JPS61141889A - Novel antibiotic sf-2197b and its preparation - Google Patents

Abstract

NEW MATERIAL:An antibiotic, SF-2197Bo having following physical and chemi cal properties: Elementary analyses in %: C 59.22, H 9.31, N 12.37, O 19.1 (remainder); molecular weight: 480 (SIMS method); melting point: 78 deg.C; [alpha]D<25>=-20.2 deg. (c=1 in methanol); soluble in methanol, chloroform, acetone, ethyl acetate, insoluble in water, n-hexane; positive to Roemer's reaction, iodine reagent, ferric chloride reagent, negative to ninhydrin and sulfuric acid reagent. USE:Antibacterial against gram-positive, gram-negative and anaerobic bacteria. PREPARATION:A strain in Microbispora such as Microbispora sp. SF-2197 (FERM P-7213) is cultured, preferably by submerged culture at 25-35 deg.C for 2-7 days.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibiotic SF-2197B substance and a method for producing the same. More specifically, it relates to a novel antibiotic SF-2197B substance and a method for producing the substance, which comprises culturing SF-2197B substance-producing bacteria belonging to the genus Microbispora and collecting SF-21978 substance from the culture. .

The present inventors searched for new and useful antibiotics that have antibacterial activity against various Durham-positive and -negative bacteria as well as anaerobic bacteria. As a result, the novel antibiotic SF- The present invention was completed by discovering that substance 2197B was produced, isolating its effective substance pure from the culture, and determining its physicochemical and biological properties.

As an example of the SF-2197B substance-producing bacteria used in the present invention, actinomycetes S
There is strain F-2197. The mycological properties of SF-2197 strain are as follows.

② Morphological properties Simply branched aerial mycelium is often attached to glucose-asparagine agar, Beneft agar, etc., and many short chains of alternating spores are formed on the aerial mycelium. When observed using an electron microscope,
Chains of spores often consist of two, but chains of three are also common, and chains of four spores are also common. The surface of the spore is smooth, the shape of the spore is oval to oval, and the size is 0.4 to 0.6.
Xo, 6 to 1.2 μm. No motility observed. The basal hyphae were well elongated and branched, with no division observed, and no other special structures such as sporangia, sclerotia, or binding filaments were observed.

The growth conditions of the SF-2197 strain on various media are shown in this table. Culture at 28℃, observation at 14-21
This was done after one day of culture.

■, Physiological properties (11 Growth temperature range: 15 to 15 in yeast malt agar)
It grows in a temperature range of 42°C and grows well at 25-30°C.

(2) Liquefaction of gelatin: Negative (3) Hydrolysis of starch: Negative (4) Peptonization of skim milk: Negative Coagulation of skim milk: Negative (5) Reduction of nitrate: Negative (6) Salt tolerance: 5% salt However, it will not grow at 7% or more.

(7) Production of melanin-like pigment: doubtful ■, availability of carbon source (Breedham-Gotzlieb agar medium) (1) Sugars used: D-glucose, D-fructose, D-xylose, glycerol, L- Arabinose, D-mannitol, raffinose, L-rhamnose, Shakrose (2) Unused li:i-inositol V, @Cell wall composition Becker et al.'s method [^pp1. Mi
crobiol.

12.421 (1964)) and Le Civallier (Le
chevalier) method (J, Lab, Cl1
n, Med,, 71.

934 (196B)), diaminopimelic acid in the whole cell hydrolyzate was DL type, and xylose and arabinose were not detected.

Based on the above mycological properties, strain SF-2197 is a member of the genus Microbispora and Microtetraspora among actinomycetes.
It is considered to be an intermediate strain of the genus, but mainly consists of two spore chains.
Considering its individuality, we decided to place it in the genus Microbispora.

Therefore, the present inventors used the SF-2197 strain as Microbispora sp. SF-2197 (Microbispora sp.
ra sp, SF-2197). In addition, this strain has been submitted to FIKEN with accession number 7213 (FERM
P-7213).

The SF-2197 strain is susceptible to changes in its properties, as seen in the case of other actinomycete strains, and can be mutated by artificial mutagenic means using, for example, ultraviolet rays, X-rays, radiation, chemicals, etc. SF-
Any substance capable of producing 2197B substance can be used in the method of the present invention.

In the method of the present invention, the strain is cultured in a medium containing nutrients that can be utilized by common microorganisms. As the nutrient source, known ones that have been conventionally used for culturing actinomycetes can be used.For example, as the carbon source, glucose, glycerol, shakerose.

Starch, starch syrup, dextrin, double starch, oatmeal, edible oil, etc. can be used. Further, as the nitrogen source, soybean flour, wheat germ, cottonseed meal, tomato ketchup, cornstarch liquor, meat extract, peptone, yeast extract, ammonium sulfate, thorium nitrate, etc. can be used. Calcium carbonate, sodium chloride, cobalt chloride as needed.

In addition to adding inorganic salts such as phosphates and magnesium sulfate, organic and inorganic substances that aid the growth of bacteria and promote the production of the SF-2197.B substance can be appropriately added.

The most suitable culture method is a liquid culture method under aerobic conditions, especially a deep culture method, similar to known methods for producing general antibiotics. The appropriate temperature for culturing is 20°C to 35°C, but in most cases it is preferable to culture at a temperature in the range of 25°C to 30°C.

The production of SF-2197B substance varies depending on the medium and culture method used, but the accumulation usually reaches its maximum within 2 to 7 days when using a shaking culture method or a deep culture method using a culture tank.

The following method is used for assaying the SF-2197B substance. Gum agar medium (Tamizu Pharmaceutical Co., Ltd.) is used as the assay medium. Bacteroides fragilis is used as the test bacterium.

The SF-2197B substance has a 0.25. Give a circular diameter of ljg/m1 to 1 (paper disk plate method).

Amberlite XAD-
2 (manufactured by Rohm and Haas), Tire Ion HP
Synthetic adsorbents such as -20 (manufactured by Mitsubishi Kasei Co., Ltd.), gel filtration agents such as Sephadex LH-20, and Laval HW-40 (manufactured by Toyo Soda Co., Ltd.).

Precipitation with hexane, solvent extraction with ethyl acetate, column chromatography with silica gel, etc. are effective, but the following method is more efficient. That is, bacterial cells and other solid substances are filtered from the culture solution using a filter aid such as diatomaceous earth, and then the active ingredients in the filtrate are filtered using Diaion H.
Adsorb onto P-20. After washing the resin part with water and pre-washing with 50% methanol water, the active ingredient is eluted with 80% acetone water.

The active fraction is concentrated under reduced pressure, and the active ingredient is extracted from the concentrated solution with acetone using ethyl acetate. After concentrating this extract under reduced pressure, n-hexane is added to precipitate the active ingredients. A pure substance of SF-2197B can be obtained by subjecting the precipitate to column chromatography such as Sephadex LH-20° silica gel, high performance liquid chromatography, etc. in an appropriate combination.

The physical and chemical properties of the SF-2197B substance are shown below.

(11 element analysis values: carbon 59.22%, hydrogen 9.31%
.

Nitrogen 12.37%, oxygen 19.1% (difference) (2) Molecular weight: m / z 481t (M
The molecular weight is thought to be 480 since a peak of +1) is observed.

(3) Melting point = 78°C (4) Specific rotation: [α]25D = -20.2° (C1
, methanol) (5) Ultraviolet absorption spectrum: Shows no characteristic absorption.

(6) Infrared absorption spectrum: The spectrum measured in the potassium bromide tablet is as shown in FIG.

(7) Solubility: methanol, chloroform, acetone,
Soluble in ethyl acetate. Insoluble in water and n-hexane.

(8) Color reaction Niremu, iodine reagent and ferric chloride reagent positive.

Ninhydrin and sulfuric acid reagents negative.

(9) Rf value of thin layer chromatography: silica gel 6
0 F254 (manufactured by Merck & Co.) Chloroform-methanol (10:1) 0.51 Benzene-acetone (1:1) 0.42 10) Distinction between neutral, acidic, and basic: Neutral 11) Color of substance: White 12) 1H-NMR spectrum in double chloroform 40
The spectrum measured at 0 MHz is shown in FIG.

