JPH0327200B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a new method for producing antibiotic OA- _6129B2 , and more specifically, the following method using fermentation method. The present invention relates to a method for selectively producing the antibiotic OA-6129B ₂ . The present inventors previously calculated the following formula In the formula, R represents a hydrogen atom, -OH or _-OSO3H .
It was discovered that the antibiotics OA-6129A (R=H) and OA-6129B ₁ were produced by fermentation of
(R=OH, 5,6-cis body), OA-6129B ₂ (R=
OH, 5,6-trans body) and OA-6129C (R=
OSO ₃ H) was proposed (European Patent Application Publication No. 48999A1, JP-A-57-62280, JP-A-57-70890 and JP-A-57-
(See Publication No. 95987). The above production strains are antibiotic OA-6129A, OA-
6129B ₁ and OA-6129B ₂ are also produced, but antibiotics
There is a tendency to produce a lot of OA-6129C. but,
When the compound of formula () above is further derived into other useful antibiotics, the compound of formula () when R=OSO ₃ H, i.e. antibiotic OA-6129C
rather than the compound of formula () when R=OH, i.e. the antibiotic OA-6129B ₁ or OA-
6129B ₂ seems to have a wider range of use. Therefore, the present inventors conducted intensive research in search of a strain capable of selectively producing the antibiotic OA-6129B ₁ and/or OA-6129B ₂ , and as a result, the above-mentioned Streptomyces sp. OA-6129
Streptomyces sp. OA-6129- obtained by mutation treatment of (Soreptomyces sp. OA-6129)
3N−504 (Streptomyces sp. OA−6129−3N−
504) is an antibiotic OA represented by the above formula ()
-6129B ₂ was discovered to be selectively produced, and the present invention was completed. According to the present invention, Streptomyces sp. OA-6129-3N-504 is cultured in a nutrient medium, and the following formula is obtained from the culture. Provided is a method for producing antibiotic OA-6129B ₂ , which comprises collecting antibiotic OA-6129B ₂ represented by: The strain used in the method of the present invention, Streptomyces sp. OA-6129-3N-504, is
This is a new strain that has not been described in conventional literature, and its mycological properties are as follows. (1) Morphology Under a microscope, straight to flexible aerial hyphae are elongated from well-branched basal hyphae, and whorled branches are not observed. A mature spore chain consists of ten or more oval to cylindrical spores, and no sporangia are observed. The size of the spores
The surface of the spore is smooth, measuring approximately 0.6-1.0 x 0.7-2.5 microns. Flagellated spores are not observed. (2) Growth conditions in various media Cultivation was performed at 28° to 30°C unless otherwise specified.
In addition, descriptions of color tones are mainly based on HDTresner and E.G. Bakas.
and EJBackus), Journal of Applied Microbiology
Applied Microbiology) Volume 11, No. 4, (1963
According to the method on pages 335-338 (2013), the codes in [ ] [CHM codes] are based on the Container Corporation of America's Color Harmony Manual (Container Corporation of America).
Color Harmony by Corporation of America
Manual) was used. (1) Seuculose/nitrate agar medium: Aerial mycelium of yellow ash [2 dc] to gray yellow pink [5 dc] grows and dissolves on medium growth of yellow ash [2 dc] to light gray yellow brown [3 ge]. No sex pigments are observed. (2) Glucose-asparagine agar medium: light yellow [2db] bright olive brown [2ge]
Bright gray [d] aerial mycelium grows on top of the good growth. This aerial mycelium later becomes grayish-yellow-pink [5 dc]. No soluble pigments are observed. (3) Glycerin-asparagine agar medium (ISP
Medium-5): Good growth of gentle yellow pink [4gc] to light brown [4ie], with light gray [d] to
Propagates bright grayish-reddish brown [5fe] aerial mycelium.
No soluble pigments are observed. (4) Starch inorganic salt agar medium (ISP medium-
4): Light gray [d] aerial mycelia are attached to the light yellow [2db] to gray [2fe] growth. No soluble dyes are produced. (5) Tyrosine agar medium (ISP medium-7): Aerial mycelium of light gray [d] to light brown gray [3fe] grows on gray-yellow [3ec] to light brown [4ie] growth. The medium will have a very slight brown tinge. (6) Nutrient agar medium: light yellow [2db] or bright yellow [2fb] ~
Aerial mycelium of light gray-reddish brown [5fe] grows on the good growth of light olive brown [2ge]. No soluble pigments are observed. (7) Yeast extract/malt extract agar medium (ISP medium-2): Gentle yellow-pink [4gc] to bright brown [4ie] with good growth, gray-yellow-pink [5dc]
Or, slightly later, bright gray [d] aerial mycelium grows. No soluble pigments are observed. (8) Oatmeal agar medium (ISP medium-3): gray-yellow [3ec] to bright orange-yellow [3ea],
Alternatively, on the good growth of light gray-yellow-brown [3ge], aerial mycelium of light brown-gray [3fe] to light gray-reddish-brown [5fe] grows, and the medium around the fungal colony takes on a slightly brown color. (9) Malate lime agar medium: dark to yellow gray [2dc] medium growth;
Aerial mycelia of light gray [d] to light gray reddish brown [5fe] are attached, and no soluble pigments are observed. A dissolution zone of calcium salts can be seen around the growing bacterial colonies. (10) Gelcose peptone gelatin medium (20
℃ culture) White [B] to gray-yellow pink [5CB] aerial mycelia grow on the pale yellow [2dB] to brown good growth. When cultured for a long period of time (about 3 weeks or more), a brown soluble pigment was produced. (3) Physiological properties (1) Growth temperature range: 10°, 20°, 25°, 30°, using yeast extract/malt extract agar medium (ISP medium-2).
As a result of experiments at temperatures of 34°, 37°, 40°, 45°, and 50°C, almost no growth occurred at 37°C. It does not grow at all above 40℃. Growth was observed at all other temperatures. The optimal growth temperature range is 20
It seems to be ~30℃. (2) Liquefaction of gelatin: Liquefaction. (3) Starch hydrolysis: decomposes. (4) Coagulation and peptonization of skim milk: It does not coagulate, but it converts into peptonization. (5) Production of melanin-like pigments: Peptone yeast iron agar medium (ISP medium-5) and tryptone yeast extract
No production of melanin-like pigment was observed in the broth medium (ISP medium-11). Although it shows a very slight brown color on tyrosine agar medium, there is only a trace of melanin production. (4) Assimilation of various carbon sources (using Pridham-Gotrib agar medium) (1) L-arabinose + (2) D-xylose + (3) D-glucose + (4) D-fructose + (5) Seuculose Doubtful (6) Inositol - (7) L-rhamnose + (8) Raffinose - (9) D-mannite ++ is assimilated, - is not assimilated. Based on the above mycological properties, the OA-6129 strain is
It is a strain belonging to the genus Streptomyces, and the shape of the aerial hyphae is Section Recti-RF (Section Recti-RF).
lexibiles), and the spore surface was smooth.
The color of aerial mycelia is light gray on most media, such as oatmeal agar, glycerin/asparagine agar, starch/inorganic salt agar, etc.
It is a strain of the Gray series. However, depending on the culture period, a red series of gray-yellow-pink [5 dc] may be exhibited on seurose/nitrate agar, yeast extract/malt extract agar, and glucose/asparagine agar media. In addition, the color of the basal hyphae is light yellow to grayish yellow in any medium at the initial stage of culture, and as the culture continues, the color becomes yellowish brown to grayish yellowish brown or brown. Melanin pigment was not observed in peptone/yeast extract/iron agar medium or tryptone/yeast extract/broth, and other water-soluble pigments were not produced in many media. However, tyrosine agar medium,
A slight brown pigment was observed in the glucose-peptone-gelatin medium and oatmeal agar medium. The present inventors have identified this strain as Streptomyces sp. OA-6129-3N-504 (Streptomyces sp.
-6129-3N-504) and was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as an international deposit pursuant to the Budapest Treaty on the Deposit of Microorganisms for the Purposes of Patent Procedures.
