JPS5846052A - Novel peptide antibiotic substance antrimycin and its preparation - Google Patents

Abstract

NEW MATERIAL:An antibiotic substance, expressed by the formula, and having the following physico-chemical properties: Appearance: Colorless powder. Elementary analysis (%): C, 43.37; H, 6.77; N, 17.64; O, 27.23. Melting point: 183 deg.C (decomposition). Specific rotatory powder:[alpha]D<26>-74.0 deg. (C: 0.5, H2O). Solubility: Soluble in water, methanol, etc. and insoluble or slightly soluble in ethanol, acetone, etc. Color reactions: Positive to the ninhydrin, Rydon-Smith reactions, etc. and negative to the Sakaguchi, Dragendorff reactions, etc. USE:An antituberculotic agent effective for Mycobacterium tuberculosis var. hominis, i.e. Mycobacterium tuberclosis H37Rv strain, and rifampicin- and capreomycin-resistant strains thereof. PROCESS:Streptomyces xanthocidicus MG125-CF1 strain (FERM-P No. 6007) is cultivated in a liquid nutrient culture medium utilizable by actinomyces aerobically, and the microbial cells are then filtered to purity and isolate the aimed antibiotic substance from the filtrate.

Description

DETAILED DESCRIPTION OF THE INVENTION - The present invention is directed to the large tuberculosis Mycobacterium tuberculosis strain Hs7Rv, and its rifampicin-resistant and cabreomycin-resistant strains.

Antrimycin (a novel peptide antibiotic represented by the structural formula below) that has growth-inhibitory effects.
The present invention relates to antrimycin) and its production method by fermentation.

The producing bacterium used in the present invention belongs to the genus Streptomyces and produces antrimycin. A representative example of this is the Streptomyces xanthocyidica strain MG125-CFI (
Streptomyces xanthocidicus
MQ 125-CF I ) (rMG125-
CFI stock. ) (Agency of Industrial Science and Technology, Institute of Microbial Technology, Application Form Accession No. 6007).

The mycological properties of MGI25-Cr2 strain are shown below.

1. Morphology The MGI25-Cr2 strain has straight hyphae (Recti-flex jibi), which is relatively longer than the one-branched basal hyphae.
Jes) Forms aerial hyphae, and spiral formation and whorled branches are not observed. Mature spore chains are found to have 10 or more chains, the size of the spores is about 0.3-0.6 x 0.6-1.8 microns, and the surface of the spores is smooth.

2. Regarding the description of growth state colors in various media, the standards shown in brackets are those of Container Fist Corporation
r Corporation of America)
Color Harmony Number Manual (Colorhar
m-one manual) was used.

(1) Sucrose/nitrate agar medium (27c culture) Growth is light yellow brown to 1, brown C6]/2 pi Redma
hogany], aerial mycelia do not attach or attach only slightly white, and soluble pigments give a brownish tinge.

(2) Glucose φ asparagine agar medium (27C
Culture) Light yellow tea to yellow tea [3ptl C1ove Brown
] white to light brown [3fe, 5ilver]
Gray to 5 ih, Lead Gray] aerial mycelia are attached, and no soluble pigment is observed.

(3) Glycerin-asparagine agar medium (ISP
-Medium 5.27C culture) Light yellow to red brown (6 pl, Deep Brown ma
-hogany) on the development of a bright teapot (3fe).

5ilver Qray ~5 fe, Ashes)
It grows on aerial mycelia and produces a pale brown soluble pigment.

(4) Starch/Inorganic Salt Agar Ground (ISP-Medium 4
．． 27C culture) on the growth of yellow tea (3pl, C1ove Brown).

White to bright tea (3fe, 5ilver Gray
~5 ih.

The soluble pigment is only brownish.

(5) Tyrosine agar medium (ISP-medium 7,27C
Culture) Light yellow to dark brown [6pnl Dk Brown ma
A bright teapot (3fe, 5i)
Aerial mycelia of lverQray ~5 fe, ashes) are attached, and the soluble pigment is brownish.

(6) Growth on nutrient agar medium (27C culture) is colorless ~
Light yellow tea (3pg, goldenBrown ~3
ng, yellow maple), no aerial mycelium was observed, and no soluble pigment was observed.

(7) Yeast/malt agar medium (ISP-medium 2°27tll' culture) Gray reddish brown (5ni, Cocoa Brown) -
Transformed i-i% brown (5pi, Deep Brown), white to bright brown (3ge, Camel)
(D) Aerial mycelia are attached, and no soluble pigment is observed.

