JPS5810392B2 - OA↓-7653 substance - Google Patents

Description

[Detailed description of the invention] The present invention relates to novel antibiotics.

The substance of the present invention is characterized in that it has the following physical and chemical properties.

(1) Appearance and properties white powder crystal (2) Solubility (3) Specific rotation (4) Elemental analysis values (5) Isoelectric point by electrophoresis method pH5-6 The measurement conditions are as follows.

That is, in advance 6.008 g of citric acid, KH2PO43,893S', H3PO41,769r in distilled water of 11.
and 5.266f of 5,5-diethylbarbic acid were dissolved, and the pH was adjusted to 3.4. with a 0.2N aqueous sodium hydroxide solution.
5. The inventive substance was electrophoresed at 300 μm for 4 hours using the buffer solutions prepared in 6.7 and 8 respectively and Whatman No. 1 filter paper (manufactured by Whatman) on which the inventive substance was spotted after being dissolved in water. Obtain the analysis diagram.

The results are shown as curve 1 in FIG. 3, which shows that the isoelectric point exists at pH 5 to 6.

(6) Infrared absorption spectrum analysis (IR) An IR analysis diagram of the KBr tablet is shown in FIG.

From Figure 4, the main absorption peaks are 3280 (S), 16
60(S), 1640(S), 1515(S), 1490(S), 1395(S), 1235(S), 1150(m), 1062(S) and 10201020(S).

(7) Ultraviolet absorption spectrum analysis (UV) (t)o,
Using IN aqueous hydrochloric acid as a solvent, 1 mg/1 of the substance of the present invention
The results of UV analysis of 0rul in one cell length box are shown in Figure 5.

From the figure, 278 (11) Distilled water is used as a solvent, and the above (1)
The results of UV analysis in the same manner as in Figure 6 are as shown in Figure 6.
(1) and income are recognized.

(ii) The results of a detailed analysis of the substance of the present invention, 0.5In9/1O1rLl, with a cell length of 1cIrL using an aqueous solution of o, IN sodium hydroxide as a solvent are shown in FIG.

is recognized.

(8) Thin layer chromatography analysis (TLC) The substance of the present invention was dissolved in water, and commercially available from Merck & Co., Ltd. [Silica gel 60F2]
The results of TLC analysis using a 54 J thin plate are as follows.

(1) Developing solvent: Butanol-acetic acid-water (ii) Developing solvent nibrobanol-2N ammonia (iii) Developing solvent: chloroform-ethanol-
Water (IV) Developing solvent: Ethanol-Water (9) Paper chromatography analysis (PC) The substance of the present invention was dissolved in water and PC analysis was performed using Whatman paper chromatography paper Whatman/161.The results are as follows. That's right.

(1) Developing solvent: butanol-acetic acid-water (11) Developing solvent: butanol-pyridine-water α0) The substance of the present invention was dissolved in water, and the TLC in the above TLC analysis
The results of the color reaction carried out on the plate are as follows.

According to the results of amino acid analysis and gas chromatography analysis, the substance of the present invention contains 0.58 μmol of glutamic acid and 1 drop of glutamic acid as one of its constituent components.
It was confirmed that 0.64 μmol was contained in yv.

The substance of the invention is also characterized in that it has antibacterial activity.

Its antibacterial activity against various bacteria is determined by determining the minimum inhibitory concentration (MIC) using the agar dilution plate method [medium Nihard Infusion (manufactured by Eiken Chemical Co., Ltd., incubation: 37°C, 18 hours]).

The determined results are shown in Table 1 below.

Furthermore, the acute toxicity of the substance of the present invention was investigated by intravenous administration using DD male mice weighing 20 to 217 mm, and the results showed that
No deaths were observed at a dose of 1500 mg/kg, and autopsy of all surviving cases revealed no significant effects on any organs.

Taking together the above-mentioned physicochemical properties, pharmacological test results, and the origin of the compound of the present invention described below, the compound of the present invention is a compound of the known antibiotic Vancomycin (Vancomycin).
CSionogis Ann, Rep, vol. 7, p. 465 (1957), Antibiotics A plate, 1957-1958. p. 906 (1958), Jo
Med, Chem, Vol. 8, p. 18 (1965), An
tibiotics Ann, 1955-1956, p. 606 (1956).

However, as mentioned below, vancomycin is used against Streptomyces orientalis (Streptomyces orientalis).
), as shown in the above-mentioned literatures, the ninhydrin color reaction and Ehritzch color reaction are negative, and the electrophoretic analysis of vancomycin hydrochloride has a pH of 7 to 7, as shown in curve 2 in Figure 3. As a result of TLC analysis using the developing solvent described in item (8) (il) to 0■, vancomycin hydrochloride has an isoelectric point at the spot shown as A in Figures 8 to 10. (In each figure, B indicates the spot when the substance of the present invention was dissolved in water and developed at the same time as above) and an acute toxicity test was conducted by intravenous administration using DD male mice weighing 20 to 21 g. The result was LD5o-400~500rn9/kg
It is clearly distinguished from the substance of the present invention.

Moreover, vancomycin is J, Me4. Chem, Vol. 8, p. 18 (1965), it has aspartic acid as a component, but it does not have glutamic acid, and from this point of view, the substance of the present invention is different from vancomycin. It was identified as a compound.

Furthermore, the present inventors compared the present invention substance with various known antibiotics that are derived from microorganisms, particularly microorganisms belonging to the genus Streptomyces, similar to the present invention substance. No antibiotics have been found that are considered to be similar.

Therefore, the substance 0A-7653 of the present invention was identified as a novel compound.

The novel substance 0A-7653 of the present invention is derived from Strepto-myces hygroscopicus (5trepto-myces hygroscopicus).
icus).

The above-mentioned bacterial strain was isolated from the soil of Hiwasa-cho, Ama-gun, Tokushima Prefecture by the present inventors, and is identified as a new strain having the following characteristics.

■ Morphology This strain was grown on a glycerin-Asuno Ragin agar medium for 28 hours.
Figure 1 shows a micrograph (magnification: 800x) of the case of culturing at 28°C for 14 days on oatmil agar medium.
FIG. 2 shows a micrograph (culture ratio: 9000 times) obtained after culturing for 4 days.

It can be seen from the 2nd grade figures that the spore-forming hyphae of this strain simply branch from the long main axis formed in the aerial hyphae, forming a spiral shape with 2 to 3 turns at the end.

In rare cases, it does not form a long main axis and has irregular and complex branches.

In addition, observation using a scanning electron microscope revealed that the spore surface structure was smooth and the spores were cylindrical to oblong.

The size of the spores is approximately 0.7-1. OXo, 7-1.6 microns, and the spores are usually in chains of 15-40.

■ Culture findings (1) Yeast/malt agar medium (b) Good growth.

Very wrinkled. (b) Aerial mycelium is usually powdery, covered tan (cover tan, 2 ge) ~
White.

(c) The back side of the subsoil hyphae is light wheat.
wheat 12ea).

) Soluble pigment is No Ni Gold (honey gold 12 ic).

(2) Oatmeal agar medium (b) Normal growth (b) Aerial mycelia are abundant, powdery, and beige. (beige, 3ge).

After that, many black wet areas appear and spread throughout the area.

(/J The back side of the soil hyphae is light beige.
t beige, 3 ec).

2) Soluble pigment is honey gold (2 ie) (3) Inorganic salt/starch agar medium (() Good growth.

(b) Aerial mycelium is abundant, powdery, covered gray (c
overt gray, 2fe).

After that, many black wet areas appear and spread throughout the area.

(/9 The back side of the soil hyphae is dull gold (dull gold)
gold, 2 ng).

) Soluble pigments are light citron gray (light
t citron gray, 1 ec).

