JP2638541B2 - Method for producing L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid which has herbicidal activity and is useful as a herbicide (JP-B-61-56210 or US Pat. Patent No. 4,265,654).

[0002]

2. Description of the Related Art As a method for producing L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid (hereinafter abbreviated as L-AMPB), L-AMPB is contained as a component in the molecule and herbicidal activity is (Also referred to as JP-B-51-639 and U.S. Pat. No. 4,309,208), namely L-2-amino-4- (hydroxymethylphosphinyl) -butyryl-L- A method of acid-decomposing alanyl-L-alanine (see JP-A-48-85538);
Alternatively, there is a method of decomposing SF-1293 substance with a microbial enzyme (see JP-A-49-31890). In addition, AMPB was first obtained in racemic form by a chemical synthesis method (see JP-A-48-91019 and JP-A-54-84529), which was optically resolved with a microbial enzyme to obtain L-AMPB. And the like are known. More recently, the present inventors have reported a method of culturing L-AMPB-producing bacteria belonging to the genus Streptomyces and directly collecting L-AMPB from the culture solution (Japanese Patent Application Laid-Open No. Sho 57-4).
No. 7485).

[0003] In order for a substance having herbicidal activity, such as L-AMPB, to be developed and manufactured as a herbicide, and to be put into practical use as a commercial product, it must be developed in accordance with the research on the development of the safety and herbicidal efficacy of the substance. Research is needed to improve the production method suitable for industrialization for mass production at low cost. AMPB produced by the above chemical synthesis method is L-AMPB
And D-AMPB, but D-AMPB itself has almost no herbicidal activity and is a non-natural substance,
When applied to soil, decomposition by soil bacteria is slow and easily remains in the soil, which may cause environmental pollution. When L-AMPB is to be produced by a synthetic method, racemic AMPB is obtained first, and then D-AMPB and L-AMPB are separately separated from this racemic form by optical resolution or the like. Separation is required, which is complicated and the yield of L-AMPB is low. However, in the method for producing L-AMPB using a microorganism or an enzyme, only L-AMPB, which is a natural substance, can be obtained. Natural type L-AMPB is ideal for weeding that does not contribute to the herbicidal action and remains in the soil without being involved in the herbicidal action. It is considered an agent.

The present inventors have paid attention to the above problems, and have an advantage of the AMPB synthesis method that it can be produced in large quantities.
L-AM using microorganisms that only AMPB can selectively produce
Attempts have been made to combine the advantages of the PB production method, and studies have been conducted to establish an improved production method of L-AMPB capable of selectively producing L-AMPB in large quantities.

[0005]

[Means for Solving the Problems] L-AMPB can be regarded as a kind of derivative of α-amino acid. On the other hand, in general, it is known that α-amino acids are usually produced by transamination from the corresponding 2-oxo acids by the action of transaminase with respect to normal naturally occurring α-amino acids constituting proteins. Thus, the present inventors have focused on 2-oxo acid corresponding to L-AMPB and conducted intensive studies. 4- (hydroxymethylphosphinyl) -2 represented by
-Oxo-butyric acid (hereinafter abbreviated as OMPB) in the presence of both glutamic acid or a salt thereof and aspartic acid or a salt thereof as an amino group donor, or glutamic acid or aspartic acid or a salt thereof alone Optically active L-AMPB can be obtained from non-optically active OMPB in a substantially short reaction time with a substantial yield by treating with a certain microorganism having an ability to produce a transaminase or a certain transaminase. Have found the surprising fact that can be generated.

Further, when glutamic acid and aspartic acid (or a salt thereof) are used in combination as amino group donors and the above-mentioned transaminase-producing microorganism or transaminase is allowed to act on OMPB in the presence of both of them, glutamic acid Also, it has been found that the production rate of the target L-AMPB is remarkably improved as compared with the case where aspartic acid or a salt thereof alone is present.

Therefore, according to the first aspect of the present invention, 4- (hydroxymethylphosphinyl) -2 represented by
Treating oxo-butyric acid with a microorganism capable of producing one or more transaminases in the presence of glutamic acid or a salt thereof and aspartic acid or a salt thereof as amino group donors, Next formula And a method for producing L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid represented by the formula:

In carrying out the first method of the present invention, OMPB
Transaminase between OMPB and the two amino group donor amino acids in an aqueous liquid reaction medium containing glutamic acid or a salt thereof acting as an amino group donor and aspartic acid or a salt thereof. A microorganism having a production ability is caused to act.

In the first method of the present invention, generally,
The transamination reaction for conversion of OMPB to L-AMPB is preferably carried out so that the reaction medium has an alkaline pH value of pH 7.5 or higher, preferably in the range of pH 8.0 to 9.0. p
Adjustment of H is performed by adding sodium hydroxide or an appropriate buffer. The transamination reaction conditions are preferably in the range of the optimal temperature and the optimal pH for the action of the transaminase-producing microorganism involved in the reaction. Usually, the reaction is carried out at room temperature to 60 ° C, preferably 20 to 50 ° C.

OMPB used as the raw material is a known substance, and its production method and physicochemical properties are described, for example, in JP-A-56-92897 or US Pat. No. 4,399,287. In the first method of the present invention, at the start of the reaction, it is usually appropriate that the initial concentration of the starting substrate OMPB dissolved in the reaction medium is in the range of 0.1 to 100 mg / ml.

The microorganism used in the first invention includes actinomycetes, bacteria, yeasts and molds. The microorganism used is Streptomyces, Streptomyces Birch shea genus, Nocardia genus, Nocardioides Opsys genus Sakkaropori Supora genus actinomycete selected from Mi Kuromonosupora genus and Streptomyces spot Rangi um spp; or Bacillus, micro A bacterium selected from the genus Coccus, Staphylococcus, Escherichia, Pseudomonas, Serratia and Corynebacterium; or a yeast selected from Saccharomyces, Candida, Cryptococcus, Debaryomyces, Trigonopsis and Hansenula; Or Aspergillus, Mucor, Aureobasidium, Chaetomium,
It can be a mold selected from the genus Penicillium and the genus Gliocladium.

An example of an actinomycete that can be used in the present invention is Streptomyces albus ( Streptom).
yces albus) , Streptomyces griseus , Streptomyces hygroscopicus ( Strepto)
myces hygroscopicus , Streptomyces lividans ( Streptomyces l)
ividans ), Streptomyces viridochromogenes ( Streptomyces viridochr)
omogenes) , Streptomyces pirosus ( S
treptomyces pilosus), Streptomyces Birch Shi helium-Shin'namoniumu (Streptov
ericillium cinnamoneum) ,
Streptomyces morokaensis ( Strepto)
myces moorokaensis ), Nocardia mediterrani
anei ), Nocardiopsis Dassonbelay (No
cardopsis dassonvillei , Saccharopolys
pora hirsuta) , Micromonospora carbono ( Micromonospora carbo)
nacae ), Streptosporangium pseudobulgari (Streptosporangium pse)
uvulgare) and the like.

One example of a bacterium which can be used in the present invention is Bacillus subtili.
s ), Micrococcus luteus
, Staphylococcus aureus
aureus), Escherichia coli (Escherichia coli)
, Pseudomonas aeruginosa (Pseudomonas aeru g
inosa ), Pseudomonas cep
acia) , Seratia marcescens
, Corynebacterium glutamicum (Coryneb acteriu
m glutamicum ).

[0014] Examples of the yeast used in the first present invention, Saccharomyces, Seribishii (Saccharomyces cere
visiae ), Candida albi
ca ns ), Cryptococcus neoformans ( Crypto
coccus neoformans ), Debaryomyces Hanseni ( De
baryomyces hansenii ), Trigonopsis variabilis , Hansenula schneji
(Hansenula schneg gi ) and the like.

[0015] Examples of fungi that can be used in the first present invention, Aspergillus flavus (Asp ergillus flavus)
, Aspergillus terreus
, Mucor spinescens , Aureobasidium pullul
ans) , Chaetomium globos
um ), Penicillium funiclosum
culosum) , Gliocladium
virens ).

The above-mentioned actinomycetes, bacteria, molds, and yeasts can be a type culture deposited at a known microorganism preservation institution, and can be purchased therefrom.

Preferred examples of the microorganism used in the first method of the present invention include Streptomyces hygroscopicus, which is known as a SF-1293 substance producing bacterium belonging to the genus Streptomyces.
s) SF-1293 strain (FERM BP-130 or ATCC 21705), JP-B-51-639 or U.S. Pat. No. 3,832,394) or a mutant thereof, Streptomyces hygroscopicus SF-1293 NP- 50 strains (FERM P-7804 or FE
RM BP-1368) (see JP-A-61-58589 or EP-A-0173327) or Streptomyces lividans 66 strain (FERM BP-737; JP-A-59-175889 or European patent application) Publication No. 0196375). In addition, any microorganism that produces an enzyme having a transaminase activity that converts OMPB to L-AMPB in the presence of glutamic acid or a salt thereof as an amino group donor can be used. The bacteriological properties of the above Streptomyces hygroscopicus SF-1293 strain are described in JP-A-51-639 or US Pat. No. 3,832,394. Although bacteriological properties of Strep preparative Mrs. hygroscopicus SF-1293 NP-50 strain (FERM P-7804) is as defined above for SF-1293 strain, deficient in the biosynthesis ability of SF-1293 substance (See Japanese Patent Application Laid-Open No. 61-58589 or European Patent Application Publication No. 0173327). In addition, 66 Hygroscopicus lividans
The bacteriological properties of (FERM BP-737) are described in JP-A-59-175889. Of these fungi, those strains with FERM BP-numbers have been deposited with the Institute of Microbial Industry and Technology under the terms of the Budapest Treaty.

According to a second aspect of the present invention, 4- (hydroxymethylphosphinyl) -2 represented by
Treating oxo-butyric acid with one or more transaminases in the presence of glutamic acid or a salt thereof and aspartic acid or a salt thereof as amino group donors, And a method for producing L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid represented by the formula:

The enzyme used in the second method of the present invention, ie, transaminase, has a transaminase activity capable of converting OMPB to L-AMPB in the presence of glutamic acid or a salt thereof and aspartic acid or a salt thereof as amino group donors. Any transaminase may be used as long as it has an enzyme.

Suitable examples of the transaminase used in the second method of the present invention include commercially available glutamate-oxaloacetate-transaminase (abbreviated as GOT) (enzyme international classification number EC 2,6,1,1). Or glutamate-pyruvate-transaminase (G
(Abbreviated as PT) (International Enzyme Classification Number EC 2,6
1,2) or an enzyme having a transaminase activity that converts OMPB to L-AMPB in the presence of L-glutamic acid as an amino group donor. These transaminases can be used alone or in combination of two or more.

Among the above, a preferred combination of transaminases is an enzyme activity for converting 2-ketoglutaric acid to glutamic acid using aspartic acid as an amino group donor, that is, a first transaminase having combined GOT activity, and an amino group. This is a combination with a second transaminase having a transaminase activity for converting OMPB to L-AMPB in the presence of glutamic acid as a donor. For example, a commercially available glutamic acid-oxaloacetic acid-transaminase (enzyme international classification number EC 2, 6, 1, 1) can be used as the transaminase having the above-mentioned GOT activity.
Alternatively, a microorganism having GOT activity, for example, Streptomyces hygroscopicus SF-1293 strain (JP-A-57-474)
No. 85; FERM BP-130; ATCC 21705). A transaminase having GOT activity and obtained by extraction by a known method can be used. These transaminases having the same GOT activity can be used alone or in combination. In addition, the above GOT
As the second transaminase used in combination with the transaminase having the same activity, any transaminase capable of converting OMPB to L-AMPB in the presence of glutamic acid may be used.

Suitable examples of glutamic acid or a salt thereof as one amino group donor used in the first or second invention include L-glutamic acid or a salt thereof, and glutamic acid or a salt thereof as the other amino group donor. A suitable example of aspartic acid or a salt thereof is L-aspartic acid or a salt thereof. Suitable examples of salts of these amino acids include alkali metal salts of the amino acids. D-amino acids corresponding to these L-amino acids can also be used as amino group donors. Total amount of glutamic acid and aspartic acid as amino group donor in the reaction medium at the start of the reaction and OM of the substrate
The molar ratio to the amount of PB can generally range from 10: 1 to 1:10.

In the first or second method of the present invention, with respect to glutamic acid and aspartic acid used together as an amino group donor, each of these amino acids may be a commercially available product, and is generally a mixture of D-form and L-form. , But is preferably composed of only the L-form. As each salt of glutamic acid and aspartic acid,
Alkali metal salts, especially sodium or potassium salts, can be used.

In the first or second method of the present invention,
Glutamic acid (or its salt) and aspartic acid (or its salt) are used in combination as amino group donors. In this case, the ratio between the concentration of each of these amino group donor amino acids and the concentration of OMPB is explained. If the molar ratio of each of these amino group donors in the reaction medium at the start of the reaction is higher than that of OMPB, the reaction rate and the conversion from OMPB to L-AMPB are increased.

However, from an economic viewpoint, an appropriate molar ratio is set. Usually, the concentration (addition amount) of glutamic acid (or its salt) in the reaction medium at the start of the reaction and the OM
The molar ratio to the PB concentration (addition amount) is preferably in the range of 0.2: 1 to 3.0: 1, and the ratio of the concentration (addition amount) of aspartic acid (or a salt thereof) to the concentration (addition amount) of OMPB is preferable. Preferably, the molar ratio ranges from 1.0: 1 to 3.0: 1.

Furthermore, in carrying out the first or second method of the present invention, the concentration of glutamic acid or a salt thereof added to the reaction medium at the start of the reaction is 1 mol of the substrate compound of the formula (II). And the concentration of the added aspartic acid or a salt thereof is a concentration corresponding to 1.0 to 3.0 mol per mol of the substrate compound of the formula (II). The pH of the reaction medium is
It is preferred that the alkaline pH be maintained between 8.0 and 9.0.

In the first method of the present invention, the microorganism is allowed to act on the OMPB substrate of the formula (II) and glutamic acid and aspartic acid (or salts thereof) as amino group donors in the following manner. Will be That is, first,
The microorganism is cultured in a medium containing nutrients used for ordinary microorganism culture. As the nutrient source, a known nutrient used for culturing a normal microorganism is used. For example, as a carbon source, glucose, starch, glycerin,
Examples include shoe cloth, syrup, molasses and the like. These may be used alone or in combination. As the nitrogen source, soybean flour, wheat germ, meat extract, peptone, dried yeast, corn steep liquor, ammonium sulfate and the like are used alone or in combination. In addition, if necessary, inorganic salts such as calcium carbonate, salt, potassium chloride, and phosphate can be added. The most suitable culture method is a liquid culture method, particularly a submerged culture method. The cultivation is performed under aerobic conditions, and a suitable temperature for culturing is 25 to 40 ° C. The cultivation days are 1 to 4 days for actinomycetes, 1 to 2 days for bacteria, 1 to 2 days for yeast, and 1 for mold.
~ 4 days is appropriate.

The culture solution of the microorganism itself may be used, or if desired, the living cells separated from the culture solution and washed may be suspended at a certain concentration in water or physiological saline or a suitable buffer. A cloudy cell suspension can also be used. Add OM to the culture solution or other cell suspension containing the cells of the microorganism used.
PB (reacting as a first substrate) is added simultaneously or sequentially with glutamic acid and aspartic acid or their salts (reacting respectively as a second substrate) as amino group donors. Thereafter, the liquid is kept so that the microorganism acts on OMPB and the two amino group donors to advance the transamination conversion reaction of OMPB. As described above, the concentration of the bacterial cells in the aqueous reaction medium solution for converting OMPB and the two amino group donors to L-AMPB by treating them with microorganisms,
The total concentration of the species amino group donors, as well as the reaction temperature and pH conditions are appropriately adjusted within a range in which the conversion reaction from OMPB to L-AMPB proceeds efficiently and the microorganisms maintain a state in which the microorganisms exert their functions. . The reaction time is also adjusted so that a substantial amount of L-AMPB is produced and accumulated in the reaction solution.

In the second method of the present invention, when OMPB and glutamic acid and aspartic acid or a salt thereof as an amino group donor are treated with a transaminase, the enzyme may be dissolved in a suitable aqueous solution of a transaminase or a buffer. OMPB and the two amino group donors are added to and dissolved in the solution, and an enzymatic transamination reaction of OMPB is performed. The concentrations of the enzyme, OMPB and the two amino group donors in the reaction system, as well as the reaction temperature and pH conditions are appropriately adjusted within an appropriate range in which the conversion reaction from OMPB to L-AMPB proceeds efficiently.

As the above-mentioned transaminase used in the method of the second invention, those in the form of a commercially available enzyme product may be used, or actinomycetes or bacteria as microorganisms usable in the first invention may be used. An aqueous solution of a crude enzyme produced directly by crushing the cells in a culture of mold, yeast or yeast, or an aqueous solution of a crude enzyme dissolved in water can be used. Further, a crude enzyme solution in the form of a disrupted cell solution may be used.

In addition, even when a crude enzyme solution is extracted from the microorganism or a culture thereof by a known method such as ultrasonic treatment or lysozyme treatment, the crude enzyme solution may be a mixture of the two amino group donors. That is, if it has the ability to produce L-AMPB from OMPB in the presence of glutamic acid and aspartic acid or a salt thereof, it can be used in the second method of the present invention. In addition, it is natural that an aqueous solution of the purified enzyme can be used.

It is known that the stability and the operability are enhanced by immobilizing an enzyme or a microorganism with an organic solvent, a cross-linking agent, a carrier, or the like. Immobilized microorganisms or immobilized enzymes can also be converted from OMPB to L-
The ability to produce AMPB can be used in the first or second method of the present invention.

For example, in the second method of the present invention, the enzymatic reaction for converting OMPB to L-AMPB using transaminase is carried out at an alkaline pH value of pH 7.5 or higher, preferably pH 8.0 to 9.0. It is preferred to do so. The reaction conditions are preferably in the range of the optimal temperature and the optimal pH of the enzyme involved in the reaction.

In a second preferred embodiment of the process according to the invention, 4- (hydroxymethylphosphinyl) -2-
Oxo-butyric acid or OMPB is treated with two or more transaminases in the presence of both L-glutamic acid or a salt thereof as an amino group donor and L-aspartic acid or a salt thereof. The transaminase used in this case is a transaminase activity for converting 2-ketoglutaric acid to glutamic acid using L-aspartic acid as an amino group donor, that is, a so-called G
A first transaminase having a transaminase activity having an activity falling within the range of the OT activity; and OMPB in the presence of L-glutamic acid as an amino group donor.
It is preferably a combination system with a second transaminase having a transaminase activity that converts to AMPB.

In a preferred embodiment of the first method of the present invention, the activity of converting 2-ketoglutaric acid to glutamic acid in the presence of L-aspartic acid as an amino group donor, so-called GOT activity, Microorganisms that produce an enzyme system having a transaminase activity that converts OMPB to L-AMPB in the presence of L-glutamic acid as a donor, such as Streptomyces hygroscopicus SF-1293 strain (FERM
BP-130 or ATCC 21705) or a mutant thereof, Streptomyces hygroscopicus SF-1293 NP-50 strain (see JP-A-61-58589; FERM P-7804 or FERM).
BP-1368) or Streptomyces lividans 66 strains (TERM
BP-737) is preferably used.

In the first or second method of the present invention,
As a result of the provision of OMPB with an amino group from L-glutamic acid (or a salt thereof) as an amino group donor by the action of the above-mentioned microorganism producing the above transaminase system or by the action of the above transaminase system, OM
PB becomes L-AMPB, and glutamic acid with amino group is 2
L-aspartic acid (or a salt thereof) as an amino group donor provides an amino group to 2-ketoglutaric acid by the action of a transaminase having GOT activity to convert it into glutamic acid. And aspartic acid itself is presumed to become pyruvate via oxaloacetate.

The first or second method of the present invention
Collection and purification of L-AMPB from a reaction solution containing L-AMPB,
It can be carried out in the same manner as the method for collecting and purifying L-AMPB from the bacterial culture in the usual L-AMPB fermentation production method. For details, refer to the description of JP-A-57-47485. That is, a method can be used in which L-AMPB is adsorbed on a cation exchange resin such as Dowex 50W (manufactured by Rohm and Haas Co.) and developed and eluted with water or dilute ammonia water. L-AMPB obtained by the method of the present invention was tested for its herbicidal efficacy, and it was found that the herbicidal efficacy was equivalent to that of L-AMPB obtained by the fermentation method of JP-A-57-47485. Was done.

Furthermore, of glutamic acid or a salt thereof and aspartic acid or a salt thereof used as an amino group donor in the first or second method of the present invention, glutamic acid or a salt thereof is omitted and aspartic acid (or Salt) alone as an amino group donor, alone as an amino group donor in the presence of aspartic acid or a salt thereof of the formula (II)
When a certain transaminase-producing microorganism or a certain transaminase is allowed to act on OMPB,
It has been found that the desired L-AMPB of formula (I) can be produced with a significant amount of production.

Therefore, according to the third aspect of the present invention, 4- (hydroxymethylphosphinyl) -2 represented by
Oxo-butyric acid, which has the ability to produce transaminase in the presence of aspartic acid or a salt thereof as an amino group donor and has the ability to produce Streptomyces, Streptoverticillium, Nocardia, Nocardiopsis , Sakkaropori Supora genus, Mi Kuromonosupora genus and actinomycetes selected from Streptomyces spot Rangi um genus or Bachirisu genus Micrococcus, Staphylococcus, Pseudomonas, bacteria selected from the genus Serratia and Corynebacterium or Saccharomyces, Genus, Candida, Cryptococcus, Debaryomyces, Yeast selected from Trigonopsis and Hansenula, or Aspergillus, Mucor,
Aureobasidium, Caetium, Penicillium and Glyocladium treated with mold, or treated with a crude enzyme solution obtained from a crushed cell of the microorganism, or one or Further transaminases, in particular glutamate-oxaloacetate-transaminase or glutamate-pyruvate-
Characterized in that it is treated with transaminase or a combination of these enzymes, And a method for producing L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid represented by the formula:

In the third method of the present invention, generally,
The transamination reaction for conversion of OMPB to L-AMPB is preferably carried out so that the reaction medium has an alkaline pH value of pH 7.5 or higher, preferably in the range of pH 8.0 to 9.0. p
Adjustment of H is performed by adding sodium hydroxide or an appropriate buffer. The transamination reaction conditions are preferably in the range of the optimal temperature and the optimal pH of the transaminase-producing microorganism or transaminase used in the reaction. Usually, the reaction is carried out at room temperature to 60 ° C., preferably 20 ° C.
It is good to carry out in the range of ~ 50 ° C.

In the third method of the present invention as well, at the start of the reaction, the initial concentration of the starting material OMPB dissolved in the reaction medium is usually in the range of 0.1 to 100 mg / ml. It is. At the start of the reaction, the molar ratio of the amount of aspartic acid as the amino group donor in the reaction medium to the amount of OMPB as the substrate can generally be in the range of 10: 1 to 1:10. In addition, the third method of the present invention can be carried out according to the first or second method of the present invention. It is also possible to use L-alanine instead of aspartic acid.

The microorganisms that can be used in the third method of the present invention include, among actinomycetes, bacteria, yeasts, and molds exemplified as usable in the first method of the present invention, those described above in the third present invention. And microorganisms belonging to the “genus” specified as described above. The transaminase that can be used in the third method of the present invention is the transaminase exemplified in the second present invention.

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.

[0044] Example 1 (1) Streptomyces hygroscopicus (Streptom
yces hygroscopicus) SF-1293 NP-50 strain (FERM-BP-1368)
The pre-culture medium (soluble starch 2.0%, polypeptone 1.0
%, Meat extract 0.3%, dipotassium hydrogen phosphate 0.05%; pH
7.0). The resulting culture was shake-cultured at 28 ° C. for 24 hours, and the resulting culture was used as a seed medium. This was used at a 2% inoculum in a production medium (glucose 7.0%, wheat germ 3.9).
%, Soluble plant protein 2.5%, monopotassium phosphate
0.3%, cobalt chloride 0.0001%; pH 6.8).
Further, aeration and stirring culture was performed at 28 ° C.

(2) OMPB and amino group donor were added to each 20 ml of a culture solution containing cells of Streptomyces hygroscopicus SF-1293 NP-50 strain obtained by culturing in the above production medium for 3 days. Of sodium L-glutamate and sodium L-aspartate at the beginning of the transamination reaction in such an amount as to give the concentration shown in Table 1 below. Further, 10 ml of 1 M Tris-HCl buffer (pH 8.5) was added. Was added to adjust the pH to 8.5, and the liquid volume was adjusted to 100 ml. That is, the above culture solution was diluted 5-fold.

The amination reaction of OMPB was carried out at 37 ° C. for 24 hours while weakly shaking a solution containing the cells and containing OMPB and two amino acids as amino group donors.

Thereafter, 100 ml of the obtained reaction solution was heated (sterilization and enzyme inactivation) to terminate the reaction. The reaction solution was filtered to remove bacterial cells, and L-AMPB in the filtrate was analyzed and quantified by an amino acid analyzer (MLC-703, manufactured by ATTO, retention time: 12 minutes). Table 1 shows the results.

[0048]

From the results in Table 1, it is found that L as an amino group donor
-When performing the amination reaction of OMPB in the presence of both sodium glutamate and sodium L-aspartate, compared to the case where the reaction is performed in the presence of either sodium L-glutamate or L-aspartic acid, It was clearly observed that the production yield of L-AMPB from OMPB was significantly increased.

Example 2 A culture solution of Streptomyces hygroscopicus SF-1293 NP-50 strain cultured under the same preculture conditions as in Example 1 was used as a seed mother in a 3 L jar fermenter.
The same production medium as in Example 1 was inoculated, and then aerated and stirred at 28 ° C. for 3 days.

Transfer 2 L of the obtained culture solution to a reaction tank,
300 g of PB were added. Also, 310 g of sodium L-glutamate (manufactured by Wako Pure Chemical Industries) and 290 g of sodium L-aspartate (manufactured by Hanoi Chemical) were added and dissolved, and NaOH was dissolved.
After adjusting the pH to 8.5 with an aqueous solution, water was added to adjust the liquid volume to 10 L. That is, at the start of the reaction, the OMPB
The concentration is 30,000 μg / ml, at which point the molar concentrations of sodium L-glutamate and sodium L-aspartate are each equal to the molar concentration of OMPB (1/6 mol). The reaction for amination of OMPB was carried out at 37 ° C. for 24 hours by adjusting the pH to 8.5 while slowly stirring a solution containing the cells, OMPB and two amino group donor compounds. Then, after adjusting the reaction solution to pH 3.0 with 50% sulfuric acid,
After adding a filter aid and filtering to remove the cells, 9
L of filtrate was obtained.

At this time, the concentration of L-AMPB in the filtrate was determined in the same manner as in Example 1, and it was 29000 μg / ml.
Then, the filtrate was passed through a column of 5 L of a cation exchange resin of Dowex 50 W (H ⁺ type) and developed with water. The obtained fraction containing L-AMPB was further added to Dowex 1 × 2
After being subjected to a column of anion exchange resin ^- (CH ₃ COO form), washed with water and eluted with aqueous acetic acid 0.3 N. The fraction containing L-AMPB as an eluent was concentrated to dryness, and the obtained white powder of L-AMPB was crystallized from methanol. L-AMPB crystal
183 g were obtained. The yield was about 70%.

The obtained crystals were analyzed by a conventional method using elemental analysis, specific rotation, melting point, infrared absorption spectrum, nuclear magnetic resonance spectrum, and mass spectrometry. As a result, it was found that the crystals completely agreed with the standard sample of L-AMPB. confirmed.

Example 3 In this example, L-AMPB was produced by causing a commercially available transaminase to act on OMPB in a 50 mM phosphate buffer solution (pH 6).

Glutamic acid as a transaminase
When oxaloacetic acid-transaminase (GOT) (Boehringer Mannheim) is used, L-aspartic acid (Asp) or L-glutamic acid (Gl
u) was added and dissolved in the reaction medium as an amino group donor (reacts as a substrate for amino group donation).

When glutamate-pyruvate-transaminase (GPT) (Boehringer Mannheim) was used, L-alanine (Ala) was added as an amino group donor as a reference example. As another reference example, glutamate dehydrogenase (GLD
When H) (Boehringer Mannheim) was used, ammonium chloride and NADH were added and dissolved as amino group donors, respectively. For comparison, 2-ketoglutaric acid (2-KG) was used as a control substrate for the amination reaction.
The production of glutamic acid (Glu) was observed in the case where was used.

After conducting the enzyme reaction at 30 ° C. for 50 minutes as described above, the reaction was stopped by heating the reaction solution at 100 ° C. for 3 minutes. After adjusting the reaction solution to pH 2 by adding diluted sulfuric acid,
The supernatant was obtained by centrifugation. The amount of L-AMPB or glutamic acid produced in the supernatant was quantified using an amino acid analyzer. The results are shown in Table 2.

[0058]

From the results in Table 2, it is clear that L-AMPB was produced from OMPB in a significant yield by enzymatic reaction, although the yield was lower than the amount of glutamic acid produced from 2-ketoglutaric acid. .

Example 4 The test bacterial strains shown in Table 3 below were inoculated into a medium consisting of 40 ml of nutrient broth (manufactured by Difco).
The culture solution obtained after the time-growing culture was used as a seed mother, and this was inoculated into 40 ml of a medium having the same composition at a 2% inoculum. After overnight culture at 28 ° C, add OM
PB was added to a concentration of 100 μg / ml. Also,
Sodium L-aspartate as an amino group donor
It was added to a concentration of 100 μg / ml. The mixture thus obtained was kept at 28 ° C. for 24 hours to carry out an OMPB conversion reaction.

Thereafter, the reaction solution was adjusted to pH 2 with 25% sulfuric acid to stop the reaction. The cells were removed by centrifugation. The amount of L-AMPB produced in the obtained supernatant was quantified using an amino acid analyzer. Table 3 shows the results.

[0062]

From Table 3, it is clear that L-AMPB was produced from OMPB.

As a reference value, a lysate obtained by sonicating the culture solution containing the cells after overnight culture was centrifuged to obtain a crude enzyme solution. Table 3 also shows the results of measuring the GOT activity. As shown in Table 3, L-
There is a considerable correlation between the amount of AMPB produced and the measured GOT activity of the crude enzyme solution. The GOT specific activities of the enzymes shown in Table 3 are values obtained by measuring the amount of protein in the crude enzyme solution by a bio-rad-protein analysis method.

[0065] Example 5 Streptomyces hygroscopicus (Streptomyces
hygroscopicus) SF-1293 strain (FERM BP-13O) in preculture medium (soluble starch 2.0%, polypeptone 1.0%, meat extract)
0.3%, 0.05% dipotassium hydrogen phosphate; pH 7.0). This was shake-cultured at 28 ° C. for 24 hours and used as a seed mother. This was used at a 2% inoculum in a production medium [glucose 7.0%, bactosoytone 4.4%, monopotassium phosphate 0.3%).
27%, disodium phosphate 0.0852%, N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (ie dotite TES) 1.15%, cobalt chloride 0.0001%; p
H6.0] and cultured at 28 ° C. with aeration and agitation. After cultivation for 4 days, the cells were collected by centrifugation and 50 mM
After washing with phosphate buffer solution (pH 6.0), sonicator (KU
The cells were treated with a BOTA INSONATOR 1.5A) for 1 minute to disrupt the cells, and the disrupted cells were centrifuged to obtain a crude enzyme solution as a supernatant.

OMPB was added to the crude enzyme solution to a concentration of 100 μg / ml and L-aspartic acid to a concentration of 200 μg / ml. For reference, a reaction using 2-ketoglutaric acid (2-KG) as a control substrate for the amination reaction was also performed.

The enzyme reaction was carried out at 30 ° C. for 2 hours.
The reaction was stopped by heating at 100 ° C. for minutes. After adjusting the pH of the reaction solution to 2 by adding dilute sulfuric acid, a supernatant was obtained by centrifugation.
Glutamate produced from L-AMPB and 2-KG in the supernatant was quantified using an amino acid analyzer. Table 4 shows the results.
It was shown to.

[0068]

[0069] Example 6 (1) Streptomyces hygroscopicus (Streptom
yces hygroscopicus) SF-1293 NP-50 strain (FERM P-7804 or FERM BP-1368) was cultured in a preculture medium (soluble starch 2.0%, polypeptone 1.0%, meat extract 0.3%, dipotassium hydrogen phosphate 0.05%; pH 7.0) 10) was inoculated. 24 at 28 ° C
The culture was shake-cultured for a period of time, and the culture broth was used as a seed. The culture medium was used as an inoculum at 2% in a production medium (7.0% glucose, 4.4% bactosoytone, 0.327% monopotassium phosphate, 0.327% disodium phosphate).
0.0852%, TES 1.15% of dootite, 0.001 cobalt chloride
01%; pH 6.0), and cultivated at 28 ° C. with aeration and agitation.

(2) Streptomyces hygroscopicus SF-1293 NP-cultured in a production medium for 3 days as described above
20 ml of a culture of 50 strains and an OMPB aqueous solution (OMPB concentration 87 mg / ml
30 ml of an aqueous L-aspartate solution (sodium L-aspartate concentration: 170 mg / ml) and 10 ml of a 1 M Tris-HCl buffer (pH 8.5). Were combined in a 250 ml Erlenmeyer flask (in this case, OMPB in the combined solution)
The concentration will be about 26 mg / ml). 37 ℃ of the mixture in the flask
For 24 hours with gentle shaking.

Thereafter, the obtained reaction mixture was centrifuged to remove bacterial cells, and the obtained supernatant solution (100 ml) was analyzed using an amino acid analyzer to detect L-AMPB in the supernatant solution. As a result, it was confirmed that 1.5 mg / ml of L-AMPB was produced.

As an amino group donor, L-alanine (concentration 170 mg / l) was used in place of sodium L-aspartate.
A similar experiment was performed using L-alanine, but 2.8 mg / ml L-AMPB was produced when L-alanine was used.

(3) A supernatant solution (100 ml) obtained when sodium L-aspartate was used as the amino group donor in the above item (2) was mixed with 400 ml of Dowex 50W × 2 (H ⁺
After applying to a cation exchange resin tower of type (1), the mixture was developed with dilute aqueous ammonia, and fractions containing L-AMPB were collected and concentrated. This was then mixed with 150 ml of Dowex 1 × 2 (CH ₃ COO).
^- subjected to anion-exchange resin column type), washed with water, 0.3
Elution with an aqueous solution of N acetic acid.

The fraction containing L-AMPB as the eluent was concentrated to dryness under reduced pressure, and further dried under vacuum to obtain a white powder of L-AMPB.
This white powder was crystallized from methanol. For the obtained crystal, elemental analysis, specific rotation, melting point,
Analysis by infrared absorption spectrum, nuclear magnetic resonance spectrum, and mass spectrometry confirmed that it completely matched the L-AMPB sample.

Example 7 (1) Cultivation Method The bacteria shown in Tables 5 to 11 below were transformed from a seed culture slant into a liquid medium (glucose 0.5%, yeast extract).
0.3%, meat extract 1.0%, peptone 1.0
%, Sodium chloride 0.3%; pH 7.0) in a large test tube (using a stainless steel coil-containing test tube only for the cultivation of actinomycetes). The cells were cultured at 28 ° C. for 24 hours (or 48 hours) with shaking.

(2) Preparation of bacterial cells 1 ml of broth after culture (0.2 ml for mold only) was extracted into micro-test tubes (manufactured by Eppendorf), and centrifuged at 12000 rpm for 3 minutes. The supernatant was discarded, and the remaining cells were collected as a sample.

(3) Amination reaction 20 μl of 1 M Tris-HCl buffer (pH 8.0) was added to the cell sample packed in the microtest tube.
And 20μl of OMPB aqueous solution (OMPB concentration 200mg / ml) (pH 8.0)
Then, 40 μl of an aqueous solution containing 1 mol / ml of sodium L-aspartate or L-alanine as an amino group donor and 120 μl of water were added, and allowed to stand at 37 ° C. for 24 hours to react. After the reaction, the reaction solution was centrifuged at 12000 rpm for 3 minutes, and then the content of L-AMPB in the supernatant was measured by an amino acid analyzer.

The results are shown in Tables 5 to 11 below.

Table 5 L-AMPB production amount (mg / ml) Bacteria (bacterium) Asp Ala Bacillus subtilis ATCC 6633 1.9 2.2 Micrococcus luteus ATCC 9341 0.2 0.3 Staphylococcus aureus 209P ATCC 6538P 0.6 0.5 Escherichia coli ATCC 10798 4.2 3.3 Pseudomonas aeruginosa ATCC 10145 3.3 3.8 Pseudomonas cepacia ATCC 17759 2.4 3.0 Serratia marcescens ATCC 13880 1.8 1.6 Corynebacterium glutamicum ATCC 13032 0.3 0.4

[0080]

[0081]

[0082]

Table 9 L-AMPB production amount (mg / ml) Bacteria (actinomycetes) Asp Ala Streptomyces viridochromogenes JCM 4977 * 5.6 3.6 * Bialaphos-producing bacteria

[0084]

Table 11 L-AMPB production amount (mg / ml) Bacteria (mold) used Asp Ala Penicillium funiculosum ATCC 9644 0.4 0.3 Gliocladium virens ATCC 9645 0.4 0.5 * The culture time was 48 hours.

In the present specification, in particular, the above-mentioned Table 3 and Tables 5 to 1
Among the microorganisms shown in 1, the ATCC-numbered strains have been deposited with the American Type Culture Collection (Washington, DC, USA), and the JCM-numbered strains have been assigned to RIKEN , Microbial strain preservation facility (Jap
An Collection of Microoganisms (Wako City, Saitama Prefecture), and the IAM numbered bacterial strain is the Institute of Applied Microbiology,
University of Tokyo) and IFO
The numbered bacterial species are from the Institute for Ferm
entation; Osaka City), and these strains are known type culture microorganisms which can be distributed and obtained from the respective depositories.

[0087]

According to the present invention, L-AMPB is produced from OMPB by the action of a microorganism having transamylase-producing ability or transamylase in the presence of glutamic acid and aspartic acid as amino group donors or in the presence of aspartic acid. It has been clarified that L-AMPB can be produced inexpensively at a high or significant yield in an appropriate reaction time by producing.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication (C12P 13/04 C12R 1: 625) (C12P 13/04 C12R 1: 365) (C12P 13/04 (C12R 1:01) (C12P 13/04 C12R 1:29) (C12P 13/04 C12R 1: 125) (C12P 13/04 C12R 1: 265) (C12P 13/04 C12R 1: 445) (C12P 13/04 (C12R 1:19) (C12P 13/04 C12R 1: 385) (C12P 13/04 C12R 1:38) (C12P 13/04 C12R 1:43) (C12P 13/04 C12R 1:15) (C12P 13/04 (C12R 1: 865) (C12P 13/04 C12R 1: 725) (C12P 13/04 C12R 1: 645) (C12P 13/04 C12R 1:78) (C12P 13/04 C12R 1:67) (C12P 13/04 C12R 1:66) (C12P 13/04 C12R 1: 785) (C12P 13/04 C12R 1:80) Accession number of microorganism FERM BP-130 Accession number of microorganism FERM BP-1368 Accession number of microorganism FERM BP-737 (72 ) Inventor Shinji Miyamichi 760 Moshioka-cho, Kohoku-ku, Yokohama-shi, Kanagawa Prefecture Inside the Pharmaceutical Research Laboratories, Meiji Seika Co., Ltd. ) Inventor Hiroyuki Anzai 760 Morioka-cho, Kohoku-ku, Yokohama-shi, Kanagawa Prefecture Inside the Pharmaceutical Research Laboratories, Meiji Seika Co., Ltd. ) Inventor Yachiyo Yoshizawa 580 Horikawa-cho, Saiwai-ku, Kawasaki-shi, Kanagawa Prefecture Inside the Drug Development Laboratory, Meiji Seika Co., Ltd. (72) Inventor Toshinori Saito 580 Horikawa-cho, Saiwai-ku, Kawasaki City, Kanagawa Prefecture, Japan (72) Inventor Hiroshi Ogawa 580 Horikawa-cho, Saiwai-ku, Kawasaki-shi, Kanagawa Prefecture Inside the Pharmaceutical Development Laboratory, Meiji Seika Co., Ltd. (72) Inventor Hidebe Takebe 580 Horikawa-cho, Saiwai-ku, Kawasaki-shi, Kanagawa Prefecture Inside the Research Laboratory (72) Inventor Atsuyuki Sato Meiji Seika Co., Ltd. 580, Horikawa-cho, Saiwai-ku, Kawasaki City, Kanagawa Prefecture Inside the Pharmaceutical Development Laboratory (72) Inventor Yukizo Nagaoka 760, Shioka-cho, Kohoku-ku, Yokohama, Kanagawa Prefecture Inside the Pharmaceutical Research Laboratories (72) Inventor Shunzo Fukatsu Meiji Seika Co., Ltd.Meiji Seika Co., Ltd. (580) Horikawacho, Kawasaki-shi, Kanagawa Pref. Inside the company's drug development laboratory

