JPH02258788A - New antibiotic hk-803c and production thereof - Google Patents

Abstract

NEW MATERIAL:A compound expressed by formula I (R is represents formulae II or III), wherein R is formula II has following physico-chemical properties. Form; colorless crystalline powder. Melting point; 191-193 deg.C (decomposition). Elementary analysis (%); C, 56.82; H, 7.78; N, 2.22. Molecular weight; 613 (with FABMS spectrum method). Mass spectrum; anion FABMS method (M-H)<->, m/z 612, cation FABMS method (M+H)<+>. m/z 614. Molecular formula; C30H48O10 NP. Specific rotatory power; [alpha] =+25.3 deg. (C=0.74, MeOH). Solubility; soluble in methanol, ethanol and alkaline water, sparingly soluble in acetone and water, hardly soluble in ether, chloroform, etc.; Color reaction; positive to 10% H2SO4 and Lemieux reagent. Amphoteric substance, etc. USE:An antibacterial agent. PREPARATION:A bacterium belonging to the genus Streptomyces such as Streptomyces.sp.-80-H-803 is cultured.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a novel antibiotic and a method for producing the same.

[Background of the Invention] The present inventor isolated a large number of microorganisms from soil for the purpose of searching for new antibiotics, isolated and searched for the antibiotics produced by the microorganisms, and developed a culture solution and cultured bacteria of microorganisms belonging to the genus Streptomyces. We have obtained new knowledge that HK-803, a new antibiotic that has not been described in any literature, is produced and accumulated in the body (Japanese Patent Application Laid-open No. 1983-
(See Publication No. 31689). As a result of further research on the products of the above-mentioned microorganisms, the present inventors discovered a new antibiotic different from the above-mentioned HK-803 and completed the present invention.

[Problems to be Solved by the Present Invention] An object of the present invention is to provide a new antibiotic and a method for producing the same.

[Means for Solving the Problems] The novel antibiotic of the present invention is obtained by culturing antibiotic HK-803C-producing bacteria belonging to the genus Streptomyces, and is isolated and collected from the culture, and is obtained by combining the following structural formula and the physical and chemical formula described below. It includes antibiotics HK-803C and HK-803C, which have physical and biological properties.

HK-803CI HK 803Cz The novel antibiotic HK-803C 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 HK-
It has the ability to produce 803C and is a bacterial species belonging to the genus Streptomyces.

As an example, Streptomyces sp.
03 (Streptomyces sp, 80-H-
The microorganism called 803) has the above characteristics and advantageously produces the antibiotic HK-803C of the present invention, and can be effectively used in the method of the present invention.

In addition, not only the natural and artificial mutant strains of Streptomyces Nα8O-H-803, but also all microorganisms belonging to the genus Streptomyces that have the ability to produce the antibiotic HK-803C described below are used in the method of the present invention. be able to.

The above Streptomyces genus Nα80-H-803 (hereinafter referred to as
It is simply referred to as "rH-803 strain." ) is a soil actinomycete discovered by the present inventor in the soil collected in Jizobara, Fukushima Prefecture, and was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology in 1982.
It was deposited on July 23, 2007, and its microorganism accession number is FERMP-6644.

H-803 strain has the following mycological properties.

(I) Morphology Growth on inorganic salt/starch agar medium and yeast malt extract agar medium was observed using a microscope and an electron microscope. Strain H-803 exhibited a morphology belonging to the genus Streptomyces. Its morphological characteristics are as follows.

1) Basal mycelia: They grow well on agar and are branched.

2) Aerial mycelium It grows well on media other than Nigelcose/Asparagine agar medium and Glycerin/Asparagine agar medium, and the length of the sporophyte is short and bent, forming many dense helical threads, with 20 to 50 Forms chains of spores. According to observation using an electron microscope, the spore surface is smooth;
It is oval or oblong, and its size is 0°7 to 0.9
X is 1.0 to 1.2 μm.

(II) Properties on various media Various media were prepared according to the Japan Patent Office industry-specific examination standards, and the results observed 3 weeks after inoculation are described below. In the description of the surface color tone, the symbols in parentheses indicate the disc ribbed color.
Names Dictionary (Descriptive)
It follows the color name symbols of the 4th edition (Color Names Dtctionary).

(1) Growth on sucrose/nitric acid agar medium:
It shows normal growth, and the color of the underside is more yellowish (2f c) than pale wheat brown (2e a).

Aerial Mycelium Forms white velvety aerial mycelium on the surface of the filament, which becomes cream colored after 4 weeks (2ba).

Soluble pigment: Not produced.

(2) Glucose-asparagine agar medium raw
Growth: Growth is poor, and the underside is a bright lemon color (3e
a).

Air bacteria do not grow.

Soluble pigment: Not produced.

