JPS5817198B2 - FR-3383 substance - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION This invention relates to a novel antibiotic PR-3383 substance.

This invention provides a new antibiotic that is particularly effective against Durham-negative bacteria.

The physicochemical properties of the FR-3383 substance of this invention are as follows.

■ Elemental analysis value (to): C; 53.5. H: 6.0, N: 12.6. S:3
, '3 ■ Molecular weight: (vapor pressure drop method) 50 ■ Specific optical rotation 2 [α] = +99 (C = 0.2, solvent: 50% methanol water) ■ Ultraviolet absorption spectrum (Figure 1): λH, 0 again'''!0°INHC/,-H,0,253
nmaX (solid line) λ0°1N"0H"".

:240nm (broken line) ax ■ Infrared absorption spectrum (Figure 2): Streak 3-'lz (α-1): 660.695.

It can be produced by culturing the 'max 3383 substance-producing bacteria in a medium, and separating and collecting the FR-3383 substance from the resulting culture.

FF? ,,= Among the 3383 substance-producing bacteria, the strain newly isolated by this inventor from the soil of Odaigahara, Nara region (412
71) has the following mycological properties.

(2) Morphological properties The results of microscopic observation of the aerial mycelium grown on the sucrose/nitrate agar medium are as follows.

(1) Branching method of spore-forming hyphae: simple branching (2) Morphology of spore-forming hyphae: straight spore-forming hyphae have relatively few branches and grow straight.

The tip also forms a chain of many conidia.

(3) Number of spores: several dozen (4) Surface structure and size of spores: smooth, 0.9 to 1
, I Properties and Physiological Properties 'Properties on various media listed below are measured at 430℃ for 10 minutes unless otherwise specified.
This is an observation after culturing for ~14 days.

720.770.810.840.880.

960.1060, 1105, 1172°1180.1
230,1260,1380゜1460.1512,1
538,1550.

1563.1600. ．． 1660, 1670°1680
．． 1690, 1710, 2870゜2930.2960
．． 3310 ■ Solubility in solvents: Easily soluble in water, dimethyl sulfoxide, and dimethylformamide, slightly soluble in methanol, insoluble in acetone, ethyl acetate, and chloroporum ■ Properties of the substance: Amphoteric substance ■ Color reaction: Biuret reaction, The Fon reaction and the ferric chloride-potassium ferricyanide reaction were each positive, and the Anthrone reaction, Dragendorff reaction, Molisch reaction, Ehrlich reaction, and Sakaguchi reaction were each negative.

■ Thin layer chromatography 278 units: Eastman cellulose film //6
6065 Solvent Rf value water saturated n-butanol 0.10 PR-3383 product of this invention @1 tel lower pp-3,
Physiological properties (1) Growth temperature range 15-40℃, optimum temperature: 28℃ (2) Liquefaction of gelatin (on glucose/peptone gelatin medium) Positive (3) Hydrolysis of starch (on starch/inorganic base medium) ) Positive (4) Both coagulation and peptonization of skim milk are positive (5) Production of melanin-like pigments (on tyrosine agar and peptone yeast iron agar) Positive 4. Assimilation of each carbon source (Pridham Gosotry) (on agar medium) ■ L-arabinose ■ D-xylose + ■ D-glucose + ■ D-fructose ■ Sucrose − ■ Inositol − ■ L-rhamnose + ■ Raffinose − ■ D-Mannitol − [Phase] Mannose + 0 Salicin + (-2 Frequently used.

Used only by a few people. -2 Do not use.

) 5. Cell wall composition (10: presence is recognized).

-The existence of two is not recognized.

) The 41271 strain with the above mycological properties is described in The Active Myces Vol. 2 (1) by Waxman.
961) and Schirling and Gottlieb 1
5P (International Streptomyces prophylaxis) Reports (1968, 1939 and 1972)
Search by year): 1. /161271 strain is particularly closely related to Actino-myces albovinaceus.
s), Actinmyces 0 candidus (Abtin
Streptomyces pseudogriseolus containing Streptomyces candidus
pseudogriseolus) and Streptomyces albolongus
However, strain 41271 differs from these bacteria in the following points.

