JPH0428276B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a novel antibiotic KMR-593 and a method for producing the same. (Structure of the Invention) The present inventors isolated bacterial strains from various soils for the purpose of searching for new antibiotics, and continued research on the metabolites produced by the strains. As a result, KMR-593 was newly isolated from soil. It was discovered that a substance having antibacterial activity against Gram-positive anaerobes was produced in the culture of the bacterial strain. Therefore, as a result of separating and purifying the antibacterial substance from the culture and investigating the physical and chemical properties of the substance, it was found that it is a new antibiotic.
It was named. The present invention was completed based on this knowledge, and provides an antibiotic KMR-593 and its salt having the physicochemical properties described below, and a method for producing the antibiotic KMR-593 and its salt using a microorganism. be. A practical example of a microorganism used to produce the antibiotic KMR-593 of the present invention is the actinomycete KMR-593 isolated from soil by the present inventors.
Stocks can be given. This bacterium was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, with the accession number ``Feikoken Bacteria Collection No. 7934''.
It has been deposited as. Its mycological properties are as follows. () Morphological properties The vegetative hyphae of this fungus develop well on starch inorganic salt agar and sucrose nitrate agar, but are underdeveloped on other media. Aerial mycelium grows slightly on sucrose nitrate agar, etc., but does not grow on other media. When observed under a microscope, one spore adhesion was observed on each vegetative hyphae, but no spore adhesion was observed on the aerial hyphae. The size of the spore is circular with a diameter of approximately 1.0 μm. The surface of the spore is smooth. No sclerotia, sporangia and zoospores are found. () Properties on various media EBShirling
and D. Gottlieb's method (International Journal of Systematic Bacteriology, Vol. 16,
Table 1 shows the culture properties of this producing bacterium, which were investigated by the authors (p. 313, 1966). 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 is written in parentheses along with the color chart name. Since this bacterium grows slowly and colonies appear around the 10th day of culture, observation was performed after 3 weeks. The following are the results of observations in each culture medium when cultured at 27°C unless otherwise specified.

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[Table] Medium selected by Mrs. Project
() Physiological properties (1) Formation of melanin pigment (a) Tyrosine agar negative (b) Peptone/yeast iron agar negative (c) Glucose/peptone/gelatin medium puncture 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 positive (6) Hydrolysis of starch positive (7) Coagulation of skim milk negative (8) Peptonization of skim milk positive (9) Growth temperature range 20-35℃ (10) Availability of carbon sources (nitrate agar medium) Used; D-glucose, D-fructose, sucrose, rhamnose, L-arabinose, D-xylose, D-mannitol, Starch, soluble starch Not used; raffinose, I-inositol, melibiose, glycerol () Chemical composition of cells Diaminopimelic acid in the cell wall is OH type, and glycine was detected. Arabinose, galactose, and xylose were observed as the sugar composition of the whole bacterial cells. The mycological properties of this bacterium can be summarized as follows. Hydroxy-
It contains diaminopimelic acid and glycine, and morphologically, it attaches one spore to each vegetative hyphae. In terms of culture properties, vegetative hyphae appear orange on most media, and slightly underdeveloped aerial hyphae grow on sucrose/nitrate agar media. From these results, this bacterial strain belongs to the genus Micromonospora. Cultivation is usually preferably carried out under aerobic conditions such as shaking or aerated agitation culture. Industrially, deep aeration agitation culture is preferred. The pH of the medium is preferably slightly acidic to near neutral. The culture temperature ranges from 20 to 40℃, but usually 24 to 30℃,
Preferably, it is kept at around 27°C. In the case of liquid culture, the culture time may generally be 1 to 8 days, but preferably the culture is terminated when the amount of the antibiotic accumulated in the culture reaches the maximum. Culture conditions such as medium composition, medium liquid properties, culture temperature, stirring speed, and aeration volume 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. When foaming occurs in liquid culture, antifoaming agents such as silicone oil, vegetable oil, and surfactants are used as appropriate. The antibiotic KMR-593 accumulated in the culture obtained in this way is contained in the bacterial cells and the culture filtrate, so it is centrifuged to separate the culture filtrate and the bacterial cells. This antibiotic KMR−
It is advantageous to collect 593. To collect this antibiotic KMR-593 from the culture filtrate, the culture filtrate can be extracted with a non-hydrophilic organic solvent such as ethyl acetate, or the culture filtrate can be extracted with activated carbon, alumina, porous synthetic polymer resin, or ion exchange resin. etc., elute with an elution solvent such as ethyl acetate, and concentrate the resulting extract or eluate under reduced pressure, or add an organic solvent such as hexane to precipitate it. The obtained crude substance is further subjected to known methods commonly used for purifying fat-soluble substances,
