JPH0310692A - Antibiotic substance om-5714 and production thereof - Google Patents

Abstract

NEW MATERIAL:A compound, expressed by the formula and having the following physico-chemical properties. Melting point; 58-62 deg.C. Elementary analysis (%); C, 66.2+ or -1.0; H, 7.6+ or -0.3; N, 9.7+ or -0.3. Molecular weight; 290.16. Molecular formula; C16H22N2O3. Specific rotation; [alpha] =+23.0 deg. (c=1.0, chloroform). Solubility; soluble in methanol, ethanol, ethyl acetate and chloroform and insoluble in water and n-hexane. Color reaction; positive to sulfuric acid, iodine, phosphorus-molybdenic acid reagent and negative to ninhydrin, the Elson-Morgan reagent. Neutral substance. Color; white to light yellow, etc. USE:Useful as an agricultural chemical, capable of exhibiting activity against plant pathogenic fungi and having also herbicidal activity. PREPARATION:A strain, such sa Streptomyces.sp. OM-5714 (FERM P-10775), belonging to the genus Streptomyces is cultures at 20-40 deg.C and about pH7 for 1-7 days.

Description

[Detailed Description of the Invention] LL 5 servings 1 The present invention relates to antibiotics, and particularly to the novel antibiotic OM-57.
14 and its manufacturing method.

As an antifungal antibiotic, for example, amphotericin B1
Medicines such as griseofulvin and virolnidoline, and agricultural chemicals such as plasticidin S, polyoxin, validamycin, tetranactin, and bialafoss are known. However, the known antifungal antibiotics still have room for improvement in terms of efficacy, toxicity, antibacterial properties, etc.
This invention is based on the knowledge that the actinomycetes that the inventors isolated from soil have the ability to produce antifungal antibiotics that have particularly strong activity against plant pathogens.

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel antibiotic OM-5714 and a method for producing the same.

According to the present invention, the antibiotic OM-
5714 is provided.

The physicochemical properties of this antibiotic are as follows.

■ Melting point: 58-62℃ ■ Elemental analysis: Actual value (%) C66,2±1.0 8 7.6±0.3 N　9.7±0.3 ■ Molecule l: 290.16 ■ Molecular formula: C, sHzNt Os8 ■ Specific rotation: [α] +23. O.

(c=1.o, chloroform) ■ Ultraviolet absorption spectrum (in methanol): As shown in Figure 1 ■ Infrared absorption spectrum (KBr method): As shown in Figure 2 ■ Proton nuclear magnetic resonance spectrum (CDCI).

(middle): As shown in Figure 3 ■ C-13 nuclear magnetic resonance spectrum (in CDC1S):
As shown in Figure 4 ■ Solubility in solvents Soluble: methanol, ethanol, ethyl acetate, chloroform Insoluble: water, n-hexane ■ Positive color reaction: H, SO, iodine, phosphorus-molybdate reagent Negative: ninhydrin, Elson-Morgan reagent ■ Acidic, neutral, basic: Neutral substance @ Color of substance:
White to pale yellow Next, the biological properties of this antibiotic are shown.

■) Minimum inhibitory concentration CM of this antibiotic by antibacterial activity agar dilution method
The IC5 unit μg/ml is as shown in Table 1.

Table 1 KB176 (NIHJ, JC-2, IFO1273
4) Pseudomonas aeruginosa 〉100
(Ploo (Pseudo 7) P-3KB105 Reference Xanthomonas oryzae 〉100 (X1
00 (Xantho crab 4 small) KB88 II Micrococcus luteus 〉100 (Mic
rocoloo(1uteus) KB40 (PCI
! 001) * Staphylococcus aureus
〉100(Sta h 1ococcus aureu
s) KB34 (FDA 209P) Nemicobacterium smegmatis 〉1100f
cobacterium KB42
(ATCC607) * Bacillus subtilis >100 (Ba
cillus 5ubtilisl KB27 (PC
I 219) *Candida albicans
〉100(Candida albicans) K
FI ** Satsukaromyces salmon 〉1
100 (Saccharoces 5ake) KF
No. 26 Aspergillus niger 〉10
0 (Drunken and above Is 11 crazy) KF103iAT
cc 6275) *Villicularia oryzae 〉100(Pi
ricularia rainbow■ average) and F180 Mucor racemosus 〉100 (Mloo(r
acea+osus) KF223+IF0 4581
1 Kasayuki Phytophthora captrum 12.5 (P
h to hthora cactoruml
KF279 *Phytophthora balashtica
50 [Ph to hthora animals] IKF265 1F04783 * Ginseng sensitive agar medium (made in Japan) 37°C, pH 7, 0, determined at 24 hours.

Issan Potato Glucose Agar Medium 27℃, H6.01
Set on the 3rd.

Note: As mentioned above, this antibiotic can be used for example in Phytophthora (P
It exhibits growth inhibitory activity against fungi of the genus H to hthora.

2) Herbicidal activity The antibiotic OM-5714 inhibited the germination of radish seeds by 20% or more at a concentration of loOppm, and the elongation of young shoots was inhibited by 90%.
% or more. The herbicidal activity test was conducted using the following method. Put absorbent cotton into two small test tubes and add lO
Absorbent cotton was soaked in 1 rsQ of water containing 0 μg of OM-5714, and five seeds of Okiwari radish were sown thereon. The mouth of the test tube was covered with a metal lid and placed under fluorescent light irradiation at 27° C. for 4 days, and the number and height of seeds germinated were measured.

The effect was determined by comparing with a control without any drug addition.

3) Acute toxicity of this antibiotic when administered to toxic mice (L D
ao) was greater than or equal to 30 mg/kg (intraperitoneal) or greater than or equal to 100 B/kg (oral).

Next, according to the present invention, a microorganism belonging to the genus Streptomyces and having the ability to produce the antibiotic OM-5714 is aerobically cultured in a medium, the antibiotic OM-5714 is accumulated in the culture, and the antibiotic OM-5714 is removed from the culture. A method for producing antibiotic OM-5714 is provided, comprising the step of harvesting.

As an example of the strain used to produce the antibiotic OM-5714 of the present invention, Streptomyces sp. OM-5714, which was newly isolated by the present inventors from the soil of Fukuyama City, Hiroshima Prefecture, Stocks are one example.

The mycological properties of this strain are as follows.

+11 Morphological properties Vegetative hyphae develop well on various agar media, and no division is observed.Aerial hyphae are abundantly attached to substrates other than glucose/nitrate agar and are white or gray in color. When observed under a microscope, the aerial mycelia have a spiral shape, and chains of 20 or more spores are observed. Spore size is 1.07X0.75μI
It is. The surface of the spore is smooth or wrinkled. No sclerotia, sporangia and zoospores are found.

(II) Properties on various media E. B. Shirli
ngl and Gottlieb (D. Gottlieb)
Method of I (International Journal of Systematic Bacteriology. Vol. 16, 313)
The color tone 0 shown on the robe is the standard color, and the culture properties of this production bacterium were investigated by the Color Harmony Manual, 4th edition (Container Corporation, 1966).
of America, Chicago, 1958), and the code is 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.

(III) Physiological properties (1) Production of melanin pigment (a) Tyrosine agar negative (11) Peptone/yeast iron agar negative (c) Glucose/
Peptone-gelatin medium (21-23℃) Negative (d) Tryptone yeast solution Negative (2) Tyrosinase reaction Negative (3) Production of hydrogen sulfide Negative (4) Reduction of nitrate
Positive (5) Liquefaction of gelatin (21-23℃
) (glucose/peptone/gelatin medium) Negative (6) Starch hydrolysis Positive (7)
Coagulation of skim milk (37℃) Negative (8) Peptonization of skim milk (37℃) @Sensitive (9) Growth temperature range
Optimum temperature for growth 15℃~38℃ 29
℃ (!0) How to use carbon sources (Bleedam-Gotlieb agar medium) Use: Use everything except sucrose (11) Decomposition of cellulose Negative (IVI) Cell wall composition Diaminopimelic acid in the cell wall is LL type.

The mycological properties of the inoculation can be 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 smooth. Regarding the properties of the culture, vegetative hyphae exhibit a brown or orange tone, and aerial hyphae exhibit a white or gray tone. No soluble pigments are produced.

From these results, this bacterial strain belongs to the genus Streptomyces, and is classified according to Bridhem and Tresner's classification (Versis Manual of Determinative Bacteriosis, 8th edition, pp. 748-829, 1974). ), it is considered to be a species belonging to the Gray series.

This strain is Streptomyces sp.
e jam ces sp, ] OM-5714 and has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (Fiber Science and Technology Research Institute No. 10775).

The medium for culturing the antibiotic OM-5714-producing microorganism according to the present invention is a synthetic medium suitable for culturing Streptomyces microorganisms, containing carbon sources, nitrogen sources, inorganic substances, and other nutrients in appropriate amounts as necessary. 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 ammonium salts of various inorganic or organic acids such as ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium nitrate, urea, peptone, NZ-amine, meat extract, yeast extract,
Nitrogen-containing organic substances such as dry yeast, corn steep liquor, casein hydrolyzate, fitschmeal or its digested product, soybean flour or its digested product, defatted soybean or its digested product, soybean hydrolyzate, as well as glycine, glutamic acid, Various amino acids such as alanine can be used.

As inorganic substances, various phosphates, magnesium sulfate, common salt, etc., and trace amounts of heavy metal salts 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 aeration-stirring submerged culture. The culture temperature is usually 20°C to 40°C, and the pH is 4.
-9 (particularly around 7), and the culture period is usually 1 to 7 days, and the antibiotic OM-5714 is produced and accumulated inside and outside the bacterial cells.

After completion of the culture, the antibiotic OM-5714 is collected from the culture by, for example, 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. The crude substance of antibiotic OM-5714 is obtained by appropriately concentrating the extract to dryness. The crude substance is further
Purification is carried out 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, concentration methods, salting-out methods, etc. alone or in appropriate combinations. Antibiotic OM-5
As a suitable example for purifying 714, using a reverse bed type packing material,
Examples include high performance liquid chromatography using a concentration gradient elution method using a mixture of acetonitrile and water as an elution solution. A purified powder of antibiotic OM-5714 can be obtained by concentrating and drying the active fraction obtained by these methods.

In this specification, the detection and emplacement of the antibiotic OM-5714 was carried out 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 = 9:l, Rf of this antibiotic (nearly 0.3) and Phytophthora var. var.
, by a bioassay using n1cotianael K F-265.

Examples are shown below.

X Arashigo Streptomyces sp.
ssp,) OM-5714 strain (Feikoken Bacterial Serial No. 10775
Transfer one platinum loop from the slant culture of 100 mfi seed medium (
@2.4% Glucose O, 1%, Peptone 03%,
Meat extract 0.3%, yeast extract 0.5%, CaCO50
, 4%, pH 7.0) in a 500 IIIQ capacity Sakaro flask, and cultured with shaking at 27°C for 2 days to obtain a seed culture.

4 g of the seed culture solution obtained in this way was transplanted into a 4002 fermenter containing antibiotic production medium 2002,
Aeration and agitation culture (aeration rate: 1OQ/min, agitation: 200 rpm) was carried out at 27° C. for 3 days. Adekanol LG-109 (manufactured by Asahi Denka Co., Ltd.) was appropriately added to suppress zero foaming. A medium having the following composition was used as a medium for producing antibiotic OM-5714.

