JPS62195399A - Novel antibiotic substance globopeptin and production thereof - Google Patents

Abstract

NEW MATERIAL:An antibiotic substance globopeptin having the following physical and chemical properties. Elemental analysis (%), C 47.8, H 5.0, N 15.3, S 12.0; melting point, 200 deg.C; [alpha]<25>D=+62 deg. (C=1.0, chloroform/methanol=1:1); color reactions, positive to Dragendorff reaction and anisaldehyde-sulfuric acid reaction and negative to ninhydrin reaction and aniline phthalate reaction; solubility, easily soluble in chloroform/methanol (1:1), soluble in water and methanol, hardly soluble in ethanol, acetone, ethyl acetate, ethyl ether and benzene; nature, neutral white substance; etc. USE:Active against phytopathogenic mycete and useful as an agricultural chemical. PREPARATION:A bacterial strain belonging to Streptomyces genus such as Streptomyces noursei (FERM P-7933) is cultured under aerobic condition.

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD The present invention relates to a new antibiotic globoptin, a method for producing it, and a new bacterial strain. Conventional technology Cultivation of Streptomyces microorganisms! #(Therefore, various antibiotics obtained are known. For example, Streptomyces sp.
) 5F-2049, a sterile antibiotic obtained by culturing the 5F2049 strain (Feikoken@Yorin No. 4486) (Special Publication No. 6.0-46958), Streptomyces subflavus subsp.
r@tomyee* aubflmvus 5ubap
. 1ruma@nals) AM-3603 strain (Irumamyel No. 5619)
n) (Japanese Unexamined Patent Publication No. 57-48996) strongly suppresses the growth of fungi such as certain plant pathogenic fungi, but has no activity against bacteria. The present invention
Based on the knowledge that a certain type of Streptomyces microorganism that the present inventor isolated from soil has the ability to produce an antibiotic (named globoiptin by the present inventor) that exhibits strong inhibitory activity against fungi. I'm there. Problems to be Solved by the Invention An object of the present invention is to provide a novel antibiotic globobutin having strong activity against fungi, a method for producing the same, and a producing strain thereof. Means for Solving the Problems The physicochemical properties of the antibiotic provided by the present invention are as follows. (11 elemental analysis: C47.8, H5.0%, N
15.3%. 812.0% (2) Molecular weight: Could not be measured using normal mass spectrometry. The molecular weight is estimated to be about 900 from the vapor pressure method, but no definite value could be obtained. (3) Melting point: 200°C (4) Specific optical rotation: [α]o +6g (C = 1.0, chloroform/methanol = 1:1) (5) Ultraviolet absorption temperature: 224°C in methanol
nm, 1% 270 nm K absorption maximum, E engineering. Each value is 5
Divided by 36.262 (Figure 1). (6) Infrared absorption spectrum: As shown in Figure 2. (KBr method) (7) Amine analysis: 6N hydrochloric acid in a sealed tube at 110°C
- After 18 hours of hydrolysis, Ami/a! As/qragic acid, threonine, proline, chrysin,
Equimolar amounts of cystinka are detected. (8) Color reaction: Positive for Dragendorff reaction and anisaldehyde-sulfuric acid reaction, negative for ninhydrin reaction and aniline phthalate reaction. +9) Solubility of eJc vs. -j: Easily soluble in chloroform/methanol (1:1), soluble in water and methanol, slightly soluble in ethanol, acetone, ethyl acetate, ethyl ether, and benzene. (10) Distinction between acidic, neutral, and basic: It is a neutral substance. (11) Color of substance: It is a white substance. This substance is 5F-2049 substance <t! in terms of ultraviolet absorption spectrum and solubility in solvents. ! ? Kosho 60-4695
8') Similar to ic, but clearly different in terms of the constituent amino acids and the distinction between basic, acidic, and neutral, as described below. That is, in the substance of the present invention, aspartic acid, threonine, proline, glycine, and cystine were detected by an amino acid analyzer after acid hydrolysis, and in addition, nin
No J-positive amino acids were detected. On the other hand, 8F-
In substance 2049, as/qragic acid, threonine, proline, and glycine are detected, cystine is not detected, and at least three other unidentified amino acids are detected. Furthermore, the substance of the present invention is a neutral substance, but 3F-204
The nine substances are acidic. In addition, both substances clearly differ in VC in elemental analysis values. This substance is considered to be a new substance, since we were unable to find any literature that describes a substance having the above-mentioned 3!11 chemical properties and the biological activity described below. Globopeptin has been named Globopeptin (G1obop@ptinl). Globopeptin contains at least five amino acids.
