JPH0533992B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing bleomycins, and more specifically, a bleomycin-producing strain belonging to the genus Streptomyces is inoculated into a medium containing a nutrient source, and if necessary, terminal amine residues of the desired bleomycins are added. In the method for producing bleomycins, which involves adding a primary amine corresponding to the group, culturing, producing and accumulating bleomycins, and isolating them, the following general formula (1) is used. (In the formula, R represents an alkyl group having 1 to 6 carbon atoms, A and B represent substituents on the alkyl group, A represents hydrogen or a hydroxyl group, and B represents hydrogen, a phenyl group, an indole group, or a substituted phenyl group. ) This invention relates to an improved fermentation method for bleomycins, which is characterized by culturing with the addition of the amino acids represented by: This is something that can be effectively reduced. By applying the method of the present invention, it becomes possible to simplify or omit the complicated cleomycin removal step during the bleomycin purification process, making it possible to easily produce high-purity bleomycin powder, which is economical. Great effect. That is, 20 to 30% when the method of the present invention is not used.
However, according to the present invention, the by-product can be reduced by more than 10%, and by selecting the compound of general formula (1), etc., the by-product can be reduced to several percent or less. You can also do that. Bleomycin is an antitumor antibiotic discovered by Umezawa et al., and is widely used in cancer chemotherapy, and is particularly effective against squamous cell carcinoma (Umezawa et al. J.
Antibiotics Vol. 19A, p. 200, 1966). Bleomycin is produced by culturing actinomycetes, such as Streptomyces verticillus belonging to the genus Streptomyces, in a liquid medium containing ordinary nutrients under aeration and agitation, and is extracted and purified from the culture solution by a known method. As a result, it is obtained as a blue powder containing one atomic copper chelate.
(Umezawa et al., J. Antibiotics Vol. 19A, p. 210, 1966) According to the usual culture method, a mixture of more than ten types of bleomycins having different terminal amine residues is produced.
In addition, a method of adding a primary amine corresponding to the terminal amine residue to the culture solution as a precursor (Japanese Patent Publication No. 1973-
32354, Japanese Patent Publication No. 48-32355), one type of bleomycin is selectively produced. Streptomyces verticillus, a bleomycin-producing bacterium, exhibits a weak ability to produce cleomycin under conditions that produce bleomycins. As can be seen from JP-A-56-25197, cleomycins are structurally very similar to bleomycins, and their physical and chemical properties are also extremely similar to the corresponding bleomycins. Therefore, mutual separation is currently being achieved using advanced technology and complicated operations. Therefore, suppressing by-produced cleomycin by improving culture conditions, for example, can simplify or omit this sophisticated and complicated operation, and is extremely useful in bleomycin production. . In view of the above, the present inventors investigated the culture conditions and found that when cultured with one type of compound represented by general formula (1) in addition to the nutrients necessary for the medium, cleomycin They discovered that production can be suppressed and completed the present invention. The microorganism used in the present invention may be any bleomycin-producing microorganism, and Streptomyces verticillus, which belongs to the genus Streptomyces, is conventionally known, and a representative example thereof is Streptomyces verticillus.
NK68−144 (Feikoken accession number 4108,
ATCCNO.31307). (Streptomyces
Verticillus is Bergeis Mannual of
Determinative Bacteriology, 8th edition proposes to classify it as Streptoverticillium verticillus, but in this specification it is classified as the genus Streptomyces as usual. ) It is well known that the ability of actinomycetes to produce antibiotics tends to change naturally or artificially.
It is possible to create and use mutant strains with high bleomycin-producing ability by applying radiation, ultraviolet rays, or mutagenic agents. The bleomycin-producing bacteria used in the present invention naturally include such natural or artificial mutant strains. Streptomyces verticillus NK68−144
is Streptomyces verticillus B80−Z2
(ATCC No. 15003) (detailed in U.S. Patent No. 3681491) was subjected to mutation treatment by UV irradiation and γ-ray irradiation, resulting in a mutant strain with increased productivity of bleomycins. The alkyl group represented by R in the general formula (1) of the present invention is methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, n
-pentyl, isopentyl, n-hexyl, 2-
It is an alkyl group such as ethylbutyl, and preferably when B represents hydrogen, R is an alkyl group having 2 or more carbon atoms, more preferably 3 or more carbon atoms. Group when A or (and) B is a group other than hydrogen

