JPH0348799B2 - - Google Patents

Description

[Detailed description of the invention]

Industrial Application Field The present invention is directed to 2-amino-4-(hydroxy)(methyl)phosphinoyl-butyryl-L-alanyl-L-alanine (hereinafter referred to as SF-1293 substance) which has herbicidal activity and is useful as a herbicide. This relates to the improvement of the culture production method for .). Conventional technology SF-1293 substance is Streptomyces hygroscopicus SF
−1293 strain (see Special Publication No. 51-639, ATCC −
21705), and later it was discovered to have strong herbicidal activity (see Japanese Patent Publication No. 59-23282), and its development as a herbicide has progressed. In order to develop it as a herbicide, its safety,
In addition to research and development on efficacy, it is essential to conduct research to improve manufacturing methods for low-cost production. As a report on the improvement of the manufacturing method, 2-amino-4-(hydroxy)(methyl)phosphinoylbutyric acid (hereinafter referred to as
It is called AMPB. ) (Japanese Unexamined Patent Application Publication No. 1983-1999)
21754), a method of adding biosynthetic intermediates of SF-1293 substance (JP-A-58-219191 and JP-A-58
-146591). In both methods, the phosphorus-containing component of SF-1293 substance
Although the focus is on AMPB, there is no example yet of an improved production method that focuses on L-alanine, which is a phosphorus-free constituent according to the present invention. Problems to be Solved by the Invention Conventional herbicides are artificially synthesized compounds that are widely used and contribute to environmental pollution, but SF-1293 substance is produced by microorganisms and can be quickly It is attracting attention as an ideal herbicide because it undergoes decomposition. To date, no herbicides produced by microorganisms have been put into practical use or widely used, and this is because it is difficult to produce secondary metabolites produced by microorganisms in good yields. The present invention focuses on these problems and attempts to solve them by establishing an improved method for producing SF-1293 substance. Means and Effects for Solving the Problems The present inventors aimed to improve the production method of SF-1293 substance by using SF-1293 substance producing bacteria.
When various compounds were added during the 1293 substance production culture and the relationship with SF-1293 substance production was investigated, it was found that alanine, a type of amino acid, had a remarkable SF-1293 substance production.
−1293 It was found that it has the effect of improving substance production. Even more surprisingly, alanine, which is a component of SF-1293, is in the L form, but DL-alanine, which is industrially produced at low cost, and D-alanine, which is a non-natural type, also has the L form. Similarly, a remarkable effect of improving production of SF-1293 substance was observed. Furthermore, the present inventors found that the effects of adding alanine to AMPB, a constituent of the SF-1293 substance,
It has also been found that it is possible to further increase the yield of SF-1293 substance by combining it with conventional methods for increasing the yield of SF-1293 substance, such as addition of biosynthetic intermediates of SF-1293 substance. Therefore, the present invention basically aims at high-yield production of SF-1293 substance, a phosphorus-containing compound, which is characterized by adding alanine to the culture of SF-1293 substance-producing bacteria belonging to the genus Streptomyces. It provides law. As alanine, one type or a combination of two or more types selected from L-form, D-form, and DL-form can be used. Also, alanine can be used alone or SF-
It can be added in combination with 1293 substance constituents such as AMPB and SF-1293 substance biosynthesis intermediates. The present invention will be explained in more detail below. As the Streptomyces strain used in the culture method of the present invention using alanine to increase the yield of SF-1293 substance, any actinomycete strain that produces SF-1293 substance can be used. An example of this is the SF-1293 strain (FERM-SP-130, ATCC-21705), which was isolated from soil and identified and named Streptomyces hygroscopicus. Streptomyces hygroscopicus SF
The mycological properties of strain -1293 are specified in Japanese Patent Publication No. 51-639. Streptomyces hygroscopicus SF-1293 strain is susceptible to changes in its properties, as seen in the case of other strains of the genus Streptomyces, and is easily mutated by artificial mutagenic means using ultraviolet rays, X-rays, chemicals, etc. There are things to do, but
Even if such a mutant strain has the ability to produce SF-1293 substance, it is possible to increase the ability to produce SF-1293 substance by adding AMPB, which is a component of SF-1293 substance, or an intermediate for biosynthesis of SF-1293 substance. Any strain of the genus Streptomyces shown can be used in the method of the present invention. In the production method of the present invention, strain SF-1293 is cultured in a medium containing nutrients that can be used by ordinary microorganisms. As the nutrient source, known nutrient sources conventionally used for culturing Streptomyces bacteria can be used. Examples of carbon sources include glucose, starch, glycerin, sucrose, starch syrup, and molasses. These may be used alone or in combination. Further, as the nitrogen source, soybean flour, wheat germ, meat extract, peptone, dry yeast, cornstarch liquor, ammonium sulfate, ammonium nitrate, etc. may be used alone or in combination.
In addition, inorganic salts such as calcium carbonate, common salt, potassium chloride, and phosphates can be added as necessary. During the production culture of SF-1293 substance as described above, alanine is used alone or as a component of SF-1293 substance,
SF-1293 added in combination with biosynthetic intermediates
It is possible to increase the yield of materials. It is more effective to add in parts rather than all at once. In addition, when alanine is added in combination with SF-1293 substance component AMPB, SF-1293 substance biosynthetic intermediate, etc., normal SF
-1293 substance production culture allows the use of a medium with a lower concentration than in the production culture, which is extremely advantageous because it contributes to a reduction in manufacturing costs. The most suitable culture method is liquid culture, especially deep culture. Cultivation was carried out under aerobic conditions,
The suitable temperature for culturing is 25-35°C, but in most cases it is cultured at around 28-32°C. The appropriate number of days for culturing is 8 to 10 days. Furthermore, in tank culture, it is desirable to control the dissolved oxygen concentration to 0.5 to 5 ppm. For other details on the culture method itself, please refer to SF-
Please refer to the description of the above-mentioned Japanese Patent Publication No. 51-639, which describes the method for producing the 1293 substance. The SF-1293 substance in the culture solution is quantified using an amino acid analyzer. In other words, the culture solution
After adjusting the pH to 2.0, the supernatant was obtained by centrifugation (3000 rpm, 15 minutes) or filtration, and separated and quantified using an amino acid analyzer (Model MLC-703 manufactured by ATTO, retention time 14 minutes). . SF-1293 from the culture solution obtained according to the present invention
The method for purifying the substance is the same as the usual method for purifying the SF-1293 substance from a culture solution, and for details, please refer to the above-mentioned Japanese Patent Publication No. 51-639. Examples Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto. Example 1 A mutant strain of Streptomyces hygroscopicus SF-1293 was cultured in a preculture medium (2.0% soluble starch, 1.0% polypeptone, 0.3% meat extract, 0.05% dipotassium hydrogen phosphate, pH 7. 0) Inoculated into 10ml. this
Cultured with shaking at 28°C for 24 hours, subcultured in 80 ml of the same medium and cultured with shaking at 28°C for 24 hours was used as a seed mother, and this was used as a seed mother to incubate with 7.0% glucose, 3.9% wheat germ, and sungrain at a ratio of 3%. 2.5%, potassium phosphate 0.3%, and cobalt chloride 0.0001%, and cultured with shaking at 28°C for 8 days.
Alanine and SF-1293 substance component AMPB were added at the concentrations and methods shown in Table 1. After making the culture solution acidic, it was centrifuged (3000 rpm, 15 minutes) to obtain a supernatant, which was then analyzed using an amino acid analyzer (MLC-703 model manufactured by ATTO, retention time 14 minutes).
The production amount of SF-1293 substance was measured. The differences are shown in Table 1.

[Table] From the data in Table 1, the effect of adding alanine is clear. Example 2 The same preculture medium as in Example 1 was used as a seed mother for jar fermenters, and the same production medium as in Example 1 and its 70% concentration medium were inoculated at a rate of 2%. In a jar fermentor (2 volumes/3 volume mini jar made by Iwashiya), culture is carried out with aeration at 28℃, and the dissolved oxygen concentration is controlled by controlling the rotation speed.
Controlled at 0.5-5.0ppm. The effects of addition of alanine, SF-1293 substance component AMPB, and its acetyl form were investigated in the same manner as in Example 1. The addition method was divided into 4, 5, and 6 days, and the DL form of alanine was used, which is inexpensive.

【table】

[Table] Shows chilled form.
From the data in Table 2, the effect of alanine on improving the production of SF-1293 substance is clear. Effects of the invention As shown in the examples, L-alanine or D-alanine or an inexpensive
By adding DL-alanine, SF-1293
It has become clear that it is possible to increase the amount of material produced. It has also been revealed that the effect of alanine addition is even more dramatically enhanced when culturing with the addition of precursors such as AMPB, a constituent component of SF-1293, and in this case, the effect of adding alanine is even greater when culturing with the addition of precursors such as AMPB, a component of SF-1293. It is extremely effective because it allows the use of a culture medium and contributes to reducing manufacturing costs. As described above, the alanine-added culture of the present invention has made it possible to dramatically increase the production amount of SF-1293 substance and to significantly reduce manufacturing costs such as medium costs.

Claims (1)

[Scope of Claims] 1. A high-yield method for producing SF-1293 substance, a phosphorus-containing compound, which comprises adding alanine to the culture of SF-1293 substance-producing bacteria belonging to the genus Streptomyces. 2. The phosphorus-containing compound according to claim 1, wherein alanine is one or a combination of two or more selected from L-form, D-form and DL-form, SF-
High-yield production method for 1293 substances.