Soluble starch 2% Glycerol 0.5% Wheat germ 1.0% Meat extract 0.3% Dried yeast 0.3% CaCO50.3% pH 7.5 Add an equal volume of ethyl acetate to culture solution 1959 to extract active substances Then, the ethyl acetate layer was concentrated to dryness to obtain 140 g of oily substance.
I got it. The oily substance was dissolved in 12 chloroform and insoluble materials were removed. A column packed with silica gel (approximately 1 kg) suspended in chloroform (approximately 22.6 c layers x 75
cm), the above oil was loaded with a small amount of silica gel and chloroform/methanol (50:
1. v/v) (3°C), chloroform/methanol (20:l.

The volume of the 0 fraction eluted with v/v) (21!,) is approximately 201
It was a flood. Collect active fractions (No. 136-190),
aIli under reduced pressure! By doing this, light brown coarse powder 2
．． 1g was obtained. This coarse powder (2.0 g) was adsorbed onto a small amount of silica gel by a conventional method. Add this to benzene
Column packed with silica gel (200 m
benzene/acetone 50:l (v/v) //
5:l(v/v)n2
:1 (while changing the elution solvent sequentially in the order of v/vl, the volume of the 6 fractions eluted at about 150 to 200 mm each was about 20
It was 1β. Collect active fractions (No. 15-26),
Antibiotic OM-5714 by concentration under reduced pressure
410 mg of yellow powder was obtained.

100 mg of this sample was dissolved in a small amount of acetonitrile and further purified by high performance liquid chromatography. Odynis (ODS)-18 (YMC0DS-5 manufactured by Yamamura Scientific Co., Ltd.) (column size 20 x 250 mm) was used as a column carrier.
), and 30% acetonitrile water was used as the solvent. The flow rate was 8 ml/min. Active substances were detected by ultraviolet absorption at 275 nm, and substances eluted around a retention time of 22.5 minutes were collected. This fraction was concentrated to dryness and the antibiotic OM-
17.5B of pale yellow purified powder of 5714 was obtained.

This sample was analyzed using silica gel thin layer chromatography using chloroform/methanol (9:1.v/v).

When developed with benzene/acetone (3:1. v/v) a single spot was given. 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 above.

The antibiotic OM-5714 is mainly active against plant pathogenic fungi and also has herbicidal activity, so it is expected to be useful, for example, as a pesticide.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum (in methanol) of the antibiotic OM-5714, and Figure 2 shows the infrared absorption spectrum (in methanol).
KBr method), Figure 3 shows the proton nuclear magnetic resonance spectrum (
CDC1, in), 'JI4 figure shows C-13 nuclear magnetic resonance spectrum (in CDels).

Claims (2)

[Claims] (1) Antibiotic OM-5714 represented by formula (I). ▲There are mathematical formulas, chemical formulas, tables, etc.▼ [1] Melting point: 58-62℃ [2] Elemental analysis: Actual value (%) C 66.2±1.0 H 7.6±0.3 N 9.7 ±0.3 [3] Molecular weight: 290.16 [4] Molecular formula: C_1_6H_2_2N_2O_3 [5
]Specific rotation: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (c=1.0, chloroform) [6] Ultraviolet absorption spectrum (in methanol): As shown in Figure 1 [7] Infrared absorption spectrum (KBr method) : As shown in Figure 2 [8] Proton nuclear magnetic resonance spectrum (in CDCI_3): As shown in Figure 3 [9] C-13 nuclear magnetic resonance spectrum (in CDCI_3): As shown in Figure 4 [10] Dissolution in solvent Soluble: methanol, ethanol, ethyl acetate, chloroform Insoluble: water, n-hexane [11] Color reaction positive: H_2SO_4, iodine, phosphorus-molybdic acid reagent Negative: ninhydrin, Elson-Morgan reagent [12] Acidic, medium Nature and basicity: Neutral substance [13] Color of substance: White to pale yellow (2) Belongs to the genus Streptomyces, and the first claim
A strain having the ability to produce the antibiotic OM-5714 described in Section 1 is cultured in a medium, and the antibiotic OM-57 is added to the culture.
A method for producing antibiotic OM-5714, which comprises producing and accumulating OM-14 and collecting OM-5714 from the culture.