JP2008074729A - Method for producing amino acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing an amino acid, with which various amino acids are produced by the same method only by changing raw materials, further, many kinds of amino acids are produced and a mixed formation ratio of D isomer and L isomer is increased in a yield of only either the D isomer or a the L isomer. <P>SOLUTION: The method for producing an amino acid comprises reacting a compound represented by general formula (1) [X is H or CH<SB>3</SB>, CH<SB>2</SB>OH, CH<SB>2</SB>SH, OCH<SB>3</SB>, SCH<SB>3</SB>, CH(CH<SB>3</SB>)<SB>2</SB>, C(CH<SB>3</SB>)<SB>3</SB>, CH(CH<SB>3</SB>)CH<SB>2</SB>CH<SB>3</SB>, CH<SB>2</SB>CH(CH<SB>3</SB>)<SB>2</SB>, CH<SB>2</SB>CH<SB>2</SB>SCH<SB>3</SB>, CH<SB>2</SB>COOH, CH<SB>2</SB>CH<SB>2</SB>COOH, CH<SB>2</SB>C<SB>6</SB>H<SB>5</SB>, CH<SB>2</SB>C<SB>6</SB>H<SB>4</SB>OH or the like] with hydroxylamine in the presence of a proton donative compound. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

In the present invention, various amino acids can be produced by the same method only by changing the raw materials, and moreover, various types of amino acids can be produced at the same time, and the mixed product ratio of D-form and L-form is only one. The present invention also relates to a method for producing an amino acid capable of improving the production yield.

Conventionally used methods for producing glycine include, for example, the Strecker method and the hydantoin method.
According to the Strecker method, glycinonitrile is synthesized from formaldehyde, hydrocyanic acid and ammonia, and this is hydrolyzed with an alkali such as sodium hydroxide to produce a metal salt of glycine, and then neutralized with an acid such as sulfuric acid. To produce glycine.
According to the hydantoin method, glycolonitrile is synthesized from formaldehyde and hydrocyanic acid, and hydantoin is produced by reacting this with ammonia and carbon dioxide in the presence of water, and then glycine is produced by hydrolysis.
According to these methods, the problem of adversely affecting the environment because a large amount of alkali and acid are used, and the neutralization salt such as sodium sulfate is by-produced by neutralization, so the purity and yield of glycine is reduced. In addition, there is a problem that the treatment of the neutralized salt is costly.

For such a problem, a method for producing glycine from glycinonitrile using a microorganism has been proposed. For example, Patent Document 1 discloses that glycine is produced by allowing a microorganism belonging to the genus Pseudomonas to act on an aqueous solution of glycinonitrile. A method is disclosed.

According to such a method, glycine can be produced with high purity and high yield, but since it cannot be applied to the production method of amino acids other than glycine, it can also be applied to production methods of various amino acids, Moreover, the manufacturing method of the amino acid which can manufacture many types of amino acids simultaneously is calculated | required.
Furthermore, when each amino acid is produced by ordinary chemical synthesis, a racemic amino acid is produced. Therefore, when only one of the optical bodies of D-form and L-form is required, production efficiency can be improved by ordinary chemical synthesis. Unfortunately, there is also a need for an amino acid production method that can efficiently produce only an amino acid of an arbitrary optical body.
JP 2001-340096 A

In view of the above situation, the present invention allows various amino acids to be produced by the same method only by changing the raw materials. Furthermore, the production of many types of amino acids at the same time, the mixed product ratio of D-form and L-form An object of the present invention is to provide a method for producing an amino acid capable of improving the production yield to only one of them.

The present invention is a method for producing an amino acid by reacting a compound represented by the following general formula (1) with hydroxylamine in the presence of a proton-feeding compound.
The present invention is described in detail below.

