JPH0272183A - Antibiotic wk-2057 and production thereof - Google Patents

Abstract

NEW MATERIAL:An antibiotic WK-2057 having the following physico chemical properties. Elementary analysis: C27H32N2O (by high resolution mass spectrum). Molecular weight: 400 (by field desorption mass spectrum). Melting point: 113-118 deg.C; Specific rotatory power: [alpha]<25>=-49 deg. (C=0.45, methanol); Solubility: insoluble in hexane and water, soluble in methanol ethanol, dichloromethane, chloroform, ethyl acetate, butyl acetate and acetone.Basic substance.color reaction: positive to sulfuric acid and iodine coloring; etc. USE:An antibacterial agent against Gram-positive bacteria and a part of mycete. PREPARATION:An antibiotic WK-2057-producing bacterium belonging to the genus Streptomyces [example; Streptomyces sp. WK-2057 (FERM P-10015] is cultured preferably at neutral pH and 27 deg.C for 4-6 days.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a novel antibiotic and a method for producing the same. More specifically, the present invention is produced by culturing antibiotic-producing bacteria belonging to the genus Streptomyces, does not exhibit antibacterial activity against Durham-negative bacteria, and exhibits antibacterial activity against Durham-positive bacteria and some fungi. The present invention relates to an antibiotic having antiproliferative activity against Sarcoma 180 cells, and a method for producing the same.

[Background Art and Problems to be Solved] Antibiotics are substances produced by microorganisms and inhibit the growth of microorganisms and other cells. In general, antibiotics have high selective toxicity, so they have the advantage of having fewer side effects compared to other chemotherapeutic agents, but have the disadvantage that their effectiveness is drastically reduced by the development of resistant bacteria. Ways to overcome this drawback include chemically modifying existing antibiotics to increase their efficacy. However, there are limits to chemical modification, and new antibiotics are needed.

Therefore, the present invention aims to provide a novel antibiotic and a method for producing the same.

[Means for Solving the Problems] The present inventors isolated bacterial strains from various soils for the purpose of searching for new antibiotics, and as a result of intensive research on the metabolites produced by the strains, the present inventors discovered new antibiotics. In the culture of the bacterial strain WK-2057 isolated from the collected soil, a substance was produced that exhibited antibacterial activity against Durham-positive bacteria and some fungi, and also inhibited the growth of Sarcoma 180 cells. I found out that Then, the effective substance was isolated and purified from the culture, and its physicochemical properties were investigated. As a result, since there is no other substance with the same physical and chemical properties, we decided to name the substance WK-2057.

The present invention was completed based on this knowledge.

The present invention provides an antibiotic WK-
2057, and culturing antibiotic WK-2057-producing bacteria belonging to the genus Streptomyces in a medium, accumulating antibiotic WK-2057 in the culture, and collecting antibiotic WK-2057 from the culture. The present invention also provides a method for producing the novel antibiotic WK-2057.

The bacteria producing antibiotic WK-2057 belongs to the genus Streptomyces. For example, the bacterial strain WK-2 isolated by the present inventors
057 is an example of a strain most effectively used in the present invention, and the mycological properties of this strain are shown below.

(I) Morphological properties Vegetative hyphae develop well on various agar media, and no division is observed. Aerial mycelia are grown on oatmeal agar medium, glucose and
It grows abundantly on asparagine agar, starch/inorganic salt agar, and glycerol/asparagine agar, and has a white to gray color. When observed under a microscope, aerial hyphae are linear and chains of 20 or more spores are observed. The size of the spore is 1.2XO, 6 μm, and cylindrical, and the surface of the spore is smooth. No sclerotia, sporangia, or fumespores are observed.

(II) Properties on various media
) and Gottlleb's method (International Journal of Systemic Bacteriology, Volume 16, Page 313, 19)
The culture properties of this production bacterium investigated by 1966 are shown below. The color tone is the color of the mark, Color Harmony Manual 4th Edition (Containerφ Corporation of America, Chicago).

(1958), and the code is written in parentheses along with the color chart base. Unless otherwise specified, 27℃,
These are the results of observation on each culture medium for 2 weeks.

Culture characteristics (1) Sucrose/nitrate agar ■Growth Medium growth Cream to mistletoe green (11/2ca to 241/2ig) ■Back side Cream to sage green (11/2
ca to 24 i g) ■Aerial mycelia Not epiphytic ■Soluble pigments Not produced (2) Glucose-asparagine agar (ISP) ■Growth Good growth Biscuit to mustard (2ec to 2Ωe) ■Back side Bamboo to light mustard tan (2fb to 21e) ■Aerial mycelium Moderately epiphytic white (a) ■Soluble pigment Not produced (3) Glycerol/asparagine agar (ISP) ■Growth Good growth Bamboo to mustard (2fb to 2Ωe) ■Back side Bamboo to light mustard Tan (2f b to 2ie) ■Aerial mycelium Moderate epiphytic white (a) ■Soluble pigment Not produced (4) Starch/Inorganic salt agar (ISP) ■Growth
Good growth Light wheat (2e a) ■Back side Cobalt tan golden olive (1 1/2N g from 2Ωe) ■Aerial mycelia Good growth Alabaster tint (13ba) ■Soluble pigment Not produced (5) Tyrosine agar ( ISP) ■Growth Moderate growth Light eye poly to mustard tan (2ca to 2Ωg) ■Back side Bamboo to camel (2f B to 3ie) ■Aerial mycelium No epiphyte ■Soluble pigment No production (6) Oatmeal agar (ISP) ) ■Growth Good growing mustard (2, Q e) ■Back surface Light eye poly (2c a) ■Aerial mycelium Medium epiphytic alabaster tint (13ba) ■Soluble pigment Not produced (7) Yeast extract/malt Extract agar (ISP) ■Growth
Good growth Dull Gold (2n g) ■Back side Mustard Gold (2pg ■Aerial mycelium
No epiphyte ■Soluble pigments no production (8) Nutrient agar (ISP) ■Growth Slight growth Patty (11/2ec) ■Back side Dusty yellow (11/2gc) ■Aerial mycelium No epiphyte ■Soluble pigments no production (9) Peptone/yeast extract agar (ISP) ■ Growth
Moderately growing bamboo (2f b) ■Back side Light eye Poly to bamboo (2 ca to 2 [b) ■Aerial mycelia No epiphyte ■Soluble pigment No production (10) Glucose nitrate agar ■Growth Slightly growing light eye Poly-(2ca) ■Back side Bamboo (2Ωe) ■Aerial mycelia No epiphyte ■Soluble pigment No production (11) Glycerol/calcium malate agar ■Growth Good growth Burl pink topaz (3ca to 3ne) ■Back side From bamboo Light mustard tongue (2Ωe to 2ie) ■Aerial mycelium Poorly epiphytic pearl (2ba) ■Soluble pigment Not produced (12) Curcose φ peptone agar ■Growth Good growth Bamboo (2Ωe) ■Back side Mustard (2Ωe) ■Aerial mycelium Does not grow young ■ Soluble pigments Not produced (III) Physiological properties (1) Production of melanin pigment (a) Tyrosine agar Negative (b) Peptone/yeast iron agar Negative (c) Glucose/peptone/gelatin medium (21-23 °C) Negative (d) Tryptone yeast solution Negative (2) Tyrosinase reaction Negative (3) Production of hydrogen sulfide Negative (4) Liquefaction of gelatin (21~
23℃) Negative (glucose-peptone-gelatin medium) (5) Starch hydrolysis Positive (6)
Coagulation of skim milk (27℃) Negative (7) Peptonization of skim milk (27℃) Positive (8) Growth temperature range 15~
36°C (optimum growth temperature 27°C) (9) Utilization of carbon source (Breedam-Gotlieb agar medium) D-glucose, L-arabinose, D-xylose,
Raffinose, melibiose, D-mannitol, D-
(10) Decomposition of cellulose using fructose, L-rhamnose, i-inositol, and sucrose Negative (IV
) Cell wall composition Diaminopimelic acid in the cell wall is of the LL type.

The mycological properties of Honkan have been described in detail above, and can be summarized as follows.

Diaminopimelic acid in the cell wall is of the LL type.

The morphology of aerial hyphae is linear, forming long spore chains, and the surface of the spores is smooth. As for various cultural properties, vegetative mycelium exhibits a gold-ish or greenish color tone, and aerial mycelium exhibits a white or gray-type color tone. No soluble pigments are produced.

From the above results, this bacterial strain is a bacterial species belonging to the genus Streptomyces, according to Bridham and Tresner's classification (Basis φ Manual of Determinative Bacteriology, 8th edition).

It is considered to be a bacterial species belonging to the White or Gray series by the authors (pp. 748-829, 1974).

This strain is Streptomyces sp. WK
-2057 (StrcptoIIyccs sp, W
K-2057), which has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (Feikoken Bikki Deposit No. 10015).

The bacteria producing antibiotic WK-2057 have been described above, but in general, the mycological properties of actinomycetes are extremely variable and not constant. Streptomyces are exposed to natural or commonly used UV irradiation, X-ray irradiation, or mutagenic agents (e.g., N-methyl-N-nitro-N-nitrosoguanidine and ethyl methanesulfonate)
It is a well-known fact that mutations occur through artificial mutation means using . Such artificial mutant strains as well as natural mutant strains belong to the genus Streptomyces, and are not suitable for the antibiotic WK.
Any strain capable of producing -2057 can be used in the present invention.

In the present invention, first, antibiotic WK-2057-producing bacteria belonging to the genus Streptomyces are cultured in an appropriate medium.

In culturing this bacterium, a method of culturing actinomycetes is generally used. As the medium, a nutrient medium containing a carbon source that can be assimilated by microorganisms, a nitrogen source that can be digested, and, if necessary, an inorganic salt is used.

As assimilable carbon sources, glucose, molasses, lees, dextrin, cellulose, corn steep liquor, glycerin, organic acids, and the like are used alone or in combination. Digestible nitrogen sources include peptone,
Organic nitrogen sources such as meat extract, yeast extract, dried yeast, soybean flour, corn staple liquor, cottonseed meal, casein, soy protein digests, amino acids, and urea, or inorganic nitrogen compounds such as nitrates and ammonium salts. are used alone or in combination. In addition, inorganic salts such as sodium salts, potassium salts, calcium salts, magnesium salts, and phosphates are added as necessary. Furthermore, if necessary, micronutrients, growth-promoting substances, and precursors that promote the growth of this plant and the production of the antibiotic WK-2057 may be appropriately added.

Cultivation is usually preferably carried out under aerobic conditions such as shaking culture or aerated agitation culture. Industrially, deep aeration agitation culture is preferred. It is preferable to culture at a pH of the medium near neutrality. Although culturing can be carried out at a temperature of 20 to 37°C, it is usually kept at 24 to 30°C, preferably around 27°C. In the case of liquid culture, the culture is usually carried out for 4 to 6 days, and the antibiotic is produced and accumulated. Preferably, the culture is terminated when the amount accumulated during the culture reaches the maximum. These culture conditions, such as culture composition, liquid properties of the medium, culture temperature, stirring speed, and aeration amount, are adjusted or selected according to the type of strain used, external conditions, etc., so as to obtain favorable results. . When foaming occurs in liquid culture, antifoaming agents such as silicone foam, vegetable oil, and surfactants are conveniently used.

Since the antibiotic WK-2057 accumulated in the culture thus obtained is mainly contained in the cultured cells, the culture may be filtered using filter aids (e.g., celite and high flow) as necessary. It is advantageous to separate the culture filtrate from the bacterial cells by adding filtrate (Supercell) or by centrifugation, and then collect the antibiotic WK-2057 from the bacterial cells.

Antibiotic WK-2057 is insoluble in hexane and water, and is also insoluble in alcoholic solvents (e.g., methanol, and ethanol), chloroform-based solvents (e.g., dichloromethane, and chloroform), and ketone-based solvents (e.g., acetone, and methyl isobutyl ketone)
Since it is a basic substance that is soluble in many organic solvents such as, to separate and purify the antibiotic WK-2057 from cultured bacterial cells, a purification method that utilizes these properties is used.

Usually, cultured bacterial cells are cultured in a hydrophilic organic solvent (e.g., acetone,
and methanol), and the extract is concentrated under reduced pressure to remove the organic solvent. Thereafter, antibiotic WK-2057 is transferred to the organic solvent layer by extraction with a non-hydrophilic solvent (eg, ethyl acetate and chloroform).

The organic solvent layer obtained in this manner is washed with a dilute aqueous solution of ethylenediamine tetrahydrochloride to remove metal ions, etc., if necessary, and then treated with various dehydrating agents (for example, anhydrous sodium sulfate, and bead gel). ) and dehydrate. The solvent in the dehydrated organic solvent layer is then removed under reduced pressure. In this concentration operation, antibiotic WK-205
Although No. 7 is stable, it is usually preferable to conduct heating at a temperature of 50° C. or lower. Add hexane and an organic solvent such as petroleum ether to the residue to prepare antibiotic WK-205.
7 is precipitated. The obtained precipitate was washed several times with hexane etc., and the antibiotic WK-2 was removed by suction filtration or centrifugation.
A crude product of 057 can be collected.

To further purify the above crude product, antibiotic WK-20
Many means can be used that utilize the difference in solubility between the 57 substance and the congested substance, the difference in distribution between two unmixed liquids, or the difference in adsorption power to various adsorption carriers. Among these, chromatography is a particularly effective method. Chromatography effective for purifying the antibiotic WK-2057 substance includes adsorption chromatography using adsorption resins such as silica gel, alumina, activated carbon cellulose, and hydroxyapatite HP-20, silanized silica gel,
and reversed phase partition chromatography using octadecylsilanized silica gel, Sephadex LH-20
, gel filtration chromatography based on molecular sieves using Toyobar, etc., DEAE-cellulose, D
Examples include ion exchange chromatography using EAE-Sephadex, DEAE Toyovar, and the like.

Antibiotic WK-2057 is separated and purified by using the above-mentioned methods such as chromatography, electrophoresis, countercurrent distribution, ultrafiltration, and distillation alone or in combination or repeatedly in any order. For example, the above crude product is dissolved in a small amount of chloroform and adsorbed on silica gel,
Column chromatography is performed using a chloroform-methanol mixed solution. After concentrating the active fraction under reduced pressure, it was dissolved in a small amount of chloroform and subjected to gel filtration chromatography based on molecular sieves using a chloroform-methanol mixed solution.
7 can be separated and purified.

The physicochemical properties and biological properties of the antibiotic WK-2057 will be described below.

■Physical and chemical properties ■Properties of the substance: Blue needle-shaped crystals ■Elemental composition: C2□H32N 20 (based on high-resolution mass spectra) ■Molecular m: 400 (field desorption (F
D) Based on mass spectrum) ■Melting point: 113-118℃ ■Specific optical rotation: [α] 25-49° (C-0,45, methanol) ■Ultraviolet absorption spectrum: As shown in Figure 1 (measured in methanol, Conditions: 5can 5peed300, Onm
/1n% Bandpass 2.00 no,)
■Infrared absorption spectrum: As shown in Figure 2 (measured by KBr disk method) ■Solubility in solvents: Insoluble in hexane and water, soluble in methanol, dichloromethane, chloroform, ethyl acetate, butyl acetate, and acetone ■Nuclear magnetism Resonance spectra (in deuterated methanol, 7MS standard): Figure 3 (I H-NMR) and Figure 4 03C-N
MR) [Phase] Color reaction: H2S 04, positive for iodine color development Silica gel normal phase chromatography: Support: Silica gel 60 (manufactured by Merck & Co., Ltd.) RfltiIo, 33 (Developing solvent ethyl acetate:methanol-40:1) Rf value 0.28 (Developing solvent Chloroform: Methanol - 10:1) ■, Biological properties ■ Antibacterial spectrum Bacillus subtilis ATCC 66336325 Bacillus subtilis 1 PO 30011, 56 Micrococcus medium Luteus ATCC 94313, 12 Mycobacterium smegmatis ATCC 607> 100 Escherichia Urn Collie NIIIJ > 100
Eshiurihia e Collie NIIIJ JC-2> 100
Klebjiira and Bunyumonie ATCC10031> t
o.

Salmonella tibimium 〉100 Proteus vulgaris lPO3187> 100 Shutomonas aeruginosa lPO3080> 100 Candida albicans > 100*
Satucharomyces 0 cerevisiae ATCC9763> 1
00* Cryptococcus Fist Neoforman
100*Microsporum Gibserum
50. O*Trichophyton mentaprophyte
100* Penicillium herque 1F04674
＞100*Botrytis cinere
100 *Screotinia cinere
25. O*Piricularia oryzae 12.5** indicates antibiotic 1 based on the results obtained using potato agar.WK-2057 shows antibacterial activity against Durham-positive plants and some fungi, but it does not show antibacterial activity against Durham-negative plants. was found to have no antibacterial activity.

(2) Anticancer activity Effect on 5180 mouse tumor 5180 cells were transplanted into the peritoneal cavity of 1×10 6 cells/mouse (ICR male, 6 weeks old), treated according to the method shown in Table 1 below, and the survival days were observed.

Table 1 Dose Administration port Survival days Survival prolongation rate 22.2 1 - 9 23 1
3011.1 1-9 21.5 11
On day 5 of administration, the day of tumor transplantation was set as day 08. Number of days to live is 1
Median values of three mice per group are shown. The life extension rate was calculated using the following formula.

Life extension rate (96) As shown in Table 1 above, the anticancer activity of antibiotic WK-2057 against 8180 ascites cancer was observed.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these.

(Example) Culture of A5 Streptomyces WK-2057 500
In a ml Sakaro flask, add 2.0% glycerin and 2 soybean flour.
．． 0%, and a liquid medium containing 0.5% sodium chloride (pH
7,0) 100 ml was prepared and sterilized. And glucose 1.0%, peptone 0.5%, meat extract 0.5%
The above 1t4C was obtained from a slant culture of Streptomyces WK-2057 (Feikoken Bacteria 10015) cultured at 27°C for 6 days on an agar slant medium containing 0.3% salt, 0.3% agar, and 1.2% agar. One platinum loop was inoculated into the body culture medium.

After that, use a reciprocating shaker (amplitude 17 cm).

A seed mother was obtained by culturing with shaking at 27° C. for 72 hours using a shaker (reciprocating 120 times per minute). Next, 100 g jarfa~mentor contains 2.0% glucose, 0.5% peptone, 0.5% meat extract, 0.3% dry yeast, 0.5% salt, and 0.3% calcium carbonate. After preparing and sterilizing the medium 61M, the seed mother 61M obtained by the above method was added to it.
0 ml was aseptically transplanted. Perform aeration agitation culture,
A culture solution (6CD) was obtained.

B. Extraction of antibiotic WK-2057 from the culture The culture solution obtained in A above was centrifuged to obtain bacterial cells. The bacterial cells were extracted with acetone, and the extract was concentrated under reduced pressure to remove the organic solvent. Ethyl acetate was added to this concentrated solution, and after thorough stirring, the ethyl acetate layer was separated. This separated ethyl acetate layer was dehydrated with anhydrous sodium sulfate and then concentrated under reduced pressure to obtain 12.5 g of an oily residue containing antibiotic WK-2057.
I got it.

C. Antibiotics by silica gel chromatography The oily residue obtained in step B above was adsorbed onto a silica gel column (inner diameter 30 mm, length 400 mm) filled with silica gel (manufactured by Merck & Co., Ltd.) using chloroform, and the developing solvent was chloroform. Chromatography was carried out while continuously changing from methanol to methanol. The obtained active fraction was concentrated under reduced pressure to obtain 450 mg of WK-2057 crude product with a purity of about 6096.

In order to obtain a pure antibiotic WK-2057 obtained in the above C, purification was performed using high performance liquid chromatography (manufactured by JASCO Corporation, model LC-20). At this time, the column had an inner diameter of 7.6 mm.

Asahlpack GS-310 with length 250mm
H (Asahi Kasei) was used. Antibiotic WK obtained in above C
-2057 crude product 20mg chloroform 200μg
A sample dissolved in the solution was injected, and chromatography was performed using chloroform-methanol (20:1, a high-performance liquid chromatography solvent) as a developing solvent.

Fractions showing a peak corresponding to antibiotic WK-2057 in ultraviolet absorption at 260 nm were collected and concentrated to dryness under reduced pressure to obtain 8 mg of pure antibiotic WK-2057.
I got it. Repeat this operation several thousand times and use antibiotic WK-20.
A total of about 200 mg of 57 was obtained. This antibiotic WK-2
The physicochemical and biological properties of 057 are as described above.

[Effects of the Invention] As mentioned above, the present invention provides novel antibiotic WK-20.
57 and methods of manufacturing the same are provided.

The antibiotic WK-2057 according to the present invention does not exhibit antibacterial activity against Durham-negative bacteria, exhibits antibacterial activity against Durham-positive bacteria and some fungi, and has growth-inhibitory activity against Sarcoma 180 cells. .

[Brief explanation of the drawing]

Figure 1 is an ultraviolet absorption spectrum diagram of antibiotic WK-2057, Figure 2 is an infrared absorption spectrum diagram, and Figure 3 is I
FIG. 4 is a 13C-NMR spectrum. Applicant's agent Patent attorney Takehiko Suzue

Claims (3)

[Claims] (1) Antibiotic WK-20 with the following physical and chemical properties
57. [1] Elemental composition: C_2_7H_3_2N_2O (according to high-resolution mass spectrum) [2] Molecular weight: 400 (according to field desorption mass spectrum) [3] Melting point: 113-118°C [4] Specific optical rotation: [α]^ 2^5-49° (C=0.45, methanol) [5] Ultraviolet absorption spectrum: As shown in Figure 1 (measured in methanol) [6] Infrared absorption spectrum: As shown in Figure 2 (KBr disk method) [7] Solubility in solvents: insoluble in hexane and water,
Soluble in methanol, ethanol, dichloromethane, chloroform, ethyl acetate, butyl acetate, and acetone [8] Nuclear magnetic resonance spectrum (in heavy methanol, TMS
Standard): As shown in Figures 3 and 4 [9] Distinction between basic, neutral, and acidic: Basic substances [10] Color reaction: H_2SO_4, positive for iodine color development (2) Antibiotic WK-2 belonging to the genus Streptomyces
057-producing bacteria were cultured in a medium, and antibiotic WK was added to the culture.
-2057 was accumulated, and the antibiotic WK-2057 was collected from the culture.
Antibiotic WK-2 characterized by collecting 2057
057 manufacturing method. (3) Antibiotic WK-2 belonging to the genus Streptomyces
The 057-producing bacterium is Streptomyces WK-2057 (
3. The manufacturing method according to claim 2, wherein the method is 10015).