JPS63280073A - Novel antibiotic yp-02978l-c and production thereof - Google Patents

Abstract

NEW MATERIAL:Antibiotic YP-02978L-C shown by the formula or a tautomer thereof or a mixture thereof, namely 1-oxo-1,2-dihydro-5-[(2,4,4-trimethyl-1- cyclohexene-1-yl)methyl]phenazine. USE:An antibiotic. PREPARATION:A bacterium such as Streptomyces sp. YP-02978L strain (FERM P-8610) belonging to the genus Streptomyces, capable of producing YP-02978L-C shown by the formula or a tautomer thereof or a mixture thereof, is cultivated and an antibiotic YP-02978L-C shown by the formula or a tautomer thereof or a mixture thereof is collected from the culture mixture.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a novel antibiotic YP-02978L-C or its tautomer or a mixture thereof and a method for producing the same by fermentation.

(Solution) The present inventors have been searching for microorganisms that produce antibiotics and screening their products.The present inventors have discovered that microorganisms isolated from the soil of Hatoma Island, Okinawa Prefecture, produce antibiotics. This led to the completion of the present invention.

The present invention comprises the antibiotic YP-02978L-C represented by formula (I) or its tautomer or a mixture thereof,
The purpose is to provide that.

The tautomer of the above formula compound (I) is represented by the following formula (n) (I
(II) The present invention includes separated isomers and mixtures thereof.

The second invention of the present application is to culture a microorganism that belongs to the genus Streptomyces and has the ability to produce the antibiotic YP-02978L-C represented by the above formula (I), its tautomer, or a mixture thereof, and Shi U mock E substance YP -0
The object of the present invention is to provide a manufacturing method characterized by collecting 2978L-C(I), its tautomer, or a mixture thereof.

Below, the microorganisms used in the production method and the microorganisms used in the production method of the present invention belong to the genus Streptomyces and are represented by the above formula: YP-02978L-C or its tautomer, or their tautomers. It is a microorganism that has the ability to produce a mixture of

Specifically, such microorganisms include Streptomyces sp.
) YP-02978L strain.

The mycological properties of this strain are as follows.

(11 Morphological Properties This strain grows on various synthetic and organic media, especially sucrose/nitrate agar, tyrosine agar.Aerial mycelium grows on sucrose/nitrate agar, glycerin/asparagine agar, Only on the tyrosine agar medium, a slight epiphyte is observed.According to optical microscopic observation, the aerial hyphae are simply branched and have a spiral shape with an open tip (0pen5piral). Sexion svirares (S) or Retinachieria perti (RA). Observation with an electron microscope shows that
A chain of about 20 spores was observed, and the spore size was 0.6
~o, s x o, s ~ 1.0 μm', and its surface structure is thorn-like. Whorled branches, spores, and sclerotia formation are not observed.

(2) Growth conditions in various media The growth conditions in various media are as shown below. Unless otherwise specified, the cells were cultured at 28° C. for 21 days and observed according to conventional methods.

The description of color tone was based on the color standard (Japan Color Research Institute).

(Note) G: Growth and bacterial flora color on the colony surface A; Aerial mycelia epiphyte and its hue R: Hue on the back S: Soluble pigment (3) Physiological properties YP -02978L 1) Growth temperature range 15-33℃ Optimal growth temperature 24-27°C2) Liquefaction of gelatin/Simple gelatin (20°C) Positive/Glucose◆
Peptone Positive Gelatin (28℃) YP -02978L Peptonization of skimmed milk Negative 4) Nitrate reduction action Weak positive 5) Starch hydrolysis action Positive 6) Melanin-like pigment formation Tryptone Yeast Extract-Pros Negative Tyrosine Agar Negative Peptone Ys) - Agar Negative (Note) The growth temperature is at each temperature (5, 10, 1
5,20,25,28,30,33゜37.40,45
．． Observation results from 7 to 21 days at 50°C.

The effects on milk are the results observed after 3 to 21 days at 37°C.Other than that, unless otherwise specified, the results are shown after 2 weeks at 28°C.

(4) Assimilation of carbon sources (Pridm Godliep agar medium.

(28℃ culture) YP-02978L L-arabinose Shiri-xylose + D-glucose + D-fructose + sucrose - inositol - rhamnose + raffinose + D-mannitol + starch + (Note) +; Growing; Growth is doubtful −; No growth (5) Diaminopimelic acid (DAP) analysis LEC
The method of HVALIER et al. (LECHVALIER, MP
, et al; PP 277-238 in DI
ETZ, A et at ed,, Aetinom
yceteTaxonomy, SIM 5pecia
The results of analysis of the acid hydrolyzate of this strain according to LL-D
AP detected.

To summarize the above properties, the YP-02978L strain is.

Aerial hyphae are observed only on sucrose/nitrate agar, glycerin/asparagine agar, and tyrosine agar. Aerial hyphae are simply branched, and their shape is section spirales (S) or retinaculia pelti (RA).
A chain of about 20 spores is observed. As for the hue, the growth is pale to yellowish brown, the aerial mycelia are dense, and the soluble pigments,
No melanin-like pigment formation was observed. Furthermore, LL-diaminopimelic acid was detected by analysis of the acid hydrolyzed product of the bacterial cells.

Based on the above properties, one strain is Streptomyces (Streptomyces
Known bacterial strains that are considered to belong to the genus ``Eptomycea'' and are similar to this one strain
(Bergy's Manual of Determinology
inative Bacteriology) F 8th edition,
1974”, [International Journal of Systematic Bacteriology (Int.
National Journal of Systems
tematic Bacteriology) No. 18
Volume, No. 2, pp. 69-189, No. 4, pp. 279-392 (1968), Vol. 19, No. 4, pp. 391-512 (
1969).

Volume 22, No. 4, pp. 265-394 (1972)
Searched from J. As a result, 9 strains were found to be similar to this strain.

Streptomyces flavovires/s (S, flIL)
vo-virens), Streptomyces griseoaura/tiacus (S, griseoaur
antiacua) and Streptomyces albovinaceus (S, albovinaceus). All strains are similar to the nine strains in terms of morphological characteristics and physiological properties such as availability of carbon sources, but there are slight differences in the color tone of growth, as well as the formation of aerial mycelia ( This strain is clearly different in that it has extremely poor colonization and does not match any other strain. Based on the above results, we decided to use this strain as Streptomyces sp. YP.
It was named strain -02978L.

This strain has been deposited with the National Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiological Research Institute No. 861O.

The microorganisms used in the present invention are characterized by the above-mentioned mycological properties, and the antibiotic YP-02978L-C(I)
or its tautomer, or a mixture thereof [hereinafter referred to as compound (I), etc.].

The microorganisms used in the present invention are 2.
Easily mutated, both artificially and naturally. Therefore 9
The microorganisms used in the present invention are not limited to the above-mentioned Streptomyces sp. This includes all strains that have been artificially mutated using , X-rays, chemical agents, etc., as well as natural mutant strains of these.

Note that the new strain of microorganism YP-02978L of the present invention was obtained from natural soil, and can be easily obtained from the Microbial Technology Research Institute.

(Production method) Cultivation is advantageously carried out using a medium containing a nutrient source utilized by the microorganism. The medium may be synthetic, semi-synthetic or natural, solid or liquid, but liquid medium containing natural nutrients is usually preferred. Among the nutrient sources added to the medium, the carbon source may be any assimilable carbon compound, such as D-xylose, D-curcose, D-7 lactose, and rhamnose.

Raffinose, D-mannitol, glycerin.

Starch, salicin, starch, glucose, dextrin, coconut oil, soybean oil, α-melibiose, etc.
These may be used alone or in combination. Furthermore, alcohols, organic acids, etc. can also be used. Inorganic and organic nitrogen sources include ammonium chloride, sulfate, ammonium nitrate, urea, etc.; organic nitrogen sources include peptone, yeast extract, dried yeast, meat extract, blood meal, feather meal, gluten meal, Corn steep liquor, soy flour.

Fish meal, peanut meal, cottonseed meal, casamino acids and various amino acids (
For example, glutamic acid and aspartic acid.

Alanine, lysine, etc.), tomato juice, etc. are used alone or in combination.

In addition, sodium, potassium, magnesium, calcium, zinc, and iron are added to the medium as necessary.

Sulfates, nitrates, and chlorides of metals such as cobalt.

Organic acid salts such as carbonates, phosphates, etc. and sodium citrate can be added.

Cultivation is best done under aerobic conditions and left standing.

Both shaking and aeration and agitation culture are possible.

Shaking or aerated culture is advantageous. The culture temperature is preferably within the range of approximately 15-33°C.

A temperature of approximately 24 DEG to 27 DEG C. is particularly advantageous. Further, the pH of the medium is preferably maintained near neutrality of about 55 to 8.5, preferably 6.0 to 8.0. The culture period varies depending on culture conditions such as medium composition and temperature.

It usually takes about 2 to 21 days, preferably about 5 to 7 days, and the culture is terminated at an appropriate time by determining the time when the produced substance reaches its maximum titer.

To isolate and purify the antibiotic accumulated in the culture thus cultivated, two commonly used isolation and purification means may be applied.

Isolation and purification of antibiotics involves centrifuging or filtering the culture to remove bacterial cells, and then determining solubility in appropriate solvents, differences in solubility, differences in precipitation rates from solutions, 2) compatibility with various adsorbents. It is preferable to apply a method that utilizes a difference in adsorption affinity, a difference in distribution between two types of liquid phases, etc. These methods can be used alone, combined in any order, or applied repeatedly as necessary.

Especially the products produced by culturing the YP-02978L strain.

Extraction with organic solvents such as ethyl acetate, Wakogel C-
Using column chromatography using 200 (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) as a carrier, thin layer chromatography using silica gel 60F2S4 (manufactured by Merck & Co., Ltd.) as a carrier, and using antibacterial activity against Bacillus subtilis ATCC 6633 as an indicator, other production methods were used. separated from other substances and purified.

(Product) The physicochemical properties of the antibiotic thus obtained are as follows.

(Appearance dark blue acicular to plate-shaped crystals (2) Melting point 156-157°C (3) Elemental analysis theoretical value Actual value C = 78.98% C = 79.48%) I =
7.07%H=7.28%N=8
．． 31% N = 8.43% (4) Ultraviolet absorption spectrum (Figure 1) (5) Infrared absorption spectrum (Figure 2) (6) Hydrogen nuclear magnetic resonance spectrum (H-NMR) Figure 3 (7) Heavy carbon ( 13C) Nuclear magnetic resonance spectrum (13C)
-NMR) Figure 4 (8) Mass spectrum (Er) (EI-Ms) Figure 5 (9) Mass spectrum (HR) (HR-MS) m/
z 332.19005 (M”) αq Molecular weight I Molecular formula % Formula % Soluble in methanol, ethanol, acetone, ethyl acetate, chloroform, acidic water, insoluble in n-hexane (13 Basic, neutral, acidic bases) exhibits sexual behavior.

I Positive color reaction: Dragendorff reaction, Negative sulfuric acid reaction: Ninhydrin reaction Thin layer chromatography Silica gel 60F,
, 14 (manufactured by Meg) Developing solvent Rf value Chloroform: Methanol = 9:1 0
．． 56 Ethyl acetate: methanol = 9:1
0.15 Developing solvent Rf value Benzene: Acetone = 1:2 0.12 Chloroform: Methanol = 20: 1
Based on the physical and chemical properties of 0.29 or more, the product of the present invention is a compound represented by the above formula (I) [1-oxo-1゜2
-Dihydro-5-[(2,4,4-)limethyl--The existence of compound (II), which is a tautomer, was confirmed.

(Effects of the Invention) The compounds of the present invention have antibacterial activity, especially antibacterial activity against Durham-positive bacteria, and are useful as prophylactic and therapeutic agents for bacterial diseases in humans and animals caused by bacteria to which the antibacterial activity belongs. .

Furthermore, the compound of the present invention is expected to be useful as an anticancer agent.

The minimum effective inhibitory concentrations (MIC) of the nine compounds of the present invention are shown below.

(Example) The present invention will be explained in detail by referring to Examples below.

Example 1゜glucose 0.5%, white dextrin/2.0%, potato starch 3.0%, blood meal 1.0%, feather meal 1.0%, tomato juice 2.0%, primary sulfide Iron 0.0
2%.

Cobal chloride) 0.002%, calcium carbonate 0.2%
Prepare medium A (PH7,2) containing
Dispense 60 ml into Erlenmeyer flasks, sterilize at 120°C for 20 minutes, and add Streptomyces sp. yp-02978L grown on Bennett's agar medium.
The hyphae of the strain are scraped off, inoculated, and cultured with shaking at 27°C for 48 hours to obtain a seed culture. Next, white dextrin 4
．． 0%, glucose 0.5%, polypeptone 0.5%.

Yeast extract 0.5%, corn stewberry strength -1.5%
.

Meat extract 0.5%, magnesium sulfate 0.2%, calcium carbonate 0.2%, Adekanol (Jiryuka fi) 0
．． Prepare a medium B 10/: (PH7,5) containing 03% and add 60 ml of this to a 500 ml Erlenmeyer flask.
The seed culture was inoculated at a rate of 3.0% after being sterilized at 120°C for 20 minutes. Continue shaking culture at 27°C for 7 days.

The antibacterial activity against Bacillus subtilis ATCC6633 strain is maximum. In the culture solution obtained in this way,
Radiolite #600 (manufactured by Showa Kagaku Kogyo) is added, stirred, and filtered to obtain P liquid 10Z.

Add 4N sodium hydroxide to this P solution and p)19
Add 10 L of ethyl acetate (stir). Separate the ethyl acetate layer.
Do it once. Next, 7 L of acidic water adjusted to pH 3 with 4N hydrochloric acid is added to this ethyl acetate and stirred well. Perform two extractions with acidic water.

Add 4N sodium hydroxide solution to this acidic water to adjust the pH.
After adjusting the temperature to 9, add 4 L of ethyl acetate and stir well. The ethyl acetate layer is separated and anhydrous sodium sulfate is added thereto for dehydration. Next, the dehydrated ethyl acetate layer is concentrated under reduced pressure to obtain a dark blue oily substance.

The obtained dark blue oily substance was dissolved in a small amount of chloroform:methanol (9:1) solution, and Wakogel C-200 (
Column chromatography was carried out using chloroform:methanol (9:1) as the developing solvent by placing 200 g of the product (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) on a column packed with chloroform:methanol (9:1), and collecting both components that showed antibacterial activity against Bacillus subtilis strain ATCC 6633 and concentrating them under reduced pressure. A dark blue oily substance is obtained. The obtained oily substance was mixed with a small amount of ethyl acetate:methanol (9:1).
The solution was dissolved in a liquid, placed on a column packed with 150 g of Wakogel C-200, and subjected to column chromatography using ethyl acetate:methanol (9:1) as a developing solvent.

It exhibits antibacterial activity against Bacillus subtilis strain ATC06633, and when both blue components are collected and concentrated under reduced pressure, a dark blue oily substance is obtained.

The obtained dark blue oily substance was dissolved in a small amount of methanol, and then applied in a strip onto a Merck & Co. silica gel 60 F, 54 thin layer plate, and mixed with chloroform:methanol (
20:1) as a developing solvent, scrape off the dark blue part around RfO, 29, and elute with chloroform:methanol (20:1). Concentrating it under reduced pressure yields a dark blue powder. After dissolving this dark blue powder in a small amount of methanol, it was applied in a strip on a silica gel 60 F, S4 thin layer plate, and thin layer chromatography was performed using ethyl acetate:methanol (9:1) as a developing solvent. Scrape off the area around 0 and 15.

When dissolved in ethyl acetate:methanol (9:1) and crystallized, needle-shaped crystals of YP-02978-C were obtained as 2501
Example 2: 0.5% glucose, 2.0% white dextrin.

Tet starch 3.0%, blood meal 1.0%, feather meal 1.0%, tomato juice 2.0%, ferrous sulfate 0.
02%.

Cobal chloride) 0.002%, calcium carbonate 0.2%
Prepare medium A (PH7,2) containing
Dispense 60ml into 0ml Erlenmeyer flasks and heat at 120°C.
Streptomyces e sp. YP-02978 grown on Bennett agar medium was sterilized for 20 minutes.
The hyphae of the L strain were scraped and inoculated, and cultured with shaking at 27°C for 48 hours to obtain seed culture solution-1. Next, add 50% of medium A.
Dispense 60 ml each into 0 ml Erlenmeyer flasks, 12
3.0% seed culture solution-1 was sterilized at 0℃ for 20 minutes.
Seed culture solution-2 was obtained by inoculating the cells at a ratio of 1 to 2 and culturing them with shaking at 27° C. for 2 days. Furthermore, a 500 L tank containing 400% of medium B was sterilized at 121° C. for 30 minutes and seed culture solution-2 was inoculated at a rate of 20%. 27℃
When cultured with shaking for 6 days, Bacillus subtilis A
Antibacterial activity against TCC6633 strain is maximum.

The culture solution thus obtained was adjusted to p)I7 with 4N hydrochloric acid. Furthermore, after adding 600 ml of radiolyte and stirring, 401 microbial cells were obtained. Ethyl acetate 401 is added to the bacterial cells and stirred, and the ethyl acetate layer is separated.

Three ethyl acetate extractions are carried out.

This extract was concentrated under reduced pressure to 20 t. Next, acidic water 151 adjusted to pH 3 with hydrochloric acid is added to this concentrated solution and stirred well. Extraction with acidic water is carried out twice. After adjusting the pH to 9 by adding 4N hydroxide solution to this acidic water, 10 L of ethyl acetate was added and stirred well.

Extraction with ethyl acetate is carried out twice. Anhydrous sodium sulfate is added to the separated extract to dehydrate it. Next, the dehydrated ethyl acetate layer is concentrated under reduced pressure to obtain a dark blue oily substance.

The obtained dark blue oily substance was dissolved in a small amount of chloroform:methanol (9:1) and Wakogel C-2003
Column chromatography was performed using chloroform:methanol (9:1) as a developing solvent.
When the blue components, which show antibacterial activity in the strain, are collected and concentrated under reduced pressure, a dark blue oily substance is obtained. The obtained oily substance was dissolved in a small amount of ethyl acetate:methanol (c+:l solution) and placed on a column packed with 300 g of Wakogel C-200.

Column chromatography was performed using ethyl acetate as a developing solvent and then ethyl acetate:methanol (9:1) as a developing solvent. Bacillus subtilis ATCC66
It shows antibacterial activity against 33 strains, and when both blue components are collected and concentrated under reduced pressure, a dark blue oily substance is obtained. The obtained dark blue oily substance was dissolved in a small amount of acetone and Wakogel C
-200 was placed on a column packed with 300 g of benzene as a developing solvent, and then benzene:acetone (1:l
) as a developing solvent. It shows antibacterial activity against Bacillus subtilis ATCC 6633 strain, and when the blue fraction is collected and concentrated under reduced pressure, a dark blue oily substance is obtained. The obtained dark blue oily substance was dissolved in a small amount of chloroform:methanol (20:1), and placed on a column packed with 250 g of Wakogel C-200, followed by chloroform:methanol (20:1).
Perform column chromatography using as a developing solvent.

It shows antibacterial activity against Bacillus subtilis ATCC 6633 strain, and the blue fraction is collected and concentrated under reduced pressure. When the concentrated solution was dissolved in ethyl acetate:methanol:acetone (1:1:2) and crystallized, 3.7g of crystals were obtained and t0

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of YP-02978L-C, Figure 2 shows its infrared absorption spectrum, and Figure 3 shows the infrared absorption spectrum of YP-02978L-C.
Sono's 'H-NMR spectrum. Figure 4 shows the 13c-NMR spectrum, and Figure 5 shows the EI
-MS respectively.

Claims (1)

[Scope of Claims] 1. Antibiotic YP-02978 L-C, its tautomer, or a mixture thereof, represented by the formula ▲ Numerical formula, chemical formula, table, etc. ▼. 2. Cultivating a microorganism that belongs to the genus Streptomyces and has the ability to produce the antibiotic YP-02978 L-C, its tautomer, or a mixture thereof, represented by the formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼, Antibiotic YP-02978 from culture
Antibiotic YP-02978 L characterized by collecting LC or its tautomer or a mixture thereof
A method for producing -C or a tautomer thereof or a mixture thereof.