Ｘは、Ｈ、ＣＨ_３、ＣＨ_２ＯＨ、ＣＨ_２ＳＨ、ＯＣＨ_３、ＳＣＨ_３、ＣＨ（ＣＨ_３）_２、Ｃ（ＣＨ_３）_３、ＣＨ（ＣＨ_３）ＣＨ_２ＣＨ_３、ＣＨ_２ＣＨ（ＣＨ_３）_２、ＣＨ_２ＣＨ_２ＳＣＨ_３、ＣＨ_２ＣＯＯＨ、ＣＨ_２ＣＨ_２ＣＯＯＨ、ＣＨ_２Ｃ_６Ｈ_５、ＣＨ_２Ｃ_６Ｈ_４ＯＨ、下記化学式（２）又は下記化学式（３）を表す。

X is H, CH ₃ , CH ₂ OH, CH ₂ SH, OCH ₃ , SCH ₃ , CH (CH ₃ ) ₂ , C (CH ₃ ) ₃ , CH (CH ₃ ) CH ₂ CH ₃ , CH ₂ CH ( CH ₃ ) ₂ , CH ₂ CH ₂ SCH ₃ , CH ₂ COOH, CH ₂ CH ₂ COOH, CH ₂ C ₆ H ₅ , CH ₂ C ₆ H ₄ OH, the following chemical formula (2) or the following chemical formula (3) .

As a result of intensive studies, the present inventors can produce an amino acid without using a large amount of acid or alkali by reacting a specific compound with hydroxylamine in the presence of a proton-feeding compound. In addition, various amino acids can be produced by the same production method only by changing the kind of the specific compound, and moreover, various kinds of amino acids can be produced at the same time only by using specific reaction conditions. The present inventors have found that the production yield can be improved by making the mixed production ratio of the isomer and the L isomer only one, and the present invention has been completed.

In the present invention, a compound represented by the following general formula (1) and hydroxylamine are used.

X is H, CH ₃ , CH ₂ OH, CH ₂ SH, OCH ₃ , SCH ₃ , CH (CH ₃ ) ₂ , C (CH ₃ ) ₃ , CH (CH ₃ ) CH ₂ CH ₃ , CH ₂ CH ( CH ₃ ) ₂ , CH ₂ CH ₂ SCH ₃ , CH ₂ COOH, CH ₂ CH ₂ COOH, CH ₂ C ₆ H ₅ , CH ₂ C ₆ H ₄ OH, the following chemical formula (2) or the following chemical formula (3) .

Specific examples of the compound represented by the general formula (1) include glyoxylic acid (H, glycine), pyruvic acid (CH ₃ , alanine), β-hydroxypyruvic acid (CH ₂ OH, serine). , Mercaptopyruvic acid (CH ₂ SH, cysteine), 3-methyl-2-oxobutyric acid (CH (CH ₃ ) ₂ , valine), DL-3-methyl-2-oxovaleric acid (CH (CH ₃ ) _CH 2 CH _3, isoleucine), 4-methyl-2-oxo-Barre Rick acid _{(CH 2 CH (CH 3)} 2, leucine), alpha-keto-.gamma.-(methylthio) - Buchirikku acid _{(CH 2} CH ₂ SCH _3, methionine), oxa Le acetic acid _(CH 2 COOH, asparagus Ginsan), alpha-keto glutarate Rick acid _{(CH 2} CH ₂ COOH, glutamic acid), Fe Rupirubin acid _(CH 2 Ph, phenylalanine), 4-hydroxyphenyl pyruvic acid _(CH 2 PhOH, tyrosine), 4-imidazole Pila big acid (Formula (2), histidine), indole-3-pyruvate (the chemical formula ( 3), tryptophan) and the like. In parentheses, the functional group represented by X and the amino acid obtained mainly when each compound is used are shown.
The compound represented by the general formula (1) may be used alone or in combination of two or more.

Hydroxylamine may be used in the form of a hydroxylamine hydrochloride complex.
The addition amount of hydroxylamine is not particularly limited, but a preferable lower limit is 0.9 mol and a preferable upper limit is 1.2 mol with respect to 1 mol of the compound represented by the general formula (1). Since the hydroxylamine and the compound represented by the general formula (1) react in equimolar amounts, exceeding the above range not only wastes raw materials but also increases the risk of contamination.

In the method for producing an amino acid of the present invention, the compound represented by the general formula (1) is reacted with hydroxylamine in the presence of a proton-feeding compound.

The proton supply compound is not particularly limited, and examples thereof include hydrochloric acid, p-trienesulfonic acid, sulfuric acid, trichloroacetic acid and the like. When a hydroxylamine hydrochloride complex is used as hydroxylamine, a proton-feeding compound may be added in consideration of the amount of hydrochloride due to ionization.

The addition amount of the proton-feeding compound is not particularly limited, but a preferable lower limit is 1 mol and a preferable upper limit is 1.5 mol with respect to 1 mol of the compound represented by the general formula (1). Since the proton-providing compound and the compound represented by the general formula (1) react in equimolar amounts, exceeding the above range not only wastes raw materials but also increases the risk of contamination.

In the method for producing an amino acid of the present invention, the compound represented by the general formula (1) and hydroxylamine may be reacted in the presence of a proton-feeding compound using an appropriate solvent.
It does not specifically limit as said solvent, For example, alcohol, such as methanol, ethanol, and propanol, water, an alkaline solvent, etc. are mentioned. Of these, alcohols such as methanol, ethanol and propanol, and protic solvents such as water are preferable. Here, the protic solvent is a solvent that dissociates and readily releases protons.
The solvent may be a fluid such as nitrogen, oxygen, helium, argon, air, etc. that is a gas at normal temperature and pressure.
Moreover, although it does not specifically limit as a reaction container, Since it is easy to enclose gas, an autoclave as shown in FIG. 1 is suitable.

FIG. 1 (a) shows a schematic diagram of a production apparatus that can be used in the method for producing an amino acid of the present invention. FIG. 1 (b) shows a schematic diagram of an autoclave in the production apparatus. In the method for producing an amino acid of the present invention, a proton-feeding compound, a compound represented by the above general formula (1), hydroxylamine, and a solvent are added in the autoclave body vertical mold 2, and the autoclave lid 3 is attached. When fixed to the autoclave main body 2 and using carbon dioxide as a solvent, carbon dioxide gas is sealed from the pressure inlet 5, and when reacting in a supercritical state or subcritical state, at a corresponding temperature for 30 to 120 minutes. The method of heating and making it react is mentioned.
If the sample is added directly to the autoclave and the reaction is performed, iron ions derived from the autoclave are eluted in an acidic state, and the resulting amino acid may change. The reaction may be performed by inserting a cylindrical tube, or the reaction may be performed by inserting a glass-lined tube.

In the method for producing an amino acid of the present invention, the compound represented by the general formula (1) and hydroxylamine are obtained by reacting at a pH of 2 or less in the presence of a proton-feeding compound. The ratio of D form among the amino acids to be obtained can be increased.
This can be confirmed by performing HPLC measurement on the obtained amino acid using, for example, a CROWNPAK chiral column (manufactured by DICEL).

In the method for producing an amino acid of the present invention, in particular, the compound represented by the general formula (1) and hydroxylamine are combined with water in a supercritical state or a subcritical state in the presence of a proton-feeding compound. When reacted in a mixed solvent with carbon, a plurality of types of amino acids can be obtained simultaneously without using a plurality of types of compounds represented by the general formula (1). This can be confirmed by, for example, amino acid analysis using an L-8500 amino acid analyzer (manufactured by Hitachi, Ltd.).
In addition to being inexpensive, water and carbon dioxide are non-toxic, so they do not adversely affect the environment and can reduce the cost of recovery and the like. Carbon dioxide has a critical temperature of 304 K and a critical pressure of 7.39 MPa. Since the critical temperature is relatively low, amino acids are not decomposed by high temperatures. Furthermore, since the critical temperature of water is 647 K and the critical pressure is 22 MPa, hydrolysis of abnormal proteins such as bacteria and prions proceeds in supercritical or subcritical water, and the resulting hydrolyzable compounds such as peptides. It is possible to prevent contamination with these germs and abnormal proteins.

Here, in this specification, the supercritical state means a state higher than a critical pressure (hereinafter also referred to as Pc) and higher than a critical temperature (hereinafter also referred to as Tc). In the case of water, it means a state of 22 MPa or more and 647 K or more, and in the case of carbon dioxide, it means a state of 7.39 MPa or more and 304 K or more.
Further, in this specification, the subcritical state is a state other than the supercritical state, where 0.1 <P / Pc <1.0 and 0.5 <T when the pressure is P and the temperature is T. / Tc, or a temperature and pressure state satisfying 0.1 <P / Pc and 0.5 <T / Tc <1.0. In the case of water, it means a state exceeding 2.2 MPa and less than 22 MPa and exceeding 324 K, or a state exceeding 2.2 MPa and exceeding 324 K and less than 647 K. In the case of carbon dioxide, 0.739 MPa is used. A state exceeding 7.39 MPa and exceeding 152K, or a state exceeding 0.739 MPa and exceeding 152K to less than 304K.

The mixing ratio of water and carbon dioxide in the mixed solvent of water and carbon dioxide in the supercritical state or subcritical state is not particularly limited, but a preferable lower limit of the water ratio is 0.1% by weight. If it is less than 0.1% by weight, the raw material may not easily react. A more preferred lower limit is 1% by weight, and a more preferred upper limit is 10% by weight.

According to the method for producing an amino acid of the present invention, monoamino monocarboxylic acids (neutral amino acids) such as glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine, methionine, phenylalanine, tryptophan, tyrosine, proline, cystine, etc. Monoamino dicarboxylic acids (acidic amino acids) such as glutamic acid, aspartic acid, glutamine and asparagine, and diaminomonocarboxylic acids such as lysine, arginine and histidine can be produced.

According to the present invention, various amino acids can be produced by the same method only by changing the raw materials. Furthermore, the production of many kinds of amino acids at the same time, and the mixed product ratio of D-form and L-form only on one side. It is possible to provide a method for producing an amino acid capable of improving the production yield.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

(Example 1)
An Erlenmeyer flask was charged with 5.52 g (0.065 mol) of pyruvic acid, 4.55 g (0.065 mol) of hydroxylamine hydrochloride, and 20 mL of ultrapure water (Milli-Q: MILLIPORE) and stirred for 5 minutes to be completely uniform and transparent. Then, the acidity was measured with a pH tester (manufactured by HORIBA, pH METER F-21), and pH = −0.20 was presented. Stirring was performed again, and after a while, the temperature rose to 38 ° C., and stirring was continued for 90 minutes, and a white precipitate was formed. After standing overnight, the mixture was stirred for 60 minutes, filtered, washed with ether, and dried in a vacuum dryer at 40 ° C. for 3 hours to obtain crystals.
When the above crystals were subjected to amino acid analysis using an L-8500 amino acid analyzer (manufactured by Hitachi, Ltd.), it was found to contain alanine. Furthermore, when ESI-MS measurement was performed with an LCT mass spectrometer (MICROMASS) and positive ions were measured, a substance having the same molecular weight 89 as alanine was detected. The results are shown in Table 1.

(Example 2)
An Erlenmeyer flask was charged with 5.52 g (0.065 mol) of pyruvic acid, 4.55 g (0.065 mol) of hydroxylamine hydrochloride, and 20 mL of ultrapure water (Milli-Q: MILLIPORE) and stirred for 5 minutes to be completely uniform and transparent. Then, the acidity was measured with a pH tester (manufactured by HORIBA, pH METER F-21), and pH = −0.20 was presented. This solution was transferred to a 50 mL autoclave main body, sealed with a lid, and 20 g of carbon dioxide was sealed from the pressure inlet. The autoclave was maintained in a supercritical state of 8.11 MPa for 2 hours while being heated in a 333 K oil bath. Then, after putting the autoclave in water and cooling to room temperature, carbon dioxide gas was released from the exhaust hole of the autoclave, the lid of the autoclave was opened, and the contents were taken out. The contents were filtered, washed with ether, and dried in a vacuum dryer at 40 ° C. for 3 hours to obtain crystals.
When the above crystals were subjected to amino acid analysis using an L-8500 amino acid analyzer (manufactured by Hitachi, Ltd.), it was found to contain alanine. Furthermore, when ESI-MS measurement was performed with an LCT mass spectrometer (MICROMASS) and positive ions were measured, a substance having the same molecular weight 89 as alanine was detected. The results are shown in Table 2.

(Example 3)
0.025 mol of glyoxylic acid monohydrate and 0.025 mol of hydroxylamine hydrochloride complex were dissolved in 2 mL of ultrapure water (Milli-Q: MILLIPORE), mixed, transferred to a 50 mL autoclave main body, and then covered. Then, 20 g of carbon dioxide was sealed from the pressure inlet. The autoclave was maintained in a supercritical state of 8.11 MPa for 2 hours while being heated in a 333 K oil bath. Then, after putting the autoclave in water and cooling to room temperature, carbon dioxide gas was released from the exhaust hole of the autoclave, the lid of the autoclave was opened, and the contents were taken out.
After the contents were extracted with ether, the solvent was removed using a rotary evaporator (NAJ-100T: manufactured by Tokyo Rika Kikai Co., Ltd.) while heating in a hot water bath at 333 K to obtain crystals. The crystals were transferred to a glass filter and then dried at 313K for 16 hours or more using a vacuum dryer (VO-420: ADVANTEC).
When the dried product was subjected to amino acid analysis using an L-8500 amino acid analyzer (manufactured by Hitachi, Ltd.), it was found that 0.7% by weight contained glycine. Furthermore, when ESI-MS measurement was performed with an LCT mass spectrometer (MICROMASS) and positive ions were measured, a substance having the same molecular weight 75 as glycine was detected.

Example 4
0.05 mol of pyruvic acid and 0.05 mol of hydroxylamine hydrochloride complex are dissolved in 2 mL of ultrapure water (Milli-Q: MILLIPORE), mixed, transferred to a 50 mL autoclave main body, sealed with a lid, and sealed. Carbon dioxide (20 g) was sealed from the inlet. The container was maintained in a supercritical state of 8.11 MPa for 2 hours while being heated in a 333 K oil bath. Then, after putting the autoclave in water and cooling to room temperature, carbon dioxide gas was released from the exhaust hole of the autoclave, the lid of the autoclave was opened, and the solution was taken out.
When the solution was subjected to amino acid analysis after hydrolysis using an L-8500 amino acid analyzer (manufactured by Hitachi, Ltd.), 4.6 mg / mL alanine and 0.023 mg / mL glycine were detected.
When HPLC was performed using a CROWNPAK chiral column (DICEL), a peak having an area of 4194236 μV seconds at a retention time of 1.653 minutes and 1293623 μV seconds at 2.311 minutes was detected. The ratio of these two peaks was 3.710: 1 (standard: D-alanine 1.670 minutes, L-alanine 2.232 minutes).

(Example 5)
Dissolve 0.05 mol of pyruvic acid and 0.05 mol of hydroxylamine hydrochloride complex in 2 mL of ultrapure water (Milli-Q: MILLIPORE), mix, and transfer to a 50 mL autoclave main body with a glass tube tube inserted. After that, it was sealed with a lid, and 20 g of carbon dioxide was sealed from the pressure inlet. The container was maintained in a supercritical state of 8.11 MPa for 2 hours while being heated in a 333 K oil bath. Then, after putting the autoclave in water and cooling to room temperature, carbon dioxide gas was discharged from the exhaust hole of the autoclave, the lid of the autoclave was opened, and the cylindrical tube was taken out. When the fluid in the cylindrical tube was left overnight, crystals precipitated.
The crystals were transferred to a glass filter and then dried at 313K for 4 hours or more using a vacuum dryer (VO-420: ADVANTEC).
The dried product was hydrolyzed and subjected to amino acid analysis using an L-8500 amino acid analyzer (manufactured by Hitachi, Ltd.). As a result, four types of amino acids were detected, alanine was 12.297 ng / mg, and glutamic acid was 11. Results including 051 ng / mg, glycine 7.316 ng / mg and serine 6.852 ng / mg were shown.

(Comparative Example 1)
The reaction was carried out in the same manner as in Example 2 except that 0.065 mol of hydrochloric acid was added instead of using hydroxylamine hydrochloride, but the target amino acid (alanine) was not obtained.

(Comparative Example 2)
The reaction was carried out in the same manner as in Example 2 except that 0.065 mol of hydroxylamine was added instead of using hydroxylamine hydrochloride, but the target amino acid (alanine) was not obtained.

According to the present invention, various amino acids can be produced by the same method only by changing the raw materials. Furthermore, the production of many kinds of amino acids at the same time, and the mixed product ratio of D-form and L-form only on one side. It is possible to provide a method for producing an amino acid capable of improving the production yield.

It is the figure which showed typically an example of the manufacturing apparatus at the time of manufacturing an amino acid in the manufacturing method of the amino acid of this invention.

Explanation of symbols

1 Autoclave 2 Autoclave body vertical 3 Autoclave lid 4 Oil bath 5 Pressure inlet 6 Exhaust hole

Claims (3)

A method for producing an amino acid, comprising reacting a compound represented by the following general formula (1) with hydroxylamine in the presence of a proton-feeding compound.

X is H, CH ₃ , CH ₂ OH, CH ₂ SH, OCH ₃ , SCH ₃ , CH (CH ₃ ) ₂ , C (CH ₃ ) ₃ , CH (CH ₃ ) CH ₂ CH ₃ , CH ₂ CH ( CH ₃ ) ₂ , CH ₂ CH ₂ SCH ₃ , CH ₂ COOH, CH ₂ CH ₂ COOH, CH ₂ C ₆ H ₅ , CH ₂ C ₆ H ₄ OH, the following chemical formula (2) or the following chemical formula (3) .

A method for producing an amino acid, comprising reacting a compound represented by the following general formula (1) with hydroxylamine in the presence of a proton-feeding compound at a pH of 2 or less.

X is H, CH ₃ , CH ₂ OH, CH ₂ SH, OCH ₃ , SCH ₃ , CH (CH ₃ ) ₂ , C (CH ₃ ) ₃ , CH (CH ₃ ) CH ₂ CH ₃ , CH ₂ CH ( CH ₃ ) ₂ , CH ₂ CH ₂ SCH ₃ , CH ₂ COOH, CH ₂ CH ₂ COOH, CH ₂ C ₆ H ₅ , CH ₂ C ₆ H ₄ OH, the following chemical formula (2) or the following chemical formula (3) .

An amino acid characterized in that a compound represented by the following general formula (1) and hydroxylamine are reacted in a mixed solvent of supercritical or subcritical water and carbon dioxide in the presence of a proton-providing compound. Manufacturing method.

X is H, CH ₃ , CH ₂ OH, CH ₂ SH, OCH ₃ , SCH ₃ , CH (CH ₃ ) ₂ , C (CH ₃ ) ₃ , CH (CH ₃ ) CH ₂ CH ₃ , CH ₂ CH ( CH ₃ ) ₂ , CH ₂ CH ₂ SCH ₃ , CH ₂ COOH, CH ₂ CH ₂ COOH, CH ₂ C ₆ H ₅ , CH ₂ C ₆ H ₄ OH, the following chemical formula (2) or the following chemical formula (3) .

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