JP4590981B2 - Method for producing optically active cyclic amino acid - Google Patents

Description

  The present invention relates to a process for producing optically active cyclic amino acids that are industrially useful.

As chemical methods for producing cyclic amino acids, a method for producing L-azetidine-2-carboxylic acid (L-azetidine-2-carboxylic acid) (Non-patent document 1, Patent document 1), a method for producing pipecolic acid (pipecolic acid) ( Non-patent document 2), 4- and 5-hydroxypipecolic acid (non-patent document 3), thiazancarboxylic acid (1,4-thiazane-3-carboxylic acid) process (non-patent document) 4, 5), production method of L-morpholine carboxylic acid (L-3-Morpholine carboxylic acid) (non-patent document 6), S-azepane-2-carboxylic acid ((S) -azepane-2-carboxylic acid) The manufacturing method (Non-Patent Document 7) was known.
As a method for producing a cyclic amino acid biochemically, a method for producing L-pipecolic acid from L-lysine using pyrroline-5-carboxylic acid reductase (EC 1.5.1.2) (Non-Patent Document 8), a method for producing L-Proline from L-ornithine using pyrroline-5-carboxylic acid reductase (EC 1.5.1.2) (Non-Patent Documents 9 and 10) ), Production of L-proline from L-ornithine by ornithine cyclodeaminase (Non-patent Document 11), various diamino acids (diamino) by ornithine cyclodeaminase
acid)) for producing various cyclic amino acids (Patent Document 2).

On the other hand, as an enzyme that reduces a cyclic amino acid having a double bond at the 1-position, for example, pyrroline-2-carboxylate reductase (EC 1.5.1.1) derived from animals or fungi is Δ- 1-pyrroline-2-carboxylic acid (Δ-1-pyrroline-2-carboxylic acid) and Δ-1-piperidine-2-carboxylic acid are reduced to proline, And pipecolic acid was known to be produced (Non-patent Document 12).

  Also, a report on the metabolism of Pseudomonas bacteria that L-pipecolic acid is produced from D-lysine using Δ-1-piperidine-2-carboxylic acid as an intermediate. Among them, there is also a report that piperideine-2-carboxylate reductase (EC 1.5.1.21) is performing a reduction reaction (Non-patent Document 13).

Ketimine-reducing enzyme (EC 1.5.1.25) derived from pig liver reduces S-aminoethylcysteine ketimine, lanthionine ketimine, and cystathionine ketimine. It has been found (Non-Patent Document 14).
However, the method reported by Fujii et al. (Non-Patent Document 8) uses L-lysine 6-aminotransferase to L-lysine to produce Δ-1-piperidine-6-carboxylic acid as an intermediate. Further, L-pipecolic acid is obtained by allowing a reductase to act on it. This method can be applied only when the raw material is L-lysine and cannot be applied to the production of other cyclic amino acids.

Furthermore, Costilow et al. (Non-patent Document 11) reported that L-proline was obtained using L-ornithine using Ornithine Cyclase, but this also relates to products other than proline. There is no description. Denis et al. (Patent Document 2) uses Ornithine Cyclase to produce L-pipecolic acid, L-thiomorpholine-2-carboxylic acid, 5-hydroxy-L. -Pipecolic acid (5-hydroxy-L-pipecolic aci
d) etc. have been reported, but there is no description of yield and optical purity.

In any of the above methods, the optical purity of the product cyclic amino acid depends on the optical purity of the raw amino acid, and no optically active cyclic amino acid can be obtained from the racemic raw material at high efficiency.
On the other hand, the method using a cyclic amino acid having a double bond at the 1-position as an intermediate is industrially advantageous because a racemic cyclic amino acid or diamino acid can be used as a raw material.
Enzymatic reactions corresponding to L-proline, L-pipecolic acid, L-thiomorphine, etc. have been confirmed, respectively. It has only been confirmed, and examples of industrial production have not been known. In addition, there is a description that animal-derived enzymes are very unstable, and it has been difficult to put these enzymes into practical use.
US 5942630 WO 02/101003 Stephen Hanessian et al., Bioorganic & Medicinal Chemistry Letters (1999) vol.9, pp.1437-1442 Concepcion F Garcia et al., Tetrahydron Asymmetry (1995) vol.6, pp.2905-2906 Roland EA Callens, et al, Bull. Soc. Chim. Belg. (1982) vol.91, pp.713-723 T. Shiraiwa, et al., Biosci. Biotechnol. Biochem., Vol62, pp2382-2387 U. Larsson et al., Acta Chemica Scandinavica (1994) vol.48 pp517-525 Y. Kogami et al., Bull. Chem. Soc. Jpn (1987) vol.60, pp2963-2965 D. Seebach et al., Liebigs. Ann. Chem. (1989) pp.1215-1232 Tadashi Fujii et al., Bioscience Biotechnology Biochem (2002) vol.66, pp.1981-1984 Janet Kenklies et al., Microbiology (1999), vol.145, pp.819-826 Ralph N Costilow et al., Journal of Bacteriology (1969) vol.100, pp.662 Ralph N Costilow et al., Journal of Biological Chemistry (1971) vol.246, pp.6655-6660 Alton Meister et al., Journal of Biological Chemistry (1957) vol.229, pp.789-800 Cecil W Payton et al., Journal of Bacteriology (1982) vol.149, pp.864-871 Mirella Nardini et al., European Journal of Biochemistry (1988) vol.173, pp.689-694

Enzymatically stable enzyme that reduces a cyclic amino acid with a double bond at the 1-position. An enzyme that reacts widely with various substrates is isolated, and its immobilized enzyme is used, or a recombinant is incorporated. If microorganisms are used, the above problem is solved, and it is considered that an optically active cyclic amino acid can be produced in good yield at an industrially low cost.
Further, the present invention provides a cyclic amino acid having a double bond at the 1-position as an intermediate from an industrially inexpensive diamino acid or a racemic cyclic amino acid, and is industrially inexpensive by using an enzyme that reduces this. An object of the present invention is to provide a method for producing various optically active cyclic amino acids.

  As a result of intensive studies to solve the above problems, the present inventors have found that N-methyl-L-amino acid dehydrogenase reduces a cyclic amino acid having a double bond at the 1-position and efficiently generates an optically active cyclic amino acid. I found. In addition, a cyclic amino acid having a double bond at the 1-position can be efficiently produced from a racemic cyclic amino acid or a diamino acid using a corresponding known enzyme, and can also be obtained chemically. Therefore, an industrially inexpensive optically active cyclic amino acid can be produced by a combination of the cyclic amino acid generation reaction having a double bond at the 1-position and the stereoselective reduction reaction. The present invention has been accomplished based on these findings.

That is, according to the present invention, the following method for producing an L-form cyclic amino acid is provided.
(1) The following general formula (I)

(In the formula, A, a linear alkyl chain of 1 to 4 carbon atoms, or _{-CHOHCH 2 -, - CH 2 CHOHCH} 2 -, - SCH 2 -, - SC 2 H 4 -, - SC 3 H 6 _{-, - OCH 2 -, -} OC 2 H 4 -, - OC 3 H 6 - to cyclic amino acid having a double bond at position 1, represented by alkyl chains showing a) containing a heteroatom selected from the group consisting of N-methyl-L-amino acid dehydrogenase derived from Pseudomonas bacteria or cells containing the same, a preparation of the cells or a culture solution obtained by culturing the cells, and the following general formula (II)

(Wherein A has the same meaning as described above), to produce an L-form cyclic amino acid.
(2) The production method according to (1), wherein the N-methyl-L-amino acid dehydrogenase derived from Pseudomonas is a polypeptide shown in the following (A), (B), or (C):
(A) a polypeptide having the amino acid sequence represented by SEQ ID NO: 1;
(B) a polypeptide having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 1 and having N-methyl-L-amino acid dehydrogenase activity Or (C) a polypeptide having an amino acid sequence having 95 % or more identity with the amino acid sequence represented by SEQ ID NO: 1 and having N-methyl-L-amino acid dehydrogenase activity;
(3) The production method according to (1), wherein the cell containing N-methyl-L-amino acid dehydrogenase is a cell transformed with DNA encoding N-methyl-L-amino acid dehydrogenase derived from Pseudomonas bacteria. .
(4) Pseudomonas bacterium derived from N- methyl -L- amino acid dehydrogenase encoding DNA is less than (A), a DNA encoding the protein shown in (B) or (C), according to (3) Production method:
(A) a protein having the amino acid sequence set forth in SEQ ID NO: 1, or (B) an amino acid sequence having 95 % or more identity with the amino acid sequence set forth in SEQ ID NO: 1, and N-methyl-L- A protein having amino acid dehydrogenase activity.
(C) A protein comprising an amino acid sequence in which 1 to 10 amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID NO: 1 and having N-methyl-L-amino acid dehydrogenase activity.

The production method according to claim 3 , wherein the DNA encoding N-methyl-L-amino acid dehydrogenase derived from a genus Pseudomonas is a DNA shown in the following (D) or (E) :
(D) a DNA having the base sequence shown in SEQ ID NO: 2; or ( E ) a base sequence in which 1 to 30 bases are substituted, deleted or added in the base sequence shown in SEQ ID NO: 2 and its complementary strand And a DNA encoding a protein having N-methyl-L-amino acid dehydrogenase activity.

(6) The following general formula (III)

(Wherein, A is as defined above), a chain α, ω-diamino acid represented by D-amino acid oxidase, L-amino acid oxidase, D-amino acid dehydrogenase, L-amino acid dehydrogenase, D-amino acid 1-position obtained after reacting an enzyme selected from the group consisting of transferase and L-amino acid transferase to produce a cyclic amino acid having a double bond at the 1-position represented by the above general formula (I) A cyclic amino acid having a double bond in the L-form cyclic amino acid represented by the general formula (II) is produced by the method according to any one of claims (1) to (5). And a method for producing an L-form cyclic amino acid.
(7) The following general formula (IV)

(Wherein A is as defined above), D-amino acid oxidase is allowed to act on the cyclic amino acid to produce a cyclic amino acid having a double bond at the 1-position represented by the above general formula (I) Then, the obtained cyclic amino acid having a double bond at the 1-position is converted to the L-form cyclic ring represented by the general formula (II) by the method according to any one of (1) to (5). A method for producing an L-form cyclic amino acid, comprising generating an amino acid.

  The present invention obtains a cyclic amino acid having a double bond at the 1-position as an intermediate from an industrially inexpensive diamino acid or a racemic cyclic amino acid, which is reduced using N-methyl-L-amino acid dehydrogenase. By doing this, it is possible to provide various optically active cyclic amino acid production methods that are industrially inexpensive and have high purity.

The present invention is described in detail below.
In the general formulas (I), (II), (III) and (IV), atoms and groups in the definition of A will be specifically described.
In the present invention, examples of the alkyl chain include —CH ₂ —, —C ₂ H ₄ —, —C ₃ H ₆ —.
_{, -C 2 H 3 CH 3 -} , - C 4 H 8 -, - C 3 H 5 CH 3 -, - CH 2 CHCH 3 CH 2 -, - C 5 H 10 -, - C 4 H 7 CH 3 - _{, -C 2 H 4 CHCH 3 CH} 2 -, - CH 2 CHCH 3 C 2 H 4 -, - C
Examples thereof include straight-chain and branched alkyl chains having 1 to 6 carbon atoms such as H ₂ C (CH ₃ ) ₂ CH ₂ — and —C ₆ H ₁₂ —. Among these, a linear alkyl chain having 2 to 4 carbon atoms capable of forming a 5-membered to 7-membered cyclic amino acid is preferable. In particular, in the case of 2 carbon atoms, L-proline (Proline),
In the case of 3 carbon atoms, a 6-membered ring amino acid such as L-pipecolic acid is formed. In the case of 4 carbon atoms, azepane-2-carboxylic acid is used. Are particularly preferred. The chemical formulas of these compounds are shown below.

