JPS60244292A - Antibiotic tan-585a and tan-585b, preparation thereof and new species of genus flexibacter - Google Patents

Abstract

PURPOSE:To prepare a novel and useful antibiotic, by cultivating a microorganism of the genus Flexibacter, and collecting the aimed antibiotic exhibiting an antimicrobial activity against Gram-negative bacteria. CONSTITUTION:A microorganism, e.g. Flexibacter alginoliquefaciens YK-49 strain, belonging to the genus Flexibacter, and capable of producing antibiotic TAN-585A and TAN-585B is cultivated in a culture medium to produce and accumulate the aimed antibiotic TAN-585A and (or) TAN-585B in the culture. The resultant antibiotic is then collected. Flexibacter alginoliquefaciens is of the undecomposing type in the oxidative fermentative test and has the ability to decompose alginic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to antibiotics TAN-585A and 'I'AN-585B which exhibit antibacterial activity mainly against Gram-negative bacteria.

BACKGROUND OF THE INVENTION Known antibiotics that are believed to have properties relatively similar to the antibiotic of the present invention include The Journal of Antibiotics.
biotics)+, XXIX, 492-500
(1976).

Problems to be Solved by the Invention The present invention aims to provide a new and useful antibiotic.

Means for Solving the Problems The present inventors isolated a large number of microorganisms from soil, and found that certain microorganisms produced novel antibiotics, and that the microorganisms belonged to the genus Flexibacter. By culturing these microorganisms in an appropriate medium, it was discovered that two types of antibiotics that exhibit antibacterial activity mainly against Gram-negative bacteria were accumulated in the medium, and these antibiotics were isolated and analyzed using physical chemistry. We confirmed that these antibiotics are new antibiotics based on their clinical and biological properties, and we confirmed that these antibiotics are new antibiotics.
I decided to call it B. The present inventors completed the present invention as a result of further research based on these findings.

That is, the present invention provides 1) antibiotic TAN-585A
, Thx-585B, or a salt thereof, 2) Antibiotics belonging to the genus Flexibacter'[' Al-585A and/or B-producing bacteria are cultured in a medium, and the antibiotic TAH-585A and/or ) An antibiotic TA characterized by producing and accumulating B and collecting it.
Method for producing N-585A and/or B, and 3
) Mentatitivist is a non-degradable type, and Frekinbacter aginorikefu 7siens has the ability to decompose 1 μg of acid.

In addition, in the present specification, antibiotic TAN-585A
and TAN-585B simply r TAN-58
Sometimes referred to collectively as 5J.

The antibiotic TAN-585-producing microorganism used in the present invention may be any microorganism that belongs to the genus Flexibacter and has the ability to produce the antibiotic TIN-585t.

Examples of antibiotic TAN-585-producing bacteria include Flexibacter 1μginorikefu 1siens YK, which was collected by the present inventors from the soil of Yoshinoyama, Yoshino District, Nara Prefecture.
-49 strain (hereinafter sometimes abbreviated as rYK-49 strain).

The mycological properties of the YK-49 strain are as follows.

(a) Morphology When observed after two days of culture on a broth agar slant at 24°C, the cells show a rod-like shape with a diameter of 0.4 to 0.7 μm, a normal length of -10 μm, but in rare cases a length of 50 to 70 μm. It may also be filamentous.

It has no flagella. It has motility due to briding. It does not form spores or microcysts, Gram staining is negative, and it does not show acid fasting properties.

Can) Growth conditions on various media The cells were cultured at 24°C and observed for 1 to 14 days.

■Juice agar plate culture: Forms circular, convex, or golden-edged colonies. The surface is smooth, translucent, and has an autochromatic to cream color. No diffusible dye is produced.

■Juice agar slant culture: Shows good spread-like growth and a glossy light brown color.

■ Broth liquid culture: Grows in a turbid state, producing a small amount of sediment,
Forms a weak bacterial ring.

■Meat juice gelatin puncture culture: Growth is weak.

■ Lidomus microri: Peptonization is false positive, white turbidity is observed at the bottom, and decolorization is observed, but acid production and coagulation are not observed.

(c) Physiological properties ■ Nitrate reduction: negative ■ Denitrification reaction: negative ■ MR (methyl red) test: negative ■ VP (Voges-Proskauer lv) test: negative ■ Formation of indo μ: negative ■ Formation of hydrogen sulfide : Negative ■ Starch hydrolysis: Positive ■ Utilization of citric acid: Positive ■ Utilization of inorganic nitrogen sources I) Potassium nitrate: Negative i) Ammonium sulfate: False positive [Phase] Production of color 8 (King A, King B and Mannitol yeast extract Agar media): Does not produce soluble pigments.

■Urease: Negative■Oxidase: Positive [Phase]Catafuse: Positive ■Growth Range+) PH: Grows at pHi+, 9 to 8.5, but the optimum pH is 5.4 to 6.6゜i) Temperature: 6 Grows at ~35°C, but the optimum temperature is 14~
31℃.

[Phase] Attitude towards oxygen: Aerobic [Phase] 0-F (Oxidatipouf amentatip)
Test [Hugh-Leifso
n) Method]: Generation of acids and gases from non-dispersed sugars: Acid Gas Availability (Peptone-Water) (De7 medium) L-arabinose ± -+ D-xylose - Ten-gum course −+ D-mannose + −+ D-fructose −−+ D-gafuctose − −+ Maltose −−+ Silo sugar −−+ Lactose −−+ Trehalone −+ D−So, Lepit − − - D-Mannitol - + Innoquat - - Glycerin - - + Starch - + [Phase] Decomposition of polysaccharides: agar, cellulose, cellulose, and coloie and chitin. Decomposes alginic acid.

[Phase] 096 Salt broth liquid culture: Growth.

@596 Salt broth liquid culture: No growth.

Percy's Manual!・Og Determinant Bacteriology (Bergey's Manual of Dete
rminative Bact, eriology)
8th Edition and Interna VM Sumu Journal Op.
Systematic Bacteriology (Internat)
ional,Tournal of 8y'Btema
tic Bacteriology) Volume 30, 215~
420 (1980), Vol. 32, pp. 146-149 (1982), it is a rod-shaped or filament-shaped Gram-negative bacterium that does not have the ability to form microcysts and exhibits motility due to briding. Agar, seven! Loin, power ~ boxymethipse/L/p-su and colloida!・
Since it does not decompose any chitin, does not require salt, and does not exhibit resistance to salt, it is appropriate to classify it as belonging to the genus Requipacter. Therefore, when we compared the YK-49 strain with known species of the genus Flexibacter, we found the following properties of the YK-49 strain: 1) Oxidatig.
It differed from known species in that Fermentatigtest was non-degradable, and 2) it had the ability to decompose alginic acid. Therefore, this bacterium was recognized as belonging to a new bacterial species, and the new bacterial species was classified as 77.3/ens (Flexibacter alginolique 77.3/ens).
faciens).

This strain has been deposited with the Institute of Microbial Technology (I"R Engineering) of the Ministry of International Trade and Industry since February 28, 1980, and has been deposited under the accession number FΣRMp-7484 on February 2, 1980.
They have been deposited with the Institute for Fermentation Research Foundation as Engineering FO14321 since the 0th.

The properties of the Flexibacter bacteria used in the present invention generally change when exposed to ultraviolet light, for example.

X-rays, chemicals such as trosoguanidine, methane
It can be easily mutated by artificial mutagenesis means such as phonic acid), and any mutant strain that has the ability to produce TAN-585, which is the subject of the present invention, is used in the present invention. be able to.

When culturing ThN-585-producing bacteria, examples of secondary sources include glucose, sugar, maltose, blackstrap molasses, glycero, oils and fats (eg, soybean oil).

Those that can be assimilated by bacteria are used as appropriate, such as olive oil (olive oil, etc.), organic acids (eg, citric acid, succinic acid, gluconic acid, etc.). Examples of sources include soybean flour, cottonseed flour, and corn staple liquor.

Dry yeast, yeast extract, meat extract, begton, urine X, v
Organic nitrogen compounds and inorganic nitrogen compounds such as ammonium acid, ammonium nitrate, ammonium chloride, and ammonium phosphate can be used. In addition, examples of inorganic salts include IJium chloride, potassium chloride, μsium chloride,
Magnesium sulfate.

Inorganic salts normally required for culturing bacteria, such as potassium phosphate and disodium phosphate, are used alone or in appropriate combinations.

In addition, heavy metals such as ferrous sulfate and copper sulfate, and vitamin D
2. Vitamins such as biotin are also added as necessary. Furthermore, antifoaming agents and surfactants such as KS/Licon Oil and Poly 1μ Kylene Glyco-μEteμ may be added to the medium. Helps the growth of bacteria, TAN-58
Organic or inorganic substances that promote the production of 5 may be added as appropriate.

The culture method may be the same as the general antibiotic production method, and solid culture or liquid culture may be used. In the case of liquid culture, static culture or agitation culture may be used.

Any method such as shaking culture 1 aerated culture may be carried out, but aerated agitation culture is particularly preferred. Also, the culture temperature is approximately 15
The temperature is preferably in the range of .degree. C. to 35.degree. C., the pH of the medium is in the range of about 4 to 8, and the culture is carried out for about 8 hours to 168 hours, preferably 24 hours to 144 hours.

In order to collect the target antibiotic TAN-585 from the culture, a separation means that is normally used to collect a surrogate produced by a microorganism from the culture of the microorganism is appropriately used. For example, antibiotics TAN-5 and TAN-85 have chemical properties as water-soluble acidic substances and are mainly contained in the culture solution. It is advantageous to use a method of removing the p-liquid, contacting the resulting culture filtrate with a suitable carrier to adsorb the active ingredient in the p-liquid, then desorbing the active substance with an appropriate solvent, and collecting it separately. As a carrier for chromatography, use the differences in activity, adsorption of compounds such as silicage μ, powdered cellulose, and adsorbent resins, or the differences in the functional groups of compounds such as anion exchange resins and anion exchange resins. Alternatively, carriers that utilize the difference in molecular weight of compounds, such as molecular sieving carriers, are advantageously used. In order to elute the target compound from these carriers, the combination differs depending on the type and nature of the carrier, but for example, aqueous solutions of water-soluble organic solvents, such as aqueous acetone, aqueous alcohols, etc., or acids, 1μ potassium , a buffer solution, an aqueous solution containing an inorganic or organic salt, etc. are used in appropriate combination.

Further, the compound of the present antibiotic obtained by these chromatographies can be further purified by subjecting it to preparative high performance liquid chromatographies.

More specifically, an anion exchange resin such as DOWEX-1 (DOW & CHEMICAL μ) is used as a carrier.
(manufactured by U.S.A.), Amberly FIR Mu-68,400,
402,410 (manufactured by Rohm and Haas, USA)
, Diaion S-21A and C (manufactured by San-ai Kasei Co., Ltd.,
(Japan) etc., the antibiotic in the furnace solution is adsorbed,
It is eluted with an aqueous solution or buffer solution containing salts or acids. Also, on anion exchange, μ-lose such as DI-3
2C Wa, Toman, UK), DEAE-7μ loin (
Braun, West Germany), or anion-exchange molecular sieving resin such as DEAE- or QAE-Cef1 Dex (77/L'Masha, Sweden), etc. Elution can be performed with an aqueous solution or buffer solution containing salts or acids. In order to remove salts, colored substances, etc. from the eluate, use active reconstitution for chromatography (Takeda Pharmaceutical Co., Ltd., Japan) or an adsorbent resin such as Diaion HP.
-20 (manufactured by Ekiseikasei Co., Ltd., Japan), Amberphyto XAI
) -1[ (manufactured by Rohm and Haas, USA) and the like are advantageously used. The fractionated elution fraction is powdered through processes such as concentration, freezing, and drying. If the purity of the powder thus obtained is poor, high performance liquid chromatography is advantageously used for further purification. Examples of the carrier used include TSK Ge/I/(manufactured by Toyo Soda Co., Ltd., Japan) and Y'MC Gemu (manufactured by Yamamura Kagaku Kenkyushin, Japan), and the mobile layer is methanol! Alternatively, a mixture of acetonitrile μ or the like and an acidic aqueous solution or a buffer solution is used.

If ThM-585A and TAN-585B are simultaneously produced in the culture solution of YK-49 strain, which is one of the TAN-585 producing bacteria, QAE-Sephadex or preparative high performance liquid is used to separate them. Each can be separated as a single component by chromatography.

The TAN-585 thus obtained is usually bound to salts used for purification and cations such as sodium, potassium, lithium, μsium, ammonium ions, etc. in the buffer solution and isolated as these salts. Obtained T
In order to obtain AN-585fi as an educt, it is common to use chromatography using a cation exchange resin to determine the activity.

Antibiotic TAN-585 forms metal salts and organic amine salts. Examples of metal salts include sodium salts, potassium salts, lithium salts, and lithium salts.

TAm-585A China) IJ obtained in Example 1 described below
The physicochemical properties of the salt are as follows.

(1) Outside 11i: White powder (2) Constituent elements: C, H, H, O, Ha (3) Elemental analysis value (4) = (sample dried under reduced pressure at 40°C for 6 hours over phosphorus pentoxide) C46, 0±2.0 H4,9±1.0 N 7.0±1. O Na 5.6 ± 1.0 (4) Molecular weight: S engineering MS (5 secondary
The molecular ion peak obtained by the Ion Mass Spectrometry method is as follows.

m/z 773,751,729.707 (5) Water content = 6.9 ± 296 (thermal balance method) (6) Estimated molecular formula: % formula % (7) Circular dichroism spectrum (in water): [θ] 228+: 2-95,500±10,000 (3
) ultraviolet absorption svec)/v (in water) (Fig. 1): 96 λ 277 ± 2 nm (El, = 20 ± 5. shoulder) maχ (9) infrared absorption svec) /i/ (Fig. 2): The main absorption (wave number) by potassium bromide tablets is as follows.

3430.3280.2950. 1760°1620
, 1515. 1405.1300°T240. 1
180. NIo, 1065°1025, 950. 8
30. 760°600, 535tx' 3 αOC-Nuclear magnetic resonance (CMR) spectrum μ (100MH
z, in heavy water) (Fig. 3): The following signals are even slightly observed.

188.46 (sL 178.09 (sj).

177.60 (s), 176.88 (s).

166.96(s), 166-75(d).

181.01(ts), 159.47(e).

133.95 (s), 132.54 (d, X2) + 13
2.43 (s), 131.33 (a, X2).

B9.89 (d, X2), B7.85 (d, X2).

103.11(d), 78.99(d), 78.17(
a).

75.95(d), 75.59(a), 74.53(a
).

74.53(s), 68.07(t), 63.57(d
).

56.22(d), 56.05(on), 32.64(t
) pP (s: singlet, d: d
oublet+t: represents triplet) (2) Solubility: Soluble: water, dimethylene oxide.

E'1klfj: Ethyl acetate, chloroform, acetone.

(2) Color reaction: Positive: Ninhydrin, Nieμlich reaction.

Negative: Brave Liebuck, Parton, Dragendorff reaction.

(To) Stability: 100μg/xt + 60° in aqueous solution
C, 3 hours pH 5 - stable pH 3: unstable pH 9: unstable (141 thin! Chromatography: spot film.

Cellulose f←manufactured by Tokyo Kasei Co., Ltd.) 4. High performance liquid chromatography (manufactured by Hitachi, Ltd. 9 Japan):
Carrier, ync A-312, C Yamamura Kagaku Kenkyusho 9 Japan), mobile phase; 5g6 methanol! 10. OTMIJy acid atr solution (pH 3,0).

2 m/min, Rt (win)-3,3aQ Specific rotation: [α) 23゛5 B1.6°±15° (cm0
156. (in water) (b) Distinction between acidic, neutral, and basic: neutral substances (edashi,
Free Thx-585AF! Acidic Substances) Next, the physicochemical properties of TAN-585B disodium salt obtained in Example 2 described below are as follows.

(1) Appearance: Two-white powder (2) Constituent elements HC, H+N, Na (3) Elemental analysis value (4): (9 samples dried under reduced pressure over phosphorus pentoxide at 40°C for 6 hours) C45,3 ± 2.0 H5,0±1. ON 7.0±1. O Na 6.1 + 1.0 (4) Molecular weight: S engineering MS (Secondary engineering
The molecular ion peak obtained by the nMass8pectromctry method is as follows.

m/z 757,735,713,691 (5) Water content 26.3 ± 396 (thermal balance method) (6) Estimated molecular formula: % formula % (7) Circular dichroism spectrum (in water): [θ] 227th 2-122.000±15,000λwax 268±
2nm(4-27+5)λmax 277±2nm(
Infrared absorption spectrum/L/ (Figure 5): The main absorption (wave number) by potassium bromide tablets is as follows.

3400.3280.3050.2900°1740,
1620, 1515. 1400°1300, 12
40,1180.1110°1060, 1020.
940. 830°750, 530c11-1 0,'13C-Nuclear magnetic resonance (CMR) spectrum μ(10
0 MHz, in heavy water) (Fig. 6): At least the following signal μ is observed.

186.8 (s), 178.40 (s) = 175
．． 95(s).

174.94 (s) = 171.17 (s), 166'
, 60(d).

161.13(s), 159.51(a)-133.9
5(s).

132.76(s), 132.97(d), 132.
51(s).

131.75(d) 1131.13(s), 119.
86(d).

118.15(d), 103.05(d), 79.08
(d).

78.20(d), 75.65(cl), 74.61
(d).

68.10(t), 63.56(d), 58.74(
d).

57.21(cl), 56.29ca), 49.36(
t).

32.67 (t) ppm (Tomodashi, 8: 8in
gletsd: doublet + t: triplet
) (b) Solubility: Soluble: water, dimethysium oxide.

Poorly soluble: ethyl acetate μ, chloroform μ, acetone.

(2) Color reaction: Positive: Reacted with ninhydrin.

Negative: Brave Liebuck, Parton, Dohugundorf reaction.

a3 mW chromatography: spot film.

Seven ρ loin f (manufactured by Tokyo Kasei Co., Ltd.) α◆ High performance liquid chromatography (manufactured by Hitachi Co., Ltd. 1 Japan)
: Carrier, YMCA-312 (Yamamura Chemical Research Institute 1 Japan)
, mobile phase; 596 methanol, +10.01M phosphate buffer (pH 3,0).

2 d/min, Rt (min)-4,223,
5 ■ Specific optical rotation: [α) -158°±15° (c-O,
51, in water) Q→ Distinction between acidic, neutral, and basic: Neutral substances (however,
The free form of TAN-585B is an acidic substance) The biological properties of the antibiotic TAN-585 disodium salt are as follows.

1) Antibacterial spectrum: Shows the antibacterial spectrum (/L/) for various test bacteria as shown in Table 1.

(bottom margin) (Note 1) As a culture medium, Bacto Antibiotic
Medium 3 (D7co Laboratories.

USA) i17.5F, Bact Yeast Extract (Difco Hubovtryz, USA); 5, Of, Pact Agar (Difco Hubovtryz, USA) f2
0F, distilled water: IQOOd (pH not adjusted) was used as the inoculum solution for about 106° colony forming unit/dt? Ta.

2) Stability test against β-lactamase
J
It is shown in the g2 table.

(Note 1 (Table 2 in the margin) The numerical values are the diameter of the inhibition circle by the vapor-guinuk method (
W) is shown. Medium: Nutrient agar medium 3) Therapeutic effect on mouse infection The therapeutic effect of TAN-585A disodium salt on experimental mouse infection is as shown in Table 3), in
Despite the weak antibacterial activity in vitro, in vitro
It is an antibiotic with excellent vo effect.

Table 3 * Intra-abdominal infection; *Hata Trypticase soy
agar (B BL Microbiology Sy
(manufactured by Stems, USA) medium and 108 colony formink units/dtI were used as the inoculum solution.

*Rice* 3 administrations 4) Toxicity test No acute toxicity was observed when TAN-555 disodium salt was subcutaneously administered to mice at 1f/#.

As is clear from these data, it can be said that TAN-585 is an antibiotic that exhibits antibacterial properties mainly against Guhum-negative bacteria and is not toxic to mammals. Therefore, TAN-585 can be used to treat bacterial infections in humans, livestock, and poultry.

To use TAw-585, for example, as a therapeutic agent for Osteobacterium infection, TAN-585 is dissolved in physiological saline and administered parenterally subcutaneously or intramuscularly as an injection.
Administer ~50q/day for 97 days, preferably 5-20 da/pass/day. In addition, as an oral agent, the antibiotic TAN-585 is mixed with lactose to make a turnip 7μ agent, and the amount of TAN-585 is 1 to 100q/#/day, preferably 5 to 50111! /#
Administer /day.

Furthermore, TAN-585 obtained according to the present invention can be used as a fungicide. For example TAN-585
0.0, OT-0.1 W/V% solution dissolved in distilled water, or based on Vaseline or Furin, containing 0.2 to 20 Da of TAN-585 per 11%, preferably tit.
It can be used as an ointment containing ~10 Da to sterilize and disinfect the hands, feet, eyes, ears, etc. of humans and animals.

The antibiotic TAN-58') is also a very promising compound as a synthetic intermediate for new pharmaceuticals.

Based on the properties described above, antibiotics rAm-585A and (
JThm-585B is thought to be a monocyclic β-tectam antibiotic, but among the known antibiotics belonging to these, it has relatively similar physicochemical properties.
Examples include Nocardicin group antibiotics. Molecular formula of antibiotics TAN-585 and TAN-585B, infrared absorption spectrum) /L
/ , , CMB2-Pectop and antibacterial Spect/v
Tjl, N-585A and 'I'Al-585B are each novel monocyclic β-thuctum antibiotics, as they are clearly different when compared with those of T. t.

EXAMPLES The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited thereto. Percentages indicate 9% weight/volume unless otherwise specified.

Example 1 Sakae@Trexipacta Kinoricare grown on agar slope, Vence YK-49 (FEBNP-7484
, Eng. FO14321) strain on glucose 296. Solp!・Stashchia*, raw soybean flour 1%, corn staple liquor 1%, lipeptone (Daigo Nutrient Chemical Co., Ltd.@f
') 0.5*, an aqueous solution containing 0.3 J of common salt (pH 7, 0
) was inoculated into a 21-volume slope flask containing 50 (is/
Culture was performed at 4°C for 48 hours with reciprocal shaking. The total volume of this culture solution was added to the above medium with Actco-μ (antifoaming agent, manufactured by KAIYO Yakuhin Kogyo Co., Ltd.) 0, -0551 in a volume containing 301 t.
01 tank was inoculated and the aeration rate was 3017 minutes at 24°C.
.

The cells were cultured for 48 hours at 200 revolutions/min. Add 6g of this culture solution to glycero/L/3%, 1 raw soybean flour, 1 raw soybean flour,
In an aqueous solution (pH 7, 0) containing 0.2% of carbonic acid, actco/I1
Volume 2001 containing 120g of medium supplemented with 0.05% gold
tank, at 24℃, with aeration rate of 1 2 0 11
7 minutes.

The cells were cultured for 66 hours at 150 revolutions/min.

Culture solution (100 J. pH 6.3) t-71 Iflo Super SE IV (manufactured by Jeans Manbiμ, USA)? The filtrate (881) was filtered using IRA-402 (C1m!
．． 4 m') column chromatography. The adsorbed antibiotics were eluted with 1.0 M saline (32 A'l), and the eluate was chromatographed with activity column chromatography (2.5
I attached it to 1. The active category is 8g 6 isobutano-water (
12.51), and the eluate was subjected to column chromatography using RA-68 (C1 type, 11). The adsorbed antibacterial substance was eluted with 1.0 M saline (81), and the eluate was subjected to activity chromatography (1.51) K for desalting. The desalinated aqueous solution was concentrated under reduced pressure and low temperature to a concentration of 1.41, and the concentrated solution was purified by QAK-Cef1 Decknu (A-25, C1
Type. 0.46> column chromatography.

After washing with 0.05M saline (41), the antibiotic was fractionated and eluted with 0.1M saline. Collect the active fraction,
The activity was subjected to cuff chromatography and eluted with 896 iso-butano-μ water. The eluate was concentrated under reduced pressure, lyophilized, and acetone was added to the lyophilized product to obtain 1.69y of crude material.
t- got it. 1.42N of the crude material obtained by culturing and purifying in the same manner as above was combined, dissolved in a small amount of water, and subjected to preparative high performance chromatography using a reverse phase carrier YMC gel. 0. (Nu phosphate buffer (pua, 0
) to elute the antibiotic, and the eluate was desalted by activated carbon chromatography. Concentrate the desalted aqueous solution under reduced pressure,
It was freeze-dried, and acetone was added to the dried product to remove all the precipitate. White powder of TAN-585A disodium salt (
1,34F) was obtained.

Example 2 Flexibacter auginorike 71 grown on a nutrient agar slope! / Ens YK-49 (FE: RMP-7484
The bacterial strain of i-tech FO14321) was isolated from Guco 7296. Soluble starch 396. Raw soybean flour 196. An aqueous solution (pH 7,
0) was inoculated into a 21-volume slope flask containing 500111 t of medium supplemented with 0.5*t of precipitated μsium carbonate,
Culture was performed at 24°C for 48 hours with reciprocal shaking. The entire amount of this culture solution was inoculated into a tank with a capacity of 2,001 cm containing 120 g of a medium prepared by adding 0.05% of Actco/I/ (antifoaming agent, manufactured by Ota Pharmaceutical Co., Ltd.) to the above medium, and heated to 24°C with an aeration volume of 1,201 cm. /
The cells were cultured for 48 hours under conditions of 150 rotations/min. This culture solution 601 was converted into an aqueous solution (p) containing 2% raw soybean flour, 1% corn gluten meal/L/1%, and 0.2% polypeptone.
I7,0'I was inoculated into a tank with a capacity of 2000 g containing medium 12001I supplemented with 0.5% precipitated subacid p-sium and 0.05% F Actco-*, and incubated at 24°C with an aeration rate of 120017 minutes. , 66 under the condition of 120 revolutions/min.
Cultured for hours. Culture solution (11501, pH5,4) t-
Filter the filtrate (1200#) using Hyflo Super SE/I/ (John's Manbi μ Co., USA) and process the filtrate (1200#).
A-402 (C1 type, 401 column chromatography)
00#), and the eluate was subjected to activated carbon column chromatography (201). The active fraction was eluted with 8% isogetanol water (Ta205), and the eluate was purified with RA-
68 (C1 type, 101 column chromatography. Adsorbed antibacterial substance k1. Eluted with OM saline (8ON), and the eluate was subjected to activated carbon chromatography (101).
K was attached and desalting operation was performed. The desalted aqueous solution was concentrated to 1.5 g under reduced pressure and low temperature, and concentrated liquid f: QAE-Sephadec y
h-25(C1'm, ag)o column chromatography. Kawomu't-0.05M saline solution (2ml)
After washing with water, the antibiotic was fractionated and eluted with 0.1M saline.

The active fractions were collected and subjected to activated carbon column chromatography to obtain 896 iso-butano.

-μ Eluted with water. The eluate was concentrated under reduced pressure and lyophilized, and acetone was added to the lyophilized product to obtain crude substance 30.3f7. Crude substance 56 obtained by culturing and purifying in the same manner as above
. When eluted with 0.08M saline, the first half of the fraction contained TAN-585A and the second half contained a mixture of TAN-585A and B. Each was desalted by activated carbon chromatography, and TAN-
Mixture of 585A22f and TAN-585A, B 15.
I got 3f.

This mixture was again mixed with QtE-Cef1dex (T, 2A
') was subjected to chromatography, eluted and fractionated with 0.08M saline, and the two-component eluate was desalted by activated carbon chromatography. The desalted aqueous solution was concentrated under reduced pressure and then freeze-dried, and acetone was added to the dried product to collect the precipitate t-p.

3.71 and 2.Of white powders of 'l'' AN-585A and TAN-585B disodium salts were obtained, respectively.

Nozomi! Antibiotics of the invention'rAN-585A and TAN-5
85B shows antibacterial activity mainly against Guhum-negative bacteria,
For example, when administered orally or parenterally to humans, livestock, poultry, etc., it can be used to treat these bacterial infections.

[Brief explanation of drawings]

Figures 1, 2, and 3 show the ultraviolet absorption spectra/L/(in water) and infrared absorption spectra/L/(KB
r method) and CMR spectrum/L/(in heavy water), respectively. In addition, FIGS. 4, 5, and 6 show Example 2.
Ultraviolet absorption spectrum of the antibiotic TAN-585B disodium salt obtained! (underwater), infrared absorption spectrum I
V (KBr method) and CMR Subek)/L' (in heavy water) are shown, respectively. Calculation 1 reduction size (nm) 4 diagram Asato (nm) Procedural amendment (鵠) August 5, 1985 1, Indication of the case 1988 Patent Application No. 100754 2, Name of the invention Antibiotic TAN ~585A, TAN-585B. Its manufacturing method and new bacterial species 3 of the genus Flexibacter, and its relationship to the amended case Patent applicant address 2-27 Doshomachi, Higashi-ku, Osaka Name (293)
Takeda Pharmaceutical Co., Ltd. Representative: Joint Education Department 4, Agent Address: 2-17-85 Jusanhonmachi, Yodogawa-ku, Osaka City Column 6 for detailed explanation of the invention in the specification to be amended, Contents of the amendment l) Details After page 25, line 18 of the book [From the physicochemical properties described above and other physicochemical properties, TA
The chemical structural formulas of N-585A and TAN-585B were determined as follows. OOH In the above formula, in TAN-585A, R1 is -NHCHO
. R2 is -OH and R3 is D-glucuronic acid, and in TAN-585B, R1 is -H, R, is -NH
C) 10 and R3 are D-glucuronic acid. ” is inserted. that's all

Claims (1)

Scope of Claims (1) Antibiotic TAN-585A, TAN-585B or salts thereof having the following physicochemical properties as a disodium salt (a) Antibiotic TAN-585A (1) Appearance: White powder. (2) Constituent elements: C+ H+ N + O+ 1ia
(3) Elemental analysis values (9), sample dried over phosphorus pentoxide at 40°C under reduced pressure for 6 hours: C46,0±2.0 H4,9±1.0 H7,0±1.0 Ha 5.6 ±1.0 (4) The molecular ion peak obtained by the S engineering MS method is as follows. m/z 773,751,729.707 (5) Circular dichroism spectrum / I / (in water): [θ] 228th 2nd 955
00±10000 (6) Ultraviolet absorption spectrum fi/(in water): 1g6 λ,, 224±2nmOE1. -312±30)1% λ 269±2nm(E25±5)maz 1al
I- 51 λ, a, 277 ± 2 nm (M 1. = 20 ± 5
*Shoulder) (7) Infrared absorption subek)/L': Main absorption by potassium bromide tablets. 3430.3280.2950.1?60°1620.
1515, 1405, 1300°1240.1180.
11+0.1065°1025, 950. 830.
760゜600, 535 arl (3) Solubility: Soluble in water, dimethy/l/μ oxide, sparingly soluble in vinegar Methyl, chloroform/l/m, acetone (9) M color ff response: ninhydrin, Positive for Nieμlich reaction, negative for Brave-Lieback, Burton, and Dragendorff reactions. -6) Antibiotic TAN-585B (1) Appearance: White powder (2) Constituent elements: C, H, N, O and Na (3) Elemental analysis value (4) '1.0' on phosphorus pentoxide C, sample dried under reduced pressure for 6 hours: c 45.3 ± 2.0 ■ 5.0 ± 1.0 H7, b ± 1. O Na 6.1 + 1.0 (4) The molecular ion peak obtained by the S engineering M8 method is as follows. m/z 757,735,713.691 (5) Circular dichroism spectrum/L/(in water): [θ)227+:2-12
2000±15000 (6) Ultraviolet absorption subek) /L
' (underwater): λ 224 + 2 nm (EE = 302
+30) lLX λ 268±2nm (E = 27±5) max λ 277±2nm (Σ1% = 21±5.shoulder) max
, 1c11 (7) Infrared absorption subek)lv: Main absorption by potassium bromide tablets. 3400.32B0,3050,2900°1740,
1620. 1515. 14.00°1300,
1240. 1180. 1110°1060, 10
20. 940. 830°750, 530cII (3) Solubility: Soluble in water, dimethysium oxide,
Poorly soluble in ethyl acetate, chloroform, and acetone (9) Color reactions: Positive for ninhydrin, NiμL reaction, negative for Brave-Liebuck, Burton, and Dragendorff reactions (II) Antibiotic belonging to the genus Frekinbacter TAx-
585A and (or) B producing bacteria were cultured in a medium, and the antibiotics TAN-585A and (or) B were added to the culture.
Antibiotic TAi-585A and/or Bo production method characterized by producing and accumulating TAi-585A and collecting the same (N) Oxidity puller 1-Mentatig test is non-degradable and has agic acid degrading ability Flexibacter auginoliquefa Vens.