JPH11103887A - Production of d-amino acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simplify the separation and purification of a D-amino acid from a reaction solution by allowing an enzyme preferentially or specifically acting on an L-amino acid to act on the L-amino acid. SOLUTION: This method for producing a D-amino acid comprises allowing an enzyme (e.g. an L-amino acid oxidase) preferentially or specifically acting on an L-amino acid to act on the D, L-amino acid to convert the L-amino acid into a compound having physical properties largely different from those of the objective D-amino acid, and subsequently separating the left D-amino acid. Only the D-amino acid useful as an intermediate for medicines or agrochemicals or as a modifying agent for antibiotics can easily be deposited.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for producing a D-amino acid useful as a pharmaceutical / pesticide intermediate or a modifier of an antibiotic.

[0002]

2. Description of the Related Art D-amino acids are unnatural optically active amino acids and are known to be difficult to produce.
Heretofore, D-amino acids have been produced from D, L-amino acids by a crystallization method utilizing a difference in physical properties from L-amino acids. However, since both are amino acids, the difference in physical properties is small, and the separation and purification of D-amino acids require extremely strict conditions.

Further, an acyl-D, L-amino acid is reacted with an L-amino acid acylase to convert the acyl-L-amino acid into an L-amino acid.
A method of hydrolyzing an acyl-D-amino acid after separation into an amino acid and separating an acyl-D-amino acid and an L-amino acid, and a method of allowing an enzyme to act on a 5-substituted hydantoin (JP-A-55-104890; 55-114292), acyl-D,
A D-amino acid acylase is allowed to act on an L-amino acid to decompose an acyl-D-amino acid into a D-amino acid.
A method for separating an L-amino acid and a D-amino acid (Japanese Patent Publication No. Sho 53)
−36035).

[0004]

However, since the difference in physical properties between amino acids and amino acid derivatives is not always sufficient, these methods utilizing the difference in physical properties between amino acids and amino acid derivatives as described above are used. In the above, the separation and purification of D-amino acids require extremely strict conditions.

Further, other methods also have problems such as the fact that the raw material substrate is expensive, the steps are complicated, the yield of the product and the optical purity are low. There has been a need for a method for producing D-amino acids.

[0006]

Means for Solving the Problems The present invention converts the physical properties of an L-amino acid which is not a target compound among D, L-amino acids into a compound which is significantly different from the physical properties of a D-amino acid which is a target compound. It is intended to provide a method for simplifying the separation and purification of D-amino acids.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The present inventors have found that L-
As a result of intensive studies on a method for converting an amino acid into a compound having greatly different physicochemical properties and improved water solubility, it was found that D, L-amino acid was used as a substrate and L-amino acid oxidase such as L-amino acid oxidase was used. -Decomposition of L-amino acids into oxo acids using an enzyme that preferentially or specifically oxidizes amino acids has been found to make it possible to easily precipitate only undecomposed D-amino acids. We have established a method for easily isolating and purifying amino acids for production.

[0008] That is, the present invention comprises reacting a D, L-amino acid with, for example, an L-amino acid oxidase that catalyzes a reaction of preferentially or specifically oxidizing an L-amino acid to convert it to a corresponding oxo acid. Thereafter, a method for producing a D-amino acid, comprising separating the remaining D-amino acid.

Hereinafter, the present invention will be described in detail, but the present invention is not limited thereto.

The D, L-amino acid which can be used as the substrate of the present invention includes, for example, D, L-glutamic acid, D, L-valine, D, L-aspartic acid, D, L-methionine, D, L-leucine , D, L-isoleucine, D, L-histidine, D, L-
Arginine, D, L-phenylalanine, D, L-tyrosine,
D, L-tryptophan, D, L-alanine, D, L-serine and the like can be mentioned. These D, L-amino acids can also be used after racemizing the L-amino acids using a suitable means. As a racemization method, for example, a heating method,
Examples include a method using racemase.

The enzyme which can be used in the present invention acts on the L-amino acid preferentially or specifically to change the L-amino acid into another compound, and to determine the physicochemical properties of the compound and the remaining D-amino acid. Any of these can be used as long as they can be separated by the difference. More preferably, an enzyme that preferentially or specifically oxidizes L-amino acids can be used. For example, L-amino acid oxidase (EC 1.4.3.2), L
-Glutamate oxidase (EC 1.4.3.11), L-lysine oxidase (EC 1.4.3.14) and the like. These enzymes are used as substrates, taking into account their substrate specificity
It can be selected corresponding to the D, L-amino acid.

More specifically, the previously reported Crot
alus adamanteus [J. Biol. Chem., 235, 2013-2018 (196
0)], genus Neurospora [J. Biol. Chem., 50, 258-2268 (1
951)], genus Proteus (JP-B-45-16789), Colletotrichu
Genus m (JP-B 62-43671), genus Streptomyces (JP-B 59-26)
267), genus Cryptococcus (Japanese Patent Publication 5-61909, Japanese Patent Publication 6-46)
939), L-amino acid oxidase of the genus Trichoderma (JP-A-8-509367), genus Streptomyces (JP-B-59-26267, JP-B-
L-glutamate oxidase of the genus Trichoderema [J. Biol. Chem., 255, 976-981 (1
980)] can be used. More preferably, L-amino acid oxidase derived from Crotalus adamanteus can be used.

The reaction conditions of the present invention vary depending on the enzyme used, but are usually carried out at a pH of 7 to 10 and a temperature of 25 to 55 ° C. The pH may be controlled by adding an acid or an alkali while controlling the pH fluctuation during the reaction, but a buffer such as Tris-hydrochloric acid may be usually used. After the reaction, the desired D-
Amino acids can be easily separated and purified from the reaction solution by methods such as concentrating the reaction solution or utilizing the difference in solubility of oxo acids and D-amino acids generated from L-amino acids in various solvents. As a separation means, a method such as filtration or centrifugation can be used.

Test Example 1 L-amino acid by HPLC
Analysis of L-amino acid and D-amino acid The L-amino acid and D-amino acid analysis was performed using 0.1 M leucine, isoleucine, methionine, valine, glutamic acid, and phenylalanine D, L-amino acids in 50 mM Tris-HCl buffer (pH 7.5). After dissolving and diluting this solution 100 times with purified water, HPLC was carried out using SUMICHIRAL AO-5000 under the following conditions, and the elution pattern, elution time and peak area were analyzed.

Solvent-A: 2 mM CuSO4 / 5% Isopropylalcohol Solvent-B: 2 mM CuSO4 / 10% Isopropylalcohol Flow rate: 0.5 mL / min Absorbance: 254 nm

As a result, it was possible to separate D-form and L-form of Solvent-A from leucine, isoleucine, methionine, valine and glutamic acid. (See FIGS. 1 to 5)
Phenylalanine could be separated by Solvent-B. (See Fig. 6)

Test Example 2 Using L-amino acid oxidase
Decomposition of L-amino acids 25 mM of various amino acids (D, L-amino acids: D, L-leucine,
D, L-isoleucine, D, L-methionine, D, L-valine,
And D, L-phenylalanine) were each dissolved in 1 ml of 50 mM Tris-HCl buffer (pH 7.5) to obtain a substrate solution.

In this substrate solution, L-amino acid oxidase (Cr
from otalus adamanteus; Sigma Type I, 0.55u / mg) 2m
g, and reacted at 30 ° C. for 17 hours.
The reaction was stopped by adding μl, and the precipitate was removed by centrifugation. The type and amount of the optical form of the amino acid remaining unreacted in the reaction solution were determined by HPLC after diluting the obtained supernatant 100-fold with purified water.

As a result, only the L-amino acid of D, L-leucine, D, L-isoleucine, D, L-methionine, D, L-valine and D, L-phenylalanine reacted with L-amino acid oxidase. It was almost completely decomposed, and it became clear that only D-amino acids remained as unreacted amino acids in the reaction completed solution. (See FIGS. 7 to 11)

Test Example 3 Using L-amino acid oxidase
Decomposition of L-glutamic acid The pH of the reaction solution was adjusted to 7 using 25 mM D, L-glutamic acid, and the reaction was carried out in the same manner as in Test Example 2 using L-glutamic acid oxidase. As a result, it became clear that only L-glutamic acid was decomposed and only D-glutamic acid remained as an unreacted compound in the reaction solution as in Test Example 2. (See FIG. 12)

According to the above Test Examples 1 to 3, various D, L-amino acids are allowed to act on amino acid oxidase which acts preferentially or specifically on L-form amino acids, whereby only L-amino acids are decomposed and D-L-amino acids are decomposed. It has become clear that amino acids can be left as unreacted compounds in the reaction solution. The reaction proceeded smoothly even if the amino acid concentration at the start of the reaction was increased to a level at which it could not be dissolved, and it was possible to leave only D-amino acids as unreacted amino acids in the reaction-terminated liquid.

In this specification, "%" means "weight / volume (g / dl)".

[0023]

EXAMPLES Example 1 0.1 M D, L-leucine was added to 50 mM Tris-HCl buffer (pH 7.
5) After dissolving in 50 ml, the pH was adjusted to 7.5 to obtain a substrate solution. L-amino acid oxidase (Crotalus a
(derived from damanteus; Sigma Type I, 0.55 u / mg), and reacted at 30 ° C. for 24 hours.

Thereafter, various D, L-amino acids corresponding to 0.1 M and 100 mg of L-amino acid oxidase (all in powder form)
Was added for 24 hours, and the reaction was continued for 4 days.

As a result, the coexisting L-leucine was almost completely decomposed by L-amino acid oxidase (100 mg) within 24 hours, and L-leucine completely disappeared even in the reaction solution reacted for 4 days. Only D-leucine remained as an unreacted amino acid. (See FIG. 13)

The residual amount of D-leucine was 98.7%, and D-leucine was hardly decomposed. In addition, of the D-leucine remaining in the reaction solution, an amount corresponding to about 50 mM has already been precipitated in a crystalline state, and the reaction solution is further concentrated to precipitate D-leucine containing no L-leucine at all. I was able to. Further, the precipitated D-leucine was collected by filtration and purified by a conventional method. as a result,
Purified D-leucine was obtained with a recovery of about 95%.

Example 2 The preparation of D-isoleucine was examined under the same conditions as in Example 1 using D, L-isoleucine as a substrate. As a result, 0.1 ML-isoleucine was almost completely decomposed by L-amino acid oxidase (100 mg) within 24 hours, L-isoleucine completely disappeared even in the reaction solution reacted for 4 days, and only D-isoleucine remained unreacted. It remained as a reactive amino acid. (See FIG. 13)

The residual amount of D-isoleucine is 100%
And D-isoleucine was not decomposed at all. In addition, of the D-isoleucine remaining in the reaction solution, about 20 mM
Was already precipitated in a crystalline form, and D-isoleucine containing no L-isoleucine could be precipitated by further concentrating the reaction solution. Further, the precipitated D-isoleucine was collected by filtration and purified by a conventional method. As a result, the purified D was recovered at a recovery rate of about 96%.
-Isoleucine could be obtained.

Example 3 The preparation of D-methionine was examined under the same conditions as in Example 1 using D, L-methionine as a substrate. As a result, 0.
1M L-methionine is also almost completely decomposed by L-amino acid oxidase (100 mg) within 24 hours, L-methionine completely disappears even in the reaction solution reacted for 4 days, and D
-Only methionine remained as unreacted amino acid.
(See FIG. 13)

The residual amount of D-methionine was 92.4%, and D-methionine was not decomposed at all. Also,
Of the D-methionine remaining in the reaction solution, the amount precipitated in a crystalline form was about 4 mM. However, by concentrating the reaction solution, D-methionine containing no L-methionine could be precipitated. Was. Further, the precipitated D-methionine was collected by filtration and purified by a conventional method. As a result, purified D-isoleucine was obtained at a recovery rate of about 86%.

Example 4 The preparation of D-valine was examined under the same conditions as in Example 1 using D, L-valine as a substrate. As a result, 0.1M L-
Valine also within 24 hours L-amino acid oxidase (100mg)
And L-valine completely disappeared even in the reaction solution reacted for 4 days, and only D-valine remained as an unreacted amino acid. (See FIG. 13)

The residual amount of D-valine was 100%, and D-valine was not decomposed at all. The amount of D-valine remaining in the reaction solution precipitated in a crystalline form was about 3 mM, but the L-valine was concentrated by concentrating the reaction solution.
D-valine containing no valine could be precipitated. Further, the precipitated D-valine was collected by filtration,
Purified by a conventional method. As a result, purified D-valine was obtained with a recovery of about 96%.

Example 5 The preparation of D-phenylalanine was examined under the same conditions as in Example 1 using D, L-phenylalanine as a substrate.
As a result, 0.1 M L-phenylalanine was almost completely decomposed by L-amino acid oxidase (100 mg) within 24 hours, L-phenylalanine almost disappeared even in the reaction solution reacted for 4 days, and D-phenylalanine was not reacted. It remained as an amino acid. (See FIG. 13)

The residual amount of D-phenylalanine is 10
0%, indicating that D-phenylalanine was not decomposed at all. In addition, of the D-phenylalanine remaining in the reaction solution, an amount corresponding to about 48 mM has already been precipitated in a crystalline form, and the reaction solution is further concentrated to precipitate D-phenylalanine containing no L-phenylalanine at all. I was able to. Further, the precipitated D-phenylalanine was collected by filtration and purified by a conventional method. as a result,
Purified D-phenylalanine was obtained with a recovery of about 95%.

Example 6 1.0 g of L-phenylalanine was added to 2N sodium hydroxide
Dissolved in 50 ml and heated in a sealed tube at 150 ° C. for 5 hours. After cooling, the solution was neutralized with 2N-hydrochloric acid, and the precipitated crystals were filtered, washed with water, and dried. The resulting crystals are racemized D, L-phenylalanine, the ratio of which is D: L = 4
It was 6:54. Using 920 mg of D, L-phenylalanine obtained here, D-phenylalanine was prepared under the same conditions as in Example 5. As a result, 415 mg of purified D-phenylalanine was obtained. (42% yield from L-phenylalanine)

Example 7 The preparation of D-glutamic acid was examined under the same conditions as in Example 1 using D, L-glutamic acid as a substrate. As a result, 0.1 M L-glutamic acid was almost completely decomposed by L-amino acid oxidase (100 mg) within 24 hours, and
Even in the reaction solution reacted for one day, L-glutamic acid completely disappeared, and only D-glutamic acid remained as an unreacted amino acid.

The residual amount of D-glutamic acid is 100%
And D-glutamic acid was not decomposed at all. In addition, of D-glutamic acid remaining in the reaction solution, about 48 mM
Was already precipitated in a crystalline state, and D-glutamic acid containing no L-glutamic acid could be precipitated by further concentrating the reaction solution. Further, the precipitated D-glutamic acid was collected by filtration and purified by a conventional method. As a result, the purified D was recovered at a recovery rate of about 92%.
-Glutamic acid could be obtained.

Example 8 By heating 3 g of inexpensive L-glutamic acid at 200 ° C. for 4 hours under reduced pressure, 2.25 g of D, L-pyroglutamic acid was obtained. This was hydrolyzed with 2N-hydrochloric acid for 5 hours to obtain completely racemic D, L-glutamic acid.
2.5 g were obtained.

2.25 g of D, L-glutamic acid obtained here
Was used to prepare D-glutamic acid under the same conditions as in Example 7. As a result, 1.03 g of purified D-glutamic acid was obtained. (34% yield from L-glutamic acid)

[0040]

As described above in detail, the method for producing a D-amino acid using an L-amino acid oxidase according to the present invention enables separation and purification from a reaction solution by conversion into a compound having different physical properties. Thus, the objective D-amino acid can be obtained efficiently, so that a practical production method is provided.

[Brief description of the drawings]

FIG. 1 is a view showing an HPLC elution pattern of D, L-leucine in Test Example 1.

FIG. 2 is a view showing an HPLC elution pattern of D, L-isoleucine in Test Example 1.

FIG. 3 is a diagram showing an HPLC elution pattern of D, L-methionine in Test Example 1.

FIG. 4 is a view showing an HPLC elution pattern of D, L-valine in Test Example 1.

FIG. 5 is a diagram showing an HPLC elution pattern of D, L-glutamic acid in Test Example 1.

FIG. 6: HPLC of D, L-phenylalanine in Test Example 1
FIG. 3 is a view showing an elution pattern of the present invention.

FIG. 7 shows the HP of Test Example 2 using D, L-leucine.
It is a figure which shows the elution pattern of LC.

FIG. 8 is a view showing an HPLC elution pattern when D, L-isoleucine of Test Example 2 is used.

FIG. 9 shows H in the case of using D, L-methionine in Test Example 2.
It is a figure which shows the elution pattern of PLC.

FIG. 10 shows the HP in Test Example 2 using D, L-valine.
It is a figure which shows the elution pattern of LC.

FIG. 11 is a view showing an HPLC elution pattern when D, L-phenylalanine of Test Example 2 is used.

FIG. 12 is a view showing an elution pattern of HPLC when D, L-glutamic acid of Test Example 3 was used.

FIG. 13 is a diagram showing the results of Examples 1 to 5.

[Explanation of symbols]

In the figure,-●-indicates the remaining amount of L-amino acid, and-○-indicates the remaining amount of D-amino acid.

Claims (4)

[Claims] (1) A method for producing a D-amino acid, which comprises reacting an enzyme acting preferentially or specifically on an L-amino acid with a D, L-amino acid, and separating the D-amino acid. 2. The enzyme according to claim 1, wherein the enzyme which acts preferentially or specifically on L-amino acids is L-amino acid oxidase.
-A method for producing amino acids. 3. The method for producing a D-amino acid according to claim 1, wherein the D, L-amino acid is produced from the L-amino acid by a racemization reaction. 4. The method according to claim 1, wherein the L-amino acid oxidase is Crotalus adamant.
The method for producing a D-amino acid according to claim 1 or 2, which is derived from eus.