JPS5948997B2 - Method for producing 5'-purine ribonucleotide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing 5'-purine ribonucleotides.

5'-purine ribonucleotides occupy an important position in the fields of medicine and food.

Therefore, several methods are known for its production, and among them, a method in which a microorganism is brought into contact with a nucleobase or nucleoside and an inorganic phosphate to produce the corresponding 5'-purine ribonucleotide is known. A method using a specific species belonging to the genus or Bacillus (Japanese Patent Publication No. 42-1186), a method using Microbacterium (Japanese Patent Publication No. 4513-1988), a method using Nitrosomonas (Japanese Patent Publication No. 47-2552), Method for culturing Corynebacterium in the presence of inosine (Special Publication 1973-
44350), a method of culturing microbial cells belonging to the genus Micrococcus, Flavobacterium, Corynebacterium, and Brevibacterium in the presence of a phenolic compound, thio compound, nitroso compound, alcohol, glucosamine, or oxime (Tokukosho 44-24314), Brevibacterium genus bacteria are treated with parachloromercuribenzoate, orthoiodosobenzoate, monoiodoacetic acid, iodoacetamide, other enzyme inhibitors, various chelating agents, and mercury, silver, zinc, manganese, nickel, etc. A method of culturing in the presence of heavy metal salts, amino acids such as histidine, tryptophan, and peptone, processed natural products, etc. (Japanese Patent Publication No. 11555/1989), or methods of culturing in the presence of heavy metal salts, amino acids such as histidine, tryptophan, and peptone, and processed products of natural products, or Brevibacterium spp., specific species of Bacillus spp., Aerobacter spp. A method for culturing specific species belonging to the genus Satucharomyces, Streptomyces, etc. (Special Publication No. 37078/1978)
etc. are known.

However, in all of these known methods, the desired enzymatic reaction is performed while the microorganism grows, and not only does it take an extremely long time to produce 5'-nucleotides, but the yield is also unsatisfactory. Moreover, depending on the method, it is necessary to use extremely harmful compounds, so it cannot be said to be an advantageous method from an industrial standpoint.

As a result of intensive research aimed at establishing an industrially more advantageous method for producing 5'-purine ribonucleotides against this technical background, the present inventors discovered that Arthrobacter
Brvibacterium (Brviba)
cterium) or Micrococcus (Mic)
By contacting the cells of a microorganism belonging to the genus P. rococcus with purine ribonucleosides in the presence of phosphate ions without substantially proliferating the cells in an aqueous medium, the purine ribonucleosides can be produced in a short period of time. It was found that it was converted to the corresponding 5'-purine ribonucleotide in high yield.

The present invention was completed as a result of further research based on this new knowledge.

In the present invention, 5'-purine ribonucleotides belonging to the genus Arthrobacter, Brevibacterium, or Micrococcus are produced from purine ribonucleosides and phosphate ions in an aqueous medium under substantially nonproliferative conditions. The cells of microorganisms that have the ability to do this are used.

Representative examples of such microorganisms include, for example, Arthrobacter citreus (Arthrobacter citreus).
reus), Arthrobacter tumesen X (Art
hrobactertumescens), Arthrobacter athrocyaneus
atrocyaneus), Brevibacterium Linens
, Brevibacterium alkanophile A (Bre
vibacterium alkanophilum)
, Prehybacterium stationi7. (Brev
ibacteriumstationis), Brevibacterium thiogenitalis (Brvibacterium thiogenitalis)
um thiogenitelis), Brevibacterium inmariophylium
ilium) (r Journal of the Japanese Society of Agricultural Chemistry, Vol. 36,
No. 2, pp. 141-159 (1962)], Micrococcus v.
arians), Micrococcus glutamicus (Mi
Crococcus glutamicus).

These may be naturally or artificially mutated mutant strains as long as they can be used for the purpose of the present invention.

In addition, the classification of microorganisms in the present invention is
Bergeys Manual of Determinative Bacteriology 8th Edition
Terminative Bacteriology
8th edition).

Cultivation for obtaining the cells of the above-mentioned microorganisms can be carried out continuously or intermittently by conventional aerated agitation culture, shaking culture, static culture, solid culture, or the like.

The medium to be used may have a composition that is normal for the growth of the microorganisms used, and the carbon dioxide source may be selected from among carbohydrates, fats and oils, fatty acids, alcohols, etc. that can be assimilated, either alone or in combination. used.

In addition, as nitrogen sources, various agricultural processing waste resources such as oil extraction residue, soaking liquid, extract, etc., microbial cells, and their melted ice products are used, and ammonia or nitrate nitrogen compounds such as ammonium sulfate, ammonium nitrate, Organic/inorganic compounds such as urea and ammonium chloride can be used.

In addition to a carbonate source and a nitrogen source, essential growth factors and growth-promoting substances such as minerals, amino acids, and vitamins necessary for growth are preferably added to the medium.

Furthermore, for the purpose of controlling pH and foam during culture, it is effective to appropriately supplement aqueous ammonia, caustic alkaline solution, calicylum salts, or add an antifoaming agent.

The culture temperature may be selected as appropriate depending on the nature of the microorganism used, but it is usually cultured at about 20 to 45°C, generally 25 to 40°C.

The culturing time may be any suitable time that allows sufficient growth of the bacteria, but it is usually cultured for about 5 to 50 hours.

The microbial cells thus obtained are collected and used by means such as centrifugation or filtration, but the material containing the microorganisms, such as the culture obtained as described above, may also be used.

In the present invention, microbial cells are brought into contact with purine ribonucleosides in the presence of phosphate ions without substantially causing them to grow again.

In order to carry out the enzymatic reaction without substantially proliferating the microbial cells, for example, the microbial cells used or their contents may be treated in advance to eliminate the growth activity of the microorganisms, or/ Alternatively, the reaction may be carried out while being brought into contact with a drug having such an effect in the reaction solution.

Examples of treatments for losing the growth activity of bacterial cells include physical treatments such as freeze-thaw treatment, heating treatment under acidic or alkaline conditions (for example, pH 4 to 10), and osmotic pressure treatment. For example, organic solvents such as toluene, xylene, and butanol, cationic, anionic, or nonionic surfactants such as sodium lauriul sulfate, polyoxyethylene amino, polyoxyethylene alkyl allyl ether, and alkyldimethylbenzyl ammonium chloride. Examples include treatment that brings the material into contact with.

As the drug added to the reaction solution, the above-mentioned organic solvents and surfactants are conveniently used.

These physical treatments and contact treatments with drugs can be performed alone or in an appropriate combination.

In particular, the viable cells or culture are once frozen and then thawed, and/or one or more surfactants are added in a range of 0.01 to 0.2% (weight/volume),
Furthermore, a heating treatment is performed at 35 to 65°C for 30 seconds to 2 hours under a pH of 4 to 10, and if necessary, 0.1 to 2
% (vol/vol) concentration of organic solvent is advantageously brought into contact.

In addition, the above-mentioned surfactant may be present at a concentration of 0.01 to 0.2% (weight/volume) in the reaction system in which the above-mentioned treated product of viable cells or normal cells exists, or/and the above-mentioned surfactant may be present at a concentration of 0.01 to 0.2% (weight/volume) It is also advantageous to have an organic solvent, such as, present in a concentration of 0.1 to 2% (vol/vol).

The reaction of the present invention is carried out by bringing the microbial cells into contact with the purine ribonucleoside in the presence of phosphate ions in an aqueous medium without propagating the microbial cells as described above.

The aqueous medium is mainly composed of water and may contain various substances as described below.

As purine ribonucleosides, adenosine, inosine, xane I-sine, guanosine, etc., as well as 8-azaguanosine and 6-mercabutobrine ribosidin derivative ribonucleosides are used.

These can be used alone or in combination.

As the phosphate ion source, any source that can be dissociated into phosphate ions in an aqueous medium may be used, such as free phosphoric acid itself, inorganic phosphates, such as salts of phosphoric acid with alkali metals (sodium salts, potassium salts, etc.),
Salts with alkaline earth metals (calcium salts, magnesium salts, etc.), ammonium salts, etc. are advantageously used.

These phosphate ion donor sources are preferably used in an amount of usually about 3 times or more, especially about 5 to 20 times the mole of purine nucleoside as phosphate ions.

This reaction is usually preferably carried out at a pH of about 5 to 10 and a temperature of about 20 to 55°C. If the pH fluctuates, it is recommended to use an appropriate acid or alkali during the reaction to correct it to the desired pH. .

Further, the yield of the corresponding 5'-purine nucleotide is maximized if the reaction is generally carried out for about 3 to 30 hours while standing still or with shaking or stirring.

In order to carry out this reaction more effectively, it is desirable to maintain the catalytic activity of the microorganisms used at its maximum level, and for this purpose it is advantageous to coexist with an energy source in the reaction solution.

Examples of energy sources include carbohydrates such as glucose, sucrose, maltose, and sorbitose.
It is advantageous to carry out the reaction by adding about 5 to 5% (weight/volume), or by adding it sequentially within a range where the concentration does not become zero.

Furthermore, in order to promote the reaction and maintain and continue its activity, salts such as magnesium, iron, manganese, etc. may be added individually or in combination, if necessary.

In order to collect the 5'-purine ribonucleotide thus produced in the reaction solution, a commonly known method for collecting 5'-purine ribonucleotide is used, such as activated carbon or ion exchange resin. Column chromatography, decolorization, desalting, separation, concentration, crystallization, precipitation,
By applying unit operations such as drying alone or in combination, 5'-purine ribonucleotides can be collected as single or mixed crystals or compositions.

EXAMPLES The content of the present invention will be explained in more detail with reference to Examples below, but these are merely illustrative of the content of the present invention and do not limit the scope of the present invention.

In addition, in the examples, numbers with IFO and ATCC are as follows:
This is the storage number of the microorganism at the Fermentation Research Institute or the American Tights 0 Culture Collection, and all of these microorganisms are preserved bacteria listed in the list of the depositary institution.

Example 1 Each microorganism listed in Table 1 grown on a nutrient agar medium was
Glucose 2 adjusted to pH 7.0 with sodium hydroxide
%, peptone 1%, yeast extract 0.5%, and corn steeple strength -0.5%. Dispense 20 ml of a medium into a 200 ml Erlenmeyer flask and inoculate the sterilized medium.
The cells were cultured at ℃ for 24 hours on a rotary shaker incubator at 200 rpm.

The culture solution was centrifuged at 400 rpm for 15 minutes to collect bacterial cells, which were frozen at -20°C and left for 24 hours.

Add this to inosine 0.5%, guanosine 0.5%, ribose 1.0%, dipotassium phosphate 3.5%, magnesium nitrate (7A (salt) 0.25%, manganese sulfate 0.15%).
% solution (pH 7.5) and melted and suspended, 0.5% toluene was added and the mixture was heated at 46℃ for 2 hours.
After heating for 0 minutes, the mixture was reacted at 28° C. for 18 hours on the shaking incubator.

For comparison, similar reactions were carried out without subjecting the microbial cells to freezing treatment, toluene treatment, or heat treatment.

The concentrations of 5'-inosinic acid and 5'-guanylic acid produced in the reaction solution were as shown in Table 1.

In addition, throughout the reaction period, not only was no bacterial growth observed in the reaction system of bacterial cells subjected to the various treatments described above, but the number of viable bacteria decreased no matter which microorganisms were used. The number of bacteria decreased dramatically, and the number was less than 1/100,000 of the number originally used.

Note that the 5'-inosinic acid produced in the reaction solution is 5'-
5'-guanylic acid is determined by an enzymatic assay combining nucleotidase, nucleoside phosphorylase, and xanthine oxidase. It was quantified using a colorimetric method that develops color.