For example, it can be purified by column chromatography using a carrier such as silica gel or alumina. To collect this antibiotic KMR-593 from bacterial cells, the bacterial cells are extracted with a hydrophilic organic solvent containing water such as aqueous acetone, the resulting extract is concentrated under reduced pressure, and the concentrate is extracted with ethyl acetate. This ethyl acetate extract can be separated and purified by combining it with the ethyl acetate extract obtained from the culture filtrate, or it can be separated and purified by the same method as above. Next, the physicochemical and biological properties of the antibiotic KMR-593 will be described. () Physical and chemical properties Elemental analysis: Consists of carbon, hydrogen, oxygen and chlorine, elemental analysis values: C58.00%, H7.22%
Cl6.60% Molecular weight: Elemental analysis value and ¹³ C, ¹ H−
It is estimated _to be _{C52H70-76Cl2O18-19 from} nmr, so _the molecular weight is 1053-1075. Melting point: 160-163°C Specific rotation: [α] ¹⁸ _D +55.0° (C = 1, methanol) Ultraviolet absorption spectrum: As shown in Figure 1, λ ^MeOH _nax nm (E ¹ % _1cn ) 230 ( 316), 245
(sh), 270 (208), 315 (102) Infrared absorption spectrum (CHCl ₃ solution method):
As shown in Figure 2, 3500, 3000, 1740,
It has absorption bands at 1710, 1660, and 1600 cm ^-1 . Solubility in solvents: Soluble in acetone, methanol, ethanol, ethyl acetate, chloroform, insoluble in water and hexane Color reaction: Positive for iodine reaction, sulfuric acid reaction, ferric chloride reaction Basic, acidic, neutral distinction : Weakly acidic Color of substance: Colorless Proton nuclear magnetic resonance spectrum (CDCl ₃
middle): As shown in Figure 3, C-13 nuclear magnetic resonance spectrum (CDCl ₃
Middle): As shown in Figure 4 Silica gel thin layer chromatogram: Rf=0.54
(Carrier: Merck silica gel 60F ₂₅₄ , thickness 0.2
mm, Art5554), developing solvent: chloroform-
Methanol (9:1) () Biological properties (1) Antibacterial spectrum Paper disc method (Toyo Seisakusho Co., Ltd., 8mm
Sample preparation using φ, thick), concentration 1000μg/
The microbial growth inhibition circle diameter in ml is as shown in Table 2.

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[Table] (2) Toxicity 100 mg/Kg of this antibiotic was intraperitoneally administered to mice and observed up to 2 weeks later, but no abnormalities were observed. In addition, the KMR-593 strain is the antibiotic KMR.
In addition to -593, it produces three other antibiotics.
These can be clearly distinguished from high performance chromatography and proton nuclear magnetic resonance spectra, but their similar ultraviolet absorption spectra, similar fragment patterns in mass spectrometry, and similar antibacterial spectra suggest that they are book antibiotic
It was found to be a substance similar to KMR-593. (Effects of the Invention) This antibiotic KMR-593 has low toxicity and has antibacterial activity mainly against certain Gram-positive aerobic and anaerobic bacteria, so it is useful as a therapeutic or preventive agent for bacterial infections in humans and animals. It is. (Example) Next, the present invention will be specifically illustrated by examples.
The present invention is not limited thereby. Dispense 100 ml of liquid medium (PH7.0) containing 0.1% glucose, 2.4% starch, 0.3% peptone, 0.3% meat extract, 0.5% yeast extract, and 0.4% calcium carbonate into a 500 ml Erlenmeyer flask, and heat at 121°C. Steam sterilize for 15 minutes and add 1.0% glycerol, 1.0% calcium malate, 0.05% ammonium chloride, 0.05% dipotassium phosphate, 1.5% agar to yeast extract 0.1%
Cultured at 27°C on agar slants containing %
One platinum loop was inoculated from a slant culture of strain KMR-593, and cultured with shaking at 27°C for 5 days using a rotary shaker to obtain seeds. Soluble starch in 50 volume jar fermenter
Liquid medium containing 2.0%, dry yeast 1.0%, sodium chloride 0.3%, calcium carbonate 0.3% (PH7.0) 30
was prepared and steam sterilized at 121°C for 15 minutes. The above-mentioned 9 seedlings were transplanted to this, and cultured with aeration at 27° C. for 155 hours under conditions of a stirring speed of 250 rpm and an aeration rate of 15/min. The culture solution was centrifuged (10,000 rpm) using a shear press centrifuge to separate the bacterial cells and the culture supernatant. 10 portions of 70% acetone water were added to the obtained bacterial cells for stirring extraction, and this was filtered to obtain an acetone extract. This extract was concentrated to 500 ml under reduced pressure. After concentration, 250 ml of benzene was added and shaking and extraction were performed three times. The obtained benzene extract was concentrated to 50ml under reduced pressure, and 500ml of hexane was added to the concentrated liquid.
was added and left overnight at 4°C. The resulting precipitate was collected by filtration and dried to obtain 490 mg of a crude product. Meanwhile, the culture supernatant was synthesized using the adsorbent Diaion HP.
