JPH0372237B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel antibiotic AC-1978 and a method for producing the same. The present inventors isolated bacterial strains from various soils for the purpose of searching for new antibiotics, and continued research on the substitute products produced by them. It was discovered that a substance with antibacterial activity against Gram-positive bacteria is produced. Therefore, as a result of separating and purifying the antibacterial substance from the culture and examining the physical and chemical properties of the substance, it was found that it had the properties as described below. A substance with such physical and chemical properties is considered to be a peptide-based substance, but since no other substance has been found, the substance was named antibiotic AC-1978.
The present invention was completed based on such knowledge. The present invention is an antibiotic having the physical and chemical properties described below.
AC-1978 or its salt and antibiotic AC-1978-producing bacteria belonging to the genus Streptomyces are cultured in a medium, and the resulting culture is used to produce antibiotic AC-1978.
Antibiotic AC-1978 characterized by collecting
or a method for producing its salt. The bacteria producing this antibiotic AC-1978 belongs to the genus Streptomyces, and for example, the bacteria isolated by the present inventors
The AC-1978 strain is an example of a strain most effectively used in the present invention, and the mycological properties of this strain are as follows. (a) Morphological characteristics The vegetative system of the AC-1978 strain develops well on various agar media and usually does not have septa. Aerial bacteria grow abundantly on starch/inorganic salt agar media, nutrient agar media, etc., and take on a velvety appearance. When observed under a microscope, the sporophyte has a spiral shape, and the spores are oval in shape, with 10 or more spores in a row, and the surface is smooth. The size of the spores is 0.9 x 1.1 μm. sclerotia,
No sporangia and zoospores are found. (b) Growth status in each of the following media Method of EBShirling and D. Gottlieb [Int.J.
Bacteriol., 16 , 313 (1966)] and cultured on each medium at 27°C for 14 days unless otherwise specified, and the observed findings are shown in Table 1.
Color display is based on Color Harmony Manual 4th edition.
1958 Container Corporation of
According to America's color classification.

【table】

【table】

[Table] (c) The following physiological properties Growth temperature range: 15-47℃ Liquefaction of gelatin (glucose, peptone,
(on gelatin medium, 21-23℃); positive; hydrolysis of starch (on starch agar medium); positive; coagulation of skim milk, peptonization (37℃); coagulation; coagulation; positive; peptonization; positive; Production: (a) Tyrosine agar medium: negative, (b) peptone yeast iron agar medium: negative, (c) glucose peptone gelatin medium (puncture, 21-23℃): negative, (d) tryptone yeast solution : negative, tyrosinase reaction; negative, : production of hydrogen sulfide; negative, reduction of nitrate; positive, decomposition of cellulose; negative, (d) Assimilability of each of the following carbon sources (on Pridham-Godlieb agar medium) ;+, slightly used; ±, not used;-) L-arabinose; + D-xylose; + D-glucose; + D-fructose; + sucrose; + i-inositol; ± L-rhamnose; + raffinose; + D-mannitol; - melibiose; - (e) Cell wall composition Becker et al.'s method [Appl. Microbiol., 13 ,
236-243 (1965)], LL
type of diaminopimelic acid was detected. Arabinose and galactose are not allowed. To summarize the above mycological properties, the cell wall composition has LL type diaminopimelic acid, the morphological characteristics are that it forms a spiral spore chain, and the surface of the spores is smooth, and the various properties on the medium are as follows: The vegetative hyphae exhibit a pearl pink or related color tone, and the aerial hyphae exhibit a gray or brown tone, exhibit wettability on certain types of media, and do not produce soluble pigments or melanin pigments. From these results, it is clear that this bacterial strain belongs to the genus Stoptomyces, and is classified according to Pridham and Tosner's classification [Bergey's Manual of Determinative
Bacteriology, 8th edition, 748-829 (1974)], it is thought to belong to the White or Gray series, but it was tentatively named Streptomyces sp. AC-1978. This strain has been deposited with the National Institute of Microbiology, Agency of Industrial Science and Technology, with deposit number No. 7452 (FERM-P No..
7452). The bacteria producing the antibiotic AC-1978 have been explained above, but as a general property of actinomycetes, their mycological properties are extremely variable and not constant, and they are naturally or normally exposed to ultraviolet irradiation, X-rays, etc. It is a well-known fact that mutations occur through artificial mutation methods such as irradiation or mutagenic agents, and not only such artificial mutant strains but also natural mutant strains,
Belongs to the genus Streptomyces, and the antibiotic AC-
Any strain capable of producing 1978 can be used in the present invention. In the present invention, first, antibiotic AC-1978-producing bacteria belonging to the genus Stoptomyces are cultured in an appropriate medium. In culturing this bacterium, a method for culturing actinomycetes is generally used. As the medium, a nutrient medium containing a carbon source that can be assimilated by microorganisms, a nitrogen source that can be digested, and inorganic salts as necessary is used. As assimilable carbon sources, carbohydrates such as glucose, fructose, maltose, xylose, mannite, galactose, ribose, glycerin, molasses, starch, dextrin, corn steep liquor, etc. are used alone or in combination. In addition, organic acids such as gluconic acid, pyruvic acid, lactic acid, and acetic acid, methanol,
Alcohols such as ethanol, non-aromatic hydrocarbons such as n-paraffin, vegetable or animal fats and oils, etc. can also be used. Digestible nitrogen sources include peptone, meat extract, yeast extract, dried yeast, soy flour, soy protein decomposition, casein, amino acids, urea, NZ-amine, corn steep liquor, fat meal, and pupa hydrolysate. Organic nitrogen sources such as nitrogen, inorganic nitrogen compounds such as nitrates, ammonium salts, etc. are used alone or in combination. In addition, inorganic salts such as sodium salts, potassium salts, calcium salts, magnesium salts, and phosphates may be added as necessary. Furthermore, if necessary, the culture medium may be supplemented with antibiotics AC to support the growth of this strain.
Trace amounts of heavy metals, micronutrients, growth promoters, and precursors that promote the production of -1978 may be appropriately added. Cultivation is usually 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 neutral. The culture temperature is
Although it can be carried out at 20 to 40°C, it is usually kept at 24 to 30°C, preferably around 27°C. Regarding the culture time, in the case of liquid culture, the culture may normally be carried out for 1 to 8 days, but preferably the culture should be terminated when the amount of the present antibiotic accumulated in the culture reaches the maximum. These medium compositions, liquid properties of the medium, culture temperature, stirring speed,
It goes without saying that culture conditions such as the amount of aeration are appropriately adjusted and selected depending on the type of bacterial strain used, external conditions, etc. so as to obtain preferable results.
When foaming occurs in liquid culture, antifoaming agents such as silicone oil, vegetable oil, and surfactants are used as appropriate. Since the antibiotic AC-1978 accumulated in the culture thus obtained is contained in the bacterial cells and the culture solution, the antibiotic AC-1978 is separated into the culture solution and the bacterial cells by centrifugation. It is advantageous to take the antibiotic AC-1978. To collect this anti-producing substance AC-1978 from the culture solution, extract the culture solution with a non-hydrophilic organic solvent such as ethyl acetate, or extract the culture solution with activated carbon, alumina, porous synthetic polymer resin, or ion exchange. It may be adsorbed onto a resin or the like, eluted with an elution solvent such as ethyl acetate, and the resulting extract or eluate may be concentrated under reduced pressure or precipitated by adding an organic solvent such as hexane. The obtained crude substance can be further purified by a known method commonly used for purifying fat-soluble substances, such as column chromatography using a carrier such as silica gel or alumina. To collect this antibiotic AC-1978 from bacterial cells,
The bacterial cells are extracted with a water-containing hydrophilic organic solvent such as aqueous acetone, the resulting extract is concentrated under reduced pressure, and the concentrate is extracted with ethyl acetate. It can be separated and purified by combining it with the ethyl extract, or it can be separated and purified by the same method as above. Next, the physicochemical and biological properties of this antibiotic AC-1978 will be described. Physical and chemical properties Elemental analysis Consists of carbon, hydrogen, nitrogen and oxygen; elemental analysis values: C57.13%, H6.06%,
N11.72%, molecular weight: 1129, M ⁺ = 1129 and (M + Na ⁺ ) = 1152 observed by FD-mass spectrum, melting point: 166-168°C, specific optical rotation: [α] ²⁶ _D = 21.6° (C=1, methanol), ultraviolet absorption spectrum: as shown in Figure 1, λ ^MeOH _nax (E1% 1cm) 279 nm, infrared absorption spectrum (KBr method): as shown in Figure 2, at least 3300, 2990 ,
Has absorption bands at 1748, 1620, 1549, 1450, and 1360 cm ^-1 Solubility in solvents; Soluble in acetone, methanol, ethanol, butanol, ethyl acetate, chloroform, Insoluble in water and hexane, Color reaction; Iodine reaction Positive for phosphomolybdic acid reaction, ninhydrin reaction, negative for sulfuric acid reaction, Basic, acidic, neutral; Neutral; 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; Rf0.5 carrier; Merck silica gel 60F ₂₅₄ , thickness 0.2
mm, Art5554), Developing solvent: Chloroform-methanol (20:1), Molecular formula: Elemental analysis value, Proton nuclear magnetic resonance spectrum (Figure 3) and C-13 nuclear magnetic resonance spectrum (Figure 4), Molecular formula C _52-54
H _60-73 N _8-11 O _16-19 is estimated. Biological properties Antibacterial spectrum The minimum inhibitory concentration (MIC) for microbial growth determined by the agar dilution method is shown in Table 2. Table 2 Test bacteria MIC (μg/ml) Staphyloccus aureus ATCC6538P ＞100 Bacillus subtilis ATCC6633 ＞100 Bacillus cereus IFO3001 12.5 Micrococcus luteus 1.56 Mycobacterium smegmatis ATCC607 ＞100 Escherichia coli NiHJ ＞100 Klebsiella pneumoniae AT CC10031 ＞100 Pseudomonas aeruginosa IFO3080 ＞100 Toxicity Book 500 mg/Kg of antibiotics was administered intraperitoneally to mice, and observations were made for two weeks after which no abnormalities were observed. As mentioned above, this antibiotic AC-1978 has low toxicity and
Since it has antibacterial activity mainly against certain Gram-positive bacteria, it is useful as a therapeutic or prophylactic agent against bacterial infections in humans and animals. Next, the present invention will be specifically illustrated with reference to Examples.
This does not limit the production of the present invention. Example 100 ml of liquid medium (PH7.0) containing 1.0% glucose, 1.0% dextrin, 0.5% yeast extract, 0.5% NZ-amine (type A), and calcium carbonate was dispensed into a 500 ml Erlenmeyer flask and incubated at 121°C. Stoptomyces sp. AC-1978 (FERM-P No. 7452) was steam sterilized for 15 minutes and cultured at 27°C on an agar slant medium containing 1.2% agar in the above composition.
One platinum loopful was inoculated from the slant culture, and cultured with shaking at 27°C for 2 days using a rotary shaker to obtain seeds. 100ml jar fermenter with glycerin
2.0%, Corn Steep Liquor 1.5%, Soy Flour
0.5%, calcium carbonate 0.3%, cobalt chloride
Prepare liquid medium (PH6.5) 70 containing 0.0002%,
Steam sterilized at 121°C for 15 minutes. Add this to the above seed mother
Transplanted 21 bottles, stirring speed 250r.pm air flow rate 20
Culture was carried out with aeration at 30°C for 69 hours under conditions of 1/min. The culture solution was centrifuged (10,000 rpm) using a shear press centrifuge to separate the cells and culture supernatant. 75% acetone water 16 was added to the obtained bacterial cells and extracted with stirring, and this was separated to obtain an acetone extract. This extract was concentrated to 1 under reduced pressure. An operation of adding 500 ml of ethyl acetate to the concentrated solution and stirring and extracting was performed 10 times. The obtained ethyl acetate extract was concentrated to 50 ml under reduced pressure, 1 portion of hexane was added to the concentrated solution, and the mixture was left at 4° C. overnight. Remove the resulting precipitate,
After drying, 2.7 g of crude product was obtained. On the other hand, 30 ml of ethyl acetate was added to the culture supernatant, stirred well, and then separated by centrifugation. The ethyl acetate layer was concentrated to 50 ml under reduced pressure, 1 portion of hexane was added to the concentrated solution, and the mixture was left at 4° C. overnight. The resulting precipitate was collected and dried to obtain 4.3 g of a crude product. This was combined with the above crude product, dissolved in as little chloroform-methanol (50:1) as possible, and this was filled with chloroform-methanol (50:1) silica gel (Merck, Kieselgel 60,
Art7734) Column chromatography was performed by charging a 200 g column and eluting with chloroform-methanol (50:1). Each fraction was tracked by silica gel thin-layer chromatography (see the physicochemical properties section) and a bioassay using Micrococcus luteus, and the fractions containing the active ingredient were collected and concentrated under reduced pressure to produce the crude substance 820.
I got mg. Dissolve this in as little chloroform-acetone (10:1) as possible and fill it with 200 g of silica gel (manufactured by Merck & Co., Kieselgel 60, Art 7734).
Charge a column of chloroform-acetone (10:1) and chloroform-acetone (5:1).
1) 1, chloroform-acetone (3:1)
Column chromatography was performed, eluting with 400 ml and then 500 ml of acetone. The eluate was fractionated into 20 ml portions, and the 48th to 100th fractions eluted with chloroform-acetone (5:1) were collected and dried under reduced pressure to obtain antibiotic AC-1978 as a colorless solid product. Yield 350mg.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of the antibiotic AC-1978, 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 AC- having the following physical and chemical properties:
1978 or its salt. Elemental analysis: Consists of carbon, hydrogen, nitrogen and oxygen, elemental analysis values: C57.13%, H6.06%,
N11.72%, molecular weight: 1129 (according to FD-mass spectrum), melting point: 166-168°C, specific rotation: [α] ²⁶ _D = 21.6° (C = 1, methanol), ultraviolet absorption spectrum: Figure 1 λ ^MeOH _nax (E1% 1cm) 279 nm, infrared absorption spectrum; the infrared absorption spectrum by KBr method is as shown in Figure 2.
At least 3300, 2990, 1748, 1620, 1549,
Has absorption bands at 1450 and 1360 cm ^-1 , Solubility in solvents; Soluble in acetone, methanol, ethanol, butanol, ethyl acetate, chloroform, Insoluble in water and hexane, Color reaction; Positive for iodine reaction, Phosphormolybdic acid Negative for reaction, ninhydrin reaction, sulfuric acid reaction, basic, acidic, neutral; color of neutral substance; colorless. 2 Antibiotic AC belonging to the genus Stoptomyces
1. A method for producing antibiotic AC-1978 or a salt thereof, which comprises culturing 1978-producing bacteria in a medium and collecting antibiotic AC-1978 from the resulting culture. 3. The production method according to claim 2, wherein the antibiotic AC-1978 producing bacterium is Streptomyces sp. AC-1978.