JPS6021000B2 - Antibiotic AM-3696A and its manufacturing method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibiotic AM-369M and a method for producing the same.

The present invention is based on the finding that certain strains of actinomycetes produce novel antibiotics. The purpose of the present invention is to develop a novel antibiotic A
An object of the present invention is to provide M-3696A and a method for producing the same.
The antibiotic provided by the present invention (named AM-369) is a white powder and has the following properties in its hydrochloride form:

01 Elemental analysis (%) C 43.3-46.6 Sun 4.45-5.09 N 4.74-5.18 0 35.4~43.4 CI 4.08-8.80 ‘2’ molecular weight It could not be measured by mass vector analysis.

Famicom molecular sieve membrane membrane filter, UM
-2 (substances with a molecular weight of 1000 or less can pass through), so the passage rate is 30%, and the same UM-05 (substances with a molecular weight of 500 or less can pass through).
The molecular weight is estimated to be in the range of 500 to 100 terms, since the passage rate is 1% for the following substances (the following substances can pass through). Also, when calculated from the elemental analysis values in '1) above, AM-
As the composition formula of 369M, C30-34 days 37-4 eggs 3
018-24 CII-2 or C20-2 is given as 24-2 eggs 2011-1 length 11-2, and the corresponding molecular weights are 810-886 or 550-595, respectively.
It is. (3' melting point 230-280 pm (begins to brown at 230 pm and 280 pm)
(turns black at 0) '4' Specific rotation [Q] 122 to 1,260 (average 124o) (
C.O. 5 water) '5) Rf value AM-369 Mochi hydrochloride was prepared using a thin layer (0
．． The following Rf values are shown in thin layer chromatography using 3 pigs).

Development solvent Rf'a} Acetone, methanol, formic acid, water (31:1:1)
0.85'b' Ethyl acetate, pyridine, water (4:3:2) 0.45'c'
n-butanol, acetic acid, water (1:1:1)
0.65'd'Ethyl acetate, pyridine, acetone, water (5:5:1:3) 0.65
{61 Ultraviolet absorption spectrum (as shown in Figure 1) Maximum absorption (blood) E Chinsui (FH4.4) 2
82-284600.01N hydrochloric acid 2
81-283540.01 Regulation caustic soda
300-30281'71 infrared absorption spectrum (KB
(as shown in Figure 2)
It does not dissolve in lower alcohols such as methanol or organic solvents such as acetone, ethyl acetate, and benzene.

'91 Color Reactions Positive for Lydon-Miss reaction, anisaldehyde-sulfuric acid reaction, and potassium permanganate-bromophe/Levreux reaction, negative for anisidine reaction, ninhydrin reaction, and Sakaguchi reaction.

00 It is a basic substance.

Next, we will show the biological properties of the AM-369 vessel.

When using the AM-369 vessel (hydrochloride) taken in the example, Hartin Fusion Urao medium (pH 7.
The minimum inhibitory concentration (MIC) at 0) is shown in Table 1 (measured by the agar dilution method). Table 1 Test Bacteria MI○ (French g/similar) Staphylococcus aureus FDA209P12.5 Staphylococcus aureus FS1277 (Penicillin resistant) 12.5 Bacillus subtilis PC1219 3.1 Bacillus cereus T 6. 3 Sarcina lutea PCIIOOI O.
8 Mycobacterium smegmatis ATCC6071
2.5/Power Ludeia Asteroides KB-49
3.1 Clostridium parfringens ATCC3
62412.5 Esierhia coli NIHU
〉100 Proteus vulgaris and 0316
7 >100 Salmonella Teifuimurium K
B-20 >100 Shigella zonnei E33
>100 Pseudomonas aeruginosa P
-3 >100 Antibiotic AM-3696 as above
A shows relatively strong antibacterial activity against Gram enterobacteria,
It is also effective against penicillin-resistant bacteria and anaerobic bacteria.

When this antibiotic was intraperitoneally administered to mice, the LD rule was 300 o/X or more. This antibiotic also has a cell wall synthesis inhibiting effect. Therefore antibiotic AM-36
It is expected that carp will be useful as a reagent for antibacterial and therapeutic agents for humans and animals. As a result of comparing the above-mentioned physicochemical properties and biological properties with those of known antibiotics, it was found that the antibiotic AM-369 Tsumugi is a new substance.

That is, similar antibiotics that are basic, water-soluble, have a weak absorption maximum around 28°C, and are effective against Gram-positive bacteria include enduracidins A, B, C, and D (Tokuko Showa). 45-171 Spot No. Publication), Vancomycin (Special Publication No. 33-18450), A-4696 (Mochiseki No. 47-20397) similar to Vancomycin
, also A-477 (Special Publication No. 49-71196), also Avoparcin (Japanese Patent Publication No. 50-135), AM-
Examples include 374 (Jikai No. 48-24752), triculamine (Hakama Kosho No. 45-9234), and amide/glycosides. However, enduracidin A,
B, C and D all have a nitrogen content of 14% or more (A
M-369 ship is 4.97%), and the ultraviolet absorption spectrum under alkaline conditions is around 250 to 26 m (4.97%).
AM-369M is different from AM-3696A because it has an absorption maximum at around 30 meters. Vancomycin is an amphoteric substance and is a polymer (1800).
-Different from 3696A.

Elemental analysis value of A-4696 (C...51.$%, H...
5.77%, N...5.46%) is different from that of AM-369M. The ultraviolet absorption spectrum of A-4696 shows a maximum absorption at 27 m under acidic conditions (AM-369 ship has 2 side dishes), and the infrared absorption spectrum shows maximum absorption at 27 m.
The absorption maximum near 20-1 is observed on the AM-369 ship, but not on the A-4696. Elemental analysis values of A-447 (C... Spot .06%, Day... 618%, N...
579%) is different from that of AM-369M. Elemental analysis value of avoparsin (C...52.36%, Day...5
．． 77%, N...5.70%) and molecular weight (A at
M-369 ships are less than 1000) are AM-369M
different from that of Elemental analysis value of AM-374 (C...
51.68%, Japanese...5.85%, N...7.02%)
and molecular weight [(1500±300)×N] is AM-3
Different from 696A. Triculamine has nitrogen content (18.42%) and molecular weight (2130) of AM-3.
It is different from that of the 69th ship. Aminoglycosides that are effective only against Gram enterobacteria include AM-369M in terms of nitrogen-containing groups (more than 10%) and negative Lydon-Smith reaction.
different from. As mentioned above, the antibiotic AM-369M is novel because it is clearly distinguished from known antibiotics. The antibiotic AM-369 vessel according to the present invention cultivates microorganisms belonging to the genus Pseudonocardia and capable of producing the antibiotic AM-369M in a medium aerobically, and deposits the antibiotic AM-369M inside and outside the bacterial cells. , produced by collecting this. The bacterial strain described in the Examples is a radiobacterial strain discovered by the present inventor and was isolated and selected from soil in Nishimurayama District, Yamagata Prefecture, and its mycological properties are as follows. 1
] Morphological properties Vegetative hyphae develop well in both synthetic and natural media, and most of them grow by penetrating the media.

This form is zigzag-like. Aerial mycelium grows abundantly on synthetic media such as glucose-beptone Aio medium and on natural turquoise, and its color is primarily white or grayish-white, but on some synthetic media it takes on a grayish-blue color. When observed under a microscope, the long spore chains (more than one spore size) are irregularly branched, and their tips are straight. Under an electron microscope, the spores are 1.1 to 3.
It has a cylindrical shape with a size of about 7×0.4rm. Also, acrobetal budding, which is seen in the process of forming so-called blastopores, is observed. The surface of the spore is smooth.
No sclerotia, sporophytes and zoospores are found. [Mouth] Properties of various tower sites (Table 2) According to the method of Evie Shearing et al. (To lnt. I used it.

