JPH0631311B2 - Antibiotics RK-1061A, RK-1061B, and RK-1061C and method for producing the same - Google Patents

Description

TECHNICAL FIELD OF THE INVENTION The present invention relates to a novel compound and a method for producing the same.

The invention is more particularly directed to the antibiotic RK-1061.
A, RK-1061B and RK-1061C, and a method for producing the same. RK-1061 of the present invention
A, RK-1061B and RK-1061C are antibiotics RK-10 belonging to the genus Streptomyces.
61A, RK-1061B and RK-1061C-producing bacteria are cultivated, and they are novel antibiotics not yet described in the literature, which are separated and collected from the culture.

(Background of the Invention) In recent years, many antibiotics have been found that can be used not only for medical purposes but also for agricultural chemicals.

For the purpose of searching for useful antibiotics as described above, the present inventors have isolated microorganisms from a large number of soils, and have been purifying, identifying and developing applications of the antibiotics produced.

As a result, a novel antibiotic not described in the literature was produced in the culture of a microorganism belonging to the genus Streptomyces,
We obtained the knowledge that it was accumulated and succeeded in its isolation and purification.

(Object of the Invention) An object of the present invention is to provide a novel antibiotic and a method for separating and collecting the antibiotic from a microorganism belonging to the genus Streptomyces of actinomycete.

(Structure of Invention) That is, the present invention relates to a compound represented by the following structural formula.

(Where R is Is. ) The present invention also relates to a method for producing the above compound, which comprises culturing the compound-producing bacterium belonging to the genus Streptomyces, and separating and collecting the compound from the culture, More specifically, the producing strain is Streptomyces sp. RK-1061 (Stre
ptomyces Sp. RK-1061).

In the above formula (I), R is RK-1061A, R is Is a compound in which RK-1061B and R are The compound represented by the formula is referred to as RK-1061C.

<Microorganism used> First, the microorganism used in the present invention is the antibiotic RK-
It has the ability to produce 1061A, RK-1061B and RK-1061C, and is a bacterial species belonging to the genus Streptomyces.

As an example, Streptomyces sp. RK-
The microorganism designated as 1061 (Streptomyces Sp. RK-1061 (hereinafter referred to as "RK-1061 strain") has the above-mentioned characteristics, and the antibiotic RK-1061A of the present invention,
RK-1061B and RK-1061C are advantageously produced and can be effectively used in the method of the present invention.

In addition to the natural and artificial mutants of the above RK-1061 strain, the strains belonging to the genus Streptomyces include antibiotics RK-1061A, RK-1061B and RK-1 described below.
Any microorganism capable of producing 061C can be used in the method of the present invention.

The RK-1061 strain is a soil actinomycete found in the soil collected by the present inventor in Misaka Town, Yamanashi Prefecture,
May 29, 1985 at the Institute of Microbial Technology, National Institute of Advanced Industrial Science and Technology
The date was deposited, and the microorganism deposit number is Microorganism Research Institute
No. 278 (FERM P-8278).

The RK-1061 strain has the following mycological properties.

I. Form RK-1061 strain is an actinomycete isolated from soil collected in Misaka Town, Yamanashi Prefecture. Only L, L-diaminopimelic acid was detected and paper meso-diaminopimelic acid was detected in the paper chromatograph of hydrochloric acid hydrolyzate of whole cells. Not done. In a growth test on various agar media, it grows on all 10 kinds of media tested, but the growth on starch yeast extract agar media is good and the aerial mycelia and spores are abundant, but
The aerial hyphae and spores did not adhere well on other agar media. In the utilization test using 11 kinds of sugars as carbon sources,
This bacterium utilizes all sugars and develops. The aerial hyphae of this bacterium are grayish and the reverse side is light brownish, and there is no feature. Growth in defatted milk first causes coagulation, but later turns into peptone giving a clear brown liquid. Hydrolyzes starch, but does not liquefy gelatin. The formation of melamine pigment is observed on peptone-yeast extract-iron agar medium and tyrosine agar medium, but the soluble pigment is pale brown or gray and no characteristic pigment formation is observed. The aerial hyphae were linear and flexible under electron microscopy, and 3-5 times spiral aerial hyphae were observed on oatmeal / nitrate agar and potato extract / yeast extract nitrate agar. It is a dense spiral, but the latter is an open spiral. On the other hand, helicoidal hyphae are not easily observed in yeast extract and malt extract grown on an agar medium. A large number of spores of this fungus are formed continuously from the hyphae tip, and the spiral hyphae are sporulated. The spore surface is smooth but wrinkled. The spore size is 0.5 to 1.0 micrometer in length and 0.5 to 0.7 micrometer in width. No sporangia and motile spores were observed.

