JPS5811838B2 - Shinko Seibutsutsu BN-165 Shinko Seizouhou - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to BN-1, which is a bacterium of the genus Pseudomonas.
65 substance-producing bacteria were cultured, and the new antibiotic BN-
The present invention relates to a method for producing BN-165 substance, which comprises separating and collecting BN-165 substance.

The present inventors have discovered that a culture of a specific bacterial strain belonging to the genus Pseudomonas contains a substance that exhibits a strong growth-inhibiting effect on Durum-positive bacteria. The present invention was completed by naming the substance BN-165.

The Pseudomonas strain used in the method of the present invention contains a sufficient amount of BN-165 to be collected in its culture.
Those that have the ability to produce substances are used.

Examples of such strains include Pseudomonas sp., BN-16, which was newly isolated from soil by the present inventors.
There are 5 strains (strain number BN-165 strain).

This BN-165 strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as FERM-P No. 3143 (FERM-P No. 31).
43).

The mycological properties of the BN-165 strain are as shown below.

(a) Morphological properties Cells cultured on broth agar are 0.4-0.6 x 1.5-
It is a 2.5 micron rod and moves with polar flagella.

It does not produce spores and shows no pleomorphism.

Durham staining is negative. (b) Culture properties■ Meat juice agar culture: The bacterial cells exhibit a pale brown-yellow color and grow in cream-like mounds.

No significant viscosity or migration was observed; No formation of diffusible dye was observed.

■ Broth liquid culture: The entire medium becomes cloudy, especially near the liquid surface.

■ Meat juice gelatin puncture culture is liquefied into two bags.

■ Milk culture: Liquefaction progresses slowly and becomes slightly acidic.

(c) Physiological properties ■ Nitrate reduction: negative ■ Denitrification reaction: negative ■ MR test: negative ■ VP test: negative ■ Indole production: negative ■ Hydrogen sulfide generation: turns lead sugar paper black ■ Starch hydration Decomposition: Negative ■ Use of citric acid: Positive ■ Use of inorganic nitrogen source: Ammonium salts can be used as the only nitrogen source.

] Pigment production: No significant pigment production was observed in King AB medium.

■ Oxidase 2 weakly positive ] Rabbit blood agar turns green.

] Lysine decarboxylation reaction: Positive 0 Auxotrophic requirement: No nutritional requirement for vitamins or amino acids.

] O-F test (Hyuleifuran method): No gas or acid generation from sugar is observed with or without a paraffin seal.

] No growth is observed under anaerobic conditions.

■ Utilization of sugar (using only ammonium salt as nitrogen source) 1) Cucrose and maltose can be used as the only carbon source.

11) Glycerin, galactose, sucrose, and mannitol cannot be used.

The BN-165 strain having the above mycological properties was selected from Bergey's Manual of Determinative Bacteriology, 8th edition.
f Determinative Bacteriol
-ogy 8th edition) (1974), and the following conclusions were obtained.

This strain is a Durham-negative bacillus that does not produce spores, has the morphological characteristics of being motile by polar hairs, and is obligately aerobic, so this strain is Pseudomonas.
) can be identified as belonging to the genus.

In order to culture the BN-165 substance-producing bacteria to produce and accumulate the BN-165 substance, various types of media commonly used for fermentation of microorganisms are used.

In other words, carbohydrates such as glucose, dextrin, and starch syrup serve as carbon sources, while peptone serves as a nitrogen source.

Meat extract, powdered bouillon, course stable liquor.

Soybean meal, ammonium sulfate, ammonium chloride, etc. are used.

Inorganic salts such as common salt and calcium carbonate may also be used together, and an antifoaming agent may be added if necessary.

Appropriate culture methods include methods using liquid media such as shaking culture and deep aeration agitation culture.

The culture temperature is selected within the range of 20 to 35°C, and the appropriate number of culture days is 1 to 3 days.

The BN-165 substance is mainly accumulated in the culture medium.

The following method is used for assaying the BN-165 substance.

As the culture medium for assay, Penatuse Agar (manufactured by Kyoei Pharmaceutical Co., Ltd.) is used.

Staphylococcus aureus 209P is used as the test bacterium.

The BN-165 substance (pure product) showed a linear relationship between the logarithm of the concentration and the diameter of the inhibition circle in the range of 8mcg/ml to 2000mcg/ml in an assay using this substance, and each
Indicates an inhibition circle of 0 to 24 mm (paper disk method)
.

Since the BN-165 substance has the physical and chemical properties described below,
It is possible to extract and purify it according to its properties, and the method shown below is efficient.

That is, the culture solution containing the active ingredient is filtered under acidic conditions (pH 2-3) to remove solids, the P solution is adjusted to pH 5-6, and the active ingredient is extracted with n-butanol.

This n-butanol layer is decolorized with activated carbon, and the n-butanol layer is concentrated to dryness to obtain a crude product of BN-165 substance.

For further purification, chromatography using an adsorbent such as silica gel, alumina, or Florisil, or a gel filtration agent in combination with an appropriate chromatography countercurrent distribution operation or a precipitation method is used to obtain a colorless powder.

When the BN-165 material thus obtained was subjected to thin layer chromatography in various solvents, each gave a single spot, indicating that this powder was a pure BN-165 material.

The physical and chemical properties of the BN-165 substance are as shown below, and were measured as a hydrochloride.

(1) Elemental analysis values: C47.58%, H7.19%, N
13.80%, C17.69%, 0.23.74% (difference), and contains no other elements.

(2) Molecular weight: Estimated to be 1000 to 1200 as a result of gel filtration using Sephadex LH-20 (manufactured by Pharmacia).

The results of the amino acid analysis described later are also almost the same. I support this.

(3) Melting point: It begins to turn brown around 215°C and foams and decomposes around 235°C.

(5) Ultraviolet absorption spectrum: As shown in FIG.

(6) Infrared absorption spectrum: As shown in FIG.

(7) Solubility in solvents: Soluble in methanol and ethanol, slightly soluble in water, but almost insoluble in chloroform, acetone, ethyl acetate, and ethyl ether.

(8) Color reaction: those showing positive, plate grain, biuret,
Those showing negative results: ferric chloride, fading, ninhydrin (9) Distinguishing between basic, acidic, and neutral 2P paper electrophoresis results show the behavior of weakly basic substances.

(10) Color of substance: colorless powder (11) Amino acid composition: 110℃2 in 6N hydrochloric acid in a sealed tube
The results of 4-hour decomposition and analysis using an amino acid analyzer revealed glycine, leucine, inleucine, serine, and threonine.

Arginine and unknown amino acids are 1 A total of 7 types of amino acids were detected. The content ratio is l:3:1:1: The ratio is 1:1:1.

(12) R4 direct of silica gel thin layer chromatography:
Ethyl acetate-acetic acid-water (60:17:17) 0.6
3n Butanol-acetic acid-water (2:1:1) 0.2
The minimum inhibitory concentration of the 48N-165 substance against various microorganisms is as shown in Table 1, and it exhibits a strong inhibitory effect on Gram-positive bacteria.

In addition, the acute toxicity of this substance was determined to be 100 mg/kg by intraperitoneal administration and 1000 mg/kg by oral administration in tests using mice.
Both sides survived.

As mentioned above, the BN-165 substance is a pegtide antibiotic consisting of seven types of amino acids.

Pegtide antibiotics produced by Pseudomonas include Comirin, Viscocin (Vi-s
Cosin) Cyfosin AB is known, but this substance has a different amino acid composition from these.

In addition, many peptide antibiotics produced by bacteria other than the genus Neudomonas or actinobacteria are known, but all of them are different from this substance.

Based on the above results, the BN-165 substance was determined to be a new antibiotic.

Examples of the present invention are shown below, but it goes without saying that the present invention includes many modifications and modifications not exemplified here.

Example 1 A liquid medium containing 2% powdered bouillon was dispensed into 20 500ml bouillon flasks in 100ml portions and capped with cotton plugs.
Autoclave sterilized at 120°C for 10 minutes to prepare Pseudomonas sp, BN.
-165 strain (FERM-No. 3143) was inoculated from a slant culture using a platinum loop.

120 mc of BN-165 substance was cultured with shaking at 32°C for 2 days.
A culture solution containing 1.81 g/ml was obtained.

When this culture solution is adjusted to pH 3 and filtered, and the F solution is neutralized with sodium hydroxide and passed through a 200 ml column of Amberlite XAD-2 (Rohm & Bath Co., Ltd.), the active ingredients pass through without being adsorbed to the resin.

When 1 g of n-butanol is added to this passed liquid and shaken, the active ingredient is transferred to the n-butanol layer. When this n-butanol layer is concentrated to dryness under reduced pressure, 180 m of coarse powder of BN-165 substance is obtained.
9 was obtained.

This powder was dissolved in n-butanol, poured into a silica gel tower previously filled with n-butanol, developed with n-butanol, and the active fraction was concentrated to dryness under reduced pressure to obtain 80 mg of a slightly yellow powder.

This powder was further dissolved in methanol and Sephadex L was added.
H-20 (Pharmacia) was applied to a column previously swollen with methanol, developed with methanol, and the active fraction was concentrated to dryness under reduced pressure to form a powder of BN-165 substance with a purity of about 80%.
I got ~.

Example 2 Culture medium 151 containing 0.5% peptone, 1% crucose, 0.2% ammonium chloride, 0.4% calcium carbonate, 0.3% table salt, and 0.03% antifoaming silico extract was prepared at 30/vol. Pseudomonas sp, BN-165 strain (FERIVI-P), which had been cultured in the same culture medium in two Sakalo flasks for 1 day before, was placed in a culture tank, sterilized at 120°C for 10 minutes, and cooled.
No. 3143) was aseptically inoculated into a culture tank.

Aerated agitation culture at 30°C for 2 days (aeration rate 15j/min),
The mixture was stirred at 200 rpm) to obtain a culture solution 131 with a concentration of 150 mcg/mg.

After adjusting the culture solution to pH 2.5 with hydrochloric acid, it was filtered using a filter aid, and the pH of solution F was returned to 5.5 with hydrochloric soda.
The active ingredients are extracted from the P solution twice with 100% of n-butanol, the extracts are combined, and passed through a column of activated carbon for chromatography (Wako Tsumugi Pharmaceutical Co., Ltd.) filled with n-butanol in advance, and this column is further extracted with methanol. The washing active fraction was concentrated to dryness under reduced pressure.
1 oy of a slightly oily crude product was obtained.

This was dissolved again in 200 mA butanol, extracted 10 times with 200 m/each of pH 2 buffer, adjusted the pH of the buffer in the active section to 5, extracted the active ingredient with n-butanol, and concentrated to dryness under reduced pressure. A yellow powder of 2.5V was obtained.

Dissolve this powder in methanol, apply it to a Florisil (Wako Tsumugi) tower filled with methanol in advance, wash the tower with methanol, elute the active ingredient with methanol adjusted to pH 2 with hydrochloric acid, and concentrate the active fraction to dryness under reduced pressure. ShiBN-16
400 mg of white powder of 5 substances was obtained.

This powder was dissolved in methanol, poured into an acidic alumina tower previously filled with methanol, the active ingredient was eluted with methanol-water-1N hydrochloric acid (2:1:0.3), and the active fraction was concentrated under reduced pressure to BN- A pure product of 150/mg of 165 substances was obtained.

Example 3 Peptone 0.75%, crucose 1.0%, ammonium chloride 0.3%, calcium carbonate 0.4%, salt 0.3
%, culture medium containing 0.01% antifoaming silicone oil 20
Pseudomonas sp. BN-165 strain (FERM-01) was placed in a 300 g culture tank, sterilized at 120°C for 10 minutes, and cooled.
Seeds of P. 3143) were inoculated into a culture tank in a non-pictured area.

24-hour aeration agitation culture at 28°C (aeration rate 2001/min agitation number 111105 rpm, 80 mcg/ml culture solution 1
I got 80j.

The culture solution was adjusted to pH 2.8, filtered with a filter press using a filter aid, 50 kg of ammonium sulfate was added to the P solution, the pH was further adjusted to 6, and n-butanol 100
The active ingredient was extracted twice with 1.5 g each, and the n-butanol layers were combined and concentrated under reduced pressure to a concentration of 2.0 g to remove the resulting impurity precipitate.

300 g of activated carbon was added to this n-butanol layer, and after stirring, the activated carbon was separated by F, and the activated carbon was further washed with methanol IOJ, and the previous n-butanol layer and this methanol layer were combined and concentrated to dryness under reduced pressure to form a brown powder. I got 741.

This powder was dissolved in methanol 500-ml to filter out insoluble impurities, and Floriseal 800ml was filled with methanol in advance.
The active ingredient was eluted with hydrochloric acid and acidic methanol (pH 2), and the active fraction was concentrated under reduced pressure to produce a white precipitate of the active ingredient.
0 mg was obtained.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum of the BN-165 substance.
It was measured by dissolving the solution in a concentration of 1 ml. FIG. 2 shows an infrared absorption spectrum of the BN-165 substance, measured as a potassium bromide tablet.

Claims (1)

[Claims] 1. A method for producing a new antibiotic BN-165 substance, which comprises culturing a BN-165 substance-producing bacterium belonging to the genus Pseudomonas to accumulate the BN-165 substance, and collecting the BN-165 substance.