JPS6366200B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an industrially advantageous method for producing antibiotics B-41D, E and G useful as anthelmintics and acaricides. B-41D, E, and G are antibiotics obtained by culturing B-41-producing bacteria belonging to the genus Streptomyces, such as the B-41-146 strain, and have the following structural formula, It has been shown in Japanese Unexamined Patent Application Publication No. 2003-12113 that it is effective in exterminating helminths that parasitize animals, especially nematodes, and mites that parasitize animals and plants.
Publication No. 56-32481, Japanese Patent Application No. 55-153141
57-77686) and Japanese Patent Application No. 56-7091 (Japanese Unexamined Patent Publication No. 57-120589). By the way, when B-41-producing bacteria are cultured in the usual manner, B-41A ₁ , A ₃ , B ₂ having a methyl group at the 25th position and B-41 having an ethyl group at the 25th position correspond to the above structural formula. To produce 41A ₄ , B ₃ , B ₂ , etc. at the same time,
The most active B- with isopropyl group at position 25
A method for producing 41D, E, and G with higher yields is desired. The present inventors have discovered that when culturing B-41-producing bacteria, by adding a specific substance to the medium,
-41D, E and G were found to be produced in high yields. The present invention relates to B-41 which belongs to the genus Streptomyces.
When producing B-41D, E and G by culturing production bacteria, from valine, isobutyric acid, isovaleric acid, 2-ketoisovaleric acid, isocaproic acid, salts or esters of these acids, isobutanol and its esters. This is a method for producing B-41D, E, and G, which is characterized by adding one or more selected types to a medium. B-41 producing bacteria, such as Streptomyces B-
Strain 41-146 has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, as FAIKEN Article No. 1072, and its mycological properties are described in detail in JP-A-50-29472. The bacteria used in the present invention include the above B-41-146 strain, for example, by X-ray irradiation,
Those that have been artificially modified using ultraviolet irradiation, radiation irradiation, or artificial mutating agents and that have the ability to produce B-41D, E, and G are included. In the method of the present invention, the L-form or DL optical isomer of valine added to the medium is used. Among valine, isobutyric acid, isovaleric acid, 2-ketoisovaleric acid, and isocaproic acid, L- or DL valine, isobutyric acid, and 2-ketoisovaleric acid are preferably used. Examples of salts of these acids include sodium salts and potassium salts, and examples of esters include lower alkyl esters such as methyl, ethyl, and n-butyl, or benzyl esters. Isobutanol and its esters can also be used, and esters include those with lower saturated fatty acids such as acetic acid and propionic acid. In an experiment using the above additive ¹³ C-labeled compound, it was confirmed that an isopropyl group was specifically incorporated into the 25th position of the product. The amount of these additives added to the medium is generally 0.001 to 1 W/V%, preferably 0.005 to 0.01 W/V%.
V% is added. The addition time may be during preparation of the medium or at an appropriate stage during culture. The medium used for culturing the B-41-producing microorganisms may be one that contains nutritional sources that can be used by the microorganisms, and in addition to the above additives, carbon sources include glucose, sucrose, starch, glycerin, and starch syrup. , molasses,
Soybean oil is used, and nitrogen sources include skim milk, soybean flour, wheat germ, meat extract, peptone,
Yeast cells, corn steep liquor, ammonium sulfate, ammonium nitrate, etc. are used. In addition, calcium carbonate, common salt, potassium chloride, and phosphates can be added as necessary. The most suitable culture method is the liquid culture method, especially the deep culture method, similar to the method for producing general antibiotics. Cultivation is carried out under aerobic conditions, and the appropriate temperature for cultivation is 22 to 30°C, but in most cases 28°C.
Culture at around ℃. The liquid quality of the medium is approximately PH5.5-8.0
It is best to have a pH between 6.5 and 7.5, preferably around neutral pH, to obtain better results. Culture is B-
The process is continued until the accumulated concentration of 41D, E, and G reaches the maximum, but the time required for this depends on the culture method, temperature,
Although it varies depending on conditions such as the composition of the medium, it usually takes about 5 to 15 days. B-41 The following method is used to test each component. That is, 3 ml of the culture is placed in a small test tube, 7 ml of acetone is added, shaken, extracted, and centrifuged. TLC 10-20μ of the supernatant obtained here.
The sample was adsorbed onto a predetermined position on a commercial plate (Merck, Kieselgel 60 F ₂₅₄ ), developed with dioxane:carbon tetrachloride (18:82) for 4 hours, and then exposed to a wavelength of 245 nm using a dual wavelength chromatography scanner. (The blank is
380nm), and the absorption amount is compared with that of the standard substance and calculated. When collecting B-41D, E, and G from cultures, adsorbents such as activated carbon, alumina, and silica gel, synthetic adsorbents such as Diaion HP-10 and HP-20 (manufactured by Mitsubishi Chemical Corporation), Avicel (manufactured by Asahi Kasei Corporation), a fixing agent such as paper, an ion exchange resin, an ion exchange gel filter, etc. can be used, but the collection method shown below is the most effective. The target product is dissolved in methanol water by separating the culture using a supernatant such as diatomaceous earth and extracting the resulting cake with methanol. After adding water to this, extraction is performed with n-hexane, and this is concentrated under reduced pressure to obtain an oily substance containing the target product. This was adsorbed on a column of silica gel (Wako Gel C-200) and eluted with n-hexane:acetone (95:5) to obtain a fraction containing B-41D.
Collect the fraction containing 41E and the fraction containing B-41G. Each fraction was concentrated under reduced pressure and the resulting residue was added to a small amount of n
- When dissolved in hexane:ethyl acetate (20:1) and allowed to stand at room temperature, B-41D, B-41E, and B-41G are obtained in crystalline form, respectively. According to the method of the present invention, the production ratio of B-41D, E, and G components in particular is very high, and the amount produced is also increased, making separation and purification easy, making it an extremely advantageous method for industrial production. The present invention will be specifically described below with reference to Examples. Example 1 Seed medium (sucrose 1%, polypeptone 0.35
%, K ₂ HPO ₄ 0.05%) was dispensed into a 500 ml Erlenmeyer flask, and after sterilization, one loopful of strain B-41-146 was inoculated and cultured at 28° C. for 48 hours to serve as a seed culture. Add 1 ml of this to main medium (4% glucose, 1 ml of soybean flour)
%, skim milk 1%, NaCl 0.3%, corn steep liquor 0.2% and CaCO ₃ 0.05%) 20ml
Inoculate a 100ml Erlenmeyer flask and incubate at 28°C.
After shaking culture for 3 days, DL-valine-2-
¹³ C was added at a concentration of 0.01 W/V%, and the cells were cultured for an additional 2 days. At the end of the culture, of the B-41 group antibiotics produced in the culture, B-41D accounted for approximately 65%, E accounted for 18%, and G accounted for 5%. When cultured without additives, the proportions of D, E, and G were 37%, 9%, and 2%, respectively. A culture cultured with the addition of DL-valine- ^2-13C was filtered, and the bacterial cells were extracted with methanol. Water was added to this to make a 50% methanol solution, and the mixture was extracted with n-hexane. The obtained n-hexane layer was dehydrated with Glauber's salt and concentrated under reduced pressure to obtain an oily substance. This oily substance was purified using a silica gel column and a Lorber column (manufactured by Merck & Co.), and B-41
Each component of the material was isolated. In each component obtained
^13C uptake was performed using ^13C -NMR at 400 MHz.
When measured by mass spectrometry, DL−
Valine-2- ¹³ C of ¹³ C is B-41D, E and G
It was found that the protein was specifically incorporated into the C-25 position. Example 2 The seed culture of Example 1 was mixed with 6% glucose, 0.3% asparagine, 0.5% leucine, 0.05% dipotassium phosphate, 0.05% magnesium sulfate, 0.05% salt,
Inoculate 1 ml each into 20 ml of a medium consisting of 0.02% calcium chloride, 0.005% zinc sulfate, 0.001% manganese sulfate, 0.002% ferrous sulfate, and trace amounts of vitamins.
L-valine, isobutyric acid, 2-ketoisovaleric acid, isocaproic acid, and isobutanol were added under the conditions shown in Table 1, and cultured with shaking at 28°C for 8 days. As a result, the results shown in Table 1 were obtained. Ta.

[Table] Example 3 Prepare the seed medium shown in Example 1, and add 600 ml of it.
was dispensed into 2000 ml Erlenmeyer flasks and sterilized. One loopful of B-41-146 strain was inoculated into this,
Culture was carried out at 28° C. for 48 hours, and the two 2000 ml Erlenmeyer flasks were transferred to a 30-jar famenta. Jiya Huamenta contains 4% glucose, 1% soy flour, 0.5% corn starch, 1% skim milk, 0.2% corn steep liquor,
Medium containing 0.3% NaCl and _0.05 % CaCO20
was prepared, the pH was adjusted to 7.2 to 7.5, and it was thoroughly sterilized. During the culture period, the temperature was 28℃ and the internal pressure was 0.5Kg/ ^cm2.
was held at After 3 days of culture, 0.01% DL-valine was added and cultured for an additional 5 days. Of the total production of group B-41 antibiotics, D accounted for 68% (weight ratio). When cultured under the same conditions without adding DL-valine, the proportion of B-41D remained at 35%.
PH of culture 20 obtained with addition of DL-valine
was adjusted to 3 with sulfuric acid, 1 kg of Celite was added, and the mixture was filtered under pressure to obtain a cake weighing approximately 3 kg. This was extracted with 15 parts of methanol and separated, 10 parts of water was added to the obtained methanol solution 15 parts, and extracted with 20 parts of n-hexane. The obtained n-hexane layer was dehydrated with Glauber's salt and concentrated under reduced pressure in a 40-45°C water bath to obtain 23 g of oil. This was dissolved in about 30 ml of n-hexane, adsorbed onto a column packed with 2 kg of silica gel in advance with n-hexane, and developed with n-hexane:acetone (95:5). As a result, a fraction containing the target substance B-41D was obtained.
Got 2.5. When these were concentrated under the same conditions as described above, crude crystals of B-41D were obtained. This was dissolved in n-hexane:ethyl acetate (20:1), left to stand at room temperature, and precipitated crystals were collected to obtain 210 mg of crystals of B-41D.

Claims (1)

[Claims] 1. Antibiotic B- belonging to the genus Streptomyces
41 When producing B-41D, E and G by culturing production bacteria, valine, isobutyric acid, isovaleric acid, 2-
A method for producing B-41D, E and G, which comprises adding one or more selected from ketoisovaleric acid, isocaproic acid, salts or esters of these acids, isobutanol and esters thereof to the medium. 2 The B-41 producing bacterium is Strepmyces B-41-
146 strain according to claim 1. 3. The manufacturing method according to claim 2 for manufacturing B-41D. 4 Streptomyces genus B-41-146 strain, L-
or B-41D is produced by culturing in a medium supplemented with one or more selected from DL-valine, isobutyric acid, 2-ketoisovaleric acid, and salts or esters of these acids. The manufacturing method described in item 1.