JPH05310766A - New antibiotic substance mi481-42f4-a and its produciton - Google Patents

Abstract

PURPOSE:To obtain a new antibiotic substance M1481-42F4-A having antimicrobial activity of strongly inhibiting the growth of Gram-positive bacteria and multi-drug resistant germs and a method for producing the antibiotic substance. CONSTITUTION:The antibiotic substance MI481-42F4-A is separated and collected from a culture solution of a strain belonging to the genus Amycolatopsis. This antibiotic substance MI481-42F4-A is obtained as a colorless powdery basic substance having 294-295C melting point (decomposition) and the molecular formula C50H51O8N15S6.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a novel antibiotic MI481.
-42F4-A and its manufacturing method.

[0002]

2. Description of the Related Art A large number of antibiotics produced by microorganisms have been discovered so far and are widely used for treating bacterial infections.

[0003]

In the treatment of bacterial infections, the emergence of resistant bacteria is unavoidable, and new antibiotics effective against resistant bacteria are constantly being sought.

[0004]

As a result of research, the present inventors succeeded in obtaining a novel antibiotic MI481-42F4-A from a culture solution of a strain belonging to the genus Amycolatopsis, and this substance was found to be It was found to have antibacterial activity.

Therefore, according to the first invention, the following characteristics are provided: (1) Color and shape: colorless powder (2) Melting point: 294-295 ° C. (decomposition) (3) Elemental analysis: C 49.46% , H 4.79%, N
16.76%, O 12.23%, S 15.72% (4) Ultraviolet absorption spectrum: lambda maximum (nm) methanol 203 309 221 221 350 (shoulder) 245 (shoulder) methanol-hydrochloric acid 205 308 222 222 353 (shoulder) 250 (shoulder) methanol-sodium hydroxide 205 310 221 353 (shoulder) 250 (shoulder) (5) Infrared absorption spectrum (KBr method): 3300,3
120, 2980, 1660, 1550, 1500, 1
250,990,940,760 cm ^-1 (6) Stability: Stable at room temperature and pH 2-9 (7) Specific rotation: [α] _D ²⁸ = + 133 ° (c 0.74
5, dimethylsulfoxide) (8) Acidic, neutral, basic classification: basic substance (9) molecular weight: 1182.44 (10) molecular formula: C ₅₀ H ₅₁ O ₈ N ₁₅ S ₆ New antibiotic MI481-
42F4-A is provided.

Further, according to the second aspect of the present invention, MI481-42 belonging to the genus Amycolatopsis.
The F4-A producing bacterium is cultivated and the antibiotic MI is extracted from the culture.
There is provided a method for producing the antibiotic MI481-42F4-A, which comprises separating and collecting 481-42F4-A.

One example of the MI481-42F4-A-producing bacterium belonging to the genus Amycolatopsis that can be used in the method of the present invention is isolated from a soil sample collected by the present inventors in Nerima Ward, Tokyo in June 1987. Strain Amycolatopsis sp. MI481-42F
There is 4. The mycological properties of this strain are as follows.

Mycological properties of MI481-42F4 strain 1. Morphology Well-branched basal hyphae often have a zigzag shape,
In addition, division is recognized. The aerial hyphae are straight or irregularly curved, with fragmentation, forming cylindrical spores. The spore size is approximately 0.6-0.7 x 1.7-1.2 microns and the surface is smooth. No helix formation, limbus or sporangia are observed.

2. Regarding the description of the growth state color in various media, the standard shown in [] is the Container Harmony Manual (Container Corporation of America).
Color harmony manual).

(1) Sucrose / nitrate agar medium (3
(0 ° C culture) On the development of light yellow [1 ea, Canary Yellow], white aerial hyphae were slightly settled and no soluble pigment was observed.

(2) Glucose / asparagine agar medium (30 ° C. culture) On the development of yellow [1 1/2 ia, Sunlight Yellow], aerial mycelia of pale yellow [1 ca, Pale Yellow] are grown and dissolved. The sex pigment is slightly yellowish.

(3) Glycerin-asparagine agar medium (ISP-medium 5, culture at 30 ° C.) In developing light yellow [1 1/2 ga, Butter Yellow], light yellow [1 1/2 ca, Cream] was added. The aerial hyphae are settled, and the soluble pigment is slightly yellowish.