(1) Actinmyces albobinaceus: Does not produce melanin-like pigments or soluble pigments.

Use D-mannit.

Spore settlement is low. (2) Actin Myces candidas does not produce nimelanin-like pigments and soluble pigments.

Use D-mannitol.

(3) Streptomyces pseudogriseolus: The shape of aerial hyphae is different.

Does not produce melanin-like pigments.

Use D-mannit. (4) Streptomyces arborongus: Aerial mycelium has some color on oatmeal agar medium and glycerin-asparagine agar medium.

Does not produce soluble pigments.

Free from rhamnose and fructose.

In addition to the above, the /V5.1271 strain also differs from the four related strains described above in that it produces the novel antibiotic PR-3383 substance described below.

From these results, the A61271 strain is recognized as a new bacterial species, and it was classified as Streptomyces audainensis (
Ssreptomyces odainensis).

This Streptomyces audainensis 1271
The strain was transferred to the Institute of Microbiological Technology, Agency of Industrial Science and Technology.
It has been deposited as No. 702.

FR belonging to the genus Streptomyces used in this invention
-3383-producing bacteria can be treated with irradiation such as X-rays and ultraviolet rays, such as nitrogen mustard, azaserin, etc.
FFt by commonly used mutagenic means such as treatment with mutagenic agents such as ronitrate, N-methyl-N'-nitro-N-nitrosoguanidine (NTG), and 2-aminopurine, transduction, transformation, and conjugation. - The production capacity of 3383 substances can be increased.

Next, the PR-3383 substance of the present invention is applied to, for example, FFj-3383 belonging to the genus Streptomyces as described above.
A case of manufacturing by culturing substance-producing bacteria in a medium will be explained.

The culture method is basically similar to that of general microorganisms, but deep culture using a liquid medium is usually advantageous.

The medium used for culture may be a decoy medium (i.e., a synthetic medium, a semi-synthetic medium or a natural medium) containing a nutrient source used by the FR-3383 substance-producing bacteria belonging to the genus Streptomyces. As for the composition, carbon sources include glucose, mannose, glycerin, sucrose, molasses, starch, liquefied starch, etc., and nitrogen sources include meat extract, casamino acids, peftone, curten meal, corn meal, and cottonseed meal. , soy flour, corn steep liquor, dried yeast, ammonium phosphate,
Ammonium sulfate, urea, etc. are used.

Inorganic salts such as metal phosphates, carbonates, and chlorides, such as disodium hydrogen phosphate, potassium dihydrogen phosphate, calcium carbonate, and magnesium chloride, may also be added as necessary.

In addition, when foaming is significant during culturing, for example, vegetable oils such as soybean oil and Nohseed oil, and higher alcohols such as octadecanol, tetradecanol, and heptatool.

An antifoaming agent such as a silicon compound may be appropriately added.6 The appropriate temperature after this is around 30.degree. C., and good results are often obtained by carrying out seed culture as appropriate as the culture volume increases.

The appropriate culture time for main culture is about 50 to 100 hours.
The culture time may be further extended as the medium becomes thicker.

The culture conditions described above are applied by selecting optimal conditions depending on the characteristics of the production strain used.

Since the FR-3383 substance accumulated in the culture in this way is mainly contained in the culture solution, after removing the tooth body from the culture by centrifugation or filtration, general antibiotics can be extracted from the filtrate. Separated, collected, and purified by conventional means used in manufacturing.

i.e. vacuum concentration, freeze drying, solvent extraction, liquid conversion,
For example, treatment with resins such as anion exchange resins, cation exchange resins, nonionic adsorption resins, etc. For example, activated carbon, silicic acid,
Treatment with adsorbents such as silica gel and alumina, crystallization,
The FR-3383 substance is separated, collected, and purified from the filtrate by using methods such as recrystallization alone or in combination in any order and repeatedly.

The biological properties of the FR-3383 substance of this invention are as follows.

■ Antibacterial activity Using Nihard infusion medium, the minimum inhibitory concentration of the following test bacteria was determined by the agar dilution method.

(Amount of inoculum: test bacteria culture solution (1o6 cell number/ml)
Minimum inhibitory concentration Test bacteria (kg/ml) Escherichia coli 323 63 Shigella flexneri 2EW-10125 Salmonella enteritetes 32 Proteus vulgaris AM
63-1025 Pseudomonas aeruginosa 63 NCTC10490 ■ Acute toxicity FFt-3383 substance was intraperitoneally administered to mice (dose: 21.9/kg), and no abnormalities were observed.

From the above-mentioned physical and chemical properties and biological properties, FR
Substance -3383 is confirmed to be a new substance.

As mentioned above, PR-3383 substance has antibacterial activity and can be used as a medicine.

Next, the present invention will be explained with reference to examples.

Example A 30 jar containing 20 tons of a medium containing 3% glycose, 2% cottonseed meal, 1% gluten meal, 0.3% calcium carbonate, and 0.2% antifoam agent Adekanol (trademark, manufactured by Mudenka Co., Ltd.) Place in a fermenter and sterilize at 120°C for 20 minutes.

This was inoculated with 3% of a culture of Streptomyces audaionsis 412 strain 1 in a medium with the same composition as above, and while passing sterile air at 20 tons per minute and stirring with a propeller at 300 revolutions per minute. Incubate at 30°C for 72 hours.

After culturing, 2% diatomaceous earth is added to the culture and filtered.

16 tons of filtrate was added to non-ionic adsorption resin Diaion HP20.
(Trademark, manufactured by Mitsubishi Kasei Corporation) through a column filled with resin to adsorb the active substance onto the resin.

The effective substance is eluted from the column with 6 tons of 50% methanol water.

6 tons of eluate was concentrated to 2 tons under reduced pressure, and the resulting concentrated solution was passed through a column filled with anion exchange resin Duolite A6 (OH-type) (trademark, manufactured by Diamond Jamrock Chemical Co.) to remove the effective substance. to be adsorbed.

0.0% of the active substance was extracted from this column. Elute with 3 t of IN aqueous sodium hydroxide solution.

The active fractions are collected, concentrated under reduced pressure, and further freeze-dried to obtain 5 g of a yellow-white powder.

This powder is packed into the top of a column filled with powdered cellulose.

Develop with 85% butanol water, collect active fractions, and concentrate under reduced pressure.

0.5ml of concentrated liquid When the pH is adjusted to 4.5 with IN hydrochloric acid, crystals begin to precipitate.

When these crystals are filtered, collected and dried, a crystalline powder of PR-
700 m9 of 3383 substance is obtained.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet absorption spectrum of the FR-3383 material of the present invention, and FIG. 2 shows its infrared absorption spectrum.

Claims (1)

[Claims] 1. Elemental analysis value (to): C; 53.5, H: 60, N:
12.6, S; 3.3 Molecular weight (vapor pressure drop method) 2950 Specific rotation: (phrase = +9.9 (C = 0.2, solvent; 5
0% methanol water) Ultraviolet absorption7. S1, Tsuyo λH20 mata&i 0・IN
HCA-HρaX + 258 nrn, λO゛”NNa0H-H2O,. 4oo.aX Nujol infrared absorption spectrum Ni (1-1) aX 660.695.720.770.810. 840.880,960,1060,1105°117
2.1180,1230,1260°1380.146
0,1512,1538°1550.1563.160
0.1660. 1670.1680, 1690.1770?2870.
2930.2960.3310 Solubility in solvents: Easily soluble in water, dimethyl sulfoxide and dimethylformamide, slightly soluble in methanol, insoluble in acetone, ethyl acetate and chloroform Properties of the substance Diamphoter Color reaction: Biuret, Folin and The FR-3383 substance is characterized in that the ferric chloride-potassium ferricyanide reaction is positive, and the Anthrone, Dragendorff, Morinsch, Ehritz and Itame reactions are negative, respectively.