JP2007254439A - Method for producing optically active amino acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for purifying a high purity optically active amino acid simply and in a good efficiency from an aqueous optically active amino acid solution containing an ammonium salt. <P>SOLUTION: This method for producing the optically active amino acid is provided by removing ammonia by stripping and then performing crystallization by adding an organic solvent to isolate the optically active amino acid as a crystal. Also, the method for producing the optically active amino acid is provided with that the aqueous solution of the optically active amino acid is produced by an enzyme reaction. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a production method for isolating an optically active amino acid from an aqueous solution containing an ammonium salt with high purity.

  Optically active amino acids are useful compounds as synthetic intermediates for pharmaceuticals and the like. As a method for producing an optically active amino acid, various methods such as an extraction method, a chemical synthesis method, a fermentation method, and an enzymatic synthesis method are known.

  The extraction method requires large-scale purification equipment from protein hydrolysates. Since amino acids produced by a chemical synthesis method are generally racemic, an expensive resolving agent, an asymmetric catalyst, or the like is required to produce an optically active substance. The fermentation method has a low product concentration, requires a large-scale purification facility like the extraction method, and is not suitable for the synthesis of unnatural amino acids. The enzymatic synthesis method can avoid these problems by using an inexpensive biocatalyst, and can provide an efficient production method at a lower cost.

  As an enzymatic synthesis method of L-amino acid, for example, a method of producing L-amino acid by allowing a racemic amino acid to act on D-amino acid oxidase, amino acid dehydrogenase, and an enzyme having a coenzyme regeneration ability (Non-patent Document) 1, Patent Document 1), a method of producing an L-amino acid by reacting a corresponding keto acid with an amino acid dehydrogenase and an enzyme having a coenzyme regeneration ability (Patent Document 2) is known. Moreover, as an enzymatic synthesis method of D-amino acid, for example, a method of producing D-amino acid by reacting a corresponding N-carbamoylamino acid with decarbamoylase (Patent Document 3) is known. Furthermore, methods for producing L-amino acids or D-amino acids by reacting a racemic amino acid amide with an amidase (Patent Documents 4 and 5) are also known.

On the other hand, when an inorganic salt or the like is mixed as an impurity in the optically active amino acid aqueous solution obtained by the above method, it is necessary to remove these impurities in order to obtain a high-quality optically active amino acid. As a method for isolating an amino acid from an aqueous solution containing an inorganic salt or the like, a method for removing an inorganic salt by electrodialysis (Patent Document 6), a method for purifying an amino acid using an ion exchange resin (Patent Documents 7 and 8), After exchanging contaminating cations with hydrogen ions using a dialysis machine, a method of purifying amino acids with a cation exchange resin (Patent Document 9), adding a precipitating agent such as chlorendic acid or the like to form a sparingly soluble adduct with amino acids Methods of forming and collecting (Patent Documents 10 and 11) are known.
International Publication No. WO05 / 090950 Japanese Patent Laid-Open No. 10-23896 Japanese Patent Laid-Open No. 62-25990 JP 59-159789 A Japanese Unexamined Patent Publication No. 63-87998 Japanese Patent Laid-Open No. 58-1000068 Japanese Patent Publication No. 48-2355 Japanese Patent Laid-Open No. 56-39792 JP-A-62-195351 Japanese Patent Publication No. 45-1056 Japanese Patent Publication No. 45-29168 Nobuyoshi Nakajima et al., “Enzymatic conversion of racemic methionine to the L-enantiomer”, Journal of the Chemical Society Chemical Communications. ), 1990, No. 13, p. 947

  However, in the method for isolating amino acids according to the methods described in Patent Documents 6 to 11, a large-scale or expensive apparatus or a step of recovering free amino acids by re-decomposing a hardly soluble adduct is necessary, and an extra step This is industrially unfavorable in that it requires equipment and facilities.

  The objective of this invention is providing the manufacturing method which isolates an optically active amino acid simply and efficiently from the optically active amino acid aqueous solution containing an ammonium salt.

  As a result of diligent research in view of the above problems, the inventors of the present invention added a base that forms an easily soluble salt in an organic solvent, such as lithium hydroxide, to an aqueous optically active amino acid solution containing an ammonium salt to control the pH. It was found that the optically active amino acid can be efficiently isolated as a high-purity crystal by removing ammonia by stripping under reduced pressure, and then adding and crystallization with an organic solvent. The stripping referred to here means an operation of transferring a gas present in the liquid into the gas phase.

In addition, the present inventors have found that the present purification method is effective, for example, in the step of isolating an optically active amino acid from an optically active amino acid aqueous solution produced by the following enzyme reactions (i) to (iv): I found.
(I) a reaction (steric inversion reaction) that converts a racemic amino acid into an L-amino acid by allowing D-amino acid oxidase, amino acid dehydrogenase, and an enzyme having a coenzyme regeneration ability to act on the racemic amino acid;
(Ii) a reaction (reductive amination reaction) that converts an amino acid dehydrogenase and an enzyme having a coenzyme regeneration ability into an L-amino acid by acting on the corresponding keto acid;
(Iii) Reaction to convert D-amino acid by allowing decarbamoylase to act on the corresponding N-carbamoylamino acid (decarbamoylase reaction, and (iv) causing amidase to act on racemic amino acid amide) To convert to L-amino acid or D-amino acid by amidase reaction (amidase reaction).

  In addition, the present inventors use an acid that forms a salt that is easily soluble in an organic solvent, such as formic acid or hydrochloric acid, as an acid for adjusting the pH in the enzyme reaction, so that the optical reaction can be performed with high yield and high purity. It has been found that active amino acids can be isolated and produced. Furthermore, in the above steric inversion reaction, it was found that by using formic acid as an acid for adding ammonia or ammonium formate or controlling pH, the reaction yield can be improved and the reaction time can be shortened. It came to do.

The present invention has one or more of the following features.
1) One feature of the present invention is that, after removing ammonia from an aqueous solution containing an optically active amino acid and an ammonium salt by stripping, the crystal of the optically active amino acid is isolated by crystallization by adding an organic solvent. This is a method for producing an optically active amino acid.
2) One feature of the present invention is to control the pH during the stripping by using a base that forms a salt that is easily soluble in the organic solvent.
4) One feature of the present invention is that an aqueous solution containing the optically active amino acid and the ammonium salt is produced by an enzymatic reaction.
6) One feature of the present invention is that an aqueous solution containing the L-amino acid and an ammonium salt acts on a racemic amino acid with a D-amino acid oxidase, an amino acid dehydrogenase, and an enzyme having coenzyme regeneration ability. It is produced by an enzyme reaction.
7) One feature of the present invention is that, in the enzyme reaction, ammonia or a salt thereof is added in an amount of 0.3 equivalent or more with respect to the racemic amino acid.
9) One feature of the present invention is that an aqueous solution containing the L-amino acid and an ammonium salt is subjected to an enzymatic reaction in which an amino acid dehydrogenase and an enzyme having a coenzyme regeneration ability are allowed to act on the corresponding keto acid. Is to manufacture.
10) One feature of the present invention is that an aqueous solution containing the L-amino acid and an ammonium salt is produced by an enzyme reaction in which an amidase is allowed to act on a racemic amino acid amide.
12) One feature of the present invention is that an aqueous solution containing the D-amino acid and an ammonium salt is produced by an enzymatic reaction in which a decarbamoylase is allowed to act on an N-carbamoylamino acid corresponding to the D-amino acid. That is.
13) One feature of the present invention is that an aqueous solution containing the D-amino acid and an ammonium salt is produced by an enzyme reaction in which an amidase is allowed to act on a racemic amino acid amide.
14) One feature of the present invention is that an acid that forms a readily soluble salt in the organic solvent is used for pH control of the enzyme reaction.
17) One feature of the present invention is that, in a reaction in which a racemic amino acid is reacted with D-amino acid oxidase, amino acid dehydrogenase, and an enzyme having a coenzyme regeneration ability to convert to L-amino acid, ammonia or a salt thereof is used. The method for producing an L-amino acid, comprising adding 0.3 equivalent or more to the racemic amino acid.
19) One feature of the present invention is that a transformant capable of producing each enzyme is used instead of each enzyme used in the enzyme reaction.
20) One feature of the present invention is a method for purifying an optically active amino acid, wherein the optically active amino acid is purified by a crystallization step in which an organic solvent is added.
(I) a step of adding a base for replacing the ammonium salt with a salt having high solubility in the organic solvent to a solution containing an optically active amino acid and an ammonium salt;
(Ii) a step of removing ammonia obtained by the substitution into the gas phase by stripping from the solution after step (i) and removing it from the solution;
(Iii) Each step of adding the organic solvent to the solution after step (ii) to dissolve the salt obtained by the substitution in the organic solvent while crystallizing the optically active amino acid A method for purifying an optically active amino acid, comprising:

  Other features of the present invention and their advantages will be apparent from the specification including the following embodiments.

  This invention consists of the above-mentioned structure, and can refine | purify a highly pure optically active amino acid simply and efficiently from the optically active amino acid aqueous solution containing an ammonium salt.

  Hereinafter, the present invention will be described in detail based on embodiments. The scope of the present invention is not limited by the embodiments and examples.

1. Preparation of Optically Active Amino Acid Aqueous Solution Containing Ammonium Salt The optically active amino acid production method as an embodiment of the present invention can be applied by any method as long as it is an optically active amino acid aqueous solution containing an ammonium salt. . For example, (i) an enzyme reaction (stereoinversion reaction) in which a racemic amino acid is converted to an L-amino acid by the action of a D-amino acid oxidase, an amino acid dehydrogenase, and an enzyme having a coenzyme regeneration ability, (ii) Since an enzyme reaction (reductive amination reaction) in which an amino acid dehydrogenase and an enzyme having a coenzyme regeneration ability are allowed to act on keto acid to convert to an L-amino acid is usually performed by adding an ammonium salt, the embodiment Thus, a highly pure L-amino acid can be produced efficiently.

  Examples of the optically active amino acid that can be purified according to the embodiment include the following general formula (1).

（式中、Ｒは置換基を有していてもよい炭素数１〜２０のアルキル基、置換基を有していてもよい炭素数７〜２０のアラルキル基、もしくは置換基を有していてもよい炭素数６〜２０のアリール基を表す。）で表される光学活性アミノ酸を挙げることができる。上記Ｒにおける置換基を有していてもよい炭素数１〜２０のアルキル基としては、特に限定されず、例えば、メチル基、イソプロピル基、イソブチル基、１−メチルプロピル基、カルバモイルメチル基、２−カルバモイルエチル基、ヒドロキシメチル基、１−ヒドロキシエチル基、メルカプトメチル基、２−メチルチオエチル基、（１−メルカプト−１−メチル）エチル基、４−アミノブチル基、３−グアニジノプロピル基、４（５）−イミダゾールメチル基、エチル基、ｎ−プロピル基、ｎ−ブチル基、ｔ−ブチル基、クロロメチル基、メトキシメチル基、２−ヒドロキシエチル基、３−アミノプロピル基、２−シアノエチル基、３−シアノプロピル基、４−（ベンゾイルアミノ）ブチル基、又は、２−メトキシカルボニルエチル基などが挙げられる。

(In the formula, R has an optionally substituted alkyl group having 1 to 20 carbon atoms, an optionally substituted aralkyl group having 7 to 20 carbon atoms, or a substituent. And an optically active amino acid represented by the above-mentioned aryl group having 6 to 20 carbon atoms. The alkyl group having 1 to 20 carbon atoms which may have a substituent in R is not particularly limited, and examples thereof include a methyl group, an isopropyl group, an isobutyl group, a 1-methylpropyl group, a carbamoylmethyl group, and 2 -Carbamoylethyl group, hydroxymethyl group, 1-hydroxyethyl group, mercaptomethyl group, 2-methylthioethyl group, (1-mercapto-1-methyl) ethyl group, 4-aminobutyl group, 3-guanidinopropyl group, 4 (5) -Imidazolemethyl group, ethyl group, n-propyl group, n-butyl group, t-butyl group, chloromethyl group, methoxymethyl group, 2-hydroxyethyl group, 3-aminopropyl group, 2-cyanoethyl group 3-cyanopropyl group, 4- (benzoylamino) butyl group, 2-methoxycarbonylethyl group, etc. It is below.

  The aralkyl group having 7 to 20 carbon atoms which may have a substituent is not particularly limited, and examples thereof include a benzyl group, an indolylmethyl group, a 4-hydroxybenzyl group, a 2-fluorobenzyl group, and a 3-fluoro group. Examples include a benzyl group, 4-fluorobenzyl group, 3,4-methylenedioxybenzyl group, and the like. Examples of the aryl group having 6 to 20 carbon atoms which may have a substituent include a phenyl group and a 4-hydroxyphenyl group. Examples of the substituent include an amino group, hydroxyl group, nitro group, cyano group, carboxyl group, alkyl group, aralkyl group, aryl group, alkanoyl group, alkenyl group, alkynyl group, alkoxyl group, or halogen atom.

  In addition, the production method of the embodiment has a relatively high solubility in water, and it is difficult to separate a target product (optically active amino acid) and impurities (such as inorganic salts) by crystallization, or an optically active amino acid having low efficiency. It is particularly effective.

The optically active amino acid having high solubility in water is not particularly limited. For example, the substituent R in the formula (1) is a methyl group, isopropyl group, isobutyl group, 1-methylpropyl group, carbamoylmethyl group, 2-carbamoylethyl group. , Hydroxymethyl group, 1-hydroxyethyl group, 2-methylthioethyl group, (1-mercapto-1-methyl) ethyl group, 4-aminobutyl group, 3-guanidinopropyl group, ethyl group, n-propyl group, n The thing which is -butyl group and t-butyl group etc. can be mentioned.

2. Enzyme used for steric inversion reaction As a method for producing an optically active amino acid aqueous solution containing the above ammonium salt, steric inversion reaction will be described as an example. However, the stereoinversion reaction illustrated below is an exemplification, and other stereoinversion reactions well-known to those skilled in the art, and (ii) reductive amination reaction, (iii) decarbamoylase reaction, (iv) The optically active amino acid production method of the present invention can also be used for an optically active amino acid aqueous solution containing the above ammonium salt by amidase reaction.

  First, the enzyme used for the steric inversion reaction will be described. D-amino acid oxidase is an enzyme that oxidizes D-amino acid to produce 2-oxoacid, hydrogen peroxide, and ammonia in the presence of oxygen. An amino acid dehydrogenase is an enzyme having the activity of reductively aminating 2-oxoacid or cyclic imine, such as phenylalanine dehydrogenase, leucine dehydrogenase, pyrroline-2-carboxylate reductase, etc. Can be mentioned. The reaction by these amino acid dehydrogenases requires a reduced coenzyme such as NADH, and the coenzyme NADH is converted to an oxidized form as the reaction proceeds.

  In a preferred embodiment, an enzyme having the ability to convert this oxidized coenzyme into a reduced form (hereinafter referred to as coenzyme regeneration ability) and a compound serving as a substrate for the enzyme having the coenzyme regeneration ability are: -The amount of coenzyme used can be greatly reduced by carrying out the reaction in the presence of amino acid oxidase and amino acid dehydrogenase. Examples of the enzyme having coenzyme regeneration ability include hydrogenase, formate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, glucose-6-phosphate dehydrogenase, or glucose dehydrogenase. . Preferably, formate dehydrogenase is used.

  As the enzyme having D-amino acid oxidase, amino acid dehydrogenase, and coenzyme regeneration ability used in the embodiment, those derived from animals, plants, and microorganisms can be used, but those derived from microorganisms are preferred for industrial use. .

2-1. Examples of microorganisms having the ability to produce D -amino acid oxidase D-amino acid oxidase include the following known microorganisms having the ability to produce the enzyme, Arthrobacter, Aspergillus, and Candida. Genus Candida, Cryptococcus, Curvularia, Exophiala, Fusarium, Gibberella, Hansenula, Kloeckera, Kloeckera (Kluyveromyces), Neurospora, Pichia, Rhodosporidium, Rhodotorula, Sporobolomyces, Trigonopsis, Verticillium Verticillium), Yarrowia, etc. It is. Preferred examples include enzymes derived from microorganisms belonging to the genus Candida. Among them, Candida intermedia is preferable, and Candida intermedia NBRC0761 strain is more preferable. The above-mentioned Candida intermedia NBRC0761 strain can be obtained from the Biological Resource Department of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818).

2-2. The microorganism having a production capacity of an amino acid dehydrogenase amino acid dehydrogenase, e.g., a microorganism having the following capacity of the known the enzyme, Brevibacterium (Brevibacterium), the genus Rhodococcus (Rhodococcus), Sporosarcina (Sporosarcina), Thermoactinomyces, Thermobacterium, Microbacterium, Halomona, Clostridium, Bacillus, Neurospora, Escherichia ), Aerobactor and the like.

  Preferably, an enzyme derived from a microorganism belonging to the genus Bacillus is used. More preferred are enzymes derived from Bacillus badius IAM11059 strain and Bacillus sphaericus NBRC3341 strain. The Bacillus badius IAM11059 is a microorganism that produces phenylalanine dehydrogenase, and is described in EP256514 and Bisci. Biotechnol. Biochem. 1995, 59, 10, 1994. The Bacillus badius IAM11059 strain was obtained from the Institute for Molecular Cell Biology (1-11-1 Yayoi, Bunkyo-ku, Tokyo 113-0032), and the Bacillus sphaericus NBRC3341 strain was It is available from the National Institute of Technology and Evaluation Biological Genetic Resources Division (2-5-8 Kazusa Kamashizu, Kisarazu City, Chiba Prefecture 292-0818).

  The Bacillus sphaericus NBRC3341 strain is a microorganism that produces leucine dehydrogenase and has the same number of amino acid residues as the known leucine dehydrogenase of Bacillus subtilis. 100% match. In the gene sequence, 14 bases are different in 1095 bases. The gene sequence of Bacillus subtilis is known in Nature, 1997, 390, 249, and NCBI database accession number CAB14339. In addition, it is known in Methods Enzymol. That pyrroline-2-carboxylic acid reductase is known in the genus Neurospora, Escherichia, and Aerobactor. 1962, volume 5, page 882.

2-3. Examples of the microorganism having the ability to produce the enzyme formate dehydrogenase having the coenzyme regeneration ability include, for example, the following known microorganisms having the ability to produce the enzyme, Candida, Kloeckera, Pichia, Lipomyces, Pseudomonas, Moraxella, Hyphomicrobium, Paracoccus, Thiobacillus, Anilobacter ( Ancylobacter) and the like.

  Preferably, enzymes derived from microorganisms belonging to the genus Thiobacillus and the genus Ancylobacter are mentioned. More preferably, an enzyme derived from Thiobacillus sp. KNK65MA strain (FERM BP-7671) and Ansilobacter aquaticus KNK607M strain (FERM BP-7335) can be mentioned. Microorganisms identified by accession numbers FERM BP-7671 and BP-7335 have been deposited with the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (Central 1-1, Higashi 1-1-1 Tsukuba City, Ibaraki Prefecture 305-8566) ing.

2-4. Obtaining Enzymes In order to obtain highly active bacteria that produce each of the above enzymes efficiently and efficiently, it is effective to create transformed microorganisms as is well known. As a preparation method, for example, after cloning a D-amino acid oxidase gene from a strain exhibiting D-amino acid oxidase activity, a recombinant plasmid with an appropriate vector is prepared, and this is used without any particular limitation. A transformant can be obtained by transforming a host microorganism such as (Escherichia coli).

  As the vector, a plasmid, phage or derivative thereof derived from a microorganism capable of autonomous replication in a host can be used. Among them, as a host microorganism, for example, Escherichia coli whose bacteriological properties are well known to those skilled in the art and provided for transformation, a vector capable of autonomous replication in the microorganism is used as a vector. Is preferred. As such a vector, for example, pUC18 (Takara Bio Inc.), pUC19 (Takara Bio Inc.), pBR322 (Takara Bio Inc.), pACYC184 (Inc. Nippon Gene), pSTV28 (Takara Bio Inc.), pSTV29 (Takara Bio Inc.), pSC101 (Funakoshi Co., Ltd.), pT7Blue (Takara Bio Inc.), or based on the description of International Publication No. WO94 / 03613. PUCNT that can be produced by those skilled in the art, or derivatives thereof. These derivatives are modified genes for promoters, terminators, enhancers, SD sequences, replication initiation sites (ori), other regulation, etc., for the purpose of increasing enzyme production and stabilizing plasmids. Drug resistance, refers to a modified restriction enzyme site at the cloning site.

  As an example of the transformant thus obtained, Escherichia coli HB101 (pCDA003) (FERM P-20597) containing a D-amino acid oxidase gene derived from Candida intermedia NBRC0761 strain. (See Japanese Patent Application No. 2005-224505), and Escherichia coli HB101 (pFT001) (FERM BP- containing a formate dehydrogenase gene derived from Thiobacillus sp. KNK65MA strain (FERM BP-7671) 7672), Escherichia coli HB101 (pFT002) (FERM BP-7673) (see International Publication No. WO03 / 031626), and the like. The transformant identified by the above-mentioned deposit number FERM number is deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (Central 6th, 1-1-1 East Tsukuba City, Ibaraki Prefecture 305-8566).

  The recombinant DNA technology used in the present invention is well known in the art, and is described in, for example, Molecular Cloning 2nd Edition (Cold Spring Harbor Laboratory Press, 1989), Current Protocols in Molecular Biology (Greene Publishing Associates and Wiley-Interscience). Are listed.

  By culturing the above transformant and the like, the enzyme can be produced in large quantities, and can be used for the production of L-amino acids. The culture of microorganisms may be performed using a normal medium. The medium used for the culture may be a normal medium containing nutrients such as a carbon source, a nitrogen source and inorganic salts. When organic micronutrients such as vitamins and amino acids are added to this, favorable results are often obtained. As the carbon source, carbohydrates such as glucose and sucrose, organic acids such as acetic acid, alcohols and the like are appropriately used. As the nitrogen source, ammonium salt, aqueous ammonia, ammonia gas, urea, yeast extract, peptone, corn steep liquor and the like are used. Examples of inorganic salts include phosphate, magnesium salt, potassium salt, sodium salt, calcium salt, iron salt, sulfate, chlorine and the like.

  Culturing can be performed in a temperature range of 25 ° C to 40 ° C, with 25 ° C to 37 ° C being particularly preferred. Further, the pH can be cultured from 4 to 8, but preferably from 5 to 7.5. In addition, either a batch or continuous culture method may be used.

If necessary, treatment for enzyme induction such as addition of isopropyl-1-thio-β-D-galactoside (IPTG), lactose or the like can be performed.

3. Stereoinversion Reaction Next, a method for producing L-amino acid from racemic amino acid by stereoinversion reaction will be described. In the stereoinversion reaction, a racemic amino acid, the aforementioned D-amino acid oxidase, amino acid dehydrogenase, an enzyme having a coenzyme regeneration ability, and a compound serving as a substrate thereof are simultaneously present and the reaction is carried out in an aqueous medium.

  Catalase may be added to the reaction. By adding catalase, hydrogen peroxide generated by the reaction of D-amino acid oxidase can be decomposed, and decomposition of the substrate or product can be suppressed. As the catalase, those derived from animals, plants and microorganisms can be used, but those derived from microorganisms are preferred for industrial use. Any microorganism can be used as long as it has the ability to produce the enzyme. For example, the following known microorganisms having the ability to produce the enzyme, such as Micrococcus, Rhodopseudomonas). Commercially available enzymes can also be used. Examples of commercially available enzymes include Catazyme 25L (manufactured by Novozyme) and CATALASE (Beef River) (manufactured by P-L Biochemcals, Inc.).

  The D-amino acid oxidase, amino acid dehydrogenase, coenzyme regenerating enzyme, and catalase used in the embodiments may be those produced by culturing microorganisms having the respective activities, or You may use a product produced by breeding and cultivating a transformant introduced with each enzyme gene, or using a product produced by breeding and culturing a transformant introduced with multiple enzyme genes. May be.

  In the embodiment, the produced D-amino acid oxidase, amino acid dehydrogenase, enzyme having coenzyme regeneration ability, and catalase can be used not only as the enzyme itself, but also as a microorganism or a processed product thereof. Here, the treated product of the microorganism means, for example, a crude extract, a cultured cell freeze-dried organism, an acetone-dried organism, or a crushed product of these cells.

  Furthermore, they can be used as an immobilized enzyme obtained by immobilizing the enzyme itself or bacterial cells with a known means. Immobilization can be performed by a cross-linking method, a covalent bonding method, a physical adsorption method, a comprehensive method, and the like, which are well known to those skilled in the art.

  Further, FAD may be added to the above reaction. FAD is a coenzyme of D-amino acid oxidase, and it can be expected that the reaction efficiency is improved by adding FAD. The concentration of FAD added is preferably 0 equivalents or more and 10 equivalents or less, more preferably 0 equivalents or more and 1 equivalents or less, and even more preferably 0 equivalents or more and 0.1 equivalents or less with respect to the substrate.

  In addition, a coenzyme such as NAD may be added to the steric inversion reaction. Even when no coenzyme such as NAD is added, the reaction may proceed with a coenzyme such as NAD present in the microorganism, but the reaction efficiency is improved by adding a coenzyme such as NAD. Can be expected. The added concentration of a coenzyme such as NAD is preferably 0 equivalents or more and 2 equivalents or less, more preferably 0.00001 equivalents or more and 0.1 equivalents or less, more preferably 0.0001 equivalents or more, relative to the substrate. 0.01 equivalent or less.

3-1. Addition of ammonia or ammonia salt Further, by adding ammonia or a salt thereof to the steric inversion reaction, the reaction yield can be improved and / or the reaction time can be shortened. The concentration of ammonia added is preferably at least 0.1 equivalent, more preferably at least 0.3 equivalent, at least 0.65 equivalent, and at least 0.8 equivalent with respect to the racemic amino acid as the substrate. The upper limit of the amount added is not particularly limited, but the amount of ammonia removed in the stripping step to be described later increases as the amount added increases, so it is usually 10 equivalents or less, preferably 2 equivalents or less, more preferably 1. Perform at 5 equivalents or less. Examples of the ammonium salt include ammonium formate, ammonium chloride, ammonium carbonate, ammonium sulfate, and the like. From the viewpoint of improving the reaction yield and shortening the reaction time, ammonium formate is particularly preferable (corresponding to Examples 5 and 6). ).

3-2. Substrate charge concentration / reaction temperature The substrate charge concentration of the above steric inversion reaction is 0.1% (w / v) or more, 90% (w / v) or less, preferably 1% (w / v) or more, 60%. (W / v) The reaction is carried out in a dissolved or suspended state, and the reaction temperature in the resolution and steric inversion reaction is 10 ° C or higher and 80 ° C or lower, preferably 20 ° C or higher and 60 ° C or lower. What is necessary is just to stand still or stir for a while, adjusting and keeping.

3-3. pH adjustment The pH of the reaction is preferably 4 or more and 12 or less, more preferably 6 or more and 11 or less. The pH can be adjusted by adding an acid or base. However, when formate dehydrogenase is used as an enzyme capable of coenzyme regeneration, formic acid is usually converted into carbon dioxide as the coenzyme is regenerated. Since it is converted and removed from the reaction solution, the pH increases with the progress of the reaction, and it is necessary to adjust the pH with an acid. The pH adjustment of the enzyme reaction or the pH control of the enzyme reaction mainly means setting or adjusting the pH of the solution to a value suitable for the enzyme reaction.

  As the acid used for pH adjustment (corresponding to “acid used for pH control of enzyme reaction”), mineral acids such as hydrochloric acid and sulfuric acid, and organic acids such as formic acid and acetic acid can be used. Acids that form a readily soluble salt in the organic solvent used in the process are preferred, and examples include hydrochloric acid and formic acid. Furthermore, when formic acid is used (corresponding to Example 1), the yield of L-amino acid in the steric inversion reaction is more improved than when hydrochloric acid is used (corresponding to Example 3).

3-4. Other reaction conditions The substrate of this reaction may be added all at once, dividedly, or continuously. The resolution and the steric inversion reaction can be performed by a batch method or a continuous method.

  The stereoinversion reaction of the present invention can also be performed using an immobilized enzyme, a membrane reactor, or the like. Examples of the aqueous medium include water, a buffer solution, an aqueous medium containing a water-soluble organic solvent such as ethanol, or an organic solvent that is difficult to dissolve in water, such as ethyl acetate, butyl acetate, toluene, chloroform, and n-hexane. A suitable solvent such as a two-layer system with an aqueous medium containing an organic solvent such as can be used. Furthermore, if necessary, an antioxidant, a surfactant, a coenzyme, a metal, and the like can be added.

Insoluble matter such as bacterial cell components present in an aqueous solution containing an L-amino acid produced by the enzymatic reaction as described above is separated by a conventional separation method, for example, a separation method such as activated carbon treatment, filtration, extraction, or a combination thereof. Can be removed.

4). Removal of ammonia by stripping A method for removing ammonia from an aqueous optically active amino acid solution containing an ammonium salt obtained by the above method will be described.

  Ammonia can be removed by stripping by adding a base to the aqueous solution and reducing the pressure while keeping the pH basic. Stripping means an operation of transferring a gas present in the liquid into the gas phase.

  The pH at the time of stripping is preferably 7 or more, more preferably pH 8 or more, and further preferably 9 or more. Although there is no restriction | limiting in particular in the upper limit of pH, Since the quantity of the acid used for pH adjustment in the crystallization process mentioned later increases as pH becomes higher, it is normally performed at pH 11 or less. The pH adjustment (pH control) in the stripping step is mainly performed in order to facilitate removal of ammonia by keeping the pH of the optically active amino acid aqueous solution containing the ammonium salt basic.

  Examples of the base used for pH adjustment (corresponding to “base for controlling pH during stripping”) include, for example, lithium hydroxide, sodium hydroxide, potassium hydroxide, cesium hydroxide, magnesium hydroxide, calcium hydroxide, etc. Alkali metal hydroxides, alkaline earth metal hydroxides, tertiary amines such as triethylamine and diisopropylethylamine, and aminoalcohols such as ethanolamine and isopropanolamine. Among the above-mentioned bases, in order to obtain a highly pure optically active amino acid crystal at the time of crystallization described later, it is preferable to use one that forms a salt that is easily soluble in the organic solvent to be added, and lithium hydroxide is an example.

  The stripping pressure is preferably 500 hPa or less, more preferably 200 hPa or less. Further, the stripping temperature is preferably 10 ° C. or higher and 100 ° C. or lower, more preferably 30 ° C. or higher and 80 ° C. or lower. The stripping may be continued until the amount of ammonium ions contained in the aqueous solution is reduced to a concentration that does not mix as a salt in the subsequent crystallization step. Specifically, it is preferable to carry out until the concentration of ammonium ions remaining in the optically active amino acid aqueous solution is 0.05 M or less, or the remaining amount of ammonium ions is 5% or less at the start of stripping. Is more preferably 0.02M or less, or until the remaining amount of ammonium ions is 2% or less at the start of stripping. Moreover, in order to adjust the optically active amino acid concentration during stripping, the addition may be performed while adding water.

  As described in the item “3-1. Addition of ammonia or ammonia salt”, in the above-described stereoinversion reaction, the reaction yield can be improved by adding ammonia or a salt thereof. On the other hand, since the inorganic salt (ammonia salt) produced during the reaction is difficult to dissolve in an organic solvent, the inorganic salt (ammonia salt) is mixed in the crystal in a later step (crystallization step by adding an organic solvent), and the optically active amino acid to be purified is purified. It was a cause of lowering the purity.

  According to the conventional technology, in order to remove such inorganic salts, before the above step (crystallization step by adding alcohol), the inorganic salts by electrodialysis or ions described in the above background art are removed. Purification using an exchange resin has been performed. That is, those skilled in the art generally use electrodialysis or ion exchange resin as described above when removing contaminants.

In this respect, the embodiment adopts a completely different approach from the prior art in which a base such as lithium hydroxide is added separately (instead of simply removing the contaminant) in order to remove the contaminant. ing. A technique for easily and efficiently purifying optically active amino acids by adopting a stripping process in the process of purifying optically active amino acids from an optically active amino acid aqueous solution containing an ammonium salt is one of the findings obtained for the first time by the present inventors. One.

5. It is possible to obtain the optically active amino acid as crystals by adding a suitable organic solvent to the optically active amino acid aqueous solution to remove ammonia by crystallization above an optically active amino acids.

  The “organic solvent” to be added is not particularly limited as long as it can be uniformly mixed with water. For example, alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, and t-butanol are used. , Dimethylformamide, dimethylsulfoxide and the like, preferably methanol, ethanol and isopropanol. Moreover, these organic solvents may be used independently and may mix and use 2 or more types as needed.

  The preferable ratio of the organic solvent that can be uniformly mixed with water and water varies depending on the type of optically active amino acid, organic solvent, and the like. Generally, the higher the organic solvent ratio, the higher the yield. The ratio of the organic solvent generally gives good results when the ratio of the organic solvent in the total solvent is 50% or more, and further 80% or more. It is preferable to determine the type and ratio of the organic solvent and the concentration of the optically active amino acid so that a yield usually exceeding 70%, preferably exceeding 80%, more preferably almost quantitative yield can be achieved. These suitable conditions can be easily selected by simple experiments.

  The above crystallization operation can be further enhanced by combining with a known crystallization method such as neutralization, cooling, and concentration as necessary.

  Neutralization means adjusting the pH of the reaction solution to a suitable range by adding a suitable acid to the optically active amino acid aqueous solution. The preferred pH range here is not particularly limited as long as the optically active amino acid crystals are precipitated, but in order to improve the yield, it is near the isoelectric point of the optically active amino acid. It is preferable to adjust, usually at a pH of 2 or more and 11 or less.

  As the acid to be used, in order to obtain a highly pure optically active amino acid crystal at the time of crystallization described later, one that forms a readily soluble salt in the organic solvent to be added is preferable. For example, mineral acid such as hydrochloric acid, formic acid, acetic acid Organic acids such as

  The temperature at which crystallization is performed varies depending on the type of optically active amino acid and the type of organic solvent to be added. In general, the lower the temperature, the higher the yield, and it is usually carried out at 40 ° C. or lower, preferably 20 ° C. or lower. The lower limit is the solidification temperature of the system.

  Moreover, in order to suppress the formation of supersaturation and perform crystallization smoothly, or from the viewpoint of improving the quality, the crystallization is preferably carried out with stirring. If necessary, seed crystals can be added to promote crystallization.

The optically active amino acid crystals obtained as described above can be obtained as a wet body by, for example, solid-liquid separation by centrifugation, pressure filtration, vacuum filtration, etc., and cake washing as necessary. it can. Moreover, it can further be dried under reduced pressure to obtain a dry body.

  Specific examples of the present invention are shown below. However, the present invention is not limited to these examples.

Less than
(Example 1) Synthesis of L-norvaline by steric reversal reaction using “formic acid” for pH adjustment D-amino acid oxidase transformant Escherichia coli HB101 (pCDA003) (FERM P-20597) ( Medium A sterilized from Japanese Patent Application No. 2005-224505) (tryptone 1.6%, yeast extract 1.0%, sodium chloride 0.5%, ampicillin 0.01%, dissolved in deionized water, pH before sterilization 7. 0, except that ampicillin is added after sterilization), and then aerobically cultured by shaking at 37 ° C. for 30 hours. The bacterial cells were collected from the obtained culture solution by centrifugation, suspended in deionized water, and then disrupted by ultrasonic waves to obtain a crude enzyme solution containing D-amino acid oxidase.

  In addition, a transformant having both leucine dehydrogenase activity and formate dehydrogenase activity bred by the method shown in Reference Example 3 described later is inoculated into sterilized medium A, and then at 33 ° C. for 48 hours. The culture was aerobically cultured with shaking. The bacterial cells are collected from the obtained culture broth by centrifugation, suspended in deionized water, and then disrupted by ultrasound to obtain a crude enzyme solution containing leucine dehydrogenase and formate dehydrogenase. It was.

  7.8 g of racemic norvaline, crude enzyme solution containing the above D-amino acid oxidase, crude enzyme solution containing leucine dehydrogenase and formate dehydrogenase, catalase (Catazyme 25L, Novozyme), NAD 4.6 mg, and ammonium formate After adding 3.36 g and further adding about 1 g of 5M aqueous ammonia to adjust the pH to 8.0, the reaction solution (hereinafter referred to as “reaction solution A” as appropriate) was added to deionized water to 100 g. Expressed).

The reaction solution was aerobically stirred at 30 ° C. while controlling the pH at 8.0 with a 3M formic acid aqueous solution and reacted for 19 hours. As a result of analyzing the yield and optical purity of the produced L-norvaline using high performance liquid chromatography (HPLC), it was found that L-norvaline having an optical purity of 100% ee was obtained with a reaction yield of 94.4% with respect to the racemate. Obtained.
(HPLC analysis conditions) Column: SUMICHIRAL OA-5000 (4.6 mm × 250 mm, manufactured by Sumika Chemical Analysis), mobile phase: 2 mM copper sulfate aqueous solution / methanol = 95/5, flow rate: 1 ml / min, column temperature: 30 ° C, detection: 254 nm

(Example 2) Purification of L-norvaline from enzyme reaction solution using “formic acid” for pH adjustment L-norvaline was purified from the enzyme reaction solution obtained in Example 1 by the following steps.
(1) Activated carbon treatment, etc. The enzyme reaction solution obtained in Example 1 was charged with 5 g of activated carbon “special white birch” (manufactured by Takeda Pharmaceutical Company Limited), 2 g of filter aid “Radiolite # 700” (manufactured by Showa Chemical Industry Co., Ltd.), 1 ml of toluene was added and stirred at 30 ° C. for 2 hours. Insoluble matter was removed by vacuum filtration and washed with 35 ml of deionized water.
(2) Ammonia stripping 1.54 g of lithium hydroxide was added to the obtained filtrate to adjust the pH to 9.5. Stripping was performed for 90 minutes with stirring at an internal temperature of about 50 ° C. and a pressure of 130 hPa while maintaining the pH at 9.5 and the total weight at 100 g by adding lithium hydroxide and deionized water.
(3) Neutralization and concentration with hydrochloric acid After adding 2.8 g of 35% hydrochloric acid to the solution after stripping to adjust the pH to 6.0, the solution was further concentrated under reduced pressure until the total weight became 20.6 g. .
(4) Crystallization by addition of alcohol and filtration of crystals After concentration under reduced pressure, the pressure was returned to normal pressure, 48.4 g of methanol was added to the solution, stirred at 25 ° C for 2.5 hours, and then cooled to 0 ° C. And stirring was continued for 1.5 hours. The precipitated crystals were filtered under reduced pressure and sufficiently drained, and then washed with 18 ml of 83% methanol. Thereafter, the obtained wet crystals were dried under reduced pressure (about 10 hPa, 50 ° C., about 5 hours) to obtain L-norvaline as crystals.

When the obtained crystals of L-norvaline were analyzed under the same conditions as in Example 1, the purity was 99.6 wt% and the recovery rate from the enzyme reaction solution obtained in Example 1 was 82.7%. It was.

(Example 3) Synthesis of L-norvaline by steric reversal reaction using "hydrochloric acid" for pH adjustment A reaction solution prepared in the same manner as in Example 1 (see "Reaction Solution A" above) was prepared using a 2M hydrochloric acid aqueous solution. The mixture was aerobically stirred at 30 ° C. while controlling the pH at 8.0 and reacted for 22 hours.

As a result of analyzing the yield and optical purity of the produced L-norvaline under the same conditions as in Example 1, L-norvaline having an optical purity of 100% ee was obtained with a reaction yield of 87.0% based on the racemate. It was.

(Example 4) Purification of L-norvaline from enzyme reaction solution using “hydrochloric acid” for pH adjustment L-norvaline was purified from the enzyme reaction solution obtained in Example 3 by the following steps.
(1) Activated carbon treatment etc. The enzyme reaction solution obtained in Example 3 was charged with 10 g of activated carbon “special white birch” (manufactured by Takeda Pharmaceutical Co., Ltd.), 2 g of filter aid “Radiolite # 700” (manufactured by Showa Chemical Industry Co., Ltd.), 1 ml of toluene was added and stirred at 30 ° C. for 2 hours. Insoluble matters were removed by filtration under reduced pressure and washed with 90 ml of deionized water.
(2) Ammonia stripping 1.84 g of lithium hydroxide was added to the obtained filtrate to adjust the pH to 9.5. While maintaining the pH at 9.5 by adding lithium hydroxide and deionized water, stripping was performed for 150 minutes with stirring at an internal temperature of about 40 ° C. and a pressure of 70 to 40 hPa.
(3) Neutralization and concentration with hydrochloric acid After adding 3.2 g of 35% hydrochloric acid to the solution after stripping to adjust the pH to 6.0, the solution was further concentrated under reduced pressure until the total weight reached 18.1 g. .
(4) Crystallization by alcohol addition and filtration of crystals After concentration under reduced pressure, the pressure was returned to normal pressure, 32.3 g of methanol was added to the solution, stirred at 25 ° C for 2.5 hours, and then cooled to 0 ° C. Stirring was continued for 2 hours. The precipitated crystals were filtered under reduced pressure, sufficiently drained, and then washed with 12 ml of 80% methanol. Thereafter, the obtained wet crystals were dried under reduced pressure (about 10 hPa, 50 ° C., about 5 hours) to obtain L-norvaline as crystals.

