JPS6261317B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a process for the production of antibiotics DJ7164A, B ₁ and B ₂ and mixtures thereof by a new species of Streptomyces minuteispiralis. With the aim of discovering new microorganisms that produce useful antibiotics, the present inventors obtained a large number of microorganisms from nature and conducted repeated studies. The present invention was completed based on the finding that the strain numbered Mrs. sp.RHS5-4 was suitable for the purpose. The antibiotic-producing bacterium Streptomyces sp.RHS5-4 used in the method of the present invention was found to be a new species of actinomycete as a result of taxonomic research, and is Streptomyces minuteispiralis sp.nov.
(Streptomyces minutispiralis sp・nov). The present inventors discovered that this strain contains antibiotic DJ7164A, which has β-lactamase inhibitory activity, in the culture medium.
DJ7164B 1 was found to produce B ₁ and B ₂ , and as a result of isolating and identifying these substances, DJ7164B ₁ was found to be a known substance, epithienamycin B [17th Interscience Conference on Antimicrobial・Agents and Chemotherapy (17th Interscience Conference on
Antimicrobial Agents and Chemotherapy)
October 12-14, 1977 New York Lecture Abstracts No.80,
No. 81 (hereinafter referred to as ICAAC lecture abstract)]. Furthermore, DJ7164A and B ₂ were estimated to be substances similar to epithienamycins based on their physicochemical properties. In the present invention, the term DJ7164 substance refers to
The mixture of DJ7164A, DJ7164B ₁ and DJ7164B ₂ is collectively referred to as DJ7164B substance.
It is a generic term for a mixture of DJ7164B ₁ and DJ7164B ₂ . Streptomyces used in the method of the present invention
sp.RHS5-4 has the following mycological characteristics. Morphological characteristics The aerial hyphae of this producing fungus extend in a straight or curved shape from the basal hyphae, and form mainly compact and closed-type spirals at the tips of conidiophores formed as simple branches. . 10 to 30 conidia
When observed using an electron microscope, the shape is oval to short cylindrical, and the size is 0.5 to 0.8 μ x 1.0 to 1.4
μ and its surface is smooth. In addition, the basal hyphae elongate in a curved or wavy manner, and no breaks are observed. Axial branches, flagellated spores, sporangia and sclerotia are not observed. Growth status on various media Table 1 shows the growth status of those cultured on various media at 28°C for 21 days. [Table] Physiological properties Table 2 shows the results of tests conducted according to conventional methods. [Table] [Table] [Table] Availability of carbon sources L-arabinose, D-xylose, D-glucose,
D-fructose, sucrose, inositol, L-rhamnose, raffinose, D-mannite and glycerin are commonly used. To summarize the above results, it belongs to the genus Streptomyces. 2 Aerial hyphae form compact and closed type spirals. 3 The surface structure of conidia is smooth. 4 The color of aerial hyphae is light brownish gray to light gray and belongs to the gray color series. 5 The color of the back side is yellowish brown. 6. Produces almost no soluble pigment in various synthetic and organic media. 7 Does not produce dark brown pigment on melanin-like pigment forming medium. 8 Does not liquefy gelatin, and coagulation and peptonization of milk are both negative. can be mentioned. Based on the above properties, Bergey's Manual of
Determinative Bacteriology, 8th Ed.
Williams and Wilkins Company, 1974), The
Actionmycetes Vol. (SAWaksman, The
William and Wilkins Company, 1962), E.B.
Shirling and D.Gottlieb's ISP report (International Journal of Systematic
Bacteriology, 18 , 69, 1968; 18 , 279, 1968;
19 , 391, 1969; 22 , 265, 1972) and other domestic and international literature, it was found that Streptomyces rivani (Str.
libani), Streptomyces purchia (Str.
pulcher), Streptomyces nigerus (Str.
nigellus) and Streptomyces antimycoticus. Therefore, Streptomyces related to the present invention
As a result of simultaneous comparison between sp.RHS5-4 and ISP strains of these similar bacterial species, the following differences were observed between these similar bacterial species. 1 Streptomyces rivani ISP5555 Succeeds well with light gray aerial mycelium on sucrose/nitrate agar medium. Produces a small amount of yellow-brown pigment on glycerin/nitrate agar. A moderate amount of white aerial mycelium grows on ammonium tsapex agar medium, and the underside is pale yellowish orange. The underside appears pale yellowish brown to light yellow on starch agar medium. Good to moderate growth of white aerial mycelium on nutrient agar medium. Peptone yeast does not grow on iron agar. It coagulates skim milk and strongly peptonizes it. Does not utilize L-arabinose. 2 Streptomyces purchia ISP5566 Grows light gray aerial mycelia well on sucrose/nitrate agar medium. Grows poorly on light gray aerial mycelia on glycerin/nitrate agar medium, with pale orange to pale yellow-orange underside. present. Ammonium. A medium to poor amount of light brownish-gray aerial mycelium is established on Tzapecus medium. The back side appears pale orange on glycerin-asparagine agar medium. On tyrosine agar medium, the back side is pale orange to pale yellowish orange and does not produce soluble pigment. Medium to poor growth of white aerial mycelium on nutrient agar medium. Strongly peptonizes skimmed milk. Strongly liquefies gelatin. Do not use sucrose or raffinose. 3 Streptomyces nigerus ISP5490 Succeeds well with light gray aerial mycelium on sucrose/nitrate agar medium. Light gray aerial mycelium grows well on glycerin/nitrate agar medium, and the underside is pale yellow-brown with a partial orange tinge. A moderate amount of white aerial mycelium grows on Ammonium tsapecus agar medium. The underside appears dull yellow on yeast/malt agar. Peptone yeast does not grow on iron agar. Liquefy gelatin. Starch hydrolysis is negative. 4 Streptomyces antimycoticus
ISP5284 The conidia surface is thorn-like. Grey-white aerial mycelium grows well on sucrose/nitrate agar medium. Glycerin/nitrate agar medium is well covered with gray-white aerial mycelium, and the underside is bright reddish yellow to pale yellow. A moderate amount of white aerial mycelium grows on Ammonium tsapecus agar medium. On starch agar medium, the underside exhibits a pale yellowish brown to pale yellow color. The underside appears dull yellow on yeast/malt agar. The underside appears pale yellow on oatmeal agar medium. Liquefy gelatin. It coagulates skim milk and strongly peptonizes it. To date, as epithienamycin-producing bacteria, for example, Streptomyces flavogrizeus (Japanese Patent Application Laid-Open No. 52-65298 and JP-A No. 52-131596), Streptomyces fulvoviridis (Japanese Patent Application Laid-Open No. 53-1988)
103401 and JP 53-109997), Streptomyces olivaceus (JP 53-109997), Streptomyces argenteolus (JP 53-10999)
109997) and Streptomyces sp. A271 (Japanese Unexamined Patent Publication No. 54-92983), but both of them clearly differ from Streptomyces sp. in that their conidial chains do not form a spiral. RHS5-4
and have different mycological properties. Based on the above findings, there is no match among the known bacterial species, and it is appropriate to recognize Streptomyces sp.RHS5-4 as a new bacterial species, and Streptomyces sp.
It was named Minuteispiralis sp.nov. This bacterial strain has been deposited with the Institute of Microbiology, Agency of Industrial Science and Technology as FERM-P No. 5079. The DJ7164 substance-producing bacteria used in the method of the present invention includes the new species Streptomyces minuteispiralis and all of its natural or artificial mutant strains. That is, the new species Streptomyces minuteispiralis produces DJ7164 substance.
Includes all strains that cannot be clearly distinguished from sp.nov. Next, a method for producing the DJ7164 substance according to the present invention will be described. Streptomyces minuteispiralis sp.nov., which is the producing bacterium of substance DJ7164, is cultured under aerobic conditions in a medium containing various nutrient sources. Media composition and culture conditions may be similar to those commonly used for antibiotic production. That is, the medium basically contains a carbon source, a nitrogen source, inorganic salts, and the like. Vitamins, precursors, etc. may be added as necessary. Examples of carbon sources include glycerin,
Glucose, sucrose, starch, starch syrup, molasses, soybean oil, etc. are used alone or as a mixture. In addition, as a nitrogen source, for example, natural or inorganic substances such as soybean flour, defatted soybean flour, cottonseed flour, wheat germ, meat extract, peptone, yeast extract, dried yeast, cornstap liquor, ammonium sulfate, and sodium nitrate can be used alone. Or used as a mixture. Examples of inorganic salts include calcium carbonate,
Sodium chloride, potassium chloride, cobalt chloride,
Examples include magnesium sulfate and various phosphates, which are added to the culture medium as necessary. In particular, the addition of cobalt ions is effective in improving productivity. The most suitable culture method is the liquid culture method generally used for antibiotic production, especially the deep aeration culture method. The pH of the medium is preferably about 6.0 to 8.0, and the culture temperature is about 20 to 40°C, preferably 30°C.
It is best to adjust it to around the same value. Furthermore, an appropriate natural or synthetic antifoaming agent may be added to the culture solution as necessary. Confirmation of accumulation of DJ7164 substance is performed using Bacillus subtilis ATCC6633 (Bacillus subtilis) as the test bacterium.
The assay was carried out using the disk plate method using Escherichia subtilis ATCC 6633) or Escherichia coli K-12, and the inhibition circle was measured after culturing at 30°C for 18 to 20 hours. Usually, the amount of DJ7164 substance accumulated reaches its maximum after 50 to 120 hours of culture. The DJ7164 substance can be collected from the culture solution using its properties in accordance with the general method for collecting antibiotics. DJ7164 substance is soluble in water and mainly exists in the liquid part of the culture medium. After culturing, bacterial cells and other solid substances are removed by filtration using diatomaceous earth as a filter aid or by centrifugation, and the DJ7164 substance present in the filtrate or supernatant has the physical and chemical properties described below. It can be extracted and purified by using it. For example, by utilizing the fact that DJ7164 behaves as an acidic substance at a stable pH around neutrality, it can be purified by adsorption and desorption onto an anion exchanger. Examples of anion exchangers include DEAE-Sephadex, QAE-Sephadex (manufactured by Pharmacia), DEAE-Cellulose (manufactured by Braun), Diaion PA-306 (manufactured by Mitsubishi Kasei), and Amberlite IRA-68 (Rohm & Co., Ltd.).・Manufactured by Haas Corporation),
Dowex 1×4, Dowex 21K (Dowwex 21K)
(manufactured by Chemical Company), Duolite A-2, A-4
(Manufactured by Diamond Syamlok Chemical Co.)
etc. are available. In addition, using the properties of acidic substances, dimethyl
A method can also be used in which a quaternary ammonium salt such as benzyl cetyl ammonium chloride is dissolved in a water-immiscible solvent, such as dichloromethane, and the DJ7164 substance is extracted from the aqueous solution as the quaternary ammonium salt. It can also be purified using an adsorbent.
As an adsorbent, for example, synthetic adsorbent diamond ion
The DJ7164 substance is adsorbed using HP-20AG, HP-20, HP-30 (manufactured by Mitsubishi Kasei Corporation), Amberlite XAD-2, XAD-4 (manufactured by Rohm and Haas Corporation), or activated carbon powder. A method of elution with acetone water, methanol water, etc. can be used. Furthermore, in order to purify the DJ7164 substance, partition chromatography using a carrier such as Cererose or silica gel such as Avicel (manufactured by Asahi Kasei Corporation), or molecular sieve purification using differences in molecular weight, molecular shape, etc. Chromatography is effective. Suitable carriers for molecular sieve chromatography include Cephadex G-10 and G-15 (manufactured by Pharmacia) and Biogel P-2 (manufactured by Bio-Rad Laboratory). DJ7164 substances have similar properties
DJ7164A, a mixture of B ₁ and B ₂ ,
DJ7164A and DJ7164B substances (DJ7164B ₁ and
For separation from _DJ7164B2 mixture), it is effective to perform column chromatography using the above-mentioned anion exchanger, such as DEAE-Sephadex or QAE-Sephadex. Also,
To separate DJ7164B ₁ and DJ7164B ₂ , it is best to perform column chromatography using the synthetic adsorbent Diaion HP-20AG.
DJ7164 substance is relatively stable in powder form if stored at low temperature in the presence of a desiccant, but in aqueous solution it is extremely unstable in acidic conditions and unstable in alkaline conditions. It is most stable between PH6.5 and 8.0, but it decomposes quickly at high temperatures even in neutral solutions. Therefore, when isolating the DJ7164 substance from the culture solution, pay close attention to maintaining the pH of the solution between 6.5 and 8.0, and handle the solution at low temperatures (below 10°C). It is important to do this quickly and effectively. Confirmation of the DJ7164 substance during the extraction and purification process is similar to confirmation of accumulation in the culture solution, as well as Bacillus subtilis ATCC6633 or Escherichia coli K.
-12 using the disk plate method. Furthermore, the inhibitory activity of β-lactamase is measured as necessary. The β-lactamase inhibitory activity was determined by Enterobacter cloacae No.19.
It is measured by the inhibitory effect on β-lactamase produced by 19), but the sample to be assayed has a pH of 7.0
Dilute to an appropriate concentration with 0.01M phosphate buffer before use. DJ7164A, B ₁
and B ₂ can be purified and isolated. DJ7164B ₁ (sodium salt) thus obtained has the following physicochemical and biological properties. 1 Color and shape of the substance: Obtained by freeze-drying
The sodium salt of DJ7164B ₁ is a slightly yellow powder. 2 Specific rotation: [α] ²² _D +1.9° (C = 0.16, water) 3 Elemental analysis (measured value): C 44.79%, H
4.61%, N 6.62% The presence of sulfur is expected from the color reaction of iodochloroplatinate. 4. Molecular weight (mass spectrometry): The molecular weight of the methyl ester form of DJ7164B ₁ is 326. 5 Ultraviolet absorption spectrum: The ultraviolet absorption spectrum in 0.01M phosphate buffer (PH7.0) (at a concentration of 12.5 μg/ml is shown in Figure 1. Maximum absorption is at 308 nm.
(E ¹ % ₁ cm 340) and 226 nm (E ¹ % ₁ cm 360). Adding an equal volume of 0.02M hydroxyamine neutral aqueous solution to a 50μg/ml solution of this substance prepared in 0.01M phosphate buffer (PH7.0) and leaving it at room temperature for 30 minutes or more will completely deactivate the antibacterial activity. , the maximum absorption at 308 nm also disappears. 6 Infrared absorption spectrum: The infrared absorption spectrum of potassium bromide tablets is shown in Figure 2. Its characteristic absorption wave number is as follows. 1750cm ^-1 (β-lactam ring -CO-) 1660cm ^-1 (amide-CO-) 1600cm ^- (-COO ^- ) 7 Proton nuclear magnetic resonance spectrum: Sodium 2,2-dimethyl-2-silapentane-5-sulfonate 200MHz for DJ7164B ₁ (sodium salt) in heavy water with (DSS) as internal standard substance
Figure 3 shows the measured proton nuclear magnetic resonance spectrum. The characteristic signals are shown below. A doublet centered at approximately 1.33ppm (J=
6.0Hz) ( CH ₃ CH) A sharp singlet centered at about 2.05 ppm ( CH ₃ CO) A multiplet centered at about 3.13 ppm (− CH ₂
-) Double doublet centered at approximately 3.63ppm (J = 6.0Hz, 9.0Hz) (CO- CH ) Multiplet centered at approximately 4.10-4.30ppm ( CH -N, CH
−OH) A pair of doublets centered at approximately 6.06 ppm and approximately 7.14 ppm (J = 14.0 Hz) (− CH = CH
-) 8 Solubility: Easily soluble in water, slightly soluble in methanol,
It is insoluble in organic solvents such as acetone, ethyl acetate, chloroform, benzene, and petroleum ether. 9 Color reaction: Ehrlich reagent reaction, iodochloroplatinate reaction positive, ninhydrin reaction negative. 10 Paper chromatography: DJ7164B ₁
(Sodium salt) is developed with the following solvent for 12 hours using Toyo Roshi No. 50 (manufactured by Toyo Roshi Co., Ltd.) by the ascending method, and when detected by bioautography, it shows the following Rf value. Developing solvent Rf value n-butanol/isopropanol/water (7:7:6) 0.52 Isopropanol/water (7:3) 0.53 11 Thin layer chromatography: DJ7164B ₁ (Sodium salt) was prepared using Eastman chromagram sheet 6065 ( When developed with the following solvent using a fluorescent agent-containing cellulose sheet (manufactured by Eastman and Kodatsu) and detected by bioautography, the following results were detected.
