JPS5831918B2 - New antibiotic polymyxin S↓1 - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibiotic polymyxin S1 and a method for producing the same.

The antibiotic polymyxin S1 according to the present invention is recognized as a polymyxin antibiotic based on its physicochemical and biological properties, and as described later, it is clearly a novel polymyxin antibiotic based on its amino acid composition and constituent fatty acids. It is a certain substance.

Polymyxin S1 is a substance useful as a medicine and veterinary drug that exerts a strong effect on Durham-negative bacteria.

Next, the physical and chemical properties of polymyxin 51(7) will be shown.

However, the physical constants shown below are for polymyxin S14 hydrochloride.

As described above, polymyxin S1 having the above-mentioned physicochemical properties is a known polymyxin antibiotic, namely polymyxin A1. A2. M.

K, B, , B2. C2Pt, P2. Dl, D
2. It differs from El (colistin A), B2 (colistin B), and circulin A and B in amino acid composition.

Details are as below.

The therapeutic effects of polymyxin are as follows.

As mentioned above, polymyxin S1 has an excellent effect on Durham-negative bacteria, and its toxicity is approximately 25 to 5 when expressed in LD5o.
0/4 (mouse, intraperitoneal administration), and can be used as a medicine, veterinary drug, or disinfectant.

When administering this compound as a pharmaceutical or veterinary drug, it can be administered orally in the form of tablets, capsules, powders, etc., or parenterally as injections, liniments, suppositories, etc.

Note that polymyxin S1 can be made into various acid addition salts depending on the purpose of use.

For example, hydrochloride, sulfate, oxalate, and succinate can be produced by conventional methods.

Bacillus (Bacillus) is a bacterium that produces polymyxin S1.
Bacterial strains belonging to the genus Bacillus polymyxa R8-6 (Ba.
ci flus polymyxa R8-6) is a polymyxin S1 producing bacterium having the following mycological properties.

1. Morphological properties (nutrient agar medium, 28°C, culture for 1-2 days) (1) Fungal membrane size 0.5-1. OX2.5~5.0μ
, most of them are 0.7X3. It is a Durham stain positive bacillus of O~4.0, has a round bacterial membrane, and occurs singly or in clusters.

(2) Spores and sporangia Most spores are 1.
2X1.6μ, oval, easily stained.

It occurs in the center of the bacterium or slightly inside the end.

The sporangium has a distinct bulge.

2. Characteristics on each culture medium (1) No. 172 agar medium' (28°C, cultured for 1 to 2 days) Forms circular colonies with a diameter of 2 to 4 squares.

It has convex ridges and is fully bound with a smooth and glossy surface.

It is rubbery or sticky and changes from translucent to opaque.

(2) Nutrient agar slant medium (28° C., 1-2 days culture) Moderate, filamentous growth is exhibited, the bacterial membrane is grayish white, and the surface is glossy on the first day but becomes flaky on the second day.

The composition is buttery and opaque. No soluble pigments or intracellular pigments were observed.

(3) Nutrient liquid medium (28°C, culture for 1 to 5 days) uniformly shows moderate growth.

Neither ring formation nor bacterial membrane formation was observed.

*The medium composition is as follows.

Soluble starch 2.0%, glucose 1.0%, casamino acids 0.5%, yeast extract 0.5%, calcium carbonate 0.1
%, agar 1.2% (weight/volume).

pH 6.8°1, Physiological properties (1) Enzyme requirement (28°C, 1-2 days culture → GPYB
In the 0-F test on agar puncture medium 78, it was facultatively anaerobic;
Forms acids and gases from glucose.

※※※ (2) Growth temperature (Gly-IM agar medium, 1 day culture) The optimum temperature is about 37°C.

It does not grow at 50°C.

(3) Utilization of citric acid (28℃, 1-2 days culture)
Does not grow on synthetic media.

(4) Hydrolysis of starch (cultured at 28°C, 1 to 3 days) positive (5) Liquefaction of gelatin (cultured at 28°C, 1 to 20 days) liquefies slowly.

(6) Casein hydrolysis (28°C, culture for 2-30 days)
Positive (7) Lydomus milli (cultured at 28°C, 2-30 days) Slowly peptonizes but does not coagulate.

(8) Reduction of nitrate (28℃, 2-4 days culture) negative (9) Production of acetylmethylcarbisol (28℃, 2-4 days culture)
(10) Hydrogen sulfide production (28°C, 2-day culture, Difco-peptone iron medium) Negative (11) Urease activity (28°C, 1-7 days culture) Negative 02) Catalase activity (28°C) , 1-day culture) positive (13) Oxidase activity (28°C, 1-day culture) positive (14) Carbohydrate utilization (28°C, 3-11 days culture) D
- Forms acids from ribose, D-glucose, D-mannose, D-galactose, D-fructose, sucrose, salicin.

A slight acid formation is observed from the full dose and raffinose.

L-arabinose, D-xylose, L-rhamnose,
lactose, dextrin, starch, glycogen,
No acid formation is observed from inulin glycerol, inositol, adonitol, mannitol, sorbitol, α-methyl glycosides.

(15) Growth in sodium chloride medium (28°C, 2-6
(day culture) No growth in 5% and 10% sodium chloride IJ medium.

※※ Medium composition Nigelcose 1.0%, peptone 0.5
%, yeast extract 0.2%, beef extract 0.3%, agar 0
．． 4% (wt/vol), pH 6.6.

※※※Medium composition: glycerol 0.5%, peptone 0.
25%, beef extract 0.25%, yeast extract 0.25%
, Bacto Soitone (manufactured by Deitsuku) 0.25%, sodium chloride 03%, agar 1.25% (weight/volume), pH 6.8°. Based on the above mycological properties, this strain is Bacillus bommyxa. p'olymyxa) (Birshi's Manual of Determinative Bacteria, 2nd Edition, 8th Edition (Williams and Willkins Company Publishing, 1975) and A Guide to the Identification of the Genera of Bacteria, 2nd Edition (See Williams & Wilkins Company Publishing, 1967).

Therefore, this strain was used as Bacillus polymyxa R8-6 (Bacillus polymyxa R8-6).
It was named I l luspolymyxa R8-6) and was submitted to the Institute of Microbiology, Agency of Industrial Science and Technology as the third
It has been deposited as No. 776.

In the present invention, all polymyxin S1-producing bacteria belonging to the genus Bacillus can be used, as well as the above-mentioned strains and natural and artificial mutant strains.

Next, a method for producing PoIJ mixin S1 of the present invention will be described.

Polymyxin S1-producing bacteria are cultured under aerobic conditions in a medium containing various nutrients.

Culture conditions and medium composition may be similar to those commonly used for antibiotic production.

That is, the medium basically contains a carbon source, a nitrogen source, an inorganic salt, etc.

Vitamins and precursor substances may be included as necessary.

Carbon sources include glucose, maltose, starch, dextrin, glycerol, mannitol, organic acids, molasses,
Examples include potatoes, etc., or mixtures thereof; examples of the nitrogen source include peptone, soybean flour, corn steep liquor, malt extract, amino sugar, rice bran, wheat hulls, urea, ammonium salts, etc., or mixtures thereof. .

The medium is preferably a liquid medium, and in the case of mass production, aerated deep culture is preferable.

The pH of the medium is preferably about 5.5 to 8.5, and the culture temperature is preferably adjusted to 20 to 40°C.

If necessary, an appropriate antifoaming agent may be added before or during culturing.

The method for collecting polymyxin S1 from the culture after completion of the culture may be carried out in accordance with the method for collecting ordinary fermentation products.

For example, polymyxin S1 can be obtained by appropriately combining extraction methods using various organic solvents, chromatography using various active adsorbents, and the like.

The polymyxin S1 thus obtained can be used after being converted into a salt, if necessary, as described above.

Next, a production example of the target compound polymyxin S1 of the present invention will be shown, but this example is not intended to limit the present invention in any way.

Example 1 Medium A Nigelcose 1.0%, peptone 0.5%, meat extract 0.5%, sodium chloride 0.1%, potassium monophosphate 0.05%, magnesium sulfate heptahydrate 0. 05
%, manganese sulfate o, ooi%, ferric sulfate 0.001
%, pH 7,00 Medium B Nigelcose 1.0%, Soy flour 4.0%, Ammonium sulfate 2.0%, Potassium monophosphate 0.2%, Magnesium sulfate heptahydrate 0.05%, Calcium carbonate i
, o%, sodium chloride 0.01%, pH 7,0500
20 ml of a liquid medium Al having the above composition is added to a mA' volume Sakaro flask, Bacillus polymixa R8-6 is inoculated, and cultured with shaking at 27° C. for 1 day.

4 mi of this culture solution is used as a seed to inoculate 120 ml of liquid medium B having the above composition in a 500 ml Sakaro flask, and cultured with shaking at 27° C. for 3 days.

After adjusting the pH of the thus obtained 6t culture solution to 2 with hydrochloric acid, Hyfloth-Purcel 1801 was added and filtered.

Oxalic acid 70% in p liquid? and then adjust the pH to 7.0 with sodium hydroxide.

The colorless precipitate of calcium oxalate is filtered out, and the filtrate is passed through a column of weakly acidic cation adsorption resin (Amberlite IRC-50, sodium form).

After washing the column with water, the adsorbed antibiotic is eluted with 0.5N hydrochloric acid.

Both components showing antibacterial activity in the E. coli plate assay were collected, adjusted to pH 11.0 with sodium hydroxide, and extracted with butanol.

The extract is re-extracted with water adjusted to pH 2.0 with hydrochloric acid.

This extract is freeze-dried to obtain 600 ml of coarse powder of the target antibiotic polymyxin S1.

Approximately 2,001 vol of this crude precipitate was subjected to paper chromatography (Higashi 4P Kamitaka 51), butanol/acetic acid/water (
4:1:2).

Confirm by ultraviolet light and ninhydrin reaction, and extract the relevant part with water.

The extract was adjusted to pH 11.0, extracted with butanol, washed with water, made weakly acidic with dilute hydrochloric acid, and concentrated under reduced pressure.

Acetone is added to the resulting syrupy residue to obtain 100 ml of polymyxin S14 hydrochloride as a colorless powder.

[Brief explanation of the drawing]

FIG. 1 shows an infrared spectrum measured on a potassium bromide tablet of PoIJ Mixin S14 hydrochloride.

Claims (1)

[Claims] 1. Polymyxin S1, an antibiotic whose tetrahydrochloride has the following properties, colorless powder, melting point 202-213°C (decomposed), 3. Elemental analysis C53H91N1501. - Calculated value as 4Hcl-H2O C, 4742; H, 728; N. 15.65; C1, 10,57 Analysis value C, 47,08; H, 7,22; N. 16.14; Cl, 10.69 2. Optical rotation 23.0 (α) -54.3 ± 17° (c, 0.541. water) E. Ultraviolet spectrum HO1% λ 2 mμ (E ) 253 (1 , 4), 259(1
, 5). maXICIrL 264 (1,2). Infrared spectrum Nu''cm' 3140, 3050, 1655° ν ax 1532.1403, 1240, 1157° 1077.
930.699°, Paper chromatography Rf - approx. 0.45 (Toyoji paper M51; butanol/acetic acid/water (4:1:2)) Thin layer chromatography Rf = approx. 0.35 (Merck Precoat) Silica gel 60F, acetone/water/acetic acid/2N ammonium hydroxide (15:5:1:2)) Amino acid composition (number of moles) L-threonine 3; D-serine 1; D-phenylalanine 1; L-2,4 -Diaminobutyric acid 5゜, fatty acid composition antiisononan acid, soluble, easily soluble in water and methanol, insoluble in acetone, ethyl acetate, chloroform, and ether. 2. A method for producing polymyxin S1, a novel antibiotic, which comprises culturing polymyxin S1-producing bacteria belonging to the genus Bacillus in a medium, and collecting polymyxin S1 from the resulting culture.