JPH0420429B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a novel antibiotic SF-2312 substance and a method for producing the same. More specifically, SF- is a new antibiotic obtained by culturing actinomycetes.
2312 Substances or their salts and their production methods. Background to the Invention The present inventors searched for new and useful antibiotics that have antibacterial activity against various Gram-positive and Gram-negative bacteria, and as a result, they cultivated actinomycetes belonging to the genus Micromonospora in a nutrient medium. new antibiotics by
We discovered that SF-2312 substance is produced by fermentation, isolated SF-2312 substance, and determined its physicochemical and biological properties. Furthermore, we have found that the production of SF-2312 substance can be significantly increased by adding vitamin _B12 to the medium when culturing this bacterium. The present invention was completed based on the above findings. Summary of the Invention Therefore, the first invention is based on the following formula: The present invention provides a new antibiotic SF-2312 substance represented by: and its salt. Furthermore, the second invention provides a new antibiotic SF-2312 substance or a salt thereof, which is characterized by culturing SF-2312 substance-producing bacteria belonging to the genus Micromonospora and collecting the SF-2312 substance or its salt from the culture. A method for producing the salt is provided. DETAILED DISCLOSURE OF THE INVENTION New antibiotic SF- used in the method of the present invention
An example of 2312 substance-producing bacteria is SF, which was newly isolated from soil in Shimizu City, Shizuoka Prefecture by the present inventors.
There are −2312 stocks. The mycological properties of SF-2312 strain are as follows. Morphology The basal hyphae are elongated and branched, with a diameter of approximately
It is 0.5 μm. Aerial hyphae are not normally formed on various agar media used for culturing actinomycetes. In rare cases, spores that appear to be spores are observed singly on the basal hyphae (observation using an optical microscope), but their formation is extremely poor in both agar culture and shaking culture, and to date, spores have not been formed. have not yet been confirmed using an electron microscope. Sporangia, flagellated spores, and sclerotia are not observed in the examined area. Growth status on various media The growth status of SF-2312 strain on various agar media is as shown in the following table. Regarding color description [ ]
The standards shown in the Container Corporation of
"Color Harmony Manual" manufactured by America was used. Observations were made after culturing at 28°C for 14 to 21 days.

