JPH06125786A - Production of l-beta-hydroxyamino acid - Google Patents

Abstract

PURPOSE:To simply and efficiently obtain L-beta-hydroxyamino acid from racemic-beta-hydroxyamino acid. CONSTITUTION:Racemic-beta-hydroxyamino acid is treated with an enzyme capable of specifically decomposing D-beta-hydroxyamino acid into glycine and a corresponding aldehyde, a cell of a microorganism to produce the enzyme or a treated material of the cell to produce L-beta-hydroxyamino acid from racemic-beta- hydroxyamino acid. The objective substance is more inexpensively and more efficiently obtained than a method of using an optically resolving agent and the decomposed substance is economically reusable.

Description

Detailed Description of the Invention

[0001]

[Industrial application] Enzyme capable of decomposing racemic β-hydroxy amino acid into D-β-hydroxy amino acid specific aldehyde derivative corresponding to glycine, treatment of a microorganism producing the enzyme or its bacterial cell The present invention relates to a method for producing L-β-hydroxyamino acid, which is known to be useful as an intermediate for medicines, medicines, agricultural chemicals, etc. by treating it with a product.

[0002]

2. Description of the Related Art Conventionally, L-threo and erythro-β-hydroxyamino acid have been synthesized by the following method. That is, if necessary, an aldehyde derivative having a protective group introduced and a glycine are condensed in the presence of a strong alkali to obtain a racemic-threo / erythro-β-hydroxyamino acid derivative, and then the threo / erythro form is subjected to mutual separation treatment. To do. Then the racemic threo or erythro obtained
After removing the protecting group of the β-hydroxyamino acid derivative and introducing a substituent into the amino group, optical resolution was performed using an optical resolving agent such as quinine or brucine, and finally the substituent of the amino group was removed. It is to remove.

[0003]

When β-hydroxyamino acid is synthesized by condensing an aldehyde derivative and glycine, the stereoselectivity of the reaction is low, and therefore threo / erythro and D / L are combined in four types of isomerism. The body inevitably produces. In the synthesis of L-threo or erythro-β-hydroxyamino acid, a complicated process for separating these isomers was required, which was a big problem in developing an industrial production method. In particular, the conventional D / L optical resolution method has a problem that not only the optical resolution agent used is expensive, but also the process and its control are complicated and the yield is low. Further, in order to reuse the unnecessary isomer obtained by these operations, it is necessary to separately racemize or decompose it, which causes the process to become more complicated.

[0004]

As a method for solving this problem, an enzymatic resolution method using D-threonine aldolase is known (Japanese Patent Laid-Open Nos. 58-116691 and 58-216691). That is, it is a method of specifically degrading the D-form by D-threonine aldolase to obtain only the L-form. However, this method is only known to be applicable to L-threonine among β-hydroxy amino acids. We have found that L-β-
As a result of intensive studies on a method for synthesizing a hydroxyamino acid, surprisingly, D-threonine aldolase was found to be not only D-threonine but also various D-threonine D
The present invention was completed by finding that it acts widely on the body and decomposes into aldehyde compounds corresponding to glycine. This allows efficient L from racemic-β-hydroxy amino acids.
Not only is it possible to produce -β-hydroxyamino acid, but also the decomposition product of the D-form can be reused as a raw material for the synthesis of the racemate. Furthermore, when a recombinant in which the productivity of D-threonine aldolase is significantly improved by genetic engineering is used, even if the target L-β-hydroxyamino acid degrading enzyme is contained in the bacterial cells, It can be used in the reaction without removing the enzyme. When such a recombinant is used, D-
Since a highly reactive β-hydroxyamino acid, which requires a large amount of enzyme to decompose the body, can be efficiently reacted, a wide range of L-β-hydroxyamino acids can be synthesized. The present invention has the general formula (I)

[0005]

[Chemical 2] (In the formula, R represents a substituted or unsubstituted aliphatic group, alicyclic group,
Represents an aromatic group or a heterocyclic group. ) Is allowed to act on the racemic-β-hydroxyamino acid represented by the formula (4) by an enzyme that specifically decomposes D-β-hydroxyamino acid into an aldehyde corresponding to glycine, a microbial cell producing the enzyme, or a treated product of the microbial cell. The present invention relates to a method for producing a characteristic L-β-hydroxyamino acid.

In the raw material racemic-β-hydroxyamino acid used in the present invention, R in the above formula (I) represents a substituted or unsubstituted aliphatic group, alicyclic group, aromatic group or heterocyclic group. . Specifically, it is a compound which is a substituent selected from an alkyl group, an alkenyl group, an alkynyl group, an alicyclic hydrocarbon group, an aromatic hydrocarbon group or a heterocyclic hydrocarbon group, and is included in the substituents. Alkyl group, alkenyl group, alkynyl group, alkoxyl group, alkylene group, alicyclic hydrocarbon group, aromatic hydrocarbon group, hydroxyl group, nitro group, or halogen group in which one or more of hydrogen is the same or different May be replaced with. When the number of carbon atoms of the substituent is a linear aliphatic group, it is preferably 20 or less, particularly preferably 10 or less, and in the case of other substituents, preferably 30 or less, particularly preferably 20 or less. When used, good results can be obtained.

The starting racemic-β-hydroxy amino acid may be a mixture of its erythro / threo isomers, or may be a threo body or an erythro body alone. Although the erythro body and the threo body are complicated, they can be separated from each other by a method such as crystallization. In the present invention, isomers other than L-β-hydroxyamino acid, that is, D-threo-β-hydroxyamino acid and D-erythro-β-hydroxyamino acid are specifically decomposed into aldehydes corresponding to glycine, The aldehyde thus obtained and the corresponding aldehyde can be reused in the synthesis.

In the present invention, as described above, D-
It is possible to use an enzyme that decomposes β-hydroxyamino acid. As such an enzyme, any enzyme that specifically decomposes D-threo and / or erythro-β-hydroxyamino acid can be used. . Particularly preferred as such an enzyme is D-threo /
Those having the ability to specifically decompose erythro-β-hydroxy amino acid into aldehyde corresponding to glycine are included. Particularly preferred are those that specifically recognize the difference in the three-dimensional structure between the D-form and the L-form, but are not affected by the structural difference between the threo form and the erythro form. Furthermore, those acting on a wide range of β-hydroxy amino acids as shown in the general formula (I) can be mentioned.
Particularly preferred as such an enzyme is D-
Threonine aldolase is an example. Examples of D-threonine aldolase include those described in JP-B No. 64-11279, and those obtained by manipulating the gene thereof (for example, JP-A-3-139283).

Unexpectedly, these D-threonine aldolases have the excellent property of acting on various D-type stereoisomers of β-hydroxyamino acids and degrading them into glycine and the corresponding aldehyde. There is. Also,
Despite having a very high specificity for the D-type conformation, for the conformation such as erythro / threo,
D-threo and D-
It has an excellent property that it acts on both erythro bodies and decomposes them.

The D-threonine aldolase used in the present invention can be obtained from a D-threonine aldolase-producing bacterium or a mutant strain thereof. Examples of the D-threonine aldolase-producing bacterium include Alcaligenes
Flyfly (Alcaligenes faecalis) IFO1266
9, Pseudomonas DK-2 (Microtechnology Research Institute, No. 6200), Alice Lobacter (Arthrobacter) DK
-19 (Microtechnical Research Institute No. 6201), Xanthomonas oryzae IAM1657 and the like are known (Japanese Patent Publication No. 64-11279 and Japanese Patent Laid-Open No. 1-279).
73586). Recombinants prepared by introducing a DNA carrying a gene involved in the synthesis of D-threonine aldolase obtained from these D-threonine aldolase-producing bacteria by using a gene recombination method are particularly suitable for the present invention. It can be preferably used. Examples of microorganisms into which such a gene can be introduced and used include Escherichia (Es
cherichia bacteria, Bacillus bacteria, Xanthomonas bacteria, Acetobacter
bacter bacteria, Pseudomonas bacteria, Gluconobacter bacteria, Azotobacter bacteria, Rhizobiu
m) bacteria, Alcaligenes bacteria, Klebsiella bacteria, Salmonella
Prokaryotic microorganisms such as bacteria of the genus Serratia and lower eukaryotes such as yeast can be used.

Among such recombinants, those belonging to the genus Xanthomonas are mentioned as an example of the recombinant having a particularly high ability to produce D-threonine aldolase. Xanthomonas cit
ri) IFO3311 (pDT648) has the ability to highly produce D-threonine aldolase (JP-A-3-139283), and is particularly preferable.

The microorganism used in the present invention can be cultured according to a conventional method. The medium used for culturing is an ordinary medium containing a carbon source, a nitrogen source, an inorganic substance and the like necessary for the growth of microorganisms. Examples of the carbon source used here include glucose, galactose, and soluble starch, and as the nitrogen source, ammonium chloride, ammonium sulfate, ammonium nitrate, peptone, meat extract, yeast extract, corn steep liquor, soybean powder, cottonseed residue. And so on. In addition to sodium chloride, potassium salt, calcium salt, ammonium salt, phosphate and the like, inorganic substances such as zinc, magnesium, iron and manganese can be used. In addition, the addition of organic micronutrients such as vitamins and amino acids often produces desirable results. The culture may be performed under aerobic conditions at a pH of 4 to 10 and a temperature of 20 to 50 ° C. in an arbitrary range for 1 to 10 days. When the recombinant is used, antibiotics such as ampicillin and chloramphenicol may be added to the culture medium depending on the strain used.

In the enzyme reaction, a culture solution of the bacterial cells, bacterial cells separated from the culture solution, dried bacterial cells by freeze-drying,
Bacterial cell lysate, crude enzyme extract, purified enzyme, further, treated products and immobilization products described above can be used,
When the enzyme solution or cells to be used contains an activity such as L-threonine aldolase or L-alothreonine aldolase that decomposes the desired L-β-hydroxyamino acid, efficient synthesis In order to carry out, it is preferable to remove the activity before use in the reaction. As a method for removing these degrading activities, 60
A method of heat treatment at 30 ° C. for about 30 minutes or a method of acetone fractionation of the enzyme solution is preferable, but the method is not limited thereto, and any method can be used as long as the decomposition activity can be selectively removed. Also, by genetic manipulation D
-A recombinant that has greatly improved the productivity of threonine aldolase can be used in the reaction without removing these degrading activities.

The method for carrying out the enzymatic reaction is as follows: racemic threo and / or erythro-β-, which are substrates in an aqueous medium.
The hydroxyamino acid may be brought into contact with the above-mentioned microorganism culture solution, cells, treated cells, enzyme, or those obtained by immobilizing these by a usual method. As the aqueous medium at the time of the reaction, for example, water, a buffer solution, a water-containing organic solvent or the like can be used.

The concentration of the enzyme, microbial cells or treated cells in the reaction is not particularly limited as long as D-threo / erythro-β-hydroxyamino acid can be completely decomposed, but D-threonine aldolase activity (1 U Represents the amount of enzyme required to produce 1 μmole of glycine per minute from D-threonine), and is preferably 0.1 U / ml or more. In particular, if it is 1 U / ml or more, the reaction can be quickly terminated.

The concentration of racemic threo and / or erythro-β-hydroxy amino acid as a substrate may be 1 mM to 100 mM as long as the reaction is not significantly inhibited, and the substrate may be supplied all at once, continuously or in a divided manner. It can also be implemented by means of.

The reaction temperature is preferably 10 to 55 ° C., 20
To 40 ° C. is more preferable. PH during the reaction is 5 to 1
It is 0, preferably 6 to 8. Pyridoxal-5 '
-Phosphoric acid is added to the reaction system at 10 nM to 10 mM, preferably 1
Preferable results are obtained by adding at a concentration of μM to 100 μM.

Further, divalent metal ions such as Mg ²⁺ , Mn ²⁺ , Co ²⁺ and Fe ^{2+ in} the form of a salt are added in an amount of 100 μM to 100 mM.
A preferable result is obtained when it is preferably added at a concentration of 1 mM to 10 mM.

The reaction system may be either a batch system or a continuous system, and thus the reaction is completed in about 0.5 to 50 hours. After the completion of the reaction, the reaction solution is sterilized and deproteinized by centrifugation, ultrafiltration or the like, and first, the formed aldehyde is separated by organic solvent extraction or the like. For extraction, any solvent can be used as long as it has a high solubility of aldehyde and almost insoluble in L-β-hydroxyamino acid and glycine, but good results can be obtained by using ether.

The aldehyde can be recovered from the solvent phase by concentration under reduced pressure or the like. The isolation and recovery of L-β-hydroxyamino acid and glycine from the aqueous phase can be carried out by chromatographic separation using an ordinary ion exchange resin.
That is, the aqueous phase is passed through a column packed with a cation exchange resin, washed with water, and then eluted with diluted ammonia water or the like.

When the purity is low, the degree of purification can be increased by further performing crystallization and water recrystallization. The above-mentioned method for isolating and purifying the reaction product exemplifies one typical method, and various separation and purification methods can be applied to the present invention without being limited thereto. Examples of such a method include alcohol solvents such as methanol and ethanol, aromatic hydrocarbon solvents such as benzene and toluene, ester solvents such as ethyl acetate, ketone solvents such as acetone and methyl ethyl ketone, and dimethyl sulfoxide. Sulfoxide solvents, amide solvents such as N, N-dimethylformamide, nitrile solvents such as acetonitrile, ether solvents such as ethyl ether, dioxane, tetrahydrofuran, halogenated hydrocarbon solvents such as methylene chloride and mixed solvents thereof. Separation methods such as solvent extraction using the above-mentioned aqueous solvent, column chromatography, reverse phase chromatography, high performance liquid chromatography, recrystallization method and the like can be mentioned.

According to the method of the present invention, (1) D-threonine aldolase is used in a wide range of D-threonine aldolase.
It is possible to synthesize L-β-hydroxy amino acid from various racemic-β-hydroxy amino acids because it can act on β-hydroxy amino acid to decompose it. (2) Since both D-threo and erythro-β-hydroxyamino acid are decomposed into glycine and aldehyde, which are synthetic raw materials, they are recovered from the reaction solution and recycled again to racemic-threo / erythro-β-hydroxyamino acid synthesis. Can be used. (3) Therefore, since the target product can be obtained in high yield without the need for racemization, the process is much simpler than the conventional optical resolution method which requires complicated and expensive optical resolving agents. Can be converted. (4) By using a strain in which the productivity of D-threonine aldolase is significantly improved by gene recombination, even if the enzyme that decomposes the target L-β-hydroxyamino acid is contained in the cells, those enzymes can be used. It can be used for the reaction without removing it, and can also efficiently perform the reaction on β-hydroxyamino acid having low reactivity such that a large amount of enzyme is required for decomposing the D-form, L
It can be widely used for producing -β-hydroxy amino acids.

[0023]

EXAMPLES Next, the method of the present invention will be described in more detail by way of examples, but the present invention is not limited to the examples. In the examples,% means% by weight.

Example 1 A medium containing 0.5% of polypeptone, 0.5% of yeast extract and 0.5% of sodium chloride and having a pH of 7.5 was prepared.
Add 3 liters to a liter culture tank,
And heat sterilized for 15 minutes. The medium was inoculated with Alysobacter DK-19 (Microtechnical Laboratory, No. 6201), and the pH was adjusted.
The culture was carried out at 30 ° C. for 20 hours with aeration and stirring while maintaining the temperature at 7.5. After completion of the culturing, cells were collected from the culture solution by centrifugation, washed with water, and then 0.01 mM pyridoxal-5'-
PH containing phosphoric acid and 1.0 mM magnesium chloride
7.5 0.2M HEPES (N-2-Hydroxyethylpipe
razine-N'-2-ethanesulfonic acid) buffer solution (500 ml), this cell suspension is subjected to ultrasonic crushing treatment at 20 kHz for 3 minutes 4 times, and suspended matter in the crushed liquid is removed by centrifugation. To prepare a crude enzyme solution. Next, cold acetone was gradually added to this crude enzyme solution while stirring to give an acetone concentration of 45
The 65% fraction precipitate was collected and dissolved in the above buffer to prepare 100 ml of an acetone fractionation enzyme solution. The D-threonine aldolase activity (1 U represents the amount of enzyme required to produce 1 μmole of glycine in 1 minute from D-threonine) of the obtained acetone fractionated enzyme solution was measured and found to be 0.36 U / ml. there were. 100 mg of D, L-threo-phenylserine (equal amount mixture of D-form and L-form) was added to the obtained acetone fractionated enzyme solution, and the mixture was added at 30 ° C. for 10 minutes.
Reacted for hours. After completion of the reaction, the phenylserine isomer and glycine in the reaction solution were derivatized with o-phthalaldehyde and N-acetyl-L-cystine, and quantified by HPLC using an ODS column. Further, benzaldehyde was reacted with 2,4-dinitrophenylhydrazine and quantified by HPLC using an ODS column. L-threo-phenylserine: 2.74 mM (residual rate 99.7%) D-threo-phenylserine: 0 mM (residual rate 0%) Glycine: 2.71 mM Benzaldehyde: 2.68 mM 1N hydrochloric acid 10 ml in the reaction solution. After the addition, the insoluble matter was removed by centrifugation. To this centrifuged supernatant, 200 ml of ethyl ether was added, and the mixture was stirred and allowed to stand, the ether phase was separated and dried under reduced pressure to give 27 mg of benzaldehyde. The aqueous phase was passed through a column (10 mmφ × 100 mm) packed with a strongly acidic cation exchange resin DOWEX 50W × 8, and after sufficient washing with deionized water, the adsorbed substance was eluted with 0.5N ammonia water, and phenyl was added. Serine and glycine fractions were collected separately. When both fractions were dried under reduced pressure,
Crystals of 48 mg and 18 mg were obtained, respectively. The crystals obtained from the phenylserine fraction were subjected to isomer analysis (after derivatization with o-phthalaldehyde and N-acetyl-L-cystine, HPLC analysis with an ODS column).
The remaining phenylserine was all L-threo body.

Example 2 In the same manner as in Example 1, Pseudomonas DK-2 (Microtechnology Research Institute, No. 6200), Alcaligenes flycaris I
FO12669 and Xanthomonas oryzae IAM
1657 was cultured to prepare the acetone fractionated enzyme solution. Using the obtained acetone fractionated enzyme solution, a reaction was carried out in the same manner as in Example 1. Table 1 shows the residual ratio of each isomer obtained by the isomer analysis of the reaction solution.

[0026]

[Table 1]

Example 3 Xanthomonas citrie IFO3311 (pDT64
8), 50 μg / ml of chloramphenicol in the medium
1 / 60th of the resulting culture solution
The same procedure as in Example 1 was carried out except that the amount was crushed, washed without further fractionation with acetone, lyophilized and used as a 100 ml suspension (0.22 U / ml) in the reaction. After completion of the reaction, the phenylserine isomer, glycine and benzaldehyde in the reaction solution were quantified by the above-mentioned method to obtain the following values. L-threo-phenylserine: 2.72 mM (residual rate 9
8.6%) D-threo-phenylserine: 0 mM (residual rate
0%) Glycine: 2.74 mM Benzaldehyde: 2.66 mM Each component was fractionated and purified from the reaction solution to obtain 26 mg of benzaldehyde, 46 mg of phenylserine crystals, and 19 mg of glycine crystals. The isomer analysis of the crystals obtained from the phenylserine fraction revealed that all the residual phenylserine was the L-threo body.

Example 4 β-in which R in the general formula (I) is an ethyl group, an octyl group, an allyl group, a cyclohexyl group, a 5-hydroxy-2-nitrophenyl group and an imidazolyl group, respectively.
Using an equal amount mixture of D, L-threo-forms of hydroxyamino acid as a substrate, the acetone fractionated enzyme solution obtained in Examples 1 and 2 or the bacterial cell suspension obtained in Example 3 was used in the same manner as in Example 1. It was made to react by the method. Tables 2 and 3 show the residual ratio of each isomer obtained by isomer analysis of the reaction solution.

[0029]

[Table 2]

[Table 3]

Example 5 R in the general formula (I) is phenyl group, ethyl group, octyl group, allyl group, cyclohexyl group, 5-
In the acetone-fractionated enzyme solution obtained in Examples 1 and 2 or in Example 3, an equal mixture of D, L-erythro isomers of β-hydroxyamino acid having a hydroxy-2-nitrophenyl group and an imidazolyl group was used as a substrate. The obtained bacterial cell suspension was reacted in the same manner as in Example 1. Tables 4 to 6 show the residual ratio of each isomer obtained by the isomer analysis of the reaction solution.

[0031]

[Table 4]

[Table 5]

[Table 6]

Example 6 D of β-hydroxyamino acid in which R in the general formula (I) is a phenyl group, an ethyl group, an octyl group, an allyl group, a cyclohexyl group, a 5-hydroxy-2-nitrophenyl group and an imidazolyl group, respectively. , L-threo / erythro isomer was used as a substrate, and the acetone fractionated enzyme solution obtained in Examples 1 or 2 or the bacterial cell suspension obtained in Example 3 was treated in the same manner as in Example 1. It was made to react with. Tables 7 to 9 show the residual ratio of each isomer obtained by the isomer analysis of the reaction solution.

[0033]

[Table 7]

[Table 8]

[Table 9]

[0034]

According to the present invention, it is not necessary to introduce or remove a substituent for optical resolution, there is no need to use an expensive optical resolution agent, and the produced glycine and aldehyde derivative can be recycled for raw material synthesis. This is an extremely advantageous method as compared with the conventional method because it can be carried out and complicated racemization is not required. Particularly, by using a bacterial cell in which the productivity of D-threonine aldolase is significantly improved by genetic recombination, L-β-hydroxyamino acid can be easily and economically produced from a wider range of racemic β-hydroxyamino acid. Therefore, it is suitable as an industrial manufacturing method.

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Claims (6)

[Claims] 1. A compound represented by the general formula (I): (In the formula, R represents a substituted or unsubstituted aliphatic group, alicyclic group,
Represents an aromatic group or a heterocyclic group. ) Is allowed to act on the racemic-β-hydroxyamino acid represented by the formula (4) by an enzyme that specifically decomposes D-β-hydroxyamino acid into an aldehyde corresponding to glycine, a microbial cell producing the enzyme, or a treated product of the microbial cell. A method for producing L-β-hydroxyamino acid, which is characterized. 2. The method according to claim 1, wherein the enzyme that specifically decomposes D-β-hydroxyamino acid into an aldehyde corresponding to glycine is D-threonine aldolase. 3. A microorganism producing an enzyme that specifically decomposes D-β-hydroxy amino acid into an aldehyde corresponding to glycine is selected from the group consisting of microorganisms belonging to the genera Alcaligenes, Pseudomonas, Alithrobacter and Xanthomonas. The method of claim 1 comprising at least one selected microorganism. 4. An enzyme having an ability to specifically decompose a D-β-hydroxyamino acid derived from a microorganism transformed by genetic recombination into an aldehyde corresponding to glycine is involved in the synthesis of D-threonine aldolase. 2. The method according to claim 1, which is obtained from a transformant carrying a recombinant DNA obtained by binding a gene-bearing DNA and a vector DNA. 5. The racemic-β-hydroxy amino acid,
A racemic-threo-β-hydroxy amino acid, said L
The method according to claim 1, wherein the -β-hydroxy amino acid is L-threo-β-hydroxy amino acid. 6. The racemic-β-hydroxy amino acid is a racemic-erythro-β-hydroxy amino acid, and the L
The method according to claim 1, wherein the -β-hydroxy amino acid is L-erythro-β-hydroxy amino acid.