On August 5th, FERM BP-
334). This strain can be created by mutagenesis of the known Stoptomyces sp. OA-6129, which is described in the European Patent Application Publications and Publications mentioned above. The mutation processing method will be explained in more detail below. That is, Streptomyces sp. OA−
By treating 6129 with conventional mutation induction methods such as ultraviolet irradiation, radiation irradiation, and treatment with chemical mutagenic agents, the biosynthesis system of antibiotic OA-6129C was deleted, and antibiotic OA-6129B ₂ was selected. It is possible to obtain a strain that can produce the desired result. Here, among the chemical mutagen treatment methods, we will explain in more detail the case in which mutation is carried out by treatment using N-methyl-N'-nitro-N-nitrosoguanidine (NTG), but other methods The desired mutant strain can also be obtained by the mutation induction method described above. (1) Mutation treatment Yeast extract/malt extract agar medium (ISP
Spores scraped from the surface of one slanted culture of Streptomyces sp. OA-6129 cultured on medium-2) at 28°C for 2 weeks were suspended in 9 ml of 0.01M sodium phosphate buffer (PH7.5) and filtered through a glass filter. (No. 2) and add 9% to 1 ml of this liquid.
After diluting with ml of the above phosphate buffer,
Add NTG to a final concentration of 1000 μg/ml and keep at 28°C for 1 hour. At this time, the sporicidal effect was 90%. NTG treated spore liquid at 2500×g
Centrifuge for 20 minutes, suspend the spores in 0.85% physiological saline, dilute appropriately, apply the diluted spore suspension on ISP medium-2, and incubate at 28°C for 2 to 30 minutes.
Culture was performed for 3 weeks to form colonies. (2) Isolation method of mutant strain Discard the colonies formed on ISP medium-2 as described above and transfer them to ISP medium-2 slant medium at 28°C.
Culture was carried out for two weeks. One platinum loop from the slant culture medium of each of these strains was added to the SE-4 seed mother medium [0.3% beef extract, 0.5% bactotryptone, 0.1% glucose, 2.4% soluble starch, 0.5% yeast extract.
%, calcium carbonate 0.4%, soy flour (Ajinomoto Co., Inc.)
The mixture was inoculated into a test tube medium containing 3 ml of 0.5% PH7.0, and cultured with shaking at 28°C for 2 days. Next, 0.5ml of this culture solution was mixed with GM medium (soybean flour 4.5%, glycerin 10%, K ₂ HPO ₄ 0.2%,
MgSO ₄ 7H ₂ O 0.1%, CaCO ₃ 0.3%, CoCl ₂ 5r/
ml, pH7.0) in a 250 ml Erlenmeyer flask, and cultured on a rotary shaker at 28°C for 4 days. This culture supernatant was spotted on 5μ silica gel 60F ₂₅₄ (Merck) and dried in a vacuum desiccator for about 10 minutes, followed by chloroform:methanol:M/100 phosphate buffer (PH7.5) = 8:
After developing with a 6:1 developing solvent, remove the solvent with cold air, soak in Ehrrich reagent, heat at 105°C for 5 minutes to develop color, and remove antibiotic OA-
6129C spots disappeared and antibiotics OA-
A mutant strain showing 6129B ₂ spots was isolated. This allows those skilled in the art to easily search for microorganisms suitable for the purpose of the present invention. According to the method of the invention, antibiotic OA-6129B ₂
is the above-mentioned strain Streptomyces sp.
