JPS62205789A - Production of phosphorus-containing compound sf-1293 substance in high yield - Google Patents

Abstract

PURPOSE:To enable the mass-production of a phosphorus-containing compound SF-1293 substance useful as a herbicide in high yield at a low cost, by culturing a SF-1293 substance-producing strain belonging to Streptomyces genus in the presence of a specific compound. CONSTITUTION:Streptomyces hygroscopicus SF-1293 strain is inoculated in a medium containing glucose, peptone, CaCo3, etc., and cultured at 25-35 deg.C for 4-7 days e.g. by submerged technique while adding 0.1-20mg/ml of 4-(hydroxymethylphosphinyl)-2-oxobutyric acid of formula, its salt, monoester, diester or amide dividedly to the medium and, if necessary, adding dl-alanine to the medium at the same time. The obtained culture product is centrifuged and the supernatant liquid is purified to obtain the objective phosphorus- containing compound SF-1293 substance.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention provides a non-amino-(hydroxymethylphosphinyl)-butyryl which has herbicidal activity and is useful as a herbicide.
This invention relates to an improvement in the culture production method of L-alanyl-L-alanine (hereinafter referred to as SF-/λP3 substance).

(Prior art) 131F-/, 2 and 3 substances are Streptomyces hygroscopicus (8treptomyces hygroscopicus).
Scopicus) Japan'y-txys strain (Tokukosho!t/
-See Publication No. 63P; Fl! :RM-BP-/711
7. It was discovered as an antibiotic produced by ATOC-2170) and later discovered to have strong herbicidal activity [Tokukosho! (see Japanese Patent Publication No. 23212), and its development as a herbicide has been progressing. For friends who use it as a herbicide.

In addition to research and development into its safety and efficacy, it is essential to research and improve manufacturing methods to produce it at low cost. As a report on the improvement of the manufacturing method, a method of adding 4-amino-hiro-(hydroxymethylphosphinyltobutyric acid (hereinafter referred to as AMFB)), which is a component of the three 8F-1 lid substances (JP-A-Sho!!- )17!Refer to the Publication No. 1], as well as SF
'-/, a method of adding biosynthetic intermediates of the three substances (see Japanese Patent Application Laid-open No. 1911-191 and jt-i≠t!ri/).

(Problems to be solved by the invention) Conventional herbicides are widely used as artificial base compounds produced by organic synthesis, which contribute to environmental pollution.
Substance 1293 is produced by microorganisms and is rapidly decomposed, so it is attracting attention as an ideal herbicide. None of the herbicides produced by microorganisms have ever been put into practical use or widely used, and one reason for this is that it is difficult to produce large quantities of secondary metabolites produced by microorganisms in good yields. It is.

The present invention focuses on the above-mentioned problems and solves the SF-/2? !
The aim is to solve this problem by establishing an improved method that enables high-yield production of substances.

(Means and effects for solving the problem) The present inventors have developed an S
Various phosphorus-containing compounds were added to the medium during cultivation of 61F-/Ivy for production of the three substances using F-/λri three-substance producing bacteria, and the relationship with SF-/lli three-substance production was investigated. , BF-/AM FB, which is a constituent of the 25P3 substance
It was discovered for the first time that the production of the E3F-12P3 substance was significantly improved by the addition of Hiro-(hydroxymethylphosphinyl)-λ-oxobutyric acid (hereinafter referred to as OMFB), which has a structure similar to OMFB. The production method and physicochemical properties of AMFB are described in, for example, Japanese Patent Application Laid-Open No. 6-?1IrW7. AMFB has stereoisomerism, and the previously proposed method produces only the L-form. In contrast, OMFB has no stereoisomerism, so it can be used completely in the method of the present invention, and the present invention is extremely advantageous from the viewpoint of the number of synthesis steps, synthetic leather yield, and cost. It's a method.

Therefore, the present invention provides 8F belonging to the genus Streptomyces.
'-/lid 3 substance producing bacteria is expressed by the following formula 2H≠-(hydroxymethylphosphinyl]-1
- To provide a high-yield production method for the three substances SF-/Ivy, which is characterized by culturing in the presence of oxobutyric acid, a salt thereof, a monoester thereof, a diester thereof, or an amide thereof. It is something.

As the Streptomyces strain used in the culture method of the present invention, any actinobacterial strain that produces all of the qlF-/J5Pj substances can be used. For example, Streptomyces hygroscopicus (ENre
ptomycep hygroscopicus), and the aforementioned bacterial strains FJIP-/, 2Pj strain (1FER
M-BP-/Jθ;ATC! O-λ/70! ) can be given.

Streptomyces hygroscopicus 5F-7293
The mycological properties of the strain are specified in Japanese Patent Publication No. 1, No. 39.

Streptomyces hygroscopicus 5F-1293
As seen in the case of other strains of the genus Streptomyces, the strain is susceptible to changes in its properties, and can be easily mutated by artificial mutagenic means using, for example, ultraviolet rays, X-rays, chemicals, etc. Even if it is a mutant strain, EIF-/no23
Rsyp-i Koku3 can be improved by adding AMFB or 8F-/JPj substance bioagia intermediates, etc., which have the ability to produce substances or are constituent components of SF-/J3 substances, or by adding OMFB of the present invention. Any strain of the genus Streptomyces that exhibits the ability to produce a substance can be used in the present invention.