Further, the above alkyl chain may contain a hetero atom such as a sulfur atom, oxygen atom or nitrogen atom in the chain or at the terminal, and a heterocycle can be formed by the alkyl chain containing these hetero atoms. . A hetero atom such as a sulfur atom, an oxygen atom, or a nitrogen atom contained in the alkyl chain may be contained in plural or plural, and the number of hetero atoms contained is preferably 1 to 3. The alkyl chain containing heteroatoms such _{-CHOHCH 2 -, - CH 2 CHOHCH} 2 -, - SCH 2 -, - SC 2 H 4 -, - SC 3 H 6 -, - OCH 2 -, - OC 2 H _{Four
-, - OC 3 H 6 -} , - NHCH 2 -, - NHC 2 H 4 -, - NHC 3 H 6 -, - NHCH 2 CHCOOH -, - C 2 H 4 NHCO -, - C 2 H 4 NHCN-, _{-C 2 H 4 CHCOOH -, -} SCH 2 CHCOOH -, - SC 2 H 4 CHCOOH -, - C 3 H 6 NHCH 2 CHCOOH -, - NHCHCOOHCH 2 -, and -CH ₂ NHCHCOOHC ₂ H ₄ -, and the like .

When A is an alkyl chain containing a sulfur atom, optically active cyclic amino acids include thioproline, 3-thiomorpholine carboxylic acid, [1,4] thiazepan-3-carboxylic acid ([1,4] thiazepane-3- carboxylic acid) and the like. When A is an alkyl chain containing an oxygen atom,
Examples of the optically active amino acid include 4-oxazolidine carboxylic acid, 3-morpholine carboxylic acid and the like. When A is an alkyl chain containing a plurality of nitrogen atoms, examples of the optically active cyclic amino acid include piperazine-2-carboxylic acid. The chemical formulas of these compounds are shown below.

    Further, the substituent of the alkyl chain or the alkyl chain containing a hetero atom is not particularly limited as long as it is a group that does not adversely influence the reaction. Specifically, an alkyl group, an aryl group, an alkoxy group, Examples include a carboxyl group, a halogen group, a cyano group, an amino group, a nitro group, and a hydroxyl group. Examples of the cyclic amino acid containing a substituent include hydroxyproline and hydroxypipecolic acid. These chemical formulas are shown below.

  Next, the N-methyl-L-amino acid dehydrogenase referred to in the present invention is, for example, reduced nicotinamide adenine nucleotide as represented by N-methyl-L-amino acid dehydrogenase derived from Pseudomonas putida ATCC12633 strain. (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) (hereinafter, both may be abbreviated as “NAD (P) H”) as a coenzyme, such as pyruvate, phenylpyruvate, and the like It is an enzyme that produces N-methyl-L-amino acids such as N-methyl-L-alanine and N-methyl-L-phenylalanine by adding methylamine to keto acid (see the following reaction formula).

Examples of the N-methyl-L-amino acid dehydrogenase used in the present invention include those having the following physicochemical properties.
(A) Action: N-alkyl-L-alanine is produced from pyruvate and alkylamine or dialkylamine using NADPH and / or NADH as a coenzyme;
(B) Substrate specificity: active against alkylamines or dialkylamines but not ammonia.
(C) the optimum pH when phenylpyruvic acid and methylamine are used as substrates is around 10; and
(D) The pH at which the enzyme is stable when treated at 30 ° C. for 30 minutes is around 5 to 10.5:
The N-methyl-L-amino acid dehydrogenase of the present invention reacts by reacting a compound containing a dicarbonyl group such as phenylpyruvic acid with ammonia, alkylamine and dialkylamine in the presence of NADPH and / or NADH. It can be obtained by analyzing sex organisms and performing screening to detect the presence and degree of dehydrogenase activity.

In the screening, a proliferating microbial cell from the culture of a microorganism having N-methyl-L-amino acid dehydrogenase activity, a crushed cell of the microbial cell, or a crude enzyme or a purified enzyme isolated therefrom by a normal operation can be used.
In addition, the characteristic of the N-methyl-L-amino acid dehydrogenase is that the enzyme has a stable pH of about 5 to 10.5 and an optimum pH of about 10.

The above N-methyl-L-amino acid dehydrogenase can be isolated from, for example, a microorganism belonging to Pseudomonas putida, particularly preferably Pseudomonas putida ATCC12633.
Specific examples of the above-mentioned N-methyl-L-amino acid dehydrogenase include a polypeptide represented by the amino acid sequence shown in SEQ ID NO: 1 and a homologue thereof having N-methyl-L-amino acid dehydrogenase activity. Can be mentioned.

As properties of the N-methyl-L-amino acid dehydrogenase represented by the amino acid sequence described in SEQ ID NO: 1, in addition to the properties shown in the above (A) to (D), the following properties are also shown.
(E) The molecular weight measured by gel filtration analysis using Superose 12HR10 / 30 (manufactured by Amersham Biosciences) is about 80 to 93 kilodaltons, and at least about 36 kilodaltons in SDS-polyacrylamide electrophoresis. The estimated polypeptide band is shown.

(F) The optimum temperature by activity measurement when phenylpyruvic acid and methylamine are used as substrates is around 35 ° C.
(G) The thermal stability when treated for 30 minutes at an optimum pH (when phenylpyruvic acid and methylamine are used as substrates = pH 10) is less than about 30 ° C.
(H) Not only pyruvic acid but also at least 2-ketohexanoic acid, phenylpyruvic acid, 2-oxobutyric acid, β-fluoropyruvic acid, 2-keto-n-valeric acid.

(I) The activity is inhibited by divalent heavy metals such as 0.01 mM mercury chloride (HgCl ₂ ) and 0.01 mM copper chloride (CuCl ₂ ).
As a homologue of the above N-methyl-L-amino acid dehydrogenase, one to plural (preferably 1 to 20, more preferably 1 to 15, more preferably 1 to 1) in the amino acid sequence represented by SEQ ID NO: 1 (10, more preferably 1 to 7, particularly preferably about 1 to 5 amino acids) are deleted, substituted and / or added and have N-methyl-L-amino acid dehydrogenase activity. Polypeptide; or the amino acid sequence represented by SEQ ID NO: 1 and 50% or more, preferably 60% or more, more preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, more preferably 95% Or more, particularly preferably a polypeptide having 97% or more homology and having N-methyl-L-amino acid dehydrogenase activity; Can be mentioned.

The homology search for the above polypeptide can be performed using, for example, the FASTA program, BLAST program, etc. for DNA Databank of JAPAN (DDBJ).
Further, N-methyl-L-amino acid dehydrogenase activity means that NAD (P) H or the like is used as a coenzyme for redox reaction, methylamine is added to α-keto acid such as pyruvic acid, phenylpyruvic acid, etc. The activity of synthesizing N-methyl-L-amino acids such as methyl-L-alanine and N-methyl-L-phenylalanine.

  As a method for obtaining the above-mentioned N-methyl-L-amino acid dehydrogenase, as described above, in addition to the method of isolating and purifying from a culture of a microorganism having dehydrogenase activity in N-methyl-L-amino acid, N-methyl-L-amino acid dehydrogenase from any microorganism having N-methyl-L-amino acid dehydrogenase activity by using a probe prepared based on a base sequence encoding part or all of the amino acid sequence of N-methyl-L-amino acid dehydrogenase -After isolating DNA encoding methyl-L-amino acid dehydrogenase, it can be obtained using genetic engineering techniques.

Alternatively, the above N-methyl-L-amino acid dehydrogenase can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method). Also, Kuwawa Trading (US Advanced Chem Tech), Perkin Elmer Jaban (US Perkin Elmer), Amersham Pharmacia Biotech (Amersham Pharmacia Biotech), Aloka (US Protein Technology Instrument), Kurabo (US Synthecell) -Vega), Nippon Perceptive Limited (US PerSeptive), Shimadzu Corporation and other peptide synthesizers can be used for chemical synthesis.

In the production method of the present invention, any microorganism can be used as a cell used for reducing a cyclic amino acid having a double bond at the 1-position as long as it is a microorganism having N-methyl-L-amino acid dehydrogenase. A microorganism transformed with DNA encoding N-methyl-L-amino acid dehydrogenase is desirable.
As DNA which codes said N-methyl-L-amino acid dehydrogenase, what contains the base sequence represented by sequence number 2 is mentioned, for example.

As a homologue of DNA encoding the above-mentioned N-methyl-L-amino acid dehydrogenase, one to plural (preferably 1 to 60, more preferably 1 to 30, more preferably 1) in the base sequence represented by SEQ ID NO: 2 A polypeptide having a dehydrogenase activity, preferably having 1-20 bases, more preferably 1-10 bases, particularly preferably about 1-5 bases deleted or substituted and / or added. Examples include DNA that encodes or encodes a polypeptide that hybridizes with a DNA having the base sequence represented by SEQ ID NO: 2 under stringent conditions and has N-methyl-L-amino acid dehydrogenase activity.

Those skilled in the art will be able to introduce site-directed mutagenesis (Nucleic) into the DNA described in SEQ ID NO: 2.
Acid Res. 10, pp. 6487 (1982), Methods in Enzymol. 100, pp. 448 (1983), Molecular Cloning
2nd Edt. Cold Spring Harbor Laboratory Press (1989) (hereinafter abbreviated as “Molecular Cloning Second Edition”), PCR-A Practical Approch IRL Press pp. 200 (1991)) or the like, and a desired homolog can be obtained by appropriately introducing substitution, deletion, insertion and / or addition mutation.

  In the present specification, “DNA that hybridizes under stringent conditions” means DNA obtained by using a DNA as a probe and using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like. After performing hybridization at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using, for example, a colony or plaque-derived DNA or a filter on which the DNA fragment is immobilized, Examples include DNA that can be identified by washing the filter under a condition of 65 ° C. using a 0.1 to 2 × SSC solution (the composition of 1 × SSC is 150 mM sodium chloride, 15 mM sodium citrate).

Hybridization can be performed according to the method described in Molecular Cloning 2nd edition.
The DNA homolog described above has 60% or more, preferably 70% or more, more preferably 80% or more, particularly preferably 90% or more homology with the polypeptide having the amino acid sequence shown in SEQ ID NO: 1. And those encoding a polypeptide having N-methyl-L-amino acid dehydrogenase activity.

The DNA encoding the above N-methyl-L-amino acid dehydrogenase can be isolated by, for example, the following method.
First, after purifying the above-mentioned N-methyl-L-amino acid dehydrogenase, the N-terminal amino acid sequence is analyzed, and further, using ordinary genetic engineering analysis techniques such as cloning from chromosomal DNA using the PCR method, A gene encoding N-methyl-L-amino acid dehydrogenase can be isolated and the base sequence thereof can be analyzed. Moreover, since the base sequence has been clarified by the present invention, a primer can be set based on the base sequence, and the target gene can be cloned, or can be synthesized by a DNA synthesizer.

