JPS6310998B2 - - Google Patents

Description

[Detailed description of the invention]

The antibiotic MSD890A ₁₀ and its pharmaceutically usable salts (hereinafter referred to as antibiotic 890A ₁₀ ) are effective against both Gram-positive and -negative bacteria. Antibiotics are produced by culturing certain Streptomyces in a suitable fermentation medium. The discovery of penicillin's remarkable antibacterial properties created great interest in the field, resulting in the discovery of many other effective antibiotics. Generally, the antibacterial activity of each of these antibiotics does not involve certain medically important pathogens. For example, some are primarily effective only against Gram-positive bacteria. The resistance acquired during the widespread use of existing antibiotics in the treatment of bacterial infections has created a serious resistance problem. Therefore, the shortcomings of known antibiotics, as well as resistant strains of certain microorganisms, have stimulated further research for the discovery of other antibiotics that are effective against a wide range of pathogens. The present invention is directed to antibiotic agents. It concerns a novel method for the production of an antibiotic, herein referred to as 890A ₁₀ . The present invention includes diluted, crude concentrate and pure antibiotics. It is an object of the present invention to provide a new method for producing useful antibiotics that very effectively inhibit the growth of various Gram-negative and positive bacteria. That is,
It is an object of the present invention to provide a method for preparing antibiotics by fermenting a nutrient medium with certain Streptomyces. Other objects will become apparent from the detailed description of the invention provided hereinafter. The antibiotic of the invention is produced by growing a new strain of Streptomyces flavogriseus under controlled conditions. Based on detailed taxonomic studies, the strain used in the present invention was identified as belonging to the species Streptomyces flavogriseus and was identified as MA-4638 (Application Receipt No. No.
4284). This strain was developed by The Noisy Regional Laboratories, The Noisy Utilization Research and Development Department, and The Agricultural Research Service.
Northern Regional Laboratories, Northern
Utilization Research and Development
Division, Agricultural Research, Service)
It is permanently deposited with the United States Department of Agriculture, Peoria, Illinois Collection of Bacteria without any restrictions on use, and is publicly available under accession number NRRL11020. This strain has been deposited with the Microbial Research Institute under the accession number FERM-P No. 4284 (FERM-P No. 4284). Streptomyces flavogriseus MA-4638 produces antibiotic 890A ₁₀ , which is isolated in substantially pure form from the culture fluid. The morphological and culture characteristics of Streptomyces flavogriseus MA-4638 are shown in the following table. Morphology Sporophytes are branched, with straight or curved spore chains, forming tufts. A spore chain has 10 or more spores. Spores are spherical or oval-shaped.
0.9μ×1.2μ (970×). Culture characteristics Oatmeal agar (ISP3 medium) Vegetative hyphae - back - yellow - tan, edges brown, wrinkled; aerial hyphae - light gray, edges medium gray; soluble pigments - none. Czapek Dox Agar (Seuclose Nitrate Agar) Vegetative mycelia - underside - brown, edges dark brown; aerial mycelia - medium gray, velvety; soluble pigments - medium becomes slightly brown. Egg Albimin Agar Vegetative hyphae - underside - yellow-dark yellow, brown edges; aerial hyphae - medium gray with yellow-gray (2 dc) and grayish yellow (2 db); soluble pigments - light yellow-brown. Glycerol Asparagine Agar Vegetative hyphae - underside - brown; aerial hyphae - velvety, light gray with yellow undertones (2 dc); soluble pigment - light tan. Inorganic salts - starch agar (ISP4 medium) Vegetative mycelia - underside - yellow-brown with green tints; aerial mycelia - velvety, medium gray with yellow undertones (3fe); soluble pigments - very light yellow-brown. Yeast extract - dextrose + salt agar. Vegetative hyphae - back - light brown; aerial hyphae - light gray mixed with dark gray; soluble pigments - none. Yeast extract - Malt extract agar (ISP2 medium) Vegetative hyphae - Back - brown; Aerial hyphae - velvety, dark gray with light gray edges; Soluble pigments - none. Skimmed milk agar Vegetative mycelia - yellowish brown; Aerial mycelium - poor, white color; Soluble pigment - makes the medium slightly brown. Hydrolysis of casein - good. Litmus milk Vegetative mycelia - medium growth ring, yellowish brown; aerial mycelia - none; color - purple; coagulation and/or peptonization - complete peptonization; becomes alkaline. Skimmed milk Vegetative mycelia - medium growth rings, yellowish brown; aerial mycelia - none; soluble pigments - light brown; coagulation and/or peptonization - complete peptonization; becomes alkaline. Nutrient tyrosine agar Vegetative hyphae - underside - dark brown; aerial hyphae - dark gray with off-white edges; soluble pigment - makes the medium slightly brown; tyrosine degradation - positive. Peptone-iron-yeast extract agar Vegetative hyphae-yellow brown; Aerial hyphae-white, medium; Soluble pigments-none; Melanin-none; _H2S production-negative. Nutrient agar Vegetative hyphae - underside - light grayish brown; aerial hyphae - light gray with dark gray edges; soluble pigments - none. Nutrient Starch Agar Vegetative mycelia - tan, gray edges; aerial mycelia - medium gray; soluble pigments - none; hydrolysis of starch - good. Nutritional gelatin agar Vegetative mycelium - yellowish brown with gray edges; Aerial mycelium - off-white; Soluble pigments - none; Liquefaction of gelatin - good. Potato plug Vegetative hyphae - yellowish brown; aerial hyphae - medium to dark gray; soluble pigments - none. loeffler´s Blood serum Vegetative hyphae - cream color; aerial hyphae - none; soluble pigments - none; liquefaction - none. Gelatin puncture Vegetative hyphae - yellowish brown; Aerial hyphae - none; Soluble pigments - none; Liquefaction of gelatin - complete. All determinations above are made at 28°C unless otherwise indicated.
This was done after 3 weeks of culture. of the medium used in this experiment.
The pH is approximately neutral, ie PH6.8-7.2. The color representation used in the explanation is from the Color Harmony Manual,
4th edition (1958), Container Corporation of
America, Chicago, Illinois, as defined. The utilization or assimilability of Streptomyces flavogriseus MA-4638 for various carbohydrates was also tested.
For this purpose, the microorganisms were cultured for 3 weeks at 28° C. on a basic synthetic medium (Pridham and Gottlieb) containing 1% carbohydrate. The pH of the medium used in the experiment is approximately neutral.
(PH6.8−7.2). Table 1 shows Streptomyces flavogriseus.
Demonstrates the utilization of these carbohydrate sources by MA-4638; + growth, ± poor or questionable growth, - no growth, compared to negative control (no carbon source).
It shows. Table Glucose + Maltose + Arabinose + Mannitol + Cellulose - Mannose + Fructose + Raffinose - Inositol - Rhamnose + Lactose + Seuclose ± Xylose + The growth rate and oxygen requirement due to temperature changes by bacteria are as follows. Temperature range (yeast extract - dextrose + salt agar; 28°C - good nutritional and aerial mycelium; 37°C - good nutritional hyphae; no aerial mycelium; 50°C - no growth; oxygen demand (yeast extract - dextrose + salt agar) aerobic The present invention is not limited to the microorganism Streptomyces flavogriseus or to microorganisms that fully respond to the cultural and microscopic characteristics mentioned above, which are given by way of example only. 890A 10 is desirable and intended to include the use of mutant strains produced from the microorganism by various methods, such as X-ray irradiation, ultraviolet irradiation, nitrogen mustard, phage infection, etc. 890A ₁₀ is a microorganism, Streptomyces It is produced during aerated cultivation under controlled conditions of a suitable hydrous nutrient medium inoculated with a strain of Streptomyces flavogriseus. Hydrous media such as those used for the production of other antibiotics are suitable for the production of 890A ₁₀ . Such a medium contains sources of carbon, nitrogen and inorganic salts that can be assimilated by the microorganisms. Carbohydrates such as sugars such as dextrose, glucose, fructose, maltose,
Starches such as sucrose, xylose, etc. and dextrin or grains such as oats, rye, corn starch, corn mill, etc. can be used alone or in combination as sources of assimilable carbon in the nutrient medium. The amount of carbohydrates is about 1 to 6
Varies between % by weight. These carbon sources can be used alone or several such carbon sources can be mixed in the medium. In general, many proteinaceous substances can be used as nitrogen sources for fermentation processes. Suitable nitrogen sources include, for example, yeast hydrolyzate, dried yeast, soybean flour, cottonseed flour, casein hydrolyzate, corn staple liquor, Distiller´s Solubles, etc., with the preferred source being Daystiller's Solubles.・It is SOLUBLEB. Nitrogen sources, alone or in combination, are used in amounts ranging from about 0.2 to 6% by weight of the aqueous medium. Nutrient inorganic salts that can be added to the medium are the usual salts that can generate ions such as sodium, potassium, ammonium, calcium, magnesium, phosphoric acid, sulfuric acid, hydrochloric acid, carbonic acid, etc. Also included are trace metals such as cobalt, manganese and iron. The media described in the Examples are merely illustrative of the wide variety of media that may be used and are not intended to be limiting. Fermentation is carried out at a temperature range of about 20°C to 37°C, although for best results it is preferred to carry out fermentation at a temperature of about 23°C to 28°C. The initial pH of the nutrient medium suitable for growing the Streptomyces flavogriseus strain and producing the antibiotic 890A ₁₀ can be varied to about 6.0-8.0. Although antibiotic 890A ₁₀ can be produced both in surface and submerged cutures, it is preferred to carry out the fermentation in submerged conditions. Small-scale fermentation of antibiotics is conveniently carried out by inoculating antibiotic-producing bacteria into a suitable nutrient medium, transferring to the production medium, and then allowing fermentation to proceed for several days at a constant temperature of 24° C. on a sheaker. Fermentation is initiated by one or more stages of seed expansion in sterile flasks containing nutrient medium. The nutrient medium for the seed stage may be any suitable combination of carbon and nitrogen sources. The seed flasks are shaken in a temperature chamber at about 28° C. for 1 day or until growth is sufficient, and the resulting culture is used to inoculate either a second seed or production medium. Intermediate stage seed flasks, if used, are expanded in essentially the same manner; ie, a portion of the contents of the last stage flask is used to inoculate the production medium. The inoculated flask is shaken at constant temperature for several days, and after the incubation period the contents of the flask are centrifuged or filtered. For large-scale cultivation, the fermentation is preferably carried out in suitable tanks equipped with a medium stirrer and aeration equipment. According to this method, the nutrient medium is prepared in a tank and sterilized by heating at temperatures up to about 120°C.
After cooling, the sterile medium is inoculated with pre-grown seeds of the production bacteria and the fermentation is allowed to proceed, for example for 1-6 days, during which time the nutrient medium is agitated and/or aerated and the temperature is maintained at about 22-26°C. This method of producing antibiotic 890A ₁₀ is particularly suitable for the preparation of large quantities of antibiotics. Physicochemical properties of antibiotic 890A ₁₀ Antibiotic 890A ₁₀ is an acidic substance and electrophoretically migrates toward the anode at neutral pH. In 30 minutes at a gradient of 50 volts/cm in 0.03M potassium phosphate buffer, PH 7.1, the 890A ₁ moves 4.0 cm while the antibiotic moves 8 cm. The disodium salt is lyophilized from an aqueous solution to a white or slightly yellow powder. Antibiotics are unstable under acidic conditions in aqueous solutions and the free acid form is not isolated. The disodium salt of antibiotic 890A ₁₀ is neutral in water.
It has an absorption maximum at 299nm and a minimum at 243nm at PH.
The most purified sample of the disodium salt has an E% of 214 at 300 nm. A ₃₀₀ /of the most purified sample
The A ₂₅₀ ratio is 3.33 and the A ₃₀₀ /A ₂₂₀ ratio is 2.05.
The presence of slight impurities in this sample suggests that the corresponding ratio of the final pure sample is somewhat higher. More than 94% of absorption at 300nm is at neutral PH
It disappears by reaction with hydroxylamine. The absorption at 250nm also decreases due to the reaction with hydroxylamine, and the absorption at 250nm decreases by 300n.
The ratio for the decrease in m is approximately 0.16. The reaction with hydroxylamine, tracked by the decrease in A ₃₀₀ under the conditions described in the “Hydroxylamine Reaction” section, is clearly a first-order reaction, with a half-life of 23−60 at room temperature.
Seconds. When measured against the standard antibiotic 890A ₁ , antibiotic 890A ₁₀ has 174 bioassay units per ₃₀₀ units of HAEA. HAEA ₃₀₀ will be discussed later in the “Hydroxylamine Reaction” section. Table _890A 100MHz at 32°C in D ₂ O
The NMR spectrum is shown. Chemical shifts are given in ppm compared to the HOD of δ4.70 at 32°C, and coupling constants are in Hertz. Table C H ₃ CH 1.55 (3H; d; 6.5Hz) CH ₃ CO 2.02 (3H; S) C ₍₆₎ −H 3.89 (1H; d, d; J _6-5 =5.4Hz; J _6-8 =
9.2Hz) C ₍₅₎ −H ~4.34 (1H; d, t; J _5-6 = 5.5Hz; J _5-1
=9.5Hz) C ₍₈₎ −H ~4.8 (partially overlaps with the HOD line) C ₍₁₎ −H ₂ ~3.14 (1H; d, d; ~9.2+18Hz) ~3.33 (1H; d , d; ~10+18
Hz) -C H ₂ NH 3.43 (2H; t; 7 Hz) -CH ₂ -S- 3.03 (2H; m) The mass spectrum of TMSi-890A ₁₀ is characterized by the fragments shown in the table. Table m/e 86 227.0224 C ₅ H ₁₅ SO ₄ Si ₂ , calculated value 227.0230 241 300/1 339.1325 C ₁₅ H ₂₅ NO ₄ Si ₂ , calculated value 339.1322 342.1444 C ₁₅ H ₂₆ N ₂ O ₃ SSi, calculated value 342.14 33 368 440 458 Antibiotic 890A ₁₀ has the following molecular structure: Antibiotic 890A ₁₀ is further characterized by the following antibacterial spectrum. A test to determine the antibacterial spectrum of antibiotic 890A ₁₀ was performed using a 9.5 μg/ml aqueous solution of the antibiotic.
A 0.015 ml drop was placed on the surface of a 100 x 15 mm Petri dish containing inoculated nutrient agar + 5 ml of 0.2% yeast extract.
The cells were cultured at 25°C. The results expressed in mm diameter of the blocking circle are shown in Table 1.

【table】

[Table] Antibiotic 890A ₁₀ is effective against inoculated Gram-positive and -negative bacteria, is a strong inhibitor of bacterial β-lactamases, and can be used for the treatment of humans and livestock. 890A ₁₀ is Gram-positive or negative bacteria,
For example, Staphylococcus aureus, Proteus mirabilis, Klebsiella pneumoniae and Salmonella pneumoniae.
can be used alone or in combination with other antibacterial agents for the treatment of infections caused by Schottmuelleri). The compounds of the invention are β-lactamases sensitive β
- It can be used to enhance the activity of β-lactam antibiotics and extend the life of the antibiotic by inhibiting lactamase activity when mixed with lactam antibiotics. Therefore, a mixture of antibiotic 890A ₁₀ and a lactamase-sensitive antibiotic is more effective in treating infections caused by β-lactamase-producing bacteria than the same amount of β-lactamase-sensitive antibiotic alone. Antibiotic 890A ₁₀ can be used as an additive in animal feed for the preservation of feed and as a fungicide. The antibiotic is used to destroy and inhibit the growth of harmful bacteria on medical and dental instruments, and as a disinfectant in industrial applications, with a water content ranging from about 0.1 to 100 parts of antibiotic per million parts of solution. , preferably at a concentration in the range of about 1-10 parts of antibiotic per million parts of solution. Antibiotic 890A ₁₀ can be used in the preparation as the sole active ingredient or in admixture with one or more other antibiotics or in admixture with one or more pharmacologically active substances. Antibiotics can be administered orally, topically, intravenously or intramuscularly. The method of administration may be any conventional method in the art, ie any method applicable to the antibiotics mentioned above. Furthermore, in addition to excipients, the composition may contain other ingredients such as well-known and commonly used stabilizers, binders, antioxidants, preservatives, lubricants, suspending agents, thickening agents or flavorants. It can also contain In veterinary medicine, such as chickens, cows, sheep, pigs, etc., the compositions can take the form of intramammary preparations, for example in long-acting or rapidly disintegrating bases. Dosage depends on the condition of the patient being treated, weight and type of infection,
Although highly dependent on the route and frequency of administration, injections are preferred for common infections, and oral administration is preferred for enteric infections. In the treatment of bacterial infections in humans, compounds according to the methods of the invention can be administered orally or parenterally in conjunction with β-lactamase sensitive antibiotics according to conventional methods of antibiotic administration. Antibiotic 890A ₁₀ is about 2-600mg/Kg/day, preferably about 5-100mg/day.
Kg/day, preferably administered in divided doses, for example 3-4 times a day. This anti-inflammatory substance is, for example, 100,
It can be administered as a dosage unit containing 330, 400 or 1000 mg of active ingredient together with suitable physiologically acceptable excipients. The dosage unit can be in the form of a liquid preparation, such as a solution or suspension, or a solid, such as a tablet or capsule. It should be understood, of course, that the optimum dosage in any case will depend on the type and severity of the infection to be treated, with lower doses being used for children, and adjustments thereof are common knowledge to those skilled in the art. Antibiotic 890A ₁₀ is non-toxic and can be used in medicine.
Also used as salt. Pharmaceutically usable salts made e.g. with inorganic and organic bases; e.g. from alkali metal or alkaline earth metal hydroxides, carbonates or bicarbonates such as sodium, potassium, ammonium and calcium salts; metal salts and monoalkylamines, dialkylamines, trialkylamines, lower alkanolamines, di-lower alkanolamines, lower alkylene amines, N,N-diaralkyl lower alkylene diamines, aralkylamines, amino-substituted lower alkanols, N,N- Included are salts derived from primary, secondary or tertiary amines such as di-lower alkylamino-substituted lower alkanols, aminopolyamino and guanidino-substituted lower alkanoic acids and nitrogen-containing heterocyclic amines. Typical examples include sodium hydroxide, ammonium hydroxide, sodium carbonate,
Sodium bicarbonate, potassium carbonate, potassium hydroxide, calcium carbonate, trimethylamine, triethylamine, piperidine, N-ethylpiperidine, morpholine, quinine, lysine, protamine, arginine, procaine, ethanolamine, morphine, benzylamine, ethylenediamine, N,N '-Dibenzylethylenediamine,
Includes salts obtained from diethanolamine, piperazine, dimethylaminoethanol, 2-amino-2-methyl-1-propanol, theophylline, N-methylglucamine, and the like. Salts of compounds according to the method of the invention can be isolated directly from the fermentation medium by using a suitable eluent during ion exchange chromatography, or prepared according to conventional methods in the art. For example, a di-salt such as the disodium salt can be obtained by reacting 1 mole of the product () with 2 equivalents of sodium hydroxide in a suitable solvent. Furthermore, a mixed salt of monovalent cations can be used to convert 1 mole of monovalent base into 1 mole of product ()
Prepared by mixing with +1 mole of other base. Alternatively, monobasic salts can be obtained by reacting one equivalent of base with a monovalent cation with one mole of product (). Additionally, salts can be obtained by adding 1 mole of product to 1 mole of base with divalent cations. The salts of the invention are non-toxic derivatives which can be used as pharmaceuticals and can be used as active ingredients in suitable unit dosage forms. They can also be mixed with other agents to provide compositions with a broad spectrum of activity. Antibiotics produced according to the method described herein
A fermentation solution containing 890A ₁₀ has a concentration of approximately 2-170% per ml when assayed by the disk diffusion method using Vibrio percolans (ATCC8461).
unit of activity. The antibiotic 890A ₁₀ contained in these fermentations can be recovered and purified in a number of ways. One way is antibiotics
It is characterized by sorbing 890A ₁₀ onto a strongly basic anion exchange resin. Typical such strongly basic anion exchange resins include those having a styrene divinylbenzene matrix, such as polystyrene core quaternary ammonium resin Dowex 1×2.
(Dow Chemical Company, Midland, Michigan)
There is a chlorine type. Other representative examples of strong base exchange resins of this type include: Duolite A-
40, A-42, A-101, A-102, and A-114 (Chemical Process, Inc., Red City, Calif.). Amberlite IRA−400, IRA
-401 and IRA-410. Alternatively, weakly basic anion exchange resins such as Amberlite IRA-68 can also be used. (Amberlite resin is manufactured by Rohm and Haas, Washington Square, Philadelphia 5, Pennsylvania). The adsorbed antibiotic can be easily eluted from the anionic resin with a salt solution in 80% (V/V) aqueous methanol. The eluate thus obtained can be further purified by other purification methods. The eluate is thus concentrated and passed through a column packed with polystyrene, non-polar, hydrophobic cross-linked divinylbenzene polymers such as XAD-1, 2 and 4, or polyacrylamide resins such as XAD-7 and 8. Can be purified. XAD-2 is preferred. (XAD−
1, 2, 4, 7, and 8 are manufactured by Rohm and Haas, Washington Square, Philadelphia 5, Pennsylvania). The method for obtaining further purified antibiotic 890A ₁₀ is to use Biogel P-2 (manufactured by Bio-Rad, Richmond,
The method is to use gel filtration through a polyacrylamide gel with a pore size that excludes molecules with a molecular weight of 1800 or higher, such as (California). Other gels such as Sephadex G-10 can also be used for desalting. A preferred method by which the antibiotic 890A ₁₀ can be obtained in high purity from the culture broth consists of: centrifuging or filtering the culture solution to remove solids; The filtrate is adsorbed in the chlorine form and eluted with 3% NaCl in 80% (V/V) aqueous methanol, which concentrates the antibiotic and
Partially purify; pass through an appropriately prepared XAD-2 column, which retards the antibiotic and therefore purifies and desalts the Dowex 1×2 eluate. 890A ₁₀ concentrated fractions are combined and further purified. Chromatography of Dowex 1×2 minus 400 mesh resin is 80% NaCl in aqueous methanol.
and/or elution with NH ₄ Cl gives a clean product with initial UV absorption (NH ₄ Cl
(used to provide some buffering capacity to the eluate) 890A ₁₀ is separated from other antibiotics; desalting with Biogel P-2 or Sephadex G-10 removes the salts introduced with Dowex 1x2 chromatography. except for most of the. For remaining impurities, dowex 1x once more.
2. It can be reduced by chromatography at minus 400 meshes, eluting with a solution containing sodium chloride and 30% methanol, and then desalting. In purification by column chromatography, generally only fractions containing at least 30% pure antibiotic compared to the purest fraction in the eluate are combined for subsequent purification. Purity criteria are biological activity/
It is the ratio of A ₂₂₀ , A ₃₀₀ /A ₂₅₀ and HAEA ₃₀₀ /A ₂₂₀ ; in desalination it is the electrical conductivity. The A ₂₂₀ , A ₂₅₀ , A ₃₀₀ and biological activity of the appropriate fractions are thus determined at each chromatographic step.
If possible, also measure HAEA ₃₀₀ , and when desalting, measure electrical conductivity. The criteria for determining which fractions should be combined for further manipulation can be adjusted to either sacrifice purity in order to obtain high yields or conversely to sacrifice yield in favor of high purity. For laboratory scale operations (less than 20 sample volumes), all chromatography except XAD-2 chromatography is performed in a cold room at 2-5°C. XAD-2 chromatography is performed at room temperature. The pH of the antibiotic solution to be stored is dilute NaOH or
Adjust to 7-8 by carefully adding HCl solution. Store the aqueous solution in the refrigerator or preferably in ice water for 50 minutes.
%-80% methanol solution is stored at -20 to -60°C. In the purification step prior to XAD-2 chromatography, the solution is generally pH adjusted by adding sodium hydroxide.
0.1M Na ₂ EDTA solution neutralized to 7.0 (0.1“neutral
Add 1/4000 amount of EDTA") to make 25 μM EDTA. The flow sheet of the purification method to obtain antibiotic 890A ₁ is shown in Figure 1.

[Table] Assay method for antibiotic 890A ₁₀ Bioassay Use the agar plate disk diffusion method using Vibrio percolans ATCC8461 as the test bacterium. A pure version of antibiotic 890A ₁ is used as a standard. Antibiotic 890A ₁ is prepared according to the method set forth in Example 6. Vibrio percorans (Vibrio
Plates containing Vibrio percolans ATCC8461 are prepared as follows:
percolans ATCC8461 was suspended in 15 ml of a sterile medium containing 8 g of Difco Nutrient Broth and 2 g of yeast extract in distilled water, "Nutrient Broth - Yeast Extract" (hereinafter referred to as NBYE). become cloudy The culture is grown overnight at 28°C on a rotating shaker. This culture solution
It was used to inoculate the surface of a slant containing 1.5% agar in NBYE, and the inoculated slant was
Incubate overnight and then store in the refrigerator. Refrigerated slants prepared from one freeze-dried culture can be used for up to 4 weeks after preparation as follows: 1ase cells from the slant are suspended in 50 ml of NBYE in a 250 ml Erlenmeyer flask. The cells were cultured overnight at 28°C on a rotary shaker, and then diluted to a concentration of 50% transmittance at 660 nm.
33.2ml of this diluted culture solution containing 15g of agar
Add to NBYE1 and keep at 46℃. Pour the inoculated agar-containing medium into a 100 x 15 mm plastic petri dish.
Pour in ml portions, cool and keep for up to 5 days before use 2-4
Keep at ℃. A 1/2 inch diameter paper disk is immersed in the solution to be assayed and placed on the agar. Alternatively, pipet 0.1 ml of the solution onto a dry disc and then place the disc onto the agar. Appropriate incubation (12 at 25°C)
-24 hours), measure the diameter of the inhibition circle. If necessary, dilution of the solution to be assayed is performed with 0.05M potassium phosphate buffer, PH 7.4 "potassium phosphate buffer" (hereinafter referred to as KPB) or deionized water. Calculation of the titer is carried out as follows; the slope is determined from the measurement of the inhibition circle diameter of a ₁₀ solution of antibiotic 890A and a 4-fold dilution of this solution in KPB.
Two discs of each concentration are assayed on one plate and the average inhibition circle diameter for each concentration is determined. The slope is equal to 1/2 the difference in mean stopping circle diameter. The titer is then calculated by the following formula: titer (units/ml) = (titer of standard) x dilution factor x 10 ([D-Ds] log ₂ /slope) where D is due to the unknown sample. The mean diameter of the inhibition circle, Ds, is the mean diameter of the standard inhibition circle, and the "dilution factor" is the factor by which the unknown sample was diluted before assaying. If you don't use standards, Biblio
Percolans (Vibrio percolans) ATCC8461
When measuring above, assume that Ds is 25 mm and (standard titer) is 1 unit/ml. Pure 890A ₁₀
250 per unit of hydroxylamine quenchable absorption at 300 nm when used as a standard.
Defined as having unit potency. Assay method for measuring “890 assay units” A simple agar plate disk diffusion method is Vibrio percolans.
Perform using ATCC8461 as the test bacteria. Cephaloridine is used as a standard. Plates containing Vibrio percolans ATCC8461 are prepared as follows. Vibrio percolans ATCC8461 cells were cultured overnight at 28°C on a rotary shaker in nutrient broth-yeast extract, then 660n
Dilute to a concentration of 60% transmittance with m. diluted culture solution
Add 33.2 ml to medium 1 consisting of nutrient agar + 0.2% yeast extract kept at 46°C. Pour 10 ml of the inoculated agar medium into a 100 x 15 mm Petri dish,
Cool and keep at 2-4°C for up to 5 days until use. A concentration of cephaloridine equal to 890A ₁ 1 units/ml is prepared as described above, but determined as follows by assay on plates containing 5 ml of inoculum medium per plate. Four concentrations of cephaloridine constitute the standard--3.12, 6.25,
12.5 and 25 μg/ml, and 12.5 μg/ml as a control solution. The standard inhibition circle diameter on a 5 ml plate is: Concentration (μg/ml) Inhibition circle diameter (mm) 3.12 16.8 6.25 22.3 12.5 25.0 25 29.9 Units are 25 mm on a 5 ml plate as stated above. Defined as the amount of antibiotic per ml that creates an inhibition zone. Therefore, in this assay, 12.5μg/
cephaloridine at a concentration of 1 unit/ml
It is considered to be equal to 890A ₁ . The slope of the straight line for cephaloridine is 4.0, so the titer of the sample is calculated using a slope of 4.0. Hydroxylamine Reaction Antibiotic 890A ₁₀ reacts with hydroxylamine to produce a substance with greatly reduced absorption at 330 nm. This provides the basis for a quantitative assay of antibiotic 890A ₁₀ . Add 0.8MK ₂ HPO ₄ and
Add 1/20 volume of solution containing 0.2MKH ₂ PO ₄ to 0.05M
Potassium phosphate, pH 7.4. Next, add 100 amounts of 1M hydroxylamine hydrochloride and measure the absorbance at 300 nm at 1/2 to 2 minute intervals. The reaction is carried out at room temperature. Assuming a first-order reaction, the half-life is determined from the decrease in absorbance during the first 10 minutes.
From this half-life, the time at which the absorbance no longer decreases is determined, and the reaction is continued beyond that time. If no decrease is seen after that time, the total decreased absorbance (corrected for dilution effects and hydroxylamine absorption) is taken as the "hydroxylamine annihilating absorption (HAEA ₃₀₀ )" at 300 nm. If the absorbance decreases after that time, calculate the rate of background absorbance decrease and correct the observed absorbance at that time for the background decrease, assuming that the background decrease is proportional to time. then enter the correct value
Record as HAEA ₃₀₀ . The value of HAEA ₃₀₀ units is equal to HAEA ₃₀₀ multiplied by the volume in ml. The following examples illustrate how to obtain products according to the process of the invention. However, it will be apparent to those skilled in the art that the examples are for illustrative purposes only and that the present invention includes methods for making functionally equivalent products. Any modification of the method described herein should therefore be considered as a similar method. The methods described are susceptible to significant variations and modifications, and any minor variations and extensions are common knowledge and within the scope of the present invention. Cassiday et al., pending U.S. patent application no.
Antibiotics described in Application No. 634300, 1975, 11, 21
The preparation of 890A ₁ is included here for reference. Example 1 Slant containing MA-4638 containing 50ml of A medium
Use to inoculate a 250ml pleated Erlenmeyer flask. A medium dextrose 10.0g Yeast autolysate [Ardamine] ^*
10.0g MgSO ₄・7H ₂ O 0.05g Phosphate buffer ^** 2.0ml Distilled water 1000ml PH6.5 *Ardamine: East Products Company** Phosphate buffer solution: KH ₂ PO ₄ 91.0g Na ₂ HPO ₄ 95.0g distilled water 1000ml flask at 28℃ on 220rpm sifter, amplitude 2
Shaking at 5.1 cm (5.1 cm) for 2 days is sufficient for growth. After 2 days, this seed is used to inoculate three 250 ml Erlenmeyer flasks containing 40 ml of B medium, 2 ml per flask (5%). B medium tomato paste 20.0g Oats (powder) 20.0g Distilled water 1000ml PH: Adjusted to 7.0 using NaOH These production flasks were also kept at 28℃ and 220rpm.
Shake on a shaker during incubation for up to 4 days while assaying. On the third day, a portion of the centrifuged culture supernatant is subjected to classification and identification experiments. This culture solution is
Using a 1/4 inch (0.63 cm) assay disk on a standard assay plate, a 22 mm inhibition circle for Proteus vulgaris and a 22 mm inhibition zone for Salmonella gallinarum.
gallinarum) provides a 15mm inhibition circle.
Bioassay of electrophoretic paper of the centrifuged culture shows the presence of two components. A small spot moving far away is 890A ₁₀
including. Example 2 Two frozen vials each containing 2 ml of MA-4638 cells are slowly thawed and the contents are aseptically transferred to two seed flasks each containing 500 ml of C medium. A flask of this type, ie a shake flask with two or three pleats equipped with side branches, is plugged with cotton plugs. C medium Autolyzed yeast (Aldamine ^* ) 10.0g Glucose 10.0g MgSO ₄ -7H ₂ O 0.05g Phosphate buffer ^** 2.0ml Distilled water 1000ml Adjust pH to 6.5 with NaOH before sterilization *Ardamine: Yeast Products Company** Phosphate Buffer Solution: 91.0 g KH ₂ PO ₄ 95.0 g Na ₂ HPO ₄ 1000 ml distilled water Place the inoculated seed flask at 28°C ± 1°C on a shearer rotating at 210 rpm, at a 2-inch (5.1 cm) amplitude.
Shake for 30 hours. The culture solution in the seed flask was converted into production medium (D medium + 0.15
Used to inoculate two 14 glass fermenters containing 10% soybean oil, V/V). D medium dextrin (CPC processed starch) 40.0g Daysteelers Solubles 7.0g Yeast extract 5.0g CoCl ₂ 6H ₂ O 50.0g Distilled water 1000ml Adjust pH to 7.3 with NaOH before sterilization Set the fermenter at 24℃ and stir at 640rpm (about Kd5.5),
Operate for 72 hours at an airflow rate of 0.5VVM. An antifoaming agent, Hodag-MF (Hodag Chemical Co.), is used as necessary but not to exceed 0.1%. When the contents of two fermenters are combined after culturing, the total amount is approximately 20
becomes. Add 200 ml of culture solution to Servall
Centrifuge at 9000 rpm for 20 minutes in an RC-2B centrifuge.
The supernatant is filtered through a 1 cm layer of Hyflo Supercell in a 13 inch (33 cm) Lapp funnel equipped with a strainer cloth. The filtrate has 20 units/ml of active substance. Dowex the culture solution (18) - 1x2
(Cl ^- ) in a column (8.2 x 29 cm) of 50−100 meshes.
Pour at 150ml/min. Next, fill the column with 1 ml of deionized water.
, followed by 0.15M NaCl + 0.01M in 50% MeOH
Tris-HCl buffer, PH7.0+25μM neutral
Wash with EDTA, 15 at 150 ml/min. Then antibiotic 890A ₁₀ 3.2% NaCl in 80% MeOH
+0.02M Tris-HCl buffer PH7.0 +25μM neutral
Elute with EDTA at 100ml/min. Collect fractions as follows: 1 fraction of 1, 4 fractions of 500 ml, and 12 fractions of 1. Biological activity of each fraction and
Measure the absorbance at 220nm and determine the biological activity/A ₂₂₀ ratio.
Fractions with 0.6 units/A ₂₂₀ units or more and 10% or more of the total recovered activity per fraction are combined and used for the next purification. Thus, fractions 4-8 together contain 22% of the total bioactivity. The combined fractions are concentrated in vacuo to 190 ml, the precipitated salts are washed with deionized water, and the wash water is added to the supernatant to give a final volume of 220 ml. Adjust the concentrated solution to PH6.5 with HCl,
Amberlite
Apply to column (6.0 x 59 cm). After loading, the column is washed with 5 x 10 ml portions of deionized water, and the antibiotic is then eluted with deionized water at 35 ml/min. Fractions were collected as follows: 1 500 ml fraction, then 7 250 ml fractions, then 4 500 ml fractions. Biological activity and HAEA ₃₀₄ values are determined for fractions 3-12. Fractions 4-9 are HAEA ₃₀₄ /A ₂₂₀ ratio
0.01 or more and will be used in the next purification. Fraction 3, which contains about 1/5 of the salt applied to the column, is also added to this combined fraction pool. The combined eluate was
469HAEA with ₃₀₄ units. Dilute the combined fractions to 4.1 and dowex 1x.
4 (Cl ^- ) minus 400 mesh column (2.15×42
cm) at a rate of 2 ml/min. Column with 50% methanol
Wash with 100ml of antibiotics 0.25M in 80% MeOH
NaCl + 0.11M NH ₄ Cl + 0.0001M NH ₃ approximately 2
Elutes at ml/min. Collect 8-12 ml fractions. Bioactivity and absorbance at 220nm, 260nm and 300nm are measured in every 5 fractions and HAEA ₃₀₀ is determined on the bioactive fraction. 890A ₁₀ biological activity appears in fractions 85-150, with a maximum in fraction 120. Fractions with HAEA ₃₀₀ /A ₃₀₀ values greater than 0.05 are combined for subsequent purification. Fractions 105-133 thus together contain a total of 130 HAEA ₃₀₀ units. combined fraction
Dilute 105−133 to 1900ml with deionized water and adjust the pH to 7.6.
Adjust to. The sample is applied to a Dowex 1 x 2 (Cl ^- ) minus 400 mesh column (2.15 x 42 cm) that has been previously washed with 3% NaCl in 50% methanol and then 50 ml of 50% methanol. After applying all samples,
Wash the column with 100ml of 30% methanol, then 30%
0.26M NaCl + 0.005M NH ₄ Cl + in methanol
Elute with 0.0002M NH ₃ at 2 ml/min. 10−12ml
Collect the fractions. The main peak of 890A ₁₀ appears in fractions 230−290;
The maximum is at fraction 260. Fractions with an A ₃₀₀ /A ₂₅₀ ratio of 1.10 or more and an A ₃₀₀ /A ₂₂₀ ratio of 0.83 or more are combined for the next purification. In this way, by combining fractions 240−275, we get
91.6HAEA Contains ₃₀₀ units. The combined fractions 240-275 are concentrated under reduced pressure to 10 ml, and the concentrate is separated from the precipitated salts with a pipette. Wash the salt with 2 ml of deionized water and add the washing water to the concentrate.
12 ml of the combined concentrate and wash water were prewashed with 30 ml of saturated NaCl in deionized water, followed by 1500 ml of deionized water and 50 ml of 0.05 mM NH ₃ in deionized water on a Biogel P-2 (200-400 mesh) column (2.15
×76cm). Deionizing the column after loading
Wash with 3 x 1 ml of 0.05mM _NH3 and elute the antibiotic with 0.05mM _NH3 in deionized water at 0.73ml/min. Collect 3.65 ml fractions. The main antibiotic peak appears in fractions 29-50, with a maximum in fraction 36. Fractions with an A ₃₀₀ /A ₂₅₀ ratio of 1.9 or more and an A ₃₀₀ /A ₂₂₀ ratio of 1.45 or more are combined for subsequent lyophilization and NMR analysis. Thus fraction 36
−45 together, it contains 50.8A ₃₀₀ units, of which
45.1 is hydroxylamine quenchable. The combined fractions 36-45 were concentrated under reduced pressure to 0.1 ml and diluted with 5 ml of D ₂ O.
Add. The sample is again concentrated to 0.835 ml, of which 0.825 ml is transferred to a glass vial and lyophilized. The freeze-dried sample has ₃₀₀ units of 48.2A, solid
Contains 4.6mg. This is named sample 9441-154. End fractions 31-34 and 46-49 are combined to obtain an "end pool" which is concentrated to 0.1 ml and quickly lyophilized.
These fractions contain ₃₀₀ units of 24A, of which 17 are hydroxylamine, quenchable. This sample
Call it 9441−155. Example 3 A frozen vial containing 2 ml of MA-4638 culture is gently thawed and the contents are aseptically transferred to a seed flask containing 500 ml of C medium. Seed flask, 2 of 3 fluted shake flasks with side spouts are plugged with cotton plugs. C medium autolyzed yeast (Aldamin ^* ) 10.0g Glucose 10.0g MgSO ₄・7H ₂ O 0.05g Phosphate buffer ^** 2.0ml Distilled water 1000ml Adjust pH to 6.5 with NaOH before sterilization *Aldamin: Yeast Products Co. * *Phosphate buffer solution: 91.0 g KH ₂ PO ₄ 95.0 g Na ₂ HPO ₄ 1000 ml distilled water Shake the inoculated seed flask for 36 hours at 28°C ± 1°C on a shaker rotating at 210 rpm with a 2 inch (5.1 cm) shaker. . The culture solution of this seed flask is used as production medium (D medium +
Used to inoculate a 14 glass fermenter containing 0.15% soybean oil, V/V) 10. D medium dextrin (CPC processed starch) 40.0g Daystellers Solubles 7.0g Yeast extract 5.0g CoCl ₂ 6H ₂ O 50.0mg Distilled water 1000ml Adjust pH to 7.3 with NaOH before sterilization Set the fermenter at 24°C with stirring at 550 rpm ( Kd3.0−3.5),
Operates at an airflow rate of 0.5VVM. Antifoaming agent Hodag MF
(Hodag Chemical Company) in the required amount not exceeding 0.1%. Culture broth is collected after 72 hours of fermentation. Centrifuge 200 ml portions of the culture solution at 900 rpm using a Serval RC-2 centrifuge. The supernatant contains 7 with 39 bioassay units per ml. The supernatant is filtered through a ratproat containing a 1 cm layer of Hyflo Supercell on a strainer. liquid
Add 3.25 ml of 0.1 M neutral EDTA to 6.5, and then apply the filtrate to a Dowex 1 x 2 (Cl ^- ), 50-100 mesh column (8.2 x 21.5 cm) at 60 ml/min. After addition, the column was washed with deionized water 1, then 50%
Wash at 50 ml/min with 0.15 M NaCl + 0.02 M Tris-HCl buffer in MeOH, PH 7.08 + 25 μM EDTA12. Then add antibiotic 890A ₁₀ to 3% NaCl in 80% MeOH.
+0.02M Tris-HCl buffer, PH7.1 +25μM
Elute with EDTA 12.3 at 60 ml/min. Collect fractions as follows: 1 fraction of 500 ml, 200 ml
4 fractions, 500ml 12 fractions and 15 fractions. Biological activity appears in fractions 3-17, and is maximal in fractions 6, 7, and 8. Fractions with a biological activity/A ₂₂₀ value of 0.9 units/A ₂₂₀ units or more and containing 8% or more of the total recovered activity per fraction are combined and used for the next purification. Thus fraction 6-
12 together contain 106,000 bioactivity units or 41% of the added bioactivity. These combined fractions are stored at -60°C until combined with fractions 6-13 from the second Dowex column described in Example 4. Example 4 Two frozen vials containing 2 ml of MA-4638 culture are gently thawed and the contents are aseptically transferred to two seed flasks each containing 500 ml of C medium. A flask of this type, a 2-3 fluted shake flask with a side arm, is plugged with cotton. C medium autolyzed yeast (Aldamin ^* ) 10.0g Glucose 10.0g MgSO ₄・7H ₂ O 0.05g Phosphate buffer ^** 2.0ml Distilled water 1000ml Adjust pH to 6.5 with NaOH before sterilization *Aldamin: Yeast Products Co. * *Phosphate buffer solution: 91.0 g KH ₂ PO ₄ 95.0 g Na ₂ HPO ₄ 1000 ml distilled water Shake the inoculated seed flask at 28°C ± 1°C on a 210 rpm rotating sheaker for 36 hours at a 2 inch (5.1 cm) amplitude. . Add the culture medium of the seed flask to 2 containing D production medium 10
Used to inoculate 14 glass fermenters. D medium dextrin (CDC processed starch) 40.0g Daystellers Solubles 7.0g Yeast extract 5.0g CoCl ₂ 6H ₂ O 50.0mg Distilled water 1000ml Adjust the pH to 7.3 with NaOH before sterilization. Two fermenters were stirred at 24°C at 550 rpm (Kd3.0−
3.5), operate at an airflow rate of 0.5VVM. Use an antifoaming agent, polyglycol 2000, as much as necessary, not exceeding 0.1%. The cultures from the two fermenters are removed after 72 hours. 10 200 ml portions of each culture solution in ServeR.
Centrifugation at 9000 rpm yields supernatant 9 from each batch.
get. The bioassay shows that the batch has 23.4 units/ml and the batch has 31.8 units/ml. Both batches are combined, cooled and filtered through a 1 cm Hyflo Supercell layer on a 13 inch (33 cm) LabTek porcelain Buchner funnel. liquid 15
Add 7.5 ml of 0.1 M neutral EDTA to the solution. Add the filtrate to a Dowex 1 x 2 (Cl ^- ), 50-100 mesh column (13.4 x 16 cm) at 100 ml/min. After addition, the column was then washed 50% with deionized water 2.
Wash with 0.15M NaCl + 0.01M Tris-HCl buffer in MeOH, PH 7.01 + 25 μM EDTA20 at 40-80 ml/min. Next, add antibiotic 890A _10% to 3% in 80% MeOH.
NaCl + 0.02M Tris-HCl, PH7.0 + 25μM EDTA
Elute with solution 24.4. Collect fractions as follows: 1 fraction of 800 ml, 4 fractions of 400 ml, 12 fractions of 1000 ml,
and 5 fractions of 2000ml. Antibiotic activity appears in fractions 5-16 and is maximal in fractions 7 and 8. The total activity of these fractions is
212,000 units, equal to 51% of the added activity. Fractions 6-13 are combined for subsequent purification. The combined fractions 6-13 from the Dowex column were added to the combined fractions 6-12 from the Dowex column of Example 3 in a steam-jacketed long tube evaporator.
Concentrate under reduced pressure to 2.6. This concentrated solution is further concentrated under reduced pressure to 300 ml using a rotary evaporator, and precipitated salt crystals are filtered off. Add 1.7ml of concentrated hydrochloric acid to the concentrated solution to adjust the pH to 6.48.
Apply to an Amberlite XAD-2 column (8.1 x 59 cm) and wash the column with 3 x 20 ml of deionized water. Elute the antibiotic with deionized water at a flow rate of 60 ml/min.
Collect fractions as follows: 11 fractions, 500ml 7
The fractions and 8 1000 ml fractions were collected and cooled on ice immediately after collection. The biological activity and HAEA ₃₀₀ values of each fraction were determined as A ₂₂₀ ,
Measured together with A ₂₆₀ and A ₃₀₀ values. HAEA ₃₀₀ /
Fractions with A ₃₀₀ values greater than 0.075 are combined for subsequent purification. Fractions 5-9 thus together have 164,000 bioassay units. The combined fractions were concentrated under reduced pressure to 100 ml and diluted with methanol.
Add 200ml. Next, place the sample in a dowex 1x4
(Cl ^- ) minus 400 mesh column (2.15 x 38cm)
at a rate of 2 ml/min. After addition, the column was washed with 100 ml of 80% methanol and the antibiotics were washed in 80% MeOH.
0.26M NaCl＋0.005M NHCl＋0.00005M _NH3
It elutes with Collect 10 ml fractions. Biological activity and A ₃₀₀ , A ₂₅₀ , A ₂₂₀ and HAEA ₃₀₀
Values are determined every 5 fractions. Fractions with a HAEA ₃₀₀ /A ₂₂₀ ratio of 0.07 or higher are combined for the next purification. Fractions 125-150 are thus combined. Combined fractions from Dowex 1×4 column
Add 125-150 to the lyophilized sample 9441-155 of Example 2 redissolved in 1 ml of deionized water. This combined sample was diluted to 1900 ml with deionized water and diluted with Dowex 1x.
2 (Cl ^- ) column (2.15 x 40 cm, minus 400 mesh) at 2 ml/min. Wash the column with 150 ml of 30% methanol, then add antibiotics in 30% MeOH.
0.25M NaCl + 0.005M NH ₄ Cl + 0.00005M NH ₃ 6
Elute at 2 ml/min. Collect each 11.1 ml fraction. Absorbance at 300nm, 250nm, and 220nm
Measure each fraction. The main peak of antibiotic 890A ₁₀ appears in fractions 360-430, with a maximum in 394-400. All fractions with A ₃₀₀ /A ₂₂₀ ratio greater than 1.30 are combined for subsequent purification. Thus, fractions 380-420 are combined. These combined fractions have ₃₀₀ units of 202A,
Of these, 197 are hydroxylamine quenchable. Example 2 The combined fractions 380-420 contain ₃₀₀ units of 30.5 HAEA dissolved in 1 ml D ₂ O and 4 ml deionized water.
Add sample 9441-154. The combined sample was diluted with 2 parts of deionized water, 1 × 2 parts of Dowex (Cl ^- ),
0.6− to 200−400 mesh column (2.15×41cm)
Apply at 2ml/min. Column 30% methanol 360
Wash with ml of antibiotic 890A ₁₀ 0.25M in 30% MeOH
1.9 with NaCl + 0.005M NH ₄ Cl + 0.00005M NH ₃
Elutes at ml/min. Collect 9.7 ml fractions. Absorbance at 300nm, 250nm, and 220nm
Measure each fraction. Antibiotics as judged by absorbance at 300nm
The main peak of 890A ₁₀ appears in fractions 270-358, with a maximum in fraction 315. Fractions with an A ₃₀₀ /A ₂₀₀ ratio of 1.75 or higher are collected and used for the next purification. Thus fraction 295
-335 includes 145A ₃₀₀ units, of which 137
is hydroxylamine quenchable. The combined fractions 295-335 are concentrated under reduced pressure to 10 ml, and the concentrated solution is adjusted to pH 7.0 with 1M NaOH. Transfer the concentrated solution to Biogel P-2, 200-400 mesh column (2.15
x 75 cm) and wash the column with 3 x 1 ml of deionized water. Elute the antibiotic with deionized water at 0.6 ml/min. Collect each 6.4 ml fraction. Electrical conductivity is measured in every two fractions along with absorbance at 300 nm, 250 nm, and 220 nm. 300n
The main peak of absorbance at m appears in fraction 9-27,
Fraction 14 is the maximum. Fractions 11-16 have electrical conductivities proportional to the A ₃₀₀ value and are subjected to mass spectrometry and NMR analysis together. The combined fraction is in 38.7ml
Contains 75.4A ₃₀₀ units. Of this, 34.7 ml is concentrated to 1.22 ml, and a 1.20 ml portion is lyophilized for NMR analysis to obtain 2.84 mg of solid material. The remaining 4 ml was converted to the NH ₄ ⁺ form through a Dowex 50W x 2 (NH ₄ ⁺ ) 200-400 mesh column (0.7 x 15 cm). The resulting ammonium salt is concentrated to 0.11 ml, a 10 μ aliquot is taken, 100 μ of deionized water is added, and four samples of 40 ml each are taken and separately lyophilized and subjected to mass spectrometry. [9441-209-2]. Biogel fractions 17-23 with ₃₀₀ units of 29.4A are concentrated to 1 ml and lyophilized to obtain 1.80 mg of solid. Example 5 Two frozen vials each containing 1 ml of MA-4638 culture were gently thawed and the contents were poured into each vial with 50 ml of C medium.
Aseptically transfer to two 250 ml three-fold Erlenmeyer flasks containing ml. Seal the flask with a cotton plug. C medium autolyzed yeast (Aldamin ^* ) 10.0g Glucose 10.0g MgSO _{4.7H 2} O 0.05g Phosphate buffer ^** 2.0ml Distilled water 1000ml Adjust pH to 6.5 with NaOH before sterilization. *Aldamin: East Products Company** Phosphate buffer solution KH ₂ PO ₄ 95.0 g Na ₂ HPO ₄ 95.0 g Distilled water 1000 ml Two seed flasks were heated at 28°C ± 1°C on a shaker rotating at 210 rpm with an amplitude of 2 inches (5.1 cm). ) for 20-24 hours. The contents are then combined and used to inoculate production flasks. D. Inoculate 10 two-fold shake flasks, each containing 300 ml of production medium, with 10 ml of culture from the seed flask per flask. Seal the upper production flask with gauze. D medium dextrin (CPC processed starch) 40.0g Daystellers Solubles 7.0g Yeast extract 5.0g CoCl ₂ 6H ₂ O 50.0mg Distilled water 1000ml Adjust pH to 7.3 with NaOH before sterilization After inoculation, store the production flask at 24℃±1 210rpm at ℃
3 on a rotating shearer with an amplitude of 2 inches (5.1 cm)
Incubate for hours. The contents of the flasks are combined and the culture fluid is subjected to bioassay. Harvesting time (hours) 72 PH 6.5 890 assay (units/ml) 30.45 Centrifuge 200 ml of the total culture solution 2 in the D production flask above at 9000 rpm to obtain 1.7 PH 6.8 supernatant. The supernatant is applied to a Dowex 1 x 2 (Cl ^- ) 50-100 mesh column (3.9 x 25 cm) at 5-10 ml/min. Fill the column with 50ml of deionized water and then add 50% (V/V)
0.15M NaCl + 0.02M Tris− in aqueous methanol
Wash with HCl buffer, pH 7.0 + 25 μM neutral EDTA2. The product was then dissolved in 80% (V/V) aqueous methanol.
30g/NaCl + 0.02M Tris-HCl buffer,
Elute with PH7.0 + 25 μM EDTA at a flow rate of 5 ml/min.
Collect 10 ml fractions. Biological activity appears in fractions 5-180 and 25-70.
has a maximum. Combine fractions 12-105 and concentrate under reduced pressure to 15 ml. The pH was adjusted to 6.5 with 1M HCl, then the concentrated solution was pre-mixed with 60% aqueous acetone, 2.5 each, and
Applied to an XAD-2 column (3.4 x 55 cm) washed with deionized water and 5% NaCl in deionized water. Wash the column with 3 x 5 ml of deionized water and rinse with 10 ml of deionized water.
Elutes in minutes. Collect 10 ml fractions. Biological activity appears in fractions 32-70, with a maximum in fractions 42-62. The total eluted bioactivity is 25% of the activity of the starting culture. Combine fractions 40-240 and use the XAD of Example 1.
Add the combined stock fractions 12-34 of -2 columns. All the combined fractions were concentrated under reduced pressure to 50 ml.
Contains 120HAEA ₃₀₄ units. Dilute the concentrated solution with 200 ml of methanol and preliminarily prepare 30 g/NaCl2 and 80% in 80% aqueous methanol.
Dowex 1x washed with 1% water-containing methanol
4 (Cl ^- ), minus 400 mesh column (2.15 x 40
cm) at 2 ml/min. After adding the sample, wash the column with 100 ml of 80% (V/V) aqueous methanol.
0.22M NaCl+ in 80% (V/V) aqueous methanol
0.01M NH ₄ Cl + 0.0002M NH ₃ 2nd 80%
(V/V) 0.31M NaCl + 0.01M in aqueous methanol
Elutes with NH ₄ Cl + 0.0002M NH ₃ 3. Adjust the flow rate to 2 ml/min and collect 10 ml fractions. Antibiotic 890A ₁₀ was separated on a Dowex 1x4 column, assayed on an ATCC 8461, and fractionated.
It is eluted at 155-195. Example 6 Antibiotic 890A ₁ Streptomyces flavogriseus MA−
The lyophilized tube of 4600NRRL8140 culture solution is opened aseptically and the contents are suspended in a test tube containing 1.5 ml of sterile E medium of the following composition. E medium yeast extract 10.0g Glucose 10.0g MgSO ₄・7H ₂ O 0.05g Phosphate buffer ^** 2ml Distilled water 1000ml **Phosphate buffer solution: KH ₂ PO ₄ 91.0g Na ₂ HPO ₄ 95.0g Distilled water 1000ml This suspension was mixed with 250ml of seed C medium containing 54ml of the following composition.
Used to inoculate 3 ml fluted Erlenmeyer flasks. C medium autolyzed yeast (Aldamin ^* ) 10.0g Glucose 10.0g MgSO ₄・7H ₂ O 0.05g Phosphate buffer ^** 2ml Distilled water 1000ml Adjust pH to 6.5 with NaOH *Aldamin: Yeast Products Co., Ltd.** Phosphate Buffer solution: KH ₂ PO ₄ 91.0g Na ₂ HPO ₄ 95.0g Distilled water 1000ml Seal this kind of flask with a cotton stopper and heat at 28°C ± 1°C.
On 220 rpm rotating shear car, amplitude 2 inches (5.1
cm) for 30 hours. Inoculate 1 ml per flask with cultures from 50 250 ml uncorrupted production flasks, each containing 40 ml of production medium. Put a cotton stopper on the production flask. F medium tomato paste 20.0g Yeast 10.0g Dextrin [Amidex] 20.0g _{CoCl2.6H2O 5.0mg} Distilled water 1000ml Adjust the pH to 7.2-7.4 with NaOH. After inoculation, the production flask was heated at 28°C ± 1°C at 220 rpm.
3 on a rotating shearer with an amplitude of 2 inches (5.1 cm)
Incubate for days. Using a 1/2 inch (1.3 cm) assay disk immersed in a centrifuged culture sample, standard Vibrio
Assay flask activity against Vibrio percolans ATCC8461 assay plate.
Dilute the sample with 0.05M phosphate buffer, pH 7.4. The results are shown in the table below. Collection time 72 PH 6.4 Vibrio percolans
(1/100 dilution) assay 23mm 890 assay, unit/ml 103 Pour the entire culture solution into 200ml portions in polycarbonate tubes.
Centrifuge at 9000 rpm for 15 minutes to obtain 1600 ml of supernatant with an activity of 104 units/ml. Add 0.5ml of 0.1M neutral EDTA to this. Centrifuge the culture solution in Dowex 1 x 2 (Cl ^- ), 50 -
Flow rate 6-20 on a 100 mesh column (3.8 x 22 cm)
Multiply by ml/min. Wash the column with 100 ml of deionized water, add 50 g of NaCl, 0.02 M Tris-HCl buffer,
Deionized water containing PH7.0 and 25 μM neutral EDTA 1
Elute at a flow rate of 6 ml/min. Collect 10 ml fractions. Antibiotic 890A ₁ is counted and fractionated from the addition of salt eluate.
It appears in fractions 13-81 and reaches a maximum in fractions 25-33. Fractions 24-41 with the highest bioactivity/A ₂₂₀ ratio are combined for subsequent purification. The combined fractions have a total of 29,000 units or 17% of the added biological activity. Concentrate the Dowex eluate to 10 ml, adjust the pH to 6.5 with dilute hydrochloric acid, and add
Washed with 60% aqueous acetone, deionized water, and 5% (w/v) sodium chloride in deionized water.
Apply to XAD-2 column (3.3 x 36 cm). The sample is eluted with deionized water at a flow rate of 6 ml/min. 40−260ml
Collect the fractions. Antibiotic activity appears in fractions 6-14 with an elution volume of 220
- over 2560 ml. The peak is in fractions 9-10,
Elution volume ranges from 370-590ml. Elution volume 370−1060ml
Fractions 9-12 have the highest HEAE ₃₀₀ /A ₂₂₀ and are combined for the next purification. These fractions are
It has 36600 units, equal to 126% of the added activity. The combined fractions 9-12 are concentrated to 100 ml and added to a Dowex 1 x 4 (Cl ^- ), minus 400 mesh column (2.2 x 41 cm) at a flow rate of 2 ml/min.
Wash the column with 50 ml of deionized water while deionizing.
0.07M NaCl + 0.005M NH ₄ Cl + 0.0001M NH ₃ 3
Elute at a flow rate of 2 ml/min. After adding eluent
Collect 10.8 ml fractions. The main peak of the antibiotic 890A ₁ appears in fractions 181-217, with a maximum in fraction 198. Fractions 186-210 with a total of 114 absorbance units at 300 nm are combined. Concentrate the combined fractions to 4.0 ml and add 16μ of 1M NaOH.
Add to adjust the pH to 7.3. Transfer the concentrated solution to Biogel P-2, 200-400 mesh, column (2.15×
70 cm), washed with 3 x 1 ml of deionized water, and eluted with deionized water at 0.96 ml/min. Collect 3.85 ml fractions. The main peak of antibiotic 890A ₁ appears in fractions 24-44, with maxima in fractions 33 and 34. Max A ₃₀₀ / A ₂₄₅
Fractions 27-38 with the ratio are combined for lyophilization. These combined fractions have a total of 72A ₃₀₀ units. Concentrate the combined fractions to 3.0 ml for lyophilization and adjust the pH to 7.5 by adding 10 µ of 0.1 M NaOH. Divide the sample into 2 parts of 1.50 ml each, each part containing 14 ml.
Rapidly cool and lyophilize in a screw cap glass vial. Each sample contained 1.73 mg of 890A ₁ , corresponding to ₃₀₀ units of 35.8A.

Claims (1)

[Claims] 1. 890A _10- producing bacteria of Streptomyces flavogriseus were cultured under deep aeration conditions in a water-containing nutrient medium containing assimilable sources of carbon, nitrogen, and inorganic salts, and the following structure was obtained: A method for producing antibiotic 890A ₁₀ , which comprises recovering antibiotic 890A ₁₀ having the following properties. 2. The production method according to claim 1, wherein the microorganism to be cultured is Streptomyces flavogriseus (Feikokenbokuyori No. 4284).