JPS60172285A - Novel antibiotic ac-1978 and preparation thereof - Google Patents

Abstract

NEW MATERIAL:An antibiotic AC-1978 or a salt thereof having the following physico-chemical properties; Elementary analysis (%); C, 57.13; H, 6.06; N, 11.72. Molecular weight 1129 [measured by the field desorption (FD)-mass spectrometry]. Melting point; 166-168 deg.C. Specific rotatory power [alpha]D<26>=21.6 deg. (C=1, methanol). Soluble in acetone, ethanol, ethylacetate, chloroform, etc. and insoluble in water, hexane, etc. Positive to the iodine reaction and negative to the phosphomolydic acid, minhydrin reactions, etc. Colorless under neutral conditions. USE:An antimicrobial agent against Gram-positive bacteria with low toxicity. PREPARATION:Streptomyces species AC-1978 (FERM-P No.7452) strain is cultivated at a somewhat acidic - neutral pH and 27 deg.C for 1-8 days preferably by the submerged spinner culture method with aeration.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibiotic WAC-1978 and its production method.

The present inventors searched for a new antibiotic.As a result of separating AC from various soils and conducting research on the growing tumors, we newly isolated AC from various soils. −
It was discovered that a substance having antibacterial activity against Gram-positive bacteria was produced in a culture of the 1978 strain. Therefore, as a result of separating and purifying the carcass 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 having such physical and chemical properties is considered to be a peptide-based substance, but since no other substance has been found, this substance was named antibiotic AC-1978. Invention 8
It was completed based on the knowledge of A#'i.

The present invention provides antibiotic AC-1 having the physical and chemical properties described below.
978t or its salts and Strep)-r Antibiotic AC-1978-producing bacteria belonging to the genus Ises are cultured in a medium, and the antibiotic AC-1978 is collected from the obtained culture. 19781? c is the method for producing the salt.

The AC-1978-producing bacterium belongs to the genus Streptomyces, but for example, AC-1 isolated by the present inventors
Strain 9781 is an example of a strain most effectively used in the present invention, and the chemical properties of this strain are as follows.

6) Morphological characteristics The vegetative hyphae of the AC-1978 strain develop well on each Dan agar medium and usually do not have septa. Aerial mycelium grows abundantly on starch/mineral salt agar plates, nutrient agar plates, etc., and has a velvety golden appearance. When observed under a microscope, the sporophyte has a spiral shape, the spores are elliptical, 10 or more in number, and the surface is smooth. Spore size is 0.9 x 1.1 μm. No sclerotia, sporangia and zoospores were found. (b) Growth status in each of the following media: E, B, Shilling and D-Gott
Liab's method (Int, J, 5yat, Bacte
riol, * 16 * 343 (1966)] and IA limit unless otherwise specified) on each medium at 27°C for 14 days.
The findings observed after culturing for days are shown in Table 1. The color display is based on Co1or Harmony Manual 4th edition, 1958, Container Corpora-
Color classification according to Lion of America was followed.

aE1 Table (c) Each of the following physiological properties ■ Growth temperature range; 15 to 47°C ■ Liquefaction of gelatin (on glucose-based peptone gelatin medium, 21 to 23°C); positive ■ Hydrolysis of starch (starch agar medium) (Tyrosine agar medium; Negative, (B) Peptone yeast iron Agar medium;
℃); Negative; (d) Each of the following carbon sources Assimilation (on Prid I. Mu Godelieve agar medium) (use; +, \use; ±, use; -) ■L-arabinose; + ■D-xylose; + ■D-glucose; 10 ■ D-7 Lactose: 10 ■ Sucrose; + ■ 1-Inositol: ± ■ L-Rhamnose; 10 ■ Raffinose; + ■ D-Mannitol 〇 Melibiose nee (e) a Composition of the cell wall Eaker et al.'s method [Appl, Microbio
As a result of the analysis based on 1965), LL-type cyamino pimelic acid was detected.

Acipinose and galactose are not allowed.

To summarize the above mycological properties, the cell wall composition has LL type regiaminopimelic acid, the morphological characteristics are that it forms a spiral spore chain, the surface of the spore is smooth, and there are various properties on the medium. As such, vegetative hyphae exhibit a pearl pink or related color IA, while aerial hyphae exhibit a gray or brown tone, exhibit wettability on certain media, and produce soluble pigments and melanin pigments. It is not allowed.

From these results, it is clear that this strain belongs to the genus Streptomyces, and is classified according to Pridum and Tresner's classification (BergeyrB Manual ofD).
eterminative Bacteriology
, 8th edition, 748-829 (1974)), but it is thought to belong to the White or Daley series, but for the time being it is Streptomyces sp. A.
It was named C-1978.

