JPS6123990B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to novel antibiotic-producing bacteria. The present inventors isolated various strains of soil microorganisms and searched for the antibiotics they produced.
It was discovered that the KC-6606 strain, isolated from the soil of Kobe City, Hyogo Prefecture, produces a new antibiotic that exhibits antibacterial activity against Gram-positive bacteria, Gram-negative bacteria, and acid-fast bacteria, and furthermore, the strain KC-6606 was isolated from the soil of Kobe City, Hyogo Prefecture. The substance was successfully isolated. This antibiotic was recognized as a new substance because its physical, chemical, and biological properties were different from those of known substances, and it was named substance KA-6606. The KA-6606 substance has a glycyl group at the end and is an aminoglycoside (KA-6606) with the molecular formula C ₁₇ H ₃₅ O ₅ N ₅ .
KA-6606 substance) and a deglycylated substance (KA-6606 substance) with the molecular formula C ₁₅ H ₃₂ O ₄ N ₄ . The antibiotic-producing bacterium of the present invention, strain KC-6606 (Feikoken Bacterial Serial No. 3912), was obtained by suspending soil collected in Kobe City, Hyogo Prefecture in sterile saline using a conventional method, and adding starch to which arginine was added.・Casein agar medium (PH
7.0) and cultured at 37°C for 7 days, the resulting colonies are collected and then purified by a conventional method. This product has the following mycological properties. Unless otherwise specified, properties on various media were obtained by culturing at 27°C for 14 days and observing according to conventional methods. In addition, the color display is based on color, harmony,
The classification according to the manual was followed. Morphological properties This fungal strain forms basal hyphae and aerial hyphae on starch mineral salt medium. Basal hyphae (diameter 0.5~
0.7 μ) are long, branched, and intricately intertwined, and usually one, rarely 2 to 3 basal spores (0.8 to 1.2 μ in diameter) are attached to the end of the sporophyte (length 2.5 μ). do. Aerial hyphae (0.6-0.9 μ in diameter) are short;
Spores are observed as chains of 20 or more. The spore chain is bent and has a hook-like or spiral shape.
Spores are oval to oblong (0.5-0.6μ x 0.7-0.9
μ), the spore surface is covered with a sheath with a hair-like structure. Properties on various media 1 Sucrose/nitrate agar medium Growth: Good Aerial hyphae: Good, powdery, shell pink (5ba) Base hyphae: Buttery, light tan to rose
Beige (3gc-4gc) Soluble pigment: Pale pink with yellowish brown 2 Growth on glycerin/nitrate agar medium: Moderate to good Aerial hyphae: Good, powdery, white (a) Base hyphae: Buttery, light ivory (2ca) Soluble pigment: Pale yellow 3 Glucose-asparagine agar medium (slope) Growth: Moderate Aerial hyphae: None Base medium hyphae: butter-like to gelatinous, buff 2fb) Soluble pigment: slightly pale yellow 4 Glycerine-asparagine agar Medium (ISP No.
5) Growth: Moderate Aerial hyphae: Poor, powdery, white (a) Base hyphae: Gelatinous, light ivory (2ca) Soluble pigment: Slightly pale yellow 5 Starch/inorganic salt agar medium (ISP No. 4) ) Growth: Good Aerial mycelium: Poor, powdery, white (a) Base mycelium: Leather-like, light ivory to buff (2ca to 2fb) Soluble pigment: None 6 Tyrosine agar medium (slope, ISP No. 7) Growth: Moderately aerial hyphae: Good, powdery, white (a) Baseline hyphae: Butter-like to gelatinous, light ivory to buff (2ca-2fb) Soluble pigment: Slightly pale yellow 7 Growth on nutrient agar medium: Moderately aerial Hyphae: None Base hyphae: Gelatinous, buff (2fb) Soluble pigment: None 8 Yeast/malt agar medium (ISP No. 2) Growth: Good Aerial hyphae: Very poor, powdery, white (a) Base hyphae: Gelatin Bamboo (2gc) Soluble pigment: Pale yellow 9 Oatmeal agar medium (ISP No. 3) Growth: Poor aerial hyphae: None Base medium hyphae: Colorless Soluble pigment: None 10 Peptone yeast iron agar medium (slope,
ISP No. 6) Growth: Moderate Aerial mycelium: Poor, powdery, white (a) Baseline mycelium: Buttery to gelatinous, light melon yellow (2ea) Soluble pigment: Slightly pale yellow 11 Growth on Bennet agar medium : Good aerial mycelium: None Base medium mycelium: Gelatinous, light ivory (2ca) Soluble pigment: Slightly pale yellow Physiological properties 1 Growth temperature range: 18-45℃ 2 Liquefaction of gelatin: Yes 3 Addition of starch hydration Decomposition effect: Yes 4 Effect on milk Coagulation effect: None Peptonization: Yes 5 Production of melanin-like pigment On tyrosine agar medium: No formation On peptone/yeast/iron agar medium: No formation 6 Production and accumulation of nitrite rather than nitrate: Yes Carbon source assimilation (on Pridham-Godelieve agar medium) Used: D-glucose, D-fructose, raffinose, sucrose,
D-mannitol, galactose, inulin, salicin. Things not used: L-arabinose, D-xylose, inositol, L-rhamnose, sorbitol, lactose, dulcitol. As described above, strain KC-6606 forms basal hyphae and aerial hyphae on the agar medium, attaches basal spores to the basal hyphae, attaches spore chains to the aerial hyphae, and the surface of the spores is hairy. It is covered with a pod with a similar structure. Analysis of the cell wall revealed that it has meso-diaminopimelic acid and contains arabinose and galactose as sugars, so the cell wall of this strain was found to belong to this type. On the other hand, when analyzing the fatty acid composition in the cell wall,
LCN-A (Lipid Characteristics)
of Nocardia). Known bacterial strains with these characteristics were identified in Virgie's Manual of Determinative
When searching in Bacteriology, 8th edition (1975), I found Micropolisvora as a similar genus, and also the genus Satzcalopolyspora (Journal of General Microbiology, reported by Lacey and Gutsudferou). Volume 88, page 75, 1975)
can be given. Although it differs from the former in that the hair-like structure of the spores and more than 20 spore chains are observed, it is morphologically very similar to the latter, so KC-6606
The strain is considered to belong to the genus Satucharopolyspora. Furthermore, there is still no established method for the systematic classification of bacterial species of the genus Satucharopolyspora.
There is only one report of one species. Table 1 shows the differences between the KC-6606 strain and Satucharopolyspora hirsuta described in the above literature.

[Table] As shown above, the KC-6606 strain differs from Satzcalopolyspora hirsuta in its ability to assimilate carbon sources, but its basic properties such as morphological properties, properties on various media, and physiological properties are similar. Since they match, it is reasonable to consider them to be the same species. Based on the above findings, the present inventors believe that this strain is a natural mutant strain of Satzcalopolyspora hirsuta, and that
It was named strain −6606. The bacterial strain belonging to Saccharopolyspora hirsuta of the present invention is easily changeable in its properties and can be easily mutated by artificial mutagenesis means using, for example, ultraviolet rays, X-rays, chemicals, and the like. In addition, Satsukalopolyspora
It has not been reported in the literature that bacteria belonging to Hirusta produce antibiotics. A normal method for culturing actinomycetes is used to culture this strain. Although various carbon sources can be used for the culture medium, it is preferable to use starch, glucose, glycerin, maltose, dextrin, sucrose, fructose, molasses, etc. alone or in combination. Furthermore, depending on the assimilation ability of the bacteria, hydrocarbons, organic acids, vegetable oils, etc. may also be used. As a nitrogen source, soybean flour, yeast extract, dried yeast, peptone, meat extract, cornstarch liquor, casamino acid, daytailers soluble, ammonium chloride, ammonium sulfate, urea, sodium nitrate, etc. are used alone or in combination. . In addition, inorganic salts such as common salt, potassium phosphate, magnesium sulfate, calcium chloride, calcium carbonate, calcium hydroxide, cobalt chloride, zinc sulfate, iron chloride, iron sulfate, and trace amounts of metals can be added as necessary. Furthermore, organic and inorganic substances that aid the growth of bacteria and promote the production of the KA-6606 substance can be added as appropriate. When using the aerated culture method, an antifoaming agent such as fatty oil, silicone oil, paraffin, etc. is further used. As a culture method, although culture on a solid medium is also possible, it is preferable to use a liquid culture method, particularly a deep culture method, as in the general antibiotic production method. The culture was carried out under aerobic conditions, and the culture temperature was
20 to 35°C, preferably around 27°C. In producing the KA-6606 substance, the active substance is produced and accumulated in the culture solution when the culture is carried out for 2 to 6 days in either shaking culture or tank culture. The culture is stopped when the production amount in the culture solution reaches the maximum, and the antibiotic of interest is isolated and purified from the culture solution. To isolate and purify this substance from culture medium, KA
Since the -6606 substance is a water-soluble basic substance that is soluble in water and sparingly soluble in common organic solvents, it is possible to use ordinary methods for isolation and purification of water-soluble basic antibiotics. Adsorption/desorption methods using ion exchange resins, activated carbon, cellulose, silica gel, alumina, etc., methods of adding higher fatty acids as auxiliary agents and extraction with butanol, amyl alcohol, etc. can be used in appropriate combinations. When the culture solution is passed through a layer of weakly acidic cation exchange resin, the KA-6606 substance is adsorbed. Set this to 0.1~
After eluting with 3.0 normal alkali or acid and freeze-drying the active ingredient, a crude powder of KA-6606 substance is obtained. The weakly acidic cation exchange resins used at this time include Amberlite IRC-50 and IRC-84.
Or CG-50 (manufactured by Rohm & Haas), Diaion WK-10 or WK-20 (manufactured by Mitsubishi Kasei), etc. Examples of the alkali include aqueous ammonia and aqueous sodium hydroxide, and examples of the acid include formic acid, hydrochloric acid, and sulfuric acid. It is also possible to use a method in which the culture solution is adjusted to pH 7 to 9, the target substance is adsorbed on activated carbon, and then eluted with acidic water or methanol with hydrochloric acid. The crude powder thus obtained is adsorbed onto a weakly acidic cation exchange resin, CM-Sephadex, CM-cellulose, etc., and eluted with a concentration gradient method or a concentration step method using aqueous ammonia, aqueous ammonium formate, etc. to release the free powder. KA- as a base
6606 substance and the same substance can be separated. The free base (and) thus obtained is further passed through a column of a strongly basic anion exchange resin, such as DOWEX 1×2 (manufactured by Dow Chemical), then eluted with, for example, water, and the active fraction is collected and lyophilized. By doing so, pure free bases (and) can be obtained. This free base can be converted into the corresponding acid addition salt in a conventional manner by adding an inorganic acid such as hydrochloric acid, sulfuric acid, hydrobromic acid or an organic acid such as acetic acid, oxalic acid, etc., respectively. KA-6606 substance has a structure with the terminal glycyl group removed from KA-6606 substance, so
It can also be produced by decomposing KA-6606 substance with alkali or acid. That is,
Add an alkaline reagent such as a 0.1 to 4.0N aqueous solution of sodium hydroxide or barium hydroxide or an acidic reagent such as hydrogen chloride or sulfuric acid to the KA-6606 substance.
KA by applying 0.1~1.0N aqueous solution
-Can be converted into 6606 substance. At this time, a strong basic anion exchange resin such as Amberlite IRA400 (OH ^- type) or Dowex 1x2 (OH ^- type) is used as the alkaline reagent, and a strong acidic cation exchange resin such as Amberlite IR120 is used as the acidic reagent.
The target substance can also be obtained by adding Dowex 50×8 (H ⁺ type) or Dowex 50×8 (H ⁺ type) and reacting in a suspended state. The reaction is usually completed in 0.5 to 3 hours at 30 to 100°C. The KA-6606 substance is a basic water-soluble white substance, and is a useful new antibiotic that exhibits antibacterial properties mainly against Gram-positive bacteria, Gram-negative bacteria, and acid-fast bacteria. The physicochemical and biological properties of this substance are as follows. Free base of KA-6606 substance: (1) Appearance White powder (2) Molecular formula C ₁₇ H ₃₅ O ₅ N ₅ (3) Elemental analysis value C H N Experimental value (%) 52.10 8.84 17.83 Calculated value (%) 52.42 9.06 17.99 (4) Molecular weight 389 (mass spectrum) (5) Melting point 125-135°C (6) Specific rotation [α] ²⁷ _D +104° (c = 1, H ₂ O) (7) Ultraviolet absorption spectrum 220-360 nm It shows no specific absorption, only terminal absorption. (8) Infrared absorption spectrum The infrared absorption spectrum of pellets in potassium bromide is shown in Figure 1. (9) Solubility in solvents Extremely soluble in water. Easily soluble in methanol, slightly soluble in ethanol and acetone. Insoluble in chloroform, ethyl acetate, ether, hexane, petroleum ether. (10) Color reaction ninhydrin reaction and Lydon-Smith reaction: positive. Sakaguchi reaction, maltol reaction, ferric chloride reaction and Fehring reaction: negative. (11) Stability: Stable at pH 3 to 8, and gradually decomposed and inactivated under basic and strongly acidic conditions. (12) Nuclear magnetic resonance spectrum δ _D2O (ppm): 1.25 (3H, d) C-C H ₃ , 3.05 (3H, s) N-C H ₃ , 3.07 (2H, s) COC H ₂ N, 3.40 ( 3H, s) OC H ₃ , 5.00 (1H, d) Anomeric H (13) Mass spectrum (m/e): 389 (M ⁺ ), 276, 258, 248, 230, 180,
143 (14) Paper chromatography Rf value: 0.53 Paper; Watmann No. 1 Solvent: Chloroform/methanol/17%
Lower layer of ammonia water (2:1:1) (15) Thin layer chromatography

[Table] The plate used was TLC aluminum sheet silica gel 60F ₂₅₄ 0.2 mm (manufactured by Merck & Co.). K.A.
Tetra-N-acetyl form of -6606 substance: (1) Appearance Crystalline white powder (2) Molecular formula C ₂₅ H ₄₃ O ₉ N ₅ (3) Elemental analysis value C H N Experimental value (%) 53, 51 7.58 12.44 Calculated value (%) 53.85 7.77 12.56 (4) Molecular weight 557 (mass spectrum) (5) Melting point 160-166℃ (6) Specific rotation [α] ²⁵ _D +112゜ (c=1,
H ₂ O) (7) Nuclear magnetic resonance spectrum δ _D2O (ppm): 1.10 (3H, d)C-C H ₃ , 1.95, 2.00, 2.05, 2.07 (3H, s) COC
H ₃ , 3.09 (3H, s) N-C H ₃ , 3.42 (3H, s) O-C H ₃ , 4.11 (2H, s) COC H ₂ N-, 4.95 (1H, d) Anomeric H (8) Mass spectrum (m/e): 557 (M ⁺ ), 342, 314, 296, 227 Free base of KA-6606 substance: (1) Appearance White powder (2) Molecular formula C ₁₅ H ₃₂ O ₄ N ₄ (3) Elemental analysis value C H N Experimental value (%) 53.97 9.51 16.55 Calculated value (%) 54.19 9.70 16.85 (4) Molecular weight 332 (mass spectrum) (5) Melting point 88-93℃ (6) Specific optical rotation [α] ²⁷ _D +139.5° (c=1,
H ₂ O) (7) Does not show specific absorption in the ultraviolet absorption spectrum from 220 to 360 nm, showing only terminal absorption. (8) Infrared absorption spectrum The infrared spectrum of the pellet in potassium bromide is shown in Figure 2. (9) Solubility in solvents Extremely soluble in water. Easily soluble in methanol, slightly soluble in ethanol and acetone. Insoluble in chloroform, ethyl acetate, ether, hexane, petroleum ether. (10) Color reaction ninhydrin reaction and Lydon-Smith reaction: positive. Sakaguchi reaction, maltol reaction, ferric chloride reaction and Fehring reaction: negative. (11) Stability Stable at pH 2 or higher, and gradually decomposed under strong acidity. (12) Nuclear magnetic resonance spectrum δ _D2O (ppm): 1.25 (3H, d) C-C H ₃ , 2.70 (3H, s) N-C H ₃ , 3.45 (3H, s) O-C H ₃ , 4.95 (1H, d) Anomeric H (13) Mass spectrum (m/e): 332 (M ⁺ ), 283, 219, 119, 143 (14) Paper chromatography Rf value: 0.86 Paper; Watmann No. 1 Solvent; Chloroform/methanol/17%
Lower layer of ammonia water (2:1:1) (15) Thin layer chromatography

[Table] The plate used was TLC aluminum sheet silica gel 60F ₂₅₄ 0.2 mm (manufactured by Merck & Co.). K.A.
Penta-N-acetyl form of -6606 substance: (1) Appearance White needle-like crystals (2) Molecular formula C ₂₃ H ₄₀ O ₃ N ₄・H ₂ O (3) Elemental analysis value C H N Experimental value (%) 52.99 7.65 10.52 Calculated value (%) 53.27 8.16 10.80 (4) Molecular weight 500 (mass spectrum) (5) Melting point 139-141℃ (6) Specific optical rotation [α] ²² _D +52゜ (c=1, H ₂ O) ( 7) Nuclear magnetic resonance spectrum δ _D2O (ppm): 1.09 (3H, d) C-C H ₃ , 1.98, 1.99, 2.01, 2.13 (3H, s) CO-
C H ₃ , 3.10 (3H, s) N-C H ₃ , 3.40 (3H, s) O-C H ₃ , 4.96 (1H, d) Anomeric H (8) Mass spectrum (m/e): 500 (M ⁺ ), 303, 271, 257, 227 With reference to the above physicochemical properties, KA−
6606 and the substance is represented by the following structural formula in free base form. Antibacterial action: The antibacterial spectrum of KA-6606 and its substances against various microorganisms is shown in Table 2.

【table】

[Table] As shown above, antibiotic KA-6606 has excellent antibacterial activity against a wide range of Gram-positive bacteria, Gram-negative bacteria, and acid-fast bacteria, and is also effective against strains resistant to various known antibiotics. It has antibacterial properties. It has the property of being particularly effective against Escherichia coli and Pseudomonas aeruginosa that are resistant to kanamycin, gentamicin, tobramycin, amikacin, and the like. Infection treatment effect of this substance obtained when Staphylococcus aureus Smith strain was inoculated into the cavity of a mouse and KA-6606 substance was injected subcutaneously.
_ED50 is 0.7mg/Kg. Furthermore, the LD ₅₀ is 50 mg/Kg or more for intravenous injection in mice. In addition, as shown in Tables 3 and 4, the results of paper chromatography and thin layer chromatography show that the KA-6606 substance is different from known antibiotics.

【table】

[Table] As mentioned above, this substance has a wide and excellent antibacterial spectrum, so it is useful as an antibacterial substance in medicine, veterinary medicine, etc. This substance is also useful as a starting material for synthesizing various derivatives. Experimental examples regarding culturing this bacterium and collecting the produced KA-6606 substance are shown below. Example 1 3% starch, 15% soybean flour, cornstap liquor
0.5%, yeast extract 0.2%, magnesium sulfate 0.05
%, salt 0.3%, calcium carbonate 0.3%, and cobalt chloride hexahydrate 0.001%, strain KC-6606 was inoculated into a sterilized medium adjusted to pH 7.0, and incubated at 27°C for about 50 hours. The cultured bacteria is called the first type of bacteria.
In a 200-volume tank, fill 100 of the same medium as the seed culture medium with 1% cottonseed oil added, and add 200 of the first type bacteria.
ml was inoculated and cultured at 27°C for 4 days with aeration and stirring (225 rpm, aeration rate 50/min). After the cultivation is completed, sulfuric acid is added to the culture solution to adjust the pH to 2.0, and then dicalite (manufactured by Dicalite Orient Co., Ltd.) is added as a supporting agent to separate the bacterial cells. Add a dilute aqueous sodium hydroxide solution to the solution to adjust the pH to 8.0, pass it through a column of cation exchange resin Amberlite IRC-50 (NH ⁺ ₄ type) (discard the effluent), wash with water, and add 1N ammonia water. elute the adsorbed active substance. The activity of the eluate is measured by the paper disk method using a Bacillus subtilis agar plate, and the active fractions are combined and concentrated to about 50 ml under reduced pressure. Lyophilization of this concentrate yields 8.8 g of crude powder of KA-6606 substance. Dissolve 8g of this coarse powder in 50ml of distilled water and adjust the pH.
Cation exchange resin Amberlite adjusted to 7.0
Pass through a CG-50 type (NH ⁺ ₄ type) column (3 x 150 cm), wash with water, and add 0.01N ammonia water 5 and 1.
Elute at a flow rate of 150 ml/hour using the concentration gradient method between 5 volumes of specified ammonia water, and fractionate every 25 ml. The activity of each fraction was assayed by the paper disc method, and the fractions containing the components corresponding to KA-6606 and the fractions containing the components corresponding to KA-6606 were collected and freeze-dried.
80 mg of free base and 16 mg of free base of KA-6606
mg is obtained. Dissolve each of these freeze-dried products in water and adjust the pH.
After adjusting to 7.0, pass through a column (1 x 150 cm) of anion exchange resin Dowex 1 x 2 (OH ^- type) and elute with water. When the active fraction is collected and lyophilized, 50 mg of the purified free base of KA-6606 is obtained.
10 mg of a purified sample of the free base of KA-6606 is obtained. Example 2 KC-6606 was added to a medium with a composition of 2% starch, 1% soybean flour, 0.5% cornstarch liquor, 0.05% magnesium sulfate, 0.3% salt, and 0.3% calcium carbonate, and was sterilized and adjusted to pH 7.0. The strain is inoculated and pre-cultured at 27°C for about 50 hours, which is considered the first type of bacteria. 20
The above medium 10 was placed in a 10-volume shear fermenter, 100 ml of the first type bacteria was inoculated, and cultured at 27°C for 4 days with aeration and stirring (250 rpm, aeration rate 10). The following treatment was carried out in the same manner as in Example 1, and 20 mg of the free base of KA-6606 and 20 mg of the free base of KA-6606 were obtained.
4 mg of the free base is obtained as a purified sample. Example 3 40 mg of the KA-6606 substance obtained above was
Dissolve in 1 ml of normal sodium hydroxide aqueous solution and heat for 1 hour. Dissolve the reaction mixture in 100ml of water, neutralize it, and then add cation exchange resin Amberlite CG.
Pass through a -50 (NH ⁺ ₄ type) column (1 x 10 cm), wash the column with 100 ml of water, and elute with 1N aqueous ammonia. Fractions that are positive for ninhydrin reaction and have antibacterial activity are collected and lyophilized to yield 23 mg of the free base of KA-6606 substance as a white powder.

[Brief explanation of the drawing]

Figure 1 shows the infrared absorption spectrum of antibiotic KA-6606, and Figure 2 shows the infrared absorption spectrum of antibiotic KA-6606.

Claims (1)

[Claims] 1. Neither D-xylose, L-rhamnose, sorbitol nor inositol is used as a carbon source on a Prudham-Godlieb agar medium,
Saccharopolyspora hirsuta KC-6606 has the property of producing and accumulating nitrite from nitrate, and has the ability to produce the antibiotic KA-6606.