KR840000935B1 - Process for preparing antibiotic c-15003 p-o - Google Patents

Abstract

The prodn. of antibiotic C-15003 P-3, a component of antibiotic C-15003, by Nocardia is stimulated by addn. of isobutanol or isobutyl aldehyde to the fermn. medium. Thus, a seed culture of Nocardia Sp. No. C-15003 was inoculated into 40 mL of a medium contg. soluble starch 3%, NH4Cl 0.2%, MgSO4 0.05%, KH2PO4 1.09%, K2HPO4 2.09%, and FeSO4 0.001%, and incubated at 28oC for 48 hrs. with aeration and stirring. Then 0.05% isobutyl aldehyde was added, and cultivation was continued for 6 days. The yield of C-15003 was 27 mg/L, with 95% as P-3, compared to 10 mg/L, with 95% as P-3, compared to 10 mg/L 65% without isobutanol.

Description

Method for preparing antibiotic C-15003 P-3

The present invention relates to a process for industrially advantageously producing antibiotic C-15003 P-3.

Antibiotic C-15003 P-3 (hereinafter sometimes abbreviated as "P-3") is a compound obtained by culturing microorganisms belonging to the genus of microorganisms isolated from samples such as soil. This is described in detail in Japanese Patent Laid-Open No. 53-130495 and Japanese Patent Laid-Open No. 53-130693 (US Pat. No. 4,162,940). However, these methods simultaneously produce each component of antibiotic C-15003. Therefore, when only the P-3 component is collected, separation and purification from the culture becomes a complicated process. In terms of advantages, it can not be said.

In the above method, the inventors further improved the method for producing the P-3 component specifically, and in culturing the microorganism belonging to the genus Nocaldia in the medium, isobutyl alcohol and / or in the medium. Alternatively, by adding isobutylaldehyde, the present inventors have found that the desired purpose can be achieved.

In the present invention, antibiotic C-15003 producing bacteria (hereinafter sometimes referred to as "C-15003 producing bacteria") belonging to the genus Nokaldia are cultured in a medium to produce and accumulate antibiotic C-15003 P-3 in culture. The method for producing antibiotic C-15003 P-3, wherein isobutyl alcohol and / or isobutylaldehyde is added to the medium.

The term "antibiotic C-15003" or "C-15003" as used in the present invention refers to a generic term including a single compound or a mixture of two or more of three compounds represented by the following general formula (I).

또는

를 나타낸다.][Wherein R is -CO-CH ₂ -CH ₃ , -CO-

or

Is displayed.]

인 화합물을 "항생물질 C-15003 P-3" 혹은단지 "P-3"로, R이

인 화합물을 "항생물질 C-15003 P-4" 혹은단지 "P-4"로, 각각 호칭하는 일도 있다.Those wherein R is -CO-CH ₂ -CH ₃ in the general formula (Ⅰ) to "antibiotic C-15003 P-2", or just "P-2", the R

The phosphorus compound is called "antibiotic C-15003 P-3" or just "P-3", where R is

Phosphorus compounds may be referred to as "antibiotic C-15003 P-4" or just "P-4", respectively.

Specific examples of microorganisms that may correspond to the method of the present invention include, for example, actinomycetes No.C-15003 strain isolated from soil [Nocaldia SP.No.C-15003: hereinafter, "No.C-15003 strain". May be abbreviated as "."

This strain No.C-15003 strain is FERM-P No.3992 to the Institute of Microbiological Engineering and Technology (Japan), and the IFO-13726 to the Fermentation Research Center (Japan), the American Type Galtuic Collection ( ATcc-31281, The American Type Culture Collectiou USA), respectively. The ATcc-31281 strain is listed in The American Type Girl Collection Collection's 14th Edition, I, 1980, available from The American Type Culture Collection. No. The mycological properties of C-15003 strain are described in Japanese Patent Laid-Open No. 53-130495, Japanese Patent Laid-Open No. 53-130693, and US Patent No. 4,162,940. In other words, the method of Charing and Gotrib [International Journal of Systemic Bacteriology, Vol. 16, pp. 313 to 340, 1966] The results were observed in a similar manner and observed over 28 ° C. for 21 days.

