JPS5915634B2 - Production method of actinomycin by fermentation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing actinomycin by fermentation, using a strain belonging to a new species of Streptomyces meleochryceus as a microorganism, and producing actinomycin D.
Alternatively, it is characterized by producing actinomycin s3.

Actinomycin is a known substance that is pharmaceutically useful as an antibiotic anticancer substance, including actinomycin D 7
C7C'2 j S 3, etc.

Conventionally, actinomycin was produced by fermentation using microorganisms such as Streptomyces antibioticus, Streptomyces clitumarus, and Streptomyces spp.
Some are known that utilize S. sp. BOP476, Streptomyces flaps, Streptomyces flaveolus, etc. (CRC! Handbook of
Microbiology vol. m, 1973)
.

The present inventors have conducted various studies on manufacturing methods for antibacterial substances.

As a result, they found that an antibacterial substance accumulates in the culture of a bacterial strain (referred to as KYI 1634) isolated from soil outside the area of Shimo-Satsuki City, Tokyo. The present invention was completed by discovering that the bacterial strain is a microorganism belonging to the genus Streptomyces, and that it is a bacterial strain different from known bacterial species.

The above-mentioned KY11634 has been deposited with the National Institute of Microbial Technology, Agency of Industrial Science and Technology (its deposit number is No. 4).
No. 673).

Furthermore, the strain has been deposited with the NRRL (its deposit number is NR, RLI 1395).

Next, the mycological properties of this strain (KY11634) will be explained.

(A) Morphological properties KY11634 successfully grows aerial mycelia on a general isolation medium, and its morphology is simple branched and spiral-shaped.
It is.

More than 10 spores are chained together, and the surface is smooth.

The shape of the spore is oval (0.4-0.5μ x o, 7-0
．． 8μ).

Table 1 shows the growth conditions, colors of the front and back surfaces of colonies, and soluble pigments when KY11634 was grown on various media.

Color display is from Co1or Harmony Manual
(Cont-ainer Corporation
of America).

(B) Physiological properties Table 2 shows the physiological properties of KY11634.

The results of observations other than temperature, effects on milk and cellulose are shown after 2 weeks at 27°C; for temperature, the results are shown after 5 days, and for effects on milk and cellulose, the results are shown after 1 month.

Table 2 Physiological properties of KY11634 (1) Assimilation of carbon source (on Pridham-Godelive agar medium) Each symbol indicates the following meaning.

1: Not assimilable, +: Assimilable, 10: Actively assimilable carbon source Assimilable D-arabinose + D-xylose - D-glucose 廿D-fructose 廿Sucrose 廿Sinositol 廿L-Rhamnose 廿Raffinose 廿D-Mannitol (2) Liquefaction of gelatin (on glucose/peptone gelatin medium) None 3) Effect on milk Liquefaction Yes Penbutonization None 4) Decomposition of fibrin Slightly present (5) Hydrolysis of starch (on starch agar medium) Yes (6) Optimal growth pH 6.8-7.5 (7) Optimal growth temperature 28-30℃ (8) Production of tyrosinase No 9) Melanoid pigment (on tyrosine agar medium and peptone yeast iron agar medium) From the above properties, K
The Y11634 bacterium is classified as an actinomycete belonging to the genus Streptomyces.

Regarding the species to which this fungus belongs, E. Kuster (In
-tern, J. System, Bacteri
This bacterium is classified as Streptomyces botroensis according to the classification by J.D. Ol., Vol. 22, A3, p. 139, 1972).

However, E, B, Shirling and D
, Gottlieb [International
J, Systematio Bacteriology
yl 9(4) 391.1969], it has been shown that Streptomyces potroensis has a weak ability to assimilate raffinose.

Furthermore, the spiral state of aerial hyphae is a normal shape with one or two spirals, and an open spiral (Open 5p-ir).
al).

However, KY11634 utilizes raffinose as well as glucose, etc., and has a very dense helical shape, taking the form of a compact 5-piral structure, with almost no open 5-piral structure.

Therefore, KY11634 is clearly Streptomyces
different from Potroensis.

On the other hand, Streptomyces flaveolus is known as a bacterium that produces actinomycin S3, but this is explained in Bergey's Manual of Determinology.
in-active Bacteriology (8th edition)
According to Hairly 5p, the spores assimilate xylose, do not produce melanin, and have numerous hair-like bodies.
It is clearly distinguished from KY11634 in that it is

Therefore, we identified KY11634 as a new fungal species and identified it as Streptomyces meleochryseus (S tr ) based on the color of its vegetative hyphae.
eptomyces me 1 leochryseus
It was named KY 11634TOMITA).

