JPH01228484A - Antibiotic substance wk-1875 and production thereof - Google Patents

Abstract

PURPOSE:To obtain an antibiotic substance WK-1875 active against phytopathogenic fungi and useful as an agricultural chemical by culturing a microbial strain belonging to genus Streptomyces and capable of producing antibiotic substance WK-1875 and treating the culture product. CONSTITUTION:A microbial strain belonging to genus Streptmyces and capable of producing antibiotic substance WK-1875 (FERM P-9859) is cultured in a proper medium under aerobic condition e.g., by shaking culture or submerged culture under aeration and agitation, at 20-40 deg.C and pH4-9 for 1-7days to accumulate the antibiotic substance WK-1875 inside and outside of the cell. The culture product is separated by centrifugal separation, the filtrate is adsorbed to a porous polymer resin, etc., and eluted, and the eluate is concentrated, dried and purified to obtain the objective antibiotic substance WK-1875 having the following properties. Elemental analysis (measured value, %), C 64.7+ or -1.0, H 9.1+ or -0.3; specific rotation, -12.1 deg. (c=0.5, methanol); melting point, 116-119 deg.C; ultraviolet absorption, infrared absorption and proton nuclear magnetic resonance spectra, exhibiting specific respective patterns; solubility, soluble in methanol, ethanol, ethyl acetate and CHCl3 and insoluble in water and n- hexane; molecular formula C40H68O12.

Description

[Detailed description of the invention] The present invention relates to antibiotics, and particularly to the novel antibiotic WK-18.
75 and its manufacturing method.

As antifungal antibiotics, for example, pharmaceuticals such as amphoterine, griseofulvin, and virolnidrine, and agricultural chemicals such as plusnacidin S1 polyoxin, validamycin, and tetranactin are known. However, there is still room for improvement in the known antifungal antibiotics, for example, in terms of efficacy, toxicity, antibacterial properties, etc. The present invention shows that actinomycetes isolated from soil by the inventors are particularly effective against plant pathogenic bacteria. It is based on the knowledge that it has the ability to produce antifungal antibiotics with strong activity.

An object of the present invention is to provide a novel antibiotic WK-1875 and a method for producing the same.

・According to the present invention, antibiotic WK-1875 is provided.
The physicochemical properties of this antibiotic are as follows.

■ Melting point: 116-119℃ ■ Elemental analysis 2 actual measurements (direct (%) C64,7±l, O H9,1±0.3 ■ Molecule 51 near 40.97 ■ Molecular formula 204゜H6゜O+X ■ Specific optical rotation : [α] -12,1' (c=0.5
．． (Methanol) ■ Ultraviolet absorption spectrum (in methanol) Nikita absorption ■ Infrared absorption spectrum (KBr method): As shown in Figure 1 ■ Proton nuclear magnetic resonance spectrum (CDC1, in middle):
As shown in Figure 2 ■ C-13 nuclear magnetic resonance spectrum + CDCl, medium):
As shown in Figure 3 [Phase] Soluble in solvents Possible 78 = methanol, ethanol, ethyl acetate, chloroform Insoluble: water, n-hexane ■ Positive color reaction: H, SO.

Negative/Ehrlich reaction, Dragendorff reaction @ Classification of acidic, neutral, and basic: Weakly acidic substance @ Color of substance: White The biological properties of antibiotics are as follows.

l) Antibacterial activity Table 1 shows the minimum inhibitory concentration (MIC, microg/m concentration) of this antibiotic by the agar dilution method.

Table 1 - MIC Escherichia coli
i) > 1 OKB+76 (NIHJ, JC-2,
IFO12734) *Pseudomonas aeruginosa > 100 (Ploo (Pseudo)
Xar + Lhomonas KB88
〉100(Microcoloo(1uteu)
sl to B2O (PcI + 00116 Staphylococcus aureus > 100 (Sta h 1oco
ccus aureus) KB34 (FDA 209
P) Mycobacterium smegmatis 〉100 (Ryo Edan 11 Ean 1 song fall) KB42 (ATCC6071
I Bacillus subtilis 〉l00(Ba
cillus 5ubtilis) KB27 (PC
I 2191 *Candida albicans
〉100(Candida aLbicansl
KFI Satsukalomyces salmon
〉1100(Saccharoces 5ake)
to F26 ** Aspergillus niger
〉100(HedanPs”) LLVLMidan l[Kudanko I
KFI port 3 (ATCC62751* No. Biricularia oryzae 5.25 (
Piricularia ρniytandanri ni F18
0 Nemcor racemosus 3
12 (Mucor racemosusl KF223
(IFo 45811 Umbrella $ Phytophytola balasitica 1.56 Note: * Sensitive agar medium (made in Japan) 37℃, pH 7,
Judgment was made at 0 and 24 hours.

$3 Potato glucose agar medium 27℃, H6, Ol
On the 3rd.

As mentioned above, the present antibiotic is, for example, Viracularia (P
Shows strong activity against fungi of the genus Iricularial.

2) Acute toxicity of this antibiotic when administered to toxic mice (L D
! . ) was 30 ff1g/kg (intraperitoneal) or more or 100 mg/kg (oral) or more.

Next, according to the present invention, a microorganism belonging to the genus Streptomyces and having the ability to produce the antibiotic WK-1875 is aerobically cultured in a medium, the antibiotic WK-1875 is accumulated in the culture, and the antibiotic WK-1875 is removed from the culture. A method for producing antibiotic WK-1875 is provided, comprising the step of harvesting.

As an example of the strain used to produce the antibiotic WK-1875 of the present invention, Streptomyces sp. WK-1875 strain, which was newly isolated by the present inventors from the soil of Tokyo Bay, is Can be mentioned.

The mycological properties of this strain are as follows.

[1 Morphological properties Vegetative hyphae develop well on various agar media, and no division is observed. Aerial hyphae grow moderately on yeast extract/malt extract agar, glycerol/asparagine agar, etc., and are white to gray in color. It has a color tone of When observed under a microscope, aerial hyphae exhibit a spiral shape, and chains of 20 or more spores are observed. The size of the spore is 1. OXo, 7 μm and cylindrical. The surface of the spore is spiny. @Nuclei, sporangia and zoospores are not found.

(Ill. Properties of various media) E. B. Shirli
ng) and Gottlieb (D, Gott, 1ie)
Method b) (International Journal of
Systematic Bacteriolower. Volume 16, 31
The culture characteristics of this producing bacterium, as investigated by the authors (p. 3, 1966), are shown on the robe. The color tone was determined as a standard color using the Color Harmony Manual, 4th edition (Container Corporation of America, Chicago, 1958), and the code was written in parentheses along with the color chart name. The following are the results of observations in each medium at 27°C for 2 weeks unless otherwise specified.

Culture properties (+111 Physiological properties (11) Production of melanin pigment (shun tyrosine agar negative (ml) peptone yeast iron agar negative (c) glucose peptone gelatin medium (21-23℃) negative (d) tryptone yeast Solution Negative (2) Tyrosinase reaction Positive (3) Production of hydrogen sulfide Negative (4) Reduction of nitrate
Negative (5) Liquefaction of gelatin (21-23℃
) (glucose/peptone/gelatin medium) Positive (6) Hydrolysis of starch Positive (7) Coagulation of skim milk (37°C) Negative (8) Peptonization of skim milk (37°C) Positive (9) Growth temperature range
17°C to 40'C (10) How to use carbon sources (Breedam-Gotlieb agar medium) Used = D-glucose, arabinose, raffinose, melibiose, D-mannitol, D-xylose, L-rhamnose, i-inositol, sufrose slightly utilized: D-fructose CIl+ Decomposition of cellulose Negative (I
VI Cell Wall Composition Cell wall diaminopimelic acid is of the LL, type.

The mycological characteristics of Japan are summarized as follows. Diaminopimelic acid in the cell wall is of the LL type. The aerial mycelium has a spiral shape and forms long spore chains. The surface of the spore is spiny. Regarding the properties of the culture, vegetative mycelium exhibits a yellow or brown tone, and aerial mycelium exhibits a white or gray tone.6 Soluble pigments produce brown pigments only on tyrosine agar medium.

From these results, this bacterial strain belongs to the genus Streptomyces, according to Bridham and Tresner's classification (Versis Manual of Determinative Bacteriology, 8th edition, pp. 748-829, 1974). It is thought to be a species belonging to the gray or red series. In addition, this strain is Stretomyces Sp. WK
-1875, deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on February 8, 1986
No. 859).

The medium for culturing the antibiotic WK-1875-producing microorganism according to the present invention is a synthetic medium containing appropriate amounts of carbon sources, nitrogen sources, inorganic substances, and, if necessary, other nutrients suitable for culturing Streptomyces microorganisms. Media or natural media can be used.

The carbon source and nitrogen source used in the culture medium may be any type as long as they are available to the strain used.

For example, various carbohydrates such as glucose, glycerol, fructose, maltose, mannitol, xylose, galactose, ribose, starch or its hydrolyzate can be used as the carbon source. Its concentration is usually preferably 0.2% to 5% (in terms of glucose) based on the medium.
In addition, various organic acids such as gluconic acid, pyruvic acid, lactic acid, and acetic acid, various amino acids such as glycine, glutamic acid, and alanine, alcohols such as methanol and ethanol, and various non-aromatic hydrocarbons such as normal paraffin,
Alternatively, various vegetable or animal fats and oils can also be used.

Nitrogen sources include ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium salts of various inorganic or organic acids such as Ii1!1M ammonium, urea, peptone, NZ-amine, meat extract, yeast extract, dried yeast, and corn starch. Cheap liquor, casein hydrolyzate, fitschmeal or its digested product, soy flour or its digested product, defatted soybean or its digested product,
Nitrogen-containing organic substances, such as hydrolyzates of soybeans, and various amino acids, such as glycine, glutamic acid, and alanine, can be used.

As inorganic substances, various phosphates, magnesium sulfate, common salt, etc., and heavy metal salts of Weisha are used.

When using a mutant strain that exhibits auxotrophy, it is naturally necessary to add substances that satisfy its nutritional needs to the culture medium, but this kind of nutrients should especially not be added when using a medium containing natural products. It may not be necessary.

Fermentation is carried out under aerobic conditions such as shaking culture or submerged culture with aeration and stirring. k@Nursing water temperature usually 20"C to 40C, pa
t is 4 to 9 (especially around 7). The culture period is usually 1~
WK-1875 is produced and accumulated inside and outside the bacterial cells in 7 days.

After completion of the culture, WK-1875 is collected from the culture, for example, by the following method.

The culture is separated into a filtrate and a precipitate by centrifugation. It is extracted from the filtrate by adsorbing it onto a porous polymer resin and eluting it. By appropriately concentrating the extract to dryness, WK
-1875 crude material is obtained. The crude substance is further purified by known methods commonly used for the purification of fat-soluble substances, such as various chromatography methods using adsorbents such as silica gel, gel filtration agents, etc., eRm method, salting-out method, etc. alone or in appropriate combinations. refine. A suitable example for purifying WK-1875 is a column chromatography method using silica gel and a linear concentration gradient elution method using a mixture of chloroform and methanol as an eluent.

A purified powder of WK-1875 can be obtained by condensing the active fraction obtained by these methods to dryness.

In this specification, WK~1875 was detected and quantified using silica gel thin layer chromatography (manufactured by Merck & Co., Ltd., silica gel, thin layer plate No. 5554, thickness 0.2 mm,
Developing solvent: chloroform/methanol = 5:1. W
K-1875 Rf value around 060) and Phytophthora parasitica var. Nicotianae (Ph to
hthorap var, n1cotianael
A biological assay using KF-265 and Mucor racemosus (Mucor racemosus) KF-223 was performed.

Examples are shown below.

5ure tom ce Ustreptomyces sp.
ssp, l WK-1875 strain (Feikoken Bacterial Serial No. 985
From a slant culture of No. 9), add 1 platinum loop to OOmρ seed medium (2% glycerol, 2% cinchona, Nacf;!.

0.3%, pH 7) was inoculated into a 500rr + 2 volume Sakalo flask, and cultured with shaking at 27°C for 2 days to obtain a seed culture solution.

400mj2 of the seed culture solution obtained in this way was transplanted into a 30Ω jar fermenter containing 20β of a medium with the same composition as the seed medium (pH 7), and cultured with aeration at 27°C for 3 days (aeration rate: 1OIl/min, Stirring: 25 rpm) was performed. Adekanol LG-109 (
(manufactured by Asahi Denka Co., Ltd.) was added as appropriate.

An equal volume of ethyl acetate was added to the culture solution 151 to extract the active substance, and the ethyl acetate layer was concentrated to dryness to obtain 45 g of an oily substance. The oily substance was dissolved in 200 mβ of chloroform and insoluble materials were removed. G4 silica gel in chloroform (
column (approx. 22.6 cm x 75 cm) packed with
m) was loaded with the above oil together with a small amount of silica gel and chloroform/methanol (5:l).

v/v) (342), chloroform/methanol (3:
The volume of the 6 fractions eluted with 1, V/V) (2 floods) was approximately 2
It was set to 0 mβ. The active fractions (No. 106-185) were collected and concentrated under reduced pressure to obtain light brown crude powder 3.
．． 5g was obtained. This coarse powder (3.0 g) was adsorbed onto a small amount of silica gel by a conventional method. This was applied to a column packed with silica gel suspended in benzene (230 mfi, 1.
1 cm x 60 cm), and benzene/acetone 50:l (v/v) tt
20: l (v/v)tt
l O: 1 (v/v) n 5 :
1 (v/v) // 2:l(v/v
) while changing the elution solvent sequentially in the order of approximately 150 to 2
00 mβ was eluted. The fraction volume T was approximately IQm°C. Among the active fractions (No. 29-78), the most active fractions (No. 46-51) were collected and concentrated under reduced pressure to obtain 440 mg of white purified powder of WK-1875.
I got it. This sample was analyzed using silica gel thin layer chromatography using chloroform/methanol (5:l, v/v).
When developed with benzene/acetone (10.1.v/v) gave river-spots. A single peak was also obtained by high-performance liquid chromatography using a reversed-phase column.

The physicochemical properties of this purified powder were as described in WI.

The antibiotic WK-1875 is mainly active against plant pathogenic fungi and is therefore expected to be useful as a pesticide, for example.

[Brief explanation of the drawing]

Figure 1 shows the infrared absorption spectrum of WK-1875 (KB
Figure 2 shows the proton nuclear magnetic resonance spectrum (CD
CI, middle), Figure 3 shows the C-13 nuclear magnetic resonance spectrum (
(in CDCIs). Patent applicant Kitasato Institute (incorporated association)

Claims (2)

[Claims] (1) Antibiotic WK1875 with the following physicochemical properties
. [1] Melting point: 116-119°C [2] Elemental analysis: Actual value (%) C64.7±1.0 H9.1±0.3 [3] Molecular weight: 740.97 [4] Molecular formula: C_4_0H_5_5O_1_2 [5 ]
Specific optical rotation: [α]^1^8_D-12.1° (c=0.
5, methanol) [6] Ultraviolet absorption spectrum (in methanol): terminal absorption [7] Infrared absorption spectrum (KBr method): as shown in Figure 1 [8] Proton nuclear magnetic resonance spectrum (in CDCl_3): as shown in Figure 2 [9] C-13 nuclear magnetic resonance spectrum (in CDCl_3): As shown in Figure 3 [10] Solubility in solvents Soluble: methanol, ethanol, ethyl acetate, chloroform Insoluble: water, n-hexane [11] Presentation Color reaction positive: H_2SO_4 Negative: Ehrlich reaction, Dragendorff reaction [12] Acidic, neutral, basic: Weakly acidic substances [13]
Material color: white (2) Belongs to the genus Streptomyces and is an antibiotic WK-18
Antibiotic W, characterized in that a strain having the ability to produce 75 is cultured in a medium, antibiotic WK-1875 is produced and accumulated in the culture, and WK-1875 is collected from the culture.
Method for producing K-1875.