JPH0353891A - New antibiotic substance sf2197c and production thereof - Google Patents

Abstract

NEW MATERIAL:The antibiotic substance SF2197C expressed by formula and having the following physical and chemical properties. Appearance, colorless acicular crystal; melting point, 54 deg.C; specific rotation, [alpha] =-37.2 deg. (C=1, methanol); elemental analysis (%), C 59.6, H 9.3, N 11.3; mass spectrometry, molecular ion peak at m/z=440(M<+>) by FD-MS; molecular formula, C22H40N4O5; solubility, soluble in methanol, chloroform, acetone and ethyl acetate and insoluble in water and n-hexane; color reaction, positive to Lemieux, iodine reagent and FeCl3 reagent and negative to ninhydrin reagent; etc. USE:Antibacterial agent. PREPARATION:A microbial strain belonging to genus Actinomadura such as Actinomadura atramentaria (FERM P-7213) is cultured by submerged culture process at 25-32 deg.C for 2-7 days.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention is directed to a new antibiotic SF2197C substance and a new antibiotic SF21 produced by a microorganism belonging to the genus Actinomadura.
This invention relates to a method for producing a 97C substance.

[Prior Art and Problems to be Solved by the Invention 1] Various antibiotics produced by microorganisms have been known and have been put to practical use in the fields of pharmaceuticals, veterinary drugs, agricultural chemicals, etc. However, due to problems such as the appearance of resistant bacteria, there is a constant demand for the emergence of new antibiotics.

[Means for Solving the Problem 1] The present inventors have discovered that by first culturing a specific microorganism, a substance with strong antibacterial activity is produced and accumulated in the culture solution, and the effective substance They succeeded in collecting the substances, named them SF2197A substance and B substance, and filed a patent application (Japanese Patent Application Laid-open No. 60-83585 and No. 61-141889). The present inventors discovered that another new effective substance was produced in the culture fluid of the same strain, isolated this substance, and used the antibiotic SF2.
It was named 197C substance.

The present invention has been completed based on the above findings.

That is, the first gist of the present invention is that the new antibiotic SF2197
This invention relates to Substance C, and the physicochemical and biological properties of this substance are as follows.

(1) Physical and chemical properties of SF2197C substance 1) Appearance: Colorless acicular crystals 2) Melting point = 54°C 3) Specific rotation: [α): 0 = − 37.2° (C1,
methanol) 4) Elemental analysis values: carbon 59.6%, hydrogen 9.3%, nitrogen 11.3% 5) Mass spectrometry: Molecular ion peak m by FD-MS
/z 440 (M”) was observed.

6) Molecular formula: C22H4oN405 7) Ultraviolet absorption spectrum: Shows no characteristic absorption.

8) Infrared absorption spectrum: As shown in FIG.

9)'H-NMR spectrum: As shown in FIG.

lO)l3C-NMR spectrum: As shown in FIG.

11) Solubility: Soluble in methanol, chloroform, acetone, and ethyl acetate. Water, rr Insoluble in hexane.

12) Color reaction: Positive for Lemieux, iodine reagent and ferric chloride reagent. Negative for ninhydrin reagent.

13) Thin layer chromatography Using a thin layer of silica gel (manufactured by Merck & Co., Ltd., Art 5714), the developing solvent was chloroform-methanol (10:1).
), Rf value 0.39, toluene acetone (1
:1) Rf value 0.44 As a result of the above-mentioned physical and chemical properties and structural analysis by NMR, etc., the structure of this substance was determined as shown in the following formula.

CH, 1 (2) S. Biological properties of the F2197G substance The minimum inhibitory concentration (MIC) of the SF2197G substance according to the present invention against aerobic bacteria is shown in Table 1, and the old C against anaerobic bacteria is shown in Table 2. Table 3 shows the MIG for As these tables show, the SF2197C substance has antibacterial activity against various Durum-positive bacteria, Durum-negative bacteria, Mycoplasma, Treponema, etc., and exhibits particularly excellent antibacterial activity against anaerobic bacteria and Campylobacter. Therefore, people who are caused by these bacteria. It can be used for the prevention and treatment of animal diseases.

=7 Table 1 Test bacteria MIC (μg7mQ) Medium Medium: Sensitivity Disk Agar-N
(Nissui) Medium 2: PPLO Broth supplemented with 15% horse serum (Difco
) 8 Table 2 Test seedling MIC (μg/m(2) Medium 5462 0.025 1 Medium 1: GAM Agar (Nissui) Medium 2 = Trypticase Soy Ag with 5% horse blood added
ar (BBL) Table 3 Test bacteria MIC (μg/m(1) Medium: Heart lnfusion supplemented with 5% horse blood
Agar (Difco) (3) Acute toxicity of SF2197C substance In acute toxicity test on mice, 100mg/kg
All patients survived after intraperitoneal administration. No abnormality was observed.

The second gist of the present invention is to culture a new antibiotic SF2197C-producing bacterium belonging to the genus Actinomadura,
The present invention provides a method for producing a new antibiotic SF2197C substance, which comprises collecting the new antibiotic SF2197C substance from the culture.

(1) Bacterium producing SF2 1 97c substance An example of a bacterium producing SF2197C substance used in the present invention is JP-A-60-8358. ．． 5 “New antibiotic SF2197
"Substance A and its manufacturing method" and JP-A-61-141889
There is an actinomycete strain SF2197 whose mycological properties are described in "New antibiotic SF2197B substance and its production method". In these patent specifications, the present inventors discovered that the SF2197 strain was identified as Microhispora among actinomycetes.
a) This strain was determined to belong to the genus Microbisvora SB SF 2 197 (Microbisbora SB).
sporasp. sF2197). but,
As a result of subsequent detailed taxonomic research, strain SF2197 was found to be a new species of the genus Actinomadura, and was found to be a new species of the genus Actinomadura.
raatramentaria). These results are published in lnlJ. Syst. Bacteriol. ．．
37:342-346 (1987), and the name of this bacterium has already been approved. Therefore, the present inventors determined the mycological properties of the SF2197 strain as described in this document, and also submitted the microbiological characteristics of the SF2197 strain as described in this document, and also the microbiological characteristics of the SF2197 strain, and
) Microbispora sp. SF2197 was renamed Actinomadura atramenta 1l rear SF2197. As seen in other actinomycetes, the SF2197 strain is susceptible to changes in its properties. For example, any mutant strain (naturally occurring or induced), transconjugant, or genetically recombinant strain derived from the SF2197 strain that produces the SF2197C substance can be used in the present invention.

(2) Cultivation method of sp2f97c-producing bacteria In the method of the present invention, the aforementioned bacteria are cultured in a medium containing nutrients that can be used by ordinary microorganisms. As a source of nutrients,
Known materials conventionally used for culturing actinomycetes can be used. For example, glucose, starch syrup, dextrin, glycerin, molasses, animal/vegetable oil, etc. can be used as the carbon source. Further, as a nitrogen source, soybean meal, wheat germ, corn steep liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea, etc. can be used. others. Sodium, potassium, as needed
It is effective to add inorganic salts capable of producing calcium, magnesium, cobalt chloride, phosphoric acid, sulfuric acid, and other ions. Further, organic and inorganic substances that aid the growth of bacteria and promote the production of SF2197C substance can be appropriately added.

The most suitable culture method is a culture method under aerobic conditions, especially a deep culture method. The appropriate temperature for culturing is 20'0-3
The temperature is 5°C, but in most cases it is cultured at 25°O-32°C. Production of SF2197C substance varies depending on the medium and culture conditions. In both shaking culture and tank culture, the accumulation usually reaches its maximum within 2 to 7 days. When the accumulated amount of the SF2197C substance during culture reaches the maximum, the culture is stopped, and the target substance is isolated and purified from the culture solution.

(3) Extraction and purification method of SF2197C substance When collecting the SF2197C substance obtained by the present invention from the culture solution, conventional separation methods utilizing its properties such as solvent extraction method, ion exchange resin method, adsorption method, etc. Alternatively, extraction and purification can be carried out using partition force chromatography, gel filtration, precipitation, etc. alone or in appropriate combinations. For example, the SF2197C substance in the culture filtrate is adsorbed to a combination adsorbent such as Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation) and eluted with acetone water or the like. To further purify the SF2197C material, use silica gel (Wako Gel C-20
0. Chromatography using an adsorbent such as alumina (manufactured by Wako Pure Chemical Industries, Ltd.), Sephadex LH-20 (manufactured by Pharmacia), Toyopearl HW-40 (manufactured by Tosoh), etc. may be performed.

Examples of the present invention are shown below, but these are merely examples and do not limit the present invention.

Of course, many variations and modifications not exemplified here may be used.

Example 1 Starch 2.0%, glucose 1.0% as seed medium
, polypeptone 0.5%, wheat germ 0.6%, yeast extract 0.3%. Soybean meal 0.2% and calcium carbonate 0.2
% (pH 7.0 before sterilization) was used.

In addition, as a production medium, soybean oil 0.7%, sodium acetate 0.5%, corn stew-3.0% defatted rice bran 1.0%, magnonium sulfate (heptahydrate) 0.2%, sulfuric acid Copper (pentahydrate) 0.001% and common salt 02% (pH before sterilization)
7.0) was used.

A 100 mff Erlenmeyer flask into which 20 ml of the seed medium was dispensed was sterilized at 120°C for 15 minutes, and Actinomadura atramentaria strain SF2197 (FE
Two to three platinum loops of slanted agar culture of RNp-7213) were inoculated and cultured with shaking at 28° C. for 5 days to form a type 1 culture.

Next, a 500 mQ Erlenmeyer flask containing 80 mff of the seed medium was sterilized at 120°C for 15 minutes, inoculated with 4 mQ of the first type culture, cultured with shaking at 28°C for 2 days, and this was used as the second type culture. . Furthermore, a 5Q volume Erlenmeyer flask into which 1Q of the seed medium was dispensed was sterilized at 120°C for 30 minutes, and 80+J of the second type culture was inoculated and cultured with shaking at 28°C for one day, which was used as the third type culture.

Inoculate the above third type culture 14 into a 50Q. jar fermenter containing a seed medium of 35 that had been previously sterilized at 120°C for 30 minutes. Aerate (20M min) and stir (20 rpm) at 28°C for 1 day. This was used as the 4th type culture.

15 100% of the above type 4 culture was placed in two 300Q capacity tanks containing 200a of production medium that had been previously sterilized at 20°C for 30 minutes. Inoculated with 10012
/min) and agitation (12 rpm). After the cultivation was completed, diatomaceous earth was added as a filter aid and the mixture was filtered to obtain 350 ml of filtrate.

Example 2 The culture filtrate 350Q obtained in Example 1 was adsorbed on Diaion HP-20 (manufactured by Mitsubishi Kakaisha) 17Q, washed with water, and then pre-washed with 50% methanol water. The effective fraction was eluted with 50I2 of 80% acetone water, and the eluate was concentrated to 15Q and extracted with 1812 of ethyl acetate. The extract was concentrated to dryness to obtain 35 g of an oil containing SF2197C substance. This oily substance was applied to a column of 300 mQ of silica gel column (Wako Gel C200, manufactured by Wako Pure Chemical Industries, Ltd.), and chloroform l. After washing with 5Q, chloroform-methanol (50
: 1) 2(2, chloroform-methanol (20:1
) 212 and fractionated into 100ml2 portions.

The active fractions were collected and concentrated to dryness to obtain 15 g of an oil containing SF2197C substance. This oil was again applied to a 200 m4 column (Wako Gel C-200), and chloroform IQ, chloroform-
Methanol (50:1) IQ and chloroform-methanol (20:1) 1 (2) were eluted in this order. The active fractions were collected and concentrated to dryness to obtain 8 g of a crude oil of substance SF2197C. 8 g of this crude oil Sephadex LH-20 (manufactured by Pharmacia) equilibrated with methanol 500 m
It was applied to a Q column and developed with the same solvent. The active fractions were collected and concentrated to dryness to obtain 3 g of oil. This oil was dissolved in 5 ml of acetone, adsorbed on a Wako Gel C-300 100 mf2 column filled with n-hexane, and developed with a mixed solvent of n-hexane-acetone (1:1) to extract the fraction containing the SF2197C substance. Obtained 600m+2. This active fraction was concentrated under reduced pressure, dissolved in a small amount of methanol, and Toyovar HW-40 equilibrated with methanol in advance.
(manufactured by Tosoh Corporation) It was applied to a 300n+I2 column and developed with the same solvent. The active fractions were collected and concentrated to dryness to obtain 820 mg of oil. Dissolve this in 3mQ methanol and
m+2 high performance liquid chromatography preparative reverse phase column S-343 (Yamamura Chemical Laboratory OD5, 20X
250 mm) and eluted with 70% methanol water at a flow rate of 5 m (2/min). Fractions showing activity against Bacillus subtilis were collected and concentrated to dryness to obtain 240 mg of a crude powder of SF2197C substance. This coarse powder was dissolved in a small amount of acetone, n-hexane was added and left to stand, and SF2197C substance 8 was dissolved.
0 mg was isolated as colorless needles.

[Effects of the Invention] Since the new antibiotic SF2197C substance of the present invention has excellent antibacterial activity against various bacteria, it can be expected to be useful as an antibacterial agent.

[Brief explanation of drawings]

FIG. 1 shows the infrared absorption spectrum of the antibiotic SF2197C substance in potassium bromide tablets. FIG. 2 shows the 'H-NMRs6 vector of the antibiotic SF2197C substance in dimethyl sulfoxide d solution.

Claims (2)

[Claims] (1) SF21, a new antibiotic with the following physical and chemical properties
97C substance 1) Appearance: Colorless acicular crystals 2) Melting point: 54℃ 3) Specific rotation: [α]^2^0_D=-37.2° (c
1. Methanol 4) Elemental analysis values: 59.6% carbon, 9.3% hydrogen, 11.3% nitrogen 5) Mass spectrometry: Molecular ion peak ￥m￥/￥￥440 (M＾+ ) was recognized. 6) Molecular formula: C_2_2H_4_0N_4O_57) Ultraviolet absorption spectrum: Shows no characteristic absorption. 8) Infrared absorption spectrum: As shown in FIG. 9)^1H-NMR spectrum: As shown in FIG. 10)^1^3C-NMR spectrum: As shown in Figure 3. 11) Solubility: methanol, chloroform, acetone,
Soluble in ethyl acetate. Insoluble in water and n-hexane. 12) Color reaction: positive for Lemieux, iodine reagent and ferric chloride reagent. Negative for ninhydrin reagent. 13) Thin layer chromatography: Using a thin layer of silica gel (manufactured by Merck & Co., Art 5714), the Rf value is 0.39 when the developing solvent is chloroform-methanol (10:1), and the developing solvent is toluene-acetone (1:1). In case Rf value 0.44 (2) SF21, a new antibiotic belonging to the genus Actinomadura
97C-producing bacteria are cultivated, and the new antibiotic SF is produced from the culture.
A method for producing a new antibiotic SF2197C substance, which comprises collecting the 2197C substance.