Claims (11)

(57) [Claims] 1. The following equation 4- (hydroxymethylphosphinyl) -2 represented by
Treating oxo-butyric acid with a microorganism capable of producing one or more transaminases in the presence of glutamic acid or a salt thereof and aspartic acid or a salt thereof as amino group donors, Next formula A method for producing L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid represented by the formula: 2. A microorganism used is Streptomyces, Streptomyces Birch shea genus, Nocardia genus, Nocardioides Opsys genus Sakkaropori Supora genus, a actinomycete selected from Mi Kuromonosupora genus and Streptomyces spot Rangi um spp claims Item 1. The method according to Item 1. 3. The microorganism used is Bacillus, Micrococcus, Staphylococcus, Escherichia,
The method according to claim 1, which is a bacterium selected from the genus Pseudomonas, Serratia and Corynebacterium. 4. The microorganism used is Saccharomyces,
The method according to claim 1, which is a yeast selected from the genus Candida, the genus Cryptococcus, the genus Debaryomyces, the genus Trigonopsis, and the genus Hansenula. 5. The microorganism used is Aspergillus,
The method according to claim 1, which is a mold selected from the genus Mucor, the genus Aureobasidium, the genus Caetium, the genus Penicillium, and the genus Gliocladium. 6. The microorganism used is Streptomyces.
Albus, Streptomyces griseus, Streptomyces hygroscopicus, Streptomyces lividans, Streptomyces viridrochromogenes, Streptomyces pirosus, Streptovertisium
Cinnamonium, Streptomyces molokaensis, Nocardia Mediterrani, Nocardiopsis
Dassonvillei, Saroporisupora-Hirusu data, Mi Kuromonosupora-Karubonashi and streptavidin spot Rangi Umm
Actinomycetes selected from Pseudobulgari, or Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Pseudomonas cepacia,
A bacterium selected from Serratia marcescens and Corynebacterium glutamicum, or Saccharomyces
Yeast selected from Seribicii, Candida albicans, Cryptococcus neoformans, Debaryomyces hanseni, Trigonopsis variabilis, Hansenula schneshi, or Aspergillus flavus, Aspergillus terreus, Mucor spinesens, Aurebasidium purans, Caetum 2. A mold selected from globosam, penicillium funiculosum and gliocladium virens.
The method described in. 7. The microorganism used includes a transaminase having a GOT activity that is an activity of converting 2-ketoglutaric acid to glutamic acid in the presence of L-aspartic acid as an amino group donor, and a transaminase as an amino group donor. Compounds of formula (II) in the presence of L-glutamic acid are converted to compounds of formula (I)
A microorganism that produces a transaminase having a transaminase activity that converts to a compound of Streptomyces hygroscopicus SF-1293 strain (FERM BP-130) or a mutant thereof Streptomyces hygroscopicus SF-1293 NP- The method according to claim 1, which is 50 strains (FERM BP-1368) or 66 strains of Streptomyces lividans (FERM BP-737). 8. The concentration of the added glutamic acid or a salt thereof is from 0.2 to 3.0 per mole of the substrate compound of the formula (II).
And the concentration of the added aspartic acid or a salt thereof is a concentration corresponding to 1.0 to 3.0 mol per 1 mol of the substrate compound of the formula (II), and the pH of the reaction medium is 8.0. The method according to claim 7, wherein the alkaline pH value is maintained at ~ 9.0. 9. The following equation 4- (hydroxymethylphosphinyl) -2 represented by
Treating oxo-butyric acid with one or more transaminases in the presence of glutamic acid or a salt thereof and aspartic acid or a salt thereof as an amino group donor, characterized by the following formula: A method for producing L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid represented by the formula: 10. An enzyme used in the presence of glutamic acid-oxaloacetic acid-transaminase or 4- (hydroxymethylphosphinyl) -2-oxo-butyric acid in the presence of glutamic acid as an amino group donor. 10. A transaminase that converts to amino-4- (hydroxymethylphosphinyl) -butyric acid, or a combination of two or more thereof.
The method described in. 11. The following equation 4- (hydroxymethylphosphinyl) -2 represented by
Oxo-butyric acid having the ability to produce transaminase in the presence of aspartic acid or a salt thereof as an amino group donor, and having the ability to produce Streptomyces, Streptoverticillium, Nocardia, Nocardiopsis , Sakkaropori Supora genus, Mi Kuromonosupora genus and actinomycetes selected from Streptomyces spot Rangi um genus or Bacillus genus, Micrococcus, Staphylococcus, Pseudomonas, bacteria selected from the genus Serratia and Corynebacterium or Saccharomyces, Genus, Candida, Cryptococcus, Debaryomyces, Yeast selected from Trigonopsis and Hansenula, or Aspergillus, Mucor,
Aureobasidium, Caetium, Penicillium and Glyocladium treated with a mold, or treated with a crude enzyme solution obtained from a crushed cell of the microorganism, or glutamate-oxalo A treatment with acetic acid-transaminase or glutamic acid-pyruvate-transaminase, or a combination of these enzymes, characterized by the following formula: A method for producing L-2-amino-4- (hydroxymethylphosphinyl) -butyric acid represented by the formula: