JPS6130558B2 - - Google Patents

Description

[Detailed description of the invention]

This invention relates to a novel method for producing saquintotricin. Xanthothricin is known as an antibiotic that has antibacterial activity against Gram-positive and Gram-negative bacteria produced by bacteria belonging to Streptomyces alpus (for example, Antibiotics and Chemotherapy, Vol. 4). Third
No., pp. 259-261). As a result of intensive research into the production of xanthothricin, the inventors discovered that a new strain belonging to a new subspecies of the genus Streptomyces, which the inventors isolated from the soil of Central Asia in the Soviet Union, produces xanthothricin. After gaining this new knowledge and continuing research, we completed this invention. Among the strains belonging to Streptomyces bruneus subsp. 1. Culture and morphological characteristics Sporophytes are erect, monopodial, and singly arranged. The spores are oval, elongated, or cylindrical with a smooth capsule. No sporangia and sclerotia. (1) Satucarose-containing synthetic medium Aerial mycelium is well developed and covered with soft hairs, and the color is pink to white or pale pink. Colonies and medium are brown or dark brown. (2) Glucose-asparagine agar Aerial mycelium grows poorly and is almost absent. Pink with a touch of white. Colonies and medium are light brown in color. (3) Glycerin-asparagine medium Colonies and medium are brown, aerial mycelium is poorly grown, velvety, and brownish-pink in color. (4) Starch agar The aerial mycelium is pink with a hint of white and is starchy. Colonies and medium are pale brown-yellow. (5) Tyrosine-containing agar Aerial mycelium is poorly grown, starchy, and pink with a hint of white. Colonies and medium are colorless. (6) Meat extract, peptone-containing agar, no aerial mycelium. Colonies are dark brown with dark brown pigment diffused into the medium. (7) Yeast malt medium Aerial mycelium grows poorly and is pink with a hint of white.
Colonies and medium are light brownish reddish brown. (8) Oatmilk agar Aerial mycelium is poorly grown and brown in color. Colonies and medium are colored dark brown. Physiological characteristics Liquefies gelatin, converts milk into peptone, converts satucalase, hydrolyzes starch, grows poorly on cellulose, reduces nitrate and does not produce hydrogen sulfide, does not produce tyrosinase, synthetic medium Forms a medium brown soluble pigment. The culture is aerobic. Assimilation of carbon sources ・Glucose ++ ・Arabinose ++ ・Xylose ＋ ・Satucarose ++ ・Raffinose ++ ・Rhamnose + ・Fructose − ・Inosyte − ・Mannite − Antibacterial properties of the strain itself None Regarding strains with the above-mentioned mycological properties ,
NA Krasilinnikov, ``Radiant Juange'' (Nauka Publishing House, 1970), GF Gauze, ``Problems of Classification of Actinomycetes - Antagonists'' (Mejitsu Publishing House, 1970) A Guide to the Classification and Identification of Actinomycetes and Their Antibiotics.
(1953) and S. Waksman, "The Actinomycetes", Volume V.11, and other documents were used for a classification search. Based on culture and morphological characteristics, as well as some biochemical properties, this strain is similar to Streptomyces.
It can be included in the species Bruneus. (“Radiant Juange” by Mr. Krasilinnikov)
(1970)), but differ from conspecifics in a range of biochemical and antimicrobial properties. For example, according to Krasilinnikov's data, strains of Streptomyces bruneus are
Bacillus idosus and Bacillus idosus
It has an antibacterial effect against Bacillus subtilis, and the bean root nodule bacterium Azotobacter chlorococacum.
chroococcum) and Azotobacter vinelandii, whereas this strain does not exhibit antagonistic properties against the above microorganisms. The following table shows the comparison results of the physiological characteristics of the above-mentioned two cultures, that is, the present strain and the Streptomyces bruneus strain.

【table】

[Table] From the above observation results, this strain and Streptomyces bruneus differ in the above points, and this strain produces xanthothricin, whereas Streptomyces bruneus does not. Considering the difference that it does not produce this strain, this strain was recognized as a mutant strain of Streptomyces bruneus, and Streptomyces bruneus subsp.
brunneus subspecies xanthothricini). This mutant strain Streptomyces bruneus
Subsp. Streptomyces bruneus subsp.
The production ability of xanthothricin can be increased by commonly used strain mutation treatment methods such as treatment with drugs such as 5-bromouracil and 2-aminopurine, transformation, transduction, and conjugation. When Streptomyces bruneus subsp. do. The culture and morphological characteristics of this mutant strain are as follows. Mutant strain No. 1: Colonies are circular, slightly swollen, smooth, and have a flat periphery. Aerial mycelium is covered with soft hair and is pink in color. The substrate is brown to dark brown. A brown soluble dye is formed. Mutant strain No. 2: The colony is smooth and oblate, with a flat periphery and covered with white to pink velvety soft aerial mycelium. The substrate is light brown to pale brown. Forms a brown soluble pigment. Mutant strain No. 3: Colonies are pleated and sporulation is weak. The aerial mycelium is pink with a hint of white, and the matrix is brownish-brown. All of these mutant strains produce xanthothricin.
Can produce up to 100μg/ml. The production of xanthothricin according to this invention is carried out by Streptomyces bruneus subsp.
It is carried out by culturing xanthotricin-producing bacteria belonging to the genus Xanthotricini in a medium. A synthetic medium, a semi-synthetic medium, or a natural medium is used as the culture medium, and the medium composition includes carbon sources such as glucose, sucrose, arabinose, xylose, raffinose, rhamnose, maltose, glycerin, starch, and liquefied starch. is used as a nitrogen source, such as meat extract, casein hydrolyzate, peptone,
Gluten meal, corn meal, cotton seed meal, soybean flour, corn steep liquor, dried yeast, yeast extract, urea, ammonium phosphate, etc. are used.
In addition, inorganic salts such as disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium chloride, magnesium sulfate, and calcium carbonate are also added to the medium as necessary. The appropriate culture temperature is around 20 to 40°C, and good results are often obtained by carrying out seed culture as appropriate as the culture volume increases. The culture time for main culture is approximately 50~
100 hours is appropriate, and the culture time may be further extended as the concentration of the medium increases. The culture conditions described above are selected and applied depending on the characteristics of the xanthothricin-producing strain used. Xanthothricin produced by culture is separated, purified, and collected by means used in the production of general antibiotics. That is, xanthothricin produced in the culture broth can be extracted from the culture broth by combining and repeating methods such as vacuum concentration, solvent extraction, PH conversion, crystallization, and recrystallization in any order. Separated, purified and collected. The xanthothricin thus obtained has antibacterial activity as described above and is useful as a medicine, but it is also a raw material for the production of leumycin, which has anticancer activity as shown in the following reaction formula. It is also useful as This reaction is carried out by treating xanthotricine with an amine and then with an acid.The amines used here include primary and secondary amines.
Preferred are lower amines, such as for example mono(or di) lower alkyl amines (e.g. mono- or di-methylamine, mono- or di-ethylamine, mono- or di-propylamine, mono- or di- isopropylamine,
mono- or di-butylamine, etc.), mono- or di-lower alkenylamines (e.g., mono- or di-allylamine, etc.), mono- or di-al(lower) alkylamines (e.g., mono- or di-benzylamine, etc.) ), cyclic amines (eg, piperidine, piperazine, pyrrolidine, morpholine, etc.). Additionally, useful amines include weakly basic ion exchange resins with primary or secondary amino groups (e.g. Amberlite IR-45
(OH ^- type) (manufactured by Rohm & Haas) and ion exchange resin AH-22 (OH ^- type) (manufactured by the Ministry of Chemical Industry of the Soviet Union). As the acid, organic acids such as formic acid and acetic acid, and inorganic acids such as hydrochloric acid and sulfuric acid are used. The reaction of xanthotricine with an amine is generally carried out in a solvent such as alcohol, acetone, water, etc., and the reaction temperature is usually 20 to 25°C and preferably carried out under conditions of pH 8 to 12. After making the reaction solution obtained in this way acidic (for example, pH 3 to 4) with the above-mentioned acid, leumycin can be isolated using a solvent such as the above-mentioned alcohol such as methanol or ethanol. . Next, the present invention will be further explained by the following examples. Example 1 A culture medium with the following composition was placed in a 750 ml Erlenmeyer flask.
Add 125ml. Dry weight% Corn staple liquor 0.25 Calcium carbonate 0.5 Sodium chloride 0.5 Ammonium sulfate 0.35 Glucose 1.5 Starch 2.0 Tap water 1 PH 6.9-7.0 After sterilizing the above medium by a conventional method, Streptomyces bruneus subsp. xanthotricini RLA-1568 was added. Inoculate agar slants incubated for 12 days. Rotate the flask at a speed of 230~
A culture is obtained by placing it on a shaker at 240 rpm and culturing at 26-29°C for 48 hours. Separately, 125 ml of a medium having the same composition as above was placed in each 750 ml Erlenmeyer flask and sterilized using a conventional method. This medium is inoculated with 2.5 ml of the above culture and cultured at 26-29°C for 72 hours. After the cultivation is completed, strain the aerial mycelium from the culture solution 6 and adjust the pH of the solution to 5.6 to 6.0 with dilute sulfuric acid.
and extract with chloroform. The extracts are dried over anhydrous sodium phosphate and evaporated in vacuo (20 mm) at 35°C. The residue was dissolved in 20 ml of isopropyl alcohol and left at 5°C for 6 hours. Collect the precipitated xanthothricin crystals. A second product of xanthothricin is recovered from the liquor by evaporation and recrystallization from isopropyl alcohol (yield 363 mg), mp 172-173°C. Example 2 A 45-volume fermentation vessel is charged with 25 culture medium of the composition described in Example 1. The medium is sterilized at 122-124° C. for 35 minutes and inoculated with a culture of Streptomyces bruneus subsp. xanthotricini in an amount of 2% by volume based on the medium. Culture is 65-72
This is carried out by adding Spine whale oil as an antifoaming agent while aerating at a rate of 0.8 to 1 volume of air per minute per volume of medium. After culturing, isolate the mycelium from the culture solution 20,
Extract the liquid with chloroform. After separating the chloroform layer, the aqueous layer is brought to pH 5.0 and extracted with ethyl acetate. After evaporating the chloroform layer and ethyl acetate layer, combine the respective residues and add 300 g of silicic anhydride.
Chromatography is performed using a column (150-200 mesh) packed with Rf eluted with ethyl acetate
= 0.08 fraction (silica gel, ethyl acetate) is collected, and 962 mg of xanthothricin is recovered from the fraction. Melting point 172-173℃. Example 3 Mycelium was separated from the culture solution 6 obtained in Example 1 by filtration, and ammonium sulfate was added to the solution.
Add 3.6Kg. Filter the precipitate and extract the liquid with chloroform. The subsequent processing is carried out in the same manner as in Example 1. Example 4 A culture of Streptomyces bruneus subsp. , and then extracted with chloroform. The extracts are dried over anhydrous sodium sulfate and evaporated at 40°C.
Distilled water basified the residue to PH8.0~10.550
Dissolve in ml. Anion exchanger in the resulting solution
Add AH-22 (OH ^- ) until the color of the solution fades. After separating the anion exchanger from this mixture, the eluate containing leumycin is obtained by washing with distilled water and treatment with 5% acetic acid. This eluate is concentrated under reduced pressure and the residue is crystallized to obtain 410 mg of leumycin (yield 76%). Melting point 242-243℃.

Claims (1)

[Claims] 1. Production of xanthothricin, which comprises culturing a strain belonging to Streptomyces bruneus subsp. Law. 2. The method according to claim 1, wherein the Streptomyces bruneus subsp. xanthotoricini is Streptomyces bruneus subsp.