JPS60237996A - Novel antibiotic and its preparation - Google Patents

Abstract

PURPOSE:To collect a novel antibiotic showing antibacterial activity against Gram-positive bacteria, and to use it as a remedy or a preventive, by cultivating a new strain belonging to the genus Streptomyces. CONSTITUTION:Strain KA-5905 belonging to the genus Streptomyces is aerobically cultivated by a culture method of common ray fungus. Novel antibiotic KA- 5905 is obtained from the culture solution by a well-known method. This novel antibiotic can be optionally made into its salt. KA-5905 has the following physical and chemical properties. Molecular weight: 220-300, melting point: 66-69 deg.C, specific rotatory power [alpha]D<20>+205 deg. (Cl, methanol), soluble in methanol, acetone, etc., and insoluble in water, hexane, etc.

Description

[Detailed description of the invention] The present invention relates to a novel antibiotic and a method for producing the same.

The Ministry of Invention and others isolated various strains of soil microorganisms and searched for their antibiotic production ability, and found that the newly isolated strain KO-5905 produced a novel antibiotic. We succeeded in isolating the substance from the culture solution and completed the present invention.

As is clear from the physicochemical and biological properties described below, the antibiotic of the present invention was recognized as a new substance different from known antibiotics, and was named KA-5905 substance.

KC- which is a producing strain of KA-5905vIJ quality of the present invention.
Strain 5905 was obtained by suspending a soil sample collected in Sydney, Australia, in sterile water and adding 2% heart infusion agar (Eiken), 0.5% glucose, 1-2% rabbit blood, and mycostatin. 50μ
f/-1' Nijiline GO24μf/ml %d
eLimixi: / B 2 pf/-, nalidixic acid 8
μ? /wls pi consisting of 0.5% agar 7.
It is obtained by plating on a medium of O and culturing at 27°C for 7 days, and isolating the resulting colony.

The KC-5905 strain has the following mycological properties. In addition, the properties of various media are maintained at 27°C for 1 hour unless otherwise specified.
The cells were cultured for 4 days and observed according to conventional methods.The expression of color tone was according to the classification of Color-Harmony Manual, i4 [(Container Corporation of America)].

(1) Morphological properties KO-5905 strain successfully grows true aerial mycelium on several types of media. The hyphae are long and simply branched, and the sporophytes are branched on the aerial hyphae, and their tips are hook-shaped, ring-shaped, or spiral-shaped. Spores are oval or short cylindrical, with 0.
It has a size of about 4 to 0.7 μm x Q, 5 to 1.2 μm, and forms a chain of 20 or more pieces. The spore surface is spiny. No flagellated spores, sporangia, sclerotia formation, or fragmentation of obstructed hyphae were observed.

(n) Properties on various media 1. Growth on sucrose/nitrate agar medium. Adherence of good aerial hyphae. Color of good aerial hyphae. White (b) to light blue (Hoe). Soluble pigment. Yellowish brown 2. Glucose/AsunoQ Ragin. Growth on agar medium Good attachment of aerial hyphae Color of good aerial hyphae Pale blue (17・a) Soluble pigments Green-brown 3, Glycerin As, Q Ragin agar medium Growth on agar medium Good attachment of aerial hyphae Color of abundant aerial hyphae Pale Blue (17・C) Soluble pigment Yellowish brown 4, Growth on starch/inorganic salt agar medium Moderate growth of aerial mycelia Good air Mycelia color Pale blue (17ee) Soluble pigment Slight brown 5, Growth on tyrosine agar medium Good air Adhesion of mycelia Good Aerial hyphae color Pale blue (17ea to 17・C) Soluble pigment Brown 6, Growth on nutrient agar medium Poor Aerial hyphae Adhesion Moderate = 7- Aerial hyphae color White (b) Obstructed hyphae Color Gray-yellow (2gc) Soluble pigment Slight brown 7, Growth on yeast malt agar medium Good attachment of aerial hyphae Color of abundant aerial hyphae Pale blue (17・C) Base tube mycelia color Brown to brownish (3ni~ 3po) Soluble pigment Yellowish brown 8, Oatmeal agar medium Growth Good Aerial hyphae Adhesion Moderate Aerial hyphae color White to pale blue (&~17・C) Blocked autologous color Brown (2 pl) Soluble pigment Slight brown 8- (11 Physiological properties 1, Growth temperature range: LO ~ 39℃ Optimum growth temperature = 28.5 ~ 34.5℃ 2, Liquefaction of gelatin: Positive 3o Hydrolysis of starch 2'@Sex 4, Defatting Coagulation of milk: Negative Bezotonization of skimmed milk: Negative 5, Production of melanin-like coloration: Positive (IV) Utilization of carbon sources As a result of examining the usability of carbon sources on Zolidham-Gotzlieb agar medium, we found the following: All carbon sources were used.

L-arabinose, D-xylose, D-glucose,
D-fructose, 7-ucrose, inositol, L-
Rhamnose, raffinose, D-mannitol ■ Analysis of diaminopimelic acid in cell wall hydrolyzate L-1L-2,6-diaminopimelic acid was observed.

Based on the above results, the jllKc-5905 strain belongs to the genus Streptomyces, and the spore-forming hyphae exhibit a hook-shaped to spiral shape. The surface of the spores is thorn-shaped and consists of chains of 20 or more spores. The color tone of aerial hyphae belongs to the blue series, and subterranean hyphae often exhibit a blackish-brown color, produce melanin-like pigments, and contain glucose and
A greenish-brown soluble pigment was observed in the Asuno (Ragin agar medium). All of the carbon sources studied were used.

Based on the above characteristics, strains similar to this strain were selected from "Birshi's Manual of Determinative Bacteriolozoans" 8th edition, various bacterial strains described by IMF by Schirling and Gottlieb, and Waxman's "Zo Actenomycetes" 2nd edition. When we searched the published literature on new species of actinomycetes, we found many strains that were identical in terms of morphology, color tone of aerial hyphae, production of melanin-like pigments, and availability of carbon sources.

However, no strain that completely matches this strain in terms of the color tone of the matrix and soluble pigments has been found, and one strain that is relatively similar is Strezotomyces fistilidochromogenes (8tre
pton+yaeaviridaahromog@n*
s) I8P 5110 strain (KCC8-0856 strain)
was recognized. Streptomyces pyridochromogenes strain 18P5110 and strain 11-KC-5905 have all the same physiological properties except for the ability to hydrolyze starch. However, the green soluble pigment characteristic of the Streptomyces pyridochromogenes strain 15p5110 was not observed in the KC-5905 strain. Based on the above results, the fiKc-5905 strain is considered to be a new strain closely related to Streptomyces pyridochromogenes, and was named Streptomyces KO-5905 strain, and was submitted to the Institute of Microbiology, Agency of Industrial Science and Technology. Bacteria number 7588
(FIRM p-7588).

In the present invention, not only the above-mentioned KC-5905 strain but also
Natural mutants and artificial mutants can also be used.

For culturing the 12-strain, a normal method for culturing actinomycetes is used. Various carbon sources can be used as the carbon source for the medium, including sugars such as starch, dextrin, glucose, fructose, maltose, mannitol, kyclose, galactose, ribose, and sucrose; alcohols such as methanol, ethanol, and glycerin. It is preferable to use organic acids such as acetic acid, NO-Q lumitonic acid, lactic acid, gluconic acid, and pyruvic acid; vegetable oils such as soybean oil and cottonseed oil; and hydrocarbons such as NO-Q rough in, either alone or in combination. Examples of nitrogen sources include organic nitrogen sources such as urea, soybean flour, dry yeast, yeast extract, peptone, lipezoton, meat extract, cornstarch liquor, casamino acids, and Daytailers Soluple; ammonium chloride, ammonium sulfate, ammonium nitrate, In addition to inorganic nitrogen sources such as sodium nitrate, amino acids such as glycine, alanine, glutamic acid, and cysteine can be used alone or in combination. Other inorganic salts and trace amounts of salt such as salt, potassium chloride, calcium chloride, cobalt chloride, iron chloride, potassium phosphate, sodium phosphate, magnesium sulfate, zinc sulfate, iron sulfate, calcium carbonate, calcium hydroxide, etc., as necessary. Metals can be added. Furthermore, organic and inorganic substances that promote bacterial growth and production of KA-5905 substance can be added as appropriate. When using the aerated culture method, it is preferable to further add an antifoaming agent such as fatty oil, silicone oil, paraffin, or polyalkylene glycol.