  As described later, the DNA encoding the above-mentioned N-methyl-L-amino acid dehydrogenase can be inserted into a known expression vector to provide an N-methyl-L-amino acid dehydrogenase expression vector. Further, N-methyl-L-amino acid dehydrogenase can be obtained from the transformant by culturing the transformant transformed with the expression vector.

The microorganism to be transformed for expressing the DNA encoding the above N-methyl-L-amino acid dehydrogenase is not particularly limited as long as the host itself does not adversely affect this reaction. Examples of the microorganism include the following microorganisms.
Escherichia, Bacillus, Pseudomonas, Serratia, Brevibacterium, Corynebacterium, Streptococcus, Lactobacillus Bacteria for which host vector systems have been developed, such as genera;
Actinomycetes for which host vector systems have been developed, such as the genus Rhodococcus and the genus Streptomyces;
The genus Saccharomyces, the genus Kluyveromyces, the genus Schizosaccharomyces, the genus Zygosaccharomyces, the genus Yarrowia, the genus Trichosporon, the genus Yeasts for which host vector systems such as Hansenula, Pichia, and Candida have been developed;
Molds for which host vector systems such as Neurospora genus, Aspergillus genus, Cephalosporium genus and Trichoderma genus have been developed.

Among the microorganisms, the host is preferably the genus Escherichia, the genus Bacillus, the genus Brevibacterium, the genus Corynebacterium, particularly preferably the genus Escherichia, It belongs to the genus Corynebacterium.
The procedure for producing a transformant and the construction of a recombinant vector suitable for the host can be carried out according to techniques commonly used in the fields of molecular biology, biotechnology, and genetic engineering (for example, molecular cloning procedures). (See 2nd edition).

  Specifically, DNA encoding the N-methyl-L-amino acid dehydrogenase is introduced into a plasmid vector or phage vector that is stably present in a cell such as a microorganism, or the above N-methyl-L A DNA encoding an amino acid dehydrogenase can be transformed into a cell by introducing it directly into the host genome by integrating it into the chromosome, and the DNA can be expressed in the cell.

At this time, it is preferable to incorporate a promoter upstream of the 5′-side of the DNA strand of the present invention, and more preferably a terminator downstream of the 3′-side. The promoter and terminator that can be used in the present invention are not particularly limited as long as the promoter and terminator are known to function in microorganisms used as hosts, and vectors, promoters, and promoters that can be used in these various microorganisms. Regarding terminators and the like, for example, “Basic Course of Microbiology 8 Genetic Engineering / Kyoritsu Publishing”, especially regarding yeast, Adv.
Biochem. Eng. 43, 75-102 (1990), Yeast 8, 423-488 (1992), and the like.

Specifically, for example, in the genus Escherichia, particularly Escherichia coli, plasmid vectors include pBR and pUC plasmids, such as lac (β-galactosidase), trp (tryptophan operon), tac, Examples include promoters derived from trc (lac and trp fusion), λ phage PL, PR and the like. Examples of the terminator include trpA-derived, phage-derived, and rrnB ribosomal RNA-derived terminators.

In the genus Bacillus, examples of the vector include a pUB110 series plasmid, a pC194 series plasmid, and the like, and they can be integrated into a chromosome. As the promoter and terminator, promoters and terminators of enzyme genes such as alkaline protease, neutral protease, α-amylase and the like can be used.
In the genus Pseudomonas, the vector is based on the general host vector system developed by Pseudomonas putida, Pseudomonas cepacia, etc., or the plasmid TOL plasmid involved in the degradation of toluene compounds. And a broad host range vector (including genes necessary for autonomous replication derived from RSF1010) pKT240.

In the genus Brevibacterium, particularly Brevibacterium lactofermentum, examples of the vector include plasmid vectors such as pAJ43 (Gene 39, 281 (1985)). As the promoter and terminator, various promoters and terminators used in E. coli can be used.
In the genus Corynebacterium, particularly Corynebacterium glutamicum, vectors include pCS11 (Japanese Patent Laid-Open No. 57-183799), pCB101 (Mol. Gen. Genet. 196, 175 (1984), etc. A plasmid vector is mentioned.

In the genus Saccharomyces, particularly Saccharomyces cerevisiae, vectors include YRp, YEp, YCp, and YIp plasmids. In addition, promoters and terminators of various enzyme genes such as alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, acid phosphatase, β-galactosidase, phosphoglycerate kinase, and enolase can be used.

In the genus Schizosaccharomyces, examples of the vector include a plasmid vector derived from Schizosaccharomyces pombe described in Mol. Cell. Biol. 6, 80 (1986). In particular, pAUR224 is commercially available from Takara Shuzo and can be easily used.
In the genus Aspergillus, Aspergillus niger and Aspergillus oryzae are the most studied among molds, and integration into plasmids and chromosomes is available. Promoters derived from external proteases or amylases are available (Trends in Biotechnology 7, 283-287 (1989)).

In addition to the above, host vector systems corresponding to various microorganisms have been developed and can be used as appropriate.
In addition to microorganisms, various host and vector systems have been developed in plants and animals, especially in insects using moths (Nature 315, 592-594 (1985)), rapeseed, corn, potatoes and other plants. A system that expresses a heterogeneous protein in large quantities and a system that uses a cell-free protein synthesis system such as an E. coli cell-free extract or wheat germ have been developed and can be suitably used.

A transformant having a DNA encoding the above N-methyl-L-amino acid dehydrogenase is cultured, and the above N-methyl-L-amino acid dehydrogenase can be isolated and purified from the culture by a known method.
The method for culturing a transformant having a DNA encoding the above N-methyl-L-amino acid dehydrogenase can be performed according to a usual method used for culturing a host.

  When the transformant used in the present invention is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the medium for culturing these microorganisms contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the microorganism. As long as the medium can efficiently culture the transformant, either a natural medium or a synthetic medium may be used. The culture is preferably performed under aerobic conditions such as shaking culture or deep aeration and agitation culture, the culture temperature is usually 15 to 40 ° C., and the culture time is usually 16 hours to 7 days. During the culture, the pH is maintained at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like. Moreover, you may add antibiotics, such as an ampicillin and tetracycline, to a culture medium as needed during culture | cultivation.

As a medium for culturing a transformant obtained using an animal cell as a host cell, a commonly used RPM11640 medium (The Journal of the American Medical Association, 199, 519 (1967)), Eagle's MEM medium (Science, 122, 501 (1952)), DMEM medium (Virology, 8, 396 (195
9)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)] or a medium obtained by adding fetal calf serum or the like to these mediums. Culture is usually at pH
It is performed for 1 to 7 days under conditions such as 6 to 8, 30 to 40 ° C. and 5% CO ₂ . Moreover, you may add antibiotics, such as kanamycin and penicillin, to a culture medium as needed during culture | cultivation.

In order to isolate and purify the above-mentioned N-methyl-L-amino acid dehydrogenase from the transformant culture, ordinary protein isolation and purification methods may be used.
For example, when the above-mentioned N-methyl-L-amino acid dehydrogenase is expressed in a dissolved state in the cells, after completion of the culture, the cells are collected by centrifugation and suspended in an aqueous buffer, A cell-free extract is obtained by crushing cells with a French press, a Menton Gaurin homogenizer, Dynomill or the like. From the supernatant obtained by centrifuging the cell-free extract, an ordinary protein isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Anion exchange chromatography using resin such as diethylaminoethyl (DEAE) Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical), Cation exchange chromatography using resin such as S-Sepharose FF (Pharmacia) Hydrophobic chromatography using resins such as butyl sepharose and phenyl sepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, electrophoresis methods such as isoelectric focusing etc. Alternatively, it can be used in combination to obtain a purified preparation.

  In addition, when the above N-methyl-L-amino acid dehydrogenase is expressed by forming an insoluble substance in the cell, similarly, the cell is recovered and then crushed, and from the precipitate fraction obtained by centrifugation, After recovering the N-methyl-L-amino acid dehydrogenase by an ordinary method, the insoluble form of the N-methyl-L-amino acid dehydrogenase is solubilized with a protein denaturant. The solubilized solution is diluted or dialyzed into a dilute solution that does not contain a protein denaturant or does not denature N-methyl-L-amino acid dehydrogenase at the concentration of the protein denaturant, and the N-methyl-L-amino acid. After dehydrogenase is configured in a normal three-dimensional structure, a purified sample can be obtained by the same isolation and purification method as described above.

When the transformant is allowed to act, the transformant is treated as it is with an organic solvent such as acetone, DMSO, toluene or a surfactant, freeze-dried, physical or enzymatic. Treated with bacterial cells such as those crushed into pieces, those obtained by removing the enzyme fraction of the present invention in the transformant as a crude product or a purified product, and further represented by polyacrylamide gel, carrageenan gel, etc. Those immobilized on a carrier can be used.

  The following general formula (I), which is a substrate for cyclic amino acid production reaction to be reduced using N-methyl-L-amino acid dehydrogenase

As the cyclic amino acid having a double bond at the 1-position represented by the formula (wherein A is as defined above), those produced by any means can be used.
For example, it can be derived biologically or chemically from the corresponding diamino acid or racemic cyclic amino acid.
When derived from a diamino acid, as shown in the following reaction formula, if the α-position amino group of the diamino acid is converted to a keto group to form an α-keto acid, the α-keto acid undergoes non-enzymatic dehydration and ring closure at the 1-position. It becomes a cyclic amino acid having a double bond. In the following reaction formula, A has the same meaning as described above.

  Usually, the α-keto acid and a cyclic amino acid having a double bond at the 1-position are present as an equilibrated mixture in an aqueous solution, and therefore these are regarded as equivalent. That is, in the reaction system of the present invention, a cyclic amino acid itself having a double bond at the 1-position, a mixture of the α-keto acid and a cyclic amino acid having a double bond at the 1-position, or the α-keto acid is added, or Any of these embodiments is included in the present invention.

  The catalyst for the above reaction is not particularly limited as long as it is a catalyst capable of converting the amino group at the α-position of the diamino acid into a keto group to produce α-keto acid. Specifically, the amino acid oxidase (D Examples include enzymes such as -amino acid oxidase, L-amino acid oxidase), amino acid dehydrogenase (D-amino acid dehydrogenase), and amino acid transferase (D-amino acid aminotransferase, L-amino acid aminotransferase).

Of these, enzymes with wide substrate specificity are preferred. Specific examples include L-amino acid oxidase described in Enzyme and Microbial Technology vol.31 (2002) p77-87, and D-amino acid oxidase manufactured by Sigma.
The amino acid oxidase, amino acid dehydrogenase and amino acid transferase used are those that react only with diamino acids, and those corresponding to the coenzyme used in this reaction can be used as an alternative to the coenzyme regeneration system. That is, when NAD (P) H is used as a coenzyme in the reduction reaction of this reaction, NAD (P) H is converted to oxidized nicotinamide adenine nucleotide (NAD ⁺ ) or oxidized nicotinamide in accordance with the reduction of this reaction. Adenine dinucleotide phosphate (NADP ⁺ )
D (P) ⁺ ”, which is sometimes abbreviated as“ (D (P) ⁺ ”). On the other hand, in producing a cyclic amino acid having a double bond at the 1-position from diamino acids, NAD (P) ⁺ is used to produce NAD (P P)
It will be converted to H.