[Table] Physiological properties (1) Growth temperature: Grows in the temperature range of 15 to 38℃, and the optimum temperature is 26 to 30℃. (2) Liquefaction of gelatin: Positive (3) Hydrolysis of starch: Positive (4) Reduction of nitrate: Negative (5) Peptonization of skim milk: Negative Coagulation of 〃: Negative (6) Salt tolerance: 1.5% salt grows in the presence of
It will not grow above 3.0%. (7) Production of melanin-like pigment: negative Availability of carbon source Basic medium: Yeast extract (Difco) 1.0g CaCO ₃ 0.2g Agar (Difco) 15g Distilled water 1000ml (1) Use: D-glucose, D -Xylo-
(2) Not used: L-arabinose, L-rhamnose, i-inositol, (3) Used in doubt: D-fructose, D-
Mannitol, sucrose, raffinose Cell wall composition Lechervalier et al. (Intern.J.Syst.Bact.20:435~
443, 1970), the cell wall composition of strain SF-2312 is type D, and the sugar pattern is type D. From the above, the SF-2312 strain has a type of cell wall composition.
It is an actinomycete with a D-type sugar pattern, is a mesophilic bacterium, does not form aerial hyphae, and forms one spore each on the basal hyphae, so it is thought to belong to the genus Micromonospora. However, due to poor sporulation, spores have not yet been confirmed by electron microscopy. Therefore, further study is required to determine the genus, but at present this bacterium is classified as Micromonospora sp. SF-2312 (Micromonospora ap.SF-2312).
2312). This strain was sent to the Institute of Microbial Technology, Agency of Industrial Science and Technology.
Since March 2, 1984, it has been entrusted as FERM-P 7492. As with other microorganisms, antibiotic SF-2312
The strain that produces this, strain SF-2312, is susceptible to changes in its properties. For example, any mutant strain (naturally occurring or induced), phenozygote or genetically recombinant of or derived from the SF-2312 strain that produces the antibiotic SF-2312 is subject to the present invention. Can be used for In the method of the present invention, the strain 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 actinomycetes can be used. For example, glucose, glycerol, starch, starch syrup, sucrose, dextrin, molasses, vegetable oil, animal oil, etc. can be used as the carbon source. Further, as the nitrogen source, soybean flour, wheat germ, meat extract, peptone, yeast extract, cornstarch liquor, cottonseed meal, fish meal, ammonium sulfate, sodium nitrate, urea, etc. can be used. In addition, it is effective to add soluble inorganic salts capable of producing sodium, potassium, magnesium, calcium, cobalt, chlorine, phosphoric acid, sulfuric acid, and other ions as necessary. In addition, it helps the growth of bacteria, and the antibiotic SF-2312
Organic and inorganic substances that promote the production of can be added as appropriate. For example, vitamin _B12 is
The antibiotic SF-2312 promotes the growth of SF-2312 strain.
It is extremely effective in significantly increasing the production of. The most suitable culture method is a culture method under aerobic conditions, especially a deep culture method. The appropriate temperature for culturing is 15 to 38°C, but in most cases, culture is carried out at around 26 to 30°C. Production of the antibiotic SF-2312 varies depending on the medium and culture conditions, but the accumulation usually reaches its maximum within 2 to 10 days in both shaking culture and tank culture. The following method is used to assay SF-2312 substances. Mycin agar is used as the assay medium. Serratia marcescens is used as the test bacterium. SF−2312
The substance is 1000 μg/ml ~ in the assay using this
At 250 μg/ml, the relationship between the logarithm of the concentration and the inhibition circle diameter shows a linear relationship, giving respectively inhibition circle diameters of 23.2 to 15.8 mm (paper disc method). The SF-2312 substance obtained according to the present invention is a water-soluble acidic substance. When collecting this from the culture, it is extracted and purified using charcoal powder, Diamond Ion HP-
Chromatography using adsorbents such as 20, anion exchange resins such as Diaion WA-10, Dowex 1x2, gel filters such as Cephadex G-10, etc. is used, but the collection method shown below is efficient. It is. That is, bacterial cells and other solid substances are separated from the culture solution using a supernatant such as diatomaceous earth, and then the active ingredients in the solution are adsorbed onto Diaion WA-10.
After washing the resin part with water, it is eluted with 1N ammonia water. This eluate was concentrated under reduced pressure, and further mixed with charcoal powder, Dowex 50W x 2 (H ⁺ ), Cephadex G-10
High purity SF-2312 substance can be obtained as a free acid or Na salt by appropriately combining column chromatography such as the following. SF−2312 substance
Na salt can be obtained as a free acid by hydrolysis with acid under mild conditions. When reacted with a base in a conventional manner, a salt of SF-2312 substance is obtained. Examples of this salt include alkali metal salts, ammonium salts, and alkaline earth metal salts. The physical and chemical properties of SF-2312 substance (Na salt) are shown below. 1 Appearance White amorphous powder 2 Melting point Gradually turns brown from around 160℃, but does not show a clear melting point. 3 Elemental analysis values C21.12%, H 3.38%, N 5.97% 4 Ultraviolet absorption spectrum shows no characteristic absorption between 220nm and 370nm. 5 Infrared absorption spectrum The spectrum measured in the potassium bromide tablet is
As shown in the figure. 6 Molecular weight SF-2312 from mass spectrometry (SI-MS)
The molecular weight of the substance (Na salt) is 219. 7 Molecular formula Nuclear magnetic resonance spectrum, mass spectrometry,
The molecular formula according to elemental analysis is C ₄ H ₇ NO ₆ PNa. 8 Hydrogen nuclear magnetic resonance spectrum of SF-2312 substance (free acid) measured in heavy water
The 400MHz, ¹ H NMR spectrum is shown in Figure 2. 9 Specific optical rotation [α] ²⁰ _D = +12° (cl.H ₂ O) 10 Solubility Soluble in water, but poorly soluble or Insoluble. 11 Color reaction Positive: Remieux, sulfuric acid, ferric chloride, ferric chloride-sulfosalicylic acid reagent Negative: Ninhydrin reagent 12 Rf value of silica gel thin layer chromatography (Merck F ₂₅₄ ) Rf 70% ethanol water 0.32 n-propanol-acetic acid-water (15:10:3:
12) 0.58 Acetonitrile-water (7:3) 0.26 13 Rm value of high-voltage paper electrophoresis Rm (glutamic acid) 1.07 Pyridine-acetate buffer PH6.4. 3500V, 15 minutes Rm (fusufomycin) 0.74 Formic acid-acetate buffer PH1 .8. 3500V for 15 minutes14 Stability Relatively stable under acidic, neutral and alkaline conditions. Next, Table 1 shows the antibacterial activity of SF-2312 substance (Na salt) against various microorganisms.