Example 2 Live cells of Brevibacterium alkanophyllum IFO12498 obtained in the same manner as in Example 1 were treated with inosine 0
．． 5%, guanosine 0.5%, glucose 2%, dipotassium phosphate 3%, magnesium sulfate 0.5%, potassium permanganate 0.3% (pH 7.5)
After suspending the suspension in 10 ml of polyoxyethylene alkyl allyl ether, 0.05% of Tsusan Nonion NS-210 (manufactured by NOF Corporation) was added and heat-treated at 50° C. for 20 minutes.

Then, after adding an amount equivalent to 0.5% toluene, the temperature was increased to 37°C.
The suspension was left for 18 hours with shaking; as the reaction time progressed, the number of viable bacteria in the suspension and the amounts of 5'-inosinic acid and 5'-guanylic acid produced were measured.

The results were as shown in Table 2.

Collect 11 of the reaction solution obtained here (after 18 hours of reaction),
After removing the precipitate by centrifugation, according to the usual method,
Necleotides were adsorbed on the activated carbon layer, washed with water, and eluted using ammonia ethanol.

After concentrating this eluate to remove ethanol, a small amount of water was added, and adsorption with ion-exchanged citrus (Dowex 1×4 chlorine type), washing with water, and elution with dilute hydrochloric acid were performed.

After elution, the liquid was adjusted to pH 7.5, concentrated, and ethanol was added. The resulting precipitate was dried.
-Inosinic acid 4.2g, 5'-guanylic acid 3.7g
(All quantitative values as free acids) were included.

Example 3 Using a live cell suspension of Brevibacterium alkanophyllum IFO12498 obtained according to Example 2, (
1) Freeze and thaw this, (2) Add 1.0% (volume/volume) toluene to this and suspend well, then (
3) To this, 0.4% polyoxyethylene alkylamine (Ni-Sannaimee S-22C: manufactured by NOF Corporation)
and polyoxyethylene alkyl allyl ether with Tsusannonion NS-210 (manufactured by NOF Corporation) 1.5%
After adding (weight/volume) and mixing, (4) 50℃
(5) After heating at 45°C for 20 minutes, add 0.1% toluene (volume/volume),
(6) After heating at 35°C for 20 minutes, toluene 0.1% (volume/volume) and the above nonionic NS-2100, 1% (
weight/volume), then inosine 0.5
%, guanosine 0.5%, glucose 2.4%, dipotassium phosphate 3.5%, magnesium sulfate heptahydrate 0.5
%, manganese sulfate 4-6 hydrate 0.3%, pH 7.5, and allowed to stand at 32°C for 24 hours with shaking to remove 5'-inosinic acid in the suspension. and 5
When '-guanylic acid was quantitatively determined, the following results were obtained.

Dainosinic acid raw Gorganylic acid raw test category Acid amount (g/J) Acid amount (g/J)
(1) 3.1 2.
1 (2) 2.8 1
．． 9(3) 3.5
2.8 (4) 2.71・6 (5) 3.6 3.
0(6) 3.8 3
．． 3 Untreated bacterial cells 0.2 0-1
During this reaction, the number of viable bacterial cells in all cases, except for the untreated bacterial cell group, had drastically decreased to less than one millionth of the amount of bacteria originally used.

Claims (1)

[Claims] 1 A microorganism belonging to the genus Arthrobacter, Brevibacterium, or Micrococcus that has the ability to produce the corresponding 5'-purine ribonucleotide from a purine ribonucleoside and a phosphate ion in an aqueous medium under conditions that do not substantially proliferate. Contacting the bacterial cells with purine ribonucleosides in the presence of phosphate ions without substantially growing the bacterial cells in an aqueous medium to produce the corresponding 5'-purine ribonucleotides, which are then collected. A method for producing a 5'-purine ribonucleotide, characterized by: