JPS62239986A - Microorganism and use thereof - Google Patents

Abstract

PURPOSE:To obtain a microorganism, capable of exhibiting antifungal action and having the ability to produce substances YP-02259 L-A, -B and -C, by culitvating and separating Actinosporangium sp. YP-02259L-strain from soil. CONSTITUTION:Actionsporangium sp. YP-02259L strain (FERM-P No. 8707) is cultivated and substances YP-02259-A, YP-02259L-B and YP-02259L-C are separated from the resultant culture. A liquid culture medium normally containing natural nutrient sources is preferred for the culture medium used for cultivating the above-mentioned microorganism. The substance YP-02259L-A can be readily separated from the substances YP-02259L-B and YP-02259L-C by carrying out thin-layer chromatography of a solution of a mixture thereof in methanol using chloroform-methanol as a developing solvent on a silica gel thin-layer plate.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention is directed to the treatment of the genus Actinosporangium.
The present invention relates to a microorganism that is capable of producing YP-02259L-A, -B, and -C substances belonging to the genus Sprangium) and exhibiting antifungal effects, and a method of using this microorganism in producing the substances.

(Prior Art) YP-02259L-A, -B and Substance 10 are compounds that exhibit antifungal activity and have the physicochemical properties described below. There have been no reports of microorganisms belonging to the genus Actinosporangium that produce such substances.

(Means for Solving the Invention) The present inventors have repeatedly searched for physiologically active substances produced by microorganisms, and found that the microorganisms collected on Zamami Island, Okinawa Prefecture, were found to contain Actinosporangium, judging from their mycological properties. It was discovered that it is a new species of microorganism belonging to the genus YP-02259L-A, -B, -C.
The present invention was completed based on this finding. Namely.

The present invention belongs to the genus Actinosporangium.

YP-02259L-A, YP-”02259L-B
and YP-02259L-A, YP-0225 from the culture.
9L-B and YP-02259L-C substances are collected, in which the genus Actinosporangium
It belongs to YP-02259L-A.

YP-02259L-B and YP-02259
This method is characterized by using microorganisms capable of producing L-C substances.

(microorganisms) Belongs to the genus Actinosporangium of the present invention.

YP-02259L-A, -B, - showing antifungal effect
Specifically, the microorganism having the ability to produce C substance is Actinospora sb.
n-gium sp,) yp-02259L strain (Feikoken Bacterial Serial No. 8707). YP
The mycological properties of the -02259L strain are as follows.

1. Morphology This fungal strain grows in various synthetic and organic media and forms aerial hyphae from bifurcated basal hyphae. The short, branched aerial hyphae extend relatively long in a straight line, forming chains of 10 or more spores.

In addition, at the tip, pseudosporangiums consisting of intertwined spore chains are abundantly observed.

Their shapes and sizes vary from spherical to spherical and from 2 to 5 μm in diameter, and pseudospore spores are nonmotile. The spores are cylindrical and the size is 0.3~0.5X0.8~1.0
μm, and its surface is smooth.

2. Properties of various agar media Properties on various agar media are as shown below.

Cultures were incubated at 28°C for 21 days unless otherwise specified.

Observations were made according to conventional methods. The description of color tone was based on two-color standards (Japan Color Research Institute).

(Note) G: Growth and bacterial flora color on the colony surface A: Epiphytion of aerial mycelia and its hue R: Hue on the back S: Soluble pigment 3, physiological properties 4゜(
Note) The growth temperature is at each temperature (5, 10, 15, 20, 25,
7 at 28,30°33,37.40.45.50°C)
5° observation results from ~21st. The effect on milk was observed at 37°C for 3 to 21 days. Otherwise, unless otherwise specified, at 28℃
Observation results after a week are shown.

Carbon source assimilation (Pridham-Godelive agar medium 2)
(Culture at 8°C) (Note) +; growth ±; growth is questionable -; non-growth Analysis of diaminopimelic acid (DAP) LECHEVAL IER et al.'s method (LECHEVA
L I ER, M.P.

et aLpp227 238 in DIETZ,
Aetal ed, ActinomyceteTax
onomy, S IM 5special public
As a result of analyzing the acid hydrolyzate of the bacterial cells of this strain according to cation NQ 6.1980), LL-DAP was detected.

To summarize the above properties, the YP-02259L strain is.

In addition to forming spore chains on aerial hyphae, pseudosporangia formed when hyphae intertwine
) is formed in abundance. Because they do not have a 5-spore membrane, they vary in shape and size, and the endospores of pseudospores are nonmotile. As for the hue, the aerial mycelia are white to grayish white, and the growth is yellowish gray to yellowish brown, and no production of soluble pigments or melanin-like pigments is observed. From the analysis of acid hydrolyzate of bacterial cells,
LL-diaminopimelic acid was detected.

Based on the above properties, one strain is of the genus Streptomyces (Streptomyces).
Although it is thought to be a strain closely related to Actinosporangium (Krasil n.
1kov and Tsi-shen 1961). Therefore, this strain was selected from a known bacterial species of the genus Actinosporangium, Actinosporangium violaceum (A, viol), which has been reported in past literature.
a-ceum and Actinosporangium vitaminophyllum A, vi taminophi turn
) was compared. As a result, Actinosporangium biocellum differs from this strain in the color tone of aerial mycelia, reducing ability of nitrous salts, melanin pigment production, and sugar utilization, whereas Actenosporangium pitaminophilum does not form spore chains in aerial mycelia. peptonization of milk and sugar doping]
Because they differ from the present strain in their utility, the nine strains were judged to be new species of the genus Actinosporangium and were classified as Actinosporangium sp.
, ) was named YP-02259L.

This strain was sent to the Institute of Microbial Technology, Agency of Industrial Science and Technology.

It has been deposited under the accession number 8707. The microorganism of the present invention is characterized by the production of YP-02259L-A, -B and -C substances in addition to the above-mentioned mycological properties. The strain of the present invention, like actinomycetes, is susceptible to artificial or natural mutations. The YP-02259L strain of the present invention includes naturally isolated strains, strains that have been artificially mutated using ultraviolet rays, X-rays, chemical agents, etc., and natural mutant strains thereof. be.

The microorganisms belonging to the new bacterial species of the present invention were obtained by isolation from natural soil, but they can be easily obtained by restoring the freeze-dried product of the strain deposited with the Microbial Technology Research Institute. .

(Culture Method) The microorganism according to the present invention is cultured using a medium containing a nutrient source used by the microorganism. The medium may be synthetic, semi-synthetic, natural, solid or liquid, but liquid medium containing natural nutrients is usually preferred. The nutrient source added to the medium may be any assimilable carbon compound as a carbon source, such as arabinose, sucrose, starch, and glucose.

Glucose, starch, dextrin, coconut oil, soybean oil, α-
Melipioses and the like are used alone or in combination.

Furthermore, alcohols, organic acids, etc. may also be used. Inorganic and organic nitrogen sources include ammonium chloride and sodium nitrate.

Ammonium sulfate, ammonium nitrate, urea, etc. are used as organic nitrogen sources, peptone, yeast extract, dried yeast, meat extract, gluten meal, cornstarch, soybean flour, fish meal, peanut flour, cottonseed meal, casamino acids, etc. Various amino acids (eg, glutamic acid, alanine, lysine, etc.) can be used alone or in combination.

In addition, sodium, potassium, magnesium, calcium, iron, and iron are added to the medium as necessary.

Sulfates, nitrates, and chlorides of metals such as cobalt.

Carbonates, phosphates, etc. can be added.

Cultivation is best done under aerobic conditions and left standing.

Both shaking and aeration and agitation cultivation are possible.

Shaking or aerated culture is advantageous. The culture temperature is preferably within the range of about 25-40°C, particularly about 27-30°C.
7°C is advantageous. In addition, the pH of the medium is approximately 5.5 to 8.
It is preferable to maintain it near neutrality of 5.

The culture period varies depending on culture conditions such as medium composition and temperature, but is usually about 2 to 14 days.

The culture is terminated at an appropriate time by determining the time when the YP-02259L-A, -B, -C substances reach their maximum titer.

In order to isolate and purify the substances accumulated in the culture thus cultivated, a commonly used isolation and purification means may be applied. Actinosporangium sp. YP
When strain -02259L is cultured under the culture conditions described in the Examples below, at least three types of active substances are accumulated in the culture solution.

The active substance is isolated and purified by centrifuging or filtering the culture to remove the bacterial cells, and then the solubility in an appropriate solvent and the difference in solubility, the ability to precipitate from the solution and the difference in the precipitation rate9. It is preferable to apply a method that utilizes the difference in adsorption affinity for the agent, the difference in distribution between two types of liquid phases, etc. These methods can be used alone, combined in any order, or applied repeatedly as necessary.

Among the products produced by culturing YP-02259L strain,
-A substance, -B, -C substance is a methanol solution of their mixture Silica gel 60 F 25 manufactured by Merck & Co.
Chloroform-methanol (9:1
) is used as the developing solvent,
Divided into spots with Rf values around 0.85 and 0.45.

By utilizing this property, it can be easily separated. The purified product separated from the former spot is -A substance, and the purified product separated from the latter spot is -B,
-C substance. Also.

For substance -B and substance -C, these methanol solutions were used on a Senshu Pack ODS-H-2101 column.

Acetonitrile-tetrahydrofuran-water-01M I
It can be separated by performing high performance liquid chromatography using J7 acid buffer (pH 5) (90:20:90:2) as a solvent.

(Product) YP-02259L-A thus obtained, -
B.

The physical and chemical properties of the -C substance are as follows.

(1) Ultraviolet absorption spectrum: The spectrum measured with methanol is as follows.

YP-02259L -A Figure 1 tt -3 Figure 5... -〇 Figure 9 (2) Infrared absorption spectrum: The spectrum measured by the potassium bromide tablet method is as follows.

YP -02259L -A Figure 2 // -13
Figure 6 // -C Figure 10 (31'H-Nuclear Magnetic Resonance Spectrum The spectrum measured in double chloroform is as follows.

YP -02259L -A Figure 3N -73Figure 7 -CFigure 11 (4) 13C-Nuclear Magnetic Resonance Svector: The signal of the spectrum measured in deuterated chloroform using TMS as an internal standard is as follows. be.

YP-02259L-A substance δ (PPM): 173.4.135.3.134
．． 7.133.0゜129.5.116.9.94.0
．． 81.9.80.1.77.8゜76.7.74.9
．． 55.1.43.5.42.0.38.0.37.3
゜35.2.34.6.33.6.32.7.27.2
．． 26.4.22.8゜19.2.17.2.15.9
．． 12.9.5.5YP-02259L-C substance δ (PPM): 216.7.173.4.157.
4.134.9゜134.5.134.0.133.0
．． 129.4.116.9゜98.2.94.1.82
．． 3.82.0.80.1.77.9.77.8°77
．． 2.75.6.75.3.72.1.67.7.48
．． 4.43.5゜42.1.37.2.36.8.36
．． 0.35.2.34.8.32.8゜29.2.27
．． 2.26.1.19.2.17.8.17.3.16
．． 0°10.8. 7.6 5.6 (5) Mass spectrum: The spectrum measured by FAB-MS is as follows.

YP -02259L -A Figure 4 // -13
Figure 8 // -C Figure 12 (6) Appearance: YP-02259L-A, -B and -C are all white powders.

(7) Solubility: YP-02259L-A, -B and -C co-K Soluble in methanol, ethanol, acetone, ethyl acetate and chloroform. Insoluble in water.

(8) Distinction between basic, neutral, and acidic: yP-02259
LA, -B and -C all exhibit the behavior of neutral substances.

(9) Color reaction: YP-02259L-p, -B
Anisaldehyde sulfate, universal and sulfuric acid reactions were positive for both and -C. Ninhydrin reaction was negative.

(101 thin layer chromatography: silica gel 60 F
The Rf values obtained using the following developing solvent using a 2.7 (Merck & Co.) plate are as follows.

αυ High performance liquid chromatography: The elution time of each substance measured under the following conditions is as follows.

YP-02259L-AI 1〃
-B 7.8, -C5
, 8 Measurement conditions: Column used Senshu Pack O'DS-H-2101
(6×100mm) Solvent Acetonitrile-Tetrahydrofuran-
Water-0.1M phosphate buffer (pH 5) (90:20:9
0:2) Detection Absorption flow rate at 207mm 1.34
ml 1 minute (effect of the invention) The microorganism of the present invention is YP-02259L-A, -
It is useful in producing B and -C substances. The substance produced by the microorganism of the present invention has antibacterial activity against Durum positive bacteria, plant pathogenic fungi, and animal skin pathogenic fungi.

It is particularly active against Trichophyton interdigitalis, which is the causative agent of athlete's foot, and is therefore useful as a medicine.

Next, the activity of YP-02259L-B and -C substances against various microorganisms will be shown.

Experimental method Dissolve YP-02259L-B and 10 substances in methanol to make a solution with a concentration of 1000 γ/ml. 3
Apply this solution to a thin paper disk (manufactured by Toyo Seisaku Shin) with a diameter of 1.0 mm for antibacterial activity measurement, and remove the excess liquid.
Paper disk assay was performed on various test bacteria. The test bacteria were cultured at 27°C overnight or for three days, and the resulting growth inhibition zone diameter (mm) was measured.

Results Note 1: Peptone-yeast medium (peptone 1%, yeast 0.1%, pH 7.0). - Night culture.

Note 2: Sapro medium. Cultured for three days.

(Example) Next, nine examples will be given to further explain the method of using the microorganism of the present invention.

Example 1 White dextrose) IJN 2.0%, glucose 0.5%,
Yeast extract 0.5%, polypeptone 05%, Frein-
A medium (pH 8.0) containing 0.52% heart infusion, 0.5% corn steep liquor, 0.3% meat extract, and 02% calcium carbonate was prepared, and this was
Dispense 60ml into 00ml Erlenmeyer flasks, 1
Actinosporangium sp. YP- grown on Bennett agar medium after sterilization at 20°C for 20 minutes.
The hyphae of the 02259L strain were scraped and inoculated, and incubated at 27℃ for 72 hours.
Perform shaking culture for hours and use as a seed culture solution. Next, add 3% potato starch, 1.5% soybean flour, 5% corn steep liquor, 0.2% yeast extract, magnesium sulfate.
Heptahydrate 0.05%, sodium chloride 0.3%, cobalt chloride hexahydrate 0.002%, Adekanol (Asahi Denka FF)
) Prepare a medium 31 (PH7,1) containing 0.03%, dispense this into 500ml Erlenmeyer flasks in 60ml portions, sterilize at 120'C for 20 minutes, and add the seed culture solution to 3.0% Inoculated at a ratio of When the shaking culture was continued for 96 hours at 27°C, Micrococcus sp. 330
Antibacterial activity against one strain is maximum. Radiolye #600 (manufactured by Showa Kagaku Kogyo) is added to the culture solution thus obtained, stirred, and then filtered to obtain 25 tons of P solution. After adjusting the pH to 7.0 by adding 4N hydrochloric acid to this stock solution, 2.51% ethyl acetate was added and stirred well. Separate the ethyl acetate layer and add anhydrous sulfuric acid to it)
Add Jum and dehydrate. Next, the dehydrated ethyl acetate layer is concentrated under reduced pressure to obtain a yellow oily substance.

The resulting yellow oily substance was dissolved in a small amount of chloroform and subjected to column chromatography using chloroform-methanol (9:1) as the developing solvent, and the fractions showing antibacterial activity against Micrococcus sp. strain 3301 were collected and evaporated under reduced pressure. A pale yellow powder is obtained.

The resulting pale yellow powder was dissolved in a small amount of methanol, applied in a strip onto a thin layer plate of silica gel 60 F254 manufactured by Merck, and mixed with chloroform:methanol (9:1).
) using thin layer chromatography as a developing solvent,
The parts showing antibacterial activity against Micrococcus sp. 3301 strain around Rfo, 85 and Rfo, 45 to 0,50 were scraped off and treated with chloroform-methanol (9:1).
The antibacterial substance is eluted. When it is concentrated under reduced pressure, Rf
o, 20mg as a white powder containing substance A from around 85
However, 40 mg of white powder containing substances B and C was obtained from around Rf O, 45 to 0.50.

YP-02259'LA thus obtained,
-B.

-C substance by high performance liquid chromatography.

further refined. That is, 20ff1 containing substance A
Dissolve g of white powder in 0.2 ml of methanol.

In addition, 40 mg of white powder containing substances B and C was added to 0.4 m
1 of methanol and fractionated by high performance liquid chromatography under the following conditions. Column: Senshu Pack 0DS-H-21016jZf X 100m
m, solvent; acetonitrile: tetrahydrofuran: water: 0.1M phosphoric acid pamphlet (pH 5) (90:20
:90:2), flow rate 1.34m11min, detection: 207
Absorption in nm. When the peaks at elution times of 11 minutes, 7.8 minutes, and 5.8 minutes were collected, YP-02259L-A-B,
-C substance is 3.8 mg and 2.4 mg, respectively.
7.7 mg was obtained. The physical and chemical properties of these substances are as described above.

[Brief explanation of drawings]

(1) Figures 1, 5 and 9 are respectively YP -0
The ultraviolet absorption spectra of 2259L-A, -B and -C substances are shown. (2) Figures 2, 6 and 10 are YP-
The infrared absorption spectra of 02259L-A, -B and 10 substances are shown. (3) Figures 3, 7 and 11 are YP -0 respectively.
1H-nuclear magnetic resonance spectra of 2259L-A, -B and -C substances are shown. (4) Figures 4, 8 and 12 are YP -0 respectively.
2259L-A, -B and -C substances mass spectra are shown.

Claims (1)

[Claims] 1. Actinosporangium spp.
angium), YP-02259L-A, YP
-02259L-B and YP-02259L-C substances 2, a microorganism belonging to the genus Actinosporangium is known as Actinosporangium sp. A microorganism 3 according to claim 1, wherein the microorganism is cultured and YP-02259L-
AYP-02259L-B and YP-02259L-
In the production method of these substances in which C substances are collected, Actinosporangium spp.
YP-02259L-A, YP-02
259L-B and YP-02259L-C substance production method 4, the microorganism is Actinosporangium sp. YP-
02259L strain (Feikoken Bibori No. 8707), the method according to claim 3.