JPH03206099A - Antibiotic ksy-5 and production thereof - Google Patents

Abstract

NEW MATERIAL:Antibiotic KSY-5 or salt thereof having the following physico chemical properties and salt thereof: Colorless plate crystal and basic peptide based antibiotic; melting point, 196-199 deg.C; elemental analysis (%), C 51.54, H 8.11, N 11.89 and Cl 5.86; molecular weight, 1500-2500 (by gel permeation method); constitutional amino acid, confirming existence of valine, leucine, lysin, isoleucine, tyrosine and ornithine each contained at a molar ratio of 4:3:3:1:1:1 by decomposing antibiotic KSY-5 with 6N HCl at 110 deg.C for 16hr and then carrying out amino acid analysis of the decomposed materials; solubil ity, soluble in water, methanol and dimethylsulfoxide and slightly soluble in ethyl acetate, chloroform, ether, benzene, etc.; color reaction, positive to ninhydrin reaction and phosphomolibdic acid reaction. USE:An antibacterial agent for Gram-positive bacterium (especially methicillin- resistant Staphylococcus). PREPARATION:Bacillus laterosporus (FERM P-11175) is cultured.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel basic bebutide antibiotic and a method for producing the same.

[Prior Art] To date, various antibiotics have already been created and used in clinical settings as therapeutic agents for infectious diseases, and various peptide antibiotics with various properties are known. ing.

[Problems to be Solved by the Invention] Diseases caused by bacteria have been largely overcome by the development of treatment methods using antibiotics.

However, due to long-term or large-dose administration of conventional antibiotics, there is an increase in diseases due to changes in the causative bacteria (microbial replacement phenomenon) and the appearance of resistant bacteria (resistance phenomenon), or opportunistic bacterial infections due to weakened immunity. The increase in the number of infectious diseases has become a major problem in the current medical field of infectious disease treatment. To overcome this problem, antibiotics that exhibit new activities are constantly being sought.

[Means for Solving the Problems] The present inventors isolated a large number of microorganisms from soil for the purpose of searching for new antibiotics, and conducted a separate search for the substances they produced. This microorganism is a species belonging to the genus Bacillus, and by culturing this microorganism in an appropriate medium, a substance that exhibits antibacterial activity mainly against Durham-positive bacteria can be added to the medium. We learned that this substance accumulates, isolated this substance, confirmed that it is a new substance from its physicochemical and biological properties, and identified it as KSY-5.
I decided to call it that.

The present invention has been made based on such findings, and the present invention consists of the constituent amino acids, in molar ratios of valine: 4, leucine: 3, lysine: 3, inleucine: 1,
Basic peptide antibiotic KSY-5 and its salts, which have tyrosine 1 and ornithine 1 and have the physicochemical properties described below, and the antibiotic KSY-5, which belongs to the genus Bacillus.
The gist of the present invention is a method for producing antibiotic KSY-5 and its salts, which comprises the steps of culturing a microorganism capable of producing KSY-5 and collecting antibiotic KSY-5 from the culture.

The present invention will be explained in detail below.

The KSY-5-producing microorganism used in the present invention may be any microorganism that belongs to the genus Bacillus and has the ability to produce the antibiotic KSY-5.

An example of a KSY-5 producing bacterium is Bacillus sp, KSY-5 strain, which was isolated from soil collected by the present inventors in Fukuoka City, Fukuoka Prefecture.

Its mycological properties are as follows.

I. Mycological characteristics (1) Morphological characteristics Morphological characteristics were based on observations of cells cultured at 37° C. for 48 hours in a broth agar medium and a broth liquid medium. The results are shown in the table below.

Broth agar medium Cell shape, rod-like size 0.5-1.0 μm Xi. 'r3. 0gm Polyplasia Not observed, motile Yes Flagella epiphytic state Presence of peritrichous spores Spore shape, spherical to elliptical size 0.5-0.7 μm ×0, 5-1.3 μm Swelling of sporangia Yes Meat juice medium rod-shaped 0.5-1.0 μm X3. 040. Oμm Not observed. Not observed. Cells cultured on normal nutrient agar at 37°C for 22 hours were slightly positive in Durham staining.

(2) Growth conditions in each medium (37° C., 48 hours) The growth conditions in the broth agar plate medium, broth liquid medium, broth gelatin puncture medium, and litmus milk medium were as follows.

Meat juice agar plate medium Growth is good, cream-colored colonies are formed, colonies are almost circular with wavy edges, glossy, and no water-soluble pigments are observed Meat juice liquid medium Overall turbid, no surface growth is observed Meat juice gelatin Puncture medium Litmus milk medium that grows but does not liquefy Reduces litmus, becomes slightly pebtonized on the 7th day (3) Physiological and biochemical properties The results of test items commonly used to identify bacteria are shown below. Note that unless otherwise specified, the results are shown after 48 hours at 37°C.

Nitrate reduction: Positive methyl red reaction: Negative V-P (V
ogues-Proskauer) reaction: Negative indole reaction: Negative production of hydrogen sulfide: Negative starch decomposition.
: Decomposition of negative fats
: Degradation of positive casein
: Positive use of citric acid Koser's medium : Negative Christensen's medium : Negative Simmons' medium : Negative use of inorganic nitrogen sources Nitrate (sodium nitrate) : Negative ammonium salt (ammonium sulfate) : Negative dye life of
:Negative urease
:Negative oxidase
:Positive Power Talase :Positive (Bow 5
0) Growth range pH 5 (28℃): No growth pH 6 (28℃)
℃): Growth pH 8 (28℃)
:Growth pH 9 (28℃)
: Growth 10℃ : No growth 1
5°C: No growth (but grows after 7 days) 200C: Growth at 28°C: Growth at 33°C
:Growth at 37℃
: Growth 45°C : No growth 50°C : No growth Reaction to oxygen : Facultative anaerobic 0-F test (Hugh-Leifson method) : Generation of acid and gas from fermented sugar L-arabinose : False positive D-xylose : False positive D-glucose
: Positive, does not produce gas D-mannose : Positive, does not produce gas D-fructose : Positive, does not produce gas D-galactose : Negative Maltose : Positive, does not produce gas
:Negative lactose
: Negative trehalose : Positive, no gas produced? -Sorbit:
False positive D-mannitol: Positive, inoscito which does not produce gas ■Negative glycerin
: Positive, starch that does not produce gas :
Positive, does not produce gas (4) Chemotaxonomic properties Major menaquinones: MK-7 Cell wall diamino acids: Mesodiaminobimelic acid II, KSY
Identification of strain KSY-5 Even when fresh bacterial cells are used in Durham staining, most of the strain KSY-5 is decolorized by alcohol and is only slightly stained. However, because of the presence of endospores and the presence of menaquinone as a quinone, it is considered to be a Gram-positive bacterium with poor Durham staining properties. Furthermore, the KSY-5 strain is a bacillus with pericytium, grows aerobically, is catalase positive, and has mesodiaminobimelic acid in its cell wall.
It is considered to belong to the genus Acil Ius).

The endospores of this strain are oval in shape and are present in the central side of the sporangia.
Swelling of sporangia due to endospores is also observed. For this reason, when spore staining is performed using the W1rtz method and observed under a microscope, the spores inside the sporangium are green, and the vegetative cells are stained red in a canoe shape on one side of the spore. This characteristic of the sporoid is similar to that of Bacillus laterosporus within the genus Bacillus.
rus).

Bergey's Manual of Systematic Bacteriology Volume 2
alof Systematic Bacteriol
When comparing the physiological and biochemical properties of Bacillus lattestosporus described in J. Ogy, Volume 2) with the properties of this strain as described above, the results of comparable properties were almost identical.

Therefore, based on the morphological and physiological and biochemical characteristics described above, this strain is considered to be Bacillus lattesporus (B.
acillus laterosporus).

When culturing KSY-5 producing bacteria, carbon sources such as glucose, sucrose, maltose, blackstrap molasses, glycerol, oils and fats (e.g. soybean oil, olive oil, etc.), organic acids (e.g. citric acid, Succinic acid, curconic acid, etc.) are used as appropriate. As a nitrogen source,
For example, soybean flour, cottonseed flour, corn stew liquor, dry yeast, yeast extract, meat extract, peptone, urea,
Organic and inorganic nitrogen compounds such as ammonium sulfate, ammonium nitrate, ammonium chloride, and ammonium phosphate can be used. As the inorganic salt, inorganic salts normally required for culturing bacteria such as sodium chloride, calcium chloride, calcium carbonate, magnesium sulfate, monopotassium phosphate, and disodium phosphate are used alone or in appropriate combinations. Further, heavy metals such as ferrous sulfate and copper sulfate, vitamins such as vitamin B1 and biotin, and the like are added as necessary. Furthermore, antifoaming agents and surfactants such as silicone oil and polyalkylene glycol ether may be added to the medium.

In addition, organic or inorganic substances that help the growth of bacteria and promote the production of KSY-5 may be added as appropriate.

The culturing method may be similar to a general antibiotic production method, and solid culture or liquid culture may be used. In the case of liquid culture, any of static culture, agitation culture, shaking culture, aeration culture, etc. may be used, but aeration agitation culture is particularly preferred. Further, the culture temperature is preferably in the range of about 10 to 40°C, and the pH of the medium is in the range of about 5 to 9. The culture time is approximately 18-144 hours, preferably 48-96 hours.

In order to collect the target antibiotic KSY-5 from the culture, separation means that are normally used to collect metabolites produced by microorganisms from the culture of the microorganisms are appropriately used.

Antibiotic KSY-5 is a basic substance with properties intermediate between water solubility and fat solubility, so it is purified using this property. Furthermore, since KSY-5 is mainly contained in the culture filtrate, a filter aid is first added to the culture i& and the solids are removed by filtration or centrifugation. A means of eluting the effective substance from the obtained culture filter with an appropriate solvent and collecting it separately is advantageously used.

As the carrier, an adsorbent such as an ion exchange resin, an adsorbent resin, silica gel, activated carbon, or cellulose, or a carrier that utilizes the difference in molecular weight of the compounds, such as a molecular sieve, is used. The elution solvent varies depending on the type and properties of the carrier, but for example, an organic solvent, a water-containing solution of a water-soluble organic solvent, such as water-containing acetone, water-containing methanol, water-containing butanol, or an aqueous solution of acid, alkali, inorganic salt, or organic salt may be used alone or in combination with these solvents. A mixture with a water-miscible organic solvent is advantageously used.

Further, KSY-5 is extracted from a neutral to weakly alkaline aqueous solution with an alcoholic organic solvent that can be separated from water. In more detail, when a cation exchange resin such as Amberlite IRC-50 or CG-50 (manufactured by Rohm and Haas, USA) is used as a carrier, the antibiotic in the filtrate is adsorbed to the resin and acid or salt It is eluted with an aqueous solution containing. In addition, if an adsorbent resin such as Amberlite XAD-II (manufactured by Rohm and Haas, USA), Diaion HP-10 or HP-20 (manufactured by Mitsubishi Kabo Industries, Ltd.) is used, the active substance in the filtrate will be adsorbed. It is eluted with a mixed wave of an organic solvent and an aqueous solution, that is, a mixture of water containing acetone, methanol, etc., or an aqueous solution or buffer solution containing salts or acids. In addition, when silica gel such as Kieselgel 60 (manufactured by Merck & Co., West Germany) or a molecular sieve carrier such as Sephadex LH-20 (manufactured by Pharmacia, Sweden) is used, the active substance is adsorbed, and an appropriate solvent such as ethyl acetate, chloroform, acetone, etc. is used. , methanol, or a mixture thereof.

In addition, KSY-5 is a neutral to alkaline aqueous solution (PH
6-9) Extracted from the inside with an organic solvent that can be separated from water, such as n-butanol, iso-butanol, iso-amyl alcohol, n-amyl alcohol, etc.

Further, examples of carriers used in reverse phase preparative high performance liquid chromatography include MMC gel (manufactured by Yamamura Kagaku Kenkyusho), TSK gel (manufactured by Tosoh Corporation), and methanol, acetonitrile, etc. as a mobile phase. and a buffer solution are used.

Since KSY-5 is a weakly basic substance, it is formed into a pharmacologically acceptable salt together with an acid. Examples of the acid used to form this salt include mineral acids such as hydrochloric acid, sulfuric acid, and phosphoric acid. Organic acids such as acids include formic acid, acetic acid, oxalic acid, etc. Formation of salts is performed by known methods.

The physicochemical properties of the KSY-5 hydrochloride obtained in the Examples described below are as follows.

(a) Colorless plate-like crystals, basic peptide antibiotic (b)
Melting point: 196-199°C (c) Constituent elements: C, H, N, 0, (1(
d) Elemental analysis values (actual value %): C51.54%, H8.11%, N 11.89%, Ci 5.86% (e) Molecular weight: 1500-2500 <by gel filtration method) (f) Ultraviolet light Absorption spectrum; ? IO λmat. (Ele■) 200 nm (370.67) 2 2 5 nm (sh.) 275-280 nm (1 1.73) The absorption spectrum is shown in FIG.

(g) Infrared absorption spectrum (measured with potassium bromide tablets)
Characteristic absorption (cm-1): 3280, 3020,
2945.16B5, 1520, 1460, 1390,
'1365,1230.1160 The absorption spectrum is shown in Figure 2.

(h) Constituent amino acids: After decomposition in 6N hydrochloric acid at 110°C for 16 hours, amino acid analysis confirmed the presence of valine 4, leucine 3, lysine 3, isoleucine 1, tyrosine 1, and ornithine 1 in molar ratio.

(1) Solubility: Slightly soluble in water, methanol, dimethyl sulfoxide, ethyl acetate, chloroform, ether, benzene, etc. (j) Color reaction: Positive ninhydrin reaction, phosphomolybdic acid reaction (k) Thin layer chromatography (Merck (manufactured by Kieselgel GF254) (7) 1H-NMR spectrum: 500MH in heavy methanol with TMS as internal standard,
The results of the measurements are shown in Figure 3.

(m)"C-NMR spectrum: The 13C-NMR spectrum measured in deuteranol is shown in FIG.

From the above results and the physicochemical properties of known compounds, it was confirmed that the present antibiotic KSY-5 is a new compound.

Next, the biological properties of KSY-5 will be described.

The antibacterial properties of KSY-5 against various bacteria are shown in the table below.

The symbol a in the table indicates a nutrient agar medium, and the symbol b indicates a potato sucrose agar medium.

KSY-5 belongs to peptide antibiotics due to the above-mentioned physicochemical and biological properties. Among known peptide antibiotics, substances containing tyrosine in the molecule, such as Brevolin, The Journal of Antibiotics, Volume 7, Series A, Page 25, 1
954), Mycosubtilin (Mycosubti
1 in, The Journal of Clinical Investigation, Vol. 28, p. 924, 1949),
Subsporin, The Journal
Of Antibiotics Vol. 22, p. 467, 19
1969), Tyrocidine (Tyrocidine,
The Journal of the American Chemical Society, Vol. 74, p. 4019, 1952), Antibiotics TA
-2590 (Antibiotic TA-25
90, Special Publication No. 45-17594 1970),
Macromomycin (Macronomycin)
In, The Journal of Antibiotics, Vol. 21, p. 44, 1968), but it does not match any of these and is a new substance.

Antibiotic KSY-5 has a strong antibacterial effect against Durham-positive bacteria, especially methicillin-resistant staphylococcus (MRSA), and is therefore useful as an antibacterial agent.

[Examples] Hereinafter, the present invention will be further explained with reference to Examples, but the present invention is not limited to these Examples.

In addition, "%" in the examples means "r weight %" unless otherwise specified.

Example 1 Glycerin 5.0%, soybean flour 2.5%, brewer's yeast 0.
KSY-5 strain (accession number: FAIKEN Bacteria No. 11175) was inoculated into a liquid culture medium (PH 7.2) 70-70% with a composition of 5% calcium carbonate and 0.2% calcium carbonate, and incubated at 30°C for 48 hours.
Cultured for hours. This culture solution was mixed into medium 16 with the same composition as above.
1 and inoculated in a 30 Il fermenter at 30°C for 90 hours.
Culture was carried out with aeration and stirring. Airflow rate is 161/min, rotation speed is 45
It is 0 rpm.

A filter aid (trade name, Radiolite 700) is added to the obtained culture solution (16i) and filtered to separate the filtrate and the bacterial cells. The filtrate was then subjected to column chromatography using a weakly acidic ion exchange resin Amberlite IRC-50 (manufactured by Rohm and Haas) in H form. 0
, IN hydrochloric acid acetone (1:1) mixture to elute the antibacterial active ingredient, and after concentrating the eluate, silica gel C-200
It was subjected to column chromatography (manufactured by Wako Pure Chemical Industries, Ltd.). Chloroform acetone-methanol (
Among the fractions eluted with 2:1:2), the highly active fractions were collected, concentrated, and subjected to chromatography on Sephadex LH-20 (manufactured by Pharmacia, Sweden) filled with methanol. The resulting active fractions were combined, concentrated and then loaded with Sephadex LH with distilled water.
-20 column chromatography. When the obtained antibacterial active fractions were combined and concentrated and recrystallized from a mixture of ethanol, ethyl acetate, and hexane, KSY
Colorless plate-like crystals (760 mg) of the hydrochloride of No. 15 were obtained.

The hydrochloride of the antibiotic KSY-5 thus obtained had the physicochemical and biological properties described above.

[Effects of the Invention] Are the antibiotic KSY-5 and its salts of the present invention antibacterial and useful as agents for preventing and treating infectious diseases? Ru.

[Brief explanation of drawings]

Figure 1 is the ultraviolet absorption spectrum of KSY-5 hydrochloride measured in aqueous solution, Figure 2 is the infrared absorption spectrum of KSY-5 hydrochloride measured with potassium bromide tablets, and Figure 3 is 500MH in deuterated methanol. Figure 4 shows the 13C-NMR spectrum measured in heavy methanol.
This is an NMR spectrum.

Claims (2)

[Claims] (1) A novel antibiotic KSY-5 and its salts, which are characterized by having the following physical and chemical properties. KSY-5 hydrochloride exhibits the following properties. (a) Colorless plate-like crystals, basic peptide antibiotic (b)
Melting point: 196-199°C (c) Constituent elements: C, H, N, O, Cl (d) Elemental analysis values (actual value %): C51.54%, H8.11%, N11.89%, Cl5. 86% (e) Molecular weight: 1500-2500 (by gel filtration method) (f) Ultraviolet absorption spectrum: λ_m_a_x (E_1_c_m) 200nE (370.67) 225nm (sh.) 275-280nm (11.73) (g) Infrared rays Absorption spectrum (measured with potassium bromide tablets)
Characteristic absorption (cm^-^1): 3280, 302
0, 2945, 1635, 1520, 1460, 139
0, 1365, 1230, 1160 (h) Constituent amino acids: After decomposition in 6N hydrochloric acid at 110°C for 16 hours, amino acid analysis revealed the presence of valine 4, leucine 3, lysine 3, isoleucine 1, tyrosine 1, and ornithine 1 in molar ratio. confirm. (i) Solubility: Soluble in water, methanol, dimethyl sulfoxide, slightly soluble in ethyl acetate, chloroform, ether, benzene, etc. (j) Color reaction: Positive for ninhydrin reaction, phosphomolybdic acid reaction (k) Thin layer chromatography ( (manufactured by Merck & Co., Ltd., using Kieselgel GF254) Solvent system Rf value n-propanol-pyridine-acetic acid-water 0.80 (15:10:3:12) n-butanol-methanol-ammonia water-water 0.61 (10:4 :3:3) n-butanol-ethanol-chloroform-ammonia water 0.57 (4:5:2:5) 0.52 n-butanol-acetic acid-water (4:1:2) (2) Cultivate Bacillus bacteria that has the ability to produce the antibiotic KSY-5, and use the culture to produce the antibiotic KSY-5.
Claim (1), characterized in that it includes a step of collecting
Method for producing the antibiotic KSY-5 and its salts.