JPH0320236B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing Adriamycin, and more specifically, Streptomyces D788 3T-23 strain is cultured in a nutrient medium, and the following formula is obtained from the culture. This is a method for producing adriamycin, which is characterized by separating and purifying adriamycin shown in the following. Adriamycin is an anthracycline antibiotic with strong antitumor activity, and has a broad anticancer spectrum and is an important chemical therapy agent for cancer treatment. Conventionally, its production method involves using a mutant strain obtained by mutagenesis from Streptomyces peuceteius, which is known as a daunomycin-producing strain, that is, Streptomyces peuceteius subsp.
Streptomyces peuceticus subsp.
caesius) ATCC27952, etc. (see Japanese Patent Publication No. 45-26713), or a method of manufacturing by chemically treating daunomycin (Japanese Patent Publication No. 47-1989).
46597) etc. are known. Although these methods or improved methods are actually used for producing adriamycin, it is desired to develop a more effective production method using microbial fermentation. Through consistent research on the biosynthesis of anthracyclines, the present inventors revealed that adriamycin is biosynthesized from daunomycin by enzymatic oxidation of its 14th carbon, and the conversion of daunomycin to adriamycin by a microbial conversion method. We have developed a production method (J.Autibiotics 34 ,
1229−1231, (981)). In addition, the results of this research show that from a strain known as a daunomycin-producing strain,
By cultivating a strain that has strong activity against carbon 14 oxidation using various strain improvement methods,
This suggests that advantageous fermentative production of adriamycin is possible, and as a result of extensive research in line with this idea, the present inventors mutated an actinomycete strain isolated from soil that can produce daunomycin (baumycin). However, they conducted a search for a strain capable of producing adriamycin, and succeeded in inducing a strain that produces a large amount of adriamycin, thereby completing the present invention. By the way, Streptomyces piuseteius subspicis described in Patent Publication No. 45-26713
Caesius ATCC2759 strain has an accumulated production of about 10 μg/ml, but the strain of the present invention shows an accumulated production amount of 10 to 15 times. The microorganism used in the present invention is an adriamycin-accumulating mutant strain derived from Streptomyces strain D788, which was newly isolated from soil, by mutation treatment. D788 3T-23 strain can be mentioned. The mutant strain can be produced by various known mutation methods, such as
Treatment with mutagenic agents such as nitrosoguanidine, diepoxyethane, and nitrite, or ultraviolet rays, α-rays, and γ
It can be obtained by means of radiation irradiation such as - rays and X-rays. The method for searching for mutant strains used in the present invention will be further explained using specific examples as follows. Streptomyces D788 strain
Inoculated on YS agar slant (0.3% yeast extract, 1% soluble starch, 1.5% agar, PH7.2) at 28°C for 10
Culture for 1 day and allow spores to settle. 1. Spores were collected from the slant culture, suspended in 5 ml of sterile physiological saline, and subjected to ultrasonication (Tomy Seiko, UR-
200P type) for 20 seconds, then passed through a small sterilized glass tube (φ1.5 mm x 20 mm) filled with absorbent cotton.
Centrifuge the liquid and collect the spores. This was added to a 0.1M Tris-HCl buffer (PH
Add 5 ml of 8.5), suspend, and shake at 28°C for 1 hour. Centrifuge again, discard the supernatant, suspend in 5 ml of physiological saline, dilute appropriately with the same saline, spread on YS agar plate (agar 1.5% as described above), and culture at 28°C for 5 days to obtain colonies. (97.5% sterilization rate).
After transplanting and culturing the colony on a YS agar slope,
Inoculate a test tube containing ml of the following seed medium and culture with shaking at 28°C for 2 days. This entire amount was inoculated into a 250 ml flask containing 20 ml of a fermentation production medium having the composition shown below, and cultured with rotary shaking at 28°C for 6 days. Seed medium Soluble starch 0.5% Glucose 0.5 Esunmeat (manufactured by Ajinomoto Co., Ltd., soybean flour) 1.0 Yeast extract 0.1 Potassium diphosphate 0.1 Salt 0.1 Magnesium sulfate, 7H ₂ O 0.1 PH 7.4 Fermentation production medium

【table】

[Table] Take 1 ml of culture solution, add 3 ml of acetone to it, and shake vigorously with a mixer. Obtain the red extracted supernatant after centrifugation. Dilute this 10 times with the following mobile phase for high performance liquid chromatography, and use 10μ
Inject adriamycin to detect adriamycin. High performance liquid chromatography conditions: Main unit: Model 655 manufactured by Hitachi, Ltd. Column: YMC-Pack A-312 Mobile phase: 36% CH ₃ CN-0.01M D-10-camphorsulfonic acid (PH4.2) Flow rate: 1.0ml /min Detection: 254nm Retention time: 5.45min In this way, strains that are recognized to produce adriamycin and accumulate a large amount are selected. An example of a strain with a high accumulation amount is a strain designated by the present inventors as strain 3T-23. This strain 3T-23 differs from the parent strain Streptomyces D788 in terms of anthracycline antibiotic production in that it does not produce baumycins and that it accumulates and produces adriamycin, but it has almost no mycological properties. It doesn't change. Streptomyces below
The mycological properties of D788 and 3T-23 strains are described. Streptomyces D788 3T−
Mycological properties of 23 strains () Morphology Straight aerial hyphae extend from branched basal hyphae, and whorled branches are not observed. A mature spore chain consists of 10 or more spores, and the size of the spores is approximately 0.6 to 0.8 x 0.9 to 2.5 microns, and the surface of the spores is smooth. Ascospores and flagellated spores are not observed. () Growth status in various media Regarding color descriptions Standard H.
System of color by D.Tresner & E.J.Backus
wheels for Streptomycete taxonomy (J.
Appl. Microbiol. Vol. 11, pp. 335-338, 1963), and supplementary "Color Standard" published by Japan Color Research Institute was also used.

【table】

[Table] () Physiological properties (1) Growth temperature range: (yeast/malt/agar medium) used, PH6.0, 20℃, 28℃, 30℃,
Experiments were conducted at temperatures of 37°C and 42°C) Growth was observed at temperatures ranging from 20°C to 37°C. It does not grow at 42℃. (2) Liquefaction of gelatin: Positive (cultured at 20℃ using glucose/peptone/gelatin medium) (3) Hydrolysis of starch: Positive (starch/inorganic salt agar medium) (4) Coagulation/coagulation of skim milk Peptonization: Initially, all results were negative, but peptonization started after 15 days of culture. (5) Production of melanin-like pigments: (tryptone, yeast, broth, peptone, yeast, iron,
agar and tyrosine agar) Positive on all media () Availability of various carbon sources: (on Fridham-Godelive agar) 1 L-arabinose Positive 2 D-xylose 〃 3 D-glucose 〃 4 D-fructose 〃 5 Sucrose 〃 6 Inositol 〃 7 L-Rhamnose Negative 8 Raffinose 〃 9 D-Mannite Positive The strain Streptomyces D788 and 3T-23 of the present invention are Streptomyces piuseteius subspicis caesius (known as an adriamycin-producing strain). Streptomyces peusetius sub.sp.caesius)
(Mutant strain derived from Streptomyces peucetius)
By comparing the mycological properties described in Japanese Patent Publication No. 45-26713, there are major mycological differences in the following points. That is, (1) Morphology Streptomyces piuseteius subspiceius (Streptomyces caesius)
peucetius sub.sp.caesius) (and its parent strain
The tip of the aerial hyphae of Streptomyces peucetius is hook-shaped or ring-shaped, and the conidia are spherical and 1.1 to 3.3 microns in size. On the other hand, the strain Streptomyces D788 3T-23 of the present invention
In the strain (and its parent strain Streptomyces D788), the tip of the aerial hyphae is linear, and the conidia are rod-shaped. (2) Physiological properties Streptomyces piuseteius subspicis caesius
peucetius sub.sp.caesius) does not seem to produce melanin pigment, and peptonization of milk is also negative. On the other hand, the 3T-23 strain was positive for melanin production in all three types of media tested, and also positive for milk peptonization. (3) Carbohydrate utilization Streptomyces piuseteius subspicis caesius
peucetius sub.sp.caesius) uses roughinose and does not use L-arabinose or inositol, but strain 3T-23 uses L-arabinose,
Inositol is used and ruffinose cannot be used. As described above, the strain of the present invention, Streptomyces D788 3T-23, differs in many mycological properties, and therefore, the strain of the present invention, Streptomyces D788 3T-23 strain, is a strain of Streptomyces peucetius subsp. sp.caesius) and its parent strain, Streptomyces peucetius. The 3T-23 strain was submitted to the Institute of Microbiology, Agency of Industrial Science and Technology on February 20, 1981.
It has been deposited as No. 7457 (FERMP7457). The cultivation of the adriamycin-producing strain according to the invention can be carried out in a culture medium composition known per se, which is commonly used as a nutrient source for actinomycetes. For example, carbon sources include glucose, glycerin,
Sucrose, starch, maltose, animal and vegetable oils, etc. can be used, and as nitrogen sources, for example, soybean flour, meat extract,
Yeast extract, peptone, cornstap liquor,
Organic substances such as cottonseed meal and fishmeal as well as inorganic nitrogen substances such as ammonium sulfate, ammonium chloride, sodium nitrate, and ammonium phosphate can be used. If necessary, salts of common salts, potassium chloride, phosphates and other divalent metals such as Mg ⁺⁺ , Ca ⁺⁺ , Zn ⁺⁺ , Fe ⁺⁺ , Cu ⁺⁺ , Mn ⁺⁺ or Ni ^++, and amino acids and In addition to adding vitamins, silicone (manufactured by Shin-Etsu Chemical KK, -
An antifoaming agent such as KM75 (trademark) can also be added as appropriate. Fermentation conditions such as temperature, PH, aeration agitation and fermentation time are selected such that the strain used accumulates the maximum amount of the compound. For example, it is advantageous to carry out the fermentation at a temperature of 20 to 40°C, preferably 28°C, a pH of 5 to 9, preferably 6 to 7, and a fermentation time of 4 to 10 days, preferably 6 days. To isolate and collect adriamycin from the culture, the culture after fermentation is centrifuged or filtered in the presence of a suitable filter aid such as diatomaceous earth to separate the bacterial cells and supernatant. Or separate into filtrate. If necessary, the bacterial cells are extracted using a water-miscible organic solvent such as acetone, methanol, ethanol, or butanol, and the concentrated solution is mixed with the filtrate. This mixture is applied to an adsorption carrier, for example,
Adriamycin can be collected in a pure form by treatment with chromatography using a synthetic adsorption resin or silica gel, treatment with an anion exchange resin or cation exchange resin, or the like, either alone or in an appropriate combination. The standard product obtained by the above method of the present invention was identified as Adriamycin in 0.1N hydrochloric acid water at 80°C.
The Rf value, ultraviolet absorption spectrum, visible absorption spectrum, infrared absorption spectrum, etc. of the aglycone obtained by hydrolysis for 30 minutes on a thin layer of silica gel (F ₂₅₄ manufactured by Merck & Co., Ltd.) using several types of solvent systems were compared with standard adriamycin. This was proved by the fact that daunosamine was detected as a result of qualitative analysis of sugar components in the acid hydrolyzate by the method described in J. Antibiotics 32 , 801-819. The physicochemical properties and instrumental analysis values of this specimen shown below were compared with literature values, and the results were consistent with adriamycin. (1) Appearance Red powder (2) Melting point 199-201℃ (dec) (as hydrochloride) (3) Ultraviolet and visual absorption spectra

[Table] (4) Infrared absorption spectrum (KBr tablet) ν ^max _cn-1 : 3350, 2950, 1720, 1620, 1580,
1530, 1420, 1280, 1230, 1210, 1110, 1080,
980, 910, 870 (5) PMR spectrum (100 MHz, in chloroform) H-1 (8.00) H-2 (7.75) H-3 (7.35) H-1' (5.50) H-7 (5.25) H- 3', H-
5' (4.15) 4-O CH ₃ (4.05) H-4' (3.45) CO CH ₂
OH (4.70) H-10 (3.05) H-8 (2.20) H-2' (1.70) H-6' (1.35) Regarding the physicochemical properties and instrumental analysis data of Adriamycin, the following documents were referred to. . Literature (1) Tetrahedron Letters, 13 , 1007 (1969) (2) Biotechnol.Bioeng, 11 , 1101 (1969) (3) Recent Results in Cancer Res.p.9 Berlior-Heidelberg-New York,
Springer Verlag, 1972 Example 1 (1) Streptomyces D788
D788) 3T-23 strain (Feikoken Joyori No. 7457)
YS (0.3% yeast extract, 1% soluble starch,
1.5% agar, PH7.2) Take a loopful from the slant culture, dispense 100ml of the following seed culture medium, and sterilize it.
The seeds were inoculated into a ml Erlenmeyer flask and cultured at 28°C with shaking in a rotary shaker (220 rpm) for 2 days to prepare a seed mother. Seed medium Soluble starch 0.5% Glucose 0.5% Esunmeat (soybean flour, manufactured by Ajinomoto Co.) 1.0% Yeast extract 0.1% NaCl 0.1% K ₂ HPO ₄ 0.1% MgSO ₄・7H ₂ O 0.1% Tap water PH7.4 (sterilized) Next, add the production medium 15 with the following composition and add the above seed culture solution to two sterilized 30-volume jar fermenters at a volume of 750 ml per one (equivalent to 5%).
Additional inoculation was carried out. Production medium Taiwan yeast 5% Soluble starch 7.5% Yeast extract 0.3% NaCl 0.2% CaCO ₃ 0.3% Mineral mixture * 0.06% Tap water PH8.2 (before heat sterilization) *CuSo ₄・5H ₂ O2.8g, FeSo ₄・7H ₂ O0.4g MnCl ₂・4H ₂ O3.2g, ZnSO ₄・7H ₂ O0.8g dissolved in 500ml of distilled water. Aeration rate 5/min, stirring 450 rpm, 28℃,
After culturing for 140 hours, the culture solution becomes dark reddish brown.
Approximately 120 μg of adriamycin was accumulated in 1 ml of culture. To quantify adriamycin in the culture solution, add 1M citrate buffer (PH3.5) to 1ml of the culture solution.
Add 1 ml of acetone to this, stir, leave at room temperature for 1 hour, collect the supernatant by centrifugation, and add 36% CH ₃ CN ₃ -0.01M D-10-camphorsulfonic acid (PH4.2 ), 10μ of the solution was subjected to high performance liquid chromatography (described above), and the amount was calculated from the standard amount of adriamycin (0.1 to 0.3γ) and measured. Example 2 After completion of fermentation, culture solution 14 was diluted approximately twice with water, and after adjusting the pH to 1.7 with concentrated sulfuric acid, a supernatant (perlite) was added and filtered. liquid 12
was adjusted to pH 2.3 with 4N sodium hydroxide, passed through a 400ml column of synthetic adsorption resin HP-20, washed with water, and the adsorbed matter was eluted with 50% acetone water. Acetone was distilled off from 850 ml of the eluate under reduced pressure, and the pH of the obtained concentrate was adjusted to 8.5 with 4N sodium hydroxide.
The red substance was extracted twice with 300 ml of chloroform. The extracts were collected, concentrated to 300 ml, and acid-transformed with the same volume of 0.1 M acetate buffer, pH 3.0, to remove the aglycone. Then the pH of acid-converted water is 4N
6.0 with sodium hydroxide, chloroform 100
After washing and extracting twice with ml, the pH of the aqueous layer was adjusted to 8.5.
The mixture was extracted twice with 150 ml of chloroform. The obtained extract was washed with water, dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to obtain 2.66 g of red coarse powder. Example 3 The entire amount of the crude powder obtained in Example 2 was applied to a silica gel column (Wakogel C-200, 80 g) prepared by suspension-packing with chloroform, and
It was developed with a methanol-water (50:10:1) solvent mixture. The eluted fractions were tracked by high performance liquid chromatography (described above), and the eluted fractions containing adriamycin were collected and concentrated to dryness to obtain a crude sample of adriamycin with a purity of 57%. The entire amount of this crude sample was added to a silica gel column (Wakogel C-200, 10
g), developed with chloroform-methanol-water (60:10:0.5), and the eluted fractions were analyzed by high performance liquid chromatography, and fractions containing only adriamycin were collected. The eluate fraction was then washed with water and subjected to acid transfer extraction with 50 ml of 0.1M acetate buffer (PH 3.0), and the extracted aqueous layer was adjusted to pH 8.5 with 4N sodium hydroxide and extracted twice with 30 ml of chloroform. The extract was washed with water, further washed with saturated saline, dried with Glauber's salt, concentrated to a small amount, and precipitated by adding excess n-hexane.
The mixture was filtered, concentrated, and dried in a vacuum desiccator to obtain 124 mg of purified adriamycin.

Claims (1)

[Scope of Claims] 1. Streptomyces D788 3T-23 (Feikoken Bibori No. 7457) strain was cultured in a nutrient medium, and the following formula was obtained from the culture. A method for producing adriamycin, which comprises separating and purifying adriamycin.