WO2005080584A1

WO2005080584A1 - PROCESS FOR, WITH USE OF α-AMINO- ϵ -CAPROLACTAM RACEMASE, PRODUCING MIXTURE OF D- AND L-AMINO ACID AMIDE, PRODUCING D- OR L-AMINO ACID, AND PRODUCING D- OR L-AMINO ACID AMIDE

Info

Publication number: WO2005080584A1
Application number: PCT/JP2005/002933
Authority: WO
Inventors: Yasuhisa Asano
Original assignee: Kansai Technology Licensing Organization Co., Ltd.
Priority date: 2004-02-24
Filing date: 2005-02-23
Publication date: 2005-09-01
Also published as: JPWO2005080584A1; JP4122416B2

Abstract

A process comprising causing α-amino-ϵ-caprolactam racemase to act on at least one amino acid amide selected from the group consisting of D-amino acid amides and L-amino acid amides to thereby obtain a mixture of D-amino acid amide and L-amino acid amide with lowered optical activity. There is further provided a process producing a D- or L-amino acid, comprising causing α-amino-ϵ-caprolactam racemase and an enzyme capable of selectively acting on D- or L-amino acid amide to thereby convert the same to a D- or L-amino acid to act on at least one amino acid amide selected from the group consisting of D-amino acid amides and L-amino acid amides. There is still further provided a process for producing a D- or L-amino acid amide, comprising causing α-amino-ϵ-caprolactam racemase and an enzyme capable of selectively acting on D- or L-amino acid amide to thereby convert the same to a D- or L-amino acid to act on a mixture of D-amino acid amide and L-amino acid amide to thereby form a D- or L-amino acid and amidating the formed D- or L-amino acid.

Description

Specification

A method for producing a mixture of D and L-amino acid amides, a method for producing D or L-amino acids, and a method for producing D or L-amino acid amides using α_amino-ε-force prolatatum racemase

Technical field

[0001] The present invention provides a method for producing a mixture of D-amino acid amide and L-amino acid amide with reduced optical activity by using an enzyme that racemizes an optically active amino acid amide, And a method for producing a D- or L-amino acid amide.

Background art

[0002] D-amino acids or D-amino acid oligomers are useful as synthetic materials for pharmaceuticals, agricultural chemicals, and other chemical substances. As a method for producing D-amino acids, there is known a method for producing D-amino acids by allowing a D-stereospecific peptide hydrolase to act on mountain D-amino acid amide. At this time, since the L-amino acid amide does not serve as a substrate for the enzyme, it remains in the reaction system. Here, when the L-amino acid amide is racemized, the D-amino acid amide generated by the racemization is converted into a D-amino acid by an enzyme, and the yield of the D-amino acid can be improved (Patent Document 1). Further, when an L-stereospecific peptide hydrolase is used in place of the D-stereospecific peptide hydrolase, L-amino acids can be obtained with high yield. Therefore, in the production of D- or L-amino acids, enzymes that racemize D- or L-amino acid amides play an important role. However, the optical activity using a racemase derived from Pseudomonas putida (NCIB 40042) and a racemase derived from Rhodococcus sp. (NCIB 12569) described as an enzyme for racemizing a D or L-amino acid amide in Patent Document 1. The production of soluble D or L-amino acids has not been practically used.

[0003] It is known that optical isomers of amino acid amides such as aminobutyric acid amide are useful as pharmaceutical raw materials and the like. As a method for producing an optical isomer of an amino acid amide, a method of optically resolving a racemic form of the amino acid amide is known, but the separation and purification steps of the desired optical isomer are complicated, so that the yield and the yield are low. Improvements are required in terms of cost. Patent Document 1: International Patent Publication WO89 / 01525

Disclosure of the invention

Problems to be solved by the invention

[0004] The present invention provides a more optically active enzyme using an enzyme that acts on D_ amino acid amide or Lama amino acid amide to generate amino acid amide or D_ amino acid amide. For producing a mixture of D-amino acid amide and L-amino acid amide (hereinafter, may be referred to as a D, L-amino acid amide mixture) with reduced D, L-amino acid, and D or L-amino acid It is intended to provide a method for producing an amino acid amide.

Means for solving the problem

[0005] The inventor of the present invention has developed a known racemase capable of racemizing a cyclic amide compound, a racemase force acting on a D-amino acid amide, and converting the cyclic amide compound into an L-amino acid amide. And found to act on L-amino acid amide to convert it to D-amino acid amide, and to complete a method for producing a D, L-amino acid amide mixture, an optically active amino acid or an optically active amino acid amide. Reached.

That is, the present invention provides the following production method.

Item 1. At least one amino acid amide selected from the group consisting of D-amino acid amide and L-amino acid amide is used as a raw material, and α-amino-ε-force prolatatam racemase is allowed to act on the raw material. A method for producing a mixture of D-amino acid amide and L-amino acid amide having reduced optical activity.

Item 2. The method for producing a mixture of amino acid amides according to Item 1, wherein the α_amino-ε-caprolatatam racemase is derived from Achromobacter obae.

Item 3. Amino acid amidoca S, alaninamide, methionamide, leucinamide, serinamide, fenylalaninamide, norinamide, prolinamide, asparagineamide, cysteinamide, cystinamide, toreininamide, 2-- Item 3. The method for producing a mixture of amino acid amides according to Item 1 or 2, wherein the mixture is at least one amide selected from the group consisting of aminobutyric amide, nonolenolinamide, nonolemic isinamide and threoninamide.

Item 4. Amino acid amidoca S, araninamide, methionamide, leucinamide, serinamide, fenylalaninamide, norinamide, 2-aminobutyric acid amide, nonolevalinamide, norguchi Item 13. The method for producing a mixture of amino acid amides according to any one of Items 13 to 13, which is at least one amide selected from the group consisting of amides and threoninamides.

Item 5. Selective action on at least one amino acid amide selected from the group consisting of D-amino acid amide and L-amino acid amide, and on amino-ε-force prolatatum racemase and D or L-amino acid amide A method for producing D or L-amino acid, wherein an enzyme for converting to D or L-amino acid is acted on.

Item 6. The method for producing a D- or L-amino acid according to Item 5, wherein the para-amino-ε-caprolatatam racemase is derived from Achromobacter obae.

Item 7. Amino acid amidoca S, alaninamide, methionamide, leucinamide, serinamide, fenylalaninamide, norinamide, prolinamide, asparaginamide, cysteinamide, cystinamide, tresininamide, 2- Item 7. The method for producing a D- or L-amino acid according to Item 5 or 6, wherein the D- or L-amino acid is at least one amide selected from the group consisting of aminobutyric acid amide, nonolenoline amide, nonoresin amide and threoninamide.

Item 8. Amino acid amide power At least one selected from the group consisting of alaninamide, methionamide, leucinamide, serinamide, phenylalaninamide, nolinamide, 2-aminobutyric acid amide, nonolevalinamide, norguchiininamide and threonamide. Item 8. The method for producing a D- or L-amino acid according to any one of Items 5 to 7, which is an amide.

Item 9. Enzymes that selectively act on D-amino acid amide and L-amino acid amide compound to α-amino-ε-force prolatamase racemase and D or amino acid amide to convert to D or L-amino acid A D- or L-amino acid is produced by reacting the compound, and the resulting D- or L-amino acid is amidated to produce a D- or L-amino acid amide.

Item 10. The method for producing a D- or L-amino acid amide according to Item 9, wherein the para-amino-ε-caprolatatam racemase is derived from Achromobacter obae.

Item 11.Amino acid amidoca S, alaninamide, methionamide, leucinamide, serinamide, fenylalaninamide, norinamide, prolinamide, asparaginamide, cysteinamide, cystinamide, trenininamide, 2-aminobutyramide Item 11. The method for producing a D- or L-amino acid amide according to Item 9 or 10, which is at least one amide selected from the group consisting of nonolenoline amide, nonolenoisinamide and threoninamide. Item 12. Amino acid amide power At least one selected from the group consisting of alaninamide, methionamide, leucinamide, serinamide, fenylalaninamide, norinamide, 2-aminobutyric acid amide, nonolevalinamide, norguchiininamide and threonamide. Item 12. The method for producing a D- or L-amino acid amide according to any one of Items 9-11, which is an amide.

[0007] (1) A method for producing a mixture of D-amino acid amide and L-amino acid amide having lower optical activity than the raw material

The production method of the present invention comprises: using at least one kind of amino acid amide selected from the group consisting of D-amino acid amide and L-amino acid amide as a raw material, and reacting the raw material with amino-ε-caprolactam lacemase; It is characterized by producing a mixture of D-amino acid amide and L-amino acid amide having reduced optical activity from the raw material.

[0008] When ACL racemase is allowed to act on at least one kind of amino acid amide selected from the group consisting of D-amino acid amide and L-amino acid amide, the optics of the amino acid amide present in the reaction system with the passage of time The activity decreases, and finally, a D, L-amino acid amide racemate (a mixture of D-form and L-form at a ratio of 1: 1) is formed. By appropriately setting conditions such as the reaction time, it is possible to produce a D, L-amino acid amide mixture having an arbitrary ratio between the D-form and the L-form.

[0009] Therefore, "a mixture of D-amino acid amide and L-amino acid amide having a lower optical activity than the raw material" refers to an equivalent compound of D-amino acid amide and L_amino acid amide (racemic amino acid amide) A D-amino acid amide, an L-amino acid amide, or a D, L-amino acid amide mixture whose optical activity is closer to the equivalent mixture than the D, L-amino acid amide mixture, which is a raw material, is also included.

[0010] For example, when ACL racemase is acted on a mixture of D-amino acid amide: L-amino acid amide of 90:10, the ratio changes from 90:10 to 50:50 over the reaction time. However, the reaction is stopped halfway, and the D, L-amino acid amide mixture in any ratio between 90:10 and 50:50 (for example, 80:20, 70:30, 60:40, etc.) is used. Obtaining is also included in the production method of the present invention.

As the ACL racemase used in the present invention, for example, an enzyme to which EC 5.1.1.15 is assigned in the enzyme classification can be used. ACL racemase is isolated by culturing Achromobacter obae, Pseudomonas sp. (CCM 3443), etc. Thus, ACL racemase from any of the bacteria can be used in the present invention. The method for obtaining Achromobacter obae (FERM_P776) ACL racemase is described in “SA

Ahmed, et al., Agric. Biol. Chem., 47 (8), 1887-1893 (1983) ".

[0012] Further, the entire gene sequence of Achromobacter obae-derived ACL racemase has been reported. For example, in FIG. 2 of JP-A-63-129984, the entire DNA sequence (SEQ ID NO: 1) and the entire amino acid sequence (sequence) were reported. Number 2) is written.

[0013] Furthermore, a protein (SEQ ID NO: 50) expressing a gene (SEQ ID NO: 49) derived from Sinorhizobium meliloti (Rhizobium meliloti) 1021 having a similar primary amino acid sequence to ACL racemase derived from Achromobacter obae, Pyrococcus iuriosus DSM A protein (SEQ ID NO: 52) expressing a 3638-derived gene (SEQ ID NO: 51), a protein (SEQ ID NO: 54) expressing a Pyrococcus horikoshii 〇T3-derived gene (SEQ ID NO: 53),

Pyrococcus abyssi-derived gene (SEQ ID NO: 55) -expressed protein (SEQ ID NO: 56; Pyndoxal phosphate-dependent aminotransferase) and Thermococcus kodakaraensis-derived gene (SEQ ID NO: 57) expressed protein (SEQ ID NO: 58; aminotransferase, class III) can also be used as an ACL racemase in the production method of the present invention.

[0014] Generally, some naturally occurring proteins have deletions of amino acid residues in the amino acid sequence due to polymorphism or mutation of the gene encoding the protein, or due to a modification reaction after protein production. In some cases, mutations such as addition, substitution, and the like may occur, but nonetheless, there are proteins having the same physiological activity as proteins in which such mutations do not occur. Alternatively, it is also possible to mutate the gene artificially using genetic engineering techniques, in which case the mutated mutation does not substantially change the physiological activity of the protein. It is possible.

[0015] The origin of the protein used as the ACL racemase in the present invention may be a natural protein, a recombinant protein, or a chemically synthesized protein. When a natural protein is desired, the target protein can be expressed using a tissue or cultured cell culture as a starting material, and then used for protein precipitation, affinity chromatography, ion-exchange chromatography, or gel filtration. Of purification It is possible to purify by appropriately combining known methods. For example, when affinity chromatography is used, the target protein can be purified by using a carrier to which an antibody against the protein of the present invention is bound.

When a recombinant protein is desired to be obtained, a recombinant expression vector obtained by cloning the gene of the present invention encoding the target protein into a suitable expression vector as described above is used as a host (E. coli). , Yeast, etc.) and culturing the transformant under suitable conditions to produce the desired protein. In order to isolate the target protein, it is generally preferable to secrete the target protein into the culture supernatant.This can be done by appropriately selecting the combination of the recombinant vector / host and the culture conditions. Can be. Furthermore, those skilled in the art can also appropriately produce a protein having a desired amino acid sequence by chemical synthesis.

[0017] Furthermore, as long as the amino acid amido racemase activity is retained, the above-mentioned protein can be appropriately modified pharmaceutically acceptable. That is, the protein having the amino acid sequence represented by SEQ ID NO: 2, 50, 52 or 54 and the protein containing the partial sequence have the ability to have a carboxyl group (-COOH) or a carboxylate (-COO-) at the C-terminal. The C-terminus may be amide (-CONH) or ester (-COOR). As R of the ester

2

Is a C-alkyl such as methyl, ethyl, n-propyl, isopropyl or n-butyl.

1-6 C cycloalkyl groups such as olequinole group, cyclopentyl, cyclohexyl, phenyl,

3-8

C-aryl groups such as naphthyl, and benzyl, phenethyl, and benzhydryl

6-12

C-alkyl or α-naphthyl-C alkyl such as α-naphthylmethyl

1-2 1-2 The strength of C aralkyl groups such as glue,

7-14

Cimethyl ester and the like. As the salt of the protein of the present invention, a salt with a pharmaceutically acceptable base (eg, an alkali metal or the like) or an acid (organic acid, inorganic acid) is used, and particularly, a pharmaceutically acceptable acid addition salt. Is preferred. Such salts include, for example, salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid) And tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid). Therefore, a protein having an amino acid sequence obtained by mutating the amino acid sequence of SEQ ID NO: 2, 50, 52 or 54 by these natural or artificial mutations can also be used in the production method of the present invention as long as it has amino acid amido racemase activity.

Accordingly, the ACL racemase used in the present invention includes (1) a protein consisting of the amino acid sequence represented by the above SEQ ID NO: 2, 50, 52, 54, 56 or 58; A protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence and having an amino acid amido racemase activity, or (3) 70% or more, preferably 85%, of the amino acid sequence. A protein consisting of an amino acid sequence having a homology of at least 90%, more preferably at least 90%, and having an amino acid amidoracemase activity is also included.

[0019] Accordingly, the ACL racemase used in the present invention can be used without any particular limitation as long as it has the required enzymatic activity. For example, enzymes and microorganism cultures obtained by purifying from microorganism cultures A crude product having the above enzyme activity, an enzyme produced by genetic engineering, and the like can be used. Examples of the microorganism culture include a culture solution and cultured cells, with the cultured cells being preferred. In the case of genetic engineering production, ACL racemase can also be obtained by a method of preparing a peptide by chemical synthesis based on the above amino acid sequence, or a method of producing the above DNA sequence by recombinant DNA technology. For example, when ACL racemase is obtained by recombinant DNA technology, RNA can be expressed in vitro by preparing RNA by in vitro transcription from a vector having this sequence or a fragment thereof and performing in vitro translation using this as a type III. . If the translation region is recombined into an appropriate expression vector by a known method, a large amount of ACL racemase encoded by cDNA can be expressed in Escherichia coli, Bacillus subtilis, yeast, animal cells and the like.

When ACL racemase is expressed in a microorganism such as Escherichia coli, the cDNA of the present invention is added to an expression vector having an origin, a promoter, a ribosome binding site, a cDNA cloning site, a terminator, and the like, which are replicable in the microorganism. A translation expression region is inserted and ligated to create a recombinant expression vector, host cells are transformed with the expression vector, and the resulting transformant is cultured to obtain the ACL racemase encoded by the cDNA. It can be mass-produced in microorganisms. Alternatively, it is developed as a fusion protein with other proteins. It can also be manifested. By cleaving the obtained fusion protein with an appropriate protease, only the protein portion encoded by the cDNA can be obtained.

[0021] In the method for producing a D, L-amino acid amide mixture of the present invention, the raw material includes any one of D-amino acid amide and L-amino acid amide, and is a mixture of D-amino acid amide and L-amino acid amide. May be. However, when the raw material is an equal mixture (racemate) of D-amino acid amide and L-amino acid amide, the content of D-amino acid amide and L-amino acid amide changes even when ACL racemase is applied. Since it is not, it is prayed from raw materials. A preferred D, L-amino acid amide mixture has a molar ratio of D-amino acid amide / (moles of D-amino acid amide + moles of L-amino amide) of 0.001 0.499 and 0.501 0.999, and more preferably , 0.001-0.49 and 0.51 0.999, more preferably 0.001-0.48 and 0.52 0.999. Amino acid amides include amides of known amino acids in which an optically active form exists, such as alaninamide, methionamide, leucinamide, serinamide, phenylalaninamide, quinamide, prolinamide, asparaginamide, and cysteine. Amide, cystinamide, threonamide, isoleucinamide, tryptophanamide, glutamine amide, tyrosine amide, lysinamide, argininamide, histidine amide, aspartic amide, ku, 'noletamic amide, Norele / phosphoramide, noreleucine amide, penicillamineamide, etc., which can be used singly or in combination of two or more, regardless of D-form or L-form. Preferably, alaninamide, methionamide, leucinamide, serinamide, pheninolealanineamide, phosphorus amide, prolinamide, Asha. Laginamide, cystinamide, cystinamide, treninninamide, 2_ amino acid amide, nonole / linamide, noroleucinamide, penicillamineamide, more preferably alaninamide, methionamide, leucinamide, serinamide, Phenylalaninamide, norinamide, 2-aminobutyric acid amide, nonolevulinamide, nonorleucinamide, threonamide, and penicillamineamide, and more preferably, alaninamide, methioninamide, leucinamide, serinamide, and phenyamide. Luraninamide, valinamide, 2-aminobutyric acid amide, threonamide, and penicillamine amide.

[0022] When the raw material is a D, L-amino acid amide mixture, the content ratio of D-form and L-form in the raw material is not particularly limited. The raw material amino acid amide is usually used at a concentration of about 500 g / liter. use. When used at a low concentration, it can be used in the form of a free base, but when used at a relatively high concentration, for example, it can be used in the form of hydrochloride or tosinoleate, etc. Considering the above, it is preferable.

As the reaction medium, water or an aqueous liquid containing acetone, acetonitrile, DMSO, DMF, etc., for example, an aqueous buffer can be used. As the buffer, for example, Tris-HCl buffer, potassium phosphate buffer and the like can be used. In addition, organic solvents that do not mix with water, such as ketones, ethers, hydrocarbons, aromatic olefins, halogenated hydrocarbons, organic acid esters, alcohols, and nitrile, can also be used. For example, methyl butyl ketone, isopropyl ether, petroleum ether, hexane, heptane, cyclohexane, carbon tetrachloride, chloroform, methylene dichloride, trichloroethane, benzene, toluene, xylene, ethyl acetate, butyl acetate, butanol, hexanol , Or Octanol, etc. can be used. Further, a mixture of these organic solvents can be used, and the reaction can be carried out as a two-layer system with a water-saturated organic solvent or an aqueous buffer, or as an emulsion. Preferred are aqueous liquids, and more preferred are aqueous buffers.

[0024] The amount of ACL racemase to be used with respect to the above-mentioned raw materials is not particularly limited, but is about 0.01 to 100 U / ml, preferably 0.05 to 100 U / ml. In this case, one unit of ACL racemase is an amount of an enzyme that catalyzes the production of 1 micromol of amino acid amide per minute. When the ACL racemase is allowed to act, the reaction temperature is usually about 20 to 70 ° C, preferably about 25 to 50 ° C, and the pH is usually about 411, preferably about pH 6 to 10.

[0025] The reaction time can be appropriately set according to the type and amount of the amino acid amide as the raw material, the amount of the enzyme, the reaction temperature, the desired D / L mixing ratio in the product, and the like. It is usually about 0.2 hours to 10 days, preferably about 0.5 hours to 12 days. By increasing the reaction time, a D, L-amino acid mixture close to the racemic amino acid amide can be obtained.

[0026] The mode of production is not particularly limited. For example, the raw material and ACL racemase can be prepared in a batch by charging them in one container, or the ACL racemase can be immobilized on a column, and a solution containing the raw material can be prepared. It can also be produced by passing the solution through the column. After the enzymatic reaction, the produced amino acid can be optionally purified and collected by a conventional method. For example, after the reaction is completed, the cells are centrifuged after adding the protein denaturant to the immobilized enzyme. If using an enzyme fused with His-Tag, etc., use a Ni column or the like to remove it by selective removal, and remove the amino acids contained in the removed solution with a solvent. It is purified by extraction or ion exchange resin and crystallized.

[0027] The embodiment of the enzyme to be used includes a cell culture solution, a culture supernatant solution, a processed product of cells isolated from the culture solution, an enzymatic agent obtained therefrom, and further, these enzymes or enzyme-containing substances. Those immobilized by the method and those having enzyme activity can be used. In industrial practice, it is advantageous to use live cells, immobilized cells and the like. As the immobilization carrier, polyacrylanolamide, photocrosslinkable resin, polyurethane resin, kappa carrageenan, sodium alginate, ion exchange resin, semipermeable membrane, and the like can be used. To immobilize an enzyme, for example, a method in which a carrier absorbs an enzyme solution, a method in which an enzyme solution is mixed with a carrier, and an enzyme is absorbed and fixed

Alternatively, a method of entrapping and immobilizing the enzyme, a method of crosslinking the enzyme with a crosslinking agent, and the like can be employed.

[0028] In this manner, a desired D-mount-amino acid amide mixture can be obtained. The resulting D, L-amino acid amide mixture is lower in optical activity than the starting material amino acid amide. The type of amino acid amide of the D, L-amino acid amide mixture to be produced is the same as the amino acid amide of the above-mentioned raw material. The D, L-amino acid amide mixture to be produced may have a D-form / L-form content ratio of 1: 1.

[0029] The obtained D, L-amino acid amide mixture can also be used as a raw material for producing D or L-amino acids. For example, the production of D or L-amino acid by a method in which an enzyme that converts D or L-amino acid amide to a D or L-amino acid by acting stereospecifically on the D-amino acid amide mixture is allowed to act. Can be.

(2) Method for producing D or L-amino acid

The method for producing a D- or L-amino acid of the present invention comprises the steps of: adding at least one amino acid amide selected from the group consisting of D-amino acid amide and L-amino acid amidoca; An enzyme that selectively acts on L-amino acid amide and converts it into D or L-amino acid (hereinafter sometimes referred to as an enzyme that converts into amino acid).

When a D-amino acid is obtained by this production method, D-amino acid amide, L-amino acid amide or An enzyme that selectively acts on α-amino-ε-force prolatatum racemase and D-amino acid amide to convert the D-amino acid amide and L-amino acid amide compound into D-amino acid . The D-amino acid amide in the reaction system is converted to D-amino acid by an enzyme that converts to D-amino acid, and the L-amino acid amide in the reaction system is converted to D-amino acid amide by ACL racemase. -Amino acid amide is converted to D-amino acid by an enzyme that converts to D-amino acid to produce D-amino acid.

When an L-amino acid is obtained by this production method, D-amino acid amide, L-amino acid amide, or a mixture of D-amino acid amide and L-amino acid amide, ACL racemase and L-amino acid amide are used. Enzymes that selectively act to convert to L-amino acids. The L-amino acid amide in the reaction system is converted to an L-amino acid by an enzyme that converts to an L-amino acid, and the D-amino acid amide in the reaction system is converted to an L-amino acid amide by an ACL racemase. Amides are converted to L-amino acids by enzymes that convert L-amino acids to produce L-amino acids.

[0033] In this production method, the amount of amino acid amide that serves as a substrate for an enzyme that converts to D or L-amino acid is reduced, but the amino acid amide that is not a substrate for an enzyme that converts to D or L-amino acid is an ACL. It is converted by the action of racemase to produce an amino acid amide that serves as a substrate for the enzyme that converts to D or L-amino acids. Enzyme that converts this amino acid amide into D- or L-amino acid.To convert it into D- or L-amino acid, it converts not only amino acid amide that is a substrate of enzyme that converts into D or L-amino acid, but also D or L-amino acid. Amino acid amides, which do not serve as substrates for the enzyme, can also be effectively used as raw materials.

[0034] In the present production method, a mixture of D-amino acid amide, L-amino acid amide, D-amino acid amide and amino acid amide (D, L-amino acid amide) is used as a raw material. The embodiment is the same as the above (1) except for the following points. The mixture of D-amino acid amide and L-amino acid amide (D, L-amino acid amide) differs from the case of (1) above in that an equal mixture of D-amino acid amide and L-amino acid amide (racemic) In this production method, the D, L-amino acid amide mixture also includes an equal mixture (racemic) of D-amino acid amide and L-amino acid amide. Further, the embodiment of the ACL racemase used in the present production method is the same as in the case of the above (1). [0035] Enzymes that selectively act on D or L-amino acid amides to convert to D or L-amino acids include enzymes that selectively act on D_ amino acid amides to generate D-amino acids, and L-amino acid amides. Any enzyme capable of selectively acting to produce an L-amino acid can be used without particular limitation.

[0036] Enzymes that selectively act on D or L-amino acid amides to convert them into D or L-amino acids are usually given names such as amino acid amidase, aminopeptidase, and protease. For example, enzymes called D-aminopeptidase, alkaline D-peptidase, D-alanine amidase, L-aminopeptidase and the like can be mentioned. Moreover, what is generally called an amidase, a peptidase, a protease, a proteinase, a stereospecific amidase, or an enantiomer-specific amidase may be used. These enzymes can be used without particular limitation as long as they have the required enzyme activity.For example, enzymes obtained by purifying from a microorganism culture, crudely purified products having the enzymatic activity of the microorganism culture, and genes can be used. Engineered enzymes can be used. Here, the genetic engineering production method is the same as the above-described ACL racemase genetic engineering production method.

[0037] More specifically, L-amino acid amidase, D-aminopeptidase, L-aminopeptidase, (S) _ and (R) _enantiomer-specific amidases derived from Comamonas acidovorans KPO_2771_4 (Hayashi, Τ · Et al., J. Ferment. Bioeng., 83 (1997), 139-145),

(S) _stereospecific amidase from Pseudomonas putida ATCC 12633 (Hermes, HFM et al., Appl. Environ. Microbiol., 59 (1993), 4330-4334), (S) _stereospecific from Ochrobactrum anthropi NCIMB 40321 Amidase (van den Tweel et al., Appl. Microbiol. Biotechnol., 39 (1993), 296-300), (S) _stereospecific amino acid amidase from Mycobacterium neoaurum ATCC 25795 (Hermes et al., Appl. Environ.

Microbiol., 60 (1994), 153-159), (S) -constituent specific amino acid amidase from Pseudomonas azotoformans IAM 1603 (Hidenobu Yoneda et al. Perazinecarboxamide hydrolase, Japanese Society for Agricultural Chemistry 2002 Annual Meeting, March 26, 2002 (Sendai City)), (R) -Conformational amino acid amidase from Ochrobactrum anthropi C1-38 (Asano et al., J. Biol. Chem.) ., 264 (1989), 14233-14239 and Asano et al., Biochemistry, 31 (1992), 2316-2328), Ochrobactrum anthropi SV3 (Komeda et al., Eur. J. Biochem., 267 (2000), 2028-2035) (R) _Stereospecific amino Acid amidase, (R) _stereospecific amino acid amidase from Arthrobacter sp. NJ-26 (Ozaki et al., Biosci. Biotech. Biochem "56 (1992), 1980-1984), (R) from Brevibacillus borstelensis BCS-1 _Stereospecific amino acid amidase (Baek et al., Appl.

Environ. Microbiol, 69 (2003), 980-986), (R) _stereospecific amino acid amidase from Pseudomonas sp. (Hidenobu Yoneda et al., (R) _body specific piperazine from Pseudomonas sp. -Butylcarboxamide hydrolase, Japanese Society for Biotechnology, 2002 Annual Meeting, October 29, 2002 (Osaka City), (R) _stereospecific amino acid amidase from Pseudomonas aeruginosa PAOl (Hidenobu Yoneda et al., From Pseudomonas aeruginosa PAOl) The properties of the amidase homologous protein PA3598, the Japanese Society of Agricultural Chemistry 2003 Annual Meeting, 2003.4.1 (Fujisawa II)) can be mentioned.

[0038] When the raw material is a D, L-amino acid amide mixture, the content ratio of D-form and L-form in the raw material is not particularly limited. The amount of ACL racemase used for the above raw materials is about 0.01-lOOOU / ml, preferably 0.5-lOOU / ml, and the amount of enzyme used to convert amino acids is 0.01-1000U / ml, preferably 0.5-100U / ml. It is. One unit of the enzyme to be converted into an amino acid is the amount of the enzyme that catalyzes the hydrolysis of 1 micromol of the amino acid amide per minute. When the above-mentioned ACL racemase and amino acid amidase are allowed to act, the reaction temperature is usually about 20-70 ° C, preferably about 25-50 ° C, and the pH is usually about 411, preferably about 610. is there.

The reaction time can be appropriately set according to the kind and amount of the amino acid amide as the raw material, the amount of the enzyme, the reaction temperature, the desired D and L mixing ratio in the product, and the like. It is usually about 0.2 hours to 10 days, preferably about 0.5 hours to 12 days.

[0039] The mode of production is not particularly limited. For example, a raw material, an ACL racemase, and an enzyme to be converted into an amino acid can be prepared in a single container by charging them in a single container, or the ACL racemase can be immobilized on a column. It can also be produced by passing a solution containing the raw materials through the column, and then reacting the solution with an enzyme that converts the solution into an amino acid. After the enzymatic reaction, the generated amino acids can be purified in the same manner as in (1). The form of the enzyme used in the reaction is the same as in (1).

[0040] The D- or L-amino acid thus produced is a known D- or L-amino acid having an optically active substance. Amino acids are included, e.g., alanine, methionine, leucine, serine, phenylalanine, valine, proline, asparagine, cystine, cystine, threonine, isoleucine, tryptophan, glutamine, tyrosine, lysine, arginine, histidine, aspartate. Gnoretamic acid, 2-aminobutyric acid, norparin, norleucine, penicillamine, etc., which can be used alone or in combination of two or more. Preferably, alanine, methionine, leucine, serine, pheninolealanine, valine, proline, asparagine, cystine, cystine, threonine, 2-aminobutyric acid, norpaline, norleucine, penicillamine, more preferably alanine, methionine, Leucine, serine, phenylalanine, valine, 2-aminobutyric acid, norparin, nonoleucine, threonine, penicillamine. More preferably, they are alanine, methionine, leucine, serine, phenylalanine, valine, 2-aminobutyric acid, threonine and penicillamine.

(3) Method for producing D or L-amino acid amide

The method for producing D- or L-amino acid amide of the present invention selectively acts on a mixture of D-amino acid amide and L-amino acid amide, α-amino-ε-force prolatatam racemase and D or L-amino acid amide. And then reacting with an enzyme that converts to D or L-amino acid to produce D or L-amino acid, and amidating the generated D or L-amino acid.

[0042] As a method for separating D or L-amino acid amide from a mixture of D, L-amino acid amides, a method of generating a diastereomer by using an optically active base and separating it by a difference in physicochemical properties, A method of obtaining an optically active substance by stereoselective hydrolysis using an enzyme is known. However, the former method is difficult to separate, and the latter method is not practical because the yield is low. However, in this production method, a mixture of D, L-amino acid amide is made to act as an enzyme for converting ACL racemase and amino acid, and D or L-amino acid is separated from the reaction solution. Up to this point, it can be performed in the same manner as (2). The D or L-amino acid amide can be separated by applying a known amino acid amidating method to the obtained D or L-amino acid.

As a method for amidating D- or L-amino acids, conventionally known methods can be widely used. For example, there is a method in which an amino acid is converted into an amino acid ester, and ammonia gas is blown.

Brief Description of Drawings FIG. 1 is a graph showing racemization of L-2-aminobutyric acid amide by ACL racemase in Example 2.

FIG. 2 is a daraf showing racemization of L-alaninamide by ACL racemase in Example 2.

FIG. 3 is a graph showing racemization of D-phenylalaninamide in Example 3.

FIG. 4 is a graph showing racemization of L-phenylalaninamide in Example 3.

FIG. 5 is a daraf showing the synthesis of D-alanine from L-alananamide in Example 4.

BEST MODE FOR CARRYING OUT THE INVENTION

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

Example

[0046] Material

Here, the following materials were used.

Recombinant plasmid host: E. coli JM109

Cloning vector: pUC18 (Takara Shuzo)

Culture medium: Calo LB medium (lOg / 1 Bacto tryptone) supplemented with ampicillin, 5 g / l Bacto enzyme extract, lOg / 1 NaCl, 80 μg / ml ampicillin, pH 7.2)

Oligonucleotide: manufactured by Hokkaido System Science

DNA polymerase: Takara Ex Taq DNA polymerase (Takara Shuzo)

Restriction enzymes: EcoRI (New England Biolabs), Sphl (Toyobo)

Phosphatase: Shrimp alkaline phosphatase (Boehringer Mannheim) Religion kit ver. 2 (Takara Shuzo)

Crown Pack CR (+) Column (Daicel Chemical)

DEAE-Toyopearl 650M and Butyle-Toyopearl 650M (Toso One)

Superdex 200 HR 10/30 and Mono Q HR 5/5 (Pharmacia)

Bacto enzyme extract and Bacto tryptone (Difco)

D, L_a-Amino-ε-caprolatatam racemase (D, L_ACL) (Tokyo Kasei)

LA then L: Brenner method (M. Brenner, HR Rickenbacher, Helv. And him. Acta. 41, (1958) 181-188), D, L-ACL was prepared by optical resolution using (S) _ (-) _ L_pyrrolidonecarboxylic acid.

Production Example 1

1. Primer

Forty-six primers were designed to include the amino acid sequence of the ACL racemase shown in SEQ ID NO: 2. These 46 primers were also referred to as A1 primers and # 46 primers, and their nucleotide sequences are shown in SEQ ID NO: 348. The design method was designed so that each primer was about 50 mer, of which 20 mer overlapped (the 5 'side of each primer was G, C). The A1 primer was provided with a start codon (atg), a stop codon (tag), a ribosome binding site (aggagg), and an EcoRI recognition site (gaattc). The A45 primer was provided with a stop codon (taa), and the A46 primer was provided with a Sphl recognition site (gcatgc).

[0048] 2. PCR

PCR was performed using these primers. Each primer (46 pieces) was adjusted to a concentration of 100 pmol // l, and 1 μl of each primer solution was mixed to obtain a mix primer. Mix primer 0.5 μ1, 0.5 μ1 Takara Ex Taq polymerase, TAKARA Ex Taq Buffer, and

PCR was performed using 5 μl of 2.5 mM dNTP to make the total amount 50 μl. The PCR conditions were one set of 30 s at 94 ° C, 30 s at 52 ° C, and 30 s at 72 ° C, and this was repeated 55 times.

[0049] The total volume of the PCR reaction solution was 1.3 μl, 0.5 μl of Ex Taq polymerase, Ex Taq buffer, 5 μl of 2.5 mM dNTP, 1 μl of Al primer solution, and 1 μl of A46 primer solution. μ1, and PCR was performed. The PCR conditions were one set of 30 s at 94 ° C, 30 s at 52 ° C, and 60 s at 72 ° C, and this was repeated 23 times. In addition, PCR was performed using PTC-200 (MJ Research).

[0050] 3. Cloning

The DNA amplified by PCR was gel-extracted around 1400 bp, and the fragment obtained by EcoRI-Sphl treatment and alkali treatment was inserted downstream of the lac promoter in the pUC18 vector, and this was inserted into E. coli JM109. The cells were cultured in an LB medium supplemented with ampicillin for 12 hours at 37 ° C. for transformation. A colony showing ACL activity was selected to obtain a transformed E. coli JM109 / pACL60 showing ACL activity. [0051] 4. Purification of ACL racemase from transformed E. coli

E. coli JM109 / pACL60 was placed in a test tube containing ampicillin-added QLB medium (5 ml), and cultured at 37 ° C for 12 hours. The obtained culture solution (5 ml) was placed in a 2 L flask containing 500 ml of LB medium supplemented with 80 / ig / ml of ampicillin and 0.5 mM of isopropylthio-β-D_galactoside (IPTG). C. The cells were cultured with shaking for 8 hours. After the culture, cells were collected by centrifugation (4 ° C, 8000 g, 5 minutes), and the cells were washed with 0.85% NaCl. Similarly, the transformed Escherichia coli was cultured in 2 L flasks (20 tubes) containing 500 ml of LB medium to obtain a total of 10 L of culture solution.

[0052] Cells were similarly fractionated from 10 L of the culture solution, and a lOOmM ΚΡΒ buffer solution (ρ Η 7.0, containing 0.25M sucrose and 2 µm of pyridoxal phosphate (hereinafter referred to as PLP). Hereinafter, the “buffer” in this Production Example (excluding the buffer used for measuring enzyme activity) means this buffer.), And is suspended in a sonicator for 10 minutes (19 KHz, Insonator model 201M, Kubota Corporation). Company). Thereafter, the suspension was centrifuged (4 ° C, 15000g, 30 minutes) to remove the cells. The solution after removing the cells was heat-treated (60 ° C, 10 minutes), and denatured proteins were removed by centrifugation to obtain a supernatant. The supernatant was applied to a DEAE-Toyopearl 650 M column (3 cm diameter × 25 cm length) equilibrated with 10 mM buffer, and the column was washed with 10 mM buffer. From 0

Elution was carried out with a 10 mM buffer having a concentration gradient of 300 mM NaCl to obtain a fraction.

The ACL racemase active fraction was saturated with ammonium sulfide 30%. This solution was adsorbed to a butyl Toyopearl 650M column (1.5 cm diameter × 13 cm length) equilibrated with a buffer saturated with 30% ammonium sulfide. The column was washed with a buffer saturated with 30% ammonium sulfide, and then eluted with a buffer containing 30% to 0% ammonium sulfide in a concentration gradient. Active fractions were collected and concentrated on Centricon 10. This concentrate was equilibrated with 10 mM buffer containing 150 mM NaCl.

Adsorbed to the top of a Superdex 200 HR 10/30 column. The column was eluted by FPLC (Pharmacia) at a rate of 0.4 ml / min. Active fractions were collected and concentrated with Centricon 10. The concentrate was adsorbed on Mono Q HR5 / 5 equilibrated with lOmM buffer and eluted with 10mM buffer containing 0-300mM NaCl. The fractions containing the activity were collected and concentrated with Centricon 10 to obtain ACL racemase. The ACL-racemer obtained The enzyme was subjected to SDS electrophoresis and confirmed to be single. The molecular weight of this enzyme was 40,000. The ACL racemase activity at each purification stage was confirmed by measuring the ACL racemase activity as follows.

A mixture (2 ml) containing lOOmM potassium phosphate buffer (KPB, pH 7.0), 2 μΜ pyridoxal phosphate (PLP), l-OmM L-ACL and 20 μl of the enzyme activity solution obtained at each step Reaction at 30 ° C for 30 minutes, add 20 μl of 2N HC10 to terminate the reaction, and generate D-ACL

Four

Determine ACL racemase activity based on amount. However, in the measurement of the final fraction that was confirmed to be single by SDS electrophoresis, the amount of enzyme active solution used was 1 μl (total volume of the mixture was 2 ml), and the reaction time was 1 minute. The amount of D-ACL produced was determined by HPLC (0.6 ml / min, HCIO with 60 mM solvent) using a Crownpak CR (+) column. Absorbance of eluate (

Four

200 nM) was measured. D-ACL was racemized and the amount of enzyme that catalyzes the production of 1 μmol L-ACL per minute was defined as 1 unit.

Example 1

L-Aminobutyric acid amide, L-threoninamide, L-alaninamide, L-serinamide, L-norparinamide, L-norleucinamide, L-methioninamide, L-valinamide, L-leucinamide by ACL racemase And racemization of L-phenylalaninamide>

A mixture (2 ml) of 2 i Mi PLP, 100 mM KPB (pH 7.0), 10 mM amino acid amide and 71.2 ng of ACL racemase obtained in Production Example 1 was reacted at 30 ° C. for 30 minutes. HC10 added

4 to stop the reaction, run the reaction mixture on a HPLC equipped with a Crown Pack CR (+) column (solvent: 60 mM HCIO (0.6 ml / min)), and determine the amount of D-amino acid amide in the reaction system at 200 nm absorbance. To

Four

The specific activity of ACL racemase for L-amino acid amide was calculated. The results are shown in Table 1. The activity of ACL racemase for L-ACL was 350 U / mg. From Table 1, it was confirmed that ACL racemase generated L-amino acid amide to D-amino acid amide and racemized.

[0055] When the amino acid amide was replaced with the following amino acid D-form or L-form and reacted with ACL racemase to quantify the product, it was confirmed that ACL racemase did not racemize the following amino acid. did. Amino acids used: alanine, palin, serine, methionine, leucine, hue D- and L-forms of dilualanine

[Table 1]

Example 2

Racemization of L-2-aminobutyric acid amide and L-arananamide by ACL racemase>

A mixture (2 ml) of 20 μM PLP, 100 mM KPB (pH 7.0), 40 mM L_2_aminobutyramide and 71.2 ng of ACL racemase obtained in Production Example 1 was reacted at 30 ° C. for 120 minutes, The abundance ratio of the D-form and the L-form of the amino acid amide in the reaction solution was measured from the absorbance at 200 nm. The measurement was carried out in the same manner except that L_2-aminobutyric acid amide was replaced with L-arananamide. The results are shown in FIG. 1 (aminobutyric acid amide) and FIG. 2 (arananamide). It was confirmed that L-2-aminobutyric acid amide and L-alaninamide were racemized over time.

Example 3

Racemization of D or L-phenylalanine amide and production of optically active phenylalanine>

Transformed E. coli (E. coli JM109 / pACL60) was placed in a test tube containing 50 ml of LB medium containing 80 _β g / ml of ampicillin, and cultured at 37 ° C for 12 hours. The cells obtained from this culture were suspended in 5 ml of a solution consisting of 0.1 M Tris / HCl (pH 8.0) and 10 mM D-phenylalaninamide and reacted at 30 ° C. for 24 hours. The Escherichia coli body contains an enzyme that converts L-phenylalaninamide to L-phenylalanine (L-phenylalanine amidase), and L-phenylalaninamide in the reaction system is converted to L-phenylalanine. Converted to luranin. The reaction was stopped by adding HCIO, and the reaction solution was mounted on a crown pack CR (+) column.

HPLC (solvent was 60 mM HCIO (0.8 ml / min), and D and L-phenylalaninamide and D and L-phenylalanine were quantified from the absorbance at 200 nm. The results are shown in FIG. Quantification was performed in the same manner by replacing L-phenylalaninamide with L-phenylalaninamide and changing the reaction time to 8 hours, and the results are shown in Fig. 4. In the E. coli body, D-phenylalaninamide was replaced with D-phenylalaninamide. It does not contain the enzyme (D-phenylalanine amidase) that converts to D-phenylamine, and D-phenylalanine amide in the reaction system is not converted to D-phenylalanine.

In FIG. 3, D-phenylalaninamide is reduced, and L-phenylalanineamide and L-phenylalanine, which were not present at the beginning of the reaction, are produced. This indicates that the cells converted D-phenylalaninamide to L-phenylalaninamide and the resulting L-phenylalaninamide was converted to L-phenylalanin by L-amino acid amidase. .

In FIG. 4, L-phenylalaninamide is reduced, and D-phenylalaninamide and L-phenylalanine, which were not present at the beginning of the reaction, are produced. This indicates that the cells converted L-phenylalaninamide to D-phenylalaninamide and the L-amino acid amidase converted L-phenylalaninamide to L-phenylalanine.

Example 4

The D_alanan was synthesized by allowing the ACL racemase obtained in Production Example 1 and the D_aminopeptidase derived from Ochrobactrum anthropi C 1-38 to act on L-arananamide. Mix 2 μΜ of PLP, 100 mM KPB (pH 7.0), 45 mM L-alaninamide, ACL racemase (0.22 U) and D-aminopeptidase (2.2 U) (2 ml) and 7 hours at 30 ° C. Reacted.

The reaction was stopped by adding HC10, and the reaction solution was mounted on a crown pack CR (+) column.

HPLC (solvent: 60mM HCIO (0.4ml / min), D and L-alaninamide and D-alanine were quantified from the absorbance at 200nm. The results are shown in Fig. 5. The D-ano was used here. Funata, ¹ -fa ~, Y. Asano, et al, Biochem. Biophys. Res. Commun., 162, 470-474 (1989).

[0062] In FIG. 5, L-alananamide decreased and D-alanine which was not present at the beginning of the reaction was observed. Amide and D-alanine are produced. This indicates that the cells converted L-alananamide into D-alananamide and the resulting D-alananamide was converted to D-alananamide by D-aminopeptidase.

Industrial applicability

[0063] The method for producing a mixture of (1) D-amino acid amide and L-amino acid amide according to the present invention is a simple method of reacting ACL racemase. In the method for producing (2) D- or L-amino acid of the present invention, the amino acid amide serving as a substrate for an enzyme that converts to D- or L-amino acid is a reducing force. Furthermore, amino acid amide is converted by the action of ACL racemase to produce amino acid amide, which is a substrate for the enzyme that converts to D or L-amino acid. Since the enzyme that converts this amino acid amide to D or L-amino acid converts it to D or L-amino acid, it can be converted to D or L-amino acid without using only the amino acid amide that is the substrate of the enzyme that converts to D or L-amino acid. Since the amino acid amide that does not serve as a substrate for the enzyme to be used can also be effectively used as a raw material, the production method (2) is a simple method with a high yield that does not require optical resolution. Furthermore, the method for producing a D- or L-amino acid amide of (3) of the present invention comprises amidating the optically active amino acid obtained by the method of (2) by a known method. It is a simple and high-yield method that does not require an optical resolution operation as in the case of the production method.

Sequence listing free text

[0064] SEQ ID NOs: 3-48 show the DNA sequences of A1-A46 primers, respectively.

Claims

The scope of the claims

[1] Using at least one amino acid amide selected from the group consisting of D-amino acid amide and L-amino acid amide as a raw material, and reacting the raw material with para-amino-ε-force prolatatum racemase; A method for producing a mixture of D-amino acid amide and L-amino acid amide having reduced optical activity.

[2] The method for producing a mixture of amino acid amides according to claim 1, wherein the para-amino-ε-caprolatatam racemase is derived from Achromobacter obae.

[3] Amino acid amide power alaninamide, methionamide, leucinamide, serinamide, phenylalanamide, valinamide, prolinamide, asparaginamide, cysteinamide, cystinamide, threonamide, 2-aminobutyramide, nonolevalinamide, nonoamide The method for producing a mixture of amino acid amides according to claim 1 or 2, wherein the method is at least one amide selected from the group consisting of releucinamide and threoninamide.

[4] Amino acid amide power At least one amide selected from the group consisting of alaninamide, methionamide, leucinamide, serinamide, phenylalaninamide, valinamide, 2-aminobutyramide, nonolevalinamide, norleucinamide and threonamide. A method for producing a mixture of amino acid amides according to any one of claims 13 to 13.

[5] selectively acts on at least one amino acid amide selected from the group consisting of D-amino acid amide and L-amino acid amide, and on amino-amino-ε-force prolatatum racemase and D or L-amino acid amide A method for producing D- or L-amino acids, wherein the enzyme is converted to D- or L-amino acids.

[6] The method for producing a D- or L-amino acid according to claim 5, wherein the para-amino-ε-caprolatatam racemase is derived from Achromobacter obae.

[7] Amino acid amidocaranamide, methionamide, leucinamide, serinamide, phenylalanamide, valinamide, prolinamide, asparaginamide, cysteinamide, cystinamide, threonamide, 2-aminobutyric acid amide, nonolevalinamide, nonolenin 7. The method for producing a D- or L-amino acid according to claim 5, which is at least one amide selected from the group consisting of leucinamide and threonamide.

[8] Amino acid amide power alaninamide, methionamide, leucinamide, serinamide, fenii The D- or L-amino acid according to any one of claims 5 to 7, which is at least one amide selected from the group consisting of ualaninamide, valinamide, 2-aminobutyric amide, nonolevalinamide, norleucinamide and threoninamide. Manufacturing method.

[9] Selectively reacts with a mixture of D-amino acid amide and L-amino acid amide to para-amino-ε-caprolatatam racemase and D or L-amino acid amide to convert to D or L-amino acid A method for producing a D or L-amino acid amide by reacting an enzyme to produce a D or L-amino acid, and amidating the produced D or L-amino acid.

[10] The method for producing a D- or L-amino acid amide according to claim 9, wherein the para-amino-ε-caprolatatam racemase is derived from Achromobacter obae.

[11] Amino acid amide caralaninamide, methionamide, leucinamide, serinamide, phenylalaninamide, valinamide, prolinamide, asparaginamide, cysteinamide, cystinamide, trenininamide, 2-aminobutyric amide, nonolenolinamide, The method for producing a D- or L-amino acid amide according to claim 9 or 10, wherein the method is at least one amide selected from the group consisting of nonoleucinamide and threoninamide.

[12] Amino acid amide power At least one amide selected from the group consisting of alaninamide, methionamide, leucinamide, serinamide, phenylalaninamide, valinamide, 2-aminobutyramide, nonolevalinamide, norleucinamide and threonamide. A method for producing a D- or L-amino acid amide according to any one of claims 9-11.