JPH062069B2 - Method for producing L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention has herbicidal activity and is useful as a herbicide.
2-amino-4- (hydroxymethylphosphinyl)-
Butyric acid (Japanese Patent Publication No. 61-56210 or U.S. Pat. No. 4,265,654)
No. specification).

(Prior Art and Problems to Be Solved) SF-1293 substance produced by Streptomyces hygroscopicus SF-1293 strain (Microtechnical Laboratory, Yosei BP-130) (JP-B-51-639) And U.S. Pat. No. 4,309,208), a substance also called bialaphos, which is useful as a herbicide, L-2-amino-4- (hydroxymethylphosphinyl) -butyryl-L-alanyl-alanine. Has been identified (see, for example, JP-A-48-85538). On the other hand, L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid, which is also a compound having herbicidal activity, is abbreviated as L-AMPB and is useful as a herbicide (Japanese Patent Publication No. 61-56210). No. 4265654 and U.S. Pat. No. 4,265,654).

Formula (1) As a method for producing L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid (L-AMPB) represented by, L-AMPB-producing bacteria belonging to the genus Streptomyces, for example, Streptomyces hygroscopicus SF -1293 strain is cultured under aerobic conditions, and a method for collecting L-AMPB from the culture solution has been published by the present inventors (JP-A-57-47).
Further, as a method for producing L-AMPB in a large amount, L-AMPB is disclosed.
4- (Hydroxymethylphosphinyl) -2-oxo-butyric acid of the following formula (2) which is a 2-keto acid corresponding to is prepared by a synthetic method, and then the compound is aminated by an enzymatic reaction. A method of converting to L-AMPB has been reported by the present inventors (see Japanese Patent Application No. 62-139933).

When developing and industrializing a herbicide, it is necessary to have a technique for mass production as described above, but at the same time, it is essential to manufacture at low cost. 2- (hydroxymethylphosphinyl) -2-oxo-butyric acid (hereinafter referred to as OMPB), which is a 2-keto acid corresponding to L-AMPB, is produced by a synthetic method and is aminated to produce L-AMPB. According to the above-mentioned method, it is clear that the cost of OMPB as a substrate greatly affects the production cost of L-AMPB as a final product.

OMPB is a known substance, and its production method is disclosed in
No. 56-92897 or U.S. Pat.No. 4,399,287, the substance has the following formula (2) As shown in (1), it has a unique structure containing a P-methyl group, and a compound having such a structure generally requires very complicated and difficult steps when chemically synthesized. Therefore, the present inventors have prepared 4- (hydroxy) phosphinoyl-2-oxobutyric acid (hereinafter, referred to as OPB), which is a compound lacking the methyl group of OMPB, in the following formula (3: ) This time, we conceived to select the compound shown by as a starting material. This substance is a known substance, and its manufacturing method and physicochemical properties are described in JP-A-58-219192. When manufacturing OPB, complicated P-
Since the methylation process can be omitted, the manufacturing cost can be significantly reduced compared to the OMPB manufacturing cost. Already by the present inventors, OPB or its derivative was
-L-AMPB using a microorganism having the ability to produce AMPB
It has been disclosed that the yield of L-AMPB can be increased by adding it during the production culture of (No. 62-253389). However, it is not known about the existence of P-methylation ability of OPB or a derivative thereof in a microorganism that does not produce L-AMPB or bialaphos. From the above technical background, the present inventors have begun to study the development of a new L-AMPB production method using inexpensive OPB or its derivative as a substrate.

(Means for Solving the Problem) The present inventors hypothesized that there is a microorganism that produces L-AMPB by performing P-methylation and amination using OPB as a substrate, and carried out diligent screening of such a microorganism. As a result, the mutant strain obtained by mutating a strain belonging to the genus Streptomyces does not produce L-AMPB in normal culture, but when OPB is used as a substrate, P-methylation and amination are performed to increase L-AMPB. We have found the surprising fact of yielding. As described above, P-methylation requires complicated steps in chemical synthesis, but it was found that it is possible to easily proceed with high yield by using such a mutant strain microorganism. As such a mutant strain microorganism, the bialaphos-producing ability and the L-AMPB-producing ability are rendered facultative by the mutation treatment of the bialaphos-producing bacterium or the L-AMPB-producing bacterium belonging to the genus Streptomyces, that is, they are eliminated during normal culture. It was found that the different mutant strains are suitable. In addition to OPB, OPB derivatives used as a substrate include 2-amino-4- (hydroxy) phosphinoylbutyric acid or a salt thereof which is an amino acid corresponding to OPB, and an N-acetyl derivative of an amino acid corresponding to OPB. It was found that 2-N-acetylamino-4- (hydroxy) phosphinoyl butyric acid or a salt thereof can also be used. The present invention has been completed based on these findings.