The present invention will be explained in detail below.

In the cultivation of the present invention, ordinary methods for culturing actinomycetes are generally used.

Various sources of nutrients can be used for culture.

As the carbon source, glucose, starch, dextrin, mannose, fructose, sucrose, molasses, etc. are used alone or in combination.

Furthermore, depending on the assimilation ability of the bacteria, hydrocarbons, alcohols, organic acids, etc. may also be used.

Inorganic and organic nitrogen sources include ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, urea, etc. Natural nitrogen sources include peptone, meat extract, yeast extract, dried yeast, corn stew liquor, soy flour, casamino Acids and the like are used alone or in combination.

In addition, inorganic salts such as table salt, potassium chloride, calcium carbonate, and phosphates can be added as necessary, and organic and inorganic substances that promote the growth of the bacteria used and the production of actinomycin D and S3 can be added as appropriate. .

As a culture method, a liquid culture method, especially a deep agitation culture method, is suitable.

The optimal culture temperature is 25-40°C, especially 28-38°C, and the pH of the culture medium is preferably 4-10, preferably 6-8 by adding aqueous ammonia or ammonium carbonate solution.

When liquid culture is carried out, usually for 1 to 7 days, the target substance is produced and accumulated in the culture.

Preferably, the culture is stopped when the production amount in the culture reaches the maximum, and the target product is purified and isolated from the culture solution obtained by separating the bacterial cells and from the organic solvent extract of the bacterial cells.

The isolation and purification of actinomycin D and S3 from the culture fluid utilizes separation and purification methods commonly used for isolating microbial metabolic products from the culture fluid.

Among actinomycin D and S3 in the present invention, actinomycin D is known to have antitumor properties, but there has been no report on the antitumor properties of actinomycin S3.

The present inventors investigated the antitumor properties of actinomycin S3 and obtained the following results.

(1) Treatment effect on Sarcoma 180 ascites-type tumor, weighing approximately 20. ? 5 x 10' Sarcoma 180 ascites-type tumor cells were subcutaneously implanted in the axillary region of each group of six ddY male mice.

Twenty-four hours after transplantation, 0.2 il of a phosphate buffered saline (PBS) solution of actinomycin S3 at various concentrations was intraperitoneally administered once.

In addition, the composition of the above PBS is Na010,8&/c
i4 K O130,029/cll, N a 2
HPO41,15&/di, KH2PO40,02&
/di, (pH 7,2).

As a comparative example, a group was provided in which PBSo, 2il containing mitomycin C was intraperitoneally administered 24 hours after tumor implantation.

Average tumor volume (7IIIl') and T/C 7 days after transplantation [T: average tumor volume of test examples, C: control example (PB
so, 2il administered intraperitoneally))
was measured, and the results shown in Table 3 were obtained.

(2) Therapeutic effect on Lymphocytic Lykemia P-388 tumor
Aeukemia) P-388 tumor cells lXl0
' were transplanted intraperitoneally.

0.2 ml of actinomycin S3 in PBS 24 hours after transplantation! was administered intraperitoneally once.

As a comparative example, a group was provided in which 0.2 ml of mitomycin C in PBS was intraperitoneally administered 24 hours after tumor cell transplantation.

Average survival days and T/C after transplantation! (T: average survival days of test examples, C: average survival days of controls) are shown in Table 4.

actinomycin 83 (LD50 is actinomycin D
When used as an antitumor agent, about 1/3 of the total amount can be administered by intravenous administration, intraperitoneal administration, or the like.

In this case - the daily dosage range is as follows:

That is, in the case of continuous intravenous and intraperitoneal administration, the dose is about 2 to 4 μg, 9 kg/day, and in the case of intermittent administration (once/7 days), the dose is about 10 to 15 μg, 7 g, 17 times.

Examples will be described below, but these are merely illustrative and do not limit the scope of the present invention in any way.

Example 1 Streptomyces meleochryseus KY as inoculum
11634 was used.

The strain was grown in a seed medium in a 24-volume Erlenmeyer flask [meat extract 1/dl.

The yeast extract was inoculated into a medium pH 7.2 (before sterilization) having a composition of 1 g of yeast extract and 5 fl/dl of NaCl, and cultured with shaking (220 rpm) at 30° C. for 24 hours.

The seed culture thus obtained is 304? Fermentation medium 157 with the following composition in a jar fermenter with a volume of
Culture was carried out at 5 Orpm; aeration volume 1513/71 ML).

Fermentation medium composition Nigelcose 2'#/l, Sucrose 36fJ/LL-Asparagine 7,5 &/13KH2
PO40,E, 51/11SK2HP0.0.21
&/LMgS04・7H200,09&/It, yeast extract 2fJ/l, vitamin B11 oiη/l, biotin 30μFit, Fe 804 ・7H20
10'mL! /L CaO1230'1l19/l
lMnSO4” H2O47119/l, Zn5O,
-7H2030'n9/l, 01804-5H2027
nV1ph6. (before sterilization) with NaOH.

Culture was carried out for 72 hours without controlling the pH of the culture medium.

Bacterial cells and precipitates were separated from the culture solution to obtain Solution F 131.

First, add liquid F to 11 nonionic porous resin (product name [HP
-20J manufactured by Mitsubishi Kasei) to adsorb the active substance,
After washing with water, wash with acetone water (V/V) for 40 hours to remove impurities.

Then elute with 80% acetone water (V/V).

The acetone fraction is concentrated to dryness, dissolved in chloroform, and the insoluble materials are separated.

This chloroform solution is subjected to silica gel chromatography along with the extract from the bacterial cells described below.

Suspend the bacterial cells in acetone and extract the target substance.

The amount of acetone used is approximately 51.

The extract was concentrated to dryness, then dissolved in chloroform, and impurities were removed.

The chloroform solution obtained here was combined with the chloroform solution previously obtained from the culture p solution (total of 15 Tll), and a silica gel (trade name: 5ilic ARO (3-4Mall-inckr) filled with chloroform as a solvent) was added.
odt Co-U, S, A, ) (500 rul
), and first add enough chloroform (
Approximately 2 A') and chloroform:methanol (9
When elution is performed at a ratio of 5:5) (V/V), an actinomycin mixture is eluted.

The active fraction was concentrated to obtain about 1 g of a crude sample.

The crude sample is a mixture containing actinomycin S3 and actinomycin D in a ratio of 7=3.

Both components were obtained from the crude sample using preparative paper chromatography.

The developing solvent is rr-butyl ether: tetr
a-chloro ethane: 10% scdi
um cresotinate (2:1:3) (V/V
), and the paper used was Toyo p paper A, 50.

After development, corresponding portions were cut out and eluted with acetone.

The eluate was concentrated to dryness and crystallized from ethyl acetate to give acanomycin S30.5,9. Actinomycin D0
．． 15.9 was obtained.

The physicochemical properties of the actinomycins S3 and D thus obtained were as follows, and were consistent with those of the standard products.

Actinomycin S3 What is interesting here is that the bacteria used in the present invention produce actinomycin S3 in overwhelmingly large quantities.

Example 2 A seed mother was prepared in exactly the same manner as in Example 1, and the culture method, culture conditions, and culture time were also carried out in exactly the same manner as in Example 1.

However, the fermentation medium used had the following composition.

Fermentation medium composition: sucrose 409/l, glucose 10g/l, fructose 10&/l, cornsteeple strength -10&/l, Na(J 5 g/IJ, M
gSO4・7H201&/131KH2PO42&/l
j.

(NH4) 280415 g/, g, vitamin B110
7vl biotin 30μ9/l, FeSO4-7H201
0'? / 13, 0aO1123Q■/13. Mn
SO4・H2O4772L! / 131Zn 804
・7 H2030'Q/l, OLI 804 ・5H
202772? / 7 OpH6,8 (before sterilization) with Na
Adjust with OH.

Actinomycin was purified in exactly the same manner as in Example 1 to obtain actinomycin S30.8J and actinomycin D 0.2 g.

Claims (1)

[Claims] 1. Cultivate a microorganism belonging to Strephtomyces meleochryseus and having the ability to produce actinomycin D or actinomycin S3 in a nutrient medium, produce and accumulate actinomycin D or actinomycin S3 in the culture, and produce it from the culture. A method for producing actinomycin by a fermentation method, which comprises recovering accumulated actinomycin D or actinomycin S3.