As a culture method, although culture on a solid medium is possible, it is preferable to use a liquid culture method, especially a deep culture method, as in the production method of general antibiotics. Cultivation is carried out under aerobic conditions at 3-35°C, preferably 25-30°C.

Production of KA-5905 substance can be carried out using either shaking culture or tank culture. Culture is carried out for 1 to 10 days, and when the production amount in the culture solution reaches the maximum, the culture is stopped, and the culture solution is Isolate the desired antibiotic and produce #.

The culture solution obtained by the above method pK-5905
As a method for isolating and purifying the substance, the known 15-Akiho method used in such cases can be adopted.

For example, first, the culture solution is centrifuged or subjected to P-filtration treatment to remove bacterial cells, and then the culture solution is subjected to any combination of the following treatments. That is, a method in which the P liquid is adsorbed on activated carbon, a porous synthetic polymer resin, etc. and the target product is extracted by elution with an aqueous liquid such as methanol, ethanol, butanol, acetone, etc., and a method in which the F liquid is adsorbed with a mineral acid, such as hydrochloric acid ,
Adjust to near pii Z with sulfuric acid, etc., and extract with a water-insoluble organic solvent, such as ethyl acetate, ether, methylene chloride, chloroform, benzene, etc. If necessary, add the desired product from this extract to an alkaline aqueous solution, For example, after dissolving in a dilute bath liquid such as sodium hydroxide, sodium carbonate, hydrogen carbonate, etc., the pH is adjusted to around 2 again, and extraction is carried out with the above-mentioned water-resistant organic solvent. By concentrating the extract obtained by arbitrarily combining these, a crude substance of KA-9505 can be obtained.

This crude product can be used, for example, as silica gel or porous synthetic polymer W.
A pure product of KA-9505 substance can be obtained by chromatography using oil or the like in a conventional manner.

The OK Mu 9505 substance of the present invention obtained as described above has the following physicochemical and biological properties.

l Physical and chemical properties (1) Elemental analysis values: Carbon 57.59% 1.0% Hydrogen 7.06% O65% Nitrogen 4.66±0.5% Sulfur 11.30±1.0% (2) Molecule i = 220-300 (3) Melting point: 66-69℃ (4) Specific rotation: (α)” +20 s° (cl, methanol) (5)
Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured in methanol is as shown in Figure 1, with maximum absorption at 238 nm and 295 nm.

(6) Infrared absorption spectrum: The infrared absorption spectrum of the pellet in potassium bromide is shown in Figure 2.

(7) Proton nuclear magnetic resonance spectrum: The spectrum measured in heavy methanol is shown in Figure 3.

(8) Solubility in bath agents: Soluble in methanol, acetone and ethyl acetate, incurable in water, petroleum ether and hexane.

(9) Color reaction: ferric chloride reaction and potassium permanganate reaction are positive;
Molisch reaction and ninhydrin reaction were negative.

CIG Basic, acidic, neutral distinction: Acidic (Ill Thin layer chromatography: Rf value: 0.45 [Plate; TLC aluminum sheet silica gel 60 F'zsa, 0.2 mm (manufactured by Merck & Co.), Solvent: Acetic acid Ethyl], 19- iR value: 0.40 (Plate: Same as above, Swamp medium: Ether-hexane (10:1)), ■ Biological properties (1) The antibacterial spectrum against various microorganisms is shown in the table below.

2O- (2) Toxicity It shows almost no susceptibility to sentinel mammals.

When searching for known antibiotics with the above-mentioned properties, thiolactomycin [Charnal Op Antibiotics, 35,391~
419 (1982)) and thiotetromycin [same above, 36, 109-114 (1983)). However, Kmu-5905 substance has a nitrogen atom as a constituent element, but the two antibiotics do not contain a nitrogen atom, and their nuclear magnetic resonance spectra are different from both antibiotics. The two substances are clearly different. Therefore, substance Kmu-5905 was recognized as a novel antibiotic.

The above-mentioned 9% K-5905 substance has low hostility and exhibits antibacterial activity mainly against Durham-positive bacteria, so it is useful as a therapeutic and preventive agent for microbial infections in humans and animals.

K-5905 material is also useful as an intermediate for producing durable antibiotics and other pharmaceuticals.

Example Cornstarch 1%, ? A liquid medium 11005de500B with a composition of 0.75% ribeptone, 0.75% meat extract, 1% glucose, 0.3% salt, 0.05% magnesium sulfate and adjusted to pH 7.0 was placed in an Erlenmeyer flask. and sterilized. This medium was inoculated with an agar slant culture of Streptomyces p. strain KC-5905. This was cultured with shaking at 220 rpm and 27°C for 72 hours to obtain a seed culture.

Corn starch 2%, polypeptone 0.75%, internal extract 2%, cottonseed oil 0.5%, glucose 0.5%, salt 0
．． 3%, magnesium sulfate 0.05%, silicone 0.
A 'fc liquid medium having a composition of 1% and adjusted to pH 7.0 was dispensed into four 201 fermenters in 101 doses each, sterilized, and then inoculated with the aforementioned seed culture solution in 100 doses each. This 2
Aeration stirring method (stirring number 32°rpm, ventilation lid; 5
t/min) for 72 hours.

This culture solution was filtered to obtain 32Lf of solution F.

Transfer this F solution to an activated carbon column (6 x 40 clL).
- Adsorbed and eluted with 50% aqueous acetone.

4t of bathing liquid was heated to 1tVc'I under reduced pressure! The concentrated solution was diluted with diluted hydrochloric acid to adjust the pH to 2.2, and diluted with ethyl acetate I.
Extracted with L. After washing this extract with water, the antibiotic was dissolved in 500 m of a 0.5% aqueous sodium bicarbonate solution. Dilute hydrochloric acid was added to this transferred solution to adjust the pH to 2.2 Km, and the mixture was extracted with 500 ml of ethyl acetate. This extract was washed with water, concentrated, and the 01km solution was transferred to a silica gel column (2X40
Ql) and eluted with hexane-ethyl acetate (4:1). The active fractions were collected and concentrated to give an oily crude product.

The obtained oily substance was poured into a plate of 7 licagles [silikab].
'! 54 s 2 mm thick, 20 x 20 m (Merck Co., Ltd.) and developed with ethyl acetate-24-xane (7:1). The portion with ultraviolet absorption with an Rf value of around 0.5 was collected, and acetic acid It was eluted with ethyl.The eluate was concentrated, washed with hexane, and then dried in vacuo to obtain pure product 580q of Mu 5905 substance as colorless crystals.

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of Nimu 5905 substance.
FIG. 2 shows the infrared absorption spectrum, and FIG. 3 shows the proton nuclear magnetic resonance spectrum. that's all

Claims (1)

[Claims] 1. The following physicochemical properties (1) Elemental analysis values: Carbon 57.59±1.0% Hydrogen 7.06±0.5% Nitrogen 4.66±0.5% Sulfur 11 .30±1.0% (2) Molecular weight: 220-300 (3) Melting point: 66-69°C (4) Specific optical rotation: [a]20+205° (EL, methanol) (5) Ultraviolet absorption spectrum: methanol The ultraviolet absorption spectrum measured in the sample was as shown in FIG. 1, with maximum K absorption at 238 nm and 295 nm. (6) Infrared absorption spectrum: The infrared absorption spectrum of the pellet in potassium bromide is shown in Figure 2. (7) Proton nuclear magnetic resonance spectrum: The spectrum measured in methanol is shown in Figure 3. (8) Solubility in solvent a = Soluble in methanol, acetone and ethyl acetate, insoluble in water, petroleum ether and hexane. (9) Color reaction: ferric chloride reaction and potassium permanganate reaction are positive;
Molisch reaction and ninhydrin reaction were negative. (6) Basic, acidic, neutral classification: acidic Ωυ Thin layer chromatography: Rf value Ho, 45C plate; TLC aluminum sheet silica gel 601Z 64.0.2 mm (manufactured by Merck & Co., Ltd.), solvent: ethyl acetate] Rf value: Human-5 to antibiotics with 0.40 [crate; same as above, solvent; ether-hexane (10:1)]
905 and its salts. 2. Antibiotic KA-5905, which is characterized by culturing a KA-5905 substance-producing bacterium belonging to the genus Streptomyces, collecting the KA-5905 substance from the culture solution, and further converting this into salts as necessary. and the manufacturing method of its salts.