JPS5818075B2 - Method for producing adeno-5'-triphosphoric acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing adenosine-5'-triphosphate (hereinafter abbreviated as ATP).

An object of the present invention is to provide a method for industrially producing ATP, which is useful as a biochemical reagent or a pharmaceutical, at low cost.

Conventionally, ATP has been produced by culturing a strain belonging to Brevibacterium ammoniagenes in an adenine-containing medium and by fermentation.

Method of manufacturing (Japanese Patent Publication No. 41-17634), 5'-
A method for biochemically phosphorylating adenylic acid using yeast (Japanese Patent Publication No. 19119/1983), Candida spp.
The enzyme source is yeast cells belonging to the genus Pletanomyces or the genus Torulopsis and capable of assimilating hydrocarbons, or a processed product thereof, which acts on a reaction solution containing adenosine or 5'-adenylic acid, a phosphate donor, and sugars. A method for producing ATP by producing ATP (Japanese Patent Publication No. 50-9.873) is known.

These conventional methods have drawbacks such as long fermentation times and the high cost of azonsin and 5'-adenylic acid used as raw materials for enzyme reactions.Furthermore, there is a desire to develop an industrially inexpensive method for producing ATP. There is.

- The present inventors conducted various studies with the aim of developing an industrially inexpensive manufacturing method for ATP.

As a result, using cheap adenine as a raw material, 1. In addition, ATP can be produced in high yield by allowing the cells of microorganisms such as Satucharomyces serepiciae ATCC 20018 or their processed products to act as an enzyme source on the reaction solution containing a phosphate donor, sugars, and surfactants. We discovered the fact that it generates a significant amount.

The present invention will be explained in detail below.

Examples of the microorganism used in the present invention include microorganisms belonging to the genus Satucharomyces, Candida, and Torulopsis, and having the ability to generate ATP from adenine, phosphate donors, and sugars in the presence of a surfactant.

Suitable strains include, for example, Satucharomyces cerevisiae ATCC 20018, Candida zeylanoides ATCC 20356, and Torulopsis cyclophylla ATCC 22163.

For culturing these microorganisms, ordinary yeast culturing methods can be applied.

That is, carbon sources for the medium include, for example, carbohydrates such as glucose, sucrose, and starch hydrolysates; organic acids such as acetic acid; alcohols such as ethanol and methanol;
- Hydrocarbons such as paraffin can be used.

Nitrogen sources include ammonium sulfate, ammonium chloride, and urine L.
Inorganic and organic nitrogen sources are available, such as yeast extract, meat extract, peftone, and corn steep liquor.

Furthermore, as inorganic salts, potassium phosphate, magnesium sulfate,
Calcium chloride etc. can be used.

Furthermore, in addition to metal ions such as iron, manganese, and zinc, substances necessary for the growth of the bacterial strain are added to the medium as necessary.

Culture is usually carried out under aerobic conditions such as shaking culture or submerged culture with aeration, at a culture temperature of 25 to 35°C and a pH of 5.0.
~9.0 for 5 to 40 hours.

(Depending on the bacteria used, culture can be performed outside the above temperature and pH ranges.

In the present invention, the culture solution itself obtained by the above culture method, bacterial cells, cultured tear fluid, crushed bacterial cells,
A bacterial cell treated with a solvent such as ethanol, toluene, ether, or acetone is used as an enzyme source.

The bacterial cells may be directly suspended in the reaction solution, or may be dried using, for example, acetone, or the bacterial cells may be immobilized using a conventional method.

The reaction in the present invention is carried out by allowing an enzyme source to act on a reaction solution containing adenine, a phosphate donor, a saccharide, and a surfactant.

As the phosphoric acid donor used in the reaction, for example, KH2PO
4, K2 HPO4+ K3 PO4, NaH2PO4
vNa2 HPO4, Na3 PO4, M,! i'a
Examples include inorganic phosphates such as (PO4)2.

Further, as the sugar, for example, glucose, starch hydrolyzate, sucrose, etc. can be used.

In the present invention, it is essential that a surfactant be present in the reaction solution; if no surfactant is present,
Almost no ATP generation was observed.

As the surfactant, any of nonionic, cationic, anionic, amphoteric, and primary to tertiary amine types can be used.

To give specific examples of these, non-ionic ones include:
Nonion 0-4, Nonion LT-221, Nonion LP
-2OR (all manufactured by Nippon Oil & Fats Co., Ltd.), cationic FB, Naimeen S, etc.
-204, cationic F2-40E (both manufactured by NOF Corporation), and anionic ones include Newrex TAB.

Rapizole B-80 (both manufactured by NOF Corporation), amphoteric ones such as Cl0-18 alkylglycine [for example, Anon LG (manufactured by NOF Corporation)], and primary to tertiary type As for cto,
18 alkyldimethylamine [for example, tertiary amine FB (manufactured by NOF Corporation)], hexadecyldimethylamine (tertiary amine), and the like.

Among these, amphoteric surfactants such as C10-18 alkylglycine, primary to tertiary surfactants such as CTO-18 alkyldimethylamine, hexadecyldimethylamine, etc.
Preferred are surfactants of the grade amine type.

