JPS6158592A - Novel antibiotic substance sf-2354, its preparation and its use - Google Patents

Abstract

PURPOSE:To prepare the SF-2354 substance capable of promoting the antibacterial activity of other antibiotic substances, by culturing a microbial strain belonging to Streptomyces genus and capable of producing Streptomyces substance. CONSTITUTION:Streptomyces sp. SF-2354 (FERM-P No.7769) belonging to Streptomyces genus is cultured in a medium containing nutrients assimilable by conventional microorganisms. The culture is carried out prefeably by liquid culture, especially by submerged culture under aerobic condition. Th objective novel antibiotic substance SF-2354 is separated from the culture product. The antibiotic substance is colorless amorphous powder having elemental composition of C50.57(wt)%, H 6.67%, N 15.14% and O 26.22%, and effective to promote the antibacterial activity of other antibiotic substances (e.g. fosfomycin, beta-lactam, etc.) in spice of its slight antibacterial activity. It is used mainly as a mixture with these antibiotic substances.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibiotic SF-no3314 substance, its production method, and its uses.

More specifically, the novel antibiotic 5F-233 obtained by whole culture of actinomycetes, the substance and its production method, and the novel antibiotic 5F-J, 3! It relates to its use mainly in preparations in which the & substance is mixed with antibiotics such as phosphaomycin, β-lactams, and aminoglycosides.

Background to the Invention The present inventors isolated a large number of microorganisms from soil for the purpose of searching for new and useful antibiotics, and when searching for the antibiotics produced by them, discovered that a certain type of microorganism produced a new antibiotic. After further detailed investigation, it was confirmed that although the antibacterial activity of this antibiotic is weak, it has the effect of enhancing the antibacterial activity of other antibiotics. It was discovered that it is a new substance based on its biological properties.

The present invention was developed based on the above findings.

Conventional technology Substances that enhance the antibacterial activity of antibiotics include S.P.
/ J 7 (M, Kikuchi et al., The Journal of Antibiotics
al of Antibiotics) 3 0
Vol. 20"i-220, 1277) is known to particularly enhance the antibacterial activity of Macroly P antibiotics. Clapulanic acid (T.

Doumon Las Antimicropial Agents Anne P.
l Agents and Ohemotherapy
) / Volume 3 1. ? /j~3/ri, 1979) is a substance that synergistically enhances the antibacterial activity of β-lactam antibiotics. On the other hand, Nonorujishi 3, g volume, 1aoo~/tt0
3.7222) is also known to enhance the antibacterial activity of β-lactam antibiotics.

SUMMARY OF THE INVENTION The first invention is a colorless amorphous powder with the following properties, and its elemental composition is carbon SO, 57% by weight.
, hydrogen 6.67%, nitrogen / j, / 4%, oxygen no IJ
, 2%, and the ultraviolet absorption spectrum in an aqueous solution is shown in Figure 7, the optical external absorption spectrum in a potassium bromide tablet is shown in Figure 2, and the hydrogen nuclear magnetic resonance spectrum in heavy water is shown in Figure 3. , carbon nuclear magnetic resonance spectra are as shown in Figure 4.
It provides a S-reactive substance.

The second invention relates to SP-,2 belonging to the genus Streptomyces.
,3! ;It substance producing bacteria? The present invention provides a method for producing a novel antibiotic 5F-2334, which is characterized by culturing and collecting a novel antibiotic SF-2334 substance from the culture.

The third invention is a novel antibiotic SF'-2,? '3 <
Based on the fact that the antibacterial activity of these antibiotics is enhanced when the substance is mixed with various antibiotics such as phosphaomycin, I-lactams, or aminoglycosides, it has been proposed as a novel antibiotic as an antibacterial activity enhancer of antibiotics. 5F-233&
It provides a use for substances.

Novel antibiotic used in the present invention 3 F-J 3 j
An example of U-substance producing bacteria is Actinomycetes 5F-23sa, which was isolated from the soil of Mitsuike Park, Yokohama by the present inventors.
There are stocks. The mycological properties of the strain SF-, 23j are as follows.

(2) Morphological properties It grows well on media such as starch agar, oatmeal agar, and yeast malt agar, but there is very little or no aerial mycelium.

The branching of the aerial hyphae is simple, with no axle branching, and the tips of the aerial hyphae are spiral-shaped.

Special structures such as sclerotia and sporangia are not observed.

When observed under an electron microscope, the surface of the spores is thorn-like.

The spores are oval or oval in shape and range in size from O1a to 0.
6 x 0.7~/, 2 microns, usually i.

Chain more than one.

+1. Growth status on various media The growth status of the 5F-23JG strain on various media is shown in the table below. Culture was carried out at -1r℃, and observation was carried out at /<(-
For the standards shown in parentheses in the description of one color performed after 21 days of culture, Color Harmony Manual manufactured by Container Corporation e of America was used.

m, physiological properties (1) Growth temperature range: in starch agar/3℃~3
It grows in a temperature range of 10°C to 30°C. α, does not grow at 1°C.

(2) Liquefaction of gelatin: Positive (3) Hydrolysis of starch: Positive (4) Coagulation of skim milk: Negative Peptonization of skim milk: Positive (5) Reduction of nitrate: Negative (6) Salt tolerance Near% Na0A addition Although it grows in a medium,
The production of water-soluble dyes is also inhibited by the addition of 3% NaCl#.

(7) Production of melanin-like pigment: Negative ■, availability of carbon sources D-glucose, L-arabinose, D-fructose, D-mannite, glycerol, muinositol,
L-rhamnose, raffinose, sucrose and D
- Utilize xylose.

(2) Cell wall composition Diaminopimelic acid in the cell wall composition was of the LL type.

To summarize the above properties, the strain 5F-2,314 belongs to the genus Streptomyces, the aerial mycelium tip is spiral-shaped, and the spore surface has spiny projections.

Although the attachment of aerial hyphae is extremely weak, the color tone of mature aerial hyphae is white to water gray. Production of melanin-like pigments is not observed, but the production of water-soluble pigments in brown threads is positive.

Based on the above results, the present inventors transferred 8F-2,334 to Streptomyces sp.
ssp,) SF-2Jj≠.

This strain was transferred to the Institute of Microbial Technology, Agency of Industrial Science and Technology.
It has been deposited since January 1, 2013, and its deposit number is FBRMP776.

The SF-, ZJj≠ strain is susceptible to changes in its properties, as seen in the case of other actinomycete strains, and can be mutated by artificial mutagenic means using, for example, ultraviolet rays, X-rays, radiation, chemicals, etc. In any mutant strain, 8F-,
Any substance capable of producing 2jj≠substances can be used in the method of the present invention.

In the method of the invention, the strain is cultured in a medium containing nutrients that can be utilized by common microorganisms. As a source of nutrients,
Known materials conventionally used for culturing actinomycetes can be used.

For example, glucose, glycerol, sucrose, starch, starch syrup, textolino, molasses, oatmeal, edible oil, etc. can be used as carbon sources. Further, as the nitrogen source, soybean flour, wheat germ, cottonseed meal, tomato ketchup, cornstarch liquor, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, etc. can be used. In addition to adding inorganic salts such as 4L PI -Th Irr +-i, 44t / Shito k l + um, particulate chloride, phosphate, and magnesium sulfate, etc. , helps the growth of bacteria, S
Suitable organic and inorganic substances may be added to promote the production of F'-3.5q substances.

The most suitable culture method is a liquid culture method under aerobic conditions, especially a deep culture method, similar to known methods for producing general antibiotics. A suitable temperature for culturing is 0°C to 38°C, but in most cases it is preferred to culture at a temperature in the range of 0°C to 30°C.

Cultivation production of 5F-2,3 ICt substances varies depending on the medium and culture method used, but in the case of shaking culture method or deep culture method using a culture tank, the accumulation usually reaches its maximum within 2 to 7 days.

In assaying the 5F-2,3A;ll substance, it is possible to measure the titer using this method since it exhibits antibacterial activity against strains of Escherichia coli susceptible to β-lactam antibiotics.

In order to collect the SF-33'l substance from the culture, it is possible to use an appropriate combination of means commonly used to collect all the substances produced by microorganisms. An example of this is as follows.

In other words, the total diatoms, including bacterial cells and other solid matter, are higher than the culture material.
It is filtered separately using a filter aid, and then the active ingredients in the F solution are adsorbed on Total Diamond Ion HP-NO 0 (manufactured by Mitsubishi Chemical Corporation), washed with water, and then eluted with an SO% acetone aqueous solution. After concentrating this under reduced pressure and removing 7 setones, it was passed through a column of activated carbon (manufactured by Wako Junkei Co., Ltd.) to adsorb the effective fraction, washed with water, stripped with SO% acetone aqueous solution, and dried by condensation. A brown coarse powder is obtained. For further purification, column chromatography using Cropretub RP-/g (manufactured by Merck & Co.) can be advantageously used. That is, after loading a solution prepared by dissolving the above coarse powder in water on a column of Ricloprep RP-/g, elution was performed with a 30% methanol solution, and all fractions containing the active ingredient were repeatedly collected to obtain a pure product. Obtainable.

The physicochemical properties of the 8F-233a substance obtained by sniffing are as follows.

/ Appearance: Colorless amorphous powder 2 Melting point = 792-193℃ (decomposition point) 3, Elemental analysis: Carbon SO, S%, water hydrogen 6L 67μ Ultraviolet absorption spectroscopy: Suiraku liquid Figure 1 shows the spectrum measured inside.

j Infrared absorption spectrum: The entire spectrum measured with potassium bromide tablets is shown in Figure 2.

6. Hydrogen nuclear magnetic resonance spectrum measured in double water7
The spectrum /L/ of ttoo MHz is shown in FIG.

2 Carbon nuclear magnetic resonance spectrum: measured in heavy water/
The spectrum at 00'MHz is shown in FIG.

B Specific rotation: [α] o-. 2g0g・(co.za, water) 9, Bath soluble: Easily soluble in water, methanol, and ethanol, but insoluble in ethyl acetate, pe/zen, and chloroform.

/ 0. Color reaction: positive Lemieux reagent, negative ferric chloride reagent, ninhydrin reagent / Rf value of silica gel thin layer chromatography
-T: Merck FJJ&Developing solvent: n-butanol-acetic acid-water (2:l:l)0
, 3 6 n-butanol-methanol-water (<t:/:J) 0.36 chloroform-methanol (g:z) 0, 0 0 Next, the biological properties of the 5F-233G substance will be described, 5F-23Sq Table 1 shows the antibacterial activity of the substances against various microorganisms.

As shown in Table 1, the 5F-2,3Jtl substance showed antibacterial activity against Escherichia 11 coli, M. 236, and Myconocterium smegmates, which are sensitive to β-lactam antibiotics. It showed no antibacterial activity against positive bacteria or Durham-negative bacteria.

Table 2 shows the antibacterial activity enhancing effect of substance 5F-233G on various antibiotics.

As shown in Table 2, the SF-co3jq substance exhibited an effect of significantly enhancing the antibacterial activity of phosphomycin, MP/&/, and dipekacin against Pseudomonas aeruginosa E-2.

Pseudomonas aerugino-t B-J except 1c x
Similar potentiating effects have been observed for Schielichia coli, Staphylococcus aureus, and Klebsiella pneumoniae, and mixed preparations with these various antibiotics have been shown to be effective against mammals (e.g., humans, cows, pigs, horses, etc.). It can be used to treat various bacterial infections in chickens and other animals.

EXAMPLES Next, examples of the method for producing the novel antibiotic 5F-2GJG substance of the present invention will be shown, but these are merely illustrative and do not limit the present invention in any way.

Example 1 Streptomyces viri r chromognes sp SF'- grown on yeast extract/starch agar slant
23J'1 strain (Feikoken Bacteria No. 776) was added to Far East Powder Bouillon with 0% N-Z amine, s%, and glucose 8'.
Contains 60- of a medium consisting of .

Next, salad oil '-'
The above-mentioned inoculum was inoculated into a 00 ml Erlenmeyer flask containing a medium consisting of 0). This total temperature 2
After culturing with shaking at ♂°C for 6 days, the culture solution was filtered, and the resulting filtrate was passed through a column packed with about 3 Zf Diaion HP-J□ (manufactured by Mitsubishi Kasei Corporation) and Yomlt to adsorb the active ingredients. . After washing the column with about 1 liter of water, 50%
Elution was carried out with an acetone water bath solution, and this was concentrated under a Shichimasu tama to remove acetone, and then passed through a column filled with activated carbon (manufactured by Wako Tsumugi Co., Ltd.) xoV to adsorb the active ingredient. After washing the whole column with 100 ml of water, the active ingredient was eluted with a %X acetone aqueous solution and lyophilized to obtain about 9 tons of brown coarse powder.

Example: Approximately ayet o- of the coarse powder obtained in Example 1 was dissolved in water, and 20%
-GQ Micron) xao? column and fractionated in 1Ov portions with a 30% methanol solution.

Both fractions and active ingredients of Sr were collected and lyophilized to obtain a crude powder of approx.

Water VCC fermentation of this i/lnl for further purification,
The above column chromatography was repeated to obtain pure αomy'e as a colorless powder.

[Brief explanation of drawings]

Figure 1 is 5F-23! The entire ultraviolet absorption spectrum of the r& substance is shown, and the solid line (□) is! ; Omcg/- aqueous solution,
Break, 1! (--------) is JOmcg/-〇〇,
IN hydrochloric acid solution, the chain line (----) is! Omcg/-〇〇, IN caustic soda solution. Figure 2 is 5F-23! The optical external absorption spectrum of LA substance in potassium bromide tablets is shown. Figure 3 shows μ00MH of 5F-23A; G substance in heavy water.
Figure q, which shows the hydrogen nuclear magnetic resonance spectrum of z, is 8F-
io of 3SA substances in heavy water. A MHz carbon nuclear magnetic resonance spectrum is shown. Procedural amendment (voluntary) December 28, 1982

Claims (1)

[Claims] 1. It is a colorless amorphous powder with the following properties, and its elemental composition is 50.57% carbon and 6.67% hydrogen by weight.
%, nitrogen 15.14%, and oxygen 26.22%. The ultraviolet absorption spectrum in an aqueous solution is shown in Figure 1, the infrared absorption spectrum in potassium bromide tablets is shown in Figure 2, and the absorption spectrum in heavy water is shown in Figure 2. A novel antibiotic SF-2354 substance, characterized in that its hydrogen nuclear magnetic resonance spectrum is as shown in FIG. 3, and its carbon nuclear magnetic resonance spectrum is as shown in FIG. 4. 2. A method for producing a novel antibiotic SF-2354 substance, which comprises culturing SF-2354 substance-producing bacteria belonging to the genus Streptomyces and collecting the SF-2354 substance from the culture. 3. An antibacterial activity enhancer of antibiotics, characterized by comprising the novel antibiotic SF-2354 substance.