When the obtained crystals of L-norvaline were analyzed under the same conditions as in Example 1, the purity was 99.3 wt% and the recovery rate from the enzyme reaction solution obtained in Example 1 was 83.2%. It was.

(Example 5) Synthesis of L-norvaline by steric inversion reaction (effect of addition of ammonia)
A crude enzyme solution containing D-amino acid oxidase prepared by the same method as in Example 1 in 50 mg of racemic norvaline, a crude solution containing leucine dehydrogenase and formate dehydrogenase prepared by the same method as in Example 1. Adjust the enzyme solution, catalase (Catazyme 25L, Novozyme), NAD 0.3 mg, and ammonium formate and sodium formate so that the total amount of formic acid is 0.65 equivalents of racemic norvaline as shown in Table 1. The reaction solution was prepared by adding deionized water to 1 ml.

  This reaction solution was placed in a test tube, shaken at 30 ° C., and reacted while being controlled at 8.0 with 2N hydrochloric acid. As a result of analyzing the yield of L-norvaline produced at 2.5 hours and 5 hours after the reaction under the same conditions as in Example 1, as shown in Table 1, the reaction rate was improved by adding ammonia. Admitted.

（実施例６）立体反転反応によるＬ−ノルバリンの合成（ギ酸アンモニウム添加量の影響）
ラセミ体ノルバリン７．８ｇに、実施例１と同様の方法にて調製したＤ−アミノ酸オキシダーゼを含む粗酵素液、実施例１と同様の方法にて調製したロイシン脱水素酵素及びギ酸脱水素酵素を含む粗酵素液、カタラーゼ（Ｃａｔａｚｙｍｅ２５Ｌ、Ｎｏｖｏｚｙｍｅ社製）、ＮＡＤ４６ｍｇ、及びギ酸アンモニウム３．３６ｇ（ラセミ体ノルバリンに対して０．８当量）又は２．７３ｇ（ラセミ体ノルバリンに対して０．６５当量）を添加し、さらに５Ｍアンモニア水約１ｇを添加してｐＨを８．０に調整した後、脱イオン水にて１００ｇになるよう反応液を調製した。

(Example 6) Synthesis of L-norvaline by steric inversion reaction (effect of addition amount of ammonium formate)
A crude enzyme solution containing D-amino acid oxidase prepared by the same method as in Example 1 and leucine dehydrogenase and formate dehydrogenase prepared by the same method as in Example 1 were added to 7.8 g of racemic norvaline. Containing crude enzyme solution, catalase (Catazyme 25L, Novozyme), NAD 46 mg, and ammonium formate 3.36 g (0.8 equivalents to racemic norvaline) or 2.73 g (0.65 equivalents to racemic norvaline) And about 1 g of 5M ammonia water was added to adjust the pH to 8.0, and then a reaction solution was prepared to 100 g with deionized water.

  The reaction solution was aerobically stirred at 30 ° C. while controlling the pH at 8.0 with a 3M aqueous sulfuric acid solution, and reacted for 22.5 hours.

As a result of analyzing the yield and optical purity of the produced L-norvaline under the same conditions as in Example 1, L-norvaline having an optical purity of 100% ee was obtained in any reaction, but the reaction yield was ammonium formate. Was 96.2% when 0.8 equivalent was added to the racemate, and 92.8% when only 0.65 equivalent was added, and the effect of adding ammonium formate was recognized. It was.

(Comparative Example 1) Synthesis of L-norvaline by steric reversal reaction using sulfuric acid for pH adjustment A reaction solution prepared in the same manner as in Example 1 (see "Reaction Solution A" above) was prepared with a 3M aqueous sulfuric acid solution. While controlling the pH at 8.0, the mixture was aerobically stirred at 30 ° C. and reacted for 19 hours.

The yield and optical purity of the produced L-norvaline were analyzed under the same conditions as in Example 1. As a result, L-norvaline having an optical purity of 100% ee was obtained with a reaction yield of 97.3% based on the racemate. It was.

(Comparative Example 2) Purification of L-norvaline without performing a stripping operation from an enzyme reaction solution using sulfuric acid for pH adjustment L-norvaline was purified from the enzyme reaction solution obtained in Comparative Example 1 by the following steps.
(1) Activated carbon treatment, etc. After adding 0.71 g of 3M sulfuric acid aqueous solution to the enzyme reaction solution obtained in Comparative Example 1 to adjust the pH to 6.0, activated carbon “special white birch” (manufactured by Takeda Pharmaceutical Co., Ltd.) 10 g Then, 2 g of filter aid “Radiolite # 700” (manufactured by Showa Chemical Industry Co., Ltd.) and 1 ml of toluene were added and stirred at 30 ° C. for 1 hour. Insoluble matter was removed by vacuum filtration and washed with 70 ml of deionized water.
(2) Concentration (no stripping operation)
The obtained filtrate was concentrated under reduced pressure until the total weight became 17.2 g.
(3) Crystallization by addition of alcohol and filtration of crystals After concentration under reduced pressure, the pressure was returned to normal pressure, 12.7 g of methanol was added to the solution, cooled to 0 ° C., and stirring was continued for 2 hours. The precipitated crystals were filtered under reduced pressure and sufficiently drained, and then washed with 12 ml of 67% methanol. Thereafter, the obtained wet crystals were dried under reduced pressure (about 10 hPa, 50 ° C., about 5 hours) to obtain L-norvaline as crystals.

When the obtained crystals of L-norvaline were analyzed under the same conditions as in Example 1, the purity was 75.8 wt% and the recovery rate from the enzyme reaction solution was 87.1%.

(Reference Example 1) Breeding of a transformant having formate dehydrogenase activity A DNA primer (Primer-1: SEQ ID NO: in Sequence Listing) using the genome of Thiobacillus sp. KNK65MA strain (FERM BP-7671) as a template 1 and Primer-2: PCR was performed using SEQ ID NO: 2 in the sequence listing. Accession No. FERM BP-7671 The specified microorganism is deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (Central 1-1, Higashi 1-1-1 Tsukuba City, Ibaraki Prefecture 305-8566).

  The DNA fragment obtained by the PCR was cleaved with restriction enzymes NdeI and EcoRI. By ligating the cleaved fragment and a vector plasmid pUCNT cleaved with the same enzyme (which can be produced by those skilled in the art based on the description in the specification of International Publication No. WO94 / 03613) using T4 DNA ligase, A plasmid designed to express a large amount of formate dehydrogenase was obtained.

  The obtained plasmid was mixed with Escherichia coli HB101 competent cell (Takara Bio Inc.) and transformed to breed a transformant having formate dehydrogenase activity.

The obtained transformant was inoculated into 6 ml of medium A sterilized in a test tube, and then aerobically cultured by shaking at 37 ° C. for 24 hours. Bacteria are collected from the obtained culture broth by centrifugation and extracted with QIAprep Spin Miniprep Kit (manufactured by QIAGEN) to recover a plasmid designed to express a large amount of formate dehydrogenase. did.

(Reference Example 2) Breeding of transformant having leucine dehydrogenase activity DNA primer (Primer-3: SEQ ID NO: 3 in the sequence listing) and Primer-4 using the genome of Bacillus sphaericus NBRC3341 strain as a template : PCR was performed using SEQ ID NO: 4) in the sequence listing. The Bacillus sphaericus NBRC 3341 strain is available from the National Institute of Technology and Evaluation Biological Genetic Resources Division (2-5-8, Kazusa Kama feet, Kisarazu City, Chiba Prefecture 292-0818).

  The DNA fragment obtained by the PCR was cleaved with restriction enzymes EcoRI and SacI. The cleaved fragment and a vector plasmid pUCT (plasmid vector in which the NdeI site of pUCNT (manufactured by a person skilled in the art based on the description of International Publication No. WO94 / 03613) was disrupted) cleaved with the same enzyme, A plasmid designed to express a large amount of leucine dehydrogenase was obtained by binding using T4 DNA ligase. By transforming the obtained plasmid with competent cells of Escherichia coli HB101 (manufactured by Takara Bio Inc.), a transformant having leucine dehydrogenase activity was bred.

The obtained transformant was inoculated into 6 ml of medium A sterilized in a test tube, and then aerobically cultured by shaking at 37 ° C. for 24 hours. Bacteria are collected from the obtained culture by centrifugation, and plasmid extraction is performed using QIAprep Spin Miniprep Kit (manufactured by QIAGEN) to recover a plasmid designed to express a large amount of leucine dehydrogenase. did.

(Reference Example 3) Breeding of a transformant having leucine dehydrogenase activity and formate dehydrogenase activity A plasmid designed to express a large amount of the leucine dehydrogenase obtained in Reference Example 2 was designated by EcoRI and PstI. After cleaving, the DNA fragment containing the leucine dehydrogenase gene was recovered using TaKaRaRECOCHIP (Takara Bio Inc.). By ligating the DNA fragment and the plasmid designed to express a large amount of the formate dehydrogenase obtained in Reference Example 1 cut with the same enzyme using T4 DNA ligase, A plasmid designed to express a large amount of enzyme and formate dehydrogenase was obtained. The resulting plasmid is mixed with Escherichia coli HB101 competent cell (Takara Bio Inc.) and transformed to produce a trait having both leucine dehydrogenase activity and formate dehydrogenase activity. The transformant was bred. The transformant was used in Example 1 and the like.

Claims (20)

  Production of an optically active amino acid characterized by removing ammonia from an aqueous solution containing an optically active amino acid and an ammonium salt by stripping and then isolating the crystal of the optically active amino acid by adding an organic solvent for crystallization. Method.   The production method according to claim 1, wherein the pH is controlled during the stripping by using a base that forms a salt that is easily soluble in the organic solvent.   The production method according to claim 2, wherein the base is lithium hydroxide.   The production method according to any one of claims 1 to 3, wherein the aqueous solution containing the optically active amino acid and the ammonium salt is produced by an enzymatic reaction.   The production method according to claim 1, wherein the optically active amino acid is an L-form amino acid (L-amino acid).   The aqueous solution containing the L-amino acid and ammonium salt is produced by an enzymatic reaction in which a racemic amino acid is allowed to act on an enzyme having D-amino acid oxidase, amino acid dehydrogenase, and coenzyme regeneration ability. The manufacturing method of Claim 5 characterized by the above-mentioned.   In the said enzyme reaction, ammonia or its salt is added 0.3 equivalent or more with respect to a racemic amino acid, The manufacturing method of Claim 6 characterized by the above-mentioned.   The production method according to claim 7, wherein the salt of ammonia is ammonium formate.   The aqueous solution containing the L-amino acid and ammonium salt is produced by an enzyme reaction in which an amino acid dehydrogenase and an enzyme having a coenzyme regenerating ability are allowed to act on the corresponding keto acid. The manufacturing method according to claim 5.   6. The production method according to claim 5, wherein the aqueous solution containing the L-amino acid and an ammonium salt is produced by an enzyme reaction that causes amidase to act on a racemic amino acid amide.   The method according to any one of claims 1 to 4, wherein the optically active amino acid is a D-form amino acid (D-amino acid).   The aqueous solution containing the D-amino acid and an ammonium salt is produced by an enzymatic reaction in which a decarbamoylase is allowed to act on an N-carbamoylamino acid corresponding to the D-amino acid. Item 12. The manufacturing method according to Item 11.   12. The production method according to claim 11, wherein the aqueous solution containing the D-amino acid and an ammonium salt is produced by an enzyme reaction that causes amidase to act on a racemic amino acid amide.   The production method according to any one of claims 4 to 13, wherein an acid that forms a readily soluble salt in the organic solvent is used for pH control of the enzyme reaction.   The production method according to claim 14, wherein an acid used for pH control of the enzyme reaction is formic acid or hydrochloric acid.   The production method according to claim 14, wherein the acid used for pH control of the enzyme reaction is formic acid.   In a reaction in which D-amino acid oxidase, amino acid dehydrogenase, and an enzyme having a coenzyme regeneration ability are allowed to act on a racemic amino acid to convert it to an L-amino acid, ammonia or a salt thereof is added to the racemic amino acid in an amount of 0. A method for producing an L-amino acid, comprising adding 3 equivalents or more.   The method according to claim 17, wherein the ammonia salt is ammonium formate.   The production method according to any one of claims 4 to 18, wherein a transformant having an ability to produce each enzyme is used instead of each enzyme used in the enzyme reaction. A method for purifying an optically active amino acid by purifying an optically active amino acid by a crystallization step in which an organic solvent is added,
(I) a step of adding a base for replacing the ammonium salt with a salt having high solubility in the organic solvent to a solution containing an optically active amino acid and an ammonium salt;
(Ii) a step of removing ammonia obtained by the substitution into the gas phase by stripping from the solution after step (i) and removing it from the solution;
(Iii) adding the organic solvent to the solution after step (ii) to dissolve the salt obtained by the substitution in the organic solvent while crystallizing the optically active amino acid;
A method for purifying an optically active amino acid, comprising the steps of: