JPS638760B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel method for producing L-2-amino-4-(hydroxymethylphosphinyl)butyric acid. L-2-amino-4-(hydroxymethylphosphinyl)butyric acid is a compound that has herbicidal and antifungal effects, and its production method is already known.
SF-1293 substance (JP-A-48-22688), a method for acid decomposition of L-2-amino-4-(hydroxymethylphosphinyl)butyryl-L-alanyl-L-alanine (JP-A-48-22688) No. 85538), or a method of decomposing it with microbial enzymes (Japanese Patent Application Laid-open No. 1973-
31890), as well as chemical synthesis methods (Japanese Unexamined Patent Publication No. 1973
91019 and JP-A-54-84529), a method is known in which a racemate is obtained and optically resolved using a microorganism. Recently, it has been discovered that L-2-amino-4-(hydroxymethylphosphinyl)butyryl-L-alanine can be collected from a culture of a bacterial strain used for the production of SF-1293 substance (Japanese Patent Application Laid-Open No. 1983-1993). −7237
issue). The present inventors conducted a detailed study on the culture of a bacterial strain capable of producing the SF-1293 substance, and found that L-2-amino-4-(hydroxymethylphosphinyl)butyric acid was directly collected from the culture. They were very successful and completed the present invention. That is, in the present invention, L-2-amino-
This method is characterized by collecting 4-(hydroxymethylphosphinyl)butyric acid. The strain belonging to the genus Streptomyces used in the present invention is one that produces a sufficient amount of L-2-amino-4-(hydroxymethylphosphinyl)butyric acid to be collected in the culture. An example of such a strain is Streptomyces hygroscopieus, which was isolated from soil and whose mycological properties are described in Patent No. 827768 (Special Publication No. 51-639).
One example is the SF-1293 strain (Feikoken Bibori No. 996, ATCC No. 21705), which was identified and named. This SF-1293 strain, like other strains of the genus Streptomyces, can be mutated by ultraviolet rays, X-rays, chemicals, etc., but even such mutant strains can be mutated by L-2-amino- 4-
Any strain capable of producing (hydroxymethylphosphinyl)butyric acid can be used in the present invention. In the method of the present invention, the SF-1293 strain described above is cultured in a medium containing nutrients that can be used by ordinary microorganisms. As the nutrient source, those conventionally used for culturing Streptomyces bacteria are used. For example, the carbon source includes glucose, starch, glycerin, sucrose, starch syrup, molasses, etc., and the nitrogen source Examples include soybean flour, wheat germ, meat extract, peptone, dried yeast, corn staple liquor, ammonium sulfate, sodium sulfate, urea, and the like. The most preferable method for culturing SF-1293 strain is a liquid culture method carried out under aerobic conditions, and the temperature at this time is preferably within the range of 25 to 35°C, but especially around 28°C. temperature is often used. In addition, the culture time is longer than that for SF-1293 substance, but in both shaking culture and tank culture, L-2-amino-4-
(Hydroxymethylphosphinyl)butyric acid extraction amount reaches maximum. In the present invention, as a method for collecting L-2-amino-4-(hydroxymethylphosphinyl)butyric acid from the culture solution obtained in this way, ion exchange resin, cellulose, silica gel, alumina or Cephadex is used. Commonly used physicochemical methods such as chromatography are applied. For example, when extracting and purifying L-2-amino-4-(hydroxymethylphosphinyl)butyric acid from a culture solution, the L-2-amino-4-(hydroxymethylphosphinyl)butyric acid is a water-soluble Since it is an amphoteric substance, Amberlite IR-120, Dowex
The L-2-amino-4-(hydroxymethylphosphinyl)butyric acid is selectively adsorbed on a cation exchange resin such as 50W or an anion exchange resin such as Amberlite IRA-400 or Amberlite IR-4B, This is eluted using an acidic solution such as hydrochloric acid, sulfuric acid, or acetic acid, an alkaline solution such as sodium hydroxide or potassium hydroxide, or a salt solution such as sodium chloride or calcium carbonate. Among these methods, a method in which the culture solution is passed through a packed column of Dowex 50W (H type) cation exchange resin and then developed with water is a method using L-2-amino-4-(hydroxymethyl phosphite). This method is effective as a method for purifying nil)butyric acid. Furthermore, in order to increase the purity of L-2-amino-4-(hydroxymethylphosphinyl)butyric acid,
In addition to the above-mentioned ion exchange resin, it is effective to apply chromatography in which a column is filled with cellulose, silica gel, alumina, Cephadex, or the like. Compared to the method of decomposing the SF-1293 substance or the chemical synthesis method, the method of the present invention can collect only the L-isomer with high purity, and the process can be significantly shortened. Therefore, L-2-amino-4 -(Hydroxymethylphosphinyl)butyric acid is useful because it has advantages such as being able to reduce the manufacturing cost. The method of the present invention will be explained in more detail below based on Examples, but the present invention is not limited to the following Examples. Example 1 One mutant strain of Streptomyces hygroscopicus SF-1293 was mixed with 2.0% soluble starch and 1.0% polypeptone.
%, meat extract 0.3%, dipotassium phosphate 0.05%, pH 7.0, and inoculated into 10 ml of preculture medium. This was cultured with shaking at 28°C for 24 hours, and then subcultured into 80 ml of the same medium and cultured with shaking at 28°C for 24 hours, which was used as a seed mother for a jar fermenter. In the Jafer Armentor, the seed mother was inoculated into production medium 4.0 consisting of 4.0% glucose, 3.0% sungrain, 2.0% wheat germ, 0.1% monopotassium phosphate, and 0.0001% cobalt chloride, and incubated at 28°C. The culture was usually stirred. After extracting the culture solution over time and adjusting it to pH 2.0, it was centrifuged (3000 rpm, 10 minutes) to obtain a supernatant, and analyzed using an amino acid analyzer (MLC-703 manufactured by Art Co., Ltd.).
L-2-amino-4-(hydroxymethylphosphinyl)butyric acid was quantified using the following method (type, retention time: 12 minutes). Culture time and L-2-amino-4- in the culture solution
The production amount (μg/ml) of (hydroxymethylphosphinyl)butyric acid is shown in the table.

[Table] The culture solution obtained by aeration and agitation culture for 192 hours.
After adjusting the pH to 2.0 and removing the bacterial strain by centrifugation, a culture solution with a pH of 2.0 was obtained. Example 2 The culture solution obtained in Example 1 was applied to 400 ml of Dowex 50W x 2 (H ⁺ type) cation exchange resin, developed with water, and L-2-amino-4-
The fraction containing (hydroxymethylphosphinyl)butyric acid was concentrated. This was then applied to 150 ml of Dowex 1x2 (CH ₃ COO ^- type) anion exchange resin, washed with water, and eluted with 0.3N acetic acid. After elution, the fraction containing L-2-amino-4-(hydroxymethylphosphinyl)butyric acid was concentrated to dryness under reduced pressure, and then truly vacuum-dried to obtain L-2-amino-4-
1.20 g of white powder of (hydroxymethylphosphinyl)butyric acid was obtained. The purity determined by this amino acid analyzer was 85%, and the yield was 71%. Example 3 A seed mother cultured under the same preculture conditions as in Example 1 was used and cultured in a 570-tank fermenter.
Culture: 2.0% glucose, 2.0% starch, 2.0% soybean flour
%, wheat germ 2.5%, monopotassium phosphate 0.1%, cobalt chloride 0.0001%, pH 7.0 production medium 300 was inoculated with seed mother at a ratio of 0.5%, and the mixture was grown at 28°C.
The mixture was stirred and aerated for about 168 hours. After adjusting the pH of the obtained culture solution to 2.0, bacterial cells were removed using an Oliver filter, and a culture solution 240 was obtained. At this time, L-2-
Amino-4-(hydroxymethylphosphinyl)
Butyric acid was quantified and found to be 580 μg/ml. Next, apply this culture solution to Dowex 50W x 2.
(H ⁺ form) in a cation exchange resin tower (50), and then developed with water. The obtained L-2-amino-4
The fraction containing -(hydroxymethylphosphinyl)butyric acid was further added to Dowex 1 x 2 (CH ₃ COO ^- type).
The mixture was applied to an anion exchange resin tower (15), washed with water, and eluted with 0.3N acetic acid. Eluent L-2-
Amino-4-(hydroxymethylphosphinyl)
After concentrating the fraction containing butyric acid, methanol was added to crystallize it to obtain 88 g of crystals of L-2-amino-4-(hydroxymethylphosphinyl)butyric acid. The yield is
It was 63%. The obtained crystals were subjected to elemental analysis using conventional methods.
Specific rotation, melting point, infrared absorption spectrum, nuclear magnetic resonance spectrum, and mass spectrometry revealed that L-2-
Amino-4-(hydroxymethylphosphinyl)
It was confirmed that the structure completely matched the structure of the butyric acid sample. As an example, an infrared absorption spectrum measured by the potassium bromide tablet method (using a Hitachi infrared spectrophotometer: model 260-10) is shown.

[Brief explanation of the drawing]

The figure shows an infrared absorption spectrum of L-2-amino-4-(hydroxymethylphosphinyl)butyric acid obtained by the method of Example 3, measured by the potassium bromide tablet method.

Claims (1)

[Claims] 1. Cultivate a strain belonging to the genus Streptomyces under aerobic conditions at 25-35°C for 6-8 days, and collect L-2-amino-4-(hydroxymethylphosphinyl)butyric acid from the resulting culture solution. A method for producing L-2-amino-4-(hydroxymethylphosphinyl)butyric acid, characterized by the following.