II. Growth state on various media (27 ° C, 3 weeks culture) Color tone is described in Descriptive Color names dictionar
y) According to the 4th edition color name symbol.

1. Sucrose / nitrate agar medium Growth: Normal air Mycelium: None Back surface: 2ba (pearl) Soluble pigment: None 2. Glucose / asparagine agar medium Growth: Normal air Mycelium: None Back surface: 2ba (pearl) Soluble pigment: None 3. 3. Glycerol-asparagine agar medium Growth: Normal air Mycelium: None Back surface: 2ba (pearl) Soluble pigment: None 4. Starch / inorganic salt agar medium Growth: good Aerobic mycelium: None Back side: 2ba (pearl) Soluble pigment: None 5. Tyrosine agar medium Growth: Normal air Mycelium: Small amount b + 3ni + 4ni (Oyster white + Covert brown + Chestnut brown) Back surface: 3pn (dark brown) Soluble pigment: 3pl (deep brown) 6. 6. Nutrient agar medium Growth: Bad bacteria Mycelia: None Back surface: 3 ng (yellow maple) Soluble pigment: 3 ng (yellow maple) 7. Yeast extract / malt extract agar medium Growth: Normal aerial Mycelia: Abundant e (gray) Back surface: 3pi (gold brown) Soluble pigment: 3pn (dark brown) 8. Oatmeal agar medium Growth: Normal air Mycelium: Normal 5ge (rosewood) Back surface: 4ge (rosebeige) Soluble pigment: None 9. Peptone / East extract / Iron agar medium Growth: Poor mycelium Mycelium: None Back side: 2ba (pearl) Soluble pigment: 5pn (dark brown) 10. Starch yeast extract agar medium Growth: good Aerobic mycelium: Abundant 4ge + 4li (rose beige + beaver) Back: 4ge (rose beige) Soluble pigment: 1ih (olive gray) III. Utilization of carbon source (Pridham-Gottlieb agar medium) (27 ° C culture) Growth status 1. L-arabianose ++ 2. D-xylose +++ 3. D-glucose ++ 4. D-fructose +5. Sucrose + 6. Inositol + 7. L-rhamnose +8. Raffinose + 9. D-mannitol +10. Lactose +++ 11. Meribiose ++ +: develops ++: develops well +++: develops very well IV. Other physiological properties (27 ° C culture) 1. Liquefaction of gelatin (glucose / peptone / gelatin medium) Do not liquefy.

2. Hydrolysis of starch (starch / inorganic salt agar) Hydrolyzes.

3. Coagulation of skim milk and peptone coagulation to peptone.

4. Formation of melanin-like pigment There is pigment formation on tyrosine agar and peptone yeast iron agar.

5. Growth temperature 20-35 ° C Streptomyces genus having the above properties, that is,
Gray-typed with spiral hyphae, produces melanoid-like pigments, has a smooth spore plane, and is a sugar-utilizing species described above in the Barjay's Manual of Definite Native Bacteriology, 8th Edition ( Bergey'S Mannua
l of Determinative Bacteriology, 8th edition (197
4)). As a result, this bacterium was found to be Streptomyces griseospore.
us) or a species closely related to this.

(Culturing method and purification method) Antibiotics RK-1061A and RK-1061B of the present invention
To obtain RK-1061C, the above-mentioned antibiotic-producing bacterium belonging to the genus Streptomyces can be cultured by a usual method for producing an antibiotic. The form of the culture may be liquid culture or solid culture, and for industrially advantageous culture, a spore suspension or culture solution of the above-mentioned production strain may be inoculated into the medium and aerated stirring culture may be performed.

The nutrient source of the medium is not particularly limited, and a carbon source, a nitrogen source and the like usually used for culturing microorganisms can be contained in the medium. As a carbon source, starch,
Dextrin, glycerin, glucose, sucrose, galactose, inositol, mannitol and the like, and nitrogen sources include peptone, soybean flour, meat extract, rice bran, malt, urea, corn steep liquor, ammonium salts, nitrates and other organic substances. Alternatively, an inorganic nitrogen compound is used. In addition, inorganic salts such as salt,
Phosphates, metal salts such as potassium, calcium, zinc, manganese, iron and the like may be appropriately added, and as necessary, antifoaming agent, plant oil, mineral oil and the like may be added. Culture conditions such as culture temperature and culture time are suitable for the growth of the bacteria used,
Moreover, RK-1061A, RK-1061B and RK-
The conditions are selected to maximize the production of 1061C.
For example, the pH of the medium is preferably 4-9, especially around 6-7,
A suitable temperature for culturing is preferably about 25-35 ° C. Moreover, the culture composition, the hydrogen ion concentration of the medium, the culture temperature, the culture conditions such as stirring conditions should be appropriately adjusted so that preferable results can be obtained depending on the type of strain to be used and external conditions. Needless to say. From the culture thus obtained, RK-1061A and RK-10
In order to obtain 61B and RK-1061C, it can be collected by appropriately utilizing the means usually used for collecting metabolites. For example, RK-1061A, RK-1061B
And any of the means utilizing the difference in solubility between RK-1061C and impurities, the means utilizing the difference in ionic binding force, the means utilizing the difference in adsorption affinity, and the means utilizing the difference in molecular weight, respectively, respectively, or, They are used in appropriate combination or repeatedly. Specifically, RK-1061A, R
Most of K-1061B and RK-1061C are present in the culture filtrate, but the active fraction present in the bacterial cells is obtained by extracting the contained water with acetone and distilling off the acetone. When this is combined with the above-mentioned culture filtrate and purified by a combination of adsorption chromatography, silica gel chromatography, liquid chromatography, ion exchange chromatography, gel filtration chromatography and the like, RK-1
A complex containing 061A, RK-1061B and RK-1061C and other active ingredients (RK-1061 complex) is obtained. As a carrier for adsorption chromatography,
Diaion HP-10, MCI GEL CHP-20P as a carrier for liquid chromatography, Dowex 50WX4 as a carrier for ion exchange chromatography, and Sephapher as a carrier for gel filtration chromatography. Dex LH-20 and the like are preferable.

When the obtained RK-1061 complex is subjected to high performance liquid chromatography, a multi-component peak is observed. The column used is advantageously of the reversed-phase partition type.

Of the obtained peaks, RK-1061A and RK-10
The peaks corresponding to 61B and RK-1061C were respectively collected, concentrated and lyophilized to obtain pure R
K-1061A, RK-1061B and RK-1061
Get C respectively.

[RK-1061A, RK-1061B and RK-10
Physical and chemical properties and biological properties of 61C] (1) Shape: RK-1061A, RK-1061B and R
All of K-1061C are white powders.

(2) Melting point: RK-1061A, RK-1061B and R
All of K-1061C decompose above 190 ° C.

(3) Elemental analysis: RK-1061A: carbon 51.13% hydrogen 6.59% nitrogen 6.45% sulfur 2.92% RK-1061B: carbon 49.31% hydrogen 6.53% nitrogen 6.61% sulfur 2.72% RK-1061C: carbon 49.52% hydrogen 6.62% nitrogen 6.63 % Sulfur 2.90% (4) Molecular weight: According to FAB / mass spectrum.

RK-1061A: 1033 [(M + H) ⁺ m / z
1034 (M + Na) ⁺ m / z 1056 (M + K) ⁺ m
/ Z 1072] RK-1061B: 1009 [(M + H) ⁺ m / z
1010 (M + Na) ⁺ m / z 1032 (M + K) ⁺ m
/ Z 1048] RK-1061C: 1009 [(M + H) ⁺ m / z
1010 (M + Na) ⁺ m / z 1032 (M + K) ⁺ m / z
z 1048] (5) Specific rotation: RK-1061A: [α] _D ²⁴ + 18.7 ° (C0.3,
Water) RK-1061B: [α] _D ²⁴ + 17.3 ° (C0.4,
Water) RK-1061C: [α] _D ²⁴ + 18.9 ° (C 0.9,
water) (7) Infrared absorption spectrum: KBr method RK-1061A: 3430, 2930, 1735, 17001640, 14
60, 1405, 12701080, 1020, 885, 820765, 575 cm
^-1 (See FIG. 4) RK-1061B: 3450, 2930, 1730, 1700, 1650,
1460, 1400, 1265, 1070, 1015, 870, 820, 765, 580 c
m ^-1 (See FIG. 55) RK-1061: 3425, 2940, 1735, 1700, 1640, 14
60, 1395, 1270, 1115, 1020, 885, 820, 775, 580 cm
^-1 (See FIG. 6) (8) Solubility: RK-1061A: readily soluble in water and dimethyl sulfoxide,
Soluble in methanol and ethanol, insoluble in acetone, ethyl acetate and chloroform.

RK-1061B: Same as above RK-1061C: Same as above (9) Nuclear magnetic resonance spectrum: 400 MHz, solvent CD ₃ OD, standard TMS RK-1061A: as shown in FIG. 7.

RK-1061B: As shown in FIG.

RK-1061C: As shown in FIG.

(10) Rf value (silica gel thin layer chromatography): (11) Color reaction: All of RK-1061A, RK-1061B and RK-1061C decolorize the potassium permanganate solution and are positive for anisaldehyde-sulfuric acid reagent, anthrone reagent and ninhydrin reagent.

(12) Distinction between basic, acidic and neutral: RK-1061A, RK-1061B and RK-106.
All of 1C are acidic substances as distinguished by the filter paper electrophoresis method.

(13) Antibacterial spectrum: The minimum inhibitory concentration (MIC) was determined by the multiple dilution method using broth agar medium.

(14) Peptidoglycan synthase inhibitory activity: The incorporation of the substrate UDP- (U- ¹⁴ C] -N-acetylglucosamine into the peptidoglycan fraction was measured using a crude peptidoglycan synthase enzyme prepared from Escherichia coli, and the ID ₅₀ was determined. I asked.

(15) Acute toxicity: A, B,
No abnormalities were observed at 180 mg / kg for each C.

As described above, the antibiotics RK-1061A and RK-10 of the present invention
Comparing the physicochemical properties and biological properties of 61B and RK-1061C with known antibiotics, it contains sulfur in the molecule, exhibits an antibacterial action against Gram-positive and Gram-negative bacteria, and is a peptidoglycan synthase. Since they show inhibitory activity, the only similar substances include the antibiotics mucopeptin A (see Japanese Patent Publication No. 57-40160) and mucopeptin C (see Japanese Patent Publication No. 58-3676). However, these are specific rotations, molecular weights,
A clear difference is observed in the ultraviolet absorption spectrum.

In addition, as an antibiotic having a similar ultraviolet absorption spectrum and antibacterial activity, Tunicamycin [Journal of antibiotics]
ibiotics) Vol. 24, No. 4, 215 (1971)]
And SF-1999 substance [Meiji Seika Co., Ltd. annual research report No.
22, 1-8 (1983)], but these do not contain sulfur in the molecule.

From the above points, the antibiotic RK-1061A R of the present invention
It was concluded that K-1061B and RK-1061C are novel substances.

RK-1061A, RK-1061B and RK of the present invention
Since -1061C has antibacterial activity against various bacteria and peptidoglycan synthase inhibitory activity, it is expected to be used as an antibacterial agent.

The present invention will be described below with reference to examples.

<Example> Composition of glucose 2%, soluble starch 1%, meat extract 0.1%, yeast 0.4%, soybean flour 2.5%, sodium chloride 0.2%, and potassium diphosphate 0.005% in a 30-volume jar fermenter. The above culture medium 18 was inoculated in advance with the RK-1061 strain (Microtechnology Research Institute No. 8278) and cultured with shaking at 28 ° C. for 48 to 72 hours 140 m
1 and inoculate and culture at 28 ° C. for 65 to 70 hours with aeration and stirring until the pH exceeds 8.4. The ventilation rate at this time is 1 per minute
8. The stirring rotation speed is 350 rpm.

After the completion of the culture, the filter aid Celite is added to the culture solution, and the mixture is centrifuged and separated to separate the cells and the filtrate. Cells are 80% acetone 15
Is extracted with, and this is concentrated under reduced pressure to distill off acetone to obtain an aqueous solution of 2.5. The pH of the filtrate 75 (6 jar fermenters) was adjusted to 7.0 with HCI, and then Diaion HP-10 was used together with the previously obtained aqueous bacterial cell extract solution.
It is passed to the resin tower 7 and adsorbed. After washing with 20 of water and elution with 30% water-containing methanol 20, impurities are eluted. Then, elution is carried out with 50% water-containing acetone 40 to elute the active portion. This is concentrated under reduced pressure to obtain Concentrate 6. Then, n-butanol (3) is added thereto and the butanol extraction is performed. This operation is repeated 3 times to obtain a butanol layer 10 containing an active portion. The butanol layer is concentrated under reduced pressure and freeze-dried to obtain 27.9 g of a crude powder of RK-1061 complex. The crude powder is chromatographed on a silica gel column (6.0 x 65 cm) using the solvent system chloroform-methanol. The activity appears in the elution section of (1: 3) from chloroform-methanol (1: 2).
The active fraction is concentrated under reduced pressure and freeze-dried to obtain 16.0 g of crude powder.

This crude powder was dissolved in a small amount of 10% water-containing acetone and preliminarily prepared in the same solution as an MCI-gel column (3.0 × 75 c).
m), and after thoroughly washing with 10% hydrous acetone, the active ingredient is eluted with 50% hydrous acetone. The active portion is collected, concentrated under reduced pressure, and freeze-dried to obtain 2.5 g of crude powder. In order to remove impurities insoluble in water, the product is dissolved in water and then centrifuged at 2800 rpm for 10 minutes at room temperature. The supernatant is concentrated under reduced pressure and freeze-dried to obtain 1.2 g of crude powder.

This crude powder was dissolved in a small amount of 0.1 M pyridine-acetic acid (pH 4.0) buffer, and the cation exchange resin Dowex 50W x 4 (100-200 mesh) column (3.0 x 80 cm) prepared in advance with the same buffer was applied. Let it pass. The active portion that had passed through the same buffer was concentrated under reduced pressure and lyophilized to 620
mg of crude powder is obtained.

Furthermore, this was applied to Sephadex LH-20 column (2.2
X 72 cm), develop with 30% water-containing methanol,
The active portion was concentrated under reduced pressure and freeze-dried to obtain RK-
320 mg of 1061 composite powder are obtained.

Purification of this RK-1061 complex was carried out by high performance liquid chromatography using a reverse phase column (Nucleozil 5C ₁₈ , φ8 × 250 mm), using acetonitrile-0.1% diethylamine / formic acid (pH 4.0) (40:60) as a solvent. When the system is run at a flow rate of 2 ml / min, a multi-component peak is split (see Fig. 10). With this solvent system, the peak with a retention time of about 17 minutes was collected, and after removing acetone, a small amount of MCI gel column was passed through to remove the mixed salts. After thorough washing with water,
The active ingredient is eluted with 50% hydrous acetone. After removing acetone, lyophilization gives 3.4 mg of white powder of RK-1061A. In some cases, purification is performed by collecting the peaks several times.

In the same manner, 15.7 mg of RK-1061B is obtained by collecting a beak having a retention time of about 19 minutes.

Further, 16.7 mg of RK-1061C is obtained by collecting the peak around the retention time of 21 minutes.

[Brief description of drawings]

1, 2 and 3 show the antibiotics RK-1061A, RK-1061B and RK-1061 of the present invention, respectively.
FIG. 4 is a drawing showing an ultraviolet absorption spectrum of C, FIG.
5 and 6 show RK-1061A and RK-, respectively.
10 is a drawing showing infrared absorption spectra of 1061B and RK-1061C, and FIGS. 7, 8 and 9 show RK-1061A, RK-1061B and RK-10, respectively.
61C is a drawing showing a nuclear magnetic resonance spectrum of 61C;
Figure 0 shows RK-10 by high performance liquid chromatography.
It is a chromatogram which shows the peak of 61A, RK-1061B, and RK-1061C.

Claims (3)

[Claims] 1. A compound represented by the following structural formula. (Where R is Is. ) 2. A method for producing a compound represented by the following structural formula, which comprises culturing the compound-producing bacterium belonging to the genus Streptomyces, and separating and collecting the compound from the culture. Manufacturing method. (Where R is Is. ) 3. A compound producing bacterium is Streptomyces sp. RK-1061 (Streptomyces Sp. RK-10).
61) The manufacturing method according to claim 2.