JPS60114194A - Antibiotic yp-0583i-alpha and its preparation - Google Patents

Abstract

PURPOSE:To obtain antibiotic YP-0583I-alpha, by cultivating a novel strain belonging to the genus Actinoplanes. CONSTITUTION:Actinoplanes sp. YP-0583I strain (FERM-P7336) is cultivated in a medium containing a bromine compound, especially calcium bromide and sodium bromide to give the desired substance. The antibiotic thus obtained has antibacterial activity shown by the table.

Description

[Detailed description of the invention] The present invention relates to an antibiotic and a method for producing the same.

More specifically, YP 0583 I- has antibacterial activity.
Regarding α. In addition, the present invention cultivates antibiotic YP-0583I-α-producing bacteria belonging to the genus Actinoplanes in a medium containing a bromine compound, and produces YP-0583I-α in the culture.
The present invention relates to a method for producing YP-0583I-α, which comprises accumulating YP-0583I-α and collecting YP-0583I-α from the culture.

The present inventors collected a large number of microorganisms from soil for the purpose of developing new antibiotics, and as a result of testing the productivity of antibiotics, we discovered that Actinoplanes was newly isolated from soil.
They noticed that when the S.P. strain YP-0583■ was cultured in a medium containing bromine compounds, it produced an antibacterial active compound, and when they isolated the active ingredient from the culture, they found that this substance was The present invention was completed by confirming that it was a new antibiotic.

Actinoplanes sp. YP-0583I strain used in the present invention is a new species of actinomycete isolated from the soil of Konosu City, Saitama Prefecture, and its mycological properties are as follows.

■ Morphological properties Although the YP-0583I strain grows well on various commonly used media, the formation of aerial mycelia is not uncommon.
.

Slight formation of spore light was observed when cultured on Tyrodino agar (■5P-7) to which soil extract was added. According to observation using an electron microscope and an electron microscope, the sporophyte grows almost at right angles on the basal hyphae.

The spore light has a spherical shape with a diameter of 5 to 10 μm and a wrinkled surface.

The spores are spherical, have a diameter of 08-12 μm x 15 μm, and are motile.

(2) Growth status on various media Table 1 shows the growth status when cultured for 280.7 to 21 days on various media.

Table 1 Yeast malt agar medium Growth is poor. The surface is reddish (A, Shikushik15P-2) L-shaped oatmeal agar medium Growth is extremely poor Orange-colored T-tough (15P-3) Scooch inorganic salt agar medium Average. The surface is hard, yellowish, and pear (ISP-4). The surface is Yaya Yellow Taitai Nashiku l5P-5) L-shaped tyrosine agar medium Good condition. The surface is yellowish
Aeshuku 15P-7) L-shaped Benenoto agar medium Normal. The surface is wrinkled. 5. Reddish. None. Physiological properties The physiological properties of the YP-05831 strain are shown in Table 2. Growth temperature range, milk, and anything other than action on cellulose, at 28C.

The observation results after 2 weeks are shown. Also, 61 ships were grown.

The observation results for 7 to 30 days are shown, and the effects on milk and cellulose are the observation results for 7 to 21 days.

Table 2 Growth temperature range: 15-37°C Optimum growth temperature: 27-33°C Optimum growth pH: 6.5-75 Effect on milk: Reduction of nitrates that do not coagulate and convert into peptonization None: Production of melanin-like pigments: None Decomposition of casein: Yes Liquefaction of gelatin: None at 20°C, slight at 28°C Decomposition of cellulose: None Hydrolysis of scouch: Yes D-pheasant rose + D-7 lactose Sucrose Inositol - L-rhamnose + raffinose - D-mannitol - ＋; I use it frequently. Soil; slightly used. −;Do not use.

■ Cell wall composition Lechevalier et al. (LEC11EVA4TE
R, M, P, et al; Int, J, 5yst
Bacterio1. , 20.435-443.197
0) and Becker et al.
tal; Appl, Microbiol, 1
2.421-423, 1964; AppllMicr
obiology, 13.236-243°19 (3
The results of analyzing the bacterial cell hydrolyzate according to method 5) are shown in Tables 3 and 4.

Table 3 Cell wall type Diaminopimelic acid Glycine body LL unit Hydroxy body 10--11 Table 4 Main sugar components of whole bacterial cells Glucose Galactose Mannose Arabinose Kinulose + 10 - Trace level Judging from trace level or higher, YP The cell wall type of the -0583I strain was confirmed to be type ■.

To summarize the above properties, the actinomycete strain YP-0583I attaches spore lights to the red stalks of spores produced from one-branched substratum hyphae, and the spore lights are spherical in shape and the spores are not motile. have Also, the type of cell wall.

■It is a type. Bergey'sM bacterial genus similar to this strain
annual of Determinatire Ba
cteriology 8th Edition (1
974) and various literature, the two strains were found to be Actinoplana 7ei (Actino-plana 7ei).
Actinop of the family (aceae)
The strain was recognized as a new species belonging to the genus Actinoplanes sp.
lanes sp, ) YP-0583I.

This Actinoplanes sp. YP-05831 strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, as Microbiology Research Institute No. 7336.

The strain used in the present invention is the above-mentioned YP-05.
All strains belonging to the genus Actinoplanes and having the ability to produce the antibiotic yp-0583I- can be mentioned, as well as natural or artificially mutated strains of the 831 strain, such as those caused by ultraviolet rays, chemicals, chemical rays, radiation, drugs, etc. . In order to obtain the antibiotic YP-0583■-α according to the present invention, this bacterial strain is cultured in various media commonly used for antibiotic production, and isolated from one of the culture fluids or from the bacterial cells. Various nutrient sources can be used as the culture medium (although some may leak, carbon sources such as glucose 2-dextrino, molasses, starch, starch syrup, etc.) can be used as appropriate.
In addition, as the nitrogen source, for example, soybean flour, fish meal, meat extract, and other organic or inorganic nitrogen sources are appropriately used. Also.

Vitamins, nucleic acid bases, amino acids, various metal salts, etc. may be added as appropriate. In the infusion method of the present invention, Actinoplanes SP yp-0583
When culturing one strain.

By adding appropriate amounts of bromine compounds, especially potassium bromide and sodium bromide to the medium, the desired antibiotic YP-0583I-α is obtained. As for the culture conditions, it is generally advantageous to culture under aerobic conditions, and the culture temperature is desirably in the range of about 18 to 35 R, preferably about 30 R.
The pH of the medium is approximately 5-10.
Good results are obtained by keeping it preferably in the range of about 5-7. The culture period is appropriately set depending on the composition of the medium, temperature conditions, etc. Targeted antibiotic Y-Koshirae-05 from culture
To isolate and collect 831-α, a conventional isolation method using antibiotics from microbial cultures is applied. Since the target substance is mainly contained in the cultured oral fluid, centrifugation or 'rJ
After removing the microbial cells by filtration, the effective aphrodisiacs are extracted from the Sawaben liquid. Isolation and purification are based on differences in solubility and solubility in appropriate solvents, differences in precipitation properties and rates of precipitation from solutions, and differences in adsorption affinity for individual adsorbents.

This method is used in the production of antibiotics, which utilizes the difference in distribution between two liquid phases.

These methods can be used alone or combined in any order as needed.

It can also be applied repeatedly. The physicochemical properties of the antibiotic yp-o5s3i-α thus obtained are as follows.

Antibiotic YP-0583 ■ Physical and chemical properties of -α ■ Appearance: White powder ■ Melting point: 136-137 c.

■Infrared absorption spectrum The infrared absorption spectra of the two substances obtained by the potassium bromide tablet method are shown in Figure 2.

(2) Nuclear Magnetic Resonance Spectrum The nuclear magnetic resonance spectra at 100 MHz in double chloroform are shown in Figure 3.

■Elemental composition: Consisting of carbon, hydrogen, oxygen, chlorine and bromine, each molecule contains one atom of chlorine and one bromine.

■Molecular weight: Negative-first atom bomb/vertement mass vector method (NEGA-FAB) A peak was observed at K-1099.

■Solubility in solvents: methanol, ethanol,
Soluble in acetonate and ethyl acetate.

Insoluble in water and hexano ■Color reaction: Ferric chloride positive (weak) 10% sulfuric acid
Positive, F, Negative, Negative, 6, φ Rf value by thin layer chromatography. was impregnated with 8 mm diameter thin paper tissue for measuring antibacterial activity (manufactured by Toyo Seisakusho F71:L), excess liquid was removed, and the mixture was dried.

The antibacterial activity of the two substances was determined using various test bacteria shown in the table. The culture medium for assay, 111 composition, is as follows.

Polypeptone yeast agar medium Medium composition Polypebut 71.0% Fumo extract 01% Agar (manufactured by Difco) 12% pH 7.0 The inhibition circle diameter after 16 hours of culture at 37C was measured and the results are shown in the table below.

Next, the present invention will be further explained with reference to Examples.

The present invention is not limited to this embodiment (
・.

Example Textli/20%, Glucose 05%.

0.5% Corn Steep Liquor, 105% Polypebute, 05% Soybean Extract, 0.3% Meat Extract, 0.2% Calcium Carbonate, 0.52% Prey Infusion Powder. Prepare a medium (pH 8,0) containing the
The hyphae of Actinoplanes sp.
Culture for 48 hours at C and use as a seed culture. Next, prepare a medium by adding potassium bromide at a ratio of 0.1% to the above medium, and add 50 mt each to 500 ml Erlenmeyer flasks.
The seed culture solution was inoculated at a rate of 3% into the culture solution which was sterilized at 120C for 20 minutes and cultured with shaking at 27C.

After 72 hours, Bacillus subtilis ATCCG (33
The antibacterial activity against the three strains is maximum. Radiolye) -11 = 600 (Showa Kagaku Kogyo I (, K)) is added to the culture solution thus obtained, stirred, and filtered to obtain 5001 ml of 2 liquids.

After adjusting the pH to 7 by adding 01 N hydrochloric acid to this :f5 solution, 5000 mZ ethyl acetate was added and stirred well. Ethyl acetate JFfj is separated and dehydrated by adding anhydrous sodium sulfate. Next, the anhydrous sodium sulfate was removed and the ethyl acetate layer was concentrated under reduced pressure to obtain 1.5 g of an orange oily substance.

Dissolve this in a small amount of Be/Zeno and use Wako Gel C-20.
0 (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) was placed on a column filled with benzene and subjected to column chromatography using benzene:acetono (5:1) as the developing medium. The mixture was collected and concentrated under reduced pressure to obtain 370,111 g of a yellow oily substance. After adding a small amount of methanol and dissolving it, it was coated in a strip on a Merck silica gel 60F254 thin layer plate, and thin layer chromatography was performed using benzene:acetono (1:1) IN as an opening solvent.Bacillus subtilis ATCC6633 The part showing antibacterial activity on the strain is scraped off, the scraped Norica gel powder is packed in a column, the antibacterial active substance is eluted with benzene:acetono (1:1), and then concentrated under reduced pressure to obtain pale yellow powder 1100III. Add a small amount of methanol to this, dissolve it, apply Merck's silica gel 60F254 thin single layer plate Takumi strip 1/C, and develop with 2 chloroform:methanol (9:1).
Medium thin layer chromatography was performed.

It showed antibacterial activity against Bacillus zazutilis ATCC6633 strain at two locations with Rf values o111 and 036. Rf value 0
．． 41 was scraped off, the scraped silica gel powder was packed in a column, the antibacterial active substance was eluted with chloroform:methanol (9:1), and the substance was concentrated under reduced pressure to produce pure antibiotic YP-0583 as a white powder. -α was obtained by 9 ■.

[Brief explanation of the drawing]

FIG. 1 shows the ultraviolet absorption spectrum of yp-05831-α, FIG. 2 shows the infrared absorption spectrum of YP-0583I-α, and FIG. 3 shows the nuclear magnetic resonance spectrum of YP-05831-α.

Claims (1)

[Claims] 1. Antibiotics having the following physical and chemical properties YP-0
583I-α (1) Appearance: White powder (2) Melting point: 136-137c (3) Ultraviolet absorption spectrum (in methanol): 1st
Figure (5) Nuclear magnetic resonance spectrum (in deuterium chloroform)
; U'F3 diagram (6) Elemental composition: Consists of carbon, hydrogen, oxygen, chlorine and bromine, and contains one atom each of chlorine and bromine in one molecule. - Recognize the problem. (8) Solubility in α7 agents: methanol, ethanol. Acetone, I[Soluble in ethyl acid, insoluble in water and hexanoate (9) Color reaction; Ferric chloride Positive (weak) 10% sulfuric acid Positive, Negative Milono Negative 2 YP-0583I-, which belongs to the genus Actinoplanes α
Method 3 for producing antibiotic YP-0583I-α, characterized by culturing production bacteria in a medium containing a bromine compound and collecting antibiotic YP-0583I-α from the culture YP- belonging to the genus Actinoplanes 0583I-
α-producing bacteria is Actinoplanes sp. YP-0583
The production method according to claim 2, which is strain I (KAIKOKEN BIYORI NO. 7336).