JPH0439477B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel antibiotic TAN-585A, its salts, and a method for producing the same. The present inventors isolated a large number of microorganisms from soil for the purpose of searching for new antibiotics, and conducted a separate search for the antibiotics they produced. We learned that the microorganism belongs to the genus Flexibacter and that by culturing the microorganism in an appropriate medium, an antibiotic that exhibits antibacterial activity mainly against Gram-negative bacteria can be accumulated in the medium. We isolated this antibiotic and confirmed that it was a new antibiotic based on its physicochemical and biological properties, and decided to call it antibiotic TAN-585A. The present inventors completed the present invention as a result of further research based on these findings. The present invention provides: 1) antibiotic TAN-585A and its salt; 2) antibiotic TAN-585A-producing bacteria belonging to the genus Flexibacter are cultured in a medium, and antibiotic TAN-585A is produced and accumulated in the culture; Antibiotic TAN− characterized by collecting
This is the manufacturing method for 585A. Antibiotic TAN-585A has the following formula [In the formula, R represents D-glucuronic acid]. In addition, in this application, the antibiotic TAN-585A may be simply referred to as "TAN-585A." The antibiotic TAN-585A producing bacteria used in the present invention is Flexi-bacter
Any microorganism may be used as long as it belongs to the genus and has the ability to produce the antibiotic TAN-585A. An example of the antibiotic TAN-585A-producing bacteria is Flexibacter alginoliquefaciens strain YK-49 (hereinafter referred to as "YK-49"), which was collected by the present inventors from soil in Yoshinoyama, Yoshino District, Nara Prefecture. KK"
It is also sometimes called. ) can be given. YK−49
The mycological properties of the strain are as follows. (a) Morphology When observed after culturing on a broth agar slant at 24℃ for 2 days, the cells were 0.4-0.7 μm in diameter and usually 2-10 μm in length.
It exhibits an elongated rod shape, but in rare cases, it may exhibit a filament shape of 50 to 70 μm. It has no flagella. It has mobility by gliding. It does not form spores or microcysts, Gram staining is negative, and it does not show acid fasting properties. (b) Growth status on various media Cultured at 24°C and observed for 1 to 14 days. Broth agar plate culture: Forms round, convex, and full-edged colonies. The surface is smooth, translucent, and white.
It has a 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, produces a small amount of precipitate, and forms a weak bacterial ring. Meat juice gelatin puncture culture: Growth is weak. Litmus milk: Peptonization is positive, white cloudiness is observed at the bottom, and decolorization is observed,
No acid formation or coagulation is observed. (c) Physiological properties Nitrate ring element: Negative Denitrification reaction: Negative MR (Methyl Red) test: Negative VP (Vouges-Proskauer) test:
Negative Production of indole: Negative Production of hydrogen sulfide: Negative Hydrolysis of starch: Positive Utilization of citric acid: Positive Utilization of inorganic nitrogen source Potassium nitrate: Negative Ammonium sulfate: Positive Coagulation Pigment production (King A, King B and Mannito yeast extract agar) ): Does not produce soluble pigments. Urease: Negative Oxidase: Positive Catalase: Positive Growth Range PH: Grows between PH4.9 and 8.5, but the optimum pH is 5.4
~6.6. Temperature: Grows between 6 and 35 degrees Celsius, but the optimum temperature is 14 degrees Celsius.
~31℃. Attitude towards oxygen: aerobic O-F test (Hugh Leifson)
Leif-son method]: Non-degradable type Generation of acid and gas from sugar:

[Table] Decomposition of polysaccharides: Does not decompose agar, cellulose, carboxymethyl cellulose, and colloidal chitin. Decomposes alginic acid. Meat juice liquid culture with 0% salt: Grows. Meat juice liquid culture with 5% salt: No growth. The YK-49 strain with the above mycological properties was used as described in Virgy's Manual of Determinative
Bacteriology (Bergey's Manual of
Determinative Bacteriology (8th edition) and International Journal of Systematic Bacteriology (International Journal of Systematic Bacteriology)
Journal of Systematic Bacteriology) Volume 30
215-420 (1980) and Vol. 32, pp. 146-149 (1982), it is a rod-shaped or filament-shaped Gram-negative bacterium that does not form microcysts and has motility by gliding. It does not degrade agar, cellulose, carboxymethyl cellulose or colloidal chitin.
It is appropriate to classify it as belonging to the genus Flexibacter because it does not require salt or exhibits tolerance to salt. Therefore, when we compared strain YK-49 with known species of the genus Flexibacter, we found that the following YK-49 strain
The properties of the 49 strains are: 1) Oxidative
The affirmative test is necessarily non-degradable, and 2)
It differed from known species in that it had the ability to decompose alginic acid. Therefore, we recognized this bacterium as a strain belonging to a new bacterial species, and identified the new bacterial species as Flexibacter alginoliquefaciens (Flexibacter alginoliquefaciens).
alginoliquefaciens). This strain has been deposited with the Ministry of International Trade and Industry, Agency of Industrial Science and Technology, Microbial Research Institute (FRI) under the accession number FERM P-7484 since February 28, 1980, and with Fermentation Research Foundation since February 20, 1980. Both have been deposited as IFO 14321 at The properties of the Flexibacter bacteria used in the present invention are generally susceptible to changes, such as exposure to ultraviolet rays, X-rays, chemicals (e.g., nitrosoguanidine,
TAN-585A, which is the object of the present invention, can be easily mutated by artificial mutagenesis means such as ethyl methanesulfonic acid), and any mutant strain can be used as TAN-585A, which is the object of the present invention.
can be used in the present invention. When culturing TAN-585A-producing bacteria, carbon sources such as glucose, cane sugar, maltose, blackstrap molasses, glycerol, oils and fats (e.g., soybean oil, olive oil, etc.), organic acids (e.g., citric acid,
Succinic acid, gluconic acid, etc.) that can be assimilated by bacteria are used as appropriate. Examples of nitrogen sources include organic and inorganic nitrogen compounds such as soy flour, cotton flour, corn stave liquor, dried yeast, yeast extract, meat extract, peptone, urea, ammonium sulfate, ammonium nitrate, ammonium chloride, and ammonium phosphate. compounds are available. In addition, as the inorganic salt, for example, inorganic salts normally required for culturing bacteria such as sodium chloride, potassium chloride, calcium carbonate, magnesium sulfate, monopotassium phosphate, and disodium phosphate are used alone or in appropriate combinations. . Further, heavy metals such as ferrous sulfate and copper sulfate, vitamins such as vitamin B ₁ and biotin, and the like are added as necessary. Furthermore, antifoaming agents and surfactants such as silicone oil and polyalkylene glycol ether may be added to the medium. Other organic or inorganic substances that aid the growth of bacteria and promote the production of TAN-585A may be added as appropriate. The culturing method may be the same as a general antibiotic production method, and solid culture or liquid culture may be used. For liquid culture, static culture, agitation culture,
Any of shaking culture, aeration culture, etc. may be carried out, but aeration agitation culture is particularly preferred. Further, the culture temperature is preferably in the range of approximately 15° C. to 35° C., the pH of the medium is in the range of approximately 4 to 8, and the culture is carried out for approximately 8 hours to 168 hours, preferably 24 hours to 144 hours. In order to collect the target antibiotic TAN-585A from the culture, separation means commonly used to collect the metabolites produced by the microorganism from the culture of the microorganism are appropriately utilized. For example, antibiotic TAN−
585A exhibits chemical properties as a water-soluble acidic substance and is mainly contained in the culture solution, so it is obtained by first adding a supplement to the culture solution and removing the bacterial cells by filtration or centrifugation. The culture solution is brought into contact with an appropriate carrier to adsorb the active ingredients in the solution, and then the active substances are desorbed with an appropriate solvent.
Means of separate collection are advantageously used. Activated carbon, silica gel,
Utilizing differences in adsorption properties of compounds such as powdered cellulose and adsorbent resins, or utilizing differences in functional groups of compounds such as anion exchange resins and anion exchange cellulose,
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,
Alkali, buffer solutions, or aqueous solutions containing inorganic or organic salts are used in appropriate combinations. Furthermore, the crude substance of the present antibiotic obtained by these chromatography methods can be further purified by subjecting it to preparative high performance liquid chromatography. More specifically, anion exchange resins such as Dowex-1 (manufactured by Dow & Chemical Co., USA) and Amberlite are used as carriers.
IRA-68, 400402, 410 (Rohm & Haas, USA), Diaion SA-21A and C
(manufactured by Mitsubishi Kasei Co., Ltd., Japan), etc., the antibiotic in the solution is adsorbed and eluted with an aqueous solution or buffer solution containing salts or acids. Also, anion exchange cellulose such as DE-32 (manufactured by Watzmann, UK), DEAE-cellulose (manufactured by Braun, West Germany), or anion exchange molecular sieving resin such as DEAE- or QAE-Sephadex (manufactured by Pharmacia, Sweden), etc. )
The antibiotic can be adsorbed onto a carrier such as, for example, and eluted with an aqueous solution or buffer solution containing salts or acids. In order to remove salts, colored substances, etc. from these eluates, activated carbon for chromatography (manufactured by Takeda Pharmaceutical Co., Ltd., Japan) or adsorbent resins such as Diaion HP-20 (manufactured by Mitsubishi Kasei Co., Ltd., Japan), Amberlite XAD- (manufactured by Rohm and Haas, USA) and the like are advantageously used. The fractionated elution fraction is concentrated, frozen,
After going through processes such as drying, it is turned into powder. If the purity of the powder thus obtained is poor, high performance liquid chromatography is advantageously used for further purification. Examples of carriers used include
Examples include TSK gel (manufactured by Toyo Soda Co., Ltd., Japan), YMC gel (manufactured by Yamamura Kagaku Kenkyujo, Japan), and the moving phase is a mixture of methanol, acetonitrile, etc., and an acidic aqueous solution or a buffer solution. The TAN-585A thus obtained is usually bound to the salts used for purification and cations such as sodium, potassium, lithium, calcium, and ammonium ions in the buffer solution and isolated as these salts. In order to obtain the obtained TAN-585A salts as an educt, it is common to use activated carbon or cation exchange resin chromatography. Antibiotic TAN-585A forms metal salts and organic amine salts. Examples of metal salts include sodium salts, potassium salts, lithium salts, calcium salts, and the like. The physicochemical properties of TAN-585A dinatrium salt obtained in Example 1 described below are as follows. (1) Appearance: White powder (2) Elemental analysis value (%): (Sample dried under reduced pressure over phosphorus pentoxide at 40℃ for 6 hours) C 46.0±2.0 H 4.9±1.0 N 7.0±1.0 O 36.5 ^* N _a 5.6 ±1.0 *(However, oxygen is calculated by subtracting other elements) (3) Molecular weight: SIMS (Secondary Ion Mass
The molecular ion peaks determined by the Spectrometry method are as follows. m/z 773, 751, 729, 707 (4) Moisture content: 6.9±2% (thermobalance method) (5) Molecular formula (molecular weight): C ₃₀ H ₃₂ N ₄ O ₁₆ Na ₂ (750.58) (6) Yen Dichroic spectrum (in water): [θ]228±2
-95500±10000 (7) Ultraviolet absorption spectrum (in water) (Figure 1):
λ _nax 224±2nm (1% 1cm=312±30) λ _nax 269±2nm (1% 1cm=25±5) λ _nax 277±2nm (1% 1cm=20±5, shoulder) (8) Infrared absorption Spectrum (Figure 2): The main absorptions (wave numbers) by potassium bromide tablets are as follows. 3430, 3280, 2950, 1760, 1620, 1515,
1405, 1300, 1240, 1180, 1110, 1065, 1025,
950, 830, 760, 600, 535 cm ^-1 (9) ¹³ C-Nuclear magnetic resonance (CMR) spectrum (100 MHz, in heavy water): At least the following signals are observed. 188.46 (s), 178.09 (s), 177.60 (s),
176.88 (s), 166.96 (s), 166.75 (d), 161.01
(s), 159.47 (s), 133.95 (s), 132.54 (d, ×
2), 132.43 (s), 131.33 (d, ×2), 119.89
(d, ×2), 117.85 (d, ×2), 103.11 (d),
78.99(d), 78.17(d), 75.95(d), 75.59(d)
，
74.53(d), 74.53(s), 68.07(t), 63.57(d)
，
56.22 (d), 56.05 (t), 32.64 (t) ppm (s: singlet, d: doublet, t: triplet) (10) Solubility: Soluble: water, dimethyl sulfoxide. Poorly soluble: ethyl acetate, chloroform, acetone. (11) Color reaction: Positive: Ninhydrin reaction, Ehrlich reagent. Negative: Greig-Lieback reaction, Burton reaction, Dragendorff reaction. (12) Stability: 100μg/ml, in aqueous solution, 60℃, 3
Time PH5-7: Stable PH3: Slightly unstable PH9: Unstable (13) Thin layer chromatography: Spot film, cellulose f (manufactured by Tokyo Kasei Co., Ltd.)

[Table] (14) High performance liquid chromatography (manufactured by Hitachi, Japan): Support, YMC A-312 (manufactured by Yamamura Chemical Research Institute,
Japan), mobile phase; 5% methanol/0.01M phosphate buffer (PH3.0), 2ml/min.Rt (min) = 3.3 (15) Specific rotation: [α] ^23.5 _D −81.6° ± 15° (c=0.5
6, in water) (16) Distinction between acidic, neutral, and basic: a neutral substance (however, the free form of TAN-585A is an acidic substance) Next, we will discuss the biological properties of TAN-585A disodium salt. The antibacterial spectrum of this compound against various microorganisms is shown in Table 1.

【table】

【table】

[Table] In addition, TAN585A disodium salt has various β-
Stable against lactames. Table 2 shows Proteus mirabilis ATCC 21100 as the test bacterium.
The results of examining the stability against two types of β-lactamase are illustrated below.

[Table] **: Indicates that there is no inhibition circle.
In addition, the therapeutic effect of TAN-585A disodium salt on experimental mouse infections is shown in Table 3, and despite its weak in vitro antibacterial activity, it is an antibiotic with excellent in vivo effects. .

【table】

[Table] In addition, add 1 g of TAN-585A disodium salt/
No acute toxicity was observed when administered subcutaneously to mice at Kg. As is clear from these data, TAN−
It can be said that 585A is an antibiotic that exhibits antibacterial properties mainly against Gram-negative bacteria and is not toxic to mammals. Therefore, TAN-585A can be used to treat bacterial infections in humans, livestock, and poultry. To use TAN-585A, for example, as a therapeutic agent for Osteobacterium infection, TAN-585A is dissolved in physiological saline and administered parenterally subcutaneously or intramuscularly at 1 to 50 mg/Kg/day as an injection. Preferably 5
Administer ~20mg/Kg/day. Further, as an oral agent, the antibiotic TAN-585A is mixed with lactose to form a capsule, and TAN-585A is administered at 1 to 100 mg/Kg/day, preferably 5 to 50 mg/Kg/day. Moreover, TAN-585A obtained by the present invention
can be used as a fungicide. for example
A solution prepared by dissolving TAN-585A in distilled water at a concentration of 0.01 to 0.1 W/V%, or a solution based on petrolatum or lanolin, containing 0.2 to 20 mg of TAN-585A per gram;
Preferably, the ointment containing 1 to 10 mg can be used for sterilizing and disinfecting the hands, feet, eyes, ears, etc. of humans and animals. The antibiotic TAN-585A is also an extremely promising compound as a synthetic intermediate for new pharmaceuticals. Based on the properties described above, the antibiotic TAN-585A is considered to be a monocyclic β-lactam antibiotic. Among the known antibiotics belonging to these, nocardicin ( Nocardicin) group antibiotics. However, the molecular formula of antibiotic TAN-585A,
TAN-585A is a novel monocyclic β-lactam antibiotic because its infrared absorption spectrum, CCMR spectrum, and antibacterial spectrum are clearly different from those of nocardicin. Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto. Percentages indicate weight/volume % unless otherwise specified. Example 1 Flexibacter alginoliquefaciens YK-49 (FERM P-) grown on nutrient agar slope
7484; IFO 14321) strain at 2% glucose,
0.5% precipitated calcium carbonate in an aqueous solution (PH7.0) containing 3% soluble starch, 1% raw soy flour, 1% corn stave liquor, 0.5% polypeptone (manufactured by Daigo Nutritional Chemical Co., Ltd.), and 0.3% salt. The cells were inoculated into a 2-volume Sakaguchi flask containing 500 ml of medium supplemented with the following, and cultured with reciprocal shaking at 24°C for 48 hours. The entire amount of this culture solution was inoculated into a tank with a capacity of 50 containing 30% of the above medium and 0.05% Actocol (antifoaming agent, manufactured by Takeda Pharmaceutical Company Limited), and heated at 24°C with an aeration rate of 30%/30%.
The cells were cultured for 48 hours under conditions of 200 rotations/min. Add this culture solution 6 to 3% glycerol and 1.5% raw soybean flour.
%, corn gluten meal 1.5%, and polypeptone 0.2% in an aqueous solution (PH 7.0) supplemented with precipitated calcium carbonate 0.5% and Actocol 0.05%.
The cells were cultured for 66 hours at a temperature of 120/min and an aeration rate of 150 revolutions/min. The culture solution (100, PH6.3) was filtered using Hyflo Super Cell (manufactured by Jones-Manville, USA), and the solution (88) was
It was subjected to column chromatography using IRA-402 (CI type, 4). The adsorbed antibiotics were eluted with 1.0 M saline (32), and the eluate was subjected to activated carbon column chromatography (2.5). The active fraction was eluted with 8% iso-butanol water (12.5), and the eluate was subjected to IRA-68 (CI type, 1) column chromatography. adsorb antibacterial substances
Elution was performed with 1.0M saline (8), and the eluate was subjected to activated carbon chromatography (1.5) for desalting. The desalted aqueous solution was concentrated under reduced pressure and low temperature to a concentration of 1.4, and the concentrated solution was passed through QAE-Sephadex (A-
25, CI type, 0.4) column chromatography. After washing the column with 0.05M saline (4), the antibiotic was fractionated and eluted with 0.1M saline. The active fractions were collected and subjected to activated carbon column chromatography and eluted with 8% iso-butanol water.
The eluate was concentrated under reduced pressure and lyophilized, and acetone was added to the lyophilized product to obtain 1.69 g of a crude substance. 1.42 g 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. The antibiotic was eluted with 0.01M phosphate buffer (PH3.0), and the eluate was desalted using activated carbon chromatography. Concentrate the desalted aqueous solution under reduced pressure,
It was lyophilized and precipitated with acetone.
TAN-585A disodium salt white powder (1.34
g) was obtained.

[Brief explanation of drawings]

Figure 1 shows the ultraviolet absorption spectrum (in water) of the antibiotic TAN-585A disodium salt, and Figure 2 shows the infrared absorption spectrum (KBr method).

Claims (1)

[Claims] 1. The following formula An antibiotic TAN-585A represented by the formula [wherein R represents D-glucuronic acid] and a salt thereof. 2 Belongs to the genus Flexibacter and has the following formula A microorganism capable of producing the antibiotic TAN-585A represented by the formula [wherein R represents D-glucuronic acid] is cultured in a medium, the antibiotic is produced and accumulated in the culture, and the antibiotic is collected. Characteristic method for producing antibiotic TAN-585A.