JPH02134394A - Preparation of adenosine-5'-triphosphoric acid - Google Patents

Abstract

PURPOSE:To efficiently provide the subject compound inexpensively on industry by specifying the concentration of NH in an aqueous medium when adenine is converted into the adenine-5'-triphosphoric acid with microorganisms in the aqueous medium containing a surfactant and an organic solvent. CONSTITUTION:When adenine, a phosphoric acid donor and an energy donor are brought into contact with the culture solution of microorganisms, the cells thereof or the treated products thereof having an ability to produce the adenosine-5'-triphosphoric acid from these raw materials in an aqueous medium containing a surfactant and/or an organic solvent to provide the objective compound, the concentration of ammonium ions in the aqueous medium at the starting point of the contact reaction is controlled to <=1g/l converted into NH3. The microorganisms are preferably Brevibacterium.ammoniagenes ATCC 21170, etc. The reaction is preferably carried out at 20-50 deg.C at a pH of 6-8 for 3-48hr under an aerobic condition.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to adenosine-5'-triphosphate (hereinafter referred to as "ATPJ").
(abbreviated as )). ATP is useful as a pharmaceutical and biochemical reagent.

Conventional methods for producing ATP include a method of biochemically phosphorylating 5'-adenylic acid (hereinafter abbreviated as rAMPJ) using yeast (Japanese Patent Publication No. 19119/1971), and a method of treatment with a surfactant. A method of phosphorylating adenosine using yeast cells that have been subjected to a freeze-thaw treatment (Japanese Patent Publication No. 46-20759), a method of phosphorylating AMP using yeast cells that have been subjected to a freeze-thaw treatment (Japanese Patent Publication No. 55-45197) ), a method of phosphorylating adenosine or AMP using yeast cells capable of assimilating hydrocarbons or a processed product thereof as an enzyme source (Japanese Patent Publication No. 1987-9873), adenosine, AMP, adenosine-5'-2 A method using yeast cells that have the ability to phosphorylate phosphoric acid (hereinafter abbreviated as rADP) and that have not been subjected to any treatment that would substantially damage the cell membrane of the cells (Japanese Patent Laid-Open No. 55-34036 Publication No.) etc. are known.

All of these methods use adenosine and AM as raw materials.
P and ADP are expensive.

As an industrially cheaper method for producing ATP using adenine as a raw material, there is a method in which microorganisms belonging to Brevibacterium ammoniagenes are cultured in a medium containing an extremely low concentration of manganese ions (Japanese Patent Publication No. 26912/1983); By adding metal complexing 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, A method for culturing bacteria capable of producing nucleotides by reducing the availability of manganese ions (Japanese Patent Publication No. 47-1311)
4), and a method of culturing microorganisms capable of producing ATP in a medium containing a surfactant (Japanese Patent Publication No. 49-28996). In addition, as an enzymatic method, in the presence of a surfactant, a method using a culture solution or cells of yeast that has the ability to produce ATP from adenine, or a processed product thereof (Japanese Patent Application Laid-Open No. 136591/1982), methanol assimilation A method using bacterial cells or cultures obtained by culturing sex yeast in a medium containing methanol as the main carbon source, or processed products thereof (Japanese Patent Application Laid-Open No. 1983-74592), adenine, phosphate group donor and Energy donor and A
A method of carrying out a reaction by adding phytic acid to an aqueous medium in the presence of a surfactant and/or an organic solvent using a microorganism that has the ability to produce TP (hereinafter referred to as a converting bacterium) 51799) and the like are known. However, all of these methods do not produce a sufficient amount of ATP.

Problems to be Solved by the Invention There is a need for the development of a method for producing ATP in high yield using adenine as a raw material.

Means to Solve the Problem As a result of studying the medium components in the contact reaction, AT
It was found that P was produced in good yield.

According to the present invention, a culture solution, bacterial cells, or a processed product thereof of a microorganism having the ability to generate ATP from adenine, a phosphate group donor, and an energy donor, and adenine, a phosphate group donor, and an energy donor are combined. When ATP is produced by contacting in an aqueous medium containing a surfactant and/or an organic solvent, the ammonium ion concentration in the aqueous medium at the start of the contact reaction is 1 g/j! in terms of N H! ATP can be produced in good yield by following the steps below.

The contact reaction is carried out under aerobic conditions at 20-50°C and pH 6-8 and is completed in 3-48 hours. At this time, it is desirable to adjust the pH to 6 to 8 using an alkali such as caustic soda or caustic potash. ATP can be obtained in higher yield by adding magnesium ions to the reaction solution.

The present invention will be explained in detail below.

As the microorganism used as the converting bacterium in the present invention, any microorganism can be used as long as it has the ability to generate ATP from adenine, a phosphate group donor, and an energy donor. Furthermore, mutant strains obtained by mutating these microorganisms can also be used as long as they have the above-mentioned ATP-producing ability.

A preferred strain is a microorganism belonging to the genus Brevibacterium or Corynebacterium, and a specific example of a strain is Brevibacterium ammoniagenes ATCC21.
1? O and Corynebacterium glutamicum AT
CC211/l is mentioned.

The medium used for culturing the microorganism contains a carbon source, a nitrogen source,
Add organic and inorganic nutrient sources (natural or artificial media may be used as long as the medium contains them).

Carbon sources used for culture include carbohydrates such as glucose, fructose, sucrose, molasses, and starch hydrolysates, alcohols such as methanol, ethanol, glycerin, and sorbitol, organic acids such as pyruvic acid, lactic acid, and acetic acid, glycine, Amino acids such as alanine, glutamic acid, and aspartic acid are used, and these
Used at a concentration of 30%.

Nitrogen sources used for culture include aqueous ammonia, ammonia gas, ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium sulfate, and various inorganic and organic ammonium salts such as urea, peptone, meat extract, protein hydrolysates, fitschmeal, etc. of nitrogen-containing substances are used.

Inorganic substances include phosphates such as potassium phosphate, dipotassium phosphate, sodium phosphate, and disodium phosphate, sulfates, hydrochlorides, and nitrates of magnesium, manganese, calcium, iron, zinc, copper, 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, but if sufficient amounts of the required substances are brought in from other medium components, there is no need to add them.

Cultivation is carried out at a temperature of 20 to 40°C under aerobic conditions such as shaking, aeration, and stirring, while adjusting the pH to around neutrality using ammonia gas, aqueous ammonia, urea, caustic soda, caustic potash, etc. The process will be completed in 1 to 8 days.

The contact reaction can be carried out by adding components necessary for the contact reaction to the obtained culture solution. The ammonium ion in the culture solution is Ig/j! If the amount exceeds this amount, the culture solution may be diluted or the bacterial cells may be separated from the culture solution and washed if necessary before use.

Most preferably, the ammonium ion concentration at the end of culture is Ig/j! If the culture solution obtained by culturing as follows is used as it is, it is economical and easy to operate.

The following method is exemplified as a method for obtaining such a culture solution.

■ During the first half of the culture, adjust the pH using ammonia gas, aqueous ammonia, urea, etc., and then switch to caustic soda, caustic potash, etc. at an appropriate time to adjust the pH.

In this case, a sufficient amount of ammonia required for growth of the microorganisms must be provided as a nitrogen source. (See Example 1) ① Mix ammonia water, urea, caustic soda, caustic potash, etc. in an appropriate ratio to adjust the pH.

■ As a nitrogen source necessary for growth, add yeast extract, ammonium sulfate, ammonium phosphate, urea, or other components to the medium, and perform pHJ with caustic soda, caustic potash, etc.

By these methods, it is possible to obtain a culture solution in which the ammonium ion concentration at the end of culture is 1 g/l or less in terms of NHs, without affecting the growth rate, growth amount, or activity of the bacterial cells.

The adenine used may be synthetic or natural.

Further, a pure product may be used as it is, or any adenine-containing product such as a crudely purified adenine product such as microbial adenine fermentation liquid may be used. Adenine is 1-10 (I
g/J! , preferably at a concentration of 1 to 50 g/l.

Energy donors include glucose, flagose,
Carbohydrates such as sucrose, molasses, and starch hydrolysates, organic acids such as pyruvic acid, lactic acid, acetic acid, and α-ketoglutaric acid, and amino acids such as glycine, alanine, aspartic acid, and glutamic acid are used. These are 5 to 20
0g/I! used at a concentration of

Phosphate donors include polyphosphoric acids such as orthophosphoric acid, birophosphoric acid, tripolophosphoric acid, tetrapolyphosphoric acid, and tetrapolymetaphosphoric acid, polymetaphosphoric acid, potassium phosphate, dipotassium phosphate, sodium phosphate, and diphosphate. Inorganic phosphates such as sodium are used.

These are approximately 3-150g/j! used at a concentration of

As the surfactant, polyoxyethylene stearylamine (e.g. Naimeen S-215, manufactured by NOF Corporation),
Cationic surfactants such as cetyltrimethylammonium bromide and cetylpyridium chloride, anionic surfactants such as sodium lauryl sulfate and sodium oleylamide sulfate, polyoxyethylene sorbitan monostearate (for example, Nonion ST221, manufactured by NOF Corporation) Examples include nonionic surfactants such as, and amphoteric surfactants such as lauryl betaine (for example, Anon BF, manufactured by NOF Corporation), and these are usually 0.1 to 50 g.
/j! , preferably at a concentration of 1 to 20 g/l.

Examples of organic solvents include toluene, xylene, aliphatic alcohols, acetone, ethyl acetate, etc., and these are usually 0.1 to 50 m1/J! , preferably 1-20m1
/j! used at a concentration of

As the magnesium ion, any of salts with inorganic acids such as sulfates, nitrates, and hydrochlorides, salts with organic acids such as magnesium citrate, and other natural products with a high content of magnesium ions can be used. The amount added is usually 0.01 to 0.
It is 20 moles.

ATP accumulated in the aqueous medium can be collected by a conventional method using activated carbon, ion exchange resin, or the like.

Examples are shown below.

Example 1 Glucose 50g/l, polypeptone (manufactured by Daigo Nutrient Chemical Co., Ltd.) 10 g/l, yeast extract (manufactured by Daigo Nutrient Chemical Co., Ltd.)
10g/β, urea 5g/l, (NH4)2sO* 5g
/l5KHtPOa 1 g/Il.

K, HPo, 3g/l, MgSO<・7Hz0
1g/fl, Ca(la・2HtOO, 1g/j!, F
e5O=・7H2010mg/l, Zn5O1・7H2
010mg/CMnSO4・4~6H2020mg/
1. L-cystine 20mg/j! SD-calcium pantothenate 10mg/A', vitamin B+5mg/j'
, nicotinic acid 5mg/j! and biotin 30μg/l
A seed culture liquid medium consisting of the following composition (adjusted to pH 7.2 with caustic soda) was placed in 10ml portions of large test tubes and sterilized by heating, and Brevibacterium ammoniagenes AT
Inoculated with CC21170 and incubated at 28°C for 24 hours at 300 r
Culture was performed with reciprocating shaking at pm.

Next, 20 ml of this culture solution was inoculated into a 21 volume Erlenmeyer flask with a buffle containing 230 ml of a liquid medium having the same composition as above, and cultured with rotational shaking at 28° C. and 19 Orpm for 24 hours.

This culture solution contains glucose 100g/j! , Meat extract (manufactured by Kyokuto Pharmaceutical Industries) log/J, Polypebuto: /10g/
i, KH2P 041 g/J! , K2HP0゜1g
/R5MgSO4・7H201g/l.

Fe50.・7H, 020mg/l, ZnS0.7H2
010g/ 1SCaC1'a' 2H, 0100mg
/j! , Mn5On・4~6Hz0 4mg/l, β-
Alanine 15mg/l, L-cysteine 20mg/l,
Biotin 100■/I11 Urea 2g/i <separate sterilization) and Vitamin B + 5 mg/J! (Separate sterilization) (Heat sterilization after adjusting pH to 7.2 with caustic soda) Inoculate into a 51-volume culture tank containing a liquid medium 2.51 with the composition of
Seed culture was carried out at 32°C, 600 rpm, and aeration rate of 2.51/min while adjusting the pH to 6.8 with concentrated ammonia water.

When the glucose in the culture supernatant was consumed, 300 ml of the culture solution was aseptically collected and 180 g/g of glucose was collected.
i', meat extract LOg/l, KH, PO.

10 g/It, HPo, 10 g/l, M g S
O4-7H2010g/i', CaCj! , -28.
0100mg/l, Fe5Oa7H2020mg/j'
.

Zn5O1・7H7Ol10ll1/i, β~alanine 15+ng/β, biotin 100■/1, nicotinic acid 5
mg/j! , glutamic acid 1g/j! , L-cysteine 20 mg/ml, carbon 2 g 1 IC separate sterilization, bacteria) and vitamin B + 5 mg/1 (separate sterilization) (heat sterilized after adjusting to pH 6.8 with caustic potash). Five 51-capacity culture tanks (A, B,
C.

D and E) were inoculated at 32°C and 600 rp.
The main culture was carried out at m and an aeration rate of 2.51/min.

The pH was initially adjusted to 6.8 using concentrated ammonia water, and the residual glucose concentration in the culture solution was A: 10%, B:
When the concentrations reached 5.0%, C: 2.5%, and D: 1.25%, pH adjustment was switched from concentrated ammonia water to 10N caustic potash and culture was continued. The culture (E), which was cultured using concentrated ammonia water from beginning to end, was used as a control.

When the glucose in the culture supernatant was consumed, put 20 ml of the culture into each 200 ml beaker and add 100 g/l of glucose, 15 g/It of adenine, 0.2 g/l of nicotinic acid, and 15 g/j of KH2P0. ! ,M
g S O4・7H*0 10g/l.

Xylene 10+nl/1 and Naimeen S-2154
g/l and stirred with a magnetic stirrer for 90 Q.
The reaction was carried out while stirring at rpm, keeping the temperature at 32° C. in a constant temperature water bath, and adjusting the pH to around 7.4 with 10N caustic potassium. During the reaction, the phosphoric acid concentration in the reaction solution Tsuchiura was maintained at around 20 g/l including KH2PO, and the magnesium concentration was adjusted to 20 g/l as M g SO4.7H20.
g/J! To maintain the same level, the glucose concentration was set at 20g/
Additions were made as needed to ensure that the amount did not become less than 1 liter. Ammonium ion concentration (NH, conversion) at the start of the reaction and 2
ATP production amount at 1st hour (ATP Naz・3H20
The results of the comparison (conversion, hereinafter the same) are as shown in Table 1.

In the case of using a culture tank, the resulting cultured cells are cells that have been cultured in a state of nitrogen starvation, and although there is no ammonia present at the start of the reaction, the amount of ATP produced is very low.

Table 1 L O, Q O, OO12, 35, 00
0,O558,2 2,500,3950,7 L, 25 0.98 36.
6 Example 2 Culture solution cultured under the conditions of Example 10B (culture solution B'),
Regarding the culture solution (culture solution E') similarly cultured under the conditions of E, the culture solution was used for direct contact reaction (indicated as "Bacterial cell isolation -"), and the bacterial cells were collected by centrifugation and the same as the centrifuged supernatant. Suspended in a volume of ion-removed water (labeled as bacterial cell separation +)
Table 2 shows the results of a reaction conducted in the same manner as in Example 1, with respect to the case in which the following was used in the contact reaction.

Table 2 Culture solution B' 0, . 09 55.3 Culture solution B' + 0. (1150,9 Example 3 Seed culture in a large test tube and a 21-volume Erlenmeyer flask with a baffle was carried out in the same manner as in Example 1, and the resulting seed culture solution was mixed with glucose 250 g/Il, meat extract Log/L,
Polypeptone 100gzl.

KHzPOa 1g/l, K2HPO41g/l.

Mg5On・7Hz○ 1g/l, Fe5On7HiO
20mg/j! , Zn5O,-TH2010g/l,
CaCj! a' 2H20100tng/l, M n
S Oa ・4～68 s 04 mg / j!
, β-752:/15■/i, L-cystine 20*/
j! , biotin 100/1, urea 2g/i (separate sterilization)
and 8.5 mg/2 vitamins (separately sterilized) (heat sterilized after adjusting pH to 7.2 with caustic soda).
, 600 rpm, air flow rate 2.51/min, 1
Culture was performed while adjusting the pH to 6.8 with 0N caustic potash. Put 20ml of the obtained culture solution into a 200ml beaker, and add 100g/j of glucose to it! , adenine 15 g/1. Nicotinic acid 0.2g/i.

KH, Po, 15g/Il, MgSO4・77H
2O10/j! , xylene 10m1/J! and Naimeen S-2154g/j! While stirring at 900 rprn with a magnetic stirrer, the temperature was kept at 32°C in a constant temperature water bath, and the pH was adjusted to 7 with 10N caustic potash.
, 4. The reaction was carried out while adjusting the temperature to around 4.

During the reaction, the phosphoric acid concentration in the reaction solution Tsuchiura was KH2PO.

20g/j when closed! In order to keep the magnesium concentration around 20g/R as M g S O4 7H70, the glucose concentration is 20g/j! Appropriate additions were made midway through to avoid the following.

The ammonium ion concentration (NH, conversion) at the start of the reaction was 0.03 g/j! and ATP is 43 in 20 hours of reaction.
．． 2g/j! generated.

Example 4 Brevibacterium ammoniagenes^TCC211
70 seed cultures were carried out in exactly the same manner as in Example 1 using a large test tube, a 21-bottle Erlenmeyer flask, and a 51-volume culture tank.

When the glucose in the culture supernatant of the 51-volume culture tank seed culture was consumed, the culture solution was aseptically collected in 300 ml portions.
Glucose 180g/j! , meat extract 10g/i, KH
2P0.10g/I! , K., HPo.

10g/l, MgS O4・7H, Olog/Il
.

Ca C12' 2820100mg/l, FeS
Oa '7H,020+ng/CZ,nSO,・7Hz
0 10 mg/l, β-alanine 15 mg/J, biotin 100 M/', nicotinic acid 5 mg/J! , glutamic acid 1g/j! , L-cysteine 20 mg/C urea 2 g/β (separate sterilization) and vitamin B + 5 mg/
J! (Separate sterilization) (heat sterilized after adjusting pH to 6.8 with caustic potash) 5 containing a liquid medium 2.51
Inoculated into two 11-capacity culture tanks (F, G), each with a 32-volume culture tank.
℃, 600 rpm, aeration rate 2.51/min, culture tank F was a mixture of concentrated ammonia water and 10N caustic potassium at a volume ratio of 4:1, and culture tank G was a concentrated ammonia water solution.
Culture was performed while adjusting the pH to 6 and 8, respectively.

Put 20 ml of the obtained culture solution into 200 ml beakers, add 100 g/β of glucose and 1 ml of adenine.
5g/j! , nicotinic acid 0.2 g/It.

KH2P0.15g/j! , M g S Os・77H
2O10/l, xylene 10m1/j! and Naimeen S-2 154g/Il were added, stirred at 90 Orpm with a magnetic stirrer, maintained at 32°C in a constant temperature water bath, and adjusted to pH 7.4 with 10N caustic potash.
The reaction was carried out while adjusting to the vicinity.

During the reaction, the phosphoric acid concentration in the reaction supernatant was KH2PO.

Magnesium concentration ftMg5O,・7H,O,! so as to maintain around 20g/β. :Glucose concentration is 20g/I to maintain around 20g/J2! Appropriate additions were made midway through to avoid the following.

The results of comparing the ammonium ion concentration (in terms of NH3) and the amount of ATP produced (in terms of ATP Na2'3H20) at the start of the reaction are shown in Table 3.

Table 3 0.08 26.3 56.22.2
1 14.1 21.2 Example 5 500ml of the culture solution in culture tank F obtained in Example 4 was
Put it in a culture tank and add 100g/j of glucose! , adenine 30g1i, nicotinic acid 0.2g/l, K H4F 0
415 g/l, MgS04・77H2O1
O/12. Xylene 10m1/j! and Naimeen S.
-2154 g/l added, 800 rpm.

The reaction was carried out at 32° C. and an air flow rate of 2.51/min while adjusting the pH to 7.4 using 10N caustic potassium.

During the reaction, the phosphoric acid concentration (KH, PO.

(converted) and magnesium concentration (MgSO, 7H20
The glucose concentration was appropriately added midway so that the glucose concentration did not fall below 20 g/β. As a result, ATP was 7 after 28 hours of reaction.
0.6g/j! generated.

Example 6 Glucose 20g/i', polypeptone 15g/! A liquid medium consisting of 15 g of yeast extract/CNaCj'2.5 g/j' and 1 g/J of urea (adjusted to pH 7.2 with caustic soda) was put into a large test tube and sterilized by heating, and then Corynebacterium・Glutamicum ATC
C211/l was inoculated and cultured with shaking at 28°C for 24 hours.

Next, add 20 ml of this culture solution to 50 g/l of sucrose.
, meat extract 10g/β, K1-12P 0.2g/β, M
g S Os 7H, 00, 5g/β, (NH,).

30.5g/j! , urea 1 g/Il, F e SO
4.7H2010mg/j! , Mn SO4・4~6H
2010+++g/β, corn stave liquor 40
g/l, biotin 50μg/l, vitamin B, lOOμ
g/l and Caco, 20 g/Il (adjusted to pH 7.2 with caustic soda and then heat sterilized) into a 211 volume Erlenmeyer flask with a baffle containing 230 ml of liquid medium was inoculated, and cultured with shaking at 28°C for 24 hours. did.

Add 250ml of this culture solution to 70g/l of sucrose and meat extract Log/j! , KH2P 042 g/J! 1
2HPO-1.2g/l, MgS○, 7H201,
7g/L (NH,)iso, 17g/LFeSOa
'7H2013mg/j! %MnSO4・4~6Hz0
13mg/j! , D-n-xf-F Liquor 6log
/j! , biotin 230mg/j! and vitamin 8.
450mg/j! (adjusted to pH 6.8 with caustic soda and then heat sterilized) was inoculated into a 51-volume culture tank containing 1.4β liquid medium, and heated at 30°C and 600 rpm.
, aeration ill vvm, pH 6 with concentrated ammonia water.
, 8. Culture was performed while adjusting the temperature. During cultivation, sucrose 374 g#! 5KHaP0゜0.7g/l and HP○s0.5g/j! A liquid 50 having the composition of
0 ml was added twice, and after the second addition, pH adjustment was switched from concentrated ammonia water to 10N caustic potash and culture was continued.

When the sugar in the culture solution Tsuchiura is consumed, 500ml of culture solution
was placed in a 21-volume culture tank, and a reaction was carried out in the same manner as in Example 5. At the start of the reaction, the ammonium ion concentration in the reaction liquid supernatant was 0. It was lOg# (NHs equivalent). As a result, 8TP was 27.5g/j in 28 hours of reaction! generated.

Effects of the Invention According to the present invention, AT is produced from adenine, a phosphate group donor, and an energy donor using a microorganism as an enzyme source.
P can be produced industrially at low cost and efficiently.

Claims (3)

[Claims] (1) Adenine, a phosphate group donor and a culture solution, bacterial cells, or processed products of microorganisms that have the ability to produce adenosine-5'-triphosphate from adenine, a phosphate group donor, and an energy donor. When producing adenosine-5'-triphosphate by contacting the body and the energy donor in an aqueous medium containing a surfactant and/or an organic solvent, the A method for producing adenosine-5'-triphosphate, which comprises reducing the ammonium ion concentration to 1 g/l or less in terms of NH_3. (2) The production method according to claim 1, wherein the culture solution after the completion of culturing the microorganism is used as it is as an aqueous medium for the contact reaction. (3) Claim 1 or 2, wherein the microorganism is a microorganism belonging to the genus Brevibacterium or the genus Corynebacterium.
Manufacturing method described.