JPH0767676A - Production of 4-chlorothreonine and agricultural/ horticultural herbicide containing the same - Google Patents

Abstract

PURPOSE:To economically obtain the subject compound having excellent herbicidal activity and useful as an agricultural/horticultural herbicide by culturing chlorothreonine-producting bacteria belonging to Streptomyces. CONSTITUTION:Bacteria capable of producing 4-chlorothreonine of the formula, belonging to Streptomyces, [pref. new microbes: Streptomyces sp. OH-5093 strain (FERM P-13514)] is cultured pref. aerobically in a medium, and, from the resultant cultured product, the objective compound is recovered, for example, by the following processes: the filtrate from the cultured product is subjected to adsorption treatment with an ion exchange resin followed by elution of the crude product with an acid or alkali, and the crude product is further prurified by e.g. gel filtration chromatography. For mass culture of 4- chlorothreonine, liquid culture is preferable.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing 4-chlorothreonine and an agricultural and horticultural herbicide containing 4-chlorothreonine as an active ingredient.

[0002]

2. Description of the Related Art As a method for producing 4-chlorothreonine, a method of converting a raw material with an enzyme (JP-A-1-31)
7391, JP-A-2-207793) and the chemical synthesis method (JP-A-59-93046), but the method of obtaining from a culture obtained by culturing a microorganism is not known.

Further, conventionally, the search for antibiotics having herbicidal activity has been carried out with high selectivity as an index. In this process, highly toxic active substances such as cycloheximide and oligomycin were isolated, but they could not be put to practical use. Bialaphos, which has low toxicity, has recently been discovered, and bialaphos has been put to practical use.

[0004]

However, these compounds still have room for improvement in terms of drug efficacy, toxicity, economical efficiency and the like. Further, the known method for producing 4-chlorothreonine has room for improvement in terms of economy and the like.

An object of the present invention is to provide an entirely new method for producing 4-chlorothreonine and an agricultural and horticultural herbicide containing 4-chlorothreonine as an active ingredient.

[0006]

Therefore, as a result of various searches for microbial metabolites, the present inventors have found that the OH-5093 strain belonging to the genus Streptomyces produces 4-chlorothreonine, and that the compound has excellent herbicidal activity. The present invention was found to have activity, and the present invention was completed based on this finding.

That is, the present invention comprises a method for producing 4-chlorothreonine, which comprises culturing a 4-chlorothreonine-producing bacterium belonging to the genus Streptomyces in a medium and collecting 4-chlorothreonine from the culture. -A herbicide for agriculture and horticulture, which contains chlorothreonine as an active ingredient.

In the present invention, 4-chlorothreonine is a culture product of the substance 4-chlorothreonine producing bacterium belonging to the genus Streptomyces, which is cultured in a medium to produce and accumulate the substance 4-chlorothreonine in the medium. Can be produced by collecting 4-chlorothreonine.

Regarding 4-chlorothreonine-producing bacteria,
There is no particular limitation as long as it belongs to the genus Streptomyces and has the ability to produce the substance 4-chlorothreonine. A preferred example of the 4-chlorothreonine-producing bacterium of the present invention is Streptomyces sp. OH-5093 newly isolated from soil by the present inventors.
Stocks. The mycological properties of this strain are shown below.

(I) Morphological Properties Vegetative hyphae develop well on various agar media, and fragmentation is not observed. The aerial mycelium abundantly grows on glucose / asparagine agar, starch inorganic salt agar, etc., and exhibits a grayish color tone. When observed under a microscope, aerial hyphae have a loose spiral shape, and a chain of 20 or more spores is observed. The spore size is 1.5 × 0.9 μm and is cylindrical. The surface of spores is smooth. No sclerotium, sporangia and zoospores are found.

(II) Properties on various media EB Shirring
g) and the method of D. Gottlieb (International Journal of Systematic Bacteriology, Vol. 16, 313).
Table 1 shows the culturing properties of the presently produced strains, which were examined by pp. 1966).

As a standard color tone, the Color Harmony Manual 4th Edition (Container Corporation
Of America Chicago, 1958), and the code is also shown in parentheses along with the color-code name. The following are the results of observation in each medium at 27 ° C. for 2 weeks unless otherwise specified.

[0013]

[Table 1]

(III) Physiological properties (1) Formation of melanin pigment (B) Tyrosine agar positive (B) Peptone yeast iron agar positive (C) Glucose peptone gelatin medium positive (D) Tryptone yeast solution Positive (2) Tyrosinase reaction Positive (3) Hydrogen sulfide production Positive (4) Nitrate reduction negative (5) Gelatin liquefaction (21-23 ° C) Negative (glucose / peptone / gelatin medium) (6) Starch hydrolysis Positive (7) Coagulation of skim milk (27 ° C) Negative (8) Peptone conversion of skim milk (27 ° C) Positive (9) Growth temperature range 13 to 45 ° C (10) Utilization of carbon source (Pleedam-Gottlieb agar medium) ) Use glucose, arabinose, xylose, mannitol and fructose. Uses raffinose, melibiose, rhamnose, inositol and sucrose a little.