It is negative for the phosphorus reaction. Therefore, it is assumed that this antibiotic has a structure consisting of a chromophore part and a Veptyl part. The biological properties of this substance are as follows: (1) Antibacterial The minimum inhibitory concentration (MCC) obtained by the active agar dilution method is as shown in Table 1.The test medium used was potato-glucose agar medium (...6), and the growth at Nail°C was carried out for 3 days after inoculation.
Judgment was made on the 8th. Table 1 Mucor Racemosus KF-2230,2 (
MuCOr racemosus) Piricularia oryzae KP-1800,2 (Piricular
aria oryzae) Poturitis cinerea
KF-2410,78 (Botrytis c.
inerea) Alternaria quicutiana KF-18
51,56 (Alternaria kikuchia
na) Aspercillus niger KF-102>Z
o. (aspergilus ni Dan! Engineering) Candida albicans KF-1 > 100 (2) When this male antibiotic was administered intraperitoneally to mice, the LDIO was 2
The antibiotic globopeptin according to the present invention is produced by culturing a microorganism belonging to the genus Streptomyces and having the ability to produce globopeptin in a medium aerobically, and accumulating globopepten in the culture. , obtained by collecting from a culture.Objective of the present invention (A microorganism of the genus Streptomyces having the ability to produce the antibiotic globopeptin can be used as a strain to be used. An actinomycete, Streptomyces nursei, was isolated from soil in Kiyose City, Tokyo by
oura*i) strain MA-23. This strain is 1
It was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on November 9, 19984 as Microtechnology Research Institute Deposit No. 7933. The mycological properties of this strain are as follows. I. Morphological properties The trophic fibers are present in synthetic and natural media.

[It develops well and usually grows by penetrating the medium.] Aerial mycelium grows abundantly on synthetic media such as glucose/asparagine agar, yeast extract/malt extract agar, and natural media, and its color ranges from white to gray; Appears pale pink in nitrate medium. When observed under a microscope, 20 or more long spore chains branch out irregularly, and their tips exhibit a spiral shape. Under an electron microscope, cylindrical spores measuring 0.8 to 0.9 μm x 0.7 μm were observed, and the surface of the spores was thorn-like. Sclerotia, sporangia and zoospores are to be found. II. Properties on various agar media E. B. Shlrll
ng) et al. [International Journal of Systematic Bacteriology (Int@r
natlonal Journal of 5yst*
matle Bae-1@rlology) Vol. 16, p. 313, 1966], and the known culture medium and experimental method were used. The color tone is a progressive color table.
armony Manual) M4 version [Container Corporation Op America (Contmln@r
Corporation of Am@ricm),
Chicago, 1958], and the coat ° is written in parentheses along with the color chart name. Unless otherwise specified, 2
Observations were made at 7°C for 2 weeks. The results of Section 7 are shown in Table 2. 2nd Tablework 8P: International Streptomyces Project (ink・national Str@p
tomyc@nProj@ct) selected medium. ■, Physiological Properties 11 Formation of Melanin Pigment 1 Tyrosine Agar Medium Negative (B) - 2 Pton, Yeast Extract, Iron Agar Medium Negative (C)
Glucose-peptone-gelatin medium puncture (27°C)
(Negative) Tryptone/Soumou extract complex medium Test +21 Tyrosinase reaction Negative (3) Hydrogen sulfide production ability
Negative (4) Nitrate reduction (glucose/nitrate medium) Positive (51
Hydrolysis of starch Positive (6) Liquefaction of gelatin (glucose/peptone/gelatin medium)
l1g Sex (7) Coagulation of skim milk (2'
7°C) Negative (8) Sterilization of skim milk (27”C) Positive (9) Cellulose content %S Negative am Growth temperature range
15-51℃ old” Carbon source utilization [Pridhmm Gottlieb
eb) Agar medium] Yo(>jI Used: D-xia-sose, 1-inositol Slightly used: D-glucose, L-arabinose, raffinose, melibiose, D-mannitol, D-fructose, Sucrose V, cells Chemical analysis of cell wall composition: J) Aminopimelic acid is of the LL type. Therefore, Leehevaller
(International Journal of Cystic Bacteriology, Volume 20, 435-44
The cell wall composition of this painting (C.11 wall type.) is type 1 according to the classification of C. In addition, since the aerial hyphae have long spore chains of 20 or more in the seventh form, this strain is recognized to belong to the genus Streptomyces. The spore chain is spiral, the shape of the spore is oval or oval, and the surface is thorn-like.The color of the aerial mycelium is white or gray. , the color tone of the underside is light * to brown. Based on the above characteristics, the Kimoe strain is Streptomyces narsei (
It is considered to be closely related to 5tre tomya*m noursai). Barshees Manual Auj Determinative Bacteriology (Barg@y'
m Manual of D@termnative*
Bae-terlogy) 7B2 pages (197
When compared with the properties of Streptomyces nursei described in 2010, there was a slight difference in sugar assimilation, but this was hard to recognize as a difference in water quality, and other properties were the same. Therefore, M person-23 strain is Streptomyces nursei C
It is determined that the strain belongs to the culprit 〃υ takashi μnours@l). The medium for culturing the bacteria producing the antibiotic globobutin according to the present invention contains carbon sources, nitrogen sources, inorganic substances, and other nutrients suitable for culturing Streptomyces microorganisms, and other nutrients as necessary. Containing synthetic or natural media can be used. The carbon source and nitrogen source used in the culture medium may be any type as long as they are available to the strain used. That is, for example, multiple carbohydrates such as glucose, glycerol, fructose, maltose, mannitol, xylose, galactose, ribose, starch, or a hydrolyzate thereof can be used as the carbon source. The concentration is usually preferably 0.2% to 5% (in terms of glucose) based on the medium. In addition, various organic acids such as gluconic acid, pyruvic acid, lactic acid, and acetic acid, various amino acids such as glycine, glutamic acid, and alanine, alcohols such as KFi methanol and ethanol, various non-aromatic hydrocarbons such as normal paraffin, and plants. Various oils and fats of animal or animal origin can be used. Nitrogen sources include ammonia, ammonium chloride,
Ammonium salts of inorganic or organic acids such as ammonium phosphate, ammonium sulfate, ammonium nitrate, urea, peptone, kJZ-7ξ, meat extract, mother extract, dried yeast, corn steep liquor, casein hydrolyzate, Fitzmeal or its digested product, soybean flour or its digested product, defatted soybean or its digested product,
Nitrogen-containing organic substances such as cartilage hydrolyzate, glycine,
Various amino acids such as glutamic acid and alanine can be used. As inorganic substances, various phosphates, magnesium sulfate, common salt, etc., and trace amounts of heavy metal salts are used. Furthermore, when using a mutant strain that exhibits auxotrophy, it is of course necessary to add substances to the culture medium that satisfy its nutritional needs, but this kind of nutrients is difficult to obtain when using a culture medium containing natural substances. (K addition may not be necessary in some cases. Ticketing is performed under aerobic conditions such as incubation culture or submerged culture with aeration and agitation. The culture temperature is usually 20 to 40'C, and the pH is 4 to 9 (and around <K7). The culture period is usually 1 to 7 days, and the antibiotic globoptin is produced and accumulated inside and outside the image body. After completion of the culture, the antibiotic globopeptin is collected from the culture using, for example, the following method. The culture is centrifuged to separate the F solution and the precipitate. It is extracted from the r-liquid by adsorbing it onto a porous polymer resin and eluting it. The extract is appropriately concentrated to dryness to obtain a crude substance of siglobopeptin. The crude material is further subjected to known methods commonly used for the purification of water-soluble substances, such as various chromatographic methods using ion exchange resins, adsorbents such as silica gel, del filtration agents, etc., concentration methods, salting-out methods, etc. They are especially purified when used alone or in appropriate combinations. An example of a method suitable for purifying globoiptin is a column chromatography method using silica gel and a linear concentration gradient elution method using a mixture of chloroform and methanol as an elution solution. By concentrating and drying the active fraction obtained by these methods, a powder of the antibiotic globopeptin can be obtained. In the present invention, the antibiotic globopeptin is detected and quantified using silica gel thin layer chromatography (manufactured by Merck & Co., Ltd., silica gel, 5554, thickness 0.2 sm).