1) Morphological features

Parasitic hyphae (基 生 菌絲) are well elongated and branched in both agar and liquid medium. Most of its diameter is 0.8-1.2 µm, and sometimes it is divided into bacilli or branched short military phase. It grows well on various sorting media, and the mycelia grow on parasitic mycelia, but often form a supernatant (50-200 μm × 200 × 1000 μm), and often grow on them. Most of the mycelia of the mycelia are curved or linear, and sometimes the helix is smooth. When the mature cultures were examined, spores were not considered to be in the form of a chain, and the microbial suspensions collected from these culture surfaces were examined, and long ovals (0.8 to 1.2 μm × 4.8 to 6.8 μm) and ellipsoids (0.8 to 8 μm) 1.2 1.0-2.0 micrometers) of many spherical spherical shapes were observed, and the surface was smooth by the observation by the electron microscope.

2) Cell composition

The strain was vibrated in a modified medium of ISP No. 1 at 28 ° C. for 66 to 90 hours, and the cells were collected and washed. The cells were treated by non-backers [Applied Microbiology, Vol. 12, p. 421, 1964] and by M. B. Letch Barrier [Journal of Laboratories and According to Clinical Medicin (Journal of Laboratory and Clinical Medicine, Vol. 71, p. 934, 1868), the diaminopimelic acid and the sugar composition in the myocytes were examined, the former being a mesobody, the latter being galactose and arabinose. The presence of a corresponding spot was confirmed.

3) Properties of the classification medium

This strain grows relatively well on various media, and the parasitic mycelia are colorless to pale yellow at the beginning of culture, and then pale brown or tan. Further, yellow to yellow brown soluble pigments are produced in various sorting media. The aerial mycelia are powdery, generally medium-grown, white to yellow or pale yellow brown. The properties of the strains on the various classification media are as shown in the first table.

[Table 1]

4) Physiological Properties

Physiological properties of this strain are shown in Table 2. That is, the growth temperature range is 12 ° C to 38 ° C, and the temperature range at which the mycelia grow well on the agar medium (ISP No. 2) is 20 ° C to 35 ° C.

[Table 2]

5) Availability of various carbon sources

The medium described in the method of Freedam and Gotrib (Journal of Bacteriology, Vol. 56, p. 107, 1948) and a basal medium containing 0.1% of yeast extract (Bacto) added thereto. The use of various carbon sources was examined, and the results are shown in Table 3 below.

[Table 3]

6) Other properties

The cells were collected by the method described in the above 2), and DNA was prepared according to the method of Jay and Mamaah (Journal of Molecular Biology, p. 208, 1961). The GC content of the DNA was about 71 mol%.

The mycelia of the strain were gram stained and were positive.

The properties of No. C-15003 as described above are published by S. Waxman, The Actinomyce-tes, Vol. 2, The Williams and Winkins, 1961. , According to R.B.Bupanan and N.Basics, Burgess Manual of Determinative Bacteriology, 8th edition, 1974 and other literature Search. It is thought that this strain belongs to group III of the genus No Cardia, but among the known strains, the kind having the above properties is not found, and it was recognized as a new species.

In general, the genus Novaldia bacteria are easily changed in their properties, for example, by means of X-ray irradiation, ultraviolet irradiation, irradiation, artificial mutants using artificial modifiers (e.g., nitrosogunidine, ethyleneimine, etc.). It can be easily mutated. Even in such a mutant strain, any one having a production function of antibiotic C-15003 can be used in the method of the present invention.

In the method of the present invention, isobutyl alcohol and / or isobutyl aldehyde added to the medium are added alone or simultaneously to the medium, but the amount is generally about 0.005 to the medium as a single amount or as a total amount of both. It is added so that the capacity / volume% or more.

In particular, it is preferable to add isobutyl alcohol and / or isobutyl aldehyde in the range of about 0.01 to about 0.5 volume / volume of% with respect to the medium. However, unless the growth of the microorganisms used or the productivity of antibiotics are suppressed, the amount of addition may be increased again.

The time for adding isobutyl alcohol and / or isobutyl aldehyde to the medium may be before inoculation of C-15003 producing bacteria after inoculation of the medium, and production of C-15003 after inoculation of C-15003 producing bacteria in the medium. You may add in the process of continuing culture.

When isobutyl alcohol and / or isobutyl aldehyde are added, these may be added as a derivative. As this derivative, the ester (for example, an acidic ester, an acetate ester, a butyric acid ester, a nitrate ester, etc.) produced | generated between various acids is mentioned, for example.

The medium used for the cultivation of antibiotic C-15003 producing bacteria may be liquid or solid as long as it contains a nutrient source that can be used by the microorganism, but it is more appropriate to use a liquid medium when processing a large amount. In addition to the additive used in the present invention, the medium contains a carbon source capable of assimilation by C-15003 producing bacteria, a digestible nitrogen source, an inorganic substance, a micronutrient, and the like. As a carbon source, for example, glucose, lactose, sucrose, maltose, dextrin, starch, glycerin, mannitol, solitol and the like, fats and oils (e.g., soybean oil, raad oil, chicken oil, etc.) and other nitrogen sources, for example Meat extract, yeast extract, dry yeast, soybean meal, corn steep liquor, peptone, cottonseed meal, fructose, urea, ammonium salts (e.g. ammonium sulfate, ammonium chloride, ammonium acetate, ammonium acetate, etc.). In addition, salts containing sodium, potassium, calcium, magnesium and the like, metal salts such as iron, manganese, zinc, cobalt and nickel, salts such as phosphoric acid and boric acid are suitably used. If necessary, vitamins, oxalate groups and the like may be contained. Of course, for the purpose of adjusting the pH of the medium, an acid or an alkali may be added at the beginning of the culture or during the culture, and an appropriate amount of oil or fat or a surfactant may be added for the purpose of defoaming.

As a means of culturing, a means such as stationary culture, vibration culture, or aeration stirring culture method may be used. It goes without saying that it is preferable to use a so-called deep-air agitation culture for a large amount of processing. Naturally, the conditions of the culture are not constant depending on the condition of the medium, the composition, the type of the strain, the means of the culture, and the like, but the liquidity of the medium is preferably between about pH 5.5 to 8.0, and preferably about 6.5 to 7.5 neutral pH. Better results can be obtained nearby. The culture temperature is about 15 ° C to 35 ° C, preferably about 20 ° C to 30 ° C. Cultivation is carried out until the accumulation concentration of P-3 reaches a maximum, but the time required for this is usually about 2 to 10 days, depending on the culture method and the composition of the temperature medium.

In order to purify P-3 accumulated and accumulated in the culture as described above, a separation and purification method commonly used for collecting such microbial metabolites is suitably used. First, since this substance is neutral fat-soluble, organic solvents that are not mixed with water, such as fatty acid esters such as ethyl acetate and amyl acetate, alcohols such as butanol, halogenated hydrocarbons such as chloroform, and methyl isobutyl ketone Used. Extraction is performed near neutral, and is preferably performed using ethyl acetate in a culture adjusted to PH7. The extract is washed with water, concentrated under reduced pressure, and a non-polar solvent such as petroleum ether or hexane is added to collect crude material containing the active ingredient. In this genus many spots other than antibiotic C-15003 are identified on TLC, which is used in the next purification process. That is, various adsorption chromatography is effective as a purification method commonly used, and carriers generally used, such as silica gel, alumina, and large porous non-ionic adsorption resins, can be used as the adsorbent. In the case of using silica gel, development starts from a nonpolar solvent such as petroleum ether and hexane, and then a polar solvent such as ethyl acetate, acetone, ethanol, and methanol is added, or from halogenated hydrocarbons such as dichloromethane and chloroform. The development is started, and P-3 is eluted, separated and collected by adding polar solvents such as alcohols such as ethanol and methanol and ketones such as acetone and methyl ethyl ketone. In the case of using a porous porous adsorbent resin, lower alcohols or lower ketones, mixtures of esters and water, etc. are used to elute P-3. As lower alcohols, for example, acetone, methyl ethyl ketone, and esters may be used as lower ketones such as methanol, ethanol, propanol, butanol, and the like. One example is to dissolve the crude in 60% ethanol water, pass it through a DIION HP-10 (Mitsubishi Chemical Co.) column, adsorb it, adsorb it, wash it with 7% ethanol water, and elute it with 90% ethanol water. -3 is eluted.

In either method, the obtained portion of P-3 is concentrated under reduced pressure, and 5-8 times of ethyl acetate is added to the dried product to prevent the crystals of P-3 from being precipitated.

Physicochemical properties of antibiotic P-3 are described in Japanese Patent Application Laid-Open No. 53-130495, Japanese Patent Application Laid-Open No. 53-130693, and US Patent No. 4,162,940. That is, it is like a 4th table | surface.

[Table 4]

According to the method of the present invention, since the production rate of P-3 is very high and the production amount also increases, inexpensive liquid raw materials are used in addition to the increase in the production yield and the ease of separation and purification, and severe odors such as isobutane, There is no corrosion of the device, and isobutyl alcohol and isobutylaldehyde are low toxicity to C-15003 producing bacteria. Therefore, the method of the present invention is an extremely advantageous method for industrial production.

The biological activity and use of antibiotic C-15003 P-3 is as described in Japanese Patent Laid-Open No. 53-130495, Japanese Patent Laid-Open No. 53-130693, and US Patent No. 4,162,940. Ie bioactive

A) Antimicrobial Activity

Trypticures soy agar medium (manufactured by Baltimore Biological Ladoratories U.S.A.) was used as a test medium, and the growth inhibiting function of the microorganisms shown below was assayed by the paper disk method. That is, when the growth inhibition function was assayed by containing 0.02 ml of a 300 µg / ml solution of C-15003 P-3 in a paper disc (Toyo Corporation, thin, 8 mm in diameter) on the following microbial plate containing medium, The following microorganisms showed no activity.

Escherichia coli, Proteus vulgaris, Proteus mirabilis, Sudmonas air munosa, Bacillus cyibacteria, Bacillus subtilis, Batylus cerius, Pneumococcal bacterium, Cerratier melessense, Mycobacterium avium

On the other hand, using agar plate of assay medium [3.5g of sodium phosphate, 0.5g of 1 potassium phosphate, 5g of yeast extract (dipco), 10g of glucose, 15g of agar, 1000ml of distilled water, PH7.0], Taratomyces abelaneus ( Talaromyces avollaneus) was used as a test bacterium, and its growth ability was assayed. C-15003 P-3 showed growth inhibition at 3 µg / ml.

In addition, the tetrafimena Pyriformis W strain was used as a test microorganism, and 20 g of assay medium (triptose pelton (Dipco), 1 g of yeast extract, 2 g of glucose, 1000 ml of distilled water, and 1 mole phosphate buffer PH7. When 0,10 ml were incubated at 28 ° C. for 44 hours to 48 hours, and the microbial growth potential of this antibiotic was assayed by the liquid dilution assay, C-15003 P-3 was obtained at 1 μg / ml. To prevent the development of

B) antitumor activity

For the therapeutic effect of C-15003 P-3 (9 days continuous intraperitoneal administration) on tumor cells P 338 (1 × 10 ⁶ cells / mouse, mice, intraperitoneal transplantation), the survival rate of mice by this substance was compared. In comparison, an anti-tumor effect of 200% at 0.00625 mg / kg / day was confirmed.

C) toxicity

In acute toxicity studies with mice, C-15003 P-3 intraperitoneally injected the antibiotics with an LD ₁₀₀ value of 0.625 mg / kg and an LD ₀ value of 0.313 mg / kg.

As described above, the antibiotic C-15003 P-3 has a strong growth potential against filamentous fungi and protozoa, and thus is also useful as a fungicide or an antigen filler. In addition, antibiotic C-15003 P-3 has a life-long effect on mammals with tumors (such as mice), and is therefore expected to be useful as an antitumor agent.

In order to use this antibiotic C-15003 P-3 as an antiseptic and an antiprotozoal agent, it can be advantageously used as a reagent for assaying bacterial conditions such as soil, activated sludge or animal body fluid. In other words, when separating useful bacteria from the soil, or testing the action of bacteria other than protozoa or fungi in the operation and analysis of the activated sludge method used for wastewater treatment, it is possible to develop fungi or protozoans in the sample without developing them. Can selectively develop bacterial ecology. Specifically, the test sample is added to a liquid or a solid medium, and 0.1 m of a 1% ethanol-containing aqueous solution of 10 to 100 µg / ml of the antibiotic is added and cultured per 1 ml of the medium. As described in US Pat. No. 4,162,940, P-3 is antifungal. That is, antifungal properties are shown in the fifth table. As is apparent from Table 5, P-3 inhibits the growth of phytopathogens. A circular filter paper moistened with 0.02 ml of 1000 μg / ml solution of P-3 was placed in a medium into which microorganisms of Table 5 were respectively implanted, and the diameter of the low support was measured.

[Table 5]

P-3 can also be used as an antimicrobial agent used in the treatment of plant diseases caused by the microorganisms shown in Table 5. As a specific application example, P-3 is a liquid in which 0.5% / ml to 5µg / ml is dissolved in 1% methanol, and can be used, for example, in cases such as S. pneumoniae, S. aureus, and rice gyomas. .

The present invention will be described in more detail with reference to the following Examples.

Example 1

40 ml of seed medium (glucose 1.0%, Bacto-Triptone (Doifc Laboratories USA) 2.0%, Bacterium-Yeast extract 1.2%, PH7.0) was dispensed into a 200 ml triangle flask, and after sterilization, Nocaldia SP.No.C- 15003 weeks were inoculated. This was incubated for 48 hours in a rotary vibration gas phase (200r.p.m) at 28 ° C to obtain species culture. After 3 times of bacteria with distilled water obtained by sterilizing the culture, the bacterial body was returned to the original amount of the sterilized distilled water, and 1 ml of this medium was used as a main medium (3% of soluble starch, 0.2% of ammonium chloride, 0.05% of magnesium sulfate, and potassium monophosphate). 1.09%, dipotassium phosphate 2.09%, ferrous sulfate 0.001%, weight / volume), inoculated into 40 ml, vibrating culture at 28 ° C for 48 hours, and then added isobutylaldehyde to 0.05% (volume / volume). Then, the culture was continued for 6 days. At the end of the culture, the total production concentration of antibiotic C-15003 in the culture was 27 mg / l, and the proportion of P-3 was about 95% (weight ratio). In addition, when incubated without additives, the percentage of total production of 10 mg / l and P-3 was only 65% (weight ratio).

In addition, the total production amount of each component of antibiotic C-15003 and the quantity for each component were measured by the following method including the following example. That is, the total production amount is composed of Pilobasidium uniguttulatum IFO 0699 as a test bacterium, 15 g of tributyase peptone (Boltimore Biological Lab USA), 5 g of phyton peptone (Boltimore Biological Lab USA), salt The agar well method using assay medium of 10 g, 10 g of glucose, 15 g of agar, 1 l of instantaneous water, (PH7.3), and compares the support diameter of the test specimen with the low support diameter using standard P-3 solution. It calculated | required in conversion amount. The amounts of each component were extracted by adding the same amount of ethyl acetate to the aqueous sample containing them, and after condensation and drying, 1/100 of the original filtrate was dissolved in ethyl acetate to prepare a sample for thin layer chromatography. Thin layer chromatography are, as a developing solvent by using a glass sheet of silica gel 60F ₂₅₄ (Mel keusa product), can used a saturated ethyl acetate. The production amount and production rate were measured at an absorption concentration and a width of 254 nm using a Shimadzu 2-wavelength chromatograph-CS-910.

Example 2

The seed medium described in Example 1 was prepared, and 500 ml of the seed medium was dispensed and sterilized into a 2000 ml sachet. Inoculated therein with Nocaldia SP.No C-15003, and incubated for 48 hours on a reciprocating vibrator (110r.p.m) at 28 ° C to adjust the seed culture for inoculation. Seed medium (200% glucose, 3.0% soluble starch, 1.0% corn steep liquor, 1.0% raw soy flour, 0.5% polypeptone, Japan) in 200l stainless steel fermentation tank 0.5%, calcium carbonate PH7.0) 100l was prepared, and after sterilizing for 20 minutes at 121 ° C, inoculated with 500 ml of seed culture. Longitudinal culture was performed for 48 hours at the temperature of 28 degreeC, air flow rate 100 l / min, and 200 rotation / min. 10 l of this seed culture is transplanted into 100 l of 200 ml main medium (5% dextrin, 3% corn steep liquor, 0.1% peptone, 0.5 calcium carbonate, (weight / volume), PH7.0) in a stainless steel fermentation tank and After incubating for 48 hours under conditions of aeration of 1 VVM and agitation 150 rpm, isobutyl alcohol was added so as to be 0.1% (volume / volume), and the culture was continued for another 48 hours. As a result, the total amount of C-15003 produced was 20 mg / l, of which P-3 had a proportion of 96% (weight ratio). In the case of culturing without adding isobutyl alcohol under the same conditions, the total production amount was 12 mg / l, and the ratio of P-3 remained at 63% (weight ratio).

Example 3

The seed culture of Example 1 was cultured with 3% soluble starch, 0.2% ammonium chloride, 0.05% magnesium sulfate, 0.05% potassium chloride, 1.09% potassium phosphate, 2.09% potassium phosphate, and 0.001% ferrous sulfate. 1 ml was inoculated in 40 ml, and isobutyl alcohol or / and isobutyl aldehyde was added on condition shown in Table 6, and it vibrated and cultured at 28 degreeC for 8 days, and the result shown in Table 6 was obtained.

[Table 6]

Example 4

In the method of Example 3, when the effect of concentration of isobutyl alcohol and isobutyl aldehyde was examined by the method of adding initially to culture, the result shown in Table 7 was obtained.

[Table 7]

Example 5

The seed cultures of Example 1 were 3% soluble starch, 0.1% proplo (traders oil Co. USA), 0.2% ammonium chloride, 0.05% magnesium sulfate, 1.09% potassium phosphate, 2.09% potassium diphosphate, 40 ml of ferrous sulfate 0.001% and 0.05% isobutanol was inoculated in 2 ml each, and vibrated at 28 ° C. for 8 days. At the end of the culture, the total production concentration of antibiotic C-15003 in the culture was 38 mg / l, of which the proportion of P-3 was about 95% (weight ratio).

Example 6

50 liters of acetone are added to 95 l of the culture obtained in Example 2, followed by stirring for 30 minutes, 2 kg of Hypro Supercell (manufactured by Jonesmanville, USA) is added and stirred well. The mixture was filtered through a pressure filter to obtain 120 l of the filtrate. 50 l of water and 90 l of ethyl acetate were added to the filtrate, followed by stirring extraction. This operation was repeated twice. The ethyl acetate layers were combined, washed twice with 80 l of water, 1 kg of anhydrous sodium sulfate was added, the mixture was concentrated under reduced pressure to 200 ml after drying, and precipitated by adding petroleum ether. (58 g) To the obtained crude material, 50 ml of ethyl acetate was added and stirred. The insoluble matter was filtered off, and 10 g of silica gel (0.05 to 0.2 mm from West Germany Melk Co., Ltd.) was added to the filtrate, followed by stirring. At the top of (500 ml), 500 ml of n-hexane, 500 ml of n-hexane ethyl acetate (3: 1), n-hexane ethyl acetate (1: 1), and 2 l of ethyl acetate saturated solution are poured, and the eluate is divided by 50 ml. . 1 ml of each fraction was concentrated to dryness, and 0.1 ml of ethyl acetate was added thereto, and spotted at a position of 2.5 cm from the bottom of the silica gel glass plate (Kiegel gel 60F ₂₅₄ 0.25 mm 20 × 20 manufactured by West Germany Melk Co., Ltd.). It develops about 17 cm with saturated ethyl acetate. After the development, the absorbed phase was irradiated under ultraviolet light (2537 kPa) to collect fractions with absorption near Rf 0,42 and concentrated under reduced pressure to about 20 ml. Then, 200 ml of petroleum ether was added to the concentrate, to obtain 1,5 g of crude crystals. After heating and dissolving this in 20 ml of ethyl acetate, it cools and precipitates the crystal which precipitates, and 1,4 g of P-3 tablet crystals are obtained. mp189-190 ° C (purity 97w / w%)

Elemental analysis

C 60.10

H 7.00

N 4.30

Cl 5.43

Example 7

In the culture of Example 3, isobutyl propionate, isobutyl butyrate or isobutyl isobutyrate was added 42 hours after the start of the culture, and the culture was continued again, and the results shown in Table 8 were obtained. .

[Table 8]

Claims (1)

When culturing antibiotic c-15003 producing bacteria belonging to the genus Nokaldia and accumulating antibiotic C-15003 P-3 in the culture, isobutyl alcohol and / or isobutyl aldehyde are added to the medium. Preparation method of antibiotic C-15003 P-3.