(4) Starch / inorganic salt agar medium (ISP
-Medium 4, culture at 30 ° C) White aerial hyphae grow slightly on the development of light yellow [1 1/2 ic, Lt Antique Gold * 2gc, Bamboo], and no soluble pigment is observed.

(5) Tyrosine agar medium (ISP-medium 7, culture at 30 ° C.) For the development of yellow [1 1/2 lc, Gold], brown white [2 ca, Lt Ivor]
y] aerial hyphae are settled, and the soluble pigment becomes yellowish.

(6) Nutrient agar medium (cultivation at 30 ° C.) White aerial hyphae grow slightly on the growth of light-weight [2 gc, Bamboo], and no soluble pigment is observed.

(7) Yeast-malt agar medium (ISP-
Medium 2, culture at 30 ° C) On the development of light yellow tea [2 ne, Mastard Gold], white aerial hyphae grew and no soluble pigment was observed.

(8) Oatmeal / agar medium (ISP-
Medium 3, culture at 30 ° C) On the development of light yellow [1 1/2 ic, Lt Antique Gold], white aerial mycelium grows faintly, and the soluble pigment is slightly yellowish.

(9) Glycerin / nitrate agar medium (30
(° C culture) On the development of light yellow [1 ga, Lt Lemon Yellow], white aerial hyphae were slightly settled and no soluble pigment was observed.

(10) Starch agar medium (cultivation at 30 ° C.) White aerial mycelium slightly grows on the development of yellow [1 la, Lemon Yellow]. No soluble dye is found.

(11) Lime malate agar medium (30 ° C. culture) On the development of light yellow [1 ga. Lt Lemon Yellow], white aerial hyphae slightly colonize and no soluble pigment is observed.

(12) Cellulose (synthetic solution containing filter paper pieces, 3
(0 ° C culture) Growth develops pale yellow and white aerial mycelium, and no soluble pigment is observed.

(13) Gelatin stab culture In a 15% simple gelatin medium (cultured at 20 ° C.), pale yellow and white aerial hyphae grow faintly and no soluble dye is observed. Glucose / peptone / gelatin medium (2
In the case of 7 ° C culture), the growth is pale yellow, and the aerial mycelium does not settle,
No soluble dye is found.

(14) Skim milk (37 ° C. and 30 ° C. culture) No growth was observed in 37 ° C. culture. In the case of culturing at 30 ° C., growth is pale yellow, aerial hyphae do not grow, and soluble pigments are not observed.

3. Physiological properties (1) Growth temperature range Glucose / asparagine agar medium (glucose 1.
0%, L-asparagine 0.05%, dipotassium phosphate 0.05%, string agar 3.0%, pH 7.0), 6 ° C, 20 ° C, 24 ° C, 27 ° C, 30 ° C, 37 ° C,
Tested at each temperature of 50 ℃, 6 ℃, 37 ℃, 50 ℃
Except that, the optimum growth temperature seems to be around 27 ° C to 30 ° C.

(2) Liquefaction of gelatin (15% simple gelatin medium, 20 ° C. culture; glucose / peptone / gelatin medium, 27 ° C. culture) In 15% simple gelatin medium, liquefaction started from about 3 days after culturing and about 3 Completed a week later. In the glucose-peptone-gelatin medium, a slight liquefaction was observed around the 7th day after the culture, but the progress was extremely slow, and the culture was not completed for one month.

(3) Hydrolysis of starch (starch / inorganic salt agar medium and starter agar medium, both at 30 ° C.)
Cultivation) In any medium, water-decomposability was observed around the 6th day after culturing, and its action was moderate.

(4) Coagulation and Peptone of Skim Milk (Skim Milk, Cultivated at 37 ° C. and 30 ° C.) Growth at 37 ° C. is not observed. In the case of culturing at 30 ° C., peptization started without coagulation from about 10 days after culturing, and was completed after culturing for about 1 month.

(5) Production of melanin-like pigment (tryptone yeast broth, ISP-medium 1; peptone yeast iron agar medium, ISP-medium 6; tyrosine agar medium, ISP-medium 7; both cultures at 30 ° C. ) All media are negative.

(6) Utilization of carbon source (Pridham-Godlieve agar medium, ISP-medium 9; 30 ° C. culture) D-glucose, L-arabinose, D-xylose,
It grows using inositol, D-mannitol and lactose, but does not use sucrose. D-fructose, rhamnose and raffinose are likely to be utilized.

(7) Dissolution of lime malate (lime malate agar medium, culturing at 30 ° C.) Solubility was observed around 10 days after culturing, and its action was moderate.

(8) Reduction reaction of nitrate (peptone water containing 0.1% potassium nitrate, ISP-medium 8, culture at 30 ° C.) is positive.

(9) Degradation of cellulose (synthetic solution containing filter paper pieces, culture at 30 ° C.) Degradation is not observed after 6 months of observation.

Summarizing the above properties, MI184-4
In the 2F4 strain, the basal hypha often show a zigzag shape due to its morphology, and fragmentation is observed. The aerial mycelium is straight or irregularly curved, with fragmentation, forming cylindrical spores, and its surface is smooth. No helix formation, limbal branch or sporangium is observed. In various media, light yellow ~ white on the development of yellow ~
The pale yellow aerial hyphae are settled, and the soluble pigment is slightly yellowish or absent. The formation of melanin-like pigment is negative, the water-degradability of starch is moderate, the reduction reaction of nitrate is positive, and the proteolytic activity is moderate.

By the way, in the bacterial cell component of the MI481-42F4 strain, 2,6-diaminopimelic acid contained in the cell wall is meso-type, and the reducing sugar in all the bacterial cells is A-type containing arabinose and galactose. In addition, it contains no mycolic acid, the phospholipid is PH type (containing phosphatidylethanolamine and does not contain phosphatidylcholine and unknown glucosamine-containing phospholipid), and the major menaquinone is MK-.
9 (H ₄ ) and MK-9 (H ₂ ) was also recognized.

[0035] From the above results, MI481-42F4 strain, Amycolatopsis (Amycolatop sis, literature, Intern
ational Journal of Systematic Bacteriology, 36
Vol., 29-37, 1988). Searching for known species of the genus Amycolatopsis,
Amycolatopsis orientalis ( Amycolatopsis or
ientalis , Reference 1, Ibid; Reference 2, International Jour
nal of Systematic Bactoriology, 37, 292-2
95, 1987) and Amycolatopsis mediterranei , 1 and 2,
Ibid.) Was listed as a closely related species.

Therefore, the MI481-42F4 strain and the above-mentioned 2
We are currently conducting a comparative study of the strains conserved by this institute. Currently, MI481-42F4 strain is
SP ( Amycolatopsis sp.) MI481-42F4
And

The MI481-42F4 strain was applied for deposit at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology on February 3, 1992.
It was entrusted as Japan Institute of Microbiology, No. 12739.

As the strain that can be used in the present invention, all of the MI484-42F4-A producing strains belonging to the genus Amycolatopsis can be used, including the above strains and mutants thereof.

Antibiotics of the present invention MI481-42F4-
4 is produced by inoculating the above strain into a medium suitable for producing the antibiotic MI481-42F4-A and culturing. The medium may be a nutrient-source-containing medium used for usual actinomycete culture.

Examples of nutrient sources include commercially available peptone, meat extract, corn steep liquor, cottonseed flour, peanut flour, soybean flour, yeast extract, MZ-amine, casein hydrolyzate, sodium nitrate, ammonium nitrate, Nitrogen sources such as ammonium sulfate and commercially available glycerin, sucrose, starch, glucose, galactose, mannose, molasses and other carbohydrates, or carbon sources such as fat, and inorganics such as salt, phosphate, calcium carbonate and magnesium sulfate. Salt can be used. In addition, if necessary, a trace amount of a metal salt, or an antifoaming agent such as animal / vegetable / mineral oil may be added. Any of these may be used as long as it can be used by the producing bacterium and is useful for producing the antibiotic MI481-42F4-A, and all known culture materials for actinomycetes can be used.

Liquid culture is preferable for mass production of the antibiotic MI481-42F4-A, and the culture temperature is the antibiotic MI.
It can be applied within the range where 481-42F4-A can be produced.
For the culture, the conditions described above were applied to MI481-42F4-
It can be performed by appropriately selecting it depending on the properties of the A-producing bacterium.

The antibiotic MI481-42F4-A can be extracted with an organic solvent which is mainly present in the cells and is miscible with water such as alcohol, acetonitrile and acetone. The cell extract can be extracted with a water-immiscible organic solvent having a relatively high polarity such as butanol. In addition to the above extraction method,
Pure collection can be performed by appropriately combining or repeating known methods such as adsorption chromatography, gel filtration chromatography, thin layer chromatography, countercurrent partition chromatography, and high performance liquid chromatography.

The physicochemical properties of the antibiotic MI481-42F4-A are as follows.

(1) Color and shape: colorless powder (2) Melting point: 294-295 ° C (decomposition) (3) Elemental analysis: C 49.46%, H 4.79%, N
16.76%, O 12.23%, S 15.72% (4) Ultraviolet absorption spectrum: lambda maximum (nm) methanol 203 309 221 221 350 (shoulder) 245 (shoulder) methanol-hydrochloric acid 205 308 222 222 353 (shoulder) 250 (shoulder) Methanol-sodium hydroxide 205 310 221 353 (shoulder) 250 (shoulder).

(5) Infrared absorption spectrum (KBr method):
3300, 3120, 2980, 1660, 1550,
1500, 1250, 990, 940, 760 cm ^-1 (6) Stability: Stable at room temperature and pH 2-9 (7) Specific rotation: [α] _D ²⁸ = + 133 ° (c 0.74
5, dimethyl sulfoxide) (8) Acidic, neutral, basic classification: basic substance (9) molecular weight: 1182.44 (10) molecular formula: C ₅₀ H ₅₁ O ₈ N ₁₅ S ₆

The biological properties of the antibiotic MI481-42F4-A are as follows. Antibiotic MI481-4
Table 1 shows the inhibitory concentration of 2F4-A on various agars on a nutrient agar medium (by agar plate dilution method). Table 1
More clearly the antibiotic MI481-42F4-A
Had a strong antibacterial activity against Gram-positive bacteria. Also methicillin-resistant Staphylococcus aureus Staphylococcus,
It also strongly inhibited the growth of multidrug-resistant bacteria such as Aureus MS9610 (MRSA).

[0047]

[0048]

[0049]

According to the acute toxicity test conducted in mice, the LD ₅₀ of the antibiotic MI481-42F4-A was
The intraperitoneal dose was 100 mg / kg or more.

Further, a therapeutic effect was confirmed in an infection treatment experiment in mice. Staphylococcus aureus (S. aureus Smith) 3.8 × 10 ⁷ cells / group in 5 female CDF ₁ mice / group
ml and 10% mucin (Dicco) solution were mixed in equal amounts, and 9.5 × 10 ⁶ mice per mouse were inoculated intraperitoneally for infection. On the other hand, the antibiotic MI481-42F4-A of the present invention
10.3 mg of 350 μl of dimethyl sulfoxide (D
After dissolving in MSO), a small amount of Tween 80 was added, and physiological saline was further added, and then intraperitoneally administered once immediately after the infection and 4 hours after the infection.

As a result of life and death judgment after about 20 hours, the ED ₈₀ of this drug was 0.6 mg / kg or more. From the above, the antibiotic MI481-42F4-A may be used for treating bacterial infections in humans and animals.

As described above, the antibiotic MI481-42
Although the physicochemical properties and biological properties of F4-A have been described in detail, the antibiotic MI481-42F4-A is a novel substance because no compound similar to such properties has been known so far.

[0054]

EXAMPLES The present invention will now be described with reference to examples, but the present invention is not limited thereto.

Example 1 2.0% glycerol, dextrin (Wako Pure Chemical Industries) 2
0%, polypeptone (Wako Pure Chemical Industries) 1.0%, yeast extract (Wako Pure Chemical Industries) 0.3%, ammonium sulfate 0.2
%, Calcium carbonate 0.2%, defoamer (KM70, Shin-Etsu Chemical) 0.01%, medium (pH 7.4) 110 ml
In a 500 ml waffle Erlenmeyer flask,
After sterilization at 0 ° C. for 20 minutes, the MI481-42F4 strain (F belonging to the genus Amycolatopsis belonging to the genus Amycolatopsis (F
1 platinum loop of ERM P-12739) was inoculated, and the culture solution obtained by shaking culture at 27 ° C. for 3 days was used as an inoculum source.
2 ml of this inoculum was transplanted to the same medium as above and cultured at 27 ° C. for 4 days to accumulate the antibiotic MI481-42F4-A.

The culture broth (pH 8.2, 10.1 liter) was centrifuged to collect the cells, and this was extracted with 5 liters of methanol. The extract was evaporated to remove concentrated methanol, added with 5 liters of water and extracted with an equal amount of butanol. After concentration, methanol was added and the precipitate was collected to obtain 4.3 g of a dried product. This was dissolved in dimethylformamide, suspended in dimethylformamide, and packed into Sephadex LH-20 (Pharmacia).
It was put in a column (outer diameter 50φ × 100 mm) and gel-filtered with dimethylformamide. Active fraction ( Bacillus thermoph
Fractions showing antibacterial activity against ilus ) were concentrated under reduced pressure, dissolved in dimethylformamide, suspended in dimethylformamide and packed into Toyopearl HW-40 (Tosoh).
The mixture was placed in a column (outer diameter 40φ × 420 mm), subjected to gel filtration with dimethylformamide, and the active fraction was concentrated and dried under reduced pressure to obtain 780 mg of a dried product.

This was dissolved in 15 ml of chloroform,
This solution was added to silica gel (Merck Art. 7734) 25
g was suspended in chloroform and charged into a packed column, and the column was washed with 200 ml of chloroform and then washed with chloroform /
Elute with a mixed solution of methanol = 10/1, collect active fractions,
Concentrated to dryness under reduced pressure and pure antibiotic MI481-42F4
232 mg of colorless powder of -A was obtained.

Example 2 12 liter of the medium used in Example 1 in a 30 liter fermentor
After sterilizing at 120 ° C. for 20 minutes, 100 ml of the seed culture solution used in Example 1 was inoculated, and aeration and stirring culture was carried out at 27 ° C. for 72 hours at an aeration rate of 12 liter / minute and a stirring rotation speed of 200 times / minute. Thereafter, extraction and purification were performed in the same manner as in Example 1, and MI48
82 mg of 1-42F4-A was obtained.

[0059]

As described above in detail, the present invention provides a novel antibiotic and a method for producing the same.

[Brief description of drawings]

1 shows the UV absorption spectrum of the antibiotic MI481-42F4-A.

FIG. 2 shows an infrared absorption spectrum (KBr method) of antibiotic MI481-42F4-A.

FIG. 3: ¹ H of antibiotic MI481-42F4-A measured at 400 MHz in deuterated dimethyl sulfoxide.
Represents the NMR spectrum.

FIG. 4: ¹³ C of antibiotic MI481-42F4-A measured in deuterated dimethyl sulfoxide at 100 MHz.
Represents the NMR spectrum.

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[Procedure amendment]

[Submission date] July 1, 1992

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Mycological properties of MI481-42F4 strain 1. Morphology Well-branched basal hyphae often have a zigzag shape,
In addition, division is recognized. The aerial hyphae are straight or irregularly curved, with fragmentation, forming cylindrical spores. The size of the spores is about 0.6 to 0.7 × 0.7 to 1.2 microns, the surface is smooth. No helix formation, limbus or sporangia are observed.

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(4) Starch / inorganic salt agar medium (ISP
-Medium 4, culture at 30 ℃) Light yellow [1 1/2 ic, Lt Antique Gold ~ 2gc, Bamboo]
White aerial hyphae grew slightly on the growth of the, and no soluble pigment was observed.

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(6) Nutrient agar medium (cultured at 30 ° C.) On the development of light yellow [2 gc, Bamboo], white aerial mycelia are slightly grown and no soluble pigment is observed.

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(11) Lime malate agar medium (cultured at 30 ° C.) On the development of light yellow [1 ga , Lt Lemon Yellow], white aerial mycelium slightly colonized and no soluble pigment was observed.

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[0026] (3) Hydrolysis of starch (starch-inorganic salt agar medium and star Ji agar, both 30 ° C.
Cultivation) In any medium, water-decomposability was observed around the 6th day after culturing, and its action was moderate.

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Summarizing the above properties, MI 481 -4
In the 2F4 strain, the basal hypha often show a zigzag shape due to its morphology, and fragmentation is observed. The aerial mycelium is straight or irregularly curved, with fragmentation, forming cylindrical spores, and its surface is smooth. No helix formation, limbal branch or sporangium is observed. In various media, light yellow ~ white on the development of yellow ~
The pale yellow aerial hyphae are settled, and the soluble pigment is slightly yellowish or absent. The formation of melanin-like pigment is negative, the water-degradability of starch is moderate, the reduction reaction of nitrate is positive, and the proteolytic activity is moderate.

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By the way, in the bacterial cell component of the MI481-42F4 strain, 2,6-diaminopimelic acid contained in the cell wall is meso-type, and the reducing sugar in all the bacterial cells is A-type containing arabinose and galactose. In addition, mycolic acid is not included, phospholipids are P type II (containing phosphatidylethanolamine and not phosphatidylcholine and unknown glucosamine-containing phospholipids), and the major menaquinone is MK-.
9 (H ₄ ) and MK-9 (H ₂ ) was also recognized.

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[0035] From the above results, MI481-42F4 strain, Amycolatopsis (Amycolatop sis, literature, Intern
ational Journal of Systematic Bacteriology, 36
, Pp. 29-37, is considered to belong to 198 six years) genus. Searching for known species of the genus Amycolatopsis,
Amycolatopsis orientalis ( Amycolatopsis or
ientalis , Reference 1, Ibid; Reference 2, International Jour
nal of Systematic Bact e riology, 37, pp. 292-
295, 1987) and Amycolatopsis mediterranei (References 1 and 2, ibid.) Were mentioned as closely related species.

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The strains usable in the present invention is the strain, including its mutants, all MI48 1 -42F4-A producing bacteria belonging to the genus Amycolatopsis can be used.

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Antibiotics of the present invention MI481-42F4-
A is produced by inoculating the above strain into a medium suitable for the production of the antibiotic MI481-42F4-A and culturing. The medium may be a nutrient-source-containing medium used for usual actinomycete culture.

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(1) Color and shape: colorless powder (2) Melting point: 294-295 ° C (decomposition) (3) Elemental analysis: C 49.46%, H 4.79%, N
16.76%, O 12.23%, S 15.72% (4) Ultraviolet absorption spectrum: lambda maximum (nm) methanol 203 309 221 221 350 (shoulder) 245 (shoulder) methanol-hydrochloric acid 205 308 222 222 353 (shoulder) 250 (shoulder) Methanol-sodium hydroxide 205 310 221 353 (shoulder) 250 (shoulder )

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The biological properties of the antibiotic MI481-42F4-A are as follows. Antibiotic MI481-4
Table 1 shows the inhibitory concentration of 2F4-A on various agars on a nutrient agar medium (by agar plate dilution method). Table 1
More clearly the antibiotic MI481-42F4-A
Had a strong antibacterial activity against Gram-positive bacteria. The methicillin-resistant Staphylococcus aureus Staphylococcus
It also strongly inhibited the growth of multidrug-resistant bacteria such as Aureus MS9610 (MRSA).

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Further, a therapeutic effect was confirmed in an infection treatment experiment in mice. Staphylococcus aureus (S. aureus Smith) 3.8 × 10 ⁷ cells / group in 5 female CDF ₁ mice / group
ml and mucin (di off co) 10% solution mixed in equal amounts to 9.5 × 10 ⁶ cells per pet mice were allowed inoculated infected intraperitoneally. On the other hand, the antibiotic MI481-42F4-A of the present invention
10.3 mg of 350 μl of dimethyl sulfoxide (D
After dissolving in MSO), a small amount of Tween 80 was added, and physiological saline was further added, and then intraperitoneally administered once immediately after the infection and 4 hours after the infection.

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Example 1 Glycerol 2.0%, dextrin (Wako Pure Chemical Industries)
2.0 %, polypeptone (Wako Pure Chemical Industries) 1.0%, yeast extract (Wako Pure Chemical Industries) 0.3%, ammonium sulfate 0.
Medium (pH 7.4) 110m containing 2%, calcium carbonate 0.2%, and antifoaming agent (KM70, Shin-Etsu Chemical) 0.01%
1 is put into a 500 ml waffle Erlenmeyer flask and 1
After sterilization at 20 ° C. for 20 minutes, 1 platinum loop of MI481-42F4 strain (FERM P-12739) belonging to the genus Amycolatopsis grown on the agar slope was inoculated, and
A culture solution obtained by shaking culture at 3 ° C. for 3 days was used as an inoculum source. 2 ml of this inoculum was transferred to the same medium as above, and 27
Cultivated at 4 ° C for 4 days and antibiotic MI481-42F4-A
Accumulated.

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The culture broth (pH 8.2, 10.1 liter) was centrifuged to collect the cells, and this was extracted with 5 liters of methanol. After concentrating the extract and distilling off methanol , water 5 lit
er was added, the mixture was extracted with an equal amount of butanol, concentrated, and then methanol was added to collect the precipitate to obtain 4.3 g of a dried product. This was dissolved in dimethylformamide, suspended in dimethylformamide and packed in a packed Sephadex LH-20 (Pharmacia) (outer diameter 50φ × 1000 mm) column, and gel filtered with dimethylformamide. Active fraction ( Bacillus t
(Fraction exhibiting antibacterial activity against hermophilus ) was concentrated under reduced pressure, dissolved in dimethylformamide, suspended in dimethylformamide and applied to a Toyopearl HW-40 (tosaw) (outer diameter 40φ × 420 mm) tower, and dimethylformamide was added. Gel filtration was performed with formamide, and the active fraction was concentrated and dried under reduced pressure to obtain 780 mg of a dried product.

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Example 2 12 liter of the medium used in Example 1 in a 30 liter fermentor
After sterilizing at 120 ° C. for 20 minutes, 100 ml of the seed culture solution used in Example 1 was inoculated, and aeration and stirring culture was carried out at 27 ° C. for 72 hours at an aeration rate of 12 liter / minute and a stirring rotation speed of 200 times / minute. Thereafter, extraction and purification were carried out in the same manner as in Example 1 to obtain MI48.
82 mg of 1-42F4-A was obtained .

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[Figure 1]

Continuation of the front page (51) Int.Cl. ⁵ Identification number Reference number within the agency FI technical display area // (C12P 1/02 C12R 1: 645) (72) Inventor Hironobu Iinuma 3-35 Shiroko, Wako-shi, Saitama No. 6-301 Sumitomo Wako House (72) Inventor Kazuo Shimanaka 3-4-7-301 Nishi-Kamata, Ota-ku, Tokyo

Claims (2)

[Claims] 1. The following characteristics: (1) Color and shape: colorless powder (2) Melting point: 294-295 ° C. (decomposition) (3) Elemental analysis: C 49.46%, H 4.79%, N
16.76%, O 12.23%, S 15.72% (4) Ultraviolet absorption spectrum: lambda maximum (nm) methanol 203 309 221 221 350 (shoulder) 245 (shoulder) methanol-hydrochloric acid 205 308 222 222 353 (shoulder) 250 (shoulder) methanol-sodium hydroxide 205 310 221 353 (shoulder) 250 (shoulder) (5) Infrared absorption spectrum (KBr method): 3300,3
120, 2980, 1660, 1550, 1500, 1
250,990,940,760 cm ^-1 (6) Stability: Stable at room temperature and pH 2-9 (7) Specific rotation: [α] _D ²⁸ = + 133 ° (c 0.74
5, dimethylsulfoxide) (8) Acidic, neutral, basic classification: basic substance (9) molecular weight: 1182.44 (10) molecular formula: C ₅₀ H ₅₁ O ₈ N ₁₅ S ₆ New antibiotic MI481-4
2F4-A. 2. MI481 belonging to the genus Amycolatopsis
A method for producing a novel antibiotic MI481-42F4-A, which comprises culturing a -42F4-A producing bacterium, and separating and collecting the novel antibiotic MI481-42F4-A from the culture.