[Table] Agar dilution method medium: Nutrientagar As shown in Table 1, SF-2312 substance has antibacterial activity against Gram-negative bacteria. SF- with the above physicochemical and biological properties
Substance 2312 was determined to be a new substance as there was no corresponding item in the literature. In other words, as an antibiotic for water-soluble acidic substances containing phosphorus, Phosphomycin (The Journal of Antibiotics
23:336-347, 1970), Fosfonochlorin (Japanese Unexamined Patent Publication No. 1983-209986),
FR−31564 (Fosmidomycin) (The Journal of
Antibiotics 33:24-35.1980), etc., but it has become clear that the SF-2312 substance is different from any of these and is a new antibiotic. Examples Examples of the present invention are shown below, but these are merely illustrative and do not limit the present invention. Example 1 Micromonospora sp. SF-2312 strain (Feikoken Bacterial Serial No. 7492) grown sufficiently on yeast malt agar slant was mixed with 1.0% glucose and 1.0% starch.
%, polypeptone 0.5%, meat extract 0.2%, yeast extract 0.3%, soy flour 0.2%, calcium carbonate 0.2
%, 20ml of seed medium with the composition of PH7.0 (before sterilization)
(using a 100ml Erlenmeyer flask) was inoculated with 3 platinum loops,
The culture was cultured with shaking at 28°C for 5 days, and this was used as a seed culture. As production medium, starch syrup 2.0%, soybean oil 0.15%,
Soybean flour 1.0%, cottonseed waste 0.5%, day stealers soluble 0.25%, calcium carbonate 0.1%,
Ferrous sulfate 0.0005%, cobalt chloride 0.00005%,
A medium with a composition of nickel chloride 0.00005% and pH 7.0 (before sterilization) was used. 80ml of this live medium (using a 500ml Erlenmeyer flask)
Vitamin B ₁₂ , which had been sterilized separately, was added aseptically at varying concentrations, and the aforementioned seed culture was inoculated at a rate of 5%, followed by shaking culture at 28°C for 6 days. The results are shown in the table below.

[Table] From the above results, it is clear that vitamin B ₁₂ significantly increases the production of the antibiotic SF-2312 by the SF-2312 strain. Example 2 Starch 2.0%, glucose 1.0 as seed medium
%, wheat germ 0.6%, peptone 0.5%, yeast extract 0.3%, soybean flour 0.2%, and charcoal calcium 0.1%. Also, starch syrup 2.0 is used as a production medium.
%, wheat germ 1.3%, cottonseed meal 0.75%, corn steep liquor 1.0%, and vitamin B ₁₂ 0.0001% (separately sterilized). Use the seed medium for pH before sterilization.
7.0, the production medium was adjusted to 7.5 and used. Micromonospora sp. SF-2312 strain (Feikoken Bibori No. 7492) that had grown sufficiently on yeast/malt agar slant was divided into two 100 ml Erlenmeyer flasks containing 20 ml each of the sterilized seed medium. ~4 platinum loops were inoculated and cultured at 28°C for 4 days to form the first type culture. Next, 500 ml of 80 ml of seed medium was dispensed.
Four ml of the above-mentioned first type culture was inoculated into two Erlenmeyer flasks, and cultured with shaking at 28°C for 3 days, followed by second type culture. Next, 80 ml each of the second type culture was inoculated into two 5-volume Erlenmeyer flasks containing one seed medium each, and cultured with shaking at 28°C for 2 days to form a third type culture. Next, this third type culture was inoculated into two 50-volume jar fermenters containing 35 sterilized production media, and cultured with aeration and stirring at 28°C for 7 days (aeration volume
35/min, rotation speed is initially 300 rpm, from 41st hour
500rpm). After culturing, strain using diatomaceous earth and remove the culture solution.
Got 52. Example 3 Collection of SF-2312 substance The culture solution 52 obtained in Example 2 was passed through a Diaion WA-10 (OH ^- ) (manufactured by Mitsubishi Chemical Corporation) column 5 to adsorb the active ingredient. After washing with 20 parts of water, 1
When eluted with normal ammonia water, the active substance is eluted in two fractions, fractions 1 to 3. This active fraction 6 was concentrated to 1, and then charcoal powder 1
The active ingredient is passed through the column. When this permeate is concentrated under reduced pressure and freeze-dried, SF-2311 is obtained.
32 g of coarse powder of material was obtained. Next, this coarse powder
16g was applied to a column of silica gel C-200 (Wako Pure Chemical Industries, Ltd.) 1 filled with 90% ethanol water in advance, washed with 90% ethanol water 2, and then eluted with 60% ethanol water, resulting in fraction 4 in 80 ml fractions. -12 active fractions were obtained. This was concentrated under reduced pressure and lyophilized to obtain 5.6 g of a crude powder of SF-2312 substance. Next, 5.6 g of this crude powder was dissolved in 5 ml of water, adjusted to pH 2.0 with hydrochloric acid, applied to a 700 ml column of coal powder filled with water in advance, washed with 1.2 ml of water, and then washed with 1N ammonia water. and acetone (1:1) to obtain active fractions in fractions 8 to 14 in 60 ml fractions, which were concentrated under reduced pressure and lyophilized to yield 1.2 g of crude powder of SF-2312 substance. (Purity approx. 20
%) was obtained. Furthermore, after dissolving 1.2 g of this crude powder in a small amount of water, it was added to a Sephadex G-10 (manufactured by Pharmacia) (800 ml) tower filled with water in advance and developed with water, resulting in fractions 18 to 24 in 20 ml fractions. The active fraction was obtained, which was concentrated and lyophilized under reduced pressure to 520%
mg of coarse powder was obtained. Next, dissolve 520mg of this coarse powder in a small amount of water and use a Dowex bag filled with water in advance.
When attached to a 50W x 2 (H ⁺ , 100-200 mesh, 500ml) tower (manufactured by Dow Chemical Company) and developed with water, 10ml
By fractionation, active fractions were obtained in fractions 21 to 25,
After adjusting the pH to 6.2 with 2N caustic soda, 120 mg of the sodium salt of SF-2312 substance was obtained by concentrating and freeze-drying under reduced pressure. This gave a single spot in the thin layer chromatography.

[Brief explanation of drawings]

Figure 1 is an infrared absorption spectrum of the sodium salt of SF-2312 substance measured in a KBr tablet. Second
The figure shows the free acid of SF-2312 substance measured in heavy water.
This is a 400MHz hydrogen nuclear magnetic resonance spectrum.

Claims (1)

[Claims] 1. The following formula: New antibiotic SF-2312 substance represented by and its salt. 2. A new antibiotic characterized by culturing SF-2312 substance-producing bacteria belonging to the genus Micromonospora and collecting SF-2312 substance or its salt from the culture.
SF-2312 Substance or its salt manufacturing method.