This strain has been deposited at the National Institute of Microbiology, Agency of Industrial Science and Technology, with deposit number No. 74-2 (FERM-PNo.).

74F2).

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 means such as irradiation or mutagenic agents, and such artificial mutant strains as well as natural mutant strains belong to the genus Streptomyces and cannot be treated with antibiotics. substance ac
-1978 can be used in the present invention.

In the present invention, first, antibiotic AC-1978-producing bacteria belonging to the genus Streptomyces are cultured in a suitable medium. In Japan, the method of culturing actinomycetes is generally used. As the medium, an honor medium containing a carbon source that can be assimilated by microorganisms, a nutrient source that can be digested, and, if necessary, an inorganic salt is used. Assimilable carbon sources include glucose, fructose, maltose, xylose, mannitol, galactose, ribose,
Carbohydrates such as glycerin, fines, starch, dextrin, corn stewed liquor, etc. may be used alone or in combination. Organic acids such as gluconic acid, pyruvic acid, lactic acid, and acetic acid; alcohols such as methanol and ethanol; non-aromatic hydrocarbons such as n-paraffin; and vegetable or animal fats and oils can also be used. Digestible nitrogen sources include peptone, meat extract, yeast extract, dried yeast, soybean flour, soybean protein decomposition product, casein, amino acids, urea, NZ-amine, corn stewed liquor, fish φ meal, and oyster hydrolysate. Organic nitrogen sources such as nitrates and inorganic nitrogen compounds such as nitrates and ammonium salts may be 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, the culture medium may contain antibiotics for the growth of this strain and antibiotics** hc-1, if necessary.
Heavy metals in advance to promote the production of 978, Weidong Song'! 4! A growth promoter, a growth promoting substance, and a precursor may be added as appropriate.

Cultivation is usually preferably carried out under aerobic conditions such as aerated agitation culture. Industrially, deep aeration agitation culture is preferred. 'It is preferable to culture at a slightly acidic or neutral pH of the medium, and the h0 culture temperature can be maintained at 20-40°C, but it is usually kept at 24-30°C, preferably around 27°C. . The culture time is usually 1 to 8 in the case of liquid culture.
The culture may be carried out for one day, but it is preferable to terminate the culture when the amount of the antibiotic antibiotic accumulated in the culture reaches the maximum.

These medium composition, medium hostility, culture temperature, stirring speed,
Culture conditions such as the amount of aeration are preferred depending on the type of 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 AC-1978 accumulated in the culture thus obtained is contained within the bacterial cells and in the J@ZenF solution, so it is centrifuged and cultured. It is advantageous to separate the IIF fluid and the bacterial cells and collect the present antibiotic AC-1978 from each.

To collect this antibiotic AC-1978 from culture F solution, extract culture P solution with a water-resistant organic solvent such as ethyl acetate, or extract culture P solution with activated carbon, alumina, or porous synthetic polymer. It may be adsorbed onto a resin, ion exchange resin, etc., eluted with an elution solvent such as ethyl acetate, and the resulting extract or eluate 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 BA-soluble substances, such as column chromatography using a carrier such as silica gel or alumina.

To collect this antibiotic AC-1978 from bacterial cells, extract the bacterial cells with a hydrophilic organic solvent such as aqueous acetone,
The obtained extract is compressed under reduced pressure, and the concentrate is extracted with ethyl acetate, and this ethyl acetate extract is separated and purified by combining it with the ethyl extract obtained from the culture P solution at 0C, or Separation and purification can be performed in the same manner as described above.

Next, the physicochemical properties and biological properties of the present antibiotic AC-1978 will be described.

■ Physical and chemical properties ■ Original analysis; Consists of carbon, hydrogen, nitrogen, and adult leaves; elemental analysis; C57, l 3chi, f (6,06chi, N1
1, 72%, ■ Molecule 1t; 1129, according to FD-mass spectrum M” = 1129 and (M+Na+) = 115
'2 was observed, ■Melting point; 166-168℃, ■Specific rotation rl: Cct), 7 = 21.6° (C =
5. ■Ultraviolet absorption spectrum; as shown in Figure 1,
1%.

λ (E) 279mm.

InaX IcIn ■Infrared absorption spectrum (KBr method); as shown in Figure 2, 3300.2990.1748.1620.15
49. Has an absorption band at 1450 s 1360 cm-'', ■ Solubility in solvents: Soluble in acetone, methanol, ethanol, butanol, ethyl acetate, chloroform;
Insoluble in water and hexane, ■Color reaction; positive for iodine reaction, negative for phosphomolybdic acid reaction, ninhydrin reaction, and sulfuric acid reaction; ■Distinguish between cyclic basic, acidic, and neutral; neutral, color of O substance: colorless , 0 proton nuclear magnetic resonance spectrum (in CDC13): As shown in Figure 3, oc-ia nuclear magnetic resonance spectrum (in CDC13); As shown in Figure 4, O silica gel thin layer chromatogram p Rf = o, s
Carrier: Silica gel 60F manufactured by Merck, % thickness 0, 2m
m, Art 5554), developing solvent; chloroform-methanol (20:1), ■ molecular formula 8 nine element analysis value, proton nuclear magnetic resonance Subek!・
From the molecular formula 〇aw, 5aHso-, yA, 11
0.6~. , 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 (4th instar) Bacillus cereus IFO300112
,5M1crococcus 1uteus 1.56
Esaherichia coli NIHJ > 1
00 ■Toxicity This antibiotic was administered intraperitoneally to 500#/he mice and observed after 2 weeks, but no abnormality was detected.

The above-mentioned common antibiotic AC-1978 has low persistence and has antibacterial activity mainly against certain Durham-positive bacteria, so it is useful as a preventive drug for the treatment of bacterial infections in humans and animals. It is.

EXAMPLES Next, the present invention will be specifically illustrated with reference to Examples, but the production examples of the present invention are not limited thereto.

Example 1.5 g of glucose in a 500 a/mL Erlenmeyer flask OL.

Dispense 100 m of a liquid medium (pH 7,0) containing dextrin i, o*, yeast extract 0.5%, NZ-amine α type) 0.51 calcium carbonate, and add 121 tl to 15
Steam for 1 minute and add 1 or 2% to the above composition.
Streptomyces sp. AC-1978 (FERM-
One platinum loop was inoculated from a slant culture of P No = 74r2) and cultured with shaking using a rotary shaker at 2.7°C for 2 days to obtain seeds.

Glycery in a 100m jar/fermenter 2.
0% s ” ” Steep pulley force - 1.5%, soybean flour 0.5%, calcium carbonate 0.3%, cobal chloride)
Liquid medium containing 0.0002% (p)I6.5)70
1 and steam sterilized at 121'C for 15 minutes. The above 21 seeds were transplanted to this, and the stirring speed was 250 r.
The culture was carried out with aeration at 30° C. for 69 hours under conditions of p, m, and aeration rate of 201/min. The culture solution was centrifuged using a Sharpless centrifuge (10,000 r, p, m, ) to separate the bacterial cells and the culture supernatant.

The obtained bacterial cells were mixed with 161 g of 75% acetone water, stirred and extracted, and separated to obtain an acetone extract. This extract was condensed to 1 mm under reduced pressure. Add 5 ethyl acetate to the trowel condensate
The operation of adding 00d and stirring and extracting was performed 10 times. The obtained ethyl acetate extract was condensed to 501 Ll under reduced pressure, hexane IA' was added to the condensate, and the mixture was left at 4°C overnight. The resulting precipitate t-F was collected and dried to obtain crude product 2.7II.
I got it.

On the other hand, 30 At of ethyl acetate was added to the culture supernatant, and the mixture was thoroughly stirred and separated by centrifugation. The ethyl acetate layer was concentrated to 50 m under reduced pressure, and hexane was added to the concentrated solution.
The mixture was allowed to stand at 4°C. The resulting sediment was removed and dried to a rough texture of 4.3 mm! I got 1. This was combined with the above crude product, and the mixture was mixed with as little chloroform-methanol (50:
1) and then diluted with chloroform-methanol (s
A column of 200 g of silica gel (manufactured by Merck & Co., Ltd., 1000-Selgel 60, Art 7734) packed with o:t) was charged, and column chromatography was performed using chloroform-methanol (50:1) as elution. Each fraction was tracked by silica gel thin J-chromatography (physical and chemical properties section @ Nateru) and a bioassay method involving Micrococcus fistulae, and the active ingredients were collected into fractions that were collected under reduced pressure and analyzed with magnetic sardines. Manufactured substance 820-2ii-Futa. Add this to as small a amount as possible of chloroform-acetate 7 (
10: l) VCf? M'l)h, silica gel (Merck, Kieselgel 60, Art 7734) pre-filled with mechloroformacetone (10:l)2
Charge the 00II column with chloroform-acetone (10:1) IA'.

Column chromatography was performed eluting with chloroform-acetone (5:1) 17, chloroform-acetone (3:1) 400d, and acetone 500-0. The eluate was fractionated into 20 d fractions, and chloroform-acetone (5
: The 48th to 100th fractions eluted in step 1) were collected and dried under reduced pressure to obtain antibiotic AC-1978 as a colorless amorphous substance. Convergence ff135σ■.

[Brief explanation of the drawing]

Figure 1 is the ultraviolet absorption spectrum of the antibiotic AC-1978, Figure 2 is the infrared absorption spectrum of the antibiotic, and Figure 3 is the infrared absorption spectrum of the antibiotic.
The figure shows the proton nuclear magnetic resonance spectrum of the antibiotic '41J, and FIG. 4 shows the C-13 nuclear magnetic resonance spectrum of the antibiotic. Patent Applicant Tomo Omura Toyo Jozo Co., Ltd. (5CJJ 5JL)""' Procedural Amendment July 21, 1985 Dear Commissioner of the Japan Patent Office 1 Indication of Case 1989 Patent Application No. 28556 3 Make amendments Patent applicant address: 5-12-7 Seta, Setagaya-ku, Tokyo Name: Satoshi Omura (and one other person) 4. Spontaneity of the agent6. Number of inventions increased by amendment l), Page 5 of the specification 1st line from the bottom "Dark Convert Gray (3ih) J" should be corrected as 1 Dark Covert Gray (2ih) J. Powder, 3rd line from the top of page 7 of the specification [Light Lie=]
(2ea) J is called “dark brown (anl) J
I am corrected. 3), 4th line from the top of the same page says "F does not grow" 1 Abundant growth, velvety, slightly damp, silver -
Correct it to Gray (3fe) J. 4) On the 6th line from the top of the same page, the text ``F Light Beige'' is corrected to ``Bisla''. 5) In the first line from the top of page 19 of the specification, the statement "F2.7°C" is corrected to "27°C." 6) In the fourth line from the top of the 20th page of the specification, the phrase ``--untreated'' is corrected to ``--night device.'' 7) The claims of the present application are amended as shown in the attached sheet. The amended claim υ is an antibiotic AC-1978 that incorporates the following physical and chemical properties.
Or that wall. ■Elemental analysis: Carbon, hydrogen, nitrogen and oxygen, elemental analysis value: c 57.1 a%, H6.06chi, N1
1,72 chi, ■Molecular t; l l 29 (according to FD-mass spectrum), ■Melting point; 166~iss℃ ■Specific optical rotation: [α)''=21.6° (C=1.methanol), ■Ultraviolet absorption spectrum: As shown in Figure 1 ■Infrared absorption spectrum: As shown in M2 Figure ■Solubility in solvents: Soluble in acetone, methanol, ethanol, butanol, ethyl acetate, RI00 form, insoluble in water and hexane , ■Color reaction; positive for iodine reaction, negative for phosphomolybdic acid reaction, ninhydrin reaction, and sulfuric acid reaction; ■distinguishing between basic, acidic, and neutral; neutral; ■color of substance; colorless. 2), Streptomyces Antibiotic AC-1 belonging to the genus Ses
7. A method for producing antibiotic AC-1978t bamboo shoot salt, which comprises collecting antibiotic AC-1978 from the obtained culture. 3) The production method according to claim 2, wherein the antibiotic AC-1978 producing bacterium is Streptomyces sp. AC-1978.

Claims (1)

[Claims] 1) Antibiotic AC-197 having the following physical and chemical properties
8 or its salt. ■Genka analysis: Consists of carbon, hydrogen, nitrogen, and oxygen; base analysis value: c 57. t a%, H6,06%, N
11.72%, ■Molecular i; 1129 (according to FD-mass spectrum)
, ■Melting point; 166-168℃ ■Specific optical rotation; [α), = 21.6° (c=i, methanol), ■Ultraviolet absorption spectrum; as shown in Figure 1, ■Infrared absorption spectrum; as shown in Figure 2 ■Solubility in solvents; soluble in acetone, methanol, ethanol, butanol, ethyl acetate, chloroform, insoluble in water and hexane; ■Color reaction; positive for iodine reaction, phospholipdic acid reaction, ninhydrin reaction, Negative for chain acid reactions, ■Distinguish between basic, M, and neutral; Color of neutral substances: Colorless. AC-19, an antibiotic belonging to the genus Streptomyces,
78 main products 1! 1. A method for producing antibiotic AC-1978t or a salt thereof, which comprises culturing the antibiotic AC-1978t in a medium and removing the antibiotic AC-1978 from the obtained culture. 3) The production method according to claim 2, wherein the antibiotic AC-1978 producing bacterium is Streptomyces sp. AC-1978.