In addition, when a cyclic amino acid having a double bond at the 1-position is derived from a racemic cyclic amino acid compound, a cyclic amino acid having a different optical activity from the optically active cyclic amino acid that is finally produced is selectively oxidized to form a double bond at the 1-position. It is possible to use a reaction that generates a cyclic amino acid having or a reaction that leads all to a cyclic amino acid having a double bond at the 1-position.
That is, as described in the following reaction formula, the desired optically active cyclic amino acid, that is, the L-form cyclic amino acid is left as it is, and the opposite optically active form, that is, the D-form, is converted into a cyclic amino acid having a double bond at the 1-position. A method of converting and reducing this using the N-methyl-L-amino acid dehydrogenase of the present invention, or by converting all of them to a cyclic amino acid having a double bond at the 1-position and isolating it for the next reduction reaction. It can also be used. In the following reaction, A has the same meaning as described above.

The catalyst for converting the optically active form of D form into a cyclic amino acid having a double bond at the 1-position is not particularly limited as long as it has the activity, but an enzyme is preferably used. Examples of the enzyme include D-amino acid oxidase, D-amino acid dehydrogenase, and D-amino acid aminotransferase.

Among these, an enzyme having a wide substrate specificity is preferable. Specific examples include D-amino acid oxidase manufactured by Sigma.
The amino acid oxidase, amino acid dehydrogenase and amino acid transferase used are those that react only with a cyclic amino acid having a double bond at the 1-position of the D-form optically active substance, and corresponding to the coenzyme used in this reaction. When it can be used, it can be used as an alternative to the coenzyme regeneration system. That is, in the reduction reaction of this reaction, when NAD (P) H is used as a coenzyme, NAD (P) H becomes NAD (P) ⁺ with the reduction of this reaction. In the production of a cyclic amino acid having a double bond at the 1-position from a cyclic amino acid having a double bond at the 1-position using the opposite optically active substance, NAD (P) ⁺ is used to produce NAD (P
P) will be converted to H.

  In addition, when various amino acid oxidases are used to obtain a cyclic amino acid having a double bond at the 1-position, hydrogen peroxide is generated with the reaction, which may adversely affect the reaction, such as a decrease in enzyme activity. It is also desirable to combine another enzyme to remove hydrogen peroxide. The enzyme that removes hydrogen peroxide is not particularly limited as long as it is an enzyme that reacts with hydrogen peroxide. Specifically, catalase and peroxidase are preferable. The addition amount is not limited as long as the generated hydrogen peroxide is efficiently removed, but specifically, it is 0.1 to 1 million times active, preferably 1 to 100,000 times active against the oxidase. Used in a range.

Further, when oxidase is used, the activity can be increased by further adding a coenzyme flavin adenine dinucleotide (FAD). The addition concentration is 0.00001 to 100 mmol concentration, preferably 0.001 to 10 mmol concentration in the reaction solution.
In the production method of the present invention, the concentration of the cyclic amino acid having a double bond at the 1-position as a reaction substrate in the reaction solution is usually 0.0001 to 90% w / v, preferably 0.01 to 30% w. / V range. These may be added all at once at the start of the reaction. However, from the viewpoint of reducing the effects of enzyme substrate inhibition and improving the accumulated concentration of the product, these may be added continuously or intermittently. It is desirable to add to.

When the reaction substrate is a diamino acid, the substrate concentration is usually in the range of 0.01 to 90% w / v, preferably 0.1 to 30% w / v.
When the reaction substrate is a racemic cyclic amino acid, the substrate concentration is usually 0.01 to 90% w / v, preferably 0.1 to 30% w / v.
In this reaction, it is preferable to add coenzyme NAD (P) ⁺ or NAD (P) H, and usually 0.001 mM to 100 mM, preferably 0.01 to 10 mM.

When the above coenzyme is added, NAD (P) ⁺ produced from NAD (P) H is converted to NAD
(P) Regeneration to H is preferable for improving production efficiency, and the above regeneration methods include 1) a method using the NAD (P) ⁺ reducing ability of the host microorganism itself, 2) NAD (P) ⁺ to NAD (P) Microorganisms having the ability to produce H and processed products thereof, or glucose dehydrogenase, formate dehydrogenase, alcohol dehydrogenase, amino acid dehydrogenase, organic acid dehydrogenase (malate dehydrogenase, etc. 3) a method of adding an enzyme (regenerative enzyme) that can be used for the regeneration of NAD (P) H to the reaction system, and 3) an enzyme that can be used for the regeneration of NAD (P) H when producing a transformant. And a method for introducing the gene for the above regenerative enzymes into the host simultaneously with the DNA of the present invention.

Among these, in the method 1), it is preferable to add glucose, ethanol, formic acid or the like to the reaction system.
In the above method 2), microorganisms containing the regenerated enzymes, microorganisms transformed with DNA encoding the regenerated enzymes, microorganisms treated with acetone, freeze-dried, Treated with bacterial cells such as those crushed by the target or enzymatically, those obtained by removing the enzyme fraction as a crude product or purified product, and further immobilized on a carrier represented by polyacrylamide gel, carrageenan gel, etc. A commercially available enzyme may be used.

In this case, the amount of the regenerative enzyme used is specifically about 0.01 to 100 times, preferably about 0.5 to 20 times the enzyme activity as compared to N-methyl-L-amino acid dehydrogenase. Add to
In addition, a compound serving as a substrate for the regenerative enzyme, for example, glucose when using glucose dehydrogenase, formic acid when using formate dehydrogenase, ethanol or isopropanol when using alcohol dehydrogenase, etc. Although addition is also required, the addition amount is usually 0.1 to 100 mol times, preferably 0.5 to 20 mol times the amount of the dicarbonyl group-containing compound as the reaction raw material.

  In the above method 3), N-methyl-L-amino acid dehydrogenase DNA and regenerative enzyme DNA are integrated into the chromosome, and both DNAs are introduced into a single vector to transform the host. The method and a method of transforming a host after both DNAs are separately introduced into a vector can be used, but in the case of a method of transforming a host after both DNAs are separately introduced into a vector, It is necessary to select a vector in consideration of incompatibility.

When introducing multiple genes into a single vector, methods such as linking promoter-terminator-related regions such as promoters and terminators to each gene, or expressing them as an operon containing multiple cistrons such as the lactose operon. Is possible.
This reaction is performed in an aqueous medium containing a reaction substrate and a transformant, various coenzymes added as necessary, and a regeneration system thereof, or in a mixture of the aqueous medium and an organic solvent.

Examples of the aqueous medium include water or a buffer solution, and examples of the organic solvent include water-soluble organic solvents such as ethanol, propanol, tetrahydrofuran, and dimethyl sulfoxide, and ethyl acetate, butyl acetate, toluene, chloroform, and n-hexane. Those having a high solubility of the reaction substrate can be used as appropriate from water-insoluble organic solvents such as the above.
This reaction is usually at a reaction temperature of 4 to 60 ° C, preferably 10 to 50 ° C, and usually pH 4 to 1
1, preferably at pH 5-10.

It is also possible to use a membrane reactor or the like.
After the reaction, the cyclic amino acid produced by this reaction is separated by centrifuging, membrane treatment, etc. after the reaction, and then extracted with an organic solvent such as ethyl acetate and toluene, distillation, column chromatography, It can be performed by appropriately combining crystallization and the like.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in more detail, this invention is not limited to this.

<Enzymatic purification of N-methyl-L-amino acid dehydrogenase derived from Pseudomonas putida ATCC 12633 strain>
The enzyme activity of N-methyl-L-amino acid dehydrogenase was measured by the following method.
Sodium phenylpyruvate (final concentration 15 mM), methylamine-sulfuric acid buffer (pH 10) (final concentration 400 mM) and NADPH (final concentration 10 mM) were added to the crude enzyme solution to be measured to make a total volume of 50 μL. The reaction was carried out at 37 ° C. for 1 hour. After completion of the reaction, 5 μL of 10% trichloroacetic acid solution was added and centrifuged at 13000 rpm for 5 minutes. 10 μL of the centrifugal supernatant was diluted with 40 μL of high-performance liquid chromatography (hereinafter abbreviated as HPLC) eluent, filtered through a 0.45 μm filter, and analyzed by high-performance liquid chromatography (hereinafter abbreviated as HPLC).

The HPLC conditions are as follows.
Column: Ultron ESPh-CD (Shinwa Chemical Co., Ltd.)
Temperature: 40 ° C
Eluent: 20% acetonitrile, 80% 20 mM KH ₂ PO ₄ , H ₃ PO ₄ 0.
4 ml / L (pH 3)
Flow rate: 0.85 ml / min.
Detector: UV detector (210 nm)
Using a reagent from Sigma as a standard sample, 1 unit of enzyme activity was defined as the amount of enzyme that produced 1 μmol of N-methyl-phenylalanine per minute.
(1-1) Preparation of crude enzyme solution 100 ml of sterilized liquid medium (methylamine hydrochloride 5 g / L, glucose 1 g / L, yeast extract 5 g / L, dipotassium hydrogen phosphate 7 g / L, potassium dihydrogen phosphate 3 g / L, containing magnesium sulfate heptahydrate 0.1g / L) x 2 (500ml Sakaguchi flask) was inoculated with Pseudomonas putida ATCC 12633 and cultured with aerobic shaking at 28 ° C for 18 hours. (Pre-culture). Next, 500 ml × 4 sterilized liquid media (2 L Sakaguchi flask) having the same composition were inoculated with 50 ml of each 2 L Sakaguchi flask of the culture solution obtained in the previous culture. The culture was performed with shaking at 28 ° C. for 8 hours under aerobic conditions (pre-culture). Sterilize 200 L of medium (hereinafter abbreviated as LB medium) supplemented with 1% polypeptone (manufactured by Nacalai Tesque), 0.5% yeast extract (manufactured by Nacalai Tesque), and 1% sodium chloride (manufactured by Nacalai Tesque). All of the 2200 ml of the culture solution obtained in the preculture was inoculated and aerobically cultured at 28 ° C. for 16 hours (main culture). The culture solution obtained after the culture was centrifuged to obtain 2.5 kg of wet cells. The cells were suspended in 5 L of 20 mM Tris-HCl buffer (pH 7.0), and 5.9 L of a crude enzyme solution was obtained by ultrasonic disruption.

When ammonium sulfate fractionation was performed on the crude enzyme, the fraction with ammonium sulfate concentration of 20 to 60% was active. This fraction was collected and dialyzed 5 times against 15 L of 20 mM Tris-HCl buffer (pH 7.0). The amount of enzyme solution was 3100 ml.
(1-2) Purification by SuperQ-TOYOPEARL (TOSOH) SuperQ-TOYOPEARL (TOSO) equilibrated with 20 mM Tris-HCl buffer (pH 7.0)
700 mL of H) was supplied with 3100 mL of an ammonium sulfate fraction 20-60% saturated fraction. Thereafter, washing was performed with 4900 mL of 20 mM Tris-HCl buffer (pH 7.0). No activity was detected in the washed fraction.

After the washing, 20 mM Tris-HCl buffer 3 containing 0.2 M sodium chloride
Elute with 500 mL. Under these conditions, the active protein was eluted. Further, elution was performed with 3500 mL of 20 mM Tris-HCl buffer containing 0.5 M sodium chloride, but no activity was detected in this fraction.
The fraction eluted with 20 mM Tris-HCl buffer containing 0.2 M sodium chloride was collected, and the amount of protein and enzyme activity were measured. 3900 mL of the solution that showed activity was dialyzed 3 times with 12 L against 20 mM Tris-HCl buffer (pH 7.0) containing 20% saturated ammonium sulfate. The enzyme solution after dialysis was 3000 mL.
(1-3) Purification by Butyl-TOYOPEARL (manufactured by TOSOH) Butyl- equilibrated with 20% tris-hydrochloric acid buffer (pH 7.0) containing 20% saturated ammonium sulfate from 3000 mL of the enzyme solution obtained in (1-2) above. The solution was poured into 500 mL of TOYOPEARL (manufactured by TOSOH). The 20% saturated ammonium sulfate-containing 20 mM Tris-HCl buffer (pH 7.0) was washed with 2800 mL. When the protein amount and enzyme activity of the washed fraction were measured, the activity was detected in this fraction. Ammonium sulfate was directly added to the enzyme solution obtained by washing, and a fraction having an ammonium sulfate concentration of 5 to 20% saturation was obtained and flowed again into Butyl-TOYOPEARL.
(1-4) Second purification with Butyl-TOYOPEARL (manufactured by TOSOH) Butyl obtained by equilibrating 5700 mL of the enzyme solution of the obtained ammonium sulfate fraction with 20 mM Tris-HCl buffer (pH 7.0) containing 30% saturated ammonium sulfate -It flowed in 500 mL of TOYOPEARL (made by TOSOH). Thereafter, washing was performed with 2300 mL of 30 mM saturated ammonium sulfate-containing 20 mM Tris-HCl buffer (pH 7.0). When the protein amount and enzyme activity of the washed fraction were measured, the activity was detected in this fraction. Subsequently, this column was eluted with 20 mM Tris-HCl buffer (pH 7.0) containing 15% saturated ammonium sulfate. As a result, activity was also detected in the eluate. Both fractions showing activity were collected, 3070 g of ammonium sulfate was added to a saturation concentration of 60%, and the mixture was stirred and concentrated. As a result, the enzyme solution became 600 mL. This was dialyzed 3 times with 14 L of 20 mM Tris-HCl buffer (pH 7.0), and the resulting enzyme solution was 1000 mL.
(1-5) Purification by DEAE-TOYOPEARL (TOSOH) DEAE-TOYOPEARL (TOSOH) equilibrated with 20 mM Tris-HCl buffer (pH 7.0) from 1000 mL of the enzyme solution obtained by dialysis in (1-4) above. 600 mL). Thereafter, washing was performed with 4000 mL of 20 mM Tris-HCl buffer (pH 7.0). No activity was observed in the washed fraction. Subsequently, elution was performed with 2500 mL of 20 mM Tris-HCl buffer (pH 7.0) containing 0.1 M sodium chloride. As a result, almost no activity was detected in this fraction. Next, the protein was eluted by applying a gradient so that the sodium chloride concentration was 0.1 to 0.3M. When the fraction having enzyme activity was collected, 1700 mL of enzyme solution was obtained. To this enzyme solution, 760 g of ammonium sulfate was added, stirred and concentrated. As a result, the enzyme solution became 60 mL. This was dialyzed twice with 8 L of 20 mM Tris-HCl buffer (pH 7.0). After dialysis, the enzyme solution became 100 mL.
(1-6) Purification by Green-sepharose CL-4B 150 ml of a commercially available swollen Sepharose CL-4B gel was placed on a glass filter and washed with suction using 1 L of distilled water. This was transferred to a 2 L Sakaguchi flask. To this, 0.75 g of Reactive Green 19 (manufactured by Sigma) dissolved in 150 mL of water (ratio of 7.5 mg dye / mL gel) was added. Further, 15 mL of a 22% aqueous sodium chloride solution (final concentration: about 2%) was added, and the mixture was shaken and stirred for about 30 minutes until the gel and the pigment were well mixed. 1.5 g of crystalline sodium carbonate was added and stirred overnight at 50 ° C. After completion of the reaction, the gel suspension is transferred to a glass filter, and coloring of the filtrate is not observed in the order of water (about 1.5 L), 1M sodium chloride aqueous solution (about 1.5 L), and water (about 3 L). The dye gel was washed until Green-sepharose CL-4B was prepared.

After 450 mL of Green-sepharose CL-4B prepared by the above method was equilibrated with 20 mM Tris-HCl buffer (pH 7.0), 100 mL of the enzyme solution obtained in (1-5) above was allowed to flow.
Thereafter, washing was performed with 3000 mL of 20 mM Tris-HCl buffer (pH 7.0). In the washed fraction, an activity of about 15% of the total activity of the enzyme solution obtained in (1-5) above was detected. Next, the protein was eluted by applying a gradient so that the sodium chloride concentration was 0 to 3M. When fractions having enzyme activity were collected, 231 mL of enzyme solution was obtained. To this, 109 g of ammonium sulfate was added to a 70% saturation concentration to precipitate the protein. The precipitate was suspended in 3 mL of 20 mM Tris-HCl buffer (pH 7.0), and dialyzed against 9 L of 20 mM Tris-HCl buffer (pH 7.0) containing 20% saturated ammonium sulfate for 8 hours. Since a precipitate was observed in the tube after dialysis, 8.5 mL of the same buffer as in dialysis was added and suspended. Since the precipitate was not dissolved by this operation, the precipitate was separated from the supernatant by centrifugation. Since most of the activity was observed in the supernatant, 13 mL of the supernatant was purified with RESOURCE PHE (Amersham Bioscience). The protein was eluted by applying a gradient with 20 mL of 20 mM Tris-HCl buffer (pH 7.0) containing 20% saturated ammonium sulfate and 20 mL of 20 mM Tris-HCl buffer (pH 7.0).

The collected enzyme solution (19 mL) was concentrated to 5 mL by ultrafiltration, and then dialyzed with 20 mM Tris-HCl buffer (pH 7.0). As a result, the enzyme solution became 6.5 mL.
(1-7) Purification by Blue-sepharose 4B (Amersham Biosciences)
After equilibrating 5 mL of Blue-sepharose 4B (Amersham Biosciences) with 20 mM Tris-HCl buffer (pH 7.0), 6.5 mL of the enzyme solution obtained in (1-6) above was passed. Thereafter, washing was performed with 50 mL of 20 mM Tris-HCl buffer (pH 7.0). Further, the active fraction was eluted by applying a gradient with 50 mL of 1 M aqueous sodium chloride solution and 50 mL of 20 mM Tris-HCl buffer (pH 7.0) so that the sodium chloride concentration was 0 to 1 M.

  Table 1 summarizes the specific activities of the enzyme fractions in the purification steps (1-1) to (1-7) above.

<Analysis of SDS-PAGE and partial amino acid sequence>
All samples of each enzyme purification step were subjected to SDS-polyacrylamide gel electrophoresis.
About 35-40 kDa, which increases in quantity with purification, was cut out by a conventional method, and the amino acid sequence was analyzed by Edman method using a protein sequencer to determine the N-terminal amino acid sequence. This sequence is represented by SEQ ID NO: 3 in the sequence listing.

  The BLAST search suggested that the protein had high homology with malate dehydrogenase (Accession No. AE004091).

<Cloning of N-methyl-L-amino acid dehydrogenase gene>
Chromosomal DNA was prepared from cells obtained by culturing Pseudomonas putida ATCC12633 strain in LB medium using DNeasy Tissue Kit (Qiagen).
Primers were synthesized based on the N-terminal amino acid sequence determined in Example 2 and the base sequence encoding the amino acid sequence of malate dehydrogenase (Accession No. AE004091) found by BLAST search. The respective nucleotide sequences are shown in SEQ ID NO: 4 (NMPDHf) and 5 (NMPDHr1) in the sequence listing.

Pseudomonas putida ATCC12633 chromosomal DNA was used as a template, NMPDHf and NMPDHr1 were used as primers, and PCR (98 ° C., 20 seconds, 68 ° C., 3 minutes) was performed for 30 cycles to obtain specific amplification samples.
The DNA fragment obtained above was introduced into a cloning vector pET21a (Takara Shuzo Co., Ltd.) according to a conventional method. Hereinafter, this plasmid is referred to as pENMadh strain.

Next, Escherichia coli BL21 (DE3) (Novagen) was transformed.
The transformed strain was grown at 37 ° C. on an LB medium plate (LB medium + 2% agar) containing ampicillin (100 μg / mL). Several white colonies that appeared were cultured and plasmids were extracted under normal conditions. The obtained plasmid was cleaved with restriction enzymes NdeI and HindIII (3 hours at 37 ° C.), and it was confirmed by agarose electrophoresis whether the target DNA fragment was inserted into the plasmid.

A colony that is considered to have the target DNA fragment inserted is cultured in a liquid LB medium containing ampicillin, IPTG is added to 0.1 mM at 14 hours of culture, and further cultured for 3 hours. Fungus. The bacterial cells were sonicated to obtain a crude enzyme.
The activity of the obtained crude enzyme was measured to confirm the expression of the target protein.
When the crude enzyme obtained from the colony containing the target fragment was used, the production of N-methyl-L-phenylalanine was confirmed. N-methyl-L-phenylalanine production was observed in the disrupted cells transformed with the plasmid alone. There wasn't.

The nucleotide sequence of the inserted fragment in the plasmid whose activity was confirmed was analyzed. The base sequence of the gene is shown in SEQ ID NO: 2 in the sequence listing, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 1 in the sequence listing.
In the amino acid sequence (SEQ ID NO: 1) of the protein encoded by the obtained DNA fragment, the sequence from the 3rd amino acid to the 18th amino acid is the purified enzyme N-methyl-L-amino acid dehydrogenase determined in Example 2 Completely matched the N-terminal sequence (SEQ ID NO: 3). From this, it was confirmed that the DNA fragment obtained in this example encoded the N-methyl-L-amino acid dehydrogenase purified in Example 1.

<N-methyl-L-amino acid dehydrogenase enzyme purification from transformant>
5 mL of LB medium was placed in a test tube and sterilized with steam at 121 ° C. for 20 minutes. After reaching room temperature, 5 μL of 100 mg / ml ampicillin aqueous solution was added. The colony of the Escherichia coli clone obtained in Example 3 was aseptically inoculated with a platinum loop and cultured with shaking at 37 ° C. for 24 hours (preculture). Next, 500 mL of LB medium was put in a 2 L Sakaguchi flask and sterilized. After reaching room temperature, 500 μL of 100 mg / mL ampicillin aqueous solution was added. These were inoculated with 0.5 mL of the culture solution of the Escherichia coli clonal strain obtained in the preculture, and cultured with shaking at 37 ° C. for 14 hours. At this stage, 500 μL of 1M IPTG aqueous solution was added, and further cultured with shaking at 37 ° C. for 3 hours. After incubation, the cells were collected by centrifugation and washed twice with 20 mM Tris-HCl buffer (pH 7.0). 3.3 g of the obtained microbial cells were suspended in 28 mL of 20 mM Tris-HCl buffer (pH 7.0) (total amount: 30 ml), and the microbial cells were crushed with ultrasonic waves. The cell fragment was removed by centrifugation to obtain 29 mL of a cell-free extract. Next, purification was performed using Green sepharose CL-4B used in Example 1.

Green sepharose CL-4B (100 ml of resin) was equilibrated with Tris-HCl buffer (pH 7.0), and then the above cell-free extract was poured.
Thereafter, washing was performed with 800 mL of 20 mM Tris-HCl buffer (pH 7.0). The washed fraction was then eluted with a gradient so that the sodium chloride concentration was 0-1 M. Fractions having enzyme activity were collected and concentrated with Centriprep (Amersham Biosciences). This was dialyzed against 20 mM Tris-HCl buffer (pH 7.0) for 8 hours. After dialysis, the amount of enzyme solution was 59 ml.

Next, purification by DEAE-TOYOPEARL (manufactured by TOSOH) was performed.
The enzyme solution obtained by dialysis was subjected to DEAE-TOYOPEARL (TOSOH) (resin 60 mL) equilibrated with 20 mM Tris-HCl buffer (pH 7.0). Thereafter, washing was performed with 500 mL of 20 mM Tris-HCl buffer (pH 7.0). Next, the protein was eluted by applying a gradient so that the sodium chloride concentration was 0 to 0.35M. Fractions having enzyme activity were collected, concentrated with Centriprep (Amersham Bioscience), and then dialyzed against 20 mM Tris-HCl buffer (pH 7.0). After dialysis, the enzyme solution became 6 mL.

  Table 2 summarizes the specific activity ratios of the enzyme fractions in each of the above purification steps.

<Substrate specificity of N-methyl-L-amino acid dehydrogenase>
A purified enzyme was obtained from the Escherichia coli clonal strain in the same manner as in Example 4.
The various keto acids shown in Table 3 were added at a final concentration of 10 mM, NADPH was added at a final concentration of 0.2 mM, and sulfuric acid was added with
340 nm of a reaction solution in which methylamine adjusted to H10 was added to a final concentration of 60 mM.
The enzyme activity was measured by examining the change in absorbance. The reaction temperature during the measurement was 37 ° C. The results of relative activity are shown in Table 3, assuming that β-phenylpyruvate is used as the substrate as 100%.

<Measurement of optimum pH>
A purified enzyme was obtained from the Escherichia coli clonal strain in the same manner as in Example 4.
To this was added 340 nm of β-phenylpyruvic acid to a final concentration of 10 mM, NADPH to a final concentration of 0.2 mM, and methylamine adjusted to each pH with sulfuric acid to a final concentration of 60 mM.
The enzyme activity was measured by examining the change in absorbance. The reaction temperature during the measurement was 37 ° C. FIG. 1 shows the results of the relationship between the number of units (u / mg) per protein amount and pH, assuming that the amount of enzyme that reacts with 1 micromole of β-phenylpyruvic acid per minute is 1 unit. The optimum pH for the reaction was 10.0.

<Optimum temperature for action>
Of the reaction conditions similar to those in Example 5, methylamine-sulfuric acid (pH 10) was used, and the activity was measured by changing only the temperature. The results are shown in FIG. The optimum temperature was 30-40 ° C.

<PH stability>
The purified enzyme obtained in Example 4 was incubated at 30 ° C. for 30 minutes while changing the pH using a buffer solution, and the residual activity was measured. The reaction conditions were the same as in Example 7, methylamine-sulfuric acid (pH 10) at 37 ° C. The results are shown as residual activity with the untreated activity as 100, and are shown in FIG. The enzyme according to the invention was most stable at pH 6-9.

<Temperature stability>
The purified enzyme obtained in Example 5 was allowed to stand at 25 ° C., 30 ° C., 35 ° C., 40 ° C., 45 ° C. and 50 ° C. for 30 minutes, and the activity was measured in the same manner as in Example 6. The results are shown in FIG. 4 as residual activity with the untreated (left in ice) activity as 100.
The enzyme according to the present invention showed 100% residual activity up to 30 ° C.

<Substrate specificity when NADH is used as a coenzyme>
A purified enzyme was obtained from the Escherichia coli clonal strain in the same manner as in Example 4.
To this, various keto acids shown in Table 4 were added at a final concentration of 40 mM (note that β-phenylpyruvic acid was 30 mM), NADH was added at a final concentration of 0.3 mM, bis-Tris propane buffer (pH 10.0) was added at a final concentration of 100 mM, and methylamine was added. The enzyme activity was measured by examining the change in absorbance at 340 nm of the reaction solution added to a final concentration of 180 mM. The reaction temperature during the measurement was 37 ° C. The results of relative activity are shown in Table 4 with 100% of pyruvate as a substrate.

<Measurement of optimum pH when NADH is used as a coenzyme>
The purified main enzyme was obtained from the Escherichia coli clonal strain in the same manner as in Example 4.
The enzyme activity was determined by examining the change in absorbance at 340 nm of the reaction solution in which pyruvic acid was added to a final concentration of 80 mM, NADH was adjusted to a final concentration of 0.3 mM, and methylamine adjusted to each pH with sulfuric acid was added to a final concentration of 180 mM. It was measured. The reaction temperature during the measurement was 37 ° C. FIG. 5 shows the results of the relationship between the number of units (u / mg) per protein amount and pH, where the amount of enzyme that reacts with 1 micromole of pyruvic acid per minute is 1 unit. The optimum pH for the reaction was 9.5.

<Examination of reverse reaction using NAD as coenzyme>
The purified main enzyme was obtained from the Escherichia coli clonal strain in the same manner as in Example 4.
Investigate changes in absorbance at 340 nm of a reaction solution in which N-methyl-L-alanine is added to a final concentration of 50 mM, NAD is added to a final concentration of 10 mM, and bis-Tris propane buffer (pH 10.0) is added to a final concentration of 100 mM. The enzyme activity was measured by The reaction temperature during the measurement was 37 ° C. The activity was 7.8 × 10 ⁻³ (u / mg protein).

<Substrate specificity of this enzyme when NADPH is used as a coenzyme>
The purified main enzyme was obtained from the Escherichia coli clonal strain in the same manner as in Example 4.
Enzyme activity was determined by examining the change in absorbance at 340 nm of the reaction solution in which various amines shown in Table 5 were added to a final concentration of 60 mM, NADPH to a final concentration of 0.2 mM, and β-phenylpyruvic acid to a final concentration of 10 mM. Was measured. The reaction temperature during the measurement was 37 ° C. The results of relative activity are shown in Table 5, with methylamine as the substrate as 100%.

<Co-reaction with D-amino acid oxidase (1)>
Purification of E. coli clonal strain This enzyme was obtained in the same manner as in Example 4.
DL-pipecolic acid (manufactured by Tokyo Chemical Industry Co., Ltd.) to a final concentration of 50 mM, final concentration of 100 mM
0.052 units of D-amino acid oxidase (derived from porcine kidney manufactured by Sigma) was added to 100 μl of the reaction solution in which NADPH and Tris-HCl buffer (pH 9) were added to a final concentration of 100 mM. At this time, the amount of the protein of the present enzyme in the reaction solution was adjusted to 26.5 μg. The reaction temperature during the measurement was 30 ° C.

After 900 minutes, 25 μl of trichloroacetic acid was added to the reaction solution to a final concentration of 2% to complete the reaction.
The reaction solution was analyzed by HPLC under the following conditions.
Column: CHIRALPAK WE 250 x 4.6mm (Daicel)
Eluent: 2 mM CuSO ₄
Flow rate: 0.5 ml / min
Temperature: 50 degrees Detection: UV254nm
As a result of the measurement, D-pipecolic acid was completely lost, and L-pipecolic acid was produced at a concentration of 45 mM.

<Co-reaction with D-amino acid oxidase (2)>
The same procedure as in Example 14 was performed except that D-proline was used instead of DL-pipecolic acid. After 600 minutes, D-proline disappeared completely and 45 mM of L-proline was produced.

<Cloning of glucose dehydrogenase gene from Bacillus subtilis>
Bacillus subtilis (Bacillus subtilis strain) was cultured on LB medium to prepare bacterial cells. Chromosomal DNA was prepared from the cells using a Qiagen kit (Qiagen) according to the method described in the attached manual.
In order to regenerate NADPH, a Bacillus subtilis-derived glucose dehydrogenase (hereinafter sometimes abbreviated as “GDH”) gene described in the literature (J. Bacteriol. 166, 238-243 (1986)) was cloned. Based on the nucleotide sequence described in the literature, the primer BsuG # S (SEQ ID NO: 6) based on the 5'-end and 3'-end sequences of the structural gene for PCR cloning of only the open reading frame part of the GDH gene, BsuG # A (SEQ ID NO: 7) was synthesized. Using the Bacillus subtilis chromosomal DNA prepared above as a template, PCR (94 ° C, 30 seconds, 54 ° C, 30 seconds, 72 ° C 1 minute) was performed for 30 cycles,
Specific amplified DNA was obtained. The obtained DNA fragment was digested with two types of restriction enzymes, EcoRI and HindIII. Plasmid vector pKK223-3 (Amersham-Pharmacia) was digested with EcoRI and HindIII, and the PCR amplified DNA fragment was ligated with T4 DNA ligase to obtain pKK223-3GDH. As a result of analyzing the base sequence of the inserted fragment, all the base sequences recorded in the database (DDBJ Accession No. M12276) matched. The base sequence of the obtained GDH gene is shown in SEQ ID NO: 8.

<Construction of GDH plasmid pSTV28-GDH capable of co-expression with N-methyl-L-amino acid dehydrogenase>
PKK223-3GDH constructed in Example 16 was digested with two types of restriction enzymes, EcoRI and PstI, to prepare a fragment containing the Bacillus subtilis GDH gene. Plasmid vector pSTV28 (manufactured by TAKARA) was used for EcoRI and PstI
The fragment containing GDH was ligated with T4 DNA ligase to obtain pSTV28-GDH.

<Coexpression of Bacillus subtilis-derived GDH and N-methyl-L-amino acid dehydrogenase in E. coli>
The Escherichia coli BL21 (DE3) clonal strain carrying pENMadh constructed in Example 2 was transformed with pSTV28-GDH. The recombinant Escherichia coli was inoculated into a liquid LB medium containing 100 ug / mL ampicillin and 25 ug / mL chloramphenicol and cultured for 17 hours. Then, 1 mM IPTG was added, and further cultured for 3 hours. After collection, the cells were suspended in 20 mM Tris-HCL (pH 7.0), and the enzyme activity of the crude cell extract obtained by ultrasonic disruption was measured.
(1) Measurement of N-methyl-L-amino acid dehydrogenase activity in the co-expression strain N-methyl-L-amino acid dehydrogenase activity in the co-expression strain was measured with 100 mM bis-trispropane buffer (pH 10.0), 0.2 mM NADPH, 30 mM. The reaction was performed at 30 ° C. with a reaction solution containing methylamine and 10 mM sodium pyruvate. 1 U was defined as the amount of enzyme that oxidizes 1 μmol of NADPH per minute under the above reaction conditions. As a result, it was 6.6 U / mg protein.
(2) Measurement of glucose dehydrogenase activity in co-expression strains Glucose was added to 10 μl of crude enzyme to a final concentration of 100 mM, NADP at a final concentration of 2 mM, and Tris-HCl buffer (pH 9.0) at a final concentration of 100 mM. The enzyme activity was measured by examining the change in absorbance at 340 nm of 1 ml of the reaction solution. The reaction temperature during the measurement was 30 ° C. The number of units per protein amount (u / mg) was determined with the amount of enzyme that reacts with 1 micromole of glucose per minute as 1 unit. As a result, it was 3.4 U per 1 mg protein.

<Synthesis of L-pipecolic acid using glucose dehydrogenase / N-methyl-L-amino acid dehydrogenase co-expression transformant>
The Escherichia coli obtained in Example 18 was cultured in the same manner as in Example 18 to obtain 100 ml of a culture solution. After culturing, the cells were collected by centrifugation, and washed twice with 40 ml of 20 mM Tris-HCl buffer (pH 7.0) containing 0.85% sodium chloride to obtain resting cells.
The resting cells were frozen once at -80 degrees. To the frozen cells, 4 ml of 20 mM Tris-HCl buffer (pH 7.0) was added to thaw the cells and stir vigorously. PMSF was added to this so as to have a final concentration of 1 mM, and sonicated to obtain a crude enzyme solution having a protein concentration of 15 g / L after centrifugation.
The resulting crude enzyme solution was added to a final concentration of 1% L-lysine (manufactured by Kishida Chemical Co., Ltd.), final concentration of 0.2 mM NADP, final concentration of 100 mM glucose, and final concentration of 1 mM FAD (manufactured by Nacalai Tesque). Add a reaction solution containing L-Lysine oxidase with a concentration of 1.5 U / ml (manufactured by Seikagaku Corporation), catalase with a final concentration of 14 u / ml (manufactured by Sigma) and a final concentration of 100 mM Tris-HCl buffer (pH 7.5). The protein amount of the crude enzyme was adjusted to 1.5 g / L. The total volume was 10 ml. The reaction was carried out at 30 ° C. with stirring while adjusting the pH to 5.1 to 7.6 with a 10N aqueous sodium hydroxide solution.
After 5 hours, 0.5% L-lysine, 7 u / ml catalase and 100 mM glucose were added, and the reaction was continued for 15 hours.
The reaction solution was analyzed by HPLC under the following conditions.
Column: CHIRALPAK WE 250 x 4.6mm (Daicel)
Eluent: 2 mM CuSO ₄
Flow rate: 0.75 ml / min
Temperature: 50 degrees Detection: UV254nm
The concentration of L-pipecolic acid after the reaction for 15 hours was 14 g / L (reaction yield: 98%). Moreover, the peak of D-pipecolic acid was not seen.

<Synthesis of L-hydroxyproline using a glucose dehydrogenase / N-methyl-L-amino acid dehydrogenase co-expression transformant>
A crude enzyme solution was obtained in the same manner as in Example 19.
To the obtained crude enzyme solution, a final concentration of 10 mM cis-D-hydroxyproline (manufactured by Watanabe Chemical), a final concentration of 1 mM NADP, a final concentration of 100 mM glucose, and a final concentration of 0.1 mM FAD (manufactured by Nacalai Tesque), Final concentration 0.5 U / ml D-amino acid oxidase (derived from Porcine kidney: Wako), final concentration 50 u / ml catalase (Sigma), final concentration 100 mM Tris
A reaction solution containing a hydrochloric acid buffer (pH 8) was added, and the crude enzyme was added in an amount of 1/10. The total volume was 0.2 ml. The reaction was carried out at 30 ° C. for 24 hours. At the same time, a reaction product without a crude enzyme solution was also reacted.

The reaction solution was analyzed by HPLC under the following conditions.
Column: MCI GEL CRS10W (4.6x500mm) (Mitsubishi Chemical Corporation)
Eluent: 0.4 mM CuSO4
Flow rate: 0.5 ml / min
Temperature: 40 degrees Detection: UV254nm
As a result, 9.5 mM L-hydroxyproline was produced when the crude enzyme solution was added. No L-hydroxyproline was produced in the case where the crude enzyme solution was not added.

<Synthesis of L-pipecolic acid using glucose dehydrogenase / N-methyl-L-amino acid dehydrogenase co-expression transformant>
It carried out similarly to Example 20 with D-lysine (made by Tokyo Chemical Industry), DL-pipecolic acid (made by Tokyo Chemical Industry), and D-ornithine (made by Tokyo Chemical Industry).
5.9 mM L-pipecolic acid was produced from D-lysine. No production was seen in those without the crude enzyme.

  10 mM of L-pipecolic acid was produced from DL-pipecolic acid. No production was seen in those without the crude enzyme.

<Synthesis of L-proline using glucose dehydrogenase / N-methyl-L-amino acid dehydrogenase co-expression transformant>
6.8 mM L-proline was produced from D-ornithine. However, production was also observed in the case where the crude enzyme was not added, and 6.1 mM L-proline was produced. This is because Δ1-pyrrolidine 2-carboxylic acid produced by D-amino acid oxidase naturally becomes racemic proline, and D-proline is again subjected to the action of D-amino acid oxidase to become Δ1-pyrrolidine 2-carboxylic acid. Proline is thought to have accumulated.

<Synthesis of an optically active cyclic amino acid containing a sulfur atom in the ring and an optically active cyclic amino acid containing an oxygen atom in the ring from a diamino acid>
A crude enzyme solution was obtained in the same manner as in Example 19.
L-lysine (manufactured by Kishida Chemical Co., Ltd.), aminoethyl-L-cysteine (manufactured by ICN), or (S)-(+)-2-amino-3- (2-aminoethoxy) propanoic acid monohydrochloride with a final concentration of 10 mM Salt ((S)-(+)-2-Amino-3- (2-amino ethoxy) propanoic acid monohydrochrolide)
(Aldrich), final concentration 5.4 mM NADP, final concentration 108 mM glucose, final concentration 0.1 mM FAD (Nacalai Tesque), final concentration 0.8 U / ml L-Lysine oxidase (manufactured by Seikagaku Corporation) ), Final concentration of 54 u / ml catalase (Sigma), and final concentration of 100
The reaction mixture containing mM Tris-HCl buffer (pH 8.2) was added with an amount of the crude enzyme of 1/10 to make a total volume of 0.2 ml. The reaction was carried out at 30 ° C. for 24 hours. At the same time, a reaction without a crude enzyme solution was also reacted as a control.

The reaction solution was analyzed by HPLC under the following conditions.
Column: MCI GEL CRS10W (4.6x500mm) (Mitsubishi Chemical Corporation)
Eluent: 0.4 mM CuSO4
Flow rate: 0.5 ml / min
Temperature: 40 degrees Detection: UV254nm
As a result, 9.9 mM of L-pipecolic acid was produced from L-lysine. No production was seen in the control without addition of the crude enzyme.

A peak was generated at a retention time of 12.1 minutes when aminoethylcysteine was added to the crude enzyme solution. This peak was not generated in the case where the crude enzyme solution was not added.
This product is considered to be L-3-thiomorpholine carboxylic acid (R-3-thiomorpholine carboxylic acid) which is an L-form cyclic amino acid generated by the following reaction formula.

In the case where 2-amino-3- (2-aminoethoxy) propanoic acid was added with the crude enzyme solution, a peak was generated at a retention time of 7.8 minutes. This peak was not generated in the case where the crude enzyme solution was not added.
This product is considered to be L-3-morpholine carboxylic acid which is an L-form cyclic amino acid generated by the following reaction formula.

<Confirmation of product by LC-Mass>
In order to confirm 3-thiomorpholine carboxylic acid and 3-morpholine carboxylic acid produced in Example 23, purification and analysis using LC-Mass were performed.
The conditions for separating cyclic amino acid and raw material amino acid were examined by silica gel TLC (Merck 1.05715). When methanol: water: acetonitrile = 1: 1: 4 was used as the developing solution, the raw material amino acid remained at the origin. It was found that cyclic amino acids were developed and can be separated efficiently. In addition, the cyclic amino acids became distinctive colors due to the color developed by ninhydrin and could be distinguished from ordinary amino acids. For example, under this condition, pipecolic acid has a purple color with Rf = 0.22, and proline has a yellow color with Rf = 0.20. When the reaction solution of aminoethylcysteine of Example 23 was analyzed under these conditions, a light blue colored spot was observed at Rf = 0.32. When the reaction solution of 2-amino-3- (2-aminoethoxy) propanoic acid was analyzed under these conditions, a magenta colored spot was observed at Rf = 0.23.

All the reaction solutions were spotted on a PLC plate (Merck ART13793), sufficiently dried, and then developed with a developing solution having a composition of methanol: water: acetonitrile = 1: 1: 4. A part of the plate was cut out and developed with ninhydrin to confirm the position of the target. The relevant part was scraped off and eluted with methanol, the silica gel was removed with filter paper, and the methanol eluate was concentrated. The concentrate is dissolved in a small amount of water, acidified with hydrochloric acid, adsorbed on a strongly acidic cation exchange resin (SK1B manufactured by Mitsubishi Chemical) regenerated to H type, washed with distilled water, and washed with 1N ammonia water. Eluted with. The eluate was concentrated and dissolved with a small amount of methanol. After confirming whether the cyclic amino acid moiety was purified by TLC, analysis was performed by LC-Mass under the following conditions.

LC-Mass device Hewlett Packard 1100MSD
HPLC column Imtakt UK-C18 (250 x 4.6 mm)
Eluent 10% acetonitrile Flow rate 0.5 ml / min
Temperature 40 degrees Pressure 91 bar
UV210nm
Ionization voltage 20V
Ionization method API-ES method Positive ion measurement mode

When the sample purified from the reaction solution of aminoethylcysteine was analyzed, the peak of retention time 6.8 showed molecular ion m / z 148.1, and since one proton was added, the molecular weight of the compound was 147.1, Consistent with the molecular weight of 3-thiomorpholine carboxylic acid.
The sample purified from the 2-amino-3- (2-aminoethoxy) propanoic acid reaction solution has a retention time of 5.7 peak with molecular ion m / z 132.1, and one proton is added. The molecular weight of was 131.1, which was consistent with the molecular weight of 3-morpholinecarboxylic acid.

<Production reaction of [1,4] thiazepane-3-carboxylic acid from aminopropyl L-cysteine>
Aminopropyl-L-cysteine was synthesized from L-cysteine and bromopropylamine using a known method (DE2217895).
The reaction was carried out in the same manner as in Example 23. This was analyzed by TLC and LC-Mass as in Example 24. In TLC, the color was magenta with Rf = 0.30. In LC-Mass, a peak of molecular ion m / z 162.10 was confirmed at rt = 6.9. This is consistent with the addition of a proton to the molecular weight 161.10 of [1,4] thiazepan-3-carboxylic acid.

<Identification of [1,4] thiazepane-3-carboxylic acid>
A crude enzyme solution was obtained in the same manner as in Example 19.
Final concentration of 85 mM aminopropyl Lcysteine, final concentration of 0.1 mM FAD (Nacalai Tesque), final concentration of 0.65 U / ml L-Lysine oxidase (Seikagaku), final concentration of 123 u / ml catalase (Sigma) Manufactured), and a final concentration of 100 mM Tris-HCl buffer (pH 8.2)
10 ml of the reaction solution containing was stirred at room temperature for 5 hours. 1 ml of crude enzyme, NADP with a final concentration of 1 mM, and glucose with a final concentration of 233 mM were added thereto. The reaction was carried out at 28 ° C. for 3 days.

To this was added 10 ml of methanol, 30 ml of acetonitrile and 1 g of activated carbon, and the mixture was stirred well and centrifuged. The supernatant was filtered through a 0.20 μm filter and concentrated. This is the same as in Example 24.
And purified with SK1B. Acetone was crystallized in a place where the purified sample was dissolved in a small amount of methanol. The crystals were white. The crystallized sample was analyzed by ¹ H-NMR and ¹³ C-NMR.
¹ H-NMR (400 MHz, D ₂ O): δ = 4.01 (1H, t, J = 6.3 Hz), 3.19-3.45 (3H, m), 3.10 (1H, dd, J = 15.9, 6.8 Hz), 2.63 -2.82 (2H, m), 2.14 (2H, dt, J = 10.9, 5.1Hz)
¹³ C-NMR (100 MHz, D ₂ O): δ = 31.9, 34.2, 34.5, 46.3, 64.8, 175.0
This data confirmed [1,4] thiazepan-3-carboxylic acid.
In order to obtain further confirmation, high-resolution mass spectrometry was performed as follows.

Ionization method; DEI (+)
JEOL JMS-700 Mass Spectrometer
ScanMode EF
Calibration material PFK
As a result, the molecular weight was 161.0509 (Err-mmu -0.1) and the molecular composition was C6 H11 O2 N1 S1. This was a composition formula indicating the structure identified by NMR analysis.

<Identification of L-3-morpholine carboxylic acid>
A crude enzyme solution was obtained in the same manner as in Example 19.
Dissolve 159 mg of L-aminoethylserine (manufactured by Wako Pure Chemical Industries) in 6 ml of distilled water, and add 12.5 U / ml of L-Lysine oxidase (manufactured by Seikagaku Corporation) containing 1 mM FAD (manufactured by Nacalai Tesque). 5 ml, 1175 units catalase (manufactured by Sigma), and 1 ml of 1M Tris-HCl buffer (pH 8.2) were added and stirred at 28 degrees for one day. 1 ml of crude enzyme and 0.2 mM of 50 mM NADP
The reaction was performed at room temperature with addition of 0.8 ml of 50% glucose. Seven hours later, 0.4 ml of L-Lysine oxidase solution was added, and the mixture was further reacted for 5 days.

20 ml of methanol, 20 ml of acetonitrile and 1 g of activated carbon were added thereto, and the mixture was stirred well and centrifuged. The supernatant was filtered through a 0.20 μm filter and concentrated. In the same manner as in Example 24, PL
Purified with C and SK1B. Acetone was crystallized from the purified sample dissolved in a small amount of methanol. The crystals were white. The crystallized sample was analyzed by ¹ H-NMR.
¹ H-NMR (400 MHz, D ₂ O): δ = 4.04 (1H, dd, J = 11.6, 3.0 Hz), 3.82 (1H, dt, J = 12.4, 3.6 Hz), 3.55-3.67 (3H, m) , 3.13 (1H, dt, J = 13.1, 3.0Hz), 2.98 (1H, ddd, J = 13.4, 10.1, 0.9Hz)
From this data, it was confirmed to be L-3-morpholinecarboxylic acid.

<Production reaction of azepane-2-carboxylic acid from L-homolysine>
L-homolysine was synthesized using a known method (Japanese Patent Laid-Open No. 60-218400).
The reaction was carried out in the same manner as in Example 23. This was purified in the same manner as in Example 24 and analyzed by TLC and LC-Mass. TLC showed a brown color with Rf = 0.28. In LC-Mass, a peak of molecular ion m / z 145.10 was confirmed at rt = 7.17. This is consistent with the addition of protons to the molecular weight 144.10 of azepane-2-carboxylic acid. Moreover, when it measured by H-NMR, it corresponded with the value described in the well-known literature (Liebigs Ann. Chem 1989, pp11215-1232).

<Production reaction of 4-hydroxypipecolic acid from 5-hydroxy-DL-lysine>
5-Hydroxy-DL-lysine manufactured by Aldrich was used.
The reaction was carried out in the same manner as in Example 23. This was purified in the same manner as in Example 24 and analyzed by TLC and LC-Mass. In TLC, Rf = 0.26, which was a purple color similar to pipecolic acid. In LC-Mass, a peak of molecular ion m / z 146.20 was confirmed at rt = 5.64. This is consistent with the addition of a proton to the molecular weight of 145.16 of 4-hydroxypipecolic acid. Moreover, when it measured by H-NMR, it corresponded with the data described in the well-known literature (Bull. Soc. Chim. Belg. (1982) vol.91, pp.713-723).
This product is considered to be L-4-hydroxypipecolic acid, which is an L-form cyclic amino acid generated by the following reaction formula.

According to the present invention, a cyclic amino acid having a double bond at the 1-position is obtained as an intermediate from an industrially inexpensive diamino acid or a racemic cyclic amino acid, and this is used with N-methyl-L-amino acid dehydrogenase. Thus, it is possible to provide a process for producing various optically active cyclic amino acids that are industrially inexpensive and highly pure.
Optically active cyclic amino acids are 5-membered cyclic amino acids such as L-proline and L-hydroxyproline, and 6-members such as L-Pipecolic acid as shown in the chemical formula below. Amino acids such as cyclic amino acids and 4-membered cyclic amino acids such as azetidine-2-carboxylic acid are well known, and are useful substances that are attracting attention as intermediate materials for pharmaceuticals or agricultural chemicals.

Heterocyclic L-thioproline (L-Thioproline), L-morpholine carboxylic acid (L-3-Morpholine carboxylic acid), and L-thiomorpholine carboxylic acid (L-3-Thiomor)
Pholine carboxylic acid) is also useful as an intermediate material for pharmaceuticals or agricultural chemicals.

例えば、６員環であるピペコリン酸の誘導体関連の医薬品としては、下記化学式に示すパリナビル（Palinavir）、

For example, as a medicine related to a derivative of pipecolic acid which is a 6-membered ring, parinavir represented by the following chemical formula,

Selfotel shown in the following chemical formula,

Argatroban shown in the following chemical formula,

(Terence P Keenan et al, Tetrahedron asymmetry vol.10 (1999) p4331-4341).
Also, derivatives of pipecolic acid have been used as TNF-α converting enzyme inhibitors (Michael A Latavic et al, Bioorganic & Medicinal Chemistry Letters (2002) vol. 12, pp1387-1390).

  As for proline derivatives, zefenopril shown in the chemical formula below,

(J Med Chem (1988) vol.31 p1148).
In azetidine carboxylic acid derivatives, nicotianamine represented by the following chemical formula as gelatinase inhibitor (gelatinase inhibitor),

(Suzuki K et al, J antibiot (1996) vol.49 p1284-) and BMS-262084 shown in the following chemical formula, which is an asthma drug

(Sutton JC et al, Bioorg Med Chem Lett. 2002 12 (21) p3229-33).
In the heterocyclic ring, anti-inflammatory agent Z-4003 (EP0254354)

Thioproline is used in

FIG. 1 is a graph showing the optimum pH of the N-methyl-L-amino acid dehydrogenase used in the examples of this specification. FIG. 2 is a graph showing the optimum temperature of N-methyl-L-amino acid dehydrogenase used in Examples of the present specification. FIG. 3 is a graph showing the pH stability of N-methyl-L-amino acid dehydrogenase used in the examples of the present specification. FIG. 4 is a graph showing the thermal stability of the N-methyl-L-amino acid dehydrogenase used in the examples of the present specification. FIG. 5 is a graph showing the effect of pH on the N-methyl-L-amino acid dehydrogenase used in the examples herein.

Claims (7)

The following general formula (I)

(In the formula, A, a linear alkyl chain of 1 to 4 carbon atoms, or _{-CHOHCH 2 -, - CH 2 CHOHCH} 2 -, - SCH 2 -, - SC 2 H 4 -, - SC 3 H 6 _{-, - OCH 2 -, -} OC 2 H 4 -, - OC 3 H 6 - to cyclic amino acid having a double bond at position 1, represented by alkyl chains showing a) containing a heteroatom selected from the group consisting of N-methyl-L-amino acid dehydrogenase derived from Pseudomonas bacteria or cells containing the same, a preparation of the cells or a culture solution obtained by culturing the cells, and the following general formula (II)

(Wherein A has the same meaning as described above), to produce an L-form cyclic amino acid. The production method according to claim 1, wherein the N-methyl-L-amino acid dehydrogenase derived from Pseudomonas is a polypeptide shown in the following (A), (B), or (C):
(A) a polypeptide having the amino acid sequence represented by SEQ ID NO: 1;
(B) a polypeptide having an amino acid sequence in which 1 to 10 amino acids are deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 1 and having N-methyl-L-amino acid dehydrogenase activity Or (C) a polypeptide having an amino acid sequence having 95 % or more identity with the amino acid sequence represented by SEQ ID NO: 1 and having N-methyl-L-amino acid dehydrogenase activity; The production method according to claim 1, wherein the cell containing N-methyl-L-amino acid dehydrogenase is a cell transformed with a DNA encoding N-methyl-L-amino acid dehydrogenase derived from Pseudomonas bacteria . The production method according to claim 3 , wherein the DNA encoding N-methyl-L-amino acid dehydrogenase derived from a genus Pseudomonas is a DNA encoding a protein shown in the following (A), (B) or (C):
(A) a protein having the amino acid sequence set forth in SEQ ID NO: 1, or (B) an amino acid sequence having 95 % or more identity with the amino acid sequence set forth in SEQ ID NO: 1, and N-methyl-L- A protein having amino acid dehydrogenase activity.
(C) A protein comprising an amino acid sequence in which 1 to 10 amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID NO: 1 and having N-methyl-L-amino acid dehydrogenase activity. The production method according to claim 3 , wherein the DNA encoding N-methyl-L-amino acid dehydrogenase derived from a genus Pseudomonas is a DNA shown in the following (D) or (E) :
(D) a DNA having the base sequence shown in SEQ ID NO: 2; or ( E ) a base sequence in which 1 to 30 bases are substituted, deleted or added in the base sequence shown in SEQ ID NO: 2 and its complementary strand And a DNA encoding a protein having N-methyl-L-amino acid dehydrogenase activity. The following general formula (III)

(Wherein, A is as defined above), a chain α, ω-diamino acid represented by D-amino acid oxidase, L-amino acid oxidase, D-amino acid dehydrogenase, L-amino acid dehydrogenase, D-amino acid A transferase and an enzyme selected from the group consisting of L-amino acid transferase are reacted, and the following general formula (I)

A cyclic amino acid having a double bond at the 1-position obtained after producing a cyclic amino acid having a double bond at the 1-position represented by (wherein A is as defined above): the method according to any one of 1-5, the following general formula (II)

(Wherein A has the same meaning as described above), to produce an L-form cyclic amino acid. The following general formula (IV)

(Wherein A is as defined above), D-amino acid oxidase is allowed to act on the cyclic amino acid represented by the following general formula (I)

A cyclic amino acid having a double bond at the 1-position obtained after producing a cyclic amino acid having a double bond at the 1-position represented by (wherein A is as defined above): the method according to any one of 1-5, the following general formula (II)

(Wherein A has the same meaning as described above), to produce an L-form cyclic amino acid.

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