It is produced by inoculating OA6129-3N-504 spores or mycelia into a nutrient-containing medium and growing aerobically. As the nutrient source, those normally used as a nutrient source for actinomycetes, such as assimilable nutrient sources such as carbohydrates, nitrogen sources, and inorganic salts, can be used. For example, carbohydrates such as glucose, glycerin, maltose, sucrose, molasses, dextrin, and starch;
Carbon sources such as oils and fats such as soybean oil, peanut oil, and lard; peptone, meat extract, soybean flour, cottonseed flour, dried yeast, corn steep liquor, yeast extract, skim milk, casein, sodium nitrate, ammonium nitrate, Nitrogen sources such as ammonium sulfate: inorganic salts such as dipotassium phosphate, common salt, calcium carbonate, and magnesium sulfate can be used, and if necessary, trace metals such as cobalt and manganese can be added. As the nutrient source, any other nutrient source can be used as long as it does not inhibit the production of the antibiotic OA-6129B ₂ , and any known culture material for actinomycetes can be used. Further, in order to suppress foaming during heat sterilization and culturing, an antifoaming agent such as silicone or vegetable oil may be added. The blending ratio of the nutrient sources as described above is not particularly restricted and can be varied over a wide range, and those skilled in the art can easily determine the composition and blending ratio of the nutrient source suitable for the strain used. This can be easily determined by small-scale experiments. In addition, the nutrient medium can be sterilized prior to cultivation, and the pH of the medium can be adjusted to 4 to 9 before or after sterilization.
It is advantageous to adjust the pH to a range of , especially a pH of 6 to 8. In such a nutrient medium, Streptomyces sp.
In principle, culturing of OA-6129-3N-504 can be carried out according to methods commonly used in the production of antibiotics using general actinomycetes. It is usually preferable to culture under aerobic conditions, which can usually be carried out with stirring and/or with aeration. Further, as a culture method, any of static culture, shaking culture, and submerged culture with aeration and agitation can be used, but submerged culture is advantageous. The culture temperature that can be used is strain OA-6129-3N-
Antibiotic OA− without substantially inhibiting the growth of 504
There is no particular restriction as long as 6129B ₂ can be produced, but generally the temperature is 20 to 40℃, preferably 25℃.
Temperatures in the range ˜35° C. are preferred. Moreover, in order to carry out the culture suitably, the pH of the culture can be adjusted to a range of 4 to 9, particularly 6 to 8, during the culture, if necessary. In the case of large-scale mass cultivation, it is advantageous to perform a seed culture as appropriate, inoculate it into a nutrient medium, and perform liquid culture. Cultivation can normally be continued until sufficient antibiotic OA-6129B ₂ has accumulated. The culture time varies depending on the composition of the medium, culture temperature, production strain used, etc., but is usually in the range of 30 to 90 hours. The amount of antibiotic OA-6129B ₂ accumulated during culture can be quantified by thin layer chromatography using silica gel as described above, and thereby the optimal amount of accumulation can be easily determined. Thus, the antibiotic OA accumulated in the culture
Since −6129B ₂ is water-soluble and mainly exists outside the bacterial cells, it is advantageous to remove the bacterial cells after culturing by a separation method known per se, such as filtration, centrifugation, or extraction. , supernatant liquid, extract liquid, etc. Recovery can be carried out by various methods known per se, and in particular methods often used for recovery of carboxylic acid type antibiotics are advantageously applied. For example, solvent extraction with ethyl acetate, n-butanol, etc. at low pH and transfer of the solvent layer to high pH water layer; activated carbon, Amberlite XAD (manufactured by Rohm and Haas), Diaion HP-20 (manufactured by Mitsubishi Kasei Corporation), etc., and elution with methanol water, acetone water, etc.; Dowex 1x2 (manufactured by Dow Chemical Company), QAE-Sephadex A-25 (manufactured by Pharmacia), DEAE-Cellulose Watman DE- Adsorption and elution using ion exchange resins such as 32 (manufactured by Watmann) and DEAE-Sephadex A-25 (manufactured by Pharmacia); Cephadex G-
Gel filtration with 10 (manufactured by Pharmacia), Bio-Gel P-2 (manufactured by Bio-Rad), etc.; column chromatography with cellulose, Avicel SF (manufactured by American Viscose); addition of solvents such as acetone A forced precipitation method; a freeze-drying method, etc. may be used alone or in appropriate combinations, and may be used repeatedly in some cases. The behavior of antibiotic OA-6129B ₂ during the recovery and purification process can also be quantitatively determined by thin layer chromatography as described above, thus showing that antibiotic OA-6129B 2
−6129B ₂ is obtained. According to the method of the present invention, depending on the culture conditions, a small amount of antibiotic OA-6129A may be produced in the culture, but antibiotics OA-6129B ₁ and OA-
6129C is virtually never produced and is an antibiotic.
OA-6129B ₂ is selectively produced. Since the salt form of this antibiotic OA-6129B ₂ is generally more stable than the free form, it can be used for the pharmaceutical purposes described below, or as an intermediate for further conversion into derivatives. , or when subjecting it to the above-mentioned purification steps, it is preferable to treat it in the form of a salt. Conversion of the antibiotic OA-6129B ₂ into its salt form can be carried out according to known methods by treating the antibiotic OA-6129B ₂ with an inorganic or organic base. Inorganic or organic bases that can be used in this salt-forming reaction include, for example, alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, and lithium hydroxide; alkaline earth bases such as calcium hydroxide and magnesium hydroxide; Metal hydroxides include primary, secondary, or tertiary organic amines such as monoethylamine, dimethylamine, trimethylamine, monoethanolamine, diethanolamine, benzathine, and procaine. Antibiotic OA- produced by the method of the present invention
6129B ₂ has a wide range of antibacterial activity as described in the European patent application publication mentioned above, and can be used as an antibacterial agent. It can also be used as an intermediate. Next, the present invention will be further explained by examples. The quantitative analysis of the antibacterial active substances used in the following examples was carried out by the above-mentioned thin layer chromatography using a known antibiotic as an indicator. Example [A] In a 500ml Erlenmeyer flask
100 ml of a seed culture medium (SE-4) having the following composition was added and sterilized at 121°C for 15 minutes by a conventional method. On the other hand, Streptomyces sp. OA−6129
(Streptomyces sp. OA−6129) (FERM BP−
11) mutant strain 3N-504 strain (FERM
BP-334) was allowed to fully adhere to spores, and one platinum loop was inoculated into the seed medium and cultured at 28° C. for 48 hours with shaking in a rotary shaker (200 rpm, amplitude 7 cm). 100ml of this seed medium was added with 5.0% of the production medium (GM-3) with the following composition.
The dissolved oxygen concentration (% saturation) in the culture medium is 10% using a dissolved oxygen meter.
The aeration and agitation culture was carried out for 90 hours at a temperature of 28° C. with an aeration rate of 5/min while controlling the rotation speed so as not to exceed the rotation speed. Silicone KM-75 as an antifoaming agent [Shin-Etsu Chemical]
Co., Ltd.] was used at 0.1%. The culture solution was sampled over time, and the antibiotic OA-6129 compound was quantified in the centrifuged supernatant using the thin layer chromatography described above. The analysis results for each culture time are shown in the table below.

[Table] Composition of seed medium (SE-4): (w/v) Glucose 0.1% Soluble starch 2.4% Beef extract 0.3% Meat 0.5% Bactotryptone 0.5% CaCO ₃ 0.2% Tap water production medium (GM-3 ) Composition: Glycerin 8.0% Soy flour 3.0% Taiwanese yeast 1.0% MgSO ₄ 0.2% K ₂ HPO ₄ 0.2% CaCO ₃ 0.3% CoCl ₂ 5 μg/ml [B] After 90 hours of culture obtained in [A] above Tap water was added to the fermentation solution 4.5 to make it 6.0, and Topcoperlite No. 34 (manufactured by Toko Perlite Co., Ltd.) was added, and the solid matter was separated using Nutsuchie to make it 4.0.
A liquid () was obtained. Next, the solid substance was dissolved in tap water again to give a concentration of 4.0, and was filtered through a Nuttsuie to obtain a liquid (2.0). Combine liquids () and () and apply Diamond Ion HP20 resin.
Glass column filled with 400ml (40mm x 320mm)
The liquid was adsorbed at a rate of 400 ml/hr. 400
After washing the column with ml of distilled water, 5%
(v/v), 10% (v/v) and 15% (v/v)
It was eluted with acetone water while increasing the acetone concentration stepwise. Each fraction was 15 ml, and the eluate was fractionated and analyzed by thin layer chromatography to collect fractions 33 to 57, which mainly contained OA-6129B ₂ , to obtain 430 ml. OA-6129A, which was produced in small quantities along with OA-6129B _2, has fractions 59 to 66.
was contained in. Next, a column (17.5 mm x 250 mm) packed with Diaion PA306S in which _{the two} OA-6129B fractions were equilibrated with 0.01 M phosphate buffer (PH8.0).
After adsorption and sufficient washing with 100 ml of distilled water, the solution was eluted with a total of 1.0 saline solution in which the salt concentration varied linearly from 0 to 4%. 5ml per section
It was fractionated as Each fraction was analyzed by thin layer chromatography, and the fraction containing OA-6129B ₂
Fractions 33 to 55 were collected to obtain 130 ml. This fraction was pre-equilibrated with 0.01M phosphate buffer (PH8.0) on a Diaion PH20 column (15.0mm x 430mm).
mm), and the eluate was eluted with a total of 600 ml of acetone water whose acetone concentration varied linearly from 0 to 20%. The eluate was fractionated, each section containing 5 ml. Each fraction was analyzed by thin layer chromatography, and the fractions containing OA-6129B ₂ from fraction 41 to fraction 61 were collected to obtain 200 ml. This was freeze-dried according to a conventional method to obtain 180 mg of pale yellow powder. The purity of this powder was 51% by thin layer chromatography. [C] 150 of the freeze-dried products obtained in [B] above
After dissolving mg in about 10ml of distilled water,
It was adsorbed onto a QAE Sephadex A25 resin column (10 mm x 900 mm) equilibrated with 0.01M phosphate buffer. After washing with about 100ml of distilled water,
Elution was carried out with a total of 600 ml of saline solution whose salt concentration varied linearly from 0 to 2%. Each section was fractionated into 5 ml portions, and the eluate was fractionated. By analyzing each fraction using thin layer chromatography,
OA-6129B ₂ fractions 100 from fraction 37 to fraction 47
Got ml. This OA−6129B ₂ fraction was added to 0.01M in advance.
Adsorb onto a Diaion CHP20 column (10 mm x 90 mm) equilibrated with phosphate buffer (PH8.0),
After washing the column with a small amount of distilled water, it was eluted with a total of 800 ml of acetone water whose acetone concentration varied linearly from 0 to 10%. Each section was fractionated into 5 ml portions, and the eluate was fractionated. Each fraction was analyzed using thin layer chromatography, and a total of 180 ml of fractions 89 to 119 were packaged using Ehritzch reagent.
I got it. This was freeze-dried to obtain 39 mg of pale yellow powder. This powder was confirmed to be antibiotic OA-6129B ₂ based on bioautography and the following NMR, UV, and IR measurement values. The purity of this substance determined from UV absorbance was approximately 98%. NMR spectrum (solvent: D ₂ O, internal standard
DSS) 〓ppm: 0.87 (3H, s,

【formula】), 0.92 (3H, s,

【formula】), 1.28 (3H, d, J = 7.0Hz,

[Formula]), 2.45 (2H, t, J=6.5Hz, NH−CH ₂ −CH ₂ −
CO), 2.75-3.60 (11H, m, C- _4H2 , 4-6H, S- _CH2 - _CH2 -NH, NH- _CH2 - _CH2-
C.O.

【formula】), 3.94 (1H, s,

[Formula]), 3.95-4.35 (2H, m, C-5H,

【formula】). UV absorption spectrum 0.01M phosphate buffer (PH8.4) max nm (ε) = 300 (5400) IR absorption spectrum ν ^KBy _nax cm ^-1 1760 (β-lactam) 1660 (amide) 1600 (galboxylate)

Claims (1)

[Claims] 1. Streptomyces sp. OA-6129-3N
-504 was cultured in a nutrient medium, and the following formula was obtained from the culture: A method for producing antibiotic OA-6129B ₂ , which comprises collecting antibiotic OA-6129B ₂ shown in .