13)”C-NMR spectrum: 10 in deuterated chloroform
The spectrum measured at 0 MHz is shown in FIG.

Next, the minimum inhibitory concentration (MIC) of the SF-2197B substance against various bacteria is shown in Table 1, and the minimum inhibitory concentration (Ml-C) against various anaerobic bacteria is shown in Table 2. As these tables show, substance SF-2197B has antibacterial activity against various Durham-positive bacteria and anaerobic bacteria, and is used for the prevention and treatment of diseases in humans and animals caused by these bacteria and anaerobic bacteria.

Based on the above-mentioned properties, SF-2197A compound ir (Japanese Patent Application No. 58-1
89591), but the SF-2197B substance was determined to be a new antibiotic as it was clearly distinguished from the SF-2197A substance based on mass spectrometry values and NMR spectra.

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

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

Example 1 Inoculum culture medium (glucose 1.0%, starch 1.0%,
Peptone 0.5%, yeast extract 0.3%, soy flour 0
．． 2%, meat extract 0.2%, calcium carbonate 0.1
%) in a 100 ml Erlenmeyer flask.

After dispensing sterilization, Microbispora sp. 5P-2197
The strain (FERM P-7213) was inoculated into 3 to 4 platinum loops from yeast starch agar slant culture, and cultured at 28°C for 3 days. 50mf of the obtained inoculum was added to the above medium IIt.
Dispense into two sterilized 1-volume Erlenmeyer flasks and inoculate.
The culture was incubated with shaking at 8°C for 24 hours.

Next, this inoculum 11 was sterilized by putting the above medium 351 into two 501-capacity jar fermenters, and then inoculated at 28°C.
It was cultured for 4 hours. A production medium obtained by sterilizing this seed culture solution 301 (6.0% starch syrup, 0.3% soybean oil, 1.0% Sungrain (manufactured by Suntory).

Cottonseed meal 1.5%, gluten meal 1.2%, wheat germ 0
．． 8%, calcium carbonate 0.2%”) 2001 3
The seeds were inoculated into two 0.001 volume stainless steel tanks, and cultured with aeration and stirring at 28°C for 4 days (aeration amount: 20,017 minutes, initial stirring: 1
00 revolutions/minute, 150 revolutions/minute from the second day).

After the culture was completed, the pH of the culture solution was adjusted to 2.0, and the culture solution was filtered using diatomaceous earth as an auxiliary agent to obtain culture solution 2801.

Example 2 After adjusting the pH of the culture solution 28 (l) obtained in Example 1 to 7.0, the active ingredient was adsorbed onto Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation) 151 by a batch method.

After collecting the resin, it was packed in a tower and washed with 30f of water.
Pre-washing was performed with 601% methanol water.

Further, elution was performed with 80% acetone water. Activity category 30
1 was collected, concentrated under reduced pressure, and diluted with acetone. Concentrate 1
10f of ethyl acetate was added to 01 to extract the effective substance.

This extract was concentrated under reduced pressure, and when the volume reached 100 ml, n-hexane IIt was added and allowed to stand, causing the effective substance to precipitate. After removing the supernatant, the precipitate was dissolved in methanol, applied to a Sephadex LH-20 (manufactured by Pharmacia) 700mf column, and developed with methanol. The effective substance was eluted in fractions 24 to 36.

The active fractions were combined and concentrated to dryness under reduced pressure to yield 7.8 g of a crude powder of SF-2197B material.

Wakogel C-2 filled with this coarse powder with chloroform
00 (manufactured by Wako Pure Chemical Industries, Ltd.) in a 250mj2 tower and developed with a chloroform-methanol (50:1) mixture, l
Effective substances were eluted in fractions 80 to 130 in the 5r+nJ fraction. The active fractions were combined and concentrated to dryness to yield 1.9 g of crude powder of SF-2197B material. Toyopearl H filled with this 1.9g with methanol
W-40 (manufactured by Toyo Soda Co., Ltd.) 500mj! The active fractions were obtained in fractions 24 to 30 in a 20 ml fraction. The active fractions were combined and concentrated to dryness under reduced pressure, resulting in SF-2
880 ml of coarse powder of 197B material was obtained.

Example 3 Coarse powder 30 of SF-2197B substance obtained in Example 2
0 h to 1 mj! YMC-PAC
High performance liquid chromatography using a KS-343 (ODS) (Yamamura Kagaku Co., Ltd.) column (20 φ x 250 mm) was developed with 70% methanol at a flow rate of 4 ml/min, and the active fraction was concentrated to dryness and purified at 80 WI. ! A crude powder of I was obtained.

Furthermore, this coarse powder was filled with acetonitrile.
It was attached to a 20,200 ml column, developed with acetonitrile, and the active fraction was concentrated to dryness under reduced pressure to obtain 17 mg of white powder of SF-2197B substance. This substance has the above-mentioned physicochemical properties.

[Brief explanation of the drawing]

FIG. 1 is an infrared absorption spectrum of SF-2197B substance in potassium bromide tablets. Figure 2 is SF-2197
It is an IHN-IR spectrum of substance B in a deuterated chloroform solution. Figure 3 is the C-NMR spectrum of the SF-2197B substance in a deuterated chloroform solution.

Claims (2)

[Claims] (1) SF-2197B substance having the following physical and chemical properties. 1) Elemental analysis values: carbon 59.22%, hydrogen 9.31%,
Nitrogen 12.37%, oxygen 19.1% (difference) 2) Molecular weight: m/z 481 by mass spectrometry (SIMS)
The molecular weight is thought to be 480 because a peak of (M^++1) is observed. 3) Melting point: 78℃ 4) Specific rotation: [α]^2^3=-20.2° (C1,
Methanol) 5) Ultraviolet absorption spectrum: Shows no characteristic absorption. 6) Infrared absorption spectrum: As shown in FIG. 7) Solubility: Soluble in methanol, chloroform, acetone, and ethyl acetate. Insoluble in water and n-hexane. 8) Color reaction: Lemieux, iodine reagent and ferric chloride reagent positive. Ninhydrin and sulfuric acid reagents negative. 9) Rf value of thin layer chromatography: silica gel 60
F254 (manufactured by Merck & Co.) Chloroform-methanol (10:1) 0.51 Benzene-acetone (1:1) 0.42 10) Distinction between neutral, acidic and basic: Neutral 11) Color of substance: White 12 )^1H-NMR spectrum: As shown in FIG. 13)^1^3C-NMR spectrum: As shown in Figure 3. (2) SF-219, an antibiotic belonging to the genus Microbispora
A method for producing a new antibiotic SF-2197B substance, which comprises culturing 7B-producing bacteria in a medium and isolating an SF-2197B substance having the following physicochemical properties from the culture. 1) Elemental analysis values: carbon 59.22%, hydrogen 9.31%,
Nitrogen 12.37%, oxygen 19.1% (difference) 2) Molecular weight: m/z 481 by mass spectrometry (SIMS)
The molecular weight is thought to be 480 because a peak of (M^++1) is observed. 3) Melting point: 78°C 4) Specific rotation: [α]^2^5_D=-20.2°(C
1. Methanol) 5) Ultraviolet absorption spectrum: Shows no characteristic absorption. 6) Infrared absorption spectrum: As shown in FIG. 7) Solubility: Soluble in methanol, chloroform, acetone, and ethyl acetate. Insoluble in water and n-hexane. 8) Color reaction: Lemieux, iodine reagent and ferric chloride reagent positive. Ninhydrin and sulfuric acid reagents negative. 9) Rf value of thin layer chromatography: silica gel 60
F254 (manufactured by Merck & Co.) Chloroform-methanol (10:1) 0.51 Benzene-acetone (1:1) 0.42 10) Distinction between neutral, acidic and basic: Neutral 11) Color of substance: White 12 )^1H-NMR spectrum: As shown in FIG. 13)^1^3C-NMR spectrum: As shown in Figure 3.