JPS59216592A - Production of phosphonoformic acid - Google Patents

Abstract

PURPOSE:To obtain phosphonoformic acid with a high productivity by breeding species to be used and changed in cultivation conditions, by cultivating a strain of the genus Streptomyces under aerobic conditions, and collecting the phosphonoformic acid from the resultant culture fluid. CONSTITUTION:A strain belonging to the genus Streptomyces, e.g. Streptomyces hygroscopicus SF-1293 strain (FERM-P No. 996), is cultivated in a culture medium used usually for cultivating a microorganism of the genus Streptomyces under aerobic conditions preferably at about 28 deg.C for 4-6 days. The resultant culture fluid is then usually subjected to chromatography, etc. to give the aimed phosphonoformic acid.

Description

[Detailed description of the invention] The present invention relates to a novel method for producing phosphonoformic acid.

Phosphono Ivia is Epstein-Barr v
irus, herpe8simplex virus
It is a substance that has inhibitory activity on the harboring of various viruses such as 1nfluenzaA NA virus [5
science Vol 201819-821 (197
8) ].

As a method for producing this substance, a chemical synthesis method is already known [Chem, Ber 57B, 10
23 (1924)).

The present inventors have discovered that 118F-1293 is useful as a herbicide.
! During biosynthesis research of i (Special Publication No. 51-639),
We have discovered that a mutant strain that produces 5F-1293 substance produces a large amount of phosphonoformic acid, and have completed the present invention.

That is, the present invention cultivates a strain belonging to the genus Streptomyces under aerobic conditions, and extracts phosphono@
This is a new method for producing phosphonoformic acid, which involves collecting the acid.

The strain of Streptomyces that can be used in the present invention may be any strain as long as it has the ability to produce sufficient phosphonoacid to be collected into the culture solution. An example of such a strain of the genus Streptomyces is 'C', which was developed by the present inventors as Streptomyces hygroscopicus strain 5F-1293 (Feikoken Bacteria Serial No. 996).
Strain No.: ATOO Deposit No. 21705) was obtained by treatment with nitronganidine
3 stocks). Streptomyces hygroscopicus NP-213 strain has been deposited with Hui Technological Research Institute (Feikoku Kenkyuu No. 7044), and its mycological properties are described in Japanese Patent Publication No. 1983-639, except that it forms few spores. Streptomyces hygroscopicus SF-1293 strain.

As seen in other Streptomyces strains, the NP-213 strain is susceptible to changes in its properties, and can be mutated by artificial mutagenic means using, for example, ultraviolet rays, X-rays, chemicals, etc. All Streptomyces strains capable of producing phosphonoformic acid, even such mutant strains, can be used in the present invention.

In the production method of the present invention, strain NP-213 is cultured in a medium containing nutrients that can be used by common microorganisms. As the nutrient source, known nutrient sources conventionally used for culturing Streptomyces bacteria are used. For example, carbon sources include glucose, starch, glycerin, sucrose, starch syrup, molasses, etc., and these may be used alone or in combination. Soy flour is also used as a nitrogen source.

Wheat germ, meat extract, peptone, dry yeast, cornstarch liquor, ammonium sulfate, sodium nitrate, etc. may be used alone or in combination. In addition to adding inorganic salts such as calcium carbonate, common salt, potassium chloride, and phosphates as necessary, organic and inorganic substances that aid the growth of bacteria and promote the production of phosphonoformic acid may be appropriately added. The most suitable culture method is a liquid culture method, especially a deep culture method. Cultivation is performed under aerobic conditions, and the temperature suitable for cultivation is 25 to 35°C, but in most cases it is cultured at around 28°C. The number of days for culturing is suitably 3 to 8 days, more preferably 4 to 6 days.

In order to purify and isolate phosphonoformic acid from the culture p solution, separation and purification methods commonly used for isolating microbial metabolites from the culture solution can be used.

Specifically, since phosphonoformic acid is a water-soluble acidic substance, Amberlite □I is used for its purification.
Cation exchange resin such as R-129, DOWEX 50W, or Amberlite nA-400. A method using an anion exchange resin such as DOWEX 1×2, Hisephadex, cellulose, and CIVAGEL.

It is preferable to perform chromatography using charcoal powder or the like in an appropriate combination. For example, DOWEX 5
0 W (H-type) IΔ fat tower, j, = lh
The method of removing sexual impurities is an effective method for purifying phosphonosaccharides. In addition, phosphonoformic acid is usually
It is known that it decomposes easily in acid t1 to form phosphorous acid and carbon dioxide [J, Ohem, 8oc.

(B)618(197], )':l, In order to purify with good yield, it is necessary to carry out the process at low temperature for a short period of time. In the production of physiologically active substances using microorganisms, it is said that it is possible to increase the productivity by breeding the bacterial strains used or changing the culture conditions, and from this point of view, the present study 14 The method has significant advantages over conventional synthesis methods. - The method of the present invention will be explained in more detail below based on Examples, but the present invention is not limited to the following Examples.

Example Streptomyces hygroscopicus strain NP-213 (Wi Koken Bacteria No. 7044) was cultured in a preculture medium (2.0% soluble starch, 1% polypeptone, 0.3% meat extract, dipotassium phosphate solution) 0.05%, pr17.o)101
nlK inoculation. This was cultured with shaking at 28°C for 24 hours,
Furthermore, 80ml of the same medium. It was subcultured and shaken at 28°C for 24 hours.

Glucose/1.0% for jar fermenter.

4. Production medium with the composition of 2.25% sungrain, 3.5% wheat germ, 0.1% potassium dihydrogen phosphate, and 0.0001% cobal chloride. Ol! The seed mother was inoculated into a cell, and cultured with aeration and stirring at 28°C. The culture solution cultured for 144 hours was adjusted to p) 13.0 at 5°C, and the bacterial cells were removed using a refrigerated centrifuge to obtain a culture solution with p) of 2.01. The obtained culture p solution was applied to a column packed with 40 Q me of Dowex 50W
(Ol type) Filled with 400ml column K, washed with water (
Approximately 600#l') After eluting with Nv, at water for 20me
(Fractions 1 to 150 are 0.5% Na
p/water, 151 et seq.3. ONapl (M) dissolved in water.

Combine the fractions A173 to 187 containing phosphonoformic acid (approximately 300 ml), adjust the pH to 7.0, concentrate, separate the precipitated NaCl crystals, and add approximately 5 ml of ν liquid.
The remaining Mail was separated by chromatography using a column packed with Sephadex G-10950m. Phosphonoformic acid was contained in fractions A39 to A41, which were separated by -JQml.

By concentrating this fraction, 150 m9 of white phosphonoformic acid powder was obtained.

The elemental analysis, melting point, infrared absorption spectrum, and nuclear magnetic resonance spectrum of the phosphonoformic acid thus obtained were measured using conventional methods, and it was confirmed that the result matched that of a phosphonomyelinated acid specimen (Sigma P-2895). .

Patent applicant: Meiji Seika Co., Ltd.

Claims (1)

[Scope of Claims] 1. A method for producing phosphonoformic acid, which comprises culturing a strain belonging to the genus Streptomyces under aerobic conditions and collecting phosphonoformic acid from the resulting culture solution. 2. The strain belonging to the genus Streptomyces is Streptomyces hygroscopicus strain Np-213 (Feikoken Bacteria 9).
1'', No. 7044).