(4) Chrycerin-asparagine agar medium (a) Good growth.

(b) Aerial mycelia are abundant, powdery, and beige. (beige, 3 ge).

After that, many black wet areas appear and spread throughout the area.

(c) The back side of the basal hyphae is gold (gold, 13Alc).

) Soluble pigment is veil yellow (pale yellow, 1 ca).

(5) Peptone yeast iron agar medium (b) Normal growth.

Slightly wrinkled. (b) Aerial hyphae are not produced.

e→ The back side of the basal hyphae is cream. (cream, 13Aca).

) No soluble pigments are produced.

(6) Tyrosine agar medium (B) Good growth.

Slightly wrinkled. (Snout Aerial hyphae abundant, powdery, covered gray (covert gray, 2 fe)
').

A black wet area then appears and spreads throughout.

(c) The back side of the basal hyphae is gold (in) Soluble pigment is no gold (honey gold, 2 ic).

(7) Sucrose/nitrate agar medium (b) Good growth.

flat. (b) Aerial hyphae are not produced.

(/J The underside of the soil hyphae is citron yellow (citron yellow)
ron yellow, 11c).

(in) Soluble pigment is canary yellow (canary yellow ea).

(8) Glucose-asparagine agar medium mark Growth is normal.

(Naka) Aerial mycelia are abundant, powdery, and covered with gray 15- (
cavert gray, 2fe).

After that, a blackish-brownish green area appears from the periphery and spreads throughout the area.

e→ The back side of the soil hyphae is gold (gold, 13Alc).

) Soluble pigments are cream (cream, 1312 c
a).

(9) Nutrient agar medium ((1) Normal growth.

(O) Aerial hyphae are not produced.

(/→ The back side of the soil hyphae is yellow tint 2! (y
yellow tint, 1 ba).

) No soluble pigments are produced.

(II Calcium maleate agar medium (a) Growth is normal.

flat. (b) Aerial mycelium is not produced.

(c) The underside of the basal hyphae is canary yellow (canary yellow).
ry yellow, 1 ea).

(in) No soluble pigments are produced.

Uυ Bennett's agar medium (b) Good growth.

It wrinkles. (b) Aerial mycelium is abundant, powdery, silver
Gray (5ilver gray, 3 fe).

A black wet area then appears and spreads throughout.

(／j The back side of the soil hyphae is mustard (mustard, 21e).

) Soluble pigment is light antique gold (l
light antique gold11%ic).

All of the above-mentioned cultures were carried out at 28°C for 21 days, and the colors shown are as follows: Co1or Harmony Manual
Container Corporation of America (C
ontainer Corporation of Am
erica)].

■ Physiological properties (1) Optimal growth temperature 2'8 to 30℃ (will not grow below 10℃ and above 45℃) (2) Optimal growth pH 6.5 to 8.5 (pH 4.0 and below and pH 11 (3) Liquefaction of gelatin negative (4) Hydrolysis of starch positive (5) Coagulation of skim milk negative (6) Peptonization of skim milk positive (acidic) (7) Nitrate reducing action negative (8) Production of melanin-like pigment Negative (9) Salt tolerance Growth at 7%, no growth at 10% (10) Carbon source utilization ■ Cell wall type Cell wall types according to the classification by Lechevalier et al. (LL-diaminopimelic acid).

The results of searching for the taxonomic position of this strain based on the above mycological properties are as follows.

Tresner et al. CH, D, Tresner and E.
, J.

Backue, Applied Microbiol
4.243-250 (1956)], this strain has three basic characteristics for belonging to Streptomyces bigroscopicus. Branches as side branches, with 2 terminal branches
The aerial hyphae form a spiral shape with more than one roll, and the mature color of the aerial hyphae is brownish-gray.
) and distinct black wetness (11y
groscopic) state.

This strain was also identified as belonging to Streptomyces bigroscopicus by searching the various literatures listed below.

0 Bar' Seeds Manual of Determinative Bacteriology 8th Edition (1974), 0 Waksmann, The Actinomycetes (The Actinomycetes)
Actinomycetes) Volume 2 (1961)
, International Journal of Systematic Bacteriology
Systematic Bacteriology)
, Vol. 18, pp. 69-189 (1968), No. 1
Vol. 8, pp. 279-392 (1968), Vol. 19, pp. 391-512 (1969), and Vol. 22, No. 26
pp. 5-394 (1972).

Therefore, this bacterial strain is a representative strain of Streptomyces.
Vigroscopicus (Janssen) Waxman and Henrich CStreptomyces hygroscopicus
us (Jansen), Waksman and
Henrici (1948) and the ISP standard strain Streptomyces vigroscopicus 15P557.
8, the results shown in Table 2 below were obtained.

In the table, + indicates positive or used, and - indicates negative or not used.

From the table above, this strain is clearly distinguished from Streptomyces vigroscopicus (Janssen) in terms of gelatin liquefaction.

This strain also differs in that it does not have the ability to attach aerial hyphae on sucrose/nitrate agar medium and nutrient agar medium, whereas the above-mentioned Janssen strain is capable of attaching aerial hyphae.

Furthermore, when this strain is compared with the ISP standard strain, it is clearly different in the state of the spore surface and the availability of carbon sources.

As mentioned above, since this strain is different from the above-mentioned known strains of Bygroscopicus species, we also conducted a comparative study with the four subspecies of Bygroscopicus species described in the above-mentioned Verses (8th edition). .

The results are shown in Table 3 below.

The physical and chemical properties of each subspecies are based on the following literature.

Species extension 1: Streptomyces hygroscopicus subspecies Angustomyceticus (S t , hygroscopicus
5ubsp.

angustmyceticus ) The Journal of Uneyebiotics Series A (The Journal of
+< Antibiotics, Ser, A)
Vol. 7, pp. 116-119 (1954).

Subspecies 2: Streptomyces hygroscopicus subspecies decoicus (St, hygroscopicus 5ubsp)
.

decoyicus ) Antibiotics and Camotherapy (A
Antibiotics and Chemothera
py) Vol. 9, pp. 427-431 (1957).

Subspecies 3: Streptomyces bygroscopicus subspecies Glebosus (St.

hygroscopicus 5ubsp, gleb
osus) The Journal of Antibiotics Series A, Volume 15, pp. 21-27 (19
62).

Subspecies 4: Streptomyces hygroscopicus subspecies hygroscopicus (St, hygroscopicus 5ubs)
p.

ossamyceticus) The Journal of Antibiotics Series A, Vol. 18, pp. 82-88 (1965).

Note that + and - in the table have the same meanings as in Table 2 above.

As shown in Table 3 above, this strain matches subspecies 4 in terms of carbon source utilization, but is clearly differentiated in the production of melanin-like pigments, peptonization of resin pores, and nitrate reducing ability. It is clearly distinguished from Nos. 1 to 3 in terms of the availability of carbon sources.

From the above, it is clear that although this strain belongs to Streptomyces bigroscopicus, it is distinct from any known species or subspecies.

Moreover, there are no examples in which the antibiotic according to the present invention has been isolated from any known species or subspecies of bacteria.

Therefore, the present inventors recognized this strain as a new strain, and identified it as Streptomyces vigroscopicus subsp. hiwasaensis F B-5 (St, hygrosc.
opicus 5ubsp.

hi wasa-ensis FB-5), and applied for consignment of microorganism storage to the Institute of Microbial Technology, Agency of Industrial Science and Technology.

This is FERM-P A4 No. 4573 (FERM-P A4).
573).

Streptomyces orientalis, a strain producing vancomycin that was considered to be most similar to the antibiotic substance 0A-7653 of the present invention, was
Journal of Systematic Bacteriology, Vol. 18, p. 154 (1968), and Diactinomycetes, Vol. 2, p. 254 (1961) by Waxman.
As described in 2010), the aerial hyphae do not take a spiral shape, and the color of the aerial hyphae is white on yeast/malt agar and glycerol/asparagine agar, and on inorganic salt/starch agar. The microorganisms used in the present invention are of a white or gray type, and the presence of aerial mycelia on oatmeal agar media is slight. be.

The antibiotic 0A-7653 of the present invention is isolated and obtained from the culture of Streptomyces bigroscopicus subsp. Hiwasaensis FB-5 by culturing it in a medium.

The above-mentioned bacterial strain is cultured by conventional liquid culture or solid culture.

Preferably, shaking culture or deep aeration agitation culture in a liquid medium can be employed.

The medium composition may be one commonly used for culturing ordinary actinomycetes.

Examples of the carbon source include starch, glucose, and glycerin, and examples of the nitrogen source include soybean flour, peptone, meat extract, cottonseed flour, ammonium sulfate, and common inorganic salts such as sodium nitrate.

Furthermore, the culture medium may contain salt, calcium carbonate,
Inorganic salts such as phosphates and magnesium sulfate can be added as appropriate.

During culture, it is preferable that the medium pH is 6.5 to 7.5 and the culture temperature is 28 to 30°C. In liquid culture, the amount of production and accumulation of the substance of the present invention reaches its maximum approximately 72 hours after the start of culture. becomes.

The substance of the present invention produced and accumulated in the culture solution can be collected by conventional methods that utilize the physical and chemical properties of the substance.

For example, ion exchange resin, silica gel, Sephadex (
Seikagaku Kogyo Co., Ltd. product), activated carbon, etc., a method that utilizes the difference in numerical affinity, a difference in the distribution rate between two liquid phases, or a combination thereof.

More specifically, the culture solution obtained above is filtered or centrifuged to remove bacterial cells, and the resulting supernatant is subjected to activated carbon column chromatography, DOWEX 50WX 4 (
Dowex 50WX 4) column chromatography, ecteora cellulose column chromatography,
The active fraction may be collected and the solvent may be distilled off by sequential elution with DOWEX 50W column chromatography and silica gel column chromatography.

In this way, the antibiotic 0A-7653 substance of the present invention can be isolated and obtained.

Examples will be given below to explain the present invention in more detail.

Example 1 Streptomyces bygroscopicus subsp. Hiwasaensis F B-5 strain, starch 1
%, glucose 0.5%, glycerin 0.5%, soybean flour 0.5%, meat extract [Wako Meat Extract (Nagasu Whale + Bonito Meat) manufactured by Wako Corporation] 0.3%, polypeptone (manufactured by Otama Nutritional Chemicals) ) 0.3%, salt 0.5%, calcium carbonate 0.3
% and distilled water was inoculated into a pH 7 liquid medium consisting of 28%
Culture at ℃ for 48 hours and use as seed culture.

Next, in a 301-volume stainless steel culture tank, the seed culture was inoculated at a rate of 1% into a medium 201 having the same composition as the seed culture, and cultured with aeration at 28° C. for 72 hours.

The ventilation rate is 201/min, and the number of rotations of the bellows is 300/min.

After completion of the culture, the culture solution is centrifuged to remove bacterial cells, and the filtrate 181 is adsorbed onto an activated carbon column, washed with water, and eluted with 1.5 J of 2N ammonia aqueous solution:methanol-1:1 (V/V).

Ammonia and methanol in the eluate were distilled off under reduced pressure, and the remaining solution was adjusted to pH=3 with a 2N aqueous hydrochloric acid solution, and then adsorbed onto DOWEX 50WX4 (Na-do type).

After washing with water, add 0.1M phosphate buffer (pH=7.0) 1
1 and 0.1 molar carbonate buffer (pH=10.0
Gradient elution is carried out with a solution of 1 mol of NaCl in ) 1 ml.

The antibacterial active fraction of the eluate was collected and diluted 2 times with distilled water.
After adjusting the pH to 3 with an aqueous N-hydrochloric acid solution, apply DOWEX 5.
Adsorb to 0WX 4 (NH, 10 type).

After washing with water, elution is carried out with 500 ml of a 0.1 molar ammonia aqueous solution.

The antibacterial active fraction of the eluate was collected, ammonia was removed under reduced pressure, and the remaining solution was diluted with IN aqueous sodium hydroxide solution to pH=8.
．． After adjusting to 0, it is adsorbed on Ecteora cellulose (C1-type) (manufactured by Whatman).

After washing with water, distilled water 11 and 1 molar saline solution g! i. Perform gradient end elution at step 11.

The antibacterial active fraction of the eluate was collected and purified with 0.2N hydrochloric acid aqueous solution.
After adjusting H=3, use DOWEX 50WX4 (NH
, 10 types).

After washing with water 0. After elution with 50,077 liters of I N ammonia aqueous solution, the antibacterial active fractions were collected, the solvent was concentrated under reduced pressure, and silica gel [Wakogel C200 (manufactured by Wako Co., Ltd.) 13
．． (Add l and dry under reduced pressure.

Next, the suspension was suspended in 29 ml of 90% ethanol aqueous solution, placed on a silica gel column (Wako Gel C200), washed with 100 ml of 90% ethanol aqueous solution, and eluted with 500 ml of 70% ethanol aqueous solution.

The antibacterial active fraction of the eluate is collected, concentrated under reduced pressure, and then lyophilized to obtain 1 g of the present substance 0A-7653 in the form of white powder crystals.

The physicochemical properties of the obtained 0A-7653 substance are as described above.

[Brief explanation of drawings]

Figures 1 and 2 are micrographs of Streptomyces bigroscopicus subsp. Figure 4 is an IR analysis diagram of vancomycin, Figure 4 is an IR analysis diagram of the substance of the present invention, Figures 5 to 7 are UV analysis diagrams of the substance of the invention, and Figures 8 to 10 are diagrams of the substance of the invention and A TLC analysis diagram of vancomycin hydrochloride is shown below.

Claims (1)

[Claims] 1 (a) 0. Slightly soluble in IN aqueous hydrochloric acid, insoluble in methanol, ethanol, butanol, acetone and ethyl acetate, 0. Soluble in IN sodium hydroxide aqueous solution, (b) Positive for iodine color reaction, ninhydrin color reaction, Molisch color reaction, and Ehrlich color reaction, showing 0.219, water), (d) Element The measured values of hydrogen, carbon and nitrogen in the analysis showed C46, 10%, H4, 47% and H7, 18%, (e) the isoelectric point by electrophoresis exists at pH 5-6, (f) KBr In the infrared absorption spectrum analysis of tablets, 3280(S), 1660(S), 1640(S)
), 1515(S), 1490(S), 1395(S), 1235(S), 1150(m), 1062(S) and 10201020(S), (g) Ultraviolet absorption spectrum analysis , 0.1 N
278 mμ using an aqueous hydrochloric acid solution as a solvent, 278 mμ using distilled water as a solvent, and 0. (h) In silica gel thin layer chromatography analysis, Rf = 0 using butanol-acetic acid-water (volume ratio 4:1:1) as a developing solvent. , Rf = 0 using a propano-2N ammonia aqueous solution (volume ratio 7:3) as a developing solvent, Rf = 0.45 and ethanol-water (volume ratio 4:2) as a developing solvent. ratio 7:3) was used as the developing solvent, Rf=0.8 was shown, and (i
) In paper chromatography analysis, Rf = 0.83 using butanol-acetic acid-water (volume ratio 4:3 near) as a developing solvent and Rf = 0 using butanol-pyridine-water (volume ratio 4:3 near) as a developing solvent. .76 is 0A-7653, which is characterized by having glutamic acid and glucose as one of its constituent components.
material.