(8) Oatmeal agar medium (ISP-Medium 3.2
7C culture) Light yellow tea to brown (4pls Dk 5pice Br
own), on the development of gray-white to light brown (5fe, a
shcs ~ 5 ih, Lead Qray:] Aerial mycelia are grown on it, and the soluble pigment is only brownish.

(9) Glycerin/nitrate agar medium (27C culture)
Growth is light brown to brown (4pl, Dk spiceB
row)-Aerial mycelia do not attach. The soluble pigment only gives a brownish tinge.

QQ Starch agar medium (27C culture) Light yellow to brown (4 pl, Dk 5pice Brown to 4 p
On the development of nl Ch (Icolate Brown), gray-white to light brown (3fe, 5ilver Qr.
ay), and the soluble pigment is only brownish.

αυ Malate lime agar medium (27C culture) Growth is colorless to light brown, no aerial mycelium attached. Soluble pigments are either not visible or have only a faint brown tinge.

03 Cellulose (27C culture) No growth was observed.

63 Gelatin puncture culture In simple gelatin medium (20C culture), colorless to pale yellowish brown growth, aerial mycelia are not attached or only slightly white, and no soluble pigments are observed.

In glucose peptone gelatin medium (27C culture), light brown to brown (5pi, Russet13rown
), and aerial mycelium does not attach. Soluble pigments are either not visible or have only a faint brown tinge.

α4 Skimmed milk (37C culture) White aerial mycelia are grown on the growth of light yellowish brown, and soluble pigments are either not visible or have only a faint brown tinge.

3. Physiological properties (11 Growth temperature range) Maltose/yeast agar (maltose 1.0%, yeast extract (Oriental) 0.4%, string agar 3.
20C, 24C, 27C using 5%, pH 6,0>
, 30C, 37R, and 50C as test results 50T
It grew at all of the temperatures except for "ll", but the optimum temperature seems to be between 27tl and 30c.

(2) Liquefaction of gelatin (15% simple gelatin, 20%
In the case of simple gelatin medium, weak liquefaction started around the third day after culture, but it hardly progressed, and the effect was moderate to moderate.
It is the weaker one.

In the case of glucose-peptone-gelatin medium, liquefaction started around 144 days after culture, and its effect was judged to be weak. (27C culture) Ice-melting properties were observed 3 to 5 days after culture, and the effect was moderate to strong.

(4) Coagulation and peptonization of skimmed milk (skimmed milk 37
r culture) Coagulation begins around the second day after culture, completes around the fourth to seventh day, and peptonization begins immediately. The effect is moderate ~
He is strong.

(5) Production of melanin-like pigment (tryptone yeast prosthesis, l5P-medium 1; peptone yeast iron agar, ISP medium 6; tyrosine agar, l5P-medium 7, both cultured at 27C). However, no melanin-like pigment formation was observed.

(6) Utilization of carbon sources (Pritno Mu Gotlieb agar medium, l5P-medium 9.27C culture) glucose,
It was determined that it grows well using L-arabinose, D-fructose, and sucrose, and probably uses D-xylose, but does not use inositol, L-rhamnose, raffinose, and D-mannitol.

(7) Dissolution of malic acid lime (malic acid lime agar 27C culture) From around the 7th day after culturing, malic acid lime around the growth is dissolved,
Its effect is moderate.

(8) Nitrate reduction reaction (peptone water containing 1.0% potassium nitrate, IMF-medium 8.27C culture) was tested several times, but was found to be negative in most cases.

To summarize the above properties, MG] 25-CF 1 strain is Streptomyces.
It belongs to the genus, and the aerial hyphae are neither whorled nor spirally formed.
The surface of the spore is smooth. Growth on various media is pale yellow to brown, aerial mycelia are white to light brown, and soluble pigments are not visible or have a brownish tinge.

Melanin-like pigment is negative. The proteolytic power is moderate, and the ice-melting ability of starch is moderate to strong. When searching for known bacterial species based on these properties, Streptomyces
ocidicus literature (]) Inter-tio
nal Journal of Systematic
Bacteriology.

Volume 22, page 372, 1972; Literature (2) Journal
al of Antibiotics, Volume 19, 19
5-199, 1966) are listed as the most closely related bacterial species.

Then actually Streptomyces fistula
A summary of the results obtained by comparing the 5P5575 strain with the MG125-CFI strain is as follows.

Note: (A) is probably +, H is probably (- As seen in the table, MG125-CFI strain and Streptomyces - According to the above-mentioned document (2), the production of melanin-like pigment by Streptomyces
e), and this difference cannot be considered problematic. Therefore, the MGI25-CFI strain is considered to be the most closely related species to Streptomyces xanthosiidicus.
Streptomyces xanthosiidicus (Strcp)
tomyces Xanthocidicus) MG
It was identified as 125-CFI.

To produce antrimycin according to the present invention, the strain is first cultivated aerobically in a medium containing nutrients available for actinomycetes. As a nutrient source, known ones that have been conventionally used for culturing actinomycetes can be used. For example, as a carbon source, glucose, galactose, maltose,
Sucrose, dextrin, starch, starch syrup (starch malt saccharide), soybean oil, etc. can be used alone or in combination. Inorganic and organic nitrogen sources include ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, peptone, meat extract, and yeast extract.

Dry yeast, corn steep liquor, soy flour, cottonseed cake, oat mill, casamino acids, bactosoitone, soluble vegetable protein, and the like can be used alone or in combination. Other salt as needed,
Inorganic salts such as magnesium sulfate, copper sulfate, iron sulfate, zinc sulfate, manganese chloride, calcium carbonate, and phosphates can be added, as well as organic substances that promote the growth of this fungus and the production of antrimycin, such as amino acids. or inorganic substances can be appropriately added.

The most suitable culture method is a liquid culture method, especially a deep agitation culture method. It is preferable to culture at a culture temperature of 25° to 30°C and at a neutral or slightly acidic pH. In liquid culture, antibiotic MG1 usually occurs after 3 to 8 days of culture.
25-CFI is produced and accumulated in the culture medium. When the production amount in the culture solution reaches the maximum, the culture is stopped, the bacterial cells are separated, and the target product is purified and isolated from the broken culture solution F.

For the purification and isolation of this substance from the culture liquid, a separation and purification method that is generally used for isolating microbial metabolites from the culture liquid is used.

Antrimycin is a substance that is soluble in water and methanol, but insoluble or hardly soluble in general organic solvents such as ethanol and acetone, and its purification is performed by a method used for purifying so-called water-soluble basic substances. That is, methods such as adsorption/desorption using activated carbon powder and porous resin, column chromatography using cation exchange resin, and ion exchange Sephadex can be used in appropriate combination.

For example, after adjusting the culture F solution to pH 6.5, it is adsorbed on an activated carbon powder column, washed with water, and then eluted using a linear concentration scent elution method using acetone to obtain a brownish brown coarse powder containing IJ mycin. .

(2) The aqueous solution is passed through a DOWEX 50W-X4 (H) column, washed with water, and eluted with ammonia water. The active fraction is collected, concentrated under reduced pressure, and then lyophilized. The obtained lyophilized product was dissolved in a small amount of water, passed through a Dawex 50W-X4 column equilibrated in advance with pyridine-acetate buffer, eluted by increasing the concentration of pyridine stepwise, and the active fraction was collected. After desalting, concentrate and freeze-dry. Further, this light brown powder was dissolved in a small amount of water, and the C
Elute by M-Sephadec gradient elution method, collect the active fraction, desalt it, and concentrate and lyophilize. Next, this powder was dissolved in a small amount of water, passed through a column of SP1-Sephadex C-25 equilibrated with citrate buffer, and then eluted with sodium chloride using the linear concentration scent elution method to determine the active fraction. gather,
After desalting, the mixture was concentrated and freeze-dried to obtain a colorless powder of antrimycin.

Next, the physicochemical properties of the antrimycin obtained as described above will be shown.

1. Appearance: Colorless powder 2. Elemental analysis value (H: C, 47,37; H, 6,77; N, 17.64; 0°27.23 Calculated value for C2[1'H47Ns Oet H20K is C, 47,79; H, 7,02; N, 17.91;
0°27, 283, melting point: clear melting point, but does not show a decomposition point yet] Gradually decomposes from 83C.

4. Specific optical rotation: ■26-74.0° (CO, 5, H2O) 5. Ultraviolet absorption spectrum: The ultraviolet absorption spectrum is shown in FIG. The ultraviolet absorption measured in an aqueous solution is λH20<B s%): 229 nm (3)
6) f al.

Extra Thick lCrn 6, Infrared Absorption Spectrum: The infrared absorption spectrum measured with potassium bromide tablets is shown in Figure 2. The maximum absorption value (wave number tTn-”) is shown in 9 figures.
55.1415.1380 (shoulder), 1340°1280
．． 3260 (shoulder), J235, 1175°1145.1
070 (shoulder), 1050, 1010°965.910,
865,820,7807, Solubility in solvents: Soluble in water, methanol, and dimethyl sulfoxide, but insoluble or poorly soluble in organic solvents such as ethanol, acetone, ethyl acetate, chloroform, and benzene.

8. Color reaction: Positive for ninhydrin, Lydon Smith, sulfuric acid, and iodine reactions, negative for Sakaguchi, Dragendorff, and Pauli reactions.

9. R, f value of thin layer chromatography Nijiri gel thin layer (KieseJgel 60 F 254).

0, 25 mm, Merck) and Avicel SF cellulose thin layer, the Rf value was determined by developing with a developing solvent system of (])]n-butanol:acetic acid:water 12:3:5:v/v%). are 0.13 and 0.
57 or (2) n-pronoquinol:pyridine:acetic acid:water (15: 10: 3: 12 v/v
%) by developing with a developing solvent system, the R and f values were 0.
73 and 0,87 are shown.

10. IH-Nuclear Magnetic Resonance Vector: Measured using tetramethylsilane in heavy water as an external reference! The H-nuclear magnetic resonance vector is shown in FIG.

Il, +3C-nuclear magnetic resonance vector: 13C measured using dioxane in heavy water (δ67.4) as an internal standard
-What is the chemical shift (δ-value) of the nuclear magnetic resonance spectrum?

] 76.7. ] 76.5. I 76.2. ]
75.0. I 72.5°] 71.2. ] 6
7.7. ．． 149.2.149.0. I 22.5°65.3 (X2), 64.1.63.2.57.9.
54.1゜53.7.50.8.50.7.48.8.
27.5.20.6°20.0. ] 8.6. I
7.5. l 7.1. ] 5.6.13.0.

12. Acid hydrolysis product: Antibiotic MG125-CFI in 6N hydrochloric acid, ]05C,
Hydrolyzed in a sealed tube for 16 hours, each amino acid (molar ratio) contained in the hydrolysis was alanine (2), serine (1
), 2-hydroxymethylserine (1), 2,3-
Diaminobutyric acid (])-.

As a result of the above properties and chemical structure studies, the following structural formula was submitted for antrimycin.

Based on the above, it was determined that the antrimycin of the present invention is a novel compound.

Next, the antibacterial spectrum of the antrimycin of the present invention was determined as follows.
Shown in Table and Table 2.

Stachylococcus aureus FDA 209P
)] 00〃 Smith strain 〉100 Micrococcus frapus FDA]6)
100 Zarutina Lutea PCI 1001
) 100 Bacillus subtilis PCI 21
9) I 00# NRRLB
-558)] 00 Escherichia coli NIHJ
) 100 Esierhia coli
K-12) 100 Shigella Decenteria JS 1191
0 :>100 Shigella flexinelli
4bJS 11811:>100 Salmonella typhi T, 36) 100
Salmonella enteritidis, Dis 189]
>100 Proteus vulgaris 0X19
)100 Proteus mirabilis IFM
0M-9) I OO Proteus rettgeri GN3]
1) 100 Serratia e marcetuscens) 100 Pseudomonas
Klebsiella pneumoniae A3) 100 Klebsiella pneumoniae PCI 602)]
OO Candida albicans 3] 47
)] 00Xanthomonas oryzai
) 100 Pilichyuralya oryzai
) 100 Aspergillus niger F-16 > 100 However, when measured in a nutrient agar medium Table 2 Mycobacterium labelculosis H37RV Strain 50 Mycobacterium labelculosis H37RV
RFPR R strain 3.1 Mycobacterium labelcrosis H37RVCPM-R strain
1 2.5 However, RFP-R: Rifampicin resistant CPM-R: Cabreomycin resistant Furthermore, medium:
Kirchner semi-solid medium supplemented with 10% calf serum Judgment = 37C5 Based on visual observation after 3 weeks of culture.

As shown in Table 1, antrimycin exhibited a growth inhibiting effect on the acid-fast bacterium Mycobacterium smegmatis ATCC607.

Furthermore, as shown in Table 2, antrimycin exhibited a growth inhibiting effect on the humanoid Mycobacterium tuberculosis H37RV strain, and a stronger growth inhibiting effect on its rifampicin-resistant and cabreomycin-resistant strains.

However, it does not show antibacterial activity against Durham-positive bacteria, Durham-negative bacteria, mold, and sterile bacteria. Antrimycin has low toxicity, with an acute toxicity value (L Dso ) of 400 m9 for mice.
7kg (iv) or more ffiL;t.

As is clear from the above results, antrimycin is promising as an antituberculous chemotherapeutic agent, particularly as a new chemotherapeutic agent for rifampicin-resistant bacteria.

Examples of the present invention will be shown below, but these are merely illustrative and do not limit the present invention in any way, and various modifications are possible.

Example 1 In a 500 ml Erlenmeyer flask, 2% galactose, 2% dextrin, 1% Bactosoitone (manufactured by Difco),
Corn steep liquor (manufactured by Ajinokisha) 0.5%, ammonium sulfate 0.2%, calcium carbonate 0.2%, silicone KM-70 (manufactured by Shin-Etsu Chemical Co., Ltd.) as an antifoaming agent, and soybean oil (Kaiho). )(
1:1) mixture 0.05%, pH 7.4, -120C%
Dispense 120 ml of medium sterilized in an autoclave for 20 minutes, and add antrimycin-producing bacteria (Feikoken Bacteria No. 60) to this.
No. 07) 1 platinum loop was inoculated and cultured for 2 days in a rotary shaker at 28 rpm at 80 rpm for 7 minutes. Transfer 3 ml of this pre-culture solution to 120 ml of the above culture medium.
The cells were transferred to a sterilized 500 ml Erlenmeyer flask, and cultured with shaking under the same conditions as described above for 6 days. The culture solution has a pH of 6.
, 5 to obtain 9.7 g of P solution. The culture titer is measured by the camp assay method using Mycobacterium 607 as the test bacterium, and the culture p solution is IU/ml.

Activated charcoal powder (Wako Tsumugi KK: for chromatography) 55
Pass through a column packed with 0 ml of antibiotic MGI25-
CFI was adsorbed and eluted using an acetone linear concentration gradient elution method consisting of 1 S00 ml each of washed water and 50% acetone water.

The active fractions were collected, concentrated under reduced pressure, and freeze-dried to obtain a brown coarse powder 21', 32'? (0.5U/1n
g) ■ was improved. Next, add this 2% aqueous solution to DOWEX 50W-X.
4 (H) Pass through a 550m column, wash with water, elute with 1N aqueous ammonia, and collect the active fraction.

Concentrate and lyophilize under reduced pressure. Obtained yellowish brown powder6.
Dissolve 081i' (1.3 U/immediately) in a small amount of water and add it in advance to a pyridine:acetate buffer (pyridine concentration 0.2 M, [
DOWEX 50W-X equilibrated at pH 4,65)
Pass through a 4600 ml column and add 1200 ml of the above buffer.
First, the same buffer solution (pyridine concentration 0.4 mol, pH 4.6
5) Elution was carried out at 1500 ml. Collect the obtained active class ■ and combine it with Downick, X 50W-X4 (H) 5
The residue was passed through a 00mlO column, washed with water, eluted with 1N aqueous ammonia, concentrated under reduced pressure, and lyophilized to obtain 739.4cm (7.4U/cm) of pale yellow powder. CM-Sephadex C-25180 was prepared by dissolving this powder in a small amount of water and equilibrating it with 0.05 mol of sodium chloride water.
0rnl column, Q, '05 mol, and 0.0rnl.
The salt was eluted using a linear concentration gradient elution method consisting of three samples of each saline solution containing 37 mol of salt, and an active classification was obtained at a salt concentration of 0.06 to 0.09 mol per mol. Collect these, Daiio” HP-40
Pass through a 365 ml column to adsorb the active substance, wash with water, and add acidic acetone water (0,002 N hydrochloric acid: acetone (1
The active fraction was collected by a desalting operation eluting with a mixed solution of 1), concentrated under reduced pressure, and lyophilized to obtain a colorless powder of 364.
7■ (10,90/■) was obtained. Next, this powder was dissolved in a small amount of water, passed through a C25800ml column in advance, and eluted using the linear concentration scent elution method using the buffer and 0.5M saline moss 2000m/+ salt. An active category was obtained at a salt concentration of 0.2 to 0.3 mol. This was collected and desalted using the same method as the desalting operation performed on the eluate from the CM-4 phadex.

A colorless powder of 247.3 square meters (12,OU/■) was obtained.

[Brief explanation of drawings]

Figure 1' shows the ultraviolet absorption spectrum of antrimycin. FIG. 2 shows the infrared absorption spectrum of antrimycin measured as a potassium bromide tablet. FIG. 3 shows the 1H-nuclear magnetic resonance spectrum of antrimycin measured with a 100 MHz device using tetramethylsilane in heavy water as an external reference. Patent applicant: Microbial Chemistry Research Foundation (Yujou) 2゜

Claims (2)

[Claims] (1) Antrimycin, a novel peptide antibiotic with the following structural formula (2) Antrimycin antibiotic-producing bacteria belonging to the genus Streptomyces is cultured, the antibiotic antrimycin is produced and accumulated, and the antibiotic antrimycin having the following structural formula is collected from this culture. A method for producing the novel peptide antibiotic antrimycin.