-20 (1) column to adsorb water (5)
After washing with 30% acetone water (3), 70
Elution was performed with % acetone water (1). 70% obtained
The acetone eluate was concentrated to 100 ml under reduced pressure, 100 ml of benzene was added to the concentrated solution, and the operation of shaking and extraction was repeated three times. Benzene extract is 50% under reduced pressure.
The mixture was concentrated to ml, 500 ml of hexane was added, and the mixture was left at 4°C overnight. The resulting precipitate was collected by filtration and dried to obtain 230 mg of a crude product. Combine this with the above crude product,
Dissolve in as little acetonitrile as possible and add 1/10th of this amount to a preparative reverse phase column for high performance liquid chromatography (manufactured by Yamamura Kagaku Kenkyusho: AM324).
(ODS; 10 x 300 mm) at a flow rate of 4 ml/at acetonitrile:water:acetic acid (63:37:1).
It eluted at min. Each fraction was tracked by bioassay using Clostridium perfringes and by UV absorbance at 300 nm (as well as the fraction containing lipiamycin).
Fractions containing KMR-593 were collected (separately) and concentrated under reduced pressure to yield 116 mg of crude material (containing KMR-593). Dissolve this in as little chloroform as possible and divide it into three batches using a preparative normal phase column for high speed chromatography (manufactured by Yamamura Kagaku Kenkyusho: A-024).
(SIL); 10 x 300 mm) and eluted with chloroform:methanol (22:1) at a flow rate of 4 ml/min. The active fractions were collected and dried under reduced pressure to obtain 39 mg of antibiotic KMR-593 as a colorless amorphous substance.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of the antibiotic KMR-593, Figure 2 shows the infrared absorption spectrum of the antibiotic, Figure 3 shows the proton nuclear magnetic resonance spectrum of the antibiotic, and Figure 4 shows the C- 13 shows nuclear magnetic resonance spectra.

Claims (1)

[Claims] 1. Antibiotic KMR- having the following physical and chemical properties
593 and its salts. Elemental analysis: Consists of carbon, hydrogen, oxygen and chlorine, elemental analysis values: C58.00%, H7.22%,
Cl6.60% Melting point: 160-163℃ Specific rotation: [α] ¹⁸ _D = 55.0゜ (C = 1, methanol) Ultraviolet absorption spectrum: λ ^MeOH _nax nm (E ¹ % _1cn )
It has absorption bands at 230 (316), 245 (sh), 270 (208), and 315 (102). Infrared absorption spectrum: 1710, 1660, 1600cm
It has an absorption band at ^-1 . Solubility in solvents: Soluble in acetone, methanol, ethanol, ethyl acetate, chloroform, insoluble in water and hexane Color reactions: Iodine reaction, sulfuric acid reaction, dichloride
Positive for iron reaction Distinction between basic, acidic, and neutral: Weakly acidic Color of substance: Colorless 2 An antibiotic that belongs to the Micromonospora genus and has the following physical and chemical properties.
An antibiotic characterized by culturing a microorganism capable of producing KMR-593 in a medium, producing and accumulating the antibiotic KMR-593, and collecting the same.
Method for producing KMR-593 and its salt. Elemental analysis: Consists of carbon, hydrogen, oxygen and chlorine, elemental analysis values: C58.00%, H7.22%,
Cl6.60% Melting point: 160-163℃ Specific rotation: [α] ¹⁸ _D = 55.0゜ (C = 1, methanol) Ultraviolet absorption spectrum: λ ^MeOH _nax nm (E ¹ % _1cn )
It has absorption bands at 230 (316), 245 (sh), 270 (208), and 315 (102). Infrared absorption spectrum: 1710, 1660, 1600cm
It has an absorption band at ^-1 . Solubility in solvents: Soluble in acetone, methanol, ethanol, ethyl acetate, chloroform, insoluble in water and hexane Color reactions: Iodine reaction, sulfuric acid reaction, dichloride
Positive for iron reaction Distinction between basic, acidic and neutral: weakly acidic Color of substance: colorless