(11) Cellulose Degradation Negative (IV) Cell Wall Composition Cell wall diaminopimelic acid is of the LL type.

The bacteriological properties of this bacterium are summarized as follows.

Diaminopimelic acid in the cell wall is of the LL type. The aerial hyphae are loose spirals and form long spore chains. The surface of spores is smooth. Regarding various properties in culture, vegetative mycelia have a yellow or brown color tone, and aerial mycelia have a gray color tone. Soluble pigments are produced by glucose / asparagine agar, yeast extract / malt extract agar, etc.

From these results, this strain is a strain belonging to the genus Streptomyces, and it is classified into Pridham and Tresner (Vergis Manual of Determinating Bacteriology, 8th Edition, 748 to 829).
Page, 1974) and is considered to belong to the gray series.

Therefore, this strain was identified as a strain belonging to the genus Streptomyces, and Streptomyces sp.
It was named OH-5093. This strain is Streptomyces sp. OH-5093 ( Streptomyces sp. OH-
5093) has been deposited with the Institute of Biotechnology, Institute of Industrial Science and Technology (FERM P-13514).

As the strain that can be used in the present invention, all the 4-chlorothreonine-producing bacteria belonging to the genus Streptomyces can be used, including the above strains and mutants thereof. Four
-Chlorothreonine is produced by inoculating the above strain into a nutrient source-containing medium suitable for 4-chlorothreonine production and aerobically culturing. As the nutrient source, one that can be used as a nutrient source for actinomycetes is used.

For example, commercially available peptone, meat extract, corn steep liquor, cottonseed flour, peanut flour,
Soybean powder, yeast extract, NZ-amine, hydrolyzate of casein, nitrogen sources such as sodium nitrate, ammonium nitrate and ammonium sulfate, carbohydrates such as glycerin, starch, glucose, galactose and mannose, or carbon sources such as fat, and salt and phosphorus. Inorganic salts such as acid salts, calcium carbonate and magnesium sulfate can be used. In addition, if necessary, a trace amount of a metal salt, or an animal / plant / mineral oil as an antifoaming agent may be added. Any of these may be used as long as it can be used by the producing bacteria and is useful for the production of 4-chlorothreonine, and all known culture materials for actinomycetes can be used. 4-
Liquid culture is preferable for large-scale culture of chlorothreonine,
The culture temperature can be applied within a range in which the producing bacterium grows and 4-chlorothreonine can be produced. The culturing can be performed by appropriately selecting it according to the properties of the 4-chlorothreonine-producing bacterium using the above-mentioned conditions.

4-Chlorothreonine is mainly present in the culture filtrate. For the purification and isolation of 4-chlorothreonine from the culture filtrate, the separation and purification method generally used for isolating microbial metabolites from the culture is used. For example, a crude material can be obtained from the culture filtrate by adsorbing it on an ion exchange resin and eluting with an acid or an alkali. The crude substance is a known method used for purification of water-soluble substances, for example, ion exchange chromatography, adsorption chromatography, gel filtration chromatography, preparative thin layer chromatography, centrifugal countercurrent partition chromatography, high performance liquid chromatography. It can be collected purely by appropriately combining or repeating the above.

The physicochemical properties of 4-chlorothreonine are as follows.

(1) Property: White powder (2) Molecular weight: 154.0287 (M + H, by fast atom bombardment mass spectrometry) (3) Molecular formula: C ₄ H ₈ O ₃ NCl (4) Melting point: 142-143 ° C (5) Specific rotation: [α] _D ²⁵ = -81 (c = 0.43,
H ₂ O) (6) Ultraviolet absorption maximum (in aqueous solution): As shown in FIG. 1, it has a maximum absorption at 245 nm (ε = 1600).

(7) Absorption maximum in infrared region (KBr tablet): FIG.
As shown in 3400, 3120, 1670, 1640, 1590, 1520,
It has maximum absorption at 1400, 1360, 1020, 770, 650, 530cm ^-1 .

(8) Proton nuclear magnetic resonance spectrum (4
(00 MHz, heavy water): The chemical shift (ppm) is as follows (d is doublet, q is quartet, H is the number of protons).

Δ (ppm) 3.67 dd (1H, J =
5.5, 11.5 Hz) 3.71 dd (1H, J = 5.5, 11.5 Hz) 3.80 d (1H, J = 5.5 Hz) 4.24 q (1H, J = 5.5 Hz) (9) The chemical shift (ppm) in the ¹³ C nuclear magnetic resonance spectrum (100 MHz, heavy water) is as follows (s is a singlet, d is a doublet, t is a triplet, and q is a quartet). Indicates).

Δ (ppm) 172.7 (C = O) 70.0 (CH) 57.4 (CH) 46.6 (CH ₂ ) (10) Solubility in solvent: methanol, ethanol,
Soluble in water. Insoluble in chloroform and acetone.

(11) Color reaction: ninhydrin positive.

(12) Distinction between acidic, neutral and basic: amphoteric substance.

From the above various physicochemical properties and spectral data, it is apparent that 4-chlorothreonine is produced by the present invention.

When 4-chlorothreonine is used as a herbicide for agricultural and horticultural use, it is generally carried out in the field of pesticide manufacturing, either as it is or according to the purpose of use, or an agrochemical auxiliary is mixed for promoting or stabilizing the effect. Depending on the method, powders, fine granules, granules, wettable powders, flowables, microcapsules, oils, aerosols, fumigants for heating (mosquito coil, electric mosquito coil, etc.), fog and other fog agents,
It can be used in any formulation form such as non-heat fumigant and poison bait.

In the actual use, these various preparations can be used as they are, or diluted with water to a desired concentration and used.

Further, these preparations contain 4
-Chlorothreonine is adjusted to a content of 0.1 to 99.9%, preferably 0.2 to 80% according to a usual formulation method.

[0035]

EXAMPLES Examples of the present invention will be shown below, but these are merely examples and do not limit the present invention at all, and various modifications are possible.

Production Example Streptomyces sp. O cultured in an agar slant medium
From H-5093 strain (FERM P-13514), glucose 0.1%, starch (soluble) [Kanto Chemical Co., Inc.]
2.4%, peptone [Kyokuto Pharmaceutical Co., Ltd.] 0.3
%, Meat extract [Kyokuto Pharmaceutical Co., Ltd.] 0.3%, yeast extract [Oriental Yeast Co., Ltd.] 0.5%, calcium carbonate 0.4%, liquid medium (pH 7.0)
500 ml Erlenmeyer flask 1 in which 100 ml was dispensed
One platinum loop was inoculated to each of 0 and cultured at 27 ° C. for 2 days with shaking. Using it as a seed culture solution, maltose 5.0%, dry yeast [Kanto Chemical Co., Inc.] 1.5%, Ebios tablets [Asahi Breweries, Ltd.] 2.5%, potassium bromide 1.
0%, 0.05% potassium dihydrogen phosphate, 0.05% magnesium sulfate heptahydrate 0.05% liquid medium (pH 7.
0) Inoculate a jar fermenter containing 40 liters,
It was cultured at 27 ° C for 4 days.

The culture solution was centrifuged, and the obtained culture filtrate was passed through a cation exchange column and eluted with hydrochloric acid. After desalting, it was passed through a cation exchange column again and eluted with a buffer solution. Active fractions were collected and concentrated under reduced pressure to obtain 8 g of crude substance I. This is dissolved in a small amount of water and swelled with water Sephadex G-
It was applied to 10 columns [200 ml, manufactured by Pharmacia] and eluted with water. Active fractions were collected and concentrated under reduced pressure to obtain 1.7 g of a crude substance II. This was dissolved in a small amount of water and subjected to high performance liquid chromatography [Capsule Pack C ₁₈ SG-120,
φ20 × 250 mm, manufactured by Shiseido Co., Ltd.]
The active fractions were collected by elution with 1% trifluoroacetic acid as the mobile phase and concentrated under reduced pressure to obtain 520 mg of crude substance III.
This was dissolved in a small amount of methanol and applied to a Sephadex LH-20 column [500 ml, manufactured by Pharmacia Co.] swollen with methanol to elute with methanol, and active fractions were collected and concentrated under reduced pressure to obtain 316 mg of crude substance IV. Obtained. This was dissolved in a small amount of water and applied to Sephadex G-10 [500 ml, manufactured by Pharmacia] swollen with 50% methanol to collect the active fractions, which were concentrated under reduced pressure.
140 mg of chlorothreonine was obtained.

Formulation Example When used as a wettable powder for agricultural and horticultural use, 4-chlorothreonine 20 is used.
And 75 parts of kaolin, 3 parts of sodium alcohol sulfate and 2 parts of sodium polyacrylate are mixed and ground to prepare a formulation.

Test Example 4 Herbicidal effect of 4-chlorothreonine A cotton tube impregnated with a dilute solution of 4-chlorothreonine at a predetermined concentration was put in a test tube having a diameter of 1 cm, and three seeds of sorghum and radish radish were sown on each seed. After 7 days, the degree of growth inhibition was investigated. Table 2 shows the herbicidal effect of 4-chlorothreonine.

[0040]

[Table 2]

[0041]

INDUSTRIAL APPLICABILITY The present invention provides a method for producing 4-chlorothreonine, and the effect of an agricultural and horticultural herbicide containing 4-chlorothreonine as an active ingredient is expected.

[Brief description of drawings]

FIG. 1 is an ultraviolet absorption spectrum of 4-chlorothreonine.

FIG. 2 is an infrared absorption spectrum of 4-chlorothreonine.

Continuation of front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display area C12R 1: 465)

Claims (3)

[Claims] 1. A 4-chlorothreonine producing strain belonging to the genus Streptomyces represented by the following formula [I] is cultured in a medium, and 4-chlorothreonine is collected from the culture. Method for producing threonine. [Chemical 1] 2. The method according to claim 1, wherein the 4-chlorothreonine-producing bacterium is an OH-5093 strain belonging to the genus Streptomyces. 3. A herbicide for agriculture and horticulture, which comprises 4-chlorothreonine as an active ingredient.