That is, according to the present invention, a mutant strain obtained by a mutation treatment of a bialaphos-producing bacterium belonging to the genus Streptomyces or a L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid-producing bacterium, wherein By the mutagenesis treatment, bialaphos-producing ability and L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid-producing ability are eliminated as a facultative compound, provided that 4- (hydroxy) phosphinoyl-2-oxo-butyric acid, 2-amino-4- (hydroxy)
Phosphinoyl butyric acid or 2-N-acetylamino-4
Mutant strains having the ability to produce L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid from 4- (hydroxy) phosphinoylbutyric acid when used as a substrate were identified as 4- (hydroxy) phosphinoyl-2- As a substrate for at least one of oxo-butyric acid or a salt thereof, 2-amino-4- (hydroxy) phosphinoylbutyric acid or a salt thereof, and 2-N-acetylamino-4- (hydroxy) phosphinoylbutyric acid or a salt thereof After culturing in an added medium under aerobic conditions, L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid or its N-acetyl derivative or a salt thereof is collected from the obtained culture solution. L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid or an N-acetyl derivative thereof or a product thereof Preparation of al the salt is provided.

Examples of the microorganism used in the present invention include 4- (hydroxy) phosphinoyl-2-oxo-butyric acid (or a salt thereof such as sodium salt), 2-amino-4- (hydroxy) phosphinoyl butyric acid (or a salt thereof such as sodium salt), 2-N-acetylamino-4- (hydroxy)
When phosphinoyl butyric acid (or a salt thereof such as sodium salt) (hereinafter referred to as OPB or a derivative thereof) is used as a substrate, any microorganism can be used as long as it has the ability to produce L-AMPB from them. It is necessary that the substrate has the ability to P-methylate and 2-aminate OPB to be able to produce L-AMPB from OPB.

Examples of such microorganisms that can be used in the method of the present invention include, by the present inventors, Streptomyces hygroscopicus SF1293 strain (see Japanese Patent Publication No. 51-639; FERM).
BP-130; ATCC 21705) is treated with nitrosoguanidine to obtain a mutant strain (strain number NP 110) lacking the ability to produce bialaphos and L-AMPB. This mutant strain, Streptomyces hygroscopicus NP 110, was apologized to JIKEN on January 27, 1988.
No.), and its mycological properties are bialaphos-producing ability and L
-It is the same as the Streptomyces hygroscopicus SF 1293 strain (FERM BP-130) described in JP-B-51-639 except that it lacks the ability to produce AMPB and has a low sporulation. The NP 110 strain also does not produce L-AMPB when OPB or its derivative is not added as a substrate. The NP 110 strain is apt to change its properties as seen in the case of other Streptomyces strains, and can be mutated by artificial mutation means using, for example, ultraviolet rays, X-rays, drugs, etc. Even when the mutant strain is
Any strain of the genus Streptomyces which does not produce -AMPB but has the ability to produce L-AMPB using OPB or a derivative thereof as a substrate can be used in the present invention.

In carrying out the production method of the present invention, L
A strain belonging to the genus Streptomyces that does not produce AMPB but has the ability to produce L-AMPB using OPB or a derivative thereof as a substrate is cultured in a medium containing a nutrient source that can be used by ordinary microorganisms. As the nutrient source, a known one conventionally used for culturing strains of the genus Streptomyces is used. For example, carbon sources include glucose, starch, glycerin, sucrose, starch syrup,
Molasses and the like can be mentioned, and these can be used alone or in combination. As the nitrogen source, soybean flour, wheat germ, meat extract, peptone, dry yeast, corn stay liquor, ammonium sulfate, sodium nitrate and the like are used alone or in combination. In addition to adding inorganic salts such as calcium carbonate, sodium chloride, potassium chloride, and phosphate, if necessary, organic substances that help the growth of bacteria and use OPB or its derivative as a substrate to promote the production of L-AMPB, Inorganic substances can be added as appropriate. In particular, the use and addition of compounds such as methionine, betaine, dimethylglycine, and sarcosine, which are known as methyl group donors, are effective, but nitrogen sources containing these methyl group donors are usually substituted. Vitamin B ₁₂ and its derivative known as coenzyme of methyl group transfer reaction, and folic acid and its derivative can be used alone or in combination.
When OPB is used as a substrate, amino acids such as glutamic acid, aspartic acid and alanine can be added as amino group donors singly or in combination, and it is preferable that the amino group donors usually contain these amino group donors. Is replaced by a nitrogen source.

The most suitable culture method is a liquid culture method, particularly a submerged culture method. The culture is carried out under aerobic conditions, and the temperature suitable for the culture is 25 to 35 ° C, but in most cases, the culture is carried out at around 28 to 32 ° C. The number of culture days is appropriately 2 to 7 days, more preferably 4 to 6 days.

OPB or its derivative used as a substrate can be added to the medium as an aqueous solution all at once at any time during the above culture, or in divided portions, but added continuously in small amounts so as not to change the culture conditions significantly. It is desirable to do. The methyl group donor and the amino group donor can be added in the same manner, but it is desirable to add them at the same time as OPB or its derivative. The methyl group donor and the amino group donor are used in a molar ratio of 0.2 to 2.0 with respect to the amount of OPB or its derivative added, and preferably 0.5 to 1.0.

In the method of the present invention for producing L-AMPB using OPB or a derivative thereof as a substrate, in addition to the method of adding the substrate during the culture of the strain used in the genus Streptomyces, a suspension of bacterial cells separated from the culture solution Alternatively, a method of performing a L-AMPB production reaction by adding a substrate to an enzyme solution extracted from a cultured bacterial cell by a known method such as ultrasonic treatment or lysozyme treatment
Such a microbial cell or enzyme solution can be used in the method of the present invention as long as it has the ability to produce L-AMPB using B or its derivative as a substrate. It is known that the stability and operability of microbial cells and enzymes can be improved by immobilizing them with an organic solvent, a cross-linking agent, a carrier, etc. The immobilized microorganisms or immobilized enzymes prepared by using
-The ability to produce AMPB can be used in the present invention.

Collection and purification of L-AMPB from a culture solution or reaction solution containing L-AMPB produced by the method of the present invention can be carried out by conventional L-AMPB.
It can be carried out in the same manner as the method for collecting and purifying L-AMPB from the culture solution in the AMPB fermentation production method. For details, refer to the description in JP-A-57-47485. That is, L-AM can be applied to cation exchange resins such as Dowex 50W.
A method in which PB is adsorbed and developed and eluted with water or diluted ammonia water, or a method in which it is adsorbed on an anion exchange resin such as Dowex 1x2 and eluted with an aqueous acetic acid solution can be used. The herbicidal efficacy of the L-AMPB obtained in the present invention was tested, and it was found that the herbicidal efficacy was equivalent to that of L-AMPB obtained by the fermentative method of JP-A-57-47485. .

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

Example 1 Streptomyces
hygroscopicus) NP 110 strain (Microtechnology Research Institute No. 9842) 10 ml of pre-culture medium (soluble starch 2.0%, polypeptone 1.0%, meat extract 0.3% dipotassium hydrogen phosphate 0.05%; pH 7.0)
Was inoculated. This was shake-cultured at 28 ° C. for 24 hours, further subcultured to 80 ml of the same medium and shake-cultured at 28 ° C. for 24 hours, and used as a seed mother. %, Wheat germ 3.9%, soluble vegetable protein 2.5%, soybean flour 1.0%, phosphate-potassium 0.3
%, Cobalt chloride 0.0001%; pH 6.8). The production medium was shaken at 30 ° C. L-AMPB during the culture
As a substrate for production, each OPB or its derivative and a methyl group donor (equimolar to OPB or its derivative) were added to the culture medium under the conditions shown in Table 1 below. After acidifying the culture solution, centrifugation (3000 rpm, 15 minutes) was performed to obtain supernatant, and an amino acid analyzer (MLC manufactured by Atto Co., Ltd. was used.
The amount of L-AMPB produced was measured with a -703 type, holding time 12 minutes). The results are shown in Table 1.

As shown in Table 1, the NP 110 strain had L when no substrate was added.
-AMPB is not produced, but when OPB or its derivative is added as a substrate, OPB is added to L-AMPB at a high conversion rate.
It is clear that it has the ability to convert into. It was also effective to use betaine or the like as the methyl group donor.

Example 2 Vitamin B ₁₂ and folic acid were added to the production medium described in Example 1 at 1 μg / ml each in the production medium described in Example 1 at a ratio of 2% using the same preculture medium as in Example 1 as the seed mother of the jar mentor. The medium thus added was inoculated. 30 for Jarfarmenter (2 charge / 3, sardine mini jar)
Aeration-agitation culture was carried out at 0 ° C., and the effectiveness of OPB or its derivative as a substrate was examined as in Example 1. However, OPB or its derivative as a substrate was semi-continuously added using a peristaltic pump and an interrupter. The results are shown in Table 2.

As shown in Table 2, it is apparent that OPJ or its derivative, which is a substrate, is efficiently converted to L-AMPB even in the jar armenter.

Example 3 In order to collect L-AMPB from the culture solution obtained in Example 2, each culture solution was adjusted to pH 3 with 25% sulfuric acid, and then a filter aid was added to filter the cells to remove the cells. , About 1.5 each
A filtrate was obtained. Then, each of the filtrates was passed through a column of 300 ml of cation exchange resin of Dowex 50W (H ⁺ type), and then developed with water. The obtained fraction containing L-AMPB was further applied to a column of 200 ml of an anion exchange resin of Dowex 1 × 2 (CH ₃ COO ⁻ type), washed with water, and eluted with a 0.3N aqueous acetic acid solution. Eluent L-AMPB
After the fraction containing was concentrated to dryness, the obtained white powder of L-AMPB was crystallized from methanol.

As crystals of L-AMPB, 1.22 g when 4- (hydroxy) phosphinoyl-2-oxo-butyric acid was used as a substrate.
(78.2%), 1.17 g (78.0%) in the case of 2-amino-4- (hydroxy) phosphinoyl butyric acid, 0.97 g (78.9% in the case of 2-N-acetylamino-4- (hydroxy) phosphinoyl butyric acid. ) Got. The obtained crystals were analyzed by elemental analysis, specific optical rotation, melting point, infrared absorption spectrum, nuclear magnetic resonance spectrum, and mass spectrometry by a conventional method, and it was confirmed that they were completely in agreement with the standard of L-AMPB. .

(Effect of the Invention) Although the present invention does not produce L-AMPB during normal culture,
A strain belonging to the genus Streptomyces having the ability to produce L-AMPB from PB or its derivative as a substrate is selected, and OPB or its derivative is added as a substrate at the time of culturing the strain, and P-methylation (and amination) It is a method for producing and collecting L-AMPB which is the compound.
According to the present invention, the substrate OP
It was revealed that B or a derivative thereof was methylated and aminated in a high yield to be converted into L-AMPB, and it was possible to provide an efficient and inexpensive mass production method. INDUSTRIAL APPLICABILITY In the present invention, P-methylation (and amination) of OPB or a derivative thereof occurs in a microorganism that does not produce L-AMPB and L-AM
Based on the discovery of the new fact that PB produces,
According to this finding, further, P-methylation in fast-growing microorganisms other than strains belonging to the genus Streptomyces, and cloning of a gene that controls the enzyme of P-methylation reaction by genetic engineering that has been developed in recent years, It is suggested that the L-AMPB production method can be fundamentally improved, for example, by expressing L.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication (C12P 13/00 C12R 1:55) (C12N 1/20 C12R 1:55) (72) Inventor Yasuaki Ogawa 580 Horikawa-cho, Sachi-ku, Kawasaki-shi, Kanagawa Meiji Confectionery Research Institute (72) Inventor Shunzo 580, Horikawa-cho, Sachi-ku, Kawasaki-shi, Kanagawa Meiji Confectionery Co., Ltd.

Claims (2)

[Claims] 1. A mutant strain obtained by mutation treatment of a bialaphos-producing bacterium belonging to the genus Streptomyces or an L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid-producing bacterium, which is further treated by the mutation treatment. However, the production capacity of bialaphos and the production capacity of L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid are lost as facultative components, provided that 4- (hydroxy) phosphinoyl-2-oxo-butyric acid and 2-amino -4- (Hydroxy) phosphinoyl butyric acid or 2-N-acetylamino-4- (hydroxy)
When phosphinoyl butyric acid was used as a substrate,
-2-amino-4- (hydroxymethylphosphinyl)
-A mutant strain having the ability to produce butyric acid is prepared as 4- (hydroxy) phosphinoyl-2-oxo-butyric acid or a salt thereof, 2-amino-4- (hydroxy) phosphinoyl butyric acid or a salt thereof, and 2-N-acetyl. L-2-amino-4 was obtained by culturing under an aerobic condition in a medium to which at least one of amino-4- (hydroxy) phosphinoyl butyric acid or a salt thereof was added as a substrate, and the obtained culture solution. -(Hydroxymethylphosphinyl) -butyric acid or its N-acetyl derivative or a salt thereof is collected, and L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid or its N- Method for producing acetyl compounds or salts thereof. 2. The method according to claim 1, wherein the strain to be used is cultivated in a medium to which both a methyl group donor and an amino group donor or only the methyl group donor is added.