phosphate as medium components, inorganic salts such as potassium, magnesium, iron, and manganese, and in some cases, inorganic salts of metals such as zinc, cobalt, and molybdenum; , ammonium salts, urea, nitrates, etc. may be used. Furthermore, a surfactant or an antifoaming agent can be added to the medium as necessary. Examples of the composition of the medium used in the present invention are as follows. Carbon source 0.05-2% by volume (NH ₄ ) ₂ HPO ₄ 5-45 g/ (3.5-32 g/ as PO ₄ ) KH ₂ PO ₄ 0.5-2.5 g/ (0.35-1.75 g/ as PO ₄ ) K ₂ HPO ₄ 0.5-2.5g/ (0.275-1.37g/ as PO ₄ ) (NH ₄ ) ₂ SO ₄ 0-1.0g/ MgSO ₄・7H ₂ O 0.5-5.0g/ FeSO ₄・7H ₂ O 0.05-0.5g/ _{CaCl2.2H2O 0-0.2g} / _{MnSO4.4H2O 0-0.2g} /The culture of the ATP-producing bacteria described above in the medium described above generally has a pH of 5.8-9.0, preferably
6.0~8.0, temperature 15~50℃, preferably 30~40℃,
Aeration volume 0.5 to 4.0VVM (air volume/culture solution/
min) with stirring at 40 to 600 rpm.
It is best to do this for 1 to 9 days. In addition, to adjust the pH of the culture medium, alkaline substances such as ammonia or acidic substances such as sulfuric acid, buffers such as phosphate buffer, urea, calcium carbonate, etc. can be used, but when using alkaline substances, ammonia This is particularly suitable since it also serves as a nitrogen source. By fermenting under the above-mentioned culture conditions, ATP can be accumulated in the medium at a high concentration of 2 to 12 g/1. After fermentation is complete, remove the bacterial cells,
The generated ATP is separated and collected using a known method. For example, ATP can be collected from the supernatant after removing bacterial cells from the fermented medium by a combination of adsorption using activated carbon and anion exchange resin, fractionation treatment by elution, and concentration precipitation treatment. According to the present invention, as shown in the examples, ATP can be reduced by 2 without adding adenine or its derivatives to the culture medium.
It can be produced and accumulated in the culture medium at a high concentration of more than g/g/g. EXAMPLES The effects of the present invention will be specifically explained below with reference to Examples. The amount of ATP produced in the medium in each Example was measured as follows using the principle of the reaction between ATP and luciferin-luciferase expressed as below. (1) Luciferin + Luciferase + ATPMg ² ---→ Adenyl-Luciferin + Pyrophosphate (2) Adenyl-Luciferin O ₂ ----→ Adenyl-
Oxyluciferin 560-580nm of fluorescence generated in the reaction of (2) above
CHEM− is the intensity of light in the wavelength range of
GLOWPHOTOMETERJ4−7441 (American
(manufactured by Instrument Company) for a certain period of time to determine the integrated amount, and compare this with a known ATP standard solution calibration curve prepared in advance to measure the amount of ATP produced in the culture solution. Example 1 (NH ₄ ) ₂ HPO ₄ 7.0 g, KH ₂ PO ₄ 1.0 g,
K ₂ HPO ₄ 1.0 g, MgSO ₄ , 7H ₂ O 1.0 g and
Dispense 100 ml of a medium prepared by dissolving 0.1 g of FeSO ₄ 7H ₂ O in 1 part of ion-exchanged water into 300 ml Erlenmeyer flasks, add 1.6 g of methanol to each medium after sterilization, and add the above composition to each medium. Methylomonas probus FERM-P cultured in advance on agar slants of
No. 3193 was inoculated and cultured with shaking at 30°C.
On the fourth day after the start of culture, 2.8 g/ATP is produced and accumulated in the culture solution. This culture solution 1 was heated to 80℃ for 5 minutes, cooled and centrifuged to remove the bacterial cells, and the supernatant was adjusted to pH 3.5 with 3N hydrochloric acid and treated with activated carbon to adsorb ATP on the activated carbon. After 1.4% ammonia content 50
Elute the ATP adsorbed on activated carbon with a % alcohol solution. This eluate is concentrated under reduced pressure at low temperature to remove excess ammonia and the pH is around 8.0. Next, ATP is adsorbed from this concentrated solution on a strongly basic anion exchange resin Dowex 1-X2 (Cl) column which has been previously adjusted to Cl type. After washing this adsorbate with ion-exchanged water, it was eluted with a mixture of hydrochloric acid and sodium chloride (NaCl was dissolved in 0.02M-HCl solution (PH1.7) to make a 0.2M-Cl solution).
Separate the ATP fraction. After neutralizing this eluate with caustic soda, it is passed through a column packed with activated carbon and eluted with 15% aqueous ammonia, and the resulting eluate is concentrated to scatter the ammonia. 2.2 Crystallization of the sodium salt of ATP by adding methanol to the concentrate
I got g. Example 2 (NH ₄ ) ₂ HPO ₄ 7.0 g, KH ₂ PO ₄ 1.0 g,
K ₂ HPO ₄ 1.0g, MgSO ₄・7H ₂ O2g, FeSO ₄・
Culture medium 20 in which 0.2 g of 7H ₂ O and 0.2 ml of antifoam agent KS-66 were dissolved in 1 part of ion-exchanged water was placed in a 30-volume jar fermenter and heated at 120°C under a pressure of 1.2 kg/cm ^2.
Sterilize for 30 minutes. After cooling, add 200 ml of methanol to the above medium, and add mother bacteria (Methylomonas probus) that was previously cultured in the same medium as above.
FERM-P No. 3193) was inoculated at 2% and incubated at 35°C for 500 r.
10/20 liquid volume/min while stirring at pm
(0.5VVM), and as the bacteria grow, increase the aeration rate to 20/20/min (1.0VVM) and culture for 96 hours. The pH during the culture is automatically controlled to 6.0-7.2 using 14% ammonia water, and methanol consumed is automatically added to 0.3-2.0% as measured by gas chromatography. into the culture medium 96 hours after the start of culture.
ATP5.3g/ is generated and accumulated. ATP in culture medium
is recovered by the same method as described in Example 1. Example 3 Culture is carried out under the same conditions as in Example 2, except that the amount of (NH ₄ ) ₂ HPO ₄ among the medium components used in Example 2 is changed to 20 g/. 12 g/ATP is produced and accumulated in the culture solution 96 hours after the start of culture.
ATP is recovered using the same method as in Example 1. Example 4 Among the culture medium components used in Example 2,
Culture was carried out under the same conditions as in Example 2 except that the amount of (NH ₄ ) ₂ HPO ₄ was 30 g/. 7g/ATP is produced and accumulated in the culture solution 96 hours after the start of culture.
ATP is recovered using the same method as in Example 1. Comparative Example 1 Among the medium components used in Example 2,
Culture was carried out under the same conditions as in Example 2 except that the amount of (NH ₄ ) ₂ HPO ₄ was 3 g/. Only 0.2 g/ATP was produced in the culture solution 96 hours after the start of culture. Comparative Example 2 Among the medium components used in Example 2,
Culture was carried out under the same conditions as in Example 2 except that the amount of (NH ₄ ) ₂ HPO ₄ was 50 g/. Only 1.0 g/ATP was produced in the culture solution 96 hours after the start of culture. Example 5 The ATP production amount when each strain shown in the table below was cultured under the same culture conditions as described in Example 3 is shown in the table below. 【table】

Claims (1)

[Scope of Claims] 1. Adenosine-5 having the ability to assimilate methanol and belonging to a genus selected from the group consisting of Methylomonas, Pseudomonas, Protaminobacter, Achromobacter, Corynebacterium, and Bacillus. Adenosine-triphosphate-producing bacteria were cultured in a medium containing methanol or a chemical substance showing the same metabolic pathway as methanol, and 4 to 35 g of inorganic phosphate as a phosphate group (PO ₄ ) as a substrate. 1. A method for producing adenosine-5'-triphosphate, which comprises producing, accumulating and collecting 5'-triphosphate. 2. The manufacturing method according to claim 1, wherein the chemical substance exhibiting the same metabolic pathway as methanol is selected from the group consisting of methane, methylamine, formaldehyde, and formic acid.