Specific examples of the formula include 1-hydroxyethyl, benzyl, phenylethyl, phenylpropyl, p-hydroxybenzyl, 1-hydroxybenzyl, 1-hydroxyhexyl, phenylbutyl, and the like. Amino acids of general formula (1) include threonine, valine, leucine, isoleucine, phenylalanine, tryptophan, β-hydroxynorvaline, phenylserine, α-aminobutyric acid, norvaline, and valine, leucine, isoleucine,
Phenylalanine, β-hydroxynorvaline, etc. are preferable because they have a good yield and suppress the by-product of cleomycin. Among these, leucine, isoleucine, norleucine, and norvaline are particularly preferred. The medium used in the present invention includes starch, starch syrup, glucose, mannose, fructose, galactose, lactose, maltose, sucrose,
One or more carbon sources such as inositol, mannitol, glycerin, dextrin, molasses, organic acids, ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, urea, peptone, meat extract, yeast extract, dried yeast, corn steep liquor , soybean flour, defatted soybean flour, one or more nitrogen sources such as casamino acids, lysine, cysteine, and sodium chloride, potassium chloride, phosphates, calcium carbonate, zinc sulfate, magnesium sulfate, copper sulfate, etc. Inorganic salts and the like are used in appropriate combinations. In addition, for the purpose of promoting the production of bleomycin and cleomycin having specific terminal amine residues, there is a method of culturing by adding an appropriate amount of a defatted primary amine having two or more basic groups in the molecule to the medium. It has been established [Patent No. 728158 (Special Publication No. 48-
32354), Patent No. 728159 (Special Publication No. 48-32355)],
It goes without saying that this method can also be applied to the present invention, and the above-mentioned amines can also be added to the culture medium if necessary. Representative examples of such amines include N-(1-phenylethylamine)-1,3-diaminopropane, N-butyl-1,3-diaminopropane, and N,N-bis-
(3-aminopropyl)-1,3-diaminopropane, 3-morpholinopropylamine, and the like. In the present invention, the amount of the amino acid of general formula (1) added during culture in the above medium is 0.02 to 5%, preferably 0.05 to 3%, based on the medium. These additives can be added to the culture medium in advance and sterilized, or they can be sterilized separately and added to the sterilized culture medium at the time of inoculation with the bleomycin-producing strain or at an appropriate time after the start of culture and before the start of bleomycin production. It's okay. Cultivation is usually carried out at 20 to 30° C. for about 6 to 15 days under aeration and agitation. The end of culture is when the maximum titer is reached using Mycobacterium 607 as the test bacterium.
After completion of the culture, a crude powder of bleomycin is obtained as a blue powder by performing chromatography using a known method such as Amberlite XAD-2, Alumina, Cephadex LH-20, etc. from the culture solution. The amount of bleomycin and cleomycin present in this crude powder can be separated and quantified by performing reverse phase high performance liquid chromatography. Some examples of bleomycins obtained in this way are 3-N-(1-phenylethyl)-aminopropylaminobleomycin 3-N-butylaminopropylaminobleomycin 3-(N-methyl-N-3-amino propyl)-aminopropylbleomycin 3-(morphorino)-propylaminobleomycin and the like. Next, a concrete explanation will be given using examples. Example 1 Culture using medium 1 Medium containing 1% starch, 1% glucose, 0.75% peptone, 0.75% meat extract, and 0.3% salt (PH7.0) 100
ml into a 500ml Erlenmeyer flask,
After sterilization, Streptomyces verticillus
NK-68-144 (Microtechnical Research Commission Acceptance No. 4108,
ATCC No. 31307) and cultured at 28°C for 48 hours with rotational shaking to prepare a seed culture. Next, dextrin 1%, fructose 1%,
Glutamic acid 0.2%, Histidine 0.1%, Cysteine 0.1%, Lysine 0.1%, Salt 0.3%, Sodium nitrate 0.2%, Potassium phosphate 0.5%, Zinc sulfate 0.05%,
3-morpholinopropylamine hydrochloride was added at a rate of 0.1 mg/ml to a medium (medium 1) having a composition of 0.01% copper sulfate and 0.5% magnesium sulfate, and 1% threonine was added as an amino acid used in the present invention. After adding 100 ml of each flask to 10 500 ml Erlenmeyer flasks and sterilizing them, the above-mentioned seed culture solution was inoculated at a rate of 5% and cultured with rotary shaking at 28° C. and 180 rpm for 10 days. After the cultivation was completed, the culture solution was filtered with 5% dicalite, a diatomaceous earth additive, to obtain 900 ml of solution. This liquid was passed through a 100 ml resin tower packed with Amberlite XAD-2 to adsorb the target product, washed with water, and eluted using 150 ml of 0.01N hydrochloric acid-methanol (1:4). After neutralizing the eluate, it was concentrated to dryness and dissolved in a small amount of water-methanol (1:4). After removing the insoluble materials, the liquid was passed through an alumina column (5 ml) packed with water-methanol (1:4) and eluted with the same solvent, resulting in a blue fraction.
Collected 20ml. After concentrating this fraction, it was dissolved in about 5 ml of water-methanol (1:4) and applied to a Sephadex LH-20 column (100 ml) packed with the same solvent.
Chromatography was carried out using the same solvent as a developing agent, and the blue fraction was collected and freeze-dried.
5.87 mg of blue powder of morpholinoaminopropylbleomycin (hereinafter referred to as MOP-BPM) was obtained. This powder was dissolved in water, and a portion thereof was analyzed by reverse phase high performance liquid chromatography under the following conditions, and the cleomycin content was found to be 0.2%. (Analysis conditions) Chromatographic column: Nucleosil 7C18 (Nagel) 6 mm x 250 mm Developer: 4% potassium acetate, 2% acetic acid aqueous solution: acetonitrile (85:15 v/v) Flow rate (pressure): 1.5 ml/min (2000 psi) Detection: UV292nm Under this condition, morpholine bleomycin,
Morpholine cleomycin 13 minutes and 15 minutes, respectively.
It is separated and eluted in minutes. The contents of both components were calculated from the peak area ratio. In this example, when culturing was performed in the same manner without the addition of threonine, 10.62 mg of MOP-BLM coarse powder was obtained.
I got it. The cleomycin content of this powder is 26%
It was hot. Example 2 Effect of leucine addition on culture using medium 1 The culturing and purification operations were performed in the same manner as in Example 1 except that 1.0% leucine was added to medium 1.
38.4 mg of MOP-BLM crude powder was obtained. Cleomycin content was 2.6%. Example 3 Effect of addition of norvaline on culture using medium 1 The cultivation and purification operations were performed in the same manner as in Example 1 except that 0.5% norvaline was added to medium 1 to obtain 38.62 mg of MOP-BLM crude powder. The cleomycin content of this powder was 2.3%. Example 4 Culture using medium 2 The seed culture solution obtained in the same manner as in Example 1 was further cultivated by
The mixture was transferred to a 5-volume Erlenmeyer flask containing medium 1 with the same composition, and cultured with rotary shaking for an additional 48 hours to prepare a seed culture. Next, 6.4% starch syrup, 0.5% glucose, and 3.5% soybean flour.
%, corn steep liquor 0.75%, sodium chloride 0.3%, dipotassium phosphate 0.5%, zinc sulfate 0.05%,
30 volumes of medium 20 with a composition of 0.01% copper sulfate, 0.2% sodium nitrate, 0.5% magnesium sulfate, and 0.03% Pronal ST-1 (medium 2), with 0.01% 3-morpholinopropylamine and 0.5% norvaline added. Add the above seed culture solution 1 to the sterilized jar fermenter and incubate at 28℃.
Culture was performed at 400 rpm for 7 days. After culturing, purification was performed using the method described in Example 1, and 1.62 g of MOP
- BLM coarse powder was obtained. The cleomycin content of this crude powder was 4%. In this example, culturing and purification were performed without adding norvaline to obtain 1.02 g of MOP-BLM crude powder.
The cleomycin content of this powder was 19.5%. In the same manner as in Example 1 or 3, MOP-BLM was cultured by adding the amino acid of general formula (1). The results of the examples are also shown in the following table.

【table】

[Table] Example 5 3-(N-(S)-1'-phenylethyl)aminopropylamino (bleomycin PEP-BLM) by culture with norvaline addition in medium 2
Production of Example 3 except that 0.1% of N-[(S)-1'-phenylethyl]-1,3-diaminopropane hydrochloride was added to medium 2 instead of MOP, and 0.5% of norvaline was also added. , all were cultured and purified in the same manner as in Example 3, and crude powder of PEP-BLM was obtained.
1.11g was obtained. The cleomycin content of this powder was 3.1%.

Claims (1)

[Claims] 1. A bleomycin-producing strain belonging to the genus Streptomyces is inoculated into a medium containing a nutrient source, and if necessary, an aliphatic primary amine corresponding to the terminal amine residue of the desired bleomycin is added. In the method for producing bleomycins, which involves culturing, producing and accumulating bleomycins, and isolating them,
For the culture medium, use the following general formula (In the formula, R represents an alkyl group having 1 to 6 carbon atoms, A and B represent substituents on the alkyl group, A represents hydrogen or a hydroxy group, and B represents hydrogen, a phenyl group, an indole group, or a substituted phenyl group. ) A method for producing bleomycins, which comprises culturing with the addition of an amino acid represented by: