JPH0733736A - Novel antibiotic azicemycin b and its production - Google Patents

Abstract

PURPOSE:To obtain a novel antibiotic exhibiting a strong antibacterial activity against gram-positive bacteria, especially acid-fast bacteria, and useful as an antibiotic for medical cares. CONSTITUTION:An antibiotic, azicemycin B, represented by the formula or its salt. The compound is obtained by culturing an antibiotic azicemycin B- producing bacterium belonging to the genus Amycolatopsis, preferably Amycolatopsis S.P.-MJ126-NF4 strain (FERM-P-13362), in a nutritive medium and subsequently collecting the produced and accumulated antibiotic from the culture product.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel antibiotic Azicemicin (Azicemicin) which is produced by a microorganism belonging to the genus Amycolatopsis and has an antibacterial activity and exhibits a strong antibacterial activity against Gram-positive bacteria, particularly acid-fast bacteria. ) B or a salt thereof, and a method for producing the same.

[0002]

2. Description of the Related Art Many useful compounds such as rifamycin, kanamycin, streptomycin, capreomycin, enviromycin and cycloserine are known as antibiotics produced by microorganisms and having antibacterial activity, especially acid-fast bactericidal activity.

[0003]

Antibiotics, such as rifamycin, which show antibacterial activity, particularly antibacterial activity against acid-fast bacilli, are widely used at present, but the appearance of resistant bacteria and side effects such as liver damage and hematological disorders are caused. There are various problems such as interference. It is awaited to provide a novel antibacterial substance having a strong antibacterial activity against rifamycin-resistant bacteria and having low toxicity.

[0004]

[Means for Solving the Problems] The inventors have promoted the development and practical application research of useful antibiotics from the past.
As one part of such research, the present inventors previously produced a novel antibiotic by a microorganism belonging to the genus Amycolatopsis, which is a kind of actinomycete isolated from soil, and having a strain number of MJ126-NF4. I found out to do. This antibiotic is isolated to determine its physicochemical properties and has the molecular formula C ₂₃
It was found that it is an amphoteric yellowish brown solid substance represented by H ₂₅ O ₉ N and has a strong antibacterial activity against Gram-positive bacteria, especially acid-fast bacteria, and named it as antibiotic MJ126-NF4A (Patent application No. 5-69072; filed on March 5, 1993).

Recent studies have shown that the antibiotic MJ126-NF4A has the following chemical structure: In addition, the antibiotic MJ126-NF4A was renamed Azicemicin A.

Further, the inventors of the present invention have conducted researches to find that the above-mentioned MJ126-NF4 strain belonging to the genus Amycolatopsis is adisemycin A.
It has been found that it produces a novel antibiotic different from that and also strongly inhibits the growth of Gram-positive bacteria, especially acid-fast bacteria. We succeeded in isolating this novel antibiotic, investigated its physicochemical properties, and found that its chemical structure was represented by the following formula (I). It was named B (Azicemicin B). The present invention has been completed based on these findings.

Therefore, according to the first aspect of the present invention, And azisemycin B or a salt thereof.

The adisemycin B according to the first aspect of the present invention has the following properties. (1) Physicochemical properties of the antibiotic adisemycin B A) Property: yellow powder B) Thin layer chromatography [Merck & Co., Art.5715, developing solvent chloroform-methanol-water (4: 1: 0.1)]: Rf value 0.42 C) It is an amphoteric substance. D) Mass spectrum (m / z): 446 (M + H) ⁺ 444 (M-
H) ^-

E) Ultraviolet absorption spectrum: (i) A UV absorption spectrum measured in methanol is shown in FIG. 1 of the accompanying drawings. λmax, nm (ε) 234 (20100), 278 (35700), 322 (5270), 3
36sh (4220), 409 (11500) (ii) The UV absorption spectrum measured in 0.1N-NaOH-90% methanol is shown in Figure 2 of the accompanying drawings. λmax, nm (ε) 242 (13700), 290 (38100), 414 (18800) (iii) A UV absorption spectrum measured in 0.1N-HCl-90% methanol is shown in FIG. 3 of the accompanying drawings. λmax, nm (ε) 234 (22500), 277 (37000), 323 (5630), 3
35sh (4510), 409 (11400)

F) Infrared absorption spectrum (KBr tablet method):
Shown in FIG. 4 of the accompanying drawings. ν max (cm ⁻¹ ), 3410, 2940, 1720, 1620, 1520, 1390 G) ¹³ C-NMR spectrum (CDCl ₃ ): shown in FIG. 5 of the accompanying drawings. H) ¹ H-NMR spectrum (CDCl ₃ / TMS): shown in FIG. 6 of the accompanying drawings.

(2) Biological properties of the antibiotic adisemycin B A) Antibacterial spectrum The minimum inhibitory concentration of the antibiotic adisemycin B against various bacteria on eutrophic agar plates is shown in Table 1 below.

[0012]

B) Acute toxicity Acute toxicity of a novel antibiotic, adisemycin B, to mice was tested. As a result, 72.5 mg / kg was given to mice by intravenous injection.
Was administered, but no abnormality was observed.

According to the second aspect of the present invention, as a method for producing a new antibiotic, adisemycin B, an adisemycin B-producing bacterium belonging to the genus Amycolatopsis is cultivated in a nutrient medium to produce and accumulate adisemycin. There is provided a method for producing adisemycin B represented by the above formula (I), which comprises collecting the antibiotic.

The bacterium to be used in the second aspect of the present invention is not limited to any genus or species as long as it is a microorganism capable of producing the antibiotic adisemycin B. A preferable specific example thereof is amicolapto. There is a bacterium belonging to the cis bacterium and having a strain number of MJ126-NF4.

This MJ126-NF4 strain is an actinomycete isolated from soil in Setagaya-ku, Tokyo at the Institute for Microbial Chemistry in September 1989, and has the strain number of MJ126-NF4. The mycological properties of this MJ126-NF4 strain are described in detail below.

1. Well-divided basal hyphae are well fragmented. The aerial hyphae are straight or irregularly curved and divide into cylindrical to oval fragments or spore-like structures. Its size is about 0.3-0.5
× 0.8 to 1.1 microns. Also, from the 18th day after culturing, aerial hyphae show distorted helix formation of 1 to 2 turns. No limbus or sporangia are observed.

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

(1) Sucrose / nitrate agar medium (30
(° C culture) On the development of light yellow [2ca, Lt Ivory], white aerial hyphae grow and no soluble pigment is observed. (2) Glucose / asparagine agar medium (30 ℃ culture) light yellow [2ca, Lt Ivory] ~ light olive [1 ie, Dusty
On the growth of Olive], white aerial mycelia settle, and the soluble pigment has a faint greenish tinge. (3) Glycerin-asparagine agar medium (ISP-medium 5, 30 ℃ culture) Light yellow daidai [3gc, Lt Tan] to yellow tea [3 le, Cinnamon]
~ 3ng, Yellow Maple], white to brown-white aerial mycelium grows, and the soluble pigment takes on a faint brownish tinge. (4) Starch / inorganic salt agar medium (ISP-medium 4, 30 ℃ culture) Light yellow daidai [3gc, Lt Tan] ~ gray yellow tea [3ni, Clove
On the development of [Brown], white aerial hyphae are settled and soluble pigments are not observed.

(5) Tyrosine agar medium (ISP-medium 7, 30
Culturing at ℃) On the development of grayish yellow tea [3ni, Clove Brown], aerial hyphae of white to grayish white [b, Oyster White] are settled, and the soluble pigment partially shows dark brown. (6) Nutrient agar medium (30 ° C culture) White aerial hyphae grow slightly on the development of light yellow daidai [3gc, Lt Tan], and the soluble pigment has a brownish tinge. (7) Yeast-malt agar medium (ISP-medium 2, culture at 30 ° C) White aerial hyphae grow on the development of light yellow tea [3 le, Cinnamon], and the soluble pigment takes on a brownish tinge. (8) Oatmeal agar medium (ISP-medium 3, cultured at 30 ° C) White aerial hyphae grew on the development of light yellow [2ca, Lt Ivory], and no soluble pigment was observed. (9) Glycerin / nitrate agar medium (30 ℃ culture) Light yellow daidai [3gc, Lt Tan] ~ Light yellow tea [3ic, Lt Am
ber], the aerial hyphae of brown-white [3ba, Pearl] settled slightly, and the soluble pigment became yellowish.

(10) Starch agar medium (cultured at 30 ° C.) For the development of colorless to light yellow radish [3ca, Pearl Pink],
Grow white aerial mycelium. No soluble dye is found. (11) Lime malate agar medium (30 ° C culture) On the development of thin olives [1 gc, Citron], white aerial hyphae grow faintly, and the soluble pigment shows thin olives. (12) Cellulose (synthetic solution with filter paper pieces added, cultivated at 30 ° C) Growth is colorless, white aerial mycelium grows, and soluble pigment is not observed. (13) Gelatin stab culture In a 15% simple gelatin medium (cultured at 20 ℃), growth is pale yellow and white aerial hyphae grow, and the soluble pigment is yellow tea.
In the case of glucose-peptone-gelatin medium (cultured at 27 ° C), the growth was pale yellow and white aerial hyphae were faintly attached, and the soluble pigment was brown. (14) Skim milk (cultured at 37 ℃) Growth is pale yellow to dark olive, aerial mycelium does not grow, and soluble dye is not observed.

3. Physiological properties (1) Growth temperature range Using yeast / starch agar medium (soluble starch 1.0%, yeast extract 0.2%, string agar 3.0%, pH 7.0), 10 ℃, 20 ℃, 24 ℃, 27 ℃, 30 ℃ As a result of testing at each temperature of ℃, 37 ℃ and 50 ℃, except for 10 ℃ and 50 ℃, it grew at any of these temperatures, but the optimum growth temperature seems to be around 30 ℃.

(2) Liquefaction of gelatin (15% simple gelatin medium, 20 ° C culture; glucose / peptone / gelatin medium, 27 ° C culture) After culturing in any of 15% simple gelatin medium and glucose / peptone / gelatin medium Liquefaction started from around the 3rd day, but the progress of liquefaction was extremely slow, and it was not completed in 3 weeks of culture.

(3) Hydrolysis of starch (starch / inorganic salt agar medium and starch agar medium, both cultures at 30 ° C.) In starch / inorganic salt agar medium, hydrolyzability was observed around 14 days after culture. Its action is weak. Also,
In starch agar medium, water degradability was observed around 20 days after cultivation, but its action was extremely weak.

(4) Coagulation / peptonization of skim milk (skim milk, 37 ° C. culture) Coagulation and peptone formation are not observed in the culture for about 3 weeks. (5) Formation 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) Positive in tryptone yeast broth and peptone yeast-iron agar medium , On tyrosine agar, it is not clear.

(6) Utilization of carbon source (Pridham-Godlieve agar medium, ISP-medium 9; 30 ° C. culture) D-glucose, D-xylose, D-fructose,
It grows using inositol and D-mannitol and does not use sucrose. The use of L-arabinose, rhamnose, and raffinose is unclear.

(7) Dissolution of lime malate (lime malate agar medium, culturing at 30 ° C.) Solubility was observed around the 7th day after culturing, and its action was moderate. (8) Nitrate reduction reaction (peptone water containing 0.1% potassium nitrate, ISP-medium 8, 30 ° C culture) Positive. (9) Decomposition of cellulose (synthesis solution with filter paper pieces added, culturing at 30 ° C) No decomposition is observed under observation for about 3 months.

To summarize the above properties, the MJ126-NF4 strain has a morphologically observable fragmentation of basal hyphae, aerial mycelia of straight or irregularly curved, cylindrical to oval fragments or spores. Divide into a structure like this. From the 18th day after culturing, distorted helix formation is observed. There are no limbus or sporangia. On various media, white aerial hyphae grow on the development of light yellow to yellow tea or light olive, and the soluble pigment has a faint brownish tinge. It is considered that melanin-like pigment is probably produced, the starch has weak hydrolyzability and proteolytic activity, and nitrate reduction reaction is positive.

By the way, the cell components of the MJ126-NF4 strain contained meso-type 2,6-diaminopimelic acid, arabinose and galactose in the cell wall and showed type IV. The reducing sugar in all cells is type A containing arabinose and galactose. The results of the glycolate test were acetyl type.
In addition, it contains no mycolic acid, the phospholipid is PII type (containing phosphatidylethanolamine and no phosphatidylcholine and unknown glucosamine-containing phospholipid), and the major menaquinone is MK-9 (H ₄ ). Fatty acids are i-16: 0,
It mainly contains 16: 0, ai-17 and 10Me-16: 0.

From the above results, the MJ126-NF4 strain was identified in the genus Amycolatopsis [ Amycolatopsis, Reference, International Jo
urnal of Systematic Bacteriology, 36, 29-37 (1986)]. When searching for known bacterial species of the genus Amycolatopsis, Amycolatopsis sulphurea [Reference 1, Id. And Reference 2, International Journal of Systematic Bacter
Biology, 37, 292-295 (1987)] was mentioned as a closely related species.

Therefore, the MJ126-NF4 strain and the above-mentioned strain preserved by this institute are currently being compared and examined. Currently MJ126-
The NF4 strain was transformed into Amycolatopsis sp.
sp.) MJ126-NF4. The MJ126-NF4 strain was applied for deposit at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science, and
Accepted as FERM P-13362 on 11th of March.

In the method of the second aspect of the present invention, for culturing the adisemycin B-producing bacterium, those which are normally used as a nutrient source for actinomycetes, for example, assimilable nutrients such as carbohydrates, nitrogen sources, inorganic salts, etc. You can use the source. For example, it is possible to use carbohydrates such as glucose, maltose, molasses, dextrin, glycerin, and starch, and carbon sources such as soybean oil and peanut oil, and peptone,
Nitrogen sources such as meat extract, cotton seed powder, soybean flour, yeast extract, casein, corn steep liquor, NZ-amine, ammonium sulfate, ammonium nitrate and ammonium chloride can be used. Furthermore, dipotassium phosphate, sodium phosphate, sodium chloride, calcium carbonate, magnesium sulfate,
An inorganic salt such as manganese chloride can be used, and if necessary, trace metals such as cobalt and iron can be added.
As the nutrient source, any other known nutrient source can be used as long as it can be utilized by the bacteria used in the production of the antibiotic adisemycin B.

The mixing ratio of the nutrient sources as described above is not particularly limited and can be varied over a wide range.
The composition and proportion of the optimum nutritional source for the azisemycin B-producing bacterium to be used can be easily determined by those skilled in the art by a simple small-scale experiment.

A nutrient medium comprising the above-mentioned nutrient source is
It can be sterilized prior to culturing, and before or after this sterilization, it is advantageous to adjust the pH of the medium to a range of 6-8, in particular a pH of 6.5-7.5.

Cultivation of the adisemycin B-producing bacterium in such a nutrient medium can be carried out according to the method usually used in the production of antibiotics by general actinomycetes. Usually, it is suitable to culture under aerobic conditions, and usually, it can be performed with stirring and / or aeration. As the culturing method, any of various methods such as static culturing, shaking culturing, and submerged culturing with aeration and stirring can be used.
Suitable for mass production.

The culture temperature that can be used is not particularly limited as long as it does not substantially inhibit the growth of the adisemycin B-producing bacterium and can produce the antibiotic, and depending on the producing bacterium used. Although it can be appropriately selected, a particularly preferable culture temperature is a temperature in the range of 25 to 30 ° C.

Culturing can usually be continued until the new antibiotic, adisemycin B, is produced in the culture and fully accumulated. The culturing time varies depending on the composition of the medium, the culturing temperature, the use temperature, the production strain used, etc.
The desired antibiotic can be obtained by culturing for 72 to 96 hours. The amount of acetomycin B, a novel antibiotic, accumulated in the culture was determined by using Micrococcus luteus, Escherichia coli, Mycobacterium coli, and Mycobacterium smegatus 607. It can be quantified by the method.

Thus, the novel antibiotic adisemycin B accumulated in the culture is filtered, if necessary after filtration,
After removing the cells by a separation method known per se such as centrifugation, the filtrate is adjusted to acidic (pH 2 to 4), solvent extraction using an alcoholic organic solvent, especially butanol, adsorption or ion Chromatography utilizing exchange ability can be used alone or in combination to isolate and purify and collect. As a carrier for chromatography having adsorption or ion exchange ability, activated carbon,
Silica gel, porous polystyrene-divinylbenzene resin or various ion exchange resins can be used. Thus, a new antibiotic, adisemycin B, having the above-mentioned properties is obtained.

In addition, the azisemycin B of the present invention is hydrochloric acid,
It forms an addition salt with a pharmaceutically acceptable inorganic acid such as sulfuric acid or a pharmaceutically acceptable organic acid such as acetic acid, and also forms a salt with an organic base such as alkylamine.

Next, the present invention will be described in more detail by way of examples.

[0041]

EXAMPLES Example 1 Production of the antibiotic adisemycin B 2% galactose, 2% dextrin, 1% bacto-soyton, 0.5% corn stapler, 1% glycene,
Liquid medium containing ammonium sulfate 0.2%, calcium carbonate 0.2%, and 1 drop of silicone oil (adjusted to pH 7.4) was dispensed into Erlenmeyer flasks by 110 ml, sterilized at 120 ° C. for 20 minutes by a conventional method, and then cultured on agar slant medium. The MJ126-NF4 strain (Microtechnology Research Institute, No. 13362) was inoculated and cultivated at 30 ° C. for 8 days with rotary shaking (180 rpm).

Using the thus obtained culture as a seed culture, glycerin 2%, dextrin 2%, bacto-soyton 1%, powdered yeast extract 0.3%, ammonium sulfate
After sterilizing 45 Erlenmeyer flasks containing 110 ml of liquid medium containing 0.2%, calcium carbonate 0.2%, and 1 drop of silicone oil (adjusted to pH 7.4) for 20 minutes at 120 ° C, inoculate 2 ml of each flask. Then, it was subjected to rotary shaking culture (180 rpm) at 27 ° C. for 5 days. The obtained culture solution was filtered, the filtrate was adsorbed on a column packed with Diaion HP-20 (320 ml), and eluted successively with 2 L of water, 1.5 L of 30% methanol water, 1.5 L of 80% methanol water, and antibacterial. Fractions showing activity were collected and concentrated to dryness. The residue was dissolved in methanol, 10 g of silica gel 60 (Merck Co., Art.7734) was added, and the mixture was concentrated and dried under reduced pressure to obtain a powder.

This powder was put on a column packed with 60 g of the above silica gel and subjected to chromatography. Chloroform (250 ml), chloroform-methanol mixed solvent (mixing ratio 20: 1) (210 ml) as elution solvent, and also chloroform-methanol mixed solvent (mixing ratio 10: 1) (220 m
l), the same (mixing ratio 7: 1) (240 ml), the same (mixing ratio 5:
1) (300 ml), the same (mixing ratio 4: 1) (250 ml) and the same (mixing ratio 2: 1) (240 ml) were sequentially eluted to collect the fractions containing no adisemycin A but containing adisemycin B. (0.205g). Further, the fraction was concentrated to dryness under reduced pressure and 56 mg of the residue was TLC (Merck Co. Art.
5715), an Rf value of 0.42 [developing solvent: chloroform-methanol-water (4: 1: 0.1)] was isolated and purified from a portion of the substance exhibiting UV (254 nm) absorption. Was given.

[Brief description of drawings]

FIG. 1 is an ultraviolet absorption spectrum curve diagram of adisemycin B in methanol.

FIG. 2 is an ultraviolet absorption spectrum curve diagram of adisemycin B in 0.1N-NaOH-90% methanol.

FIG. 3 is an ultraviolet absorption spectrum curve diagram of adisemycin B in 0.1N-HCl-90% methanol.

FIG. 4 is an infrared absorption spectrum curve diagram of adisemycin B.

FIG. 5 is a ¹ H-NMR spectrum curve diagram of adisemycin B.

FIG. 6 is a ¹³ C-NMR spectrum curve diagram of adisemycin B.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yoshikazu Takahashi 3-2-3, Sakuragaoka, Tama-shi, Tokyo (72) Inventor Hiroshi Naganawa 3-17 Denenchofuhonmachi, Ota-ku, Tokyo (72) Inventor Riki Sawa 4-6-7 Ayanishi, Ayase City, Kanagawa Prefecture (72) Inventor, Masaru Hamada 26, Hidewa Residence No. 405, 1-26 Naitocho, Shinjuku-ku, Tokyo

Claims (2)

[Claims] 1. The following formula The antibiotic adisemycin B or a salt thereof represented by: 2. A method for producing the adisemycin B antibiotics according to claim 1, which belongs to the genus Amycolatopsis, is cultured in a nutrient medium to produce and accumulate adisemycin B, and the antibiotic is collected from the culture. A process for producing the antibiotic adisemycin B characterized by the following: