JPS597438B2 - New antibiotic K-710B and its manufacturing method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibiotic -710B and a method for producing the same.

J More specifically, the K-710B substance of the present invention can be obtained by culturing antibiotic K-710B-producing bacteria belonging to the genus Streptomyces, and separating and collecting the antibiotic K-710B from the culture. This is a new antibiotic that has not been described in any literature.

The present inventor isolated a large number of microorganisms from soil for the purpose of searching for new antibiotics, and as a result of separating and searching for the antibiotics produced by the microorganisms, the inventor first discovered a culture solution and cultured bacterial cells of a microorganism belonging to the genus Streptomyces. A new antibiotic K that has not been published in the literature.
-710 was isolated and collected (Patent application No. 56-211, September
As a result of further research, new findings were obtained that K-710B, a novel antibiotic that differs in physicochemical properties from the K-710 substance, is produced and accumulated in the culture. We succeeded in separating and collecting it, and completed the present invention.

The novel antibiotic K-710B of the present invention and its manufacturing method will be described in detail below.

First, the microorganism used in the present invention is an antibiotic K-7.
It has a production ability of 10B and is a species belonging to the genus Streptomyces.

One example is the genus Streptomyces. /1680-K
-710(StreplOmycessp-/F68O
The microorganism designated as K-710) has the above characteristics, advantageously produces the antibiotic K-710B of the present invention, and can be effectively used in the method of the present invention.

In addition to the natural and artificial mutant strains of the Streptomyces genus A68O-K-710, all microorganisms belonging to the Streptomyces genus that have the ability to produce the antibiotic K-710B described below can be used in the method of the present invention. can do.

The above Streptomyces genus. /F6.8O-K-710(
Hereinafter, it will simply be referred to as "K-710 stock."

) is a soil actinomycete discovered by the present inventor in soil collected in Aizuwakamatsu City, Fukushima Prefecture, and was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on December 19, 1981.
The microorganism accession number is Microken Bacteria No. 6277 (FER
MP-6277). K-710 strain has the following mycological properties. (1) Morphology The morphology of strain K-710 was observed on oatmeal agar medium and glucose asparagine agar medium on the 14th day.

As a result of microscopic observation, the basal hyphae are well developed and branched. Aerial hyphae are short, branched and curved, 3-
A spiral thread with 7 turns is formed. The chain of spores is 20-40. No whorled branches are formed. According to observation using an electron microscope, the spore surface is linear, oval or oblong, and the size of the spore is (0.8 to 1.1 μm) x (1.1 to 1.5 μm).
μm). ) Properties on various media We prepared various media in accordance with the Japan Patent Office's industry-specific examination standards. The results observed 3 weeks after seeding are described below.

In addition, when describing color tones, the symbols in parentheses refer to Disc Rib Tape Color Names Dictionary (Descr).
iptiveCOIOrNamesDictiOnar
y) It follows the color name symbols of the 4th edition. (1) Growth on shakerose/nitric acid agar medium: Although slow, good growth can be seen spreading on the agar surface after culturing for 3 to 4 weeks.

The color of the bacterial cells on the underside is pale yellow.

Aerial mycelium: Not formed.

Soluble pigment: Not produced.

(2) Growth on glucose-asparagine agar medium: Growth is slow and normal growth is observed, and the color of the bacterial cells on the back side is white to cream color (2ba).

Aerial hyphae: Slightly pale white aerial hyphae are observed.

Soluble pigment: Not produced.

(3) Growth on glycerin/asparagine agar medium: Growth similar to that on glucose/asparagine agar medium is observed.

Aerial mycelium: Not formed.

Soluble pigment: Not produced (4) Growth on inorganic salt/starch agar medium: Growth is slow, spreading and flat medium growth? The color of the bacterial cells on the underside is a bright wheatish color.

Aerial hyphae: Slightly pale white aerial hyphae are observed and turn gray when cultured for a long period of time (5fe).

Soluble pigment: Not produced.

(5) Growth on tyrosine agar medium: Poor growth was observed, and the color of the bacterial cells on the back side was a bright wheat color (2ea).

Aerial hyphae: Slightly white aerial hyphae are observed.

Soluble pigment: Not produced.

(6) Growth on nutrient agar medium: Poor growth was observed, and the bacterial cells on the back side were colorless.

Aerial mycelium: Not formed.

Soluble pigment: Not produced.

(7) Yeast extract, malt extract agar medium Growth: Good growth was observed, and the color of the bacterial cells on the back side was golden yellow (2ee).

Aerial hyphae: Often forms thin white aerial hyphae.

Soluble pigment: Not produced. (8) Growth on oatmeal agar medium: Good, the color of the bacterial cells on the back side is wheatish (2ea) to golden yellow (21e), and when cultured for a long period of time, the center becomes blackish brown (24 ml).

Aerial mycelium: Short aerial mycelium grows all over the surface soil, and when cultured for a long time, it becomes white or blackish brown (3 m0 to black (10 m0) depending on the culture condition.

Soluble pigment: Not produced.

(9) Peptone/yeast extract/iron agar medium Growth: No growth or poor growth.

Aerial mycelium: Not formed.

Soluble pigment: Not produced.

(Auto) Physiological properties (1) Growth temperature: Grows at 37°C rather than 23°C, but at 27°C
Therefore, 33°C is the optimum temperature for growth.

(2) Starch decomposition ability: positive (3) Skim milk powder: Peptonize.

(4) Production of melanin-like pigment: Not produced.

(5) Gelatin liquefying power: weak. (6) Nitrate reduction: Reduce.

Utilization of various carbon sources The results of culturing the K-710 strain by adding various sugars to a sugar utilization medium using Pridaham (manufactured by Difco) are as follows.

The characteristic mycological properties of strain K-710 are summarized as follows.

(1) It grows well in oatmeal medium and forms gray aerial mycelia.

(2) Aerial hyphae form short spiral filaments, and the spore surface is lance-shaped.

(3) It does not produce melanoid pigments or characteristic soluble pigments on various media.

(4) Make good use of various sugars.

Strepetomyces species with the above properties were identified in the Journal of Fermentation by Mr. Nonomura.
Actinomycetes described in Technology Vol. 52, No. 2 1. S. P. Stock 4
Description of the taxonomy of 58 bacterial species (Key classification and identification of 458 species of Streptomyces included in S.P.) (KeyfOrCl
aSSifiCati(]1andidentific
ati0n0f458species0fstrept
0mycesinciudedinI. S''. ), Actinomyces malachitichus (A.
malachiticus), Actinomyces viridO-Dlastic
us), Streptomyces sparsogenes (S. sp.
It is considered to be a bacterial species closely related to the arginus βb).

Next, in carrying out the method of the present invention, antibiotic K-710B-producing bacteria belonging to the genus Streptomyces can be cultured by a conventional method for producing antibiotics.

The form of culture may be liquid culture or solid culture, and for industrially advantageous culturing, a spore suspension or culture solution of the above-mentioned producing bacteria may be inoculated into a medium and culture with aeration and agitation may be performed.
The nutrient source of the medium is not particularly limited, and the medium may contain carbon sources, nitrogen sources, and the like that are commonly used for culturing microorganisms.

Carbon sources include starch, dextrin, glycerin, glucose, shakerose, galactose, inositol, mannitol, etc. Nitrogen sources include peptone, soybean flour, meat scratches, rice bran, wrinkles, urea, cornstarch, etc. , ammonium salts, nitrates, and other organic or inorganic nitrogen-containing substances.

In addition, inorganic salts such as common salt, phosphates, calcium, zinc, manganese, iron and other metal salts may be added as appropriate, and if necessary, anti-foaming agents such as dynamic, vegetable or mineral oils may be added. May be added. Culture conditions such as culture temperature and culture time are selected to be suitable for the growth of the bacteria used and to maximize production of the K-710B substance.

For example, the pH of the medium is preferably around neutrality, and the appropriate culture temperature is preferably about 25°C to 35°C. In addition, the culture time is usually 2
~7 days is best. However, these culture conditions such as culture composition, medium liquid properties, culture temperature, and stirring conditions should be adjusted and selected as appropriate to obtain favorable results depending on the type of bacterial strain used and external conditions. Needless to say.

From the culture thus obtained, antibiotic K-71
In order to obtain OB, it can be collected using any means commonly used for collecting metabolites. For example, K-71
Each of these methods utilizes the solubility difference between the 0B substance and the impurity, the adsorption affinity difference, the ionic bonding force difference, and the distribution difference between the organic solvent and the organic solvent. Used alone, in combination, or repeatedly. Specifically, the K-710B substance exists in the culture solution and the cultured bacterial cells, but it can be extracted from the culture solution under acidic conditions with an organic solvent such as ethyl acetate, butyl acetate, butanol, or chloroform.

The K-710B substance present in the cultured bacterial cells can be extracted from the bacterial cells using aqueous acetone, aqueous methanol, or the like. The K-710B substance extracted in this way can be purified by a combination of adsorption chromatography, ion exchange chromatography, gel filtration chromatography, etc. by taking advantage of its acidic and fat-soluble properties. I can do it. As the carrier for adsorption chromatography, adsorbents such as silica gel and Diaion HP-20 are used. The primary carrier for ion exchange chromatography is D
Anion exchangers such as EAE-cellulose and DEAE-cephalose can be used. In addition, for gelation, a related soil using methanol as a solvent, Cephadex LH-
20 may be advantageously used. High performance liquid chromatography is used for more advanced purification. In this case, a reverse phase distribution type column can be advantageously used. The K-710B substance purified in this manner precipitates as a highly pure white powder when ether, petroleum ether, etc. is added to the methanol solution, and this can be collected by filtration. The antibiotic K-710B thus obtained
The physicochemical and biological properties of
(According to the physicochemical and biological properties of antibiotic K-710B) Contained amino acids: Acid decomposition yields aspartic acid, serine, and several unidentified amino acids.

Color reactions: Lydon-Smith reaction is positive, ninhydrin and Dragendorff reactions are negative.

Distinction between basic, acidic, and neutral: Considered to be an amphoteric substance.

Antibacterial spectrum (by agar dilution method): As shown below, it exhibits growth inhibitory activity against plant pathogenic molds accompanied by swelling of hyphae, but within the tested range, it exhibits almost no inhibitory activity against bacteria and yeasts. do not have.

Comparing the above physicochemical properties and biological properties of antibiotic K-710B with conventional antibiotics, antibiotic AC-
69 (see JP-A-55-33445) is mentioned as a similar substance, but it is clearly different from this substance in terms of amino acid composition, specific rotation, PKa' value, etc. A direct comparison using layer chromatography showed that they were clearly different substances.

Further, in comparison with the novel antibiotic K-710 according to the invention of the present inventor, it was proved that the substance is clearly different in elemental analysis values, specific optical rotation, molecular weight, etc. Therefore, it was concluded that K-710B is a new antibiotic that has not been described in any literature. In addition, the inhibitory activity of the antibiotic K-710B against various plant pathogenic fungi was confirmed in the InvivO test (pot test), so K-710B is highly expected to be put to practical use as an antifungal agent for agriculture and horticulture. It is something that will be done.

The method of the present invention will be explained in detail below with reference to examples, but the method of the present invention is not limited thereto.

Example glucose 2%, soluble starch 1%, meat processing scratches 0.1%,
The above-mentioned K-710 strain (Feikoken Bacterium 6th grade
48-7 after inoculating with No. 277 (FERMP-6277)
300 ml of culture solution cultured with shaking at 27°C for 2 hours is inoculated and cultured with aeration at 310°C for 72 hours with aeration.

Final pH is 7.5-8.0. The culture solution is separated into bacterial cells and liquid by centrifugal filtration, and the bacterial cells are mixed with 60% acetone 101.
Extract twice. The culture fluid and bacterial cell extract were combined and passed through a Diaion HP-1061 column. After washing the column with 10% acetone 101, elution is performed using 70% acetone 141. The active fractions are collected and concentrated under reduced pressure to obtain a concentrated aqueous solution of 1.31. Butanol 0.6
1 was extracted twice, and the butanol layer was concentrated under reduced pressure to obtain a residue 8.
Get 9. In order to further purify this, silica gel column chromatography is performed.

φ6X30c prepared with chloroform! n column, chloroform 42, 10% methanol-chloroform 21, 20% methanol-chloroform 41, 50%
Elution is performed sequentially with methanol-chloroform 21, and the eluate is fractionated into 15WL portions. The active fraction (fraction 113-170) was collected and concentrated under reduced pressure to obtain a crude powder 13.
Get 19. For further purification, 1,500 η of the crude powder was dissolved in a small amount of methanol, applied to a Sephadex LH-20 (1009) column prepared with methanol, and then eluted with methanol.

The active fractions were collected and concentrated under reduced pressure to obtain a crude powder of 315η. For final purification, fractionation is performed using high performance liquid chromatography.

Hitachi RP-18 (NucleOsll5cl8, φ41
Using a 1×150fn) column and 65% methanol as a solvent, fractionation was repeated, and a single K-710 substance 16η (retention time: 12 to 16 minutes) was obtained from the 160η crude powder.
K-710B substance separated separately (retention time: 16-1
8 min) 5η was obtained as a white powder.

[Brief explanation of drawings]

The attached drawing is a graph showing the infrared absorption spectrum of potassium bromide tablets of the antibiotic K-710B of the present invention.

Claims (1)

[Claims] 1. Shape: White powder. Melting point: Gradually decomposes at temperatures above 200°C. Elemental analysis: Carbon 53.96%, Hydrogen 7.12%, Nitrogen 1
2.24% specific optical rotation: [α]^2^0_D=-28.6
° (C=0.124, methanol) Ultraviolet absorption spectrum: Infrared absorption spectrum showing terminal absorption at 211 mμ in methanol: (in KBr) 3400, 2930, 2850, 1740, 1650,
1530, 1400, 1200, 1060, 700, 5
It has a characteristic absorption band at 50cm^-^1. Solubility: Well soluble in methanol, slightly soluble in water and lower alcohols. Poorly soluble in acetone and ether. R_f value: (by silica gel thin layer chromatography plate) Developing solvent R_f value propanol-1N ammonia water 0.52 (7:3) butanol-acetic acid-water 0.33 (4:1:1) chloroform-methanol 0.53 (1:1) Molecular weight: 1189 (according to secondary ion mass spectrometry) Amino acids contained: Acid decomposition yields aspartic acid, serine, and several unidentified amino acids. Color reactions: Lydon-Smith reaction is positive, ninhydrin and Dragendorff reactions are negative. Distinction between basic, acidic, and neutral: Considered to be an amphoteric substance. Antibacterial spectrum (by agar dilution method): Shows growth inhibiting activity against plant pathogenic molds accompanied by swelling of mycelia. K-710B_o2, a new antibiotic with the above-mentioned physicochemical and biological properties.
Antibiotic K-710B-producing bacteria belonging to the genus Ptomyces were cultured, and the new antibiotic K-710 was extracted from the culture.
A novel antibiotic K- characterized by separating and collecting B.
710B manufacturing method. 3 Antibiotic K-710B belonging to the genus Streptomyces
The producing bacterium is Streptomyces sp. 80-K-71
0 (Streptomyces sp.80-K-710
) The method according to claim 2.