In the production method of the present invention, EIF-/, 213 strain 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, pezoton, dried yeast, cornstarch liquor, ammonium sulfate, ammonium nitrate, etc. are used alone or in combination. In addition, inorganic salts such as calcium carbonate, common salt, potassium chloride, and phosphates can be added as necessary.

According to the present invention, SF'
-/U can increase the yield of three substances. OMFB
It is more effective to add in portions during culture rather than all at once at the beginning of production culture. The amount of OMFB added is not critical, but approximately 0.1-
It may be added at a rate of 20 .mu./d1, preferably about 0.times.-j .mu./d.

In addition, when adding OMPBf, normal SF-12
This method is extremely advantageous because it allows the use of a medium with a low concentration during the production culture of 93 substances, contributing to a reduction in manufacturing costs. Furthermore, in the known method of adding AMPB during the production and culture of the three 5F-l ivy substances, the addition of alanine at the same time significantly improved the production of the three SF-/J substances. -/? 7
01 according to the present invention, although it is described in the specification of No. 7yo3.
In the case of addition of 4FB, the production of F3F-1273 substance can be further increased by simultaneously adding inexpensive dl-alanine.

The most suitable culture method is liquid culture, especially deep culture. Cultivation is carried out under aerobic conditions, and the temperature suitable for cultivation is NO! ~3! ℃, but in many cases ko r ~ 32℃
Cultivate nearby. The number of culture days can be shorter than the usual 87-/J3-substance production culture, and the appropriate number of culture days is usually 7 days. For other details of the culture method itself, please refer to the above-mentioned Tokko Shoti-
Please refer to the description in Publication No. t32.

Quantification of the three SF-l ivy substances in the culture solution is performed using an amino acid analyzer. That is, after adjusting the culture solution pH to 2.0, the supernatant was obtained by centrifugation (3000 rpm, /j min) or filtration, and the supernatant was analyzed using an amino acid analyzer (ATTO! pMX,
, type 0-703, retention time l≠min).

The purification method of 8F-l ivy 3 substances from the culture solution obtained according to the present invention is the same as the purification method from the usual EIF-/core 3 culture solution.
-Please refer to the description in Publication No. t3.

(Examples) Hereinafter, the present invention will be explained and explained in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 Strephtomyces hygroscopicus EIF'-l Kota 3 strains/mutant strains were cultured in a preculture medium (2.0% soluble starch,
Polypeptone 1.0%, meat extract 0.3%, dipotassium hydrogen phosphate 0.0! %, pH7,0) / Orttl
was inoculated. This was cultured with shaking for 21 hours in t-xr''c), then subcultured to the same medium romi and cultured with shaking at 2t℃ for 2 hours. Germ 3.2 excellent, Sungraine, !%
, potassium phosphate 0.3%, potassium chloride 0-00
The cells were inoculated into a production medium consisting of 0.07% group ①, and cultured with shaking at 21r°C. OMFB and alanine were added 6 times and in the manner shown in Table 1 below. After making the culture solution acidic, it was centrifuged (30θOrpm, It min) to obtain a supernatant, and the production amount of 5F-l ivy 3 substances was measured using an amino acid analyzer (Atto Shachi MLO-70j type, retention time 1 Komin). The results are shown in Table 1.

As shown in Table 1, the effect of adding OMFB or its ester is obvious.

Example The same preculture medium as in Example 1 was used as the seed mother of jar fermenter (31 capacity mini jar made by Iwashiya), and the same production medium as in Example 1 and its 7θ concentration medium ( In a Jar Fermentor, aerated agitation culture was carried out at 2rC, and the effect of adding OMFB and alanine was investigated in the same manner as in Example 1.However, in this example, OMFB and alanine were inoculated. was added semi-continuously using a peristaltic pump and an interlayer. The results are shown in Table 2.

S'IP-l by addition of OMFB as shown in Table 2
The effect of the three ivy substances on improving production is clear.

(Effects of the invention) As shown in the examples, by adding oMPBt or its salt or ester during the production culture of the F3F-1293 substance, the production amount of the three substances p, sF'-tx was increased. It has become clear that it is possible to measure

It was also revealed that the effect of adding OMFB was further enhanced when alanine was added.

In the case of culture with addition of OMPB, the usual 5F-12
This method is extremely advantageous because a medium with a lower concentration can be used during production and culture of substances, and it also contributes to reducing raw material costs. In addition, since the production lap is faster than the normal production culture of SF-I 3 substances, the culture time can be shortened, and utility costs such as electricity and steam required for tank operation can be reduced. At the same time, it is possible to increase the annual production of υ per culture tank. This shows that the present invention is an extremely effective method for producing herbicides, which are inexpensive and need to be produced in large quantities.

Claims (1)

[Claims] SF-1293 substance-producing bacteria belonging to the genus Streptomyces is defined as 4-(hydroxymethylphosphinyl)-2 represented by the following formula: ▲ Numerical formula, chemical formula, table, etc. are available▼
- A method for producing a phosphorus-containing compound SF-1293 substance in high yield, which comprises culturing in the presence of oxobutyric acid, a salt thereof, a monoester thereof, a diester thereof, or an amide thereof.