The color tone is Color 1 Harmony as a standard color table. The color was determined using the 4th edition of the manual (Container 1, Corporation of America, Chicago, 195 Ikioh), and the code was written in parentheses along with the color chart name. Unless otherwise specified, the following are the observation results on each tower's ground during the second week of 270. Table 2 ISP: Media selected by the International Strebtomyces Project [m] Biological properties '11 Melanin pigment formation Tyrosine agar Negative Beptone Yeast Iron agar Negative N-glucose Beptone Gelatin・Puncture (270)
Negative A Trypsin/yeast solution Negative {2
1 Tyrosinase reaction negative {31
Hydrogen sulfide production hall Negative [4' Reduction of silver nitrate (glucose/nitrate beach)
Positive■ Hydrolysis of starch
Enteric {6} Liquefaction of gelatin (glucose-veptone-gelatin medium) Positive '71 Coagulation of skim milk (270) Positive [81 Beptonization of skim milk (270) Enteric [91 Decomposition of cellulose Negative 00 Growth temperature range ( ℃)
20-36 (11) Utilization of carbon sources (Pridham-Gottlieb agar tower) Commonly used: D-glucose, L-arapinose, D-mannitol, sucrose, i-inositol, raffinose, D
-Xylose, melibiose, salicin, fructose Some use: L-rhamnose [W] Chemical analysis of cells Composition of cell wall: Diaminopimelic acid is in the meso form, does not have glycine, and has arabinose.

Contains galactose.

Sugar composition of whole bacterial cell: Contains arabinose, galactose, lipose, and glucose.

Therefore, Rechevalle (Inter.J.System
．． Bact. The cell wall composition of this bacterium (Cellwall
type) is N type, and the bran composition of the whole bacterial body (Wholece
llsugdrpattern) becomes type A.

This bacterium belongs to the genus Mycobacterium, Nocardia, Pseudonocardia, Thermomonospora, or Micropolyspora, as the cell wall composition and sugar composition of the entire bacterial body are NA type. it is conceivable that.

However, in its morphology, the vegetative hyphae have a zigzag shape, the aerial hyphae have spores longer than 1q, and the spores are also characterized by the formation of blastopores.
Pseudonocardia (Arch. Microbio
It is reasonable to think that it belongs to 2 Tora et al., pp. 373-414, 1957). Now, the conventional box species belonging to the genus Pseudonocardia is Pseudonocardia spinoza (Inter.J.System.mother ct
．． , vol. 21, pp. 29-43, 1971), Pseudonochaldeia thermophila (Inter. J. Sys
tem. Gender ct. 21, pp. 29-43, 1971) and Pseudonocardia fastidiosa (US Pat. No. 4,031,206, 1977). However, when comparing the above-mentioned properties of this strain with literature on these known bacterial species or standard strains,
The growth temperature range of Pseudonocardia spinoza is 20 to 30 qo (this strain is 20 to 3 qo), and the spore size is 2.5 to 4.5 x 0.4 to 1.01 m. (This strain is 1.1 to 37 x 0.4 French m) It is distinguished from this strain by the fact that the surface of the spores is thorn-like (this strain is smooth). The temperature range is 28-60°C, and the spore size is 2.5 x 1.
Pseudonocardia fastidiosa is distinguished from this strain in that it does not grow on oatmeal agar and has a spore size of 9 to 10 x 1.0 m. It is mountain m, yeast malt agar tower place,
It is distinguished from this strain in that it grows in bulges on starch inorganic salt substrates, nutrient agar media, etc., and aerial mycelium does not attach, or even if it attaches, it is very sparse and white in color.
This strain was thus recognized to belong to a new bacterial species that is distinct from the three known species of the genus Pseudonocardia, and was therefore named Pseudonocardia azurea. However, this strain is Syudonochaldeia sp.
Deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under the name 3696 (
Recruitment Research Institute No. 4738). Mutant strains of the above-mentioned strains can also be used in the method of the invention.

Other strains belonging to the genus Pseudonocardia that have the ability to produce the present antibiotic can also be used. As the medium, a synthetic medium or a natural medium containing an appropriate amount of carbon source, nitrogen source, inorganic substances, and other nutrients as necessary can be used.

The carbon source and nitrogen source used in the culture medium may be any type that can be used by the strain used. That is, various carbohydrates such as glucose, glycerol, fructose, maltose, mannite, xylose, galactose, lactose, ribose, starch or its hydrolyzate can be used as the carbon source. The concentration is usually preferably 0.1% 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 methanol and ethanol, and various non-aromatic hydrocarbons such as normal paraffin, Various vegetable or animal fats and oils can also be used. Examples of nitrogen sources include ammonium salts of various inorganic or organic acids such as ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate, and ammonium nitrate, urea, beptone, NZ-amine, meat extract,
Yeast extract, dried yeast extract, corn steep liquor,
Nitrogen-containing organic substances such as casein hydrolyzate, fish meal or its digested product, soybean flour or its digested product, defatted soybean or its digested product, ant hydrolyzate, and various amino acids such as glycine, glutamic acid, and alanine are used. It is possible. As the inorganic substance, various phosphates, magnesium sulfate, common salt, etc., and also some heavy metal salts are used.

Furthermore, when using a mutant strain that exhibits auxotrophy, it is of course necessary to add substances to the medium that satisfy its nutritional needs, but this kind of nutrients should not be added especially when using a medium containing natural substances. It may not be necessary.

Fermentation is carried out under aerobic conditions such as Zhen Liao culture or aerated deep culture.

The culture temperature is usually 20-4000C. The culture period is usually 1 to 8 days, and the antibiotic AM-369 is present inside and outside the bacterial cells.
Ships generate and accumulate. Antibiotic A from the culture after completion of culture
For example, the M-369 ship is collected using the following method. The culture is separated into fermentation liquid and precipitate by centrifugation. It is extracted from the furnace liquid by adsorbing it onto activated carbon, porous synthetic resin, ion exchange resin, etc. and eluting it. The precipitate is extracted with an organic solvent such as aqueous acetone or aqueous methanol. A crude powder of AM-369M is obtained by appropriately concentrating and drying the extract. The coarse powder can be further processed by known methods commonly used in the purification of water-soluble substances, such as various chromatography methods using adsorbents such as ion exchange resin and silica gel, or gel filters, concentration methods, salting-out methods, etc. It is purified by appropriately combining. One example is a column chromatography method using carboxymethyl cellulose (Nichiju type) and a linear concentration gradient melting method using an ammonium formate solution as an elution solution. Powder of antibiotic AM-369M can be obtained by concentrating and drying the active fraction obtained by these purification methods. In the present invention, the detection and quantification of AM-3696 is performed using microorganisms that are susceptible to AM-369 (e.g., Staphylococcus aureus FDA20, Bacillus spusi IJS PC1219, or Sarcina lutea PCI).
A biological method using IOOI) as a target bacterium or a chemical method using a color reaction were used, and these types of methods are well known.

Example Pseudo/Cardia Sp. One platinum loop from a slant culture of AM-3696 strain (NRRLI1412) was inoculated with 500' of seed stock (glycerin 2.0%,
2 containing 2.0% soy flour, 0.3% salt, pH 7.0)
The customer's Erlenmeyer flask was inoculated and cultured on a shaker at 270℃ for 2 days to obtain a seed culture.

This seed culture was added to the fermentation tower site [Dexrin (2.0
%), soy flour (1.0%), yeast extract (0.3%),
KH2P04 (0.1%), K2HP04 (0.1%)
, Mtaka S04.7 is 0 (0.1%); pH 7.0] was inoculated to 2% in a 200 tank fermenter.
270 and 4 self-aerated rope culture (aeration rate 50 evenings/min,
Key Azusa 20 enemies. p. m. ) was carried out. 180% supernatant liquid obtained by centrifuging the culture solution to remove bacterial bodies and other precipitates;
90 pieces of activated carbon, which had been degassed in advance, was added and lit for 30 minutes. Thereafter, the activated carbon was separated in a furnace, washed with water, and eluted with 60% acetone containing 0.01N sulfuric acid, and the active fractions of the eluate were combined and concentrated. Neutralize the concentrate with barium hydroxide,
The generated precipitate was poured into a furnace. The furnace solution was passed through a column (
The column was washed with water, eluted with 0.1N hydrochloric acid, and the active fractions were combined and neutralized with caustic soda. Pass the neutralized solution through an activated carbon column (1800'),
After washing with water, it was eluted with 60% acetone containing 0.01N hydrochloric acid, and the active fractions were combined and concentrated. Weakly basic anion exchange resin Amberlite IR-45 (OH-type) is added to the concentrated solution to neutralize it, and after the resin is filtered out, the furnace solution is freeze-dried to produce AM.
- Two females of coarse powder with 369 needles were obtained. 10 pieces of this coarse powder
Column (300ml) dissolved in water and packed with activated carbon
After washing the column with water, add 10% pyridine solution 6
00, 0.1N hydrochloric acid, 600, and 0.01N hydrochloric acid, 200, were sequentially passed through the column, and in each case, the liquid that passed through the column was discarded.

Next, it was eluted with a 50% acetone solution containing 0.01N hydrochloric acid, and the active fractions were combined and concentrated.
After neutralization by adding 5 (OH-type), the resin was separated by furnace. Cellulose powder Avicel (trade name) was added to the furnace solution, and the mixture was dried to dryness. Apicel (40 g) was suspended in n-butanol and packed into a column, and the above-mentioned Tsuru Avicel suspended in n-butanol was loaded onto the top of the column. This column (11
4 of the fractions (1.88 cc each) were eluted with an eluent consisting of n-butano, acetic acid, and water (5:1:2).
Nos. 1 to 60 were collected and concentrated, and the concentrated solution was neutralized with caustic soda. After neutralization, the column was passed through an activated carbon column (20'), washed with water, eluted with a 50% acetone solution containing 0.01 N hydrochloric acid, and the active fraction was collected and concentrated.
After neutralization with (OH-type), the resin was separated in a furnace, and the furnace liquid was freeze-dried to obtain 150 samples of AM-3696A powder (hydrochloride). Dissolve this in 5M water and phosphorize with 0.5N caustic soda.
Adjusted to H7.5, weakly acidic cation-exchanged cellulose carboxymethylcellulose (manufactured by Whatman) (Nichiju type)
After washing the column with water, water (750 [)] and 0.5 molar ammonium formate (7
It was eluted by linear gradient elution with an elution solution consisting of 50') and 147 to 17 fleas of each fraction (7') were collected. Pass the collected active fraction through an activated carbon column (10')*
After washing with water, it was eluted with a 50% acetone solution containing 0.01N hydrochloric acid, the active fraction was collected and concentrated, and after neutralization with Amberlite IR-45 (OH-type), the resin was separated in a furnace. Freeze-dry the furnace liquid to obtain AM-3696A powder (hydrochloride) 41
I got the o. The properties of this product were as described from page 4, line 1 to page 7, line 1 of this specification.

[Brief explanation of drawings]

Figure 1 shows the antibiotic AM-369 secret purified powder (hydrochloride)
Figure 2 shows the infrared absorption spectrum (KB subtracted). Fins 1, 2, 2

Claims (1)

[Claims] 1. Antibiotic AM-36, which is a white powder in the form of hydrochloride and is characterized by having the following physical and chemical properties:
96A. (a) Elemental analysis C 43.5% H 4.46% N 4.97% (b) Melting point 230-280°C (c) Specific optical rotation [α]^2^8_D-122--1
26° (average -124°) (c0.5, water) (d) Ultraviolet absorption spectrum As shown in Figure 1 (E) Infrared absorption spectrum As shown in Figure 2 (F) Rf value Silica gel G manufactured by Merck & Co., West Germany (Art 7731) ) shows the following Rf values in chromatography using a thin layer (0.3 mm) prepared using: Developing solvent Rf Acetone/methanol/formic acid/water (3:1:1:1) 0.85 Ethyl acetate/pyridine/water (4:3:2) 0.45 n-butanol/acetic acid/water (1:1: 1) 0.65 Ethyl acetate/pyridine/acetone/water (5:5:1:3) 0.65 (g) Solubility in solvents Soluble in water, sparingly soluble in dimethyl sulfoxide,
It does not dissolve in lower alcohols such as methanol or organic solvents such as acetone, ethyl acetate, and benzene. (H) Color reaction Positive for Lydon-Smith reaction, anisaldehyde-sulfuric acid reaction, and potassium permanganate-bromophenol blue reaction, and negative for anisidine reaction, ninhydrin reaction, and Sakaguchi reaction. (li) Others It is a basic substance. 2. Belongs to the genus Pseudonochaldeia and is an antibiotic AM-
Production of antibiotic AM-3696A, which comprises aerobically cultivating a microorganism capable of producing 3696A in a medium, thereby accumulating the antibiotic AM-3696A inside and outside the bacterial body, and recovering the accumulated antibiotic AM-3696A. Method.