(3) Growth on glycerin/asparagine agar medium:
It shows normal growth, and the color of the underside is bright yellow (1% βa).
.

Aerial fungi: Thin white aerial mycelium grows on the surface of the filament.

Soluble pigment: Forms a pale yellow soluble pigment.

(4) Growth on inorganic salt/starch agar medium: Shows good growth, and the color of the underside is bright wheat brown (2ea) to pale yellow (2iC).

Aerial mycelium: Abundant fluff-like aerial mycelia grow on the plant, and the color changes from white to gray (5fe).

Soluble pigment: Not produced.

(5) Growth on tyrosine agar medium: Shows good growth, and the underside is bright orange <4
IC).

Aerial mycelium: It grows velvety, white aerial mycelia.

Soluble pigment: Not produced.

(6) Growth on nutrient agar medium: Shows V-shaped growth, and the color of the underside is bright wheat brown (2ea).

Aerial mycelium: White, velvety aerial mycelium grows all over.

Soluble pigment: Not produced.

(7) Yeast extract, malt extract agar medium growth: Extremely good growth, with yellow-orange color (3ia) on the underside.

Aerial mycelia: Abundant fluff-like aerial mycelium grows on the entire surface, and after 30 days of culture, it becomes grayish (5th) and later becomes grayish brown (4ig).

Soluble pigment: Gives a yellowish tinge.

(8) Growth on oatmeal agar medium: Extremely good growth is shown, and the color of the underside is light wheat brown (2ea) to light yellowish brown.

Aerial mycelium: Abundant fluff-like aerial mycelia grow on the plant and are white to gray (2fe) in color. The center of the village takes on a blackish color when cultivated for a long period of time.

Soluble pigment: Not produced.

(9) Peptone/yeast extract/iron agar medium Growth: Shows poor growth, and the color of the underside is a bright wheat color (2ea).

Aerial fungal thread: White, thin aerial fungal thread grows on the surface.

Soluble pigment: Not produced.

(I[r) Physiological properties (1) Growth temperature: Grows at 23°C to 37°C.

(2) Starch decomposition crab decomposes.

(3) Skimmed milk powder: coagulation and negative properties.

Peptonization positivity.

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

(5) Gelatin liquefaction crab negative property.

(6) Nitrate reduction: Positive.

(IV) Utilization of various carbon sources The results of culturing strain H-803 by adding various sugars to a sugar medium prepared by Pridaham (manufactured by Difco) are as follows.

L-Arabinose D-Xylose D-Glucose D-Fructose Sucrose - Inositol - Rhamnose Raffinose D - No mannitol Added → Grows well + Grows ± Hardly grows Mycological properties of strain H-803 As mentioned above, the characteristics of this strain are summarized as follows.

l) Aerial mycelia are often attached, and the sporophyte is bent and well branched. Forms a dense helical thread, forming a chain of spores,
When cultured for a long time, it turns gray. The spore surface is smooth.

It has a morphology belonging to Streptomyces.

2) No distinctive color tone is seen on the back side of the bacterial colony.

3) No soluble dye is formed.

4) Chromochenic negative.

5) Use various sugars except arabinose, xylose, and mannitol.

Actinobacteria r, s, p, described by Mr. Nonomura in Journal of Fermentation Technology, Vol. 52, No. 2.
Description of taxonomy of 458 bacterial strains (key classification and identification)
of 458 Species of the Streptomyces
Included In, 1. S, P, ) (
key for classi-fication a
nd tdent4fication of 4585
pecies of the Streptomyces
1 included in 1. H- by S, P,)
A search for the taxonomic status of strain 803 reveals that there are some differences in sugar utilization, but it is similar to Streptomyces sioyaensis.
It is concluded that it is the most closely related strain to .ensis).

However, since a clear difference was observed in the ability to produce antibiotics, it was concluded that the H-803 strain is a new strain belonging to Streptomyces sioyaensis.

Next, in carrying out the method of the present invention, it is sufficient to culture the antibiotic HK-803C-producing bacteria belonging to the genus Streptomyces using a conventional method for producing antibiotics. Solid culture may be used, and for industrially advantageous cultivation, a spore suspension or culture solution of the above-mentioned producing bacteria may be inoculated into a medium and cultured with aeration and stirring.

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, sucrose, galactose, inositol, mannitol, etc. Nitrogen sources include peptone, soybean flour, meat extract, rice bran, koji, urea, cornstap liquor, and ammonium. Salts, nitrates, and other organic or inorganic nitrogen-containing substances are used. Other basic salts such as table salt, phosphates, calcium, zinc,
Metal salts such as manganese and iron may be added as appropriate.
If necessary, antifoaming agent, mineral oil, etc. 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 HK-803C substance. For example, the p++ 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 about 2 to 7 days.

However, these culture compositions, medium liquid properties, culture aK degree,
It goes without saying that culture conditions such as stirring conditions should be adjusted and selected as appropriate to obtain preferable results depending on the type of bacterial strain used and external conditions. To obtain the antibiotic HK-803C from the culture thus obtained, any means commonly used to collect metabolites can be used as appropriate. For example, HK-803
Each of these methods utilizes the solubility difference between the C 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 HK-803CTjjyJ substance is present in the culture filtrate and the cultured bacterial cells, but it is appropriate to extract it from the culture filtrate with butanol under acidic conditions. The HK-803C1+3 substance present in the cultured bacterial cells can be extracted from the bacterial cells using aqueous acetone, aqueous methanol, or the like. The HK-803C substance extracted in this manner can be purified by a combination of adsorption chromatography, ion exchange chromatography, gel filtration chromatography, etc. by utilizing its acidic and fat-soluble properties.

As a carrier for adsorption chromatography, adsorbents such as Diaion HP-10 and activated carbon are used. As a carrier for ion exchange chromatography, anion exchangers such as DEAE-cellulose and DEAE-Sepharose can be used.

Furthermore, since methanol is used as a solvent for gel filtration, Sephadex LH-20 can 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. HK-80 purified in this way
When ether, petroleum ether, etc. is added to the 3C substance and its methanol solution, it precipitates as a highly pure white powder, which can be collected by filtration. This white powder is recrystallized with water-containing acetone or the like to obtain colorless crystals. When this colorless crystal was subjected to high performance liquid chromatography, HK803C+ and HK-
It is fractionated into 803C2. HK-80 thus obtained
3C, and HK-803C2 have the following physical and chemical properties and biological properties.

(M+H)”, m/z 614(6) Molecular formula 203°I(4so+.NP(7) Specific optical rotation: [
α〕. +25.3" (C0,74, MeOH) Physicochemical and biological properties of antibiotic HK-803C (1) Shape: Colorless crystalline powder (2) Melting point: 191-193℃ (decomposed) ( 3)
Elemental analysis: Actual value Carbon 56.82%, Hydrogen 7.
78%, Nitrogen 2.22% Calculated value (C5°) I4sO+. NP -H2 (as 1) Carbon 57.05%, Hydrogen 7.92%, Nitrogen 2.
22% (4) Molecular weight: 613 (by FABMS spectroscopy) (5) Quality analysis: Anion FABMS method: (MH)-
1 m/z 612 Cation F A B M S method: E, c, 7 6 4 (9) Infrared absorption spectrum: (KBr) 3400
．． 3275.2975.2950.2880.1725
．． 1630.1460, 385.1255.1185
．． 1055.975.735 555cm-'0φ Solubility: Soluble in methanol, ethanol, alkaline water Slightly soluble in acetone, water Slightly soluble in ether, chloroform, benzene, ethyl acetate 0υ [value Nijiri gel thin layer chromatography n-proper tool ;0,INNlI40H=7:3
(V/V) Rf value 0.51 0 Color reaction: 10% Hz S Oa, 0 positive for Lemieux reagent Distinction between basic, acidic, and neutral: Amphoteric substance α Antibacterial spectrum; Test bacteria inhibition zone Diameter (x, 2 μg/disc) Pyricularia oryzae Botrytis cinerea Alternaria
Alternaria mali antibiotic HK-
Physical and biological properties of 803C (1) Shape: Colorless powder (2) Melting point: 189-192°C (decomposition) (3) Elemental analysis: Actual values Carbon 56.79%, Hydrogen 7.85 %
, Nitrogen 2.35%, Calculated value (as C: iJlngO+oNP ・IIzO) Carbon 57.05%, Hydrogen 7.92%, Nitrogen
2.22%, (4) Molecular weight: 613 (by FABMS spectroscopy) [5) Mass spectrometry: Anion FABMS method: (M-HP,
m/z 612 Cation FABMS method: (M+H)'', m/z 614 (6) Molecular formula: C30H480to NP (force
Specific optical rotation: [α]. +22.1° (C0,5, MaO
H) Elc, 748 (9) Infrared absorption spectrum: (KBr) 3400
．． 3275.2975.2950.2880.1725
．． 1630.1460.1385.1255.1185
．． 1055.975.735 555cr'α0 Solubility: Soluble in methanol, ethanol, alkaline water Slightly soluble in acetone, water Slightly soluble in chloroform, benzene, ethyl acetate Ql) Rf value Nijiri gel thin layer chromatography n-proper tool: 0. I N NH40H=7
: 3 (V/V) Rf value 0.51 Color reaction: 10% H2SO, positive for Lemieux reagent Basic, acidic, neutral distinction: Amphoteric substance 0◇ Antibacterial spectrum: Test bacteria inhibition zone Diameter (2 μg/disc) Comparing the physicochemical and biological properties of Pyricularia oryzae and larger antibiotics HK-803C and HK-803C2 with known antibiotics, it was found that Comamycin A and B (Japanese Patent Publication No. 45-8636) are similar substances in that they contain phosphorus. However, Comamycin A is clearly different in that it is described as a neutral substance and this substance is an acidic substance, and Comamycin B is described as a weakly acidic substance, but it is clearly different from this substance in elemental analysis. There is a difference. Furthermore, comamycin A is described as a polypeptide containing arginine, threonine, alanine, and valine, and B is a polypeptide containing arginine, glutamic acid, lysine, and valine, but HK-803C and HK-
The 803C2 analog does not contain any arginine or glutamic acid. Furthermore, there is clearly a significant difference between comamycin A and B in the infrared absorption spectrum.

Furthermore, it is clearly distinguished from HK-803 in terms of melting point, specific rotation, molecular weight, etc. Therefore, it was concluded that HK-s03c+ and HK-803C2 are new compounds.

The method of the present invention will be explained in detail below using Examples, but the method of the present invention is not limited thereto.

Example 3 Glucose 2% in ON volume jar fermenter,
Soluble starch 1%, meat extract O, 1%, yeast 0.4%, soy flour 2.5%, salt 0.2%, dibasic potassium phosphate 0.005
% of culture medium 18e, the same medium was previously inoculated with the aforementioned H-803 strain (Feikoken Bacteria No. 6644) and cultured with shaking at 27°C for 48-72 hours.
mf was inoculated and cultured with aeration at 27°C for 48 to 72 hours.

Final pH is 7.5-8.0. journo mentor
A filter aid, Celite, is added to the culture solution for 6 groups, and the mixture is centrifugally filtered to separate the bacterial cells and the filtrate. The bacterial cells were extracted using 60% acetone 2ON, and this was concentrated under reduced pressure to distill off the acetone.
An aqueous solution of 101 is obtained. Combine the culture filtrate and bacterial cell extract and prepare Diamond HP-1010j! The column is washed with 81% water and 40% acetone 81, and then eluted with 60% acetone 81. The active fractions are collected, acetone is distilled off under reduced pressure, and the aqueous solution is extracted twice with 41 butanol. After concentrating the extract to a small amount under reduced pressure, 500 mA of water is added to separate it into a viscous precipitate and a supernatant. The precipitate is dissolved in a small amount of methanol and applied to an activated carbon column (diameter: 400 mm, length: 300 mm). water, 30
% methanol, 70% methanol, 40% acetone, 7
When elution is performed in the order of 0 acetone, the activity range is 40 to 70.
% acetone elution area. The active fraction was collected, concentrated under reduced pressure and refrigerated to yield about 30 g of crude powder. 4 g of this crude powder is suspended in aqueous acetone, placed on a column of MIC-GEL (28 x 800 fins) packed with 50% acetone, and developed with a 50% acetone solution. Divide into four active fractions and analyze each by HPLC. Reversed phase column, Senshu Bank (ODS-H, 10
When analyzed using acetonitrile: 1% triethylamine-phosphate (pH 7) buffer (82:18) as a solvent system, the HK-803C substance was detected at RT,
Detected at 17.4 minutes. Both fractions containing HK-803C material are collected and concentrated to obtain 2010 mg0 crude powder. Suspend this 100μ in aqueous acetone and transfer LH20 (1011X 40
01n) and developed with 50% acetone water. The eluate was fractionated and HK-8 was determined by HPLC.
Fill the 03C substance! I approve. I- obtained from these two parts
(K-803C material is also an approximately 1:1 mixture of HK 803C+ material and HK803 C material.

When this active fraction is collected and left at room temperature, HK-803C
I Precipitates in the form of animal crystals. This was collected by filtration and dried to obtain 15 quartz of HK-803C+ substance. Further, when the mother liquor portion is concentrated and freeze-dried, 18 μg of powder is obtained. This was combined with a reverse phase column (Senshu Bank, 0DI-Ca,
10φ×250mm, acetonitrile; methanol: water: 1% diethylamine-carbonic acid (pH 7,2) = 45:1
0:35:10). The active peak was collected, concentrated, and lyophilized to yield HK-803C.

13 ■ are obtained.

Procedural amendment (formality) 1.Display of the incident 1999 Patent Application No. 80999 3.? I! person who corrects Relationship with the incident outgoing person name (679) RIKEN 4. Agent

Claims (3)

[Claims] (1) Novel antibiotic HK-803C represented by the following formula
. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (2) Streptomyces
A method for producing antibiotic HK-803C, which comprises culturing antibiotic HK-803C-producing bacteria belonging to the genus, and separating and collecting antibiotic HK-803C from the culture. (3) The antibiotic HK-803C-producing bacterium is Streptomyces sp.
Claim (2) which is ces sp, 80-H-803)
The manufacturing method described in.