; In the reaction in the present invention, adenine 1-59/13 and phosphate donor (as PO4) 30-59/13 are added to the reaction solution.
2001/l, sugars (glucose) 30-100
．． ! It is preferable to add it to the reaction solution at the start of the reaction or at the early stage of the reaction, although it varies depending on the concentration of il/l, surfactant and surfactant concentration of 0.5 to 2.

The amount of the enzyme source to be used to act on the reaction solution varies depending on how the enzyme source is used (bacterial cells, culture solution, processed material of microbial cells) and the concentration of the reaction raw materials.
150 g/l (as dry bacterial cells) is suitable.

The reaction is usually carried out at a pH of 4. 0~9.0, reaction temperature 15~
40°C is suitable.

The reaction is carried out under aeration and stirring conditions, and is completed in 3 to 20 hours.

- To give an example of specific reaction conditions and ATP production yield, dry yeast cells (water content approximately 3.0%) 20-100C9/
l), adenine 1-5 (&/l), phosphate donor (
As PO4) 30-200! l/1. glucose 30
~1100 (/l), surfactant 0.5 ~ 259/11
Approximately 3-1 at pH 6-8.5 and temperature 25-37℃
After 0 hours, ATP can be produced with a molar yield of 90-99.

Purification and isolation of ATP from the reaction solution can be carried out by using activated carbon, ion exchange resin, etc., as exemplified in Examples.

Next, examples of the present invention will be shown.

Example 1 Satucharomyces cerevisiae (ATCC2) was used as the inoculum.
0 0 1 8 ), Torulopsis cyclophisi (ATCC 22163), and Candida zeylanoides (ATCC 20356), and these strains were treated with 3Elldl of glucose and 0.5ji/dl of ammonium sulfate.
, KH2PO40,1,! i'/ dir, MEI
SO4・7H200, 0597dl, yeast extract 0.3
9/lU, CaCO319/dl, 1
Glucose at a rate of 0% (volume) 13. ill/dmu ammonium sulfate 1.0 g/ait.

KH2PO40, 1g/di, M, 9SOa ・7
H200, 05g/dl, corn steeple force -0,
5 g/d11 pl (6,5 (before sterilization)) was inoculated into a 51 jar fermenter containing 31 bacterial cell production medium (before sterilization),
Aerated agitation culture was carried out at a temperature of 48 hours at a stirring speed of 600 rpQ and a volume of 31/-.

During the course of culture, the pH is adjusted to 6 to 6.5 with aqueous ammonia.

The cells obtained by centrifuging the culture solution were dried in a fluidized bed dryer at 45° C. for 1 hour, and the dried cells (moisture content: 3 parts) were subjected to the reaction.

The dried bacterial cells 5 (1, adenine 2g, glucose 30
g, n-hexadecyldimethylamine (tertiary amine pB
1,000 ml of a 0.3M phosphate buffer (pH 8,0) containing 0.10% (Nippon Oil & Oil Co., Ltd.) was aerated and stirred at 30° C. for 4 hours.

The amount of ATP produced in the reaction solution after the reaction was as shown in the following table.

Each of the above reaction solutions was heat-treated at 85° C. for 20 minutes, and solid matter was removed by centrifugation to obtain a supernatant.

This supernatant liquid was adjusted to pH 3.5 with 2SO4, 18 g of activated carbon was added to it to adsorb ATP, and after washing with water, a mixture of 50% ethanol and ammonia [28% ammonia aqueous solution and ethanol (99%) were mixed in 1 part] 1 (V/V)] and concentrate the eluate to around pH 8.

Next, the concentrated solution was passed through a strong basic anion exchange resin Dowex-1 x 4 which had been adjusted to C1 type in advance, and after washing with water, it was poured into a mixture of hydrochloric acid and common salt (0.02N-HC6 solution (pH 1.7)). A 0.2M NaCl solution obtained by dissolving NaCl] was used to collect the ATP fraction.

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, crystals of the sodium salt of ATP are obtained.

The yield was as shown in the table below.

Example 2 Same conditions as Example 1 except that the three strains used in Example 1 were used and 0.20% of alkylglycine (Anon LG, NOF) was used instead of n-hexadecyldinthylamine. The mixture was aerated and stirred for 6 hours.

The amount of ATP produced in the reaction solution after the reaction was as shown in the following table.

Each of the above reaction solutions was purified in the same manner as in Example 1 to obtain the sodium salt of ATP as shown in the following table.

Example 3 Same as Example 1 except that the three strains used in Example 1 were used and 0.25% of alkyldimethylglycine (tertiary amine FB, NOF) was used instead of n-hexadecyldimethylamine. The mixture was aerated and stirred under the same conditions for 8 hours.

ATP generation zone JI-+Jiyi trs in the reaction solution after reaction
Each of the above reaction solutions was purified in the same manner as in Example 1 to obtain the sodium salt of ATP as shown in the following table.

Claims (1)

[Scope of Claims] 1. Azone belonging to the genus Satucharomyces, Candida or Torulopsis from adenine, a phosphate donor and a saccharide in the presence of a surfactant. Adenosine-5'-triphosphate is produced by allowing a culture solution or cells of a microorganism capable of producing syn-5'-triphosphate, or a treated product thereof, to act on a reaction solution containing adenine, a phosphate donor, a saccharide, and a surfactant. An adenosylene which produces 5'-triphosphoric acid. Method for producing no-5'-triphosphoric acid.