JPS6129717B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an antifungal substance neoenactin and a method for producing the same. More specifically, the present invention relates to a method for aerobically culturing the antifungal agent neoenactin and bacteria belonging to the genus Streptovertercillium, and isolating and purifying the antifungal agent neoenactin. Diseases caused by fungi are generally considered incurable, and there has been a rapid increase in the number of fungal infections in recent years. Polyene antibiotics are currently commonly used as therapeutic agents for deep-seated mycoses. However, there is a narrow gap between the effective dose and the toxic dose, which poses a safety issue. Therefore, the present inventors developed a new screening method using antagonism between polyene antibiotics (e.g. trichomycin) and cholesterol [Chemical and
Pharmaceutical Bulletin) Volume 21, page 2057,
1973] and searched for non-polyene antibiotics that have antifungal effects or substances that enhance the action of polyene antibiotics. By this method we have obtained Streptomyces roseoviridis H 646
−SY3 is enactin [Journal of Antibiotics, No. 30]
Vol. 182, 1977]. Enactin itself does not have strong antibacterial activity against fungi, but it has been found to significantly enhance the antibacterial activity of trichomycin or amphotericin B. Furthermore, when we searched for microbial cultures using this screening method, we discovered that a strain belonging to Streptoverticillium produced neoenactin, a new substance with strong antifungal activity. Neoenactin of the present invention is a useful antifungal strain, exhibiting a minimum inhibitory concentration of 0.313 μg/ml against the fungus Candida albicans Yu-1200, and enhances the antibacterial activity of the polyene antifungal agent tricomycin. Also,
It enhances the antifungal activity of trichomycin even in the presence of cholesterol, which is said to weaken polyene antifungal agents. The present invention provides neoenactin or its functional derivatives and salts thereof. In addition, the present invention also relates to strains belonging to the genus Streptovertercilium, such as Streptovertercilium
Oribole Teikiyuri Subsp Neoenacteicus H829−MY10 (Streptoverticillium
An object of the present invention is to provide a method for culturing the antifungal substance neoenactin (subsp. Neoenactin is a white amorphous powder.
Shows a melting point of 60.5-64.5°C. Neoenactin is soluble in lower alcohols, ethyl acetate, ether, and chloroform, and insoluble in water, n-hexane, and petroleum ether. The percentage composition (average value) of enactin based on elemental analysis is as follows. C, 63.47%, H, 10.04%, N, 7.44%, (0, 19.05%) Halogen and sulfur were not detected. The ultraviolet absorption of neoenactin in methanol solution, 0.1N hydrochloric acid-methanol solution, and 0.1N caustic soda-methanol solution is shown in attached Figure 1. 208 nm in methanol solution (E ¹ % ₁ cm = 166) 0.1N hydrochloric acid methanol solution 208 nm (E ¹ % cm =
161) 0.1N, caustic soda methanol solution at 240 nm (E ¹ % cm = 138) The infrared absorption spectrum of neoenactin potassium bromide tablets is shown in attached Figure 2. Specific optical rotation [α] ²⁰ _D = -14.2° (C = 3%, in methanol) Color reaction of neoenactin Ninhydrin reaction + Naphresorcinol-phosphoric acid reaction + KMnO ₄ + Anthrone-phosphoric acid reaction - α-naphthol-phosphorus Acid reaction - Components of neoenactin Neoenactin was hydrolyzed with 1N hydrochloric acid at 110°C for 17 hours, and the hydrolyzate was extracted with ether and then ethyl acetate. The aqueous layer was evaporated to dryness, dissolved in water, passed through Amberlite IR-45, the passing liquid was collected and evaporated under reduced pressure to reduce the volume, and ethanol was added. The obtained white precipitate was identified as L-serine by paper chromatography and infrared absorption spectrum. Paper chromatography and silica gel G thin layer chromatography of neoenactin and enactin Candida albicans as the detected microorganism
The results of various paper chromatography using Yu-1200 and silica gel G thin layer chromatography are shown in Tables 1 and 2. For detection, a ninhydrin reaction and a method of spraying and heating 40% sulfuric acid were used as supplementary methods.

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[Table] Specific examples of producing microorganisms include Streptovertercilium oliboreteikilium subsp. neoenateicus H829-MY10, which belongs to the genus Streptovertercilium. This strain was isolated by the present inventors from soil in Nagasaki Prefecture, and its mycological properties are as follows. (1) Morphology: This strain forms well-branched basal hyphae under the microscope, but the growth of aerial hyphae is slow, and on most synthetic or organic media, aerial hyphae are not produced or are not produced at all. is also very poor. The sporophyte is mostly straight and produces primary whorls or rarely secondary whorls. The shape of the spore is cylindrical and monophalangeal, and the size is 1
The surface of the spore is smooth at ~1.2 microns. Ball-like bodies found in Streptomyces luteocolor or Streptoverticillium verticillus are sometimes observed at the tip of the sporophyte. (2) Growth status on various media;

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[Table] (3) Physiological properties (i) Growth temperature range: Good growth in the temperature range of 20 to 37°C on maltose-yeast extract agar medium. (ii) Liquefaction of gelatin; has moderate liquefaction ability. (iii) Hydrolysis of starch; negative (iv) Coagulation of skim milk; positive Peptonization of skim milk; positive (v) Production of melanin pigment; negative (4) Availability of carbon sources (Pridham, Gotsdriep agar) ( i) Used; mehu-inositol, D-galactose, dextrici, maltose, glycerol (ii) Used in doubt; L-arabinose, D-
Fructose, flynose, D-trehalose (iii) Not used; D-xylose, sucrose, D-mannitol, lactose To summarize the mycological properties of strain H829-MY10, they produce whorled branches, grow pale yellow-brown, and are white. aerial mycelia and soluble pigment non-production. These characteristics of the H829-MY10 strain are related to Streptovertercilium of the genus Streptovertercilium.
Streptoverticillium (Streptoverticillium)
olivoreticuli). In other words, Streptovertercilium oriboleteikilily produces primary and secondary whorled branches, extends pale yellow-brown basal hyphae, and produces spherical structures with unknown physiological functions. However, Streptovertercilium oliboreteikilily belongs to the chromodienic type and hardly uses inositol.
Different from H829-MY10 strain. From the above, H829−
The MY10 strain is considered to be a subsp. of Streptoverticillium olivoreticuli subsp. .
neoenacticus). The H829-MY10 strain is susceptible to changes in its properties, such as UV rays,
The method of the present invention can be used for all mutant strains that can be mutated by artificial means using X-rays, high frequencies, radiation, chemicals, etc., and that have the ability to produce neoenactin or neoenactin components. I can do it. This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, and its accession number is
No. 4376 (FERM-P4376). In the method of the present invention, the above-mentioned bacterial strain is usually cultured in a medium containing nutrients that can be utilized by microorganisms. As the nutrient source, known nutrient sources conventionally used for culturing Streptomyces bacteria can be used.
For example, gelose, starch, glycerin, dextrin, sucrose, starch syrup, molasses, soybean oil, etc. can be used as the carbon source. Also, as a nitrogen source, soya meal, dry dry mother, cornstarch liquor, wheat germ, cotton seed meal, ammonium sulfate, sodium nitrate, etc. can be used. Calcium carbonate, if necessary,
In addition to adding inorganic salts such as sodium chloride, potassium chloride, phosphates, manganese chloride, copper sulfate, ferrous sulfate, and zinc sulfate, appropriate organic and inorganic substances are added that help the growth of bacteria and promote the production of neoenactin. I can do it. As a culture method, liquid culture is generally suitable, as is the case with antibiotic production methods. Cultivation is carried out under aerobic conditions, and the appropriate temperature for culturing is
The temperature is 25-35°C, but in most cases it is cultured at around 28°C. Neoenactin production reaches its maximum in 1 to 3 days in both shaking culture and tank culture. The culture conditions described above can be applied by selecting the optimal conditions depending on the characteristics of the production strain used. Neoenactin is contained in both the bacterial cells and the culture solution, so it is possible to extract it from both. It is. For example, the culture solution is extracted with ethyl acetate at pH 8, or the culture solution is adsorbed onto Amberlite XAD- and eluted with aqueous methanol. Furthermore, methanol extraction is performed from the bacterial cells, and after methanol is distilled off under reduced pressure, ethyl acetate extraction is performed at pH 8. Neoenactin contained in both ethyl acetate extracted fractions can be back-transferred to the aqueous layer at pH 2. Neoenactin transferred to the aqueous layer is extracted again into ethyl acetate fraction at pH 8. This ethyl acetate extracted fraction is concentrated under reduced pressure to obtain a highly viscous oily substance. oily substance 1/
20M phosphate buffer (PH8.0) was dissolved in saturated ethyl acetate, cellulose powder soaked in 1/20M phosphate buffer (PH8.0) and air-dried was developed in a column prepared using the same ethyl acetate solution. do. Collect the fraction containing neoenactin and wash with a small amount of water.
Evaporate to dryness under reduced pressure and dissolve 1/20M phosphate buffer in saturated ethyl acetate. This is again developed in a similar column of cellulose powder. The fractions containing neoenactin are collected, washed with a large amount of water, and evaporated to dryness under reduced pressure to obtain neoenactin powder. The antibacterial spectrum of neoenactin against various microorganisms is shown in Tables 4 and 5. The minimum inhibitory concentration was determined on plain agar medium containing glucose at 37°C.

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[Table] As is clear from the above table, neoenactin exhibits almost no antibacterial activity against bacteria, but it has the property of exhibiting antibacterial activity against yeast and mold. It also enhances the antibacterial activity of known polyene antibiotics, trichomycin, and amphotericin B in the presence and absence of cholesterol. No other known substances possess the above-mentioned physicochemical and physical properties, and it is recognized as a new antibiotic with unique properties. Furthermore, neoenactin not only has an effect on yeast and mold, but also suppresses the uptake of 3 _H -thymidine or 3 _H -leucine against cancer cells (eg, Ehrlichi's ascites cancer cells), thereby exhibiting an antitumor effect. Example 1 Streptovertercilium oriboleteikilily subsp. Neoenacteicus H829−
Bacterium 1ase was inoculated from MY10 preservation slanted agar into 100 ml of a liquid medium (PH7.0) containing 1% maltose, 0.2% yeast extract, and 0.2% polypeptone, and cultured with shaking at 27°C for 24 hours, which was used as a starter. 1.5% soluble starch, 1% in a 30 volume jar fermentor
Glucose, 2% Soya Meal, 0.5% Ebios, 0.25% NaCl, 0.3% _CaCO3 , 0.0008%
_MnCl2・_4H2O , 0.0007% _CuSO4・_5H2O ,
0.0002% _ZnSO4・_7H2O , 0.0001% _FeSO4・
A medium containing 7H ₂ O (PH 7.6, before sterilization) 15 was charged and the medium was sterilized by a conventional method. Transfer 300 ml of seed culture, aeration rate 10 per minute, stirring number 250 rpm, 36 at 27℃
The cultured cells were cultured with stirring for a period of time to obtain 1 kg of cultured bacteria. The bacterial cells were extracted three times with twice the volume of methanol, and the extracts were combined and dried under reduced pressure to form a syrup. PH syrup
Extraction was carried out with an equal volume of ethyl acetate. Neoenactin extracted into the ethyl acetate layer was back-extracted with 200 ml of water at pH 2.0. This aqueous layer was adjusted to pH 8 again and extracted with an equal amount of ethyl acetate. The ethyl acetate layer was washed with water and then evaporated to dryness under reduced pressure. The amount of crude enactin was 804 mg. Example 2 500 mg of crude neoenactin was soaked in 1/20M phosphate buffer (PH8.0), air-dried, and saturated with 1/20M phosphate buffer (PH8.0) in ethyl acetate (abbreviated below).
A column of cellulose powder prepared using
2 cm), and the active fraction was collected.
The activated fraction was washed with water and concentrated to dryness under reduced pressure.
93.6 mg of neoenactin was obtained. This neoenactin was added again to a similar column of cellulose powder (87x
1.5 cm), separate the active fraction,
After washing with water, it was concentrated to dryness under reduced pressure. The yield was 43.3 mg.

[Brief explanation of the drawing]

Figure 1 shows the UV absorption of neoenactin. Figure 2 shows the IR absorption of neoenactin.

Claims (1)

[Scope of Claims] 1. Antifungal substance neoenactin and its functional derivatives and salts thereof having the following physical and chemical properties. (a) Elemental analysis value The elemental analysis value (percentage composition (average value) % by elemental analysis is as follows. C: 63.47 H: 10.04 N: 7.44 (O: 19.05) However, halogen elements and sulfur are Not detected. (b) Melting point The melting point of neoenactin is 60.5 to 64.5°C. (c) Ultraviolet absorption spectrum The ultraviolet absorption spectrum of neoenactin is as shown in Figure 1. Also, the absorption maximum in each solution The values of E ¹ % 1 cm and E 1 % ₁ cm are as follows. [Table] (d) Infrared absorption spectrum The infrared absorption spectrum of neoenactin measured by the potassium bromide tablet method is shown in Figure 2. ( e) Specific rotation The specific rotation of neoenactin in methanol is as follows: [α] ¹² _D = -14.2° (C = 3%) (f) Solubility in solvent The solubility of neoenactin is as follows: Soluble: lower alcohol, ethyl acetate, ether, chloroform Insoluble: water, N-hexane, petroleum ether (g) Color reaction The color reaction of neoenactin is as follows: Ninhydrin reaction + naphtresorcinol - Phosphoric acid reaction + KMnO ₄ + Anthrone-phosphoric acid reaction - α-naphthol-phosphoric acid reaction - (h) Rf value Single spot obtained with various developing solvents in neoenactin paper chromatography and silica gel G thin layer chromatography. Rf
The values are as follows. () Paper chromatography [Table] () Silica gel G thin layer chromatography [Table] (i) Appearance Neoenactin appears as a white amorphous powder. (j) Stability against acids and alkalis When Neoenactin is heated at 100℃ for 5 minutes at pH 9.0, 80% of Neoenactin is inactivated. However, approximately 90% of neoenactin's activity is retained even when neoenactin is heated at 100°C at PH2.6 for 5 minutes. 2. Production of the antifungal substance neoenactin or its constituent factors, which comprises culturing neoenactin-producing bacteria belonging to the genus Streptovertercilium and collecting the antifungal substance neoenactin or its constituent factors from the culture solution and bacterial cells. Law.