JPS6120278B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an improved method for producing SF-1293, an antibiotic useful as a non-polluting antifungal agent and herbicide, by culturing. SF-1293 substance is disclosed in Japanese Patent Publication No. 51-639 (Patent No.
It is an antibiotic produced for the first time by the production method described in No. 827768), and has the following formula: It has the structure of SF-1293 substance is Special Publication No. 51-639
As described in the publication, in addition to having a wide range of antifungal activity, it is a particularly useful substance as a herbicide that exhibits herbicidal activity against a wide range of plants.
67026, Special Publication No. 59-23282 (Patent No.
1270965)]. Conventionally, herbicides have been widely used as artificially synthesized compounds based on organic synthesis, and since these are non-natural products, they decompose slowly and may become a cause of environmental pollution. However, the SF-1293 substance is produced by microorganisms and is rapidly decomposed, so it is considered an ideal herbicide that does not pollute the environment. The reason for this is that the SF-1293 substance is a natural product that is optically active in the L form, and is in a form that is easily metabolized by soil bacteria. A method for producing SF-1293 substance by organic synthesis is also known (Meika Annual Report
(Vol. 13, p. 54, 1973), the synthesis yield is low, and what is obtained is a mixture of optically inactive D and L isomers, of which the non-natural D isomer cannot be degraded by soil bacteria. There may also be a drawback that it is slow. On the other hand, considering the biosynthesis process of SF-1293 substance, the formula: It is presumed that compounds having a hydroxymethylphosphinoyl group represented by
The present invention was established by discovering that when added to the culture of SF-1293 substance-producing bacteria, it was converted to SF-1293 substance at a high rate. Therefore, the present invention provides L-2-amino-4-(hydroxymethylphosphinoyl)butyric acid and DL-2-amino-4 as SF-1293 substance precursors to SF-1293 substance-producing bacteria belonging to the genus Streptomyces. −
(Hydroxymethylphosphinoyl) A hydroxymethylphosphinoyl group-containing substance selected from butyric acid, methylphosphinic acid, and their salts is added to culture, and SF-1293 substance is collected from the culture. The present invention provides a high-yield production method for antibiotic SF-1293 substance. The structural formula of the basic compound used as the hydroxymethylphosphinoyl group-containing substance in the method of the present invention is as follows. 2-amino-4-(hydroxymethylphosphinoyl)butyric acid Methylphosphinic acid According to the method of the present invention, utilizing the above phenomenon,
This is extremely advantageous because it can dramatically increase the production yield of SF-1293 substance and contribute to reducing manufacturing costs. Furthermore, in the method of the present invention, the present inventors
L-2-amino-4 as SF-1293 substance precursor
-(Hydroxymethylphosphinoyl)butyric acid allows SF- with almost 100% conversion at low concentrations.
1293 substance, the DL form of the compound obtained by synthesis has a conversion rate of about 50% compared to the L form. It was found that the present invention can be applied to the optical resolution of -4-(hydroxymethylphosphinoyl)butyric acid or its salt. Specifically, such optical resolution is performed by injecting DL-2-amino-4-(hydroxymethylphosphinoyl) into SF-1293 substance-producing bacteria belonging to the genus Streptomyces as an SF-1293 substance precursor.
L-2-amino-
4-(Hydroxymethylphosphinoyl)butyric acid or its salt is selectively consumed, and D-2-amino-4-(hydroxymethylphosphinoyl)butyric acid or its salt remaining in the culture solution is removed by a method known per se. This can be achieved by isolation by method. The microorganism used in the culture method of the present invention is any strain that produces the SF-1293 substance belonging to the genus Streptomyces, and one representative strain is Streptomyces hygroscopicus SF-1293.
(Streptomyces hygroscopicus SF-1293), and was designated as Streptomyces sp. This is a strain internationally deposited as (transferred from No. 996). Furthermore, regarding the production of SF-1293 substance 2-
Various mutant strains can also be used, such as amino-4-(hydroxymethylphosphinoyl)butyric acid dependent mutants. As the medium, various nutrient sources commonly used in microbial fermentation production can be used.
For example, carbon sources include starch, glucose, glycerin, soybean oil, etc., and nitrogen sources include soybean flour, wheat germ, polypeptone, soluble vegetable protein, etc. As with general antibiotic production and conversion, the most suitable culture method is liquid culture, especially deep culture. Cultivation and conversion are carried out aerobically, with suitable culturing temperatures ranging from 20 to 37°C, but often 28°C.
It will be held nearby. Culture and conversion usually reach the maximum titer in 2 to 5 days for both shaking and tank culture. For details of the culture method itself, please refer to the above-mentioned Japanese Patent Publication No. 51-639. SF-1293 substance assay is Bacillus subtilis mutant strain No.8193
By microbial assay using strains. i.e. glucose 1.0%, meat extract 0.5%,
_{NH4H2PO4 0.1} %, _NaCl 0.5%, _MgSO4 .
_7H2O0.02 %, _{K2HPO4 0.3} %, agar 1.5% (PH7.0)
Inoculate the spore suspension into a medium, prepare an agar plate, and assay using the paper disc method. The SF-1293 substance exhibits a linear relationship between logarithm of concentration and inhibition diameter within the range 3-50 μg/ml. When DL-2-amino-4-(hydroxymethylphosphinoyl)butyric acid is used in the additive culture, which is a feature of the present invention, SF-
1293 substance and remaining unused D-2-
Although amino-4-(hydroxymethylphosphinoyl)butyric acid coexists, chromatography using an ion exchange resin is particularly effective for separating the two. For example, culture Amberlite IRA−
D-2-
Amino-4-(hydroxymethylphosphinoyl)butyric acid eluted after the SF-1293 substance, allowing both substances to be isolated in their respective pure forms. The recovered D-2-amino-4-(hydroxymethylphosphinoyl)butyric acid can be easily racemized by dissolving it in water or a dilute alkaline aqueous solution and heating it to a temperature of 150 to 250°C in a closed container. It can be converted into DL form and used again for supplementation culture.
In particular, in the present invention, 2-amino-4-
It has the advantage that the DL form of (hydroxymethylphosphinoyl)butyric acid can be converted to SF-1293 substance with high utilization efficiency. Next, the present invention will be explained with reference to examples. Example 1 Streptomyces hygroscopicus SF
-1293 strain (Feikoken Joyori No. 130) was inoculated into a medium consisting of 2.0% soluble starch, 1.0% polypeptone, 0.3% meat extract, and 0.05% K ₂ HPO ₄ (PH 6.0 to 7.0). Incubate the cells with shaking at 28°C for 24 hours. Glycerin 2.75
%, wheat germ 3.26%, sungrain 0.5%, soybean oil 0.2%, _{CoCl2.6H2O0.001 %} , _NiCl2 .
6H ₂ O 0.0001%, NaH ₂ PO ₄ 0.005%, and a medium supplemented with precursors at the concentrations shown in Table 1 were inoculated and incubated at 28°C.
Incubate with shaking for 96 hours. The culture solution was centrifuged (3000 rpm, 15 minutes) to obtain a supernatant, which was diluted and assayed. The results are shown in Table 1.

[Table] As shown in Table 1, the effect of adding the precursor is clear. Example 2 Culture was carried out under the same preculture conditions as in Example 1, and 200 μg/ml of DL-2-amino-4-
Add (hydroxymethylphosphinoyl)butyric acid and incubate with shaking for 96 hours. The pH of the obtained culture solution
Adjust to 2.0, remove bacterial cells by centrifugation, and 1.2
A culture solution was obtained. The culture titer was 600 μg/ml. After decolorizing through a 50ml activated carbon column, 500ml
Adsorb it on Dowex 50W (H ⁺ type) and wash it with 500ml of water. Elution is 0.05N
The antibacterial fractions were collected and concentrated to dryness to obtain about 2.3 g of crude powder, which had a specific activity of SF-1293 substance of 285 _μg /mg. Next, dissolve this coarse powder in about 10ml of water,
The column was adsorbed onto 100 ml of Dowex (CH ₃ COO ^- type) and the column was washed with water. Elution with 0.2N acetic acid yielded 940 mg of a fraction containing SF-1293 substance. The specific activity of SF-1293 substance is 600μg/mg
It was hot. D-2-amino-4-(hydroxymethylphosphinoyl)butyric acid was separated by successively eluting the column with 0.5N acetic acid, collecting the ninhydrin-positive fractions, and concentrating to dryness.
150 mg of coarse powder was obtained. This powder was dissolved in 0.5 ml of water and adjusted to pH 5.5, then adsorbed onto a 50 ml Dowex 1×2 (CH ₃ COO ^- type) 200-400 mesh, and the column was washed with water and 0.2 N acetic acid. Then, 23 mg of SF-1293 substance was recovered with 0.2N acetic acid. Next, the column was eluted with 0.5N acetic acid, and the fractions positive for ninhydrin reaction were collected and concentrated to dryness.92
mg of D-2-amino-4-(hydroxymethylphosphinoyl)butyric acid crystals were obtained. The properties of this crystal are: melting point 152℃, optical rotation [α] _D
= -26.2° (c = 1.0, 1N HCl), and the elemental analysis value was calculated from the molecular formula C ₅ H ₁₂ NO ₄ P. The measured value was C32, compared to the calculated value of C33.16% H6.68% N7.73%. .87% H6.71% N7.45%. Dissolve 50 mg of this D-2-amino-4-(hydroxymethylphosphinoyl)butyric acid in 0.2 ml of water,
Heated in a sealed tube at 150°C for 25 hours, decolorized the resulting reaction solution with charcoal powder, dried it, and recrystallized from water-ethanol to obtain DL-2-amino-4-(hydroxymethylphosphinoyl)butyric acid. 42 mg of crystals were obtained.

Claims (1)

[Claims] 1 L-2-amino-4-(hydroxymethylphosphinoyl) was used as an SF-1293 substance precursor in SF-1293 substance-producing bacteria belonging to the genus Streptomyces.
A substance containing a hydroxymethylphosphinoyl group selected from butyric acid, (-DL-2-amino-4-(hydroxymethylphosphinoyl)butyric acid,)-methylphosphinic acid and salts thereof is added and cultured. A method for producing an antibiotic SF-1293 substance with high yield, which comprises collecting the SF-1293 substance from a substance.