Developing solvent; chloroform/methanol = 2:1, Rf value of antibiotic globopeptin (around 0.45) and Mucor racemosus
By bioassay using IP-223. Next, examples of the present invention will be shown. the below described! ! In the examples, the pH of the medium was adjusted to 7 before use. Example 1 Streptomyces nursei (5tre ton ee)
mm) From a slant culture of strain MA-23 (Feikoken Bacteria No. 7933), a loopful of platinum was added to 100μ of seed medium (2.0% glucose, 0.5% phthalate, 0.5% meat extract, chloride). 5001LI with 0.5% sodium, 0.3% dry yeast, 0.3% calcium carbonate)! ! The mixture was inoculated into a Sakaro flask and cultured with shaking at 27°C for 1 day to obtain a harvested culture. Added 600*j of this seed culture to production medium (2.0% starch, 1.0% glycerol, 1.5% soybean flour)
, sodium chloride 0.2%, magnesium phosphate 0.5
%) into a 5OL jar fermenter,
Cultured at 271C for 4 days with air agitation (aeration volume 15 t/
mtn, stirring 200 r, p, m, ). 28 t of the culture solution was centrifuged using a Sharpless centrifuge to remove bacterial cells, and 20 t of supernatant liquid was obtained. Globobutin was extracted from this supernatant with 18 L of n-butanol, and the extract was concentrated to dryness and then dissolved in 2 L of water. Add this liquid to 5001 adsorption type synthetic resin Diaion H.
After passing through a column packed with P-20 (manufactured by Mitsubishi Kasei Corporation), it was eluted with 50% acetone water 31I, and approximately 200 yLt
It was fractionated. The active fractions (Fractions 12 to 14) were collected and concentrated under reduced pressure to remove acetone, and then freeze-dried to obtain 1.32 g of a crude substance. This crude substance was dissolved in 20-ml water and 80stj of acetone was added to the solution, and the resulting precipitate was collected.Precipitate 370M
9t 2d Dissolved in chloroform/methanol (3:1), pre-prepared with chloroform/methanol (3:1)
The sample was loaded onto the top of a column (3,2X 70σ) packed with silica gel (952, manufactured by Merck & Co., Ltd.) suspended in silica gel. Coat the column with chloroform/methanol (3:1) 70
Elution was carried out at a resistance of 0, and then the elution solvent was changed to chloroform/methanol (2:1)K, and each fraction was fractionated by about 10. The active fractions (Nn 100-150) were collected and concentrated under reduced pressure to obtain 46 wq of white purified powder of the antibiotic globodubutin. This sample gave a single spot on thin chromatography, and the physical and chemical properties of JXFi
It was as described above. Example 2-4 Streptomyces narsei MA- used in Example 1
23 strains (Feikoken Bokuyori No. 7933) were used as seed bacteria. The production medium (pH 7.0) was prepared by adding 3% each of glycerol, glucose, or starch as a carbon source to a basal medium consisting of 1% soybean flour, 2% N*CI O-2%, and 0.5% magnesium phosphate. Each IJ was dispensed into a 500 ml Sakaro flask and sterilized. 2N of the 8Ii culture solution obtained in the same manner as in Example 1 was transplanted into each of the 7 laths of each plate, and cultured with reciprocal shaking at 27° C. for 3 days in a conventional manner. After the culture was completed, an equal amount of ethanol was added to the culture solution and stirred. After centrifugation, the amount of Globojuku butin produced in the supernatant was measured, and the results were as shown in Table 3. The determination of globopeptin was determined by a known microbiological quantitative method using Mucor rae@mau* as a test bacterium, and the standard sample was globobezotin obtained by K as in Example 1. A pure product was used. Table 3 Glycero/I/35 3 Glucose 304 Starch 110 Effects of the Invention The antibiotic globo butin has low toxicity and is mainly active against plant pathogenic fungi, so it is expected to be useful as a pesticide, for example.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum (measured in methanol solution) of the antibiotic globopeptin, and Figure 2 shows the infrared absorption spectrum (KBr method). ” 1st mouth

Claims (1)

[Scope of Claims] [1] Globopeptin, an antibiotic having the following physical and chemical properties. (1) Elemental analysis: C47.8%, H5.0%, N15.
3%, S12.0% (2) Melting point: 200℃ (3) Specific optical rotation: [α]^2^5_D+62℃ (c=1
．． 0, chloroform/methanol = 1:1) (4) Ultraviolet absorption spectrum (in methanol): As shown in Figure 1. (5) Infrared absorption spectrum (KBr method): As shown in Figure 2. (6) Amino acid analysis: 1 mol of aspartic acid, 1 mol of threonine, 1 mol of proline, 1 mol of glycine, 1 mol of cystine (7) Color reaction: Positive for Dragendorff reaction, anisaldehyde-sulfuric acid reaction, ninhydrin reaction, aniline phthalate reaction is negative. (8) Solubility in solvents: easily soluble in chloroform/methanol (1:1), soluble in water and methanol, slightly soluble in ethanol, acetone, ethyl acetate, ethyl ether, and benzene. (9) Distinction between acidic, neutral, and basic: It is a neutral substance. (10) Color of substance: It is a white substance. [2] A strain that belongs to the genus Streptomyces and has the ability to produce the antibiotic globopeptin and has the following physicochemical properties is aerobically cultured in a medium, and the antibiotic globopeptin is accumulated in the culture and collected. A method for producing the antibiotic globopeptin, characterized by: (1) Elemental analysis: C47.8%, H5.0%, N15.
3%, S12.0% (2) Melting point: 200℃ (3) Specific optical rotation: [α]^2^5_D+62° (c=1
．． 0, chloroform/methanol = 1:1) (4) Ultraviolet absorption spectrum (in methanol): As shown in Figure 1. (5) Infrared absorption spectrum (KBr method): As shown in Figure 2. (6) Amino acid analysis: 1 mol of aspartic acid, 1 mol of threonine, 1 mol of proline, 1 mol of glycine, 1 mol of cystine (7) Color reaction: Positive for Dragendorff reaction, anisaldehyde-sulfuric acid reaction, ninhydrin reaction, aniline phthalate reaction is negative. (8) Solubility in solvents: easily soluble in chloroform/methanol (1:1), soluble in water and methanol, slightly soluble in ethanol, acetone, ethyl acetate, ethyl ether, and benzene. (9) Distinction between acidic, neutral, and basic: It is a neutral substance. (10) Color of substance: It is a white substance. (3) A microorganism belonging to the genus Streptomyces having the following mycological properties. I, Morphological properties Vegetative hyphae develop well in both synthetic and natural media and usually grow penetrating into the media. Aerial mycelium grows abundantly on glucose/asparagine agar, yeast extract/malt agar, and natural media, and its color ranges from white to gray, but becomes pale pink on sucrose/nitrate media. When observed under a microscope, 20 or more long spore chains branch out irregularly, and their tips exhibit a spiral shape. Under an electron microscope, the size was 0.8 to 0.9 μm×0.
Cylindrical spores measuring 7 μm in size were observed, and the surface of the spores was thorn-like. No sclerotia, sporangia and zoospores are found. II. Properties on various agar media are as shown in the table below. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (Note) (I) ISP: International Streptomyces Project
eject) selected media. (II) E.B. Shirl
ing) et al.'s method [International Journal
of Systematic Bacteriology (InternationalJ
Our own Systematic Bact
eriology) Volume 16, page 313, 1966]
Accordingly, known media and experimental methods were used. Unless otherwise specified, observations were made at 27°C for 2 weeks. (III) Color tones are determined using the Color Harmony Manual as a standard color table.
) 4th edition [Container Corporation of America
tionof America), Chicago, 1958], and the code is written in parentheses along with the color chart name. III. Physiological properties (1) Formation of melanin pigment (a) Tyrosine agar medium negative (b) Peptone/yeast extract/iron agar medium negative (c)
Glucose-peptone-gelatin medium puncture (27℃)
Negative (d) Tryptone/yeast extract liquid medium Negative (2) Tyrosinase reaction Negative (3) Hydrogen sulfide production ability Negative (4) Nitrate reduction (glucose/nitrate medium) Positive (5) Starch hydrolysis Positive (6) Gelatin Liquefaction (glucose-peptone-gelatin medium) Negative (7) Coagulation of skim milk (27℃) Negative (8) Peptonization of skim milk (27℃) Positive (9) Decomposition of cellulose Negative (10) Growth temperature range 15 ~51℃ (11) Utilization of carbon source [Priedum Gottlieb (
Pridham Gottlieb) Agar medium] Often used: D-xylose, 1-inositol Slightly used: D-glucose, L-arabinose,
Raffinose, melibiose, D-mannitol, D-fructose, sucrose IV, chemical analysis of cells Cell wall composition: Diaminopimelic acid is LL type.