KR830001245B1 - Method of preparing antibiotic mycoplanesin - Google Patents

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Description

Method of preparing antibiotic mycoplanesin

1, 2 and 3 show the ultraviolet absorption spectrum, infrared absorption spectrum and nuclear magnetic resonance spectrum of mycoplanesin, respectively.

The present invention is a microbial actinomycetes strain No.41042 and Actinoplanes (Actinoplanes) that can produce the mycoplanecin mycoplanecin (mycoplanecin) Mycoplanesin-producing microorganisms by culturing It relates to a manufacturing method.

Among the new antibiotics, mycoplanesin can be characterized by the following physical and chemical properties:

1. Color and shape of material: White powder

2. Melting Point: 161-167 ℃

3. Specific light intensity: [α] _D ²¹ -66 ° (C = 0.4, Chloroform)

4. Elemental analysis: C 61.78% H 8.48% N 11.75% (weight schedule after drying)

5. UV Absorption Spectrum: Only terminal absorption as shown in FIG. 1 was shown when measured at a concentration of 20 µg / ml in 50% aqueous methanol solution.

6. Infrared Absorption Spectrum: Spectra measured on a KBr disc are shown in Figure 2.

7. Nuclear magnetic resonance spectrum: The nuclear magnetic resonance spectrum measured using tetramethylsilane (TMS) as an internal reference in heavy chloroform as a solvent is shown in FIG.

8. Solubility: Soluble in methanol, ethanol, ethyl acetate, acetone and chloroform, poorly soluble in benzene, insoluble in water.

9. Coloring Reaction: A color reaction was performed on oxo and potassium permanganate on brown, silica gel thin layer chromatography with 50% v / v sulfuric acid, and no coloration reaction on ninhydrin and 2,4-dinitrophenylhydrazine.

10. Amino acid composition: hydrolyzed with 6N hydrochloric acid at 105 ° C. for 20 hours, followed by glycine, proline, leucine, 2-amino-5-methylhexanoic acid, N-methylthreonine, N-methyllosin, methylproline and ethylproline 1 mole and 2 moles of N-methylvaline were detected.

11. Antibacterial activity: It has strong antibacterial activity against various bacteria in mycobacterium.

In addition, mycoplanesin exhibited an Rf value of 0.15 on silica gel thin layer chromatography (F254, 0.25 mm, N0.5715, manufactured by Merck) when developed with ethyl acetate, and developed with a mixture of chloroform and methanol 95: 5 by volume ratio. 0.64 was shown at that time.

The novel microorganism of the present invention is an actinomycete strain No. 42422, which was isolated from soil samples collected from Hyogo Sumo City, Japan. This strain was deposited with the Japan Institute of Commerce, Industry and Technology, Microbiological Technology Research Center, and received accession number FERM-4504. This strain also received accession number NRRL-11462, deposited with the United States Department of Agriculture and Forestry.

A method for producing the new antibiotic mycoplanesin is to cultivate mycoplanesin-producing microorganisms of Actinomycetes and then isolate mycoplanesin from the culture.

Morphological characteristics and physiological characteristics of actinomycete No. 4042 are described below.

1. Morphological features

Strain 41042 by incubation for 14 days at 28 ° C. on an ISP4 medium defined by the International Streptomyces Project (ISP) has the following morphological characteristics.

Spores are spherical or globular, spores 7-15μ, spores 0.8μ × 1.3μ, flagella, with pH7.0, 1 / 15M phosphate buffer Forty minutes the appearance of Yuju (游走 性). The growth results in various media for 14 days at 28 ° C. are shown in Table 1.

TABLE 1

Note: SM: Parasitic Hyphae AM: Aerobic Hyphae SP: Soluble Pigment SG: Spores

2. Physiological Characteristics

The physiological characteristics of strain No. 4042 are shown in Table 2.

TABLE 2

The medium used is as follows.

Medium A: Tryptophan-Yeast Extract (ISP 1)

Medium B: peptone-yeast extract-iron agar (ISP 6)

Table 3 shows the carbon source utilization pattern of strain No. 4042 after 14 days at 28 ° C. on a Pridham-Gottlieb's ager medium.

TABLE 3

++: Used well. +: Used. -: Not used.

The following medium was used.

Badge C: Freedam-Gottlibo Agar

Badge D: Freedam-Gotliv Agar + Yeast Extract 0.5%

3. Cell Components

ratio. M. Applied Microbiology 12, pp. 421-423 (1964) and 13, 236-243 (1965), by B. Becker et al. blood. M.P. Lechevalier and H. Table 4 and 5 show the results of analysis of acid hydrolyzate of cells by paper chromatography by H. Prauser by the method described in Actinomycetals, pages 311-316 (1970).

TABLE 4

Cell wall shape

From this result, the cell wall form was confirmed to be II.

TABLE 5

Sugar component in whole cell

As is evident from the above constitution, strain No. 4422 belongs to the genus Actinoplanes of Actinoplanassiaceae in the actinomycetes. This is because the strain forms globular to sub-globule spores, is oily and cell wall type II. Therefore, this new strain is referred to as Actinoplanes strain No. 4422.

As is known, the properties of actinomycetes containing actinoprenes are not constant but can be rapidly changed naturally and artificially.

The present invention relates to a method for producing a novel antibiotic mycoplanesin by culturing the actinoplanes strain No. 4042, and to produce a mycoplanesin to obtain a variant strain of the microorganism, generally this antibiotic. Use of strains belonging to the genus Actinoplanes is also within the scope of the present invention.

Cultivation of mycoplanesin-producing bacteria by the method of the present invention can be carried out under the conditions normally used for culturing Actinoplanes genus. Shake stirring or aeration stirring culture in liquid medium is suitable.

As a nutrient medium, what is normally used for culturing actinomycetes can be used. Examples include assimilable carbon sources (e.g., glucose, arabinose, galactose, mannose, sucrose, maltose, dextrin, starch, glycerin, vegetable oils (e.g. soybean oil, cottonseed oil, corn oil), animal fats (chicken) Oil, lard) or fish oil), assimilable nitrogen sources (e.g., soybean meal, peanut oil, cottonseed meal, fishmeal, corn stequily, oatmeal, skim milk, peptone, meat extract, raw yeast, yeast extract Minoic acid, sodium nitrate, ammonium nitrate or ammonium sulfate) and inorganic salts such as sodium chloride, potassium chloride, phosphate, magnesium carbonate, calcium carbonate or calcium chloride. The nutrient medium may also contain small amounts of other metal salts, such as ferrous sulfate, copper sulfate, magnesium sulfate, zinc sulfate or cobalt sulfate.

Cultivation in a liquid medium is preferably carried out aerobic under agitation, and in this case, an antifoaming agent may be added to the medium, and as the antifoaming agent, silicone oil, vegetable oil or surfactant may be used.

Cultivation is suitably performed at a pH of near neutral and 18 to 30 ° C, preferably at a temperature of about 28 ° C.

The titer of mycoplanesin produced in the culture medium during the cultivation can be quantitatively determined by cup assay using Mycobacteri-um smegmatis ATCC 607 strain as a test bacterium. Mycoplanesin production peaks after 3-5 days of culture.

Mycoplanesin is present in both the liquid and cell parts of the culture medium prepared by the method of the present invention. After the end of the culture, in order to recover the antibiotic from the culture, the cells and other solid parts are recovered from the liquid phase, for example, by using diatomaceous earth as a filter aid or by centrifugation. Mycoplanesin in the cell part, filtrate or supernatant is then isolated and purified using conventional methods suitable for its physicochemical properties.

For example, mycoplanesin in the cell part can be extracted by adding a solvent that can be miscible with water, such as methanol, ethanol, isopropanol or acetone.

The solvent is then removed from this extract and the residue is reextracted using a solvent that is incompatible with water, such as chloroform, ethyl acetate, methylisobutylketone or methylene chloride. Mycoplanesin in the liquid part of the culture is extracted with a solvent that is incompatible with water, mixed with the extract extracted from the cell part, and then concentrated to obtain a crude mycoplanesin extract.

Alternatively, mycoplanesin can be extracted by directly adding a solvent which is miscible with water as described above directly to the culture without separating the cells from the liquid portion, and the resulting extract is filtered and concentrated to remove the solvent. The residue is reextracted using a solvent that cannot be miscible with water in this manner.

The mycoplanesin thus obtained can be further purified by well-known methods for purifying compounds having similar physicochemical properties, but it is suitable to use a purification method using an adsorbent or a countercurrent distribution method. Among the suitable adsorbents, alumina, silica gel, Sephadex (trademark of polysaccharide-derived organic compounds) or cellulose can be used. It is suitable to use a column chromatography separation method using silica gel as a carrier and methanol, ethyl acetate, chloroform or mixtures thereof as eluent. Further refining

Purified mycoplanesin thus obtained shows a single point in the color reaction of sulfuric acid, potassium permanganate or oxo on thin layer chromatography of silica gel, and exhibits the following physicochemical properties.

The minimum inhibitory concentration (MIC) of mycoplanesin for various microorganisms is shown in Table 6. Evaluation is performed as follows. In case of Mycobacteria, incubate for 5 weeks at 37 ° C by dilution using 10% bovine serum added Dubos liquid medium, and for 37 hours at 37 ° C by agar dilution method using Hert Infusion agar medium for general bacteria. After incubation, and in the case of fungi and yeast, each was evaluated after incubation at 26 ° C. for 48 hours by agar dilution method using agar medium as a mortal.

TABLE 6

Medium DM: 10% bovine serum was added to the other strains except Dubos medium, Mycobacterium smegmatis ATCC607 and Mycobacterium Frei 2 strains.

Badge H: Hert Infusion Agar Badge.

Badge S: Saboru Agar Badge.

Toxicity of the antibiotic mycoplanesin was tested by intraperitoneal administration to mice at 100 mg / kg body weight, with no deaths.

The preparation of mycoplanesin is described in detail in the Examples below.

Example 1

To 500 ml of sagaguchi hulask was added to 100 ml of a seed medium having a pH of 7.0 before sterilization and having the following composition (percentage w / v).

This medium was inoculated by culturing Actinoplanes strain No. 4204 and reciprocating shaking was carried out at 28 ° C. for 96 hours. The resulting medium was divided by 5 ml, and each portion was placed in each of the saga gucci hulask containing 100 ml of the production medium having a pH of 7.0 before sterilization and having the following composition (percentage w / v).

Subsequently, reciprocating shaking culture was performed at 28 ° C. for 96 hours. The resulting medium was mixed.

5% w / v celite 545 (trademark of Johns Manvillle Product Corporation) was added to 4 l of the combined medium (pH 7.2), and the culture medium was filtered to separate the liquid from the cell-containing filter cake. The filtrate was extracted with 2 liters of ethyl acetate to recover the mycoplanesin component, while the cell cake was extracted with 2 liters of 20% v / v water containing acetone and the acetone was distilled off under reduced pressure, and the residue was extracted with 2 liters of ethyl acetate. Extracted using. Ethyl acetate was extracted from the filtrate, and the cell cake was combined to obtain 4 L of the combined extract.

This compound extract was washed twice with 1 L of saturated aqueous sodium chloride solution each time. The washing was dehydrated with anhydrous sodium sulfate and then evaporated to concentration under reduced pressure to afford 1.07 g of an oil.

This oily substance was dissolved in a small amount of chloroform, and adsorbed to a chelum containing 20 g of silica cal made of chloroform in advance. This column was then washed with chloroform and the impurities eluted using a chloroform and edyl acetate 1: 1 elution mixture followed by only edyl acetate. The desired mycopolylanesine was then eluted using a mixed solvent of 95 vol% edyl acetate and 5 vol% methanol. 1 L of the active fraction was separated from the eluted fractions and concentrated by evaporation under reduced pressure to obtain 130 mg of white powder.

110 mg of this white powder was dissolved in a small amount of chloroform, and the resulting solution was passed through a 200 ml column containing Sephadex LH-20 (manufactured by Macaque, Sweden) filled with chloroform, and then eluted with chloroform. The collected active fractions were concentrated by evaporation under reduced pressure to obtain 90 mg of mycoplanesin white powder. This purified product showed a single point when oxo, sulfuric acid and potassium permanganate were used on thin layer chromatography on silica gel.

Example 2

Ten 500 ml of sagaguchi hulas, each filled with 100 ml of the seed culture liquid having the same composition as described in Example 1, were inoculated by incubating Actinoplanes strain No. 41042, and reciprocating shaking was performed at 28 ° C. for 96 hours. .

15 l of the production medium having the same composition as in Example 1 was added to a 30 l fermentor, and then inoculated by adding 750 ml of the culture solution prepared as described above, followed by shaking for 25 hours at 25 ° C. while passing 15 l of air per minute at 28 ° C. Stirred.

15 L of acetone was added to the resulting culture, the mixture was stirred, and the product was filtered by adding 500 g of a filter aid. 25 L of this filtrate was concentrated by evaporation under reduced pressure to distill acetone. Methylene chloride 7 L was added to the remaining liquid (15 L), and the mixture was extracted under agitation. The residue was further extracted with 7 liters of methylene chloride, and the extract was combined, and then the solvent was distilled off to obtain 1.8 g of an oily substance.

A solution of this oily substance dissolved in a small amount of chloroform was adsorbed onto a column filled with 50 g of silica gel and chloroform, and mycoplanesin was eluted using a solvent having 99% by volume of chloroform and 1% by volume of methanol.

400 ml of the active fractions were collected and concentrated by evaporation under reduced pressure to obtain 390 mg of white powder. A solution of a white powder dissolved in a small amount of chloroform was developed using a preparative thin layer chromatography using silica gel plate (5717, manufactured by Merck) using a mixture of chloroform and methanol in a volume ratio of 9: 5. The activation band was extracted with ethyl acetate to obtain 250 mg of mycoplanesin as a white powder having a purity of 90% (biological evaluation).

Claims (1)

A method for producing antimycotic mycoplanesin by culturing Actinoplanesnoven strain No. 41042 (NRRL-11462) as a mycoplanesin producing bacterium and isolating mycoplanesin from the resulting culture.

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