KR840000378B1 - Process for preparing"mildiomycin" - Google Patents

Abstract

Mildiomycin is produced by fermn. with Streptoverticillium rimofaciens. Thus, one loopful of s. rimofaciens FERM-P2549 (IFO 13592) was inoculated into 25mL medium contg. glucose 10%, Proflo 3%, corn ext. soln. 1%, NaCl 0.5%, FeSO4 0.001%, CaCO3 0.5%, and choline 3-70mM and incubated in rotary shaker (200rpm) at 28≰C fro 120hr. The broth (1 L) was filtered, treated with anion exchange chromatog., and concd. After addn. of MeOH to the conc., 1.1g mildiomycin was obtained. Mildiomycin inhibited the powder milew and has acaricidal properties.

Description

Preparation of Mildiomycin

The present invention relates to a method for preparing Mildiomycin.

That is, in the production method of Mildiomycin, in which a radioactive bacterium having Mildiomycin production ability is cultured in a medium to produce and accumulate Mildiomycin in the medium, the presence of one or two or more kinds of N-methyl compounds in the medium is present. It is a method characterized by.

Mildiomycin has been called antibiotic B-98891 and has been discovered since its appearance, structural formula

On the other hand, it is an antibiotic that has been noted for its effect as a prophylactic, therapeutic or acaricide for powdery mildew in a wide range of crops. (Kiso 51 (1976) -9718, East 50 (1975) -126892, The Journal of Antibiotics, Vol. 31, No. 6, pages 511-518, pages 519-524) , 1978).

Against this backdrop, the present inventors have intensively studied the production conditions for the purpose of supplying Mildiomycin in large quantities and at low cost, and as a result, they have been cultivating radiation bacteria having Mildiomycin production capacity. At that time, it was found that a significant amount of Mildiomycin can be produced by the presence of an N-methyl compound in the medium, and finally the present invention was completed.

That is, the present invention is a method for producing Mildiomycin, which comprises culturing a radiation bacterium having Mildiomycin production ability in a medium containing 3 mM or more of N-methyl compound, thereby producing and accumulating Mildiomycin in the culture. .

Examples of the radiation bacterium with Mildiomycin production ability used in the method of the present invention include, for example, radiation bacteria belonging to the genus Streptoverticillium, in particular, Straptoverticillium rimofaciens FERM-. P2549 (IFO 13592) is used.

This bacterium is based on the classification criteria in the Burgy's Manual of Determinative Bacteriology 7th Edition and is identical to the Streptomyces rimofaciens. Although it was defined (Special Place 50 (1975) -126892), the classification criteria were subsequently revised, and according to the Burj's Manual of Determinant Bacteriologici-8th Edition (1974), the bacterium It has to belong to the inside.

(The Journal of Antibiotics, Vol. 31, No. 6, pages 511 to 518) In addition, as the N-methyl compound, a compound having one or more nitrogen atoms substituted with 1 to 4 methyl groups in a molecule is used.

Among them, for example, one having a nitrogen atom substituted with a methyl group in the molecule is preferable, and a fourth ammonium salt having trimethylammonio: t2 of nitrogen atoms substituted with three methyl groups is particularly suitable.

Also, those which can be changed to> N-CH ₃ in the medium, such as N, N-methylenebisacrylamide, are also used as N-methyl compounds. The molecular weight of the N-methyl compound is usually 50 to 1000, preferably 90 to 130. Although used in spite of being water-soluble and water-insoluble, an easily water-soluble N-methyl compound is easy to use.

Examples of such N-methyl compounds include N-methyl acid amide, N-methyl amino compound, N-methylamine, N-methylammonium compound, polymethylenediamine, N, N-methylenebisacrylamide, and the like. -methyl amide such as, for example, N, N- dimethyl C ₂ -6 aliphatic acid amides (eg N, N- dimethylacetamide, etc.), N- methyl-alkenyl-carbonyl amides (eg N- methylacrylamide Similar to N-methylamino compounds, for example monoditriol tetramethyl urea (eg N-methyl urea, 1,1,3,3-tetramethyl urea, etc.), dimethylaminoalcohol (e, g, Such as N-methylamine, for example N-methylammonium compound, similar to monodioltrimethylamine (e, g, trimethylamine, dimethylamine, etc.) Phosphatidylethanol-mono, dioltrimethylammonium (e, g, lecithin, etc.) trimethylammonium alcohol (e, g, corin, etc.), trimethylammonium Ethylenediamine, tetramethylene, similar to carboxylic acids (e, g, betaine, etc.), alkyltrimethylammonium (e, g, tetramethylammonium, etc.), ethylenebis (trimethylammonium) chloride and like polymethylene diamine Good results are obtained when N-methylammonium, which can be effectively used such as diamine and hexamethylenediamine, and especially choline, betaine and tetramethylammonium are used. These N-methyl compounds may be used alone or in combination of two or more.

Also, N-methyl compounds, such as betaine molasses containing betaine, soy flour containing lecithin, or eggs containing choline and lecithin are added to the medium, and the content of N-methyl compound is 3 mM or more. The method of making is also included in the claims of the present patent.

What is necessary is just to select suitably the density | concentration of the N-methyl compound contained in a culture medium in the range which does not suppress the growth of the microorganism to be used, and it is contained normally 3mM or more.

Preferably, the addition of 4 mM to 200 mM, more preferably 7 to 50 mM, is effective, and when it exceeds 200 mM, the progress of the effect is slowed down. In addition, the addition amount of the natural product containing the N-methyl compound may be selected so that the concentration of the N-methyl compound contained may be in the above-mentioned range, but when the amount of the conventional addition exceeds the conventional amount, a large amount is used. Depending on the effect of the components, the growth of microorganisms is remarkably affected, and the production of Mildiomycin may be inhibited. In that case, it is preferable to use an N-methyl compound in combination.

In addition, as the timing for incorporating these N-methyl compounds into the medium, it is the easiest and most effective method to add the medium before inoculating the microorganisms, but it can be appropriately added during the culture.

Moreover, as a carbon source of the medium used for cultivation, for example, starch, dextrin, glucose, maltose, sugar, sorbitol and the like, in addition to sugars that can use microorganisms used in molasses, coon syrup, starch syrup, for example acetic acid, Organic acids such as succinic acid and fats and oils can also be used.

As the nitrogen source, in addition to various ammonium salts, acetates, and urea, yeast extract, casein, meat juice, cottonseed foil, corn steep liquor, and organic natural substances such as soybean meal can be used.

As the inorganic salts, for example, salts such as iron, magnesium, manganese, cobalt, copper, sodium, potassium, calcium, and zinc are suitably used as necessary. If the microorganisms to be used require specific nutrients such as amino acids, vitamins and nucleic acid bases, they can be added as appropriate.

Cultivation is applied to the chungchi culture method, aeration stirring culture method, shaking culture method, etc., but usually by aeration stirring culture method is advantageous.

As culture temperature, it is 20 degreeC-40 degreeC, Preferably it is 24 degreeC-34 degreeC, As a liquidity of a batch, it is preferable to maintain normally in the range of pH 4-9, For this, for example, caustic alkali, ammonia water, Alternatively, the mixture may be cultured with a dilute aqueous solution such as sulfuric acid or hydrochloric acid while correcting, or an alkali salt such as calcium carbonate or sodium carbonate may be added to the medium. The incubation time can be achieved by culturing until the production of Mildiomycin sufficiently, the maximum yield can usually be obtained between 4 to 12 days.

When it is necessary to isolate and extract Mildiomycin from the culture thus obtained, a known method generally used as a means for separating basic water-soluble antibiotics is suitably applied.

For example, the insoluble components in the cells and cultures are removed by filtration or centrifugation, and then contacted with activated carbon or an adsorbent resin to adsorb Mildiomycin, which is a water-containing organic solvent, salt or acid-containing water, or buffer solution. And the like can be eluted.

In addition, since the antibiotic is a basic substance, it is well absorbed by the cation exchange resin, and it can be eluted with a suitable acid, alkali or buffer solution. Mildiomycin can be obtained by selecting, combining, and concentrating these methods suitably as needed.

Mildiomycin obtained in this way can be used safely as an active ingredient of plant fungicide. (U.S. Patent 4,007,267, United Kingdom Patent 1,507,193)

Although the content of this invention is demonstrated further more concretely using an Example below, with this example, the content of this invention is not restrict | limited.

Example 1

(1) 10% glucose, cottonseed germ meal (trade name Propro, Traderoil Mill Co. USA) 3% corn steep liquor 1%, salt 0.5%, ferrous sulfate sulfate 0.001%, calcium carbonate 0.5% Is added to the concentration shown in the first table, and then the pH is adjusted to 7.0 by dropwise adding 20% aqueous sodium hydroxide solution.

Next, it was sterilized and inoculated with a platinum loop of a culture medium of FERM-P2549 (IFO 13592), and incubated at 28 ° C. for 120 hours on a 200 rpm rotating shaker. It was.

Mildiomycin in the culture was quantified according to a known method (The Journal of Antibiotics, Vol. 31, No. 6, pages 519-524, 1978). The results shown in Table 1 were obtained.

[Table 1]

(2) In the above (1), 1 l of the culture using a medium added with choline 14 mM was collected and centrifuged to obtain a liquid floating on the surface, which was obtained by ion exchange resin Amberlite IRC-50 (H). ⁺ -Form) (made by Rohm & Haas Co. USA) was passed through a packed column.

The column was washed with water, eluted with 500 ml of 0.5% ammonia water, and the sulfur fraction was passed through a column of 50 ml of activated carbon for chromatography (Tadaya quenching agent) and adsorbed.

250 ml of acetone-water (3: 7) mixed solvent was used to collect and concentrate the eluted active fractions, and acetone was added dropwise to the concentrate to precipitate dense dimycin.

1.3 g of crude powder was dissolved in water and passed through a 25 ml column of Ambarite CG-50 (H ⁺ -) (manufactured by Rohm & Hass Co. USA), and then the column was washed with 10 ml of water, followed by 250 ml of 0.5% ammonia water. Eluted using.

The active fractions were collected, passed through a column of 50 ml of activated carbon, adsorbed, fractional eluted with 250 ml of acetone-0.1N formic acid (2: 8), and the active fractions were concentrated.

Methanol was added to the concentrate to precipitate Mildiomycin.

271nm 및 280nm에서의 자외선부 흡광도계수는 완전히 문헌에 기재된 값과 일치되었다. (The Journal of Antibiotics, 제31권, 제6호 523페이지, 1978년)It was dried under reduced pressure to obtain 1.1 g of Mildiomycin Formate: in its physicochemical properties, at its melting point, its fluorescence (Cl, O, H ₂ O and Cl, O, 0.1 N HCl).

The UV absorbance coefficients at 271 nm and 280 nm were in full agreement with the values described in the literature. (The Journal of Antibiotics, Vol. 31, No. 6, page 523, 1978).

Example 2

In the same manner as in Example 1, various N-methyl compounds shown in Table 2 instead of choline were added to the medium at a concentration of 7 mM to culture and quantitatively obtain the results shown in Table 2.

[Table 2]

Example 3

In the same manner as in Example 1, petane was added to the medium instead of choline to have various concentrations in the table below, followed by culturing and quantification, and the results shown in Table 3 were obtained.

[Table 3]

Example 4

In the same manner as in Example 1, choline and tetramethylammonium hydrochloride were combined in the medium, mixed and added so as to have various concentrations in the table below, and cultured and quantified, and the results shown in Table 4 were obtained.

[Table 4]

Example 5

In the method of Example 1, except that choline was used instead of 1,1,3,3-tetramethylurea and the time of addition to the culture medium was changed as shown in the table below, and the result was incubated and quantified by the same method. Is shown in Table 5.

The addition amount was 7 mM in the culture solution.

[Table 5]

Claims (1)

Mildiomycin-producing bacteria belonging to the strap variety silium. A method for producing Mildiomycin, characterized by culturing N-methyl compound in a medium containing 3 mM or more, thereby producing and accumulating Mildiomycin in the culture.