Indicates Rf value. Developing solvent Rf value n-butanol/isopropanol/water (7:7:6) 0.61 isopropanol/water (7:3) 0.60 n-propanol/water (4:1) 0.56 acetonitrile/water (9:1) 0.25 12 High pressure Filter paper electrophoresis: When electricity is applied for 30 minutes at 75 Volt/cm in 0.1 M Tris-HCl buffer (PH7.5), the behavior on Toyo Roshi No. 51 is that it moves to the anode side,
When the mobility of brome phenol blue used as a standard substance is 1.0, the relative mobility of DJ7164B ₁ is 0.75. 13 Stability: Freeze-dried powder has -
It is relatively stable if stored at a low temperature below 10℃, but its aqueous solution is extremely unstable in acidic conditions and unstable in alkaline conditions, and is most stable around pH 6.5 to 8.0, but even neutral solutions are unstable at high temperatures. Disassemble immediately. 14 Antibacterial spectrum: Table 3 shows the results of measuring the antibacterial spectrum of DJ7164B ₁ (sodium salt) using the paper disc method using a regular agar medium "Eiken" (manufactured by Eiken Chemical Co., Ltd.). [Table] 15 β-lactamase inhibitory activity [Measurement method] β-lactamase inhibition value I ^CEZ ₅₀ is
β
- Represents the amount of substance required to inhibit the hydrolysis of cefazolin by lactamases by 50% in 10 minutes. The rate at which cefazolin is hydrolyzed can be determined by measuring the decrease in absorbance at 270 nm. As the β-lactamase, a partially purified β-lactamase produced by Enterobacter cloacae No. 19 is used. The potency of this crude enzyme against cephaloridine is 33 units per mg of enzyme protein as determined by iodometry. In this case, 1 unit refers to the enzyme titer that decomposes 1 μmol of cephaloridine in 1 minute at 30° C. and pH 7.0. The reagents used are as follows. [Reagents] Substrate solution: PH7.0, 0.01M phosphate buffer containing 50 μg/ml of cefazolin. Enzyme solution: for 10 minutes without inhibitors
PH 7.0, 0.01M phosphate buffer containing an enzyme that reduces absorbance at 270 nm by approximately 0.15 (contains approximately 1.4 units of β-lactamase). Inhibitor dilution solution: Inhibitor sample at PH7.0,
Dilute appropriately with 0.01M phosphate buffer. Trichloroacetic acid solution: 9.0ml of 50% (w/v) trichloroacetic acid solution, 1.0M sodium acetate solution
Mix 55.0ml and 82.5ml of 1.0M acetic acid solution to make a total volume of 1000ml. [Reaction] Enzyme solution 0.2ml and inhibitor diluted solution 0.2ml
Place in a test tube, shake gently for 5 minutes in a constant temperature bath at 30°C, add 3.0 ml of substrate solution, and continue shaking at 30°C for an additional 10 minutes. Immediately after exactly 10 minutes, add 2.0 ml of trichloroacetic acid solution.
Add to stop the reaction. At the same time, prepare a control sample without inhibitor, a blind sample without enzyme solution, and a blind sample without inhibitor and enzyme solution using the same method (control and blind samples are all prepared in 5.4
Adjust the pH to 7.0 with 0.01M phosphate buffer. ). After stopping the reaction, the change in absorbance at 270 nm was measured, and the absorbance of the sample solution and control was corrected by the absorbance of each blind sample to determine the amount of inhibitor required to inhibit 50% of the hydrolysis of cefazolin in 10 minutes ( ^ICEZ) . ₅₀ μg/ml). [Results] According to the above measurement method, DJ7164B ₁ showed ^{an ICEZ} ₅₀ of 0.004 μg/ml for β-lactamase produced by Enterobacter cloacae No. 19. Since the above-mentioned physicochemical and biological properties of DJ7164B ₁ were in good agreement with epithienamycin B reported in the ICAAC abstract, DJ7164B ₁ obtained in the present invention was identified as epithienamycin B. Next, target compounds of the present invention DJ7164A, B ₁ and
A production example of _B2 is shown, but this example does not limit the present invention in any way. Example (1) Culture of the new actinomycete Streptomyces minuteispiralis Streptomyces minuteispiralis sp.
nov. (Feikoken Bibori No. 5079) by slant into a 500 ml Erlenmeyer flask containing 100 ml of the following medium A.
The seeds were inoculated with platinum hooks, and seed culture was carried out at 30°C for about 72 hours in a rotary shaking incubator (160 rpm). A Medium Glycerin 2.0% (w/v) Glucose 0.1 Soybean flour 0.5 Dried yeast 0.5 Calcium carbonate 0.3 Sodium chloride 0.3 Cobalt chloride hexahydrate 0.0002 Antifoaming agent (Adekanol EX885, manufactured by Asahi Denka)
0.04 (v/v) (PH7.0 before sterilization) B medium: 0.5% (w/v) corn staple liquor was used instead of soybean flour in A medium. Inoculate 2 ml each of this seed culture into two 500 ml Erlenmeyer flasks containing 100 ml of the above A medium, and store at 30°C for 72 hours.
Time-rotated shaking culture (160 rpm) was performed. 200 ml of this secondary seed culture was inoculated into a 30 volume jar fermentor containing 20 parts A medium, and the aeration rate was 10/1.
Aerated agitation culture was performed at 30° C. for 72 hours at a stirring speed of 250 rpm. Contain 20% of this seed culture solution and 1000% of the above B medium.
Inoculate a 1500 volume fermentation tank, aeration volume 500/
Main culture was carried out at 30° C. for 90 hours with aeration and stirring at a stirring speed of 160 rpm. As a result, 950 culture fluids containing DJ7164 substance were obtained. Subsequently, 20 kg of a filter aid Radiolite 900 (manufactured by Showa Kagaku Kogyo Co., Ltd.) was added to this culture solution, and the mixture was filtered using a filter press to obtain a culture filtrate 850. (2) Separation and purification of DJ7164 substance The above culture filtrate was immediately cooled to 10°C, and a Diaion PA-306 (Cl type) 50 column (30 cm x 75
cm) at a flow rate of SV=5 to adsorb the DJ7164 substance. 5% (w/v) containing 50% (v/v) salt
v) The active ingredient was eluted with 100 portions of an aqueous methanol solution. The active fractions were collected and concentrated at an internal temperature of 30° C. or below to remove methanol and obtain a concentrated solution 120. Transfer this concentrated solution 120% to a Diaion HP-20 column (40%
cm x 70 cm) to adsorb the active ingredients. Subsequently, it was washed with deionized water for 30 minutes and eluted with 0.01M phosphate buffer (PH7.0) containing 20% acetone for 80 minutes. The eluate was fractionated into 20 fractions. The active ingredient was eluted in fractions 4, 5, and 6, which were combined and concentrated under reduced pressure at an internal temperature of 34° C., and acetone was distilled off to obtain a concentrated solution 50. This concentrated solution was then heated for 50 min at 4°C.
The mixture was passed through a DEAE-Sephadex A-25 column (40 cm x 20 cm) that had been sufficiently equilibrated with 0.01M phosphate buffer (PH7.0) to adsorb the active ingredient.
After washing the column with 0.01M phosphate buffer (PH7.0) 20, wash the column with 0.01M phosphate buffer (PH7.0) containing 1% NaCl.
The eluate was fractionated into nine fractions. The active component eluted between fractions 3 and 11. fraction 4 to 1
Column of Diamondion HP-20 (40
cm x 70 cm) to adsorb the active ingredient, then washed with 30 ml of deionized water and eluted with 10% acetone water.
The eluate was fractionated into 10 fractions, and the active ingredient was obtained in fractions 7 and 8. The active fractions were collected and concentrated under reduced pressure at an external temperature of 30°C to remove acetone, yielding concentrated liquid 17.
Next, at 4°C, this concentrated solution 17 was mixed with DEAE- which had been sufficiently equilibrated with 0.01M phosphate buffer (PH7.0).
The active ingredients were adsorbed onto a Sephadex A-25 column (10 cm x 55 cm). Wash the column with 0.01M phosphate buffer (PH7.0) containing 0.5% salt.
Elution was performed with 0.01M phosphate buffer (PH7.0), and the eluate was fractionated into 400ml portions. At this stage, DEAE-Sephadex A-25 column chromatography completely separates DJ7164B from DJ7164A, a similar substance. That is, DJ7164A was eluted in fractions 2 to 8,
DJ7164B substance eluted between fractions 11 and 29. DJ7164A and DJ7164B substances were analyzed using paper chromatography (Toyo Roshi No. 50) using developing solvent n.
-Butanol/isopropanol/water (7:7:
6), when expanded using the 12-hour ascending method, Rf=
Active spots at Rf = 0.40 and Rf = 0.52 were observed and distinguished by bioautography. Collect fractions 11 to 29 containing DJ7164B substance,
Diaion HP-20 column (15cm x
48 cm) to adsorb the active ingredients, then add 2 ml of deionized water.
After washing with water, it was eluted with 10% acetone water. The eluate was fractionated into 400ml portions, and the active ingredient was separated from fractions 8 to 1.
I got it between 3 and 3. Collect fractions 8 to 13 and use an external temperature of 30°C.
Concentrate under reduced pressure to remove acetone and concentrate 2.
I got it. The concentrated solution was then freeze-dried to obtain about 43 g of DJ7164B substance as a brown coarse powder. For the above-mentioned fractions 2 to 8 containing DJ7164A, desalination was performed in the same manner as for the DJ7164B substance.
Approximately 27 g of DJ7164A was obtained as a brown freeze-dried crude powder. 3 Isolation of DJ7164B ₁ and DJ7164B ₂ The following operations were performed to further purify the DJ7164B substance. 22.5g of the coarse powder of DJ7164B substance obtained in Example (2) was added to 0.01M phosphate buffer (PH7.0) at 4°C.
Dissolve in 100ml of 0.01M phosphate buffer (PH
DEAE equilibrated with 7.0)
The active ingredients were adsorbed onto 25 columns (6 cm x 38 cm). Then 0.01M phosphate buffer (PH7.0) 500
After washing with 0.5% NaCl-containing phosphate buffer (PH7.0)
It was eluted. The eluate was fractionated into 15 ml portions, and active fractions were obtained between fractions 275 and 420. The active fractions were collected and applied to a Diaion HP-20AG column (4.5 cm x 60 cm) at 4°C to adsorb the active ingredient, washed with 300 ml of deionized water, and eluted with 10% acetone water. The eluate was fractionated into 12 ml portions, and the active ingredient was separated into fraction 5.
Obtained between 2 and 90. Active fractions 52 to 90 were collected, concentrated to 50 ml under reduced pressure at an external temperature of 30°C or below, and lyophilized to yield 3.46 g of light brown powder.
I got it. Next, 3.46 g of this light brown powder was dissolved in 10 ml of deionized water at 4°C, mixed with 10 g of Avicel, lyophilized, and layered on an Avicel column (2.5 cm x 35 cm) prepared in advance with 90% acetonitrile. After washing with a small amount of acetonitrile, it was developed with 90% acetonitrile. The eluate was fractionated into 20 ml portions, detected by ultraviolet absorption and bioassay, and divided into fraction 6.
I collected numbers 1 to 66. By concentrating this product under reduced pressure at an external temperature of 30℃ or less and freeze-drying it, 48mg of
A pale brown powder was obtained. In order to further purify 48 mg of this powder, dissolve it in a small amount of 0.01M phosphate buffer (PH7.0) at 4℃ and preliminarily
Adsorb it onto a Biogel P-2 column (25cm x 38cm) prepared with 0.01M phosphate buffer (PH7.0),
Developed with the same solvent. The eluate was fractionated into 6 ml portions.
Active fractions 18, 19 and 20 were obtained by detection using ultraviolet absorption and bioassay. Active fractions 18 to 20 were collected and directly lyophilized without desalting to obtain 66 mg of lyophilized powder containing phosphate. Next, 66 mg of this freeze-dried powder was dissolved in a small amount of deionized water at 4°C, applied to a Diaion HP-20AG column (1.5 cm x 52 cm) to adsorb the active ingredient, and developed with deionized water. The eluate was fractionated into 6 ml portions and detected by ultraviolet absorption and bioassay.
As a result, the active fractions form two peaks, with the main active fraction forming a peak in fractions 18 to 28 with fraction 25 as the peak, and the other peak in fraction 35 as the main active fraction. 45. Fractions 18 to 28
10 mg of the sodium salt of DJ7164B ₁ was obtained as a slightly yellow powder by concentrating under reduced pressure at an external temperature of 30° C. or lower and freeze-drying. This purified specimen
The ultraviolet absorption spectrum in 0.01M phosphate buffer (PH7.0) is 308 nm (E ¹ % ₁ cm340) and 226 nm.
It showed maximum absorption at (E ¹ % ₁ cm360). The physicochemical and biological properties of DJ7164B ₁ are as described above. On the other hand, for the active fraction distinguished from DJ7164B ₁ on the HP-20AG column, fractions 30 to 40 were collected, concentrated at an external temperature of 30°C or less, and then lyophilized to form the sodium salt of DJ7164B ₂ into a slightly yellow powder. 2 mg was obtained. Purified specimen DJ7164B ₂ obtained in this way
The physicochemical and biological properties of (sodium salt) are as follows. 1 Ultraviolet absorption spectrum: Maximum absorption in 0.01M phosphate buffer (PH7.0) is 306nm (E ¹ % ₁ cm300)
and 226 nm (E ¹ % ₁ cm330). 2 High-pressure filter paper electrophoresis: The behavior on Toyo Roshi No. 51 when electricity was applied for 30 minutes at 75 volt/cm in 0.1M Tris-HCl buffer (PH7.5) was that the bromine used as a standard substance moved to the anode side.・If the mobility of phenol blue is 1.0, the relative mobility of DJ7164B ₂ is 0.75. 3. Infrared absorption spectrum: The infrared absorption spectrum of the potassium bromide tablet shows absorption based on the carbonyl of the β-lactam ring at 1750 cm ⁻¹ . 4 Proton nuclear magnetic resonance spectrum: 90M for DJ7164B ₂ (sodium salt) in heavy water using tetramethylsilane (TMS) as an external standard substance.
As a result of measuring the Hz proton nuclear magnetic resonance spectrum, the main characteristic signals were a doublet (J = 6.0 Hz) centered at approximately 1.53 ppm, approximately
A sharp singlet at 2.25ppm, a pair of doublets (J-
14Hz) was observed. 5 β-lactamase inhibitory activity: DJ7164B ₂ is a β-lactamase inhibitor produced by Enterobacter cloacae No.19.
It showed an ^ICEZ ₅₀ of 0.008 μg/ml for lactamase. From the above physicochemical and biological properties,
DJ7164B ₂ is presumed to be a substance similar to epithienamycin D [see ICAAC presentation abstract]. 4 Isolation of DJ7164A Approximately 27 g of the crude powder of DJ7164A obtained in Example (2) was purified in the same manner as in Example (3) to obtain 4 mg of a slightly yellow powder as the sodium salt of DJ7164A. The physicochemical and biological properties of this purified sample DJ7164A (sodium salt) are as follows. 1 Paper chromatography: DJ7164A (sodium salt) uses Toyo Roshi No. 50 and is developed with the following solvent for 12 hours by the ascending method, and when detected by bioautography, it shows the following Rf value. Developing solvent Rf value n-butanol/isopropanol/water (7:7:6) 0.40 isopropanol/water (7:3) 0.50 2 Ultraviolet absorption spectrum: Maximum absorption in 0.01M phosphate buffer (PH7.0) is 298nm (E ¹ % _1cm240 )
exists in 3 High-pressure filter paper electrophoresis: The behavior on Toyo Roshi No. 51 when electricity was applied for 30 minutes at 75 volt/cm in 0.1M Tris-HCl buffer (PH7.5) was that the bromine used as a standard substance moved to the anode side.・If the mobility of phenol blue is 1.0, the relative mobility of DJ7164A is 0.75. 4 Infrared absorption spectrum: The infrared absorption spectrum of potassium bromide tablets shows an absorption at 1750 cm ^-1 attributed to the carbonyl of the β-lactam ring. 5 Proton nuclear magnetic resonance spectrum: 90MHz for DJ7164A (sodium salt) in heavy water using tetramethylsilane (TMS) as an external standard.
As a result of measuring the proton nuclear magnetic resonance spectrum, the main characteristic signals were a doublet (J = 6.0Hz) centered at approximately 1.60ppm, approximately
A sharp single line was seen at 2.20ppm. 6 β-lactamase inhibitory activity: DJ7164A showed ^{an ICEZ} ₅₀ of 0.063 μg/ml against β-lactamase produced by Enterobacter cloacae No. 19. From the above physicochemical and biological properties,
DJ7164A is epithienamycin A or C, or a mixture thereof [see ICAAC presentation abstract]
It is estimated to be a substance similar to.

[Brief explanation of the drawing]

FIG. 1 is an ultraviolet absorption spectrum, FIG. 2 is an infrared absorption spectrum, and FIG. 3 is a nuclear magnetic resonance spectrum.

Claims (1)

[Claims] 1. Cultivate Streptomyces minuteispiralis and use antibiotics DJ7164A,
A method for producing an antibiotic, which comprises collecting DJ7164B ₁ , DJ7164B ₂ or a mixture thereof.