JP5005293B2 - A method for producing D-amino acid oxidase and L-amino acid, 2-oxoacid, or cyclic imine. - Google Patents

Description

  The present invention relates to novel D-amino acid oxidases produced by microorganisms, DNAs encoding them, and D-amino acid oxidases using microorganisms or transformants having the ability to produce these D-amino acid oxidases. The present invention also relates to a production method and an efficient production method of L-amino acid, 2-oxoacid or cyclic imine using D-amino acid oxidase.

  D-amino acid oxidase (enzyme number [EC1.4.3.3]) is an enzyme that oxidizes D-amino acid to produce hydrogen peroxide, 2-oxo acid, and ammonia in the presence of oxygen. This D-amino acid oxidase is known to have industrially significant activities such as stereoselective oxidation of racemic amino acids to produce L-amino acids and 2-oxo acids. L-amino acids are compounds useful as synthetic intermediates and sweeteners for pharmaceuticals and the like.

  Examples of microorganisms that produce D-amino acid oxidase include Trigonopsis variabilis (Patent Document 1), Fusarium oxysporum (Patent Document 2), and Candida tropicalis (Non-Patent Document 1). Etc., and each enzyme has been purified and isolated, and its properties have been elucidated. Regarding the expression of the D-amino acid oxidase gene in the transformant, for example, Trigonopsis variabilis (Patent Document 3), Fusarium solani (Patent Document 4), Rhodotorula gracilis (Rhodotorula gracilis) Patent Document 5), a gene derived from Candida boidinii (Non-Patent Document 2) is known. In order to produce D-amino acid oxidase with these transformants at a high rate, in many cases, a method of increasing the enzyme production by means such as adding an inducer after culturing in a state in which expression is suppressed is known. ing.

  It is known that Candida intermedia contained in the genus Candida has D-amino acid oxidase activity in the state of a crude enzyme solution (Non-patent Document 3).

  Conventionally known D-amino acid oxidases are inferior in terms of enzyme productivity and titer.

Furthermore, D-amino acid oxidase derived from porcine kidney or Trigonopsis variabilis is allowed to act on racemic amino acid in the presence of an amino acid dehydrogenase and an enzyme capable of coenzyme regeneration. Reactions which are converted into L-amino acids via oxo acids or imino acids are already known (Non-patent Documents 4 and 5).
Japanese Examined Patent Publication No. 62-501777 JP 11-318439 A JP-A-63-71180 European Patent Application Publication No. EP364275 International Publication No. WO97 / 040171 Pamphlet Some Properties of Peroxisome associated D-Amino Acid Oxidase from Candida tropicalis. Agricul. And Biol. Chemistry, 1986, 50, 10, 2637 Characterization and High-level Production of D-Amino Acid Oxidase in Candida bodyinii. Biosci. Biotechnol. Biochem., 2001, 65, 3, 627 Production of D-Amino Acid Oxidase by Candida tropicalis. J. et al. Ferment. Technol. 1977, 55, 1, 13 Enzymatic Conversion of Racemeticine to the L-Enantiomer. J. et al. Chem. Soc. Chem. Commun. 1990, 13, 947 Enzymatic synthesis of chiralintermediates for Omaparilat, an antihypertensive drug. Biomolecular Engineering, 2001, 17, 167

  The D-amino acid oxidase obtained in the present invention is an enzyme that oxidizes a D-amino acid in the presence of oxygen to produce hydrogen peroxide, 2-oxoacid and ammonia. Further, by combining this D-amino acid oxidase with an amino acid dehydrogenase, or an amino acid dehydrogenase and an enzyme having a coenzyme regeneration ability, the racemic amino acid is converted into an L-amino acid via keto acid or imino acid. It is a useful enzyme that can be used in a stereoinversion reaction that is quantitatively converted into a thiol.

  An object of the present invention is to provide a novel D-amino acid oxidase. Another object of the present invention is to clarify the amino acid sequence of the D-amino acid oxidase, the DNA sequence of the gene, a microorganism or transformant capable of producing the enzyme, and the D-amino acid oxidase using them. It is in providing the manufacturing method of. Furthermore, this invention is providing the manufacturing method of the efficient L-amino acid, 2-oxo acid, or cyclic imine using the said D-amino acid oxidase.

  In view of the above problems, the present inventors have achieved isolation of two novel D-amino acid oxidase genes from Candida intermedia and expression in host microorganisms, and breeded transformants. A 2-oxo acid or L-amino acid is produced by allowing a D-amino acid oxidase obtained in the present invention to act on a racemic amino acid alone or in combination with an enzyme having amino acid dehydrogenase and coenzyme regeneration ability. As a result, the present invention has been completed.

The present invention has one or more of the following features.
1) One feature of the present invention is the following polypeptide (a), (b) or (c):
(A) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing;
(B) a polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, inserted, deleted and / or added in the amino acid sequence shown in SEQ ID NO: 1 and having D-amino acid oxidase activity;
(C) A polypeptide comprising an amino acid sequence having 69% or more homology with the amino acid sequence shown in SEQ ID NO: 1 and having D-amino acid oxidase activity.
2) One feature of the present invention is the following polypeptide (d), (e) or (f):
(D) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing;
(E) a polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, inserted, deleted and / or added in the amino acid sequence shown in SEQ ID NO: 2 and having D-amino acid oxidase activity;
(F) A polypeptide comprising an amino acid sequence having 69% or more homology with the amino acid sequence shown in SEQ ID NO: 2 and having D-amino acid oxidase activity.
6) One feature of the present invention is the following DNA (g), (h) or (i):
(G) DNA comprising the base sequence shown in SEQ ID NO: 3 in the sequence listing;
(H) A polypeptide having a base sequence in which one or several bases are substituted, inserted, deleted and / or added in the base sequence shown in SEQ ID NO: 3 and having D-amino acid oxidase activity Encoding DNA:
(I) A DNA comprising a base sequence having 67% or more homology with the base sequence shown in SEQ ID NO: 3 and encoding a polypeptide having D-amino acid oxidase activity.
7) One feature of the present invention is the following DNA (j), (k) or (l):
(J) DNA consisting of the base sequence shown in SEQ ID NO: 4 in the sequence listing;
(K) A polypeptide having a base sequence in which one or several bases are substituted, inserted, deleted and / or added in the base sequence shown in SEQ ID NO: 4 and having D-amino acid oxidase activity Encoding DNA:
(1) DNA encoding a polypeptide having a base sequence having 67% or more homology with the base sequence shown in SEQ ID NO: 4 in the sequence listing and having D-amino acid oxidase activity.
8) One feature of the present invention is a recombinant plasmid containing the DNA.
12) One feature of the present invention is a transformant obtained by transforming a host microorganism with the recombinant plasmid.
16) One feature of the present invention is a microorganism having the ability to produce the polypeptide and belonging to the genus Candida.
17) One feature of the present invention is that D-amino acid oxidase is produced by culturing a microorganism capable of producing the polypeptide, accumulating the polypeptide in the culture, and collecting the polypeptide. Is the method.
19) One feature of the present invention is that the D-amino acid oxidase (polypeptide having D-amino acid oxidase activity), a transformant, or a microorganism,
General formula (1)

(In the formula, R has an optionally substituted alkyl group having 1 to 20 carbon atoms, an optionally substituted aralkyl group having 7 to 20 carbon atoms, or a substituent. Or an aryl group having 6 to 20 carbon atoms).
General formula (2)

(Wherein R is the same as defined above), or
General formula (3)

(Wherein, R is the same as described above).
20) One feature of the present invention is that the D-amino acid oxidase (polypeptide having D-amino acid oxidase activity), a transformant, or a microorganism,
General formula (4)

(Wherein, R ′ is an alkyl chain having 2 to 7 carbon atoms which may have a substituent).
General formula (5)

(Wherein R ′ is the same as above), or
General formula (6)

(Wherein R ′ is the same as described above).
21) One feature of the present invention is that the D-amino acid oxidase (polypeptide having D-amino acid oxidase activity), a transformant, or a microorganism is treated in the presence of an amino acid dehydrogenase and an enzyme capable of coenzyme regeneration. so,
General formula (1)

(In the formula, R has an optionally substituted alkyl group having 1 to 20 carbon atoms, an optionally substituted aralkyl group having 7 to 20 carbon atoms, or a substituent. Or an aryl group having 6 to 20 carbon atoms).
General formula (2)

(Wherein R is the same as described above).
22) One feature of the present invention is that the D-amino acid oxidase (polypeptide having D-amino acid oxidase activity), a transformant, or a microorganism is treated in the presence of an amino acid dehydrogenase and an enzyme capable of coenzyme regeneration. so,
General formula (4)

(Wherein, R ′ is an alkyl chain having 2 to 7 carbon atoms which may have a substituent), and the cyclic amino acid represented by
General formula (5)

(Wherein R ′ is the same as described above).

  This invention consists of the above-mentioned structure and can manufacture novel D-amino acid oxidase efficiently. Moreover, L-amino acid, 2-oxo acid, or cyclic imine can be efficiently produced by using the D-amino acid oxidase, a transformant that produces the D-amino acid oxidase, or a microorganism. .

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

1. Polypeptide First, a polypeptide as an embodiment of the present invention will be described. The polypeptide as an embodiment is a polypeptide having D-amino acid oxidase activity, and is capable of oxidizing a D-amino acid stereoselectively. Compared with other D-amino acid oxidases, for example, the following points are different.
(A) When compared with the D-amino acid oxidase of Trigonopsis variabilis (Japanese Examined Patent Publication No. Sho 62-501677, JP-A 63-71180) in terms of amino acid sequence, the amino acid sequence shown in SEQ ID NO: 1 has a homology of 38. 9%, the amino acid sequence shown in SEQ ID NO: 2 has 29.7% homology.
(B) D-amino acid oxidase and amino acid sequence of Candida boidinii (Biosci. Biotechnol. Biochem., 2001, 65, 3, 627, Yeast, 2000, 16, 1217) When compared, the amino acid sequence shown in SEQ ID NO: 1 has a homology of 31.1%, and the amino acid sequence shown in SEQ ID NO: 2 has a homology of 59.8%.
(C) When compared with the D-amino acid oxidase of Rhodotorula gracilis (International Publication No. WO97 / 040171, Protein expression and purification, 1998, Vol. 14, p. 289), it is shown in SEQ ID NO: 1. The amino acid sequence obtained has 28.9% homology, and the amino acid sequence shown in SEQ ID NO: 2 has 30.8% homology.
(D) When compared with the D-amino acid oxidase of Fusarium solani (European Patent Application Publication No. EP364275) in terms of amino acid sequence, the amino acid sequence shown in SEQ ID NO: 1 has a homology of 36.7%, SEQ ID NO: The amino acid sequence shown in 2 has 27.4% homology.

  The “homology” in the embodiment can be determined using a method well known to those skilled in the art, sequence analysis software, and the like. Here, as an example, GENETYX Ver. The homology search of 7 Genetic Information Processing Software / Network Edition (Genetics) was used.

2. Measurement of D-amino acid oxidase activity In an embodiment, the measurement of D-amino acid oxidase activity of a polypeptide can quantitate hydrogen peroxide produced by the reaction by an enzymatic method using peroxidase. The determination of hydrogen peroxide by the enzymatic method is carried out as follows.

  The enzyme solution was appropriately diluted, and 25 mM D-amino acid, 0.80 mM 4-aminoantipyrine, 1.31 mM N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine, 6 u / ml peroxidase ( TOYOBO Inc.) and a coloring solution consisting of 50 mM Tris-hydrochloric acid buffer (pH 8.0), mixed 1: 5, reacted at 30 ° C. for 30 minutes, measured for absorbance at 555 nm, and prepared in advance. Based on the line, the amount of hydrogen peroxide produced in the reaction solution is quantified.

3. Microorganism The polypeptide of the embodiment can be obtained from a microorganism having D-amino acid oxidase activity. Although it will not specifically limit if it is microorganisms which produce the polypeptide, For example, the microorganisms which belong to Candida (Candida) are mentioned, Especially Candida intermedia (Candida intermedia) is preferable, More preferably, Candida intermedia ( Candida intermedia) NBRC0761 strain.

  Note that the microorganism that produces the polypeptide of the embodiment may be a wild strain of the above-described microorganism, or may be a mutant strain improved in mutation. Mutant strains can be obtained by methods well known to those skilled in the art, such as UV irradiation and treatment with drugs such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and ethylmethanesulfonate (EMS). it can.

  The medium for culturing the microorganism producing the polypeptide of the present invention is not particularly limited as long as the microorganism can grow. For example, as a carbon source, carbohydrates such as glucose and sucrose, alcohols such as ethanol and glycerol, fatty acids such as oleic acid and stearic acid and esters thereof, oils such as rapeseed oil and soybean oil, and ammonium sulfate as a nitrogen source , Sodium nitrate, peptone, casamino acid, corn steep liquor, bran, yeast extract, etc. As inorganic salts, magnesium sulfate, sodium chloride, calcium carbonate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, etc. as other nutrient sources Ordinary liquid media containing malt extract, meat extract and the like can be used.

  Furthermore, a substance that enhances the production of D-amino acid oxidase, for example, an amino acid or an amino acid derivative may be added in a small amount. The concentration of these D-amino acid oxidase production-enhancing substances in the medium is selected from the range of 0.001 wt% or more and 10 wt% or less, preferably 0.01 wt% or more and 1 wt% or less.

  The culture is usually carried out aerobically, and the temperature ranges from 10 ° C. to 60 ° C., preferably from 20 ° C. to 50 ° C., and the pH ranges from 3 to 11, preferably from 5 to 9. It is used, and the culture time can be 1 day or more and 5 days or less. In addition, either a batch or continuous culture method may be used.

  After completion of the culture, the cells are collected from the culture solution by centrifugation or the like, and the cells are disrupted by means such as ultrasonic disruption to obtain a crude enzyme solution. The polypeptide of the present invention can be obtained by purifying the crude enzyme solution by a salting-out method, a column chromatography method or the like.

4). Amino acid sequence The polypeptide of the present invention may be a natural enzyme obtained from a microorganism as described above, or may be a recombinant enzyme produced using a gene recombination technique. As a natural enzyme, the polypeptide shown by sequence number 1 or 2 of a sequence table can be mention | raise | lifted.

  The polypeptide as an embodiment of the present invention comprises an amino acid sequence in which one or several amino acids are substituted, inserted, deleted and / or added in the amino acid sequence shown in SEQ ID NO: 1 or 2 in the sequence listing, And may be a polypeptide having D-amino acid oxidase activity, 69% or more of the amino acid sequence shown in SEQ ID NO: 1 or 2, preferably 70% or more, 75% or more, 80% or more, 85% or more, It may be a polypeptide having an amino acid sequence having homology of 90% or more, 95% or more, more preferably 99% or more, and having D-amino acid oxidase activity. The “several amino acids” is not limited as long as the D-amino acid oxidase activity is not lost, but is preferably 20 amino acids or less, more preferably 15 amino acids or less, still more preferably 10 amino acids or less, most preferably Preferably, it is 5, 4, 3, or 2 or less.

  The “polypeptide having D-amino acid oxidase activity” may be any polypeptide as long as D-amino acid oxidase activity can be detected under the above-mentioned activity measurement conditions, but preferably the amino acid represented by SEQ ID NO: 1 or 2 This refers to a polypeptide having an activity of 10% or more, more preferably 40% or more, more preferably 60% or more, and still more preferably 80% or more when a polypeptide comprising a sequence is used.

5. DNA
“DNA” as an embodiment of the present invention will be described. The DNA of the present invention may be any DNA that encodes a polypeptide as described above. The DNA may be the DNA represented by SEQ ID NO: 3 or 4 in the sequence listing, and 1 or several bases may be substituted, inserted, deleted and / or added in the base sequence represented by SEQ ID NO: 3 or 4 in the sequence listing. It may also be a DNA encoding a polypeptide having a base sequence and having D-amino acid oxidase activity. The term “several bases” means that the number thereof is not limited as long as the polypeptide encoded by DNA does not lose D-amino acid oxidase activity, but is preferably 50 bases or less, more preferably 30 bases or less, More preferably, it is 20 amino acids or less, and most preferably 10, 9, 8, 7, 6, 5, 4, 3, or 2 or less.

  Further, it is 67% or more, preferably 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, more preferably 99% or more with the base sequence represented by SEQ ID NO: 3 or 4. It may be a DNA encoding a polypeptide consisting of a homologous base sequence and having D-amino acid oxidase activity.

  The DNA (D-amino acid oxidase gene) of the present invention can be obtained from a microorganism having D-amino acid oxidase activity as described above. In order to obtain the target DNA, for example, the following method can be used.

  First, a DNA primer designed based on a plurality of homologous sequence sites of known D-amino acid oxidase is synthesized.

  Next, chromosomal DNA is isolated from the microorganism that is the source of D-amino acid oxidase. Chromosomal DNA is obtained from cultured cells using a method such as the Marmur method (see J. Mol. Biol., 3, 208 (1961)). Using this chromosomal DNA as a template and performing PCR using the above DNA primers, a part of the target gene can be obtained.

  Next, DNA fragments encoding the N-terminal side and the C-terminal side of the already acquired partial gene can be acquired by an inverse PCR method (see, for example, Nucleic Acids Res. 16, 8186 (1988)). The base sequence of this DNA fragment is determined and combined with the base sequence of the partial gene to obtain the full-length base sequence of the D-amino acid oxidase gene presumed to include the translation start site to the stop codon.

  When the presence of an intron is estimated in the D-amino acid oxidase gene obtained from the chromosomal DNA from the analysis of the obtained base sequence, the D-amino acid oxidase gene from which the intron has been removed is obtained by the following method. be able to.

  First, total RNA was extracted from cells obtained by culturing a microorganism that is the source of D-amino acid oxidase using ISOGEN (manufactured by Nippon Gene), and TaKaRa RNA LA PCR Kit (AMV) Ver.1.1 (TaKaRa) The complementary strand DNA (cDNA) is prepared using total RNA as a template. Primers are designed from the base sequences of the N-terminal and C-terminal portions of the full-length D-amino acid oxidase gene obtained by the inverse PCR method. Using the designed primer, PCR is carried out using cDNA as a template, and an open reading frame is determined from the full-length sequence containing no intron.

6). Transformant / vector By using the DNA obtained by the above method or a recombinant plasmid obtained by incorporating the DNA into a vector, a host microorganism can be transformed to obtain a transformant.

  As the host and vector, the host-vector system described in “Guidelines for Recombinant DNA Experiments” (Science and Technology Agency, Research and Development Bureau, Life Science Division, revised on March 22, 1996) can be used. For example, as a host, Escherichia genus, Pseudomonas genus, Flavobacterium genus, Bacillus genus Serratia genus, Corynebacterium genus, Corynebacterium genus, Brevibacterium ( Microorganisms belonging to the genus Brevibacterium, Agrobacterium, Acetobacter, Gluconobacter, Lactobacillus, Streptococcus or Streptomyces Can be used.

  As the vector, a plasmid, phage or derivative thereof derived from a microorganism capable of autonomous replication in the above host can be used. Among them, it is preferable to use Escherichia coli as a host microorganism and a vector capable of autonomous replication in the microorganism as a vector. Examples of such vectors include pUC18, pUC19, pBR322, pACYC184, pSTV28, pSTV29, pSC101, pT7Blue, or pUCNT, or derivatives thereof. These derivatives are modified genes for promoters, terminators, enhancers, SD sequences, replication initiation sites (ori), other regulation, etc., for the purpose of increasing enzyme production and stabilizing plasmids. Drug resistance, refers to a modified restriction enzyme site at the cloning site.

  As an example of a transformant, a recombinant plasmid pCDA001 or pCDA006 in which DNA obtained as described above from Candida intermedia NBRC0761 strain was incorporated into pUCNT (see International Publication No. WO94 / 03613) was used. By transforming Escherichia coli HB101 or JM109, transformants Escherichia coli HB101 (pCDA001) or JM109 (pCDA006) can be obtained. The bacteriological properties of Escherichia coli HB101 or JM109 are described in "BIOCHEMICALS FOR LIFE SCIENCE" (Toyobo Co., Ltd., 1993, pages 116-119) and various other known literatures. Is well known. Escherichia coli HB101 (pCDA001) or JM109 (pCDA006) is similar to Escherichia coli HB101 or JM109 except that it can produce a specific enzyme by genetic recombination. Have

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

7). Cultivation of transformants, etc. By culturing the transformants etc. that can produce the D-amino acid oxidase of the present invention, the enzyme can be produced in large quantities and used for the production of L-amino acids or 2-oxo acids. can do.

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

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

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

8). Production method of L-amino acid and the like An efficient production method of L-amino acid, 2-oxoacid, or cyclic imine by using D-amino acid oxidase obtained in the embodiment will be described. 2-Oxo acids can be obtained by allowing D-amino acid oxidase to act on racemic amino acids to oxidize D-amino acids (resolution reaction). In this reaction, it is presumed that imino acid is generated as an intermediate from D-amino acid to 2-oxo acid, but imino acid is immediately converted to 2-oxo acid by hydrolysis reaction, and finally Only 2-oxo acids are obtained. Cyclic imines can be obtained by allowing D-amino acid oxidase to act on cyclic amino acids. An L-amino acid can be obtained as a residue of the resolution reaction, and the 2-oxo acid, imino acid, or cyclic imine produced by the reaction is further brought into contact with an amino acid dehydrogenase and reacted. Can also be converted to an L-amino acid (stereoinversion reaction).

9. Amino acid dehydrogenase Here, the amino acid dehydrogenase is an enzyme having the activity of reductively aminating 2-oxo acid or cyclic imine. For example, phenylalanine dehydrogenase, leucine dehydrogenase, pyrroline-2- Carboxylic acid reductase can be mentioned. As the amino acid dehydrogenase used in the present invention, those derived from animals, plants and microorganisms can be used, but those derived from microorganisms are preferred for industrial use.

  Any microorganism can be used as long as it has the ability to produce the enzyme. For example, the following known microorganisms having the ability to produce the enzyme, Brevibacterium, Rhodococcus ( Rhodococcus), Sporosarcina, Thermoactinomyces, Microbacterium, Halomona, Clostridium, Bacillus, Neurospora , Escherichia, Aerobactor and the like.

  Preferably, an enzyme derived from a microorganism belonging to the genus Bacillus is used. More preferable examples include enzymes derived from Bacillus badius IAM11059 and Bacillus sphaericus NBRC3341. Bacillus badius IAM11059 is a microorganism that produces phenylalanine dehydrogenase and is disclosed in European Patent Application Publication No. EP256514 and Bisci. Biotechnol. Biochem. 1995, 59, 10, 1994.

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

  In order to obtain a highly active bacterium that efficiently and highly produces amino acid dehydrogenase, it is effective to create a transformed microorganism as is well known. As a preparation method, for example, after cloning an amino acid dehydrogenase gene from a strain exhibiting amino acid dehydrogenase activity, a recombinant plasmid with an appropriate vector is prepared and used to transform an appropriate host fungus. Can be obtained. Recombinant DNA technology is well known in the art.

10. Coenzyme regeneration system Reaction with amino acid dehydrogenase requires a reduced coenzyme such as NADH,
As the reaction proceeds, coenzyme NADH is converted to an oxidized form. An enzyme having the ability to convert this oxidized coenzyme into a reduced form (hereinafter referred to as coenzyme regeneration ability) and a compound serving as a substrate for the enzyme coexist with D-amino acid oxidase and amino acid dehydrogenase. In this way, the amount of coenzyme used can be greatly reduced.

  Examples of the polypeptide having coenzyme regeneration ability include hydrogenase, formate dehydrogenase, alcohol dehydrogenase, aldehyde dehydrogenase, glucose-6-phosphate dehydrogenase, or glucose dehydrogenase.

  Preferably, formate dehydrogenase is used. As formate dehydrogenase, those derived from plants and microorganisms can be used, but those derived from microorganisms are preferred for industrial use. Any microorganism can be used as long as it has the ability to produce the enzyme. For example, the following known microorganisms having the ability to produce the enzyme, Candida, Kloeckera (Kloeckera) ), Pichia, Lipomyces, Pseudomonas, Moraxella, Hyphomicrobium, Paracoccus, Thiobacillus, Ansilobacter There is a genus (Ancylobacter).

  Preferably, enzymes derived from microorganisms belonging to the genus Thiobacillus and the genus Ancylobacter are mentioned. More preferable examples include enzymes derived from Thiobacillus sp. KNK65MA (FERM BP-7671) and Ancilobacter aquaticus KNK607M strain (FERM BP-7335).

  In order to obtain a highly active bacterium that efficiently produces formate dehydrogenase with high efficiency, it is effective to produce a transformed microorganism as is well known. As a preparation method, for example, as described in International Publication No. WO03 / 031626, after cloning a formate dehydrogenase gene from a strain exhibiting formate dehydrogenase activity, a recombinant plasmid with an appropriate vector was prepared, It can be obtained by transforming a suitable host bacterium using this. Recombinant DNA technology is well known in the art.

  As a transformant that highly produces formate dehydrogenase thus obtained, formate dehydrogenase derived from Thiobacillus sp. KNK65MA (FERM BP-7671) described in International Publication No. WO03 / 031626 An Escherichia coli HB101 (pFT001) (FERM BP-7672) or Escherichia coli HB101 (pFT002) (FERM BP-7673) containing the gene, or WO 02/46427 Examples include Escherichia coli HB101 (pFA001) (FERM BP-7334), which contains a formate dehydrogenase gene derived from Ancylobacter aquaticus KNK607M strain (FERM BP-7335). Can.

  Production of amino acid dehydrogenase and formate dehydrogenase by these transformants, production of amino acid dehydrogenase by the aforementioned strain showing amino acid dehydrogenase activity, or formate dehydrogenase by the strain showing formate dehydrogenase activity For the production, for example, culture may be performed using a normal nutrient medium described in International Publication No. WO03 / 031626, and treatment for enzyme induction may be performed as necessary.

11. Catalase Addition Catalase may be added to the D-amino acid oxidase reaction. Even when catalase is not added, the reaction proceeds and a product can be obtained. However, the substrate or product may be decomposed by hydrogen peroxide generated by the D-amino acid oxidase reaction, which may reduce the yield. . By adding catalase, such decomposition of the substrate or product can be suppressed, and an improvement in yield can be expected. Catalase is an enzyme that catalyzes the reaction of decomposing hydrogen peroxide into water and oxygen.

  As the catalase, those derived from animals, plants and microorganisms can be used, but those derived from microorganisms are preferred for industrial use. Any microorganism can be used as long as it has the ability to produce the enzyme. For example, the following known microorganisms having the ability to produce the enzyme, such as Micrococcus, Rhodopseudomonas). Commercially available enzymes can also be used. Examples of commercially available enzymes include Catazyme 25L (manufactured by Novozyme) and CATALASE (Beef River) (manufactured by P-L Biochemcals, Inc.).

12 Transformants, etc. The D-amino acid oxidase, amino acid dehydrogenase, coenzyme regenerating enzyme, and catalase used in the present invention are produced by breeding, culturing, and cultivating transformants into which the respective enzyme genes have been introduced. The product obtained by breeding and culturing a transformant introduced with a plurality of enzyme genes may be used.

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

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

13. Production of L-amino acid and the like Production of L-amino acid, 2-oxoacid or cyclic imine by the resolution reaction of the present invention, or production of L-amino acid by steric inversion reaction can be performed by the following method.

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

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

  In the cyclic amino acid represented by the general formula (4), R ′ is an alkyl chain having 2 to 7 carbon atoms which may have a substituent. Examples of the substituent include an amino group, hydroxyl group, nitro group, cyano group, carboxyl group, alkyl group, aralkyl group, aryl group, alkanoyl group, alkenyl group, alkynyl group, alkoxyl group, or halogen atom.

14 Split reaction In the split reaction, the aforementioned D-amino acid oxidase is allowed to act on the substrate, and the reaction is carried out in an aqueous solvent. Catalase may be added to the above reaction. By adding catalase, hydrogen peroxide generated by the reaction of D-amino acid oxidase can be decomposed, and decomposition of the substrate or product can be suppressed. Further, FAD may be added to the reaction. FAD is a coenzyme of D-amino acid oxidase, and it can be expected that the reaction efficiency is improved by adding FAD. The concentration of FAD added is preferably 0 equivalents or more and 10 equivalents or less, more preferably 0 equivalents or more and 1 equivalents or less, and even more preferably 0 equivalents or more and 0.1 equivalents or less with respect to the substrate.

15. Stereo-inversion reaction In the stereo-inversion reaction, the aforementioned D-amino acid oxidase, the aforementioned amino acid dehydrogenase and the enzyme having the ability to regenerate coenzyme, and the compound serving as the substrate are simultaneously present in the substrate, and the reaction is performed in an aqueous medium. . Catalase may be added to the reaction. By adding catalase, hydrogen peroxide generated by the reaction of D-amino acid oxidase can be decomposed, and decomposition of the substrate or product can be suppressed.

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

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

  In addition, ammonia or a salt thereof may be added to the stereoinversion reaction. The concentration of ammonia added is preferably 0 equivalents or more and 10 equivalents or less, more preferably 0 equivalents or more and 2 equivalents or less, still more preferably 0.1 equivalents or more and 1.5 equivalents or less with respect to the substrate.

  The concentration of the substrate used for the resolution and the steric inversion reaction is 0.1% (w / v) or more and 90% (w / v) or less, preferably 1% (w / v) or more and 60% (w / v). The reaction is carried out in a dissolved or suspended state below, and the reaction temperature in the resolution and steric inversion reaction is adjusted to an appropriate temperature of 10 ° C. or higher and 80 ° C. or lower, preferably 20 ° C. or higher and 60 ° C. or lower, pH 4 or higher. , 12 or less, preferably pH 6 or more and 11 or less, while standing or stirring for a while. The substrate may be added all at once, dividedly or continuously. The resolution and steric inversion reaction can be performed in a batch method or a continuous method.

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

16. Product Isolation The L-amino acid, 2-oxoacid, or cyclic imine produced can be isolated by conventional separation methods, for example, separation methods such as extraction, concentration, crystallization, column chromatography, and combinations thereof. Can be separated and purified.

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

(Example 1) Isolation of D-amino acid oxidase 1 gene Candida intermedia strain NBRC0761 was sterilized in vitro in 6 ml of medium (glucose 1.65%, NH ₄ H ₂ PO ₄ 0. 5%, KH ₂ PO ₄ 0.25%, MgSO ₄ 0.1%, FeCl ₃ .6H ₂ O 0.002%, corn steep liquor 0.1%, pH 5.2 before sterilization and inoculated at 30 ° C. And cultured with shaking aerobically for 14 hours. 1 ml of this culture solution was sterilized in a 500 ml Sakaguchi flask, 100 ml of medium (sucrose 1.65%, NH ₄ H ₂ PO ₄ 0.5%, KH ₂ PO ₄ 0.25%, MgSO ₄ 0.1%, FeCl ₃ .6H ₂ O 0.002%, corn steep liquor 0.1%, FAD 0.0005%, D-alanine 0.089%, pre-sterilization pH 5.2, except for FAD and D-alanine on 0.02 μm filter Sterilized and added separately.) And inoculated aerobically at 30 ° C. for 24 hours.

  After completion of the culture, chromosomal DNA was prepared from the cells obtained by centrifugation using an UltraClean Microbial DNA Isolation Kit (manufactured by MO BIO Laboratories, Inc.). Next, DNA primers designed based on known D-amino acid oxidase sequence homologous sites (Primer-1: SEQ ID NO: 5 in the sequence listing and Primer-2: SEQ ID NO: 6 in the sequence listing) were obtained in advance. PCR was performed using chromosomal DNA as a template. As a result, a DNA fragment having a part of the target D-amino acid oxidase gene and a base number of about 370 bp (referred to as a partial gene) was obtained.

  Next, the following operation was performed to obtain the full length of the target gene. In the partial gene, based on the nucleotide sequences corresponding to the N-terminal side and the C-terminal side of the enzyme, a DNA primer (Primer-3: SEQ ID NO: 7 in the sequence listing and Primer) directed toward the outside of the partial gene. -4: SEQ ID NO: 8 in the sequence listing was synthesized. Using this primer, inverse PCR was performed using DNA obtained by cyclization of the previously obtained chromosomal DNA digested with restriction enzymes HincII or HhaI using T4 DNA ligase as a template.

  As a result, a DNA fragment having a base number of about 1.0 to 2.5 kbp containing a gene portion further outside the already obtained partial gene was obtained. After determining the base sequence of this DNA fragment, the entire base sequence of the gene of D-amino acid oxidase 1 including the start codon to the stop codon shown in SEQ ID NO: 3 in the sequence listing is combined with the base sequence of the previous partial gene. Were determined. The gene for D-amino acid oxidase 1 did not contain introns. Next, a primer having a sequence in which cleavage sites of restriction enzymes NdeI and SacI are bound to the N-terminal and C-terminal parts of the gene of D-amino acid oxidase 1 (Primer-5: SEQ ID NO: 9, Primer-6: arrangement), respectively. A DNA fragment containing the open reading frame shown in SEQ ID NO: 3 was obtained by amplifying the DNA in the meantime by PCR using chromosomal DNA as a template using SEQ ID NO: 10) of the table.

(Example 2) Isolation of D-amino acid oxidase 2 gene DNA primers designed based on known D-amino acid oxidase sequence homologous sites (Primer-7: SEQ ID NO: 11 in Sequence Listing and Primer-8: Sequence Listing) PCR was performed using the chromosomal DNA prepared in the same manner as in Example 1 as a template. As a result, a DNA fragment having a part of the target D-amino acid oxidase gene and a base number of about 350 bp (referred to as a partial gene) was obtained.

  Next, the following operation was performed to obtain the full length of the target gene. In the partial gene, based on the nucleotide sequence corresponding to each of the N-terminal side and the C-terminal side of the enzyme, a DNA primer (Primer-9: SEQ ID NO: 13 in the sequence listing and Primer 9) directed toward the outside of the partial gene. -10: SEQ ID NO: 14) in the sequence listing was synthesized. Using this primer, inverse PCR was performed using as a template the DNA obtained by cyclization of the previously obtained chromosomal DNA digested with the restriction enzyme EcoRV using T4 DNA ligase.

  As a result, a DNA fragment having a base number of about 1.5 kbp containing a gene portion further outside the already obtained partial gene was obtained. After determining the base sequence of this DNA fragment, the entire base sequence of the D-amino acid oxidase gene including the start codon to the stop codon shown in SEQ ID NO: 15 in the sequence listing was determined by combining with the base sequence of the previous partial gene. . From the analysis of the base sequence, the D-amino acid oxidase gene obtained from chromosomal DNA contained 2 exons and 1 intron.

  In order to obtain the gene of D-amino acid oxidase 2 excluding introns, the following operation was performed. First, from the cells obtained by culturing Candida intermedia NBRC0761 strain in the same manner as in Example 1, using total RNA prepared using ISOGEN (manufactured by Nippon Gene) as a template, TaKaRa RNA LA Complementary strand DNA (cDNA) was prepared using PCR Kit (AMV) Ver.1.1 (TaKaRa). A primer having a sequence in which a cleavage site of the restriction enzyme NdeI is bound to the N-terminus of the D-amino acid oxidase gene (Primer-11: SEQ ID NO: 16 in the sequence listing), a cleavage site of the restriction enzyme SacI in the C-terminal portion; In addition, a primer having a sequence in which the stop codon is changed from TAG to TAA (Primer-12: SEQ ID NO: 17 in the Sequence Listing) is used to amplify the DNA by PCR using cDNA as a template, thereby removing introns. A DNA fragment containing the open reading frame shown in SEQ ID NO: 4 was obtained. D-amino acid oxidase 2 had 29.2% homology with D-amino acid oxidase 1 in terms of amino acid sequence and 48.2% in terms of the base sequence of the gene encoding them.

(Example 3) Preparation of recombinant plasmid pCDA001 expressing the gene of D-amino acid oxidase 1 The DNA fragment obtained in Example 1 was cleaved with restriction enzymes NdeI and SacI, and vector plasmid pUCNT (international) cleaved with the same enzyme. PCDA001 expressed by the restriction enzyme map of FIG. 1 and designed to express the gene of D-amino acid oxidase 1 was obtained by binding with T4 DNA ligase.

(Example 4) Preparation of recombinant plasmid pCDA006 expressing the gene of D-amino acid oxidase 2 The DNA fragment obtained in Example 2 was digested with restriction enzymes NdeI and SacI, and vector plasmid pUCNT (international) digested with the same enzyme. PCDA006 represented by the restriction enzyme map of FIG. 2 and designed to express the gene of D-amino acid oxidase 2 was obtained by binding with T4 DNA ligase.

(Example 5) Preparation of transformant using recombinant DNA containing gene of D-amino acid oxidase 1 Plasmid pCDA001 obtained in Example 3 is mixed with competent cells of Escherichia coli HB101. The medium (tryptone 1.0%, yeast extract 0.5%, sodium chloride 1.0%, agar 1.5%, ampicillin 0.010%, dissolved in deionized water, pH 7 before sterilization) 0.0, but ampicillin is added after sterilization) to obtain a transformant Escherichia coli HB101 (pCDA001) containing a recombinant DNA containing a D-amino acid oxidase gene as a colony.

  The obtained transformant colony was dissolved in a sterilized medium (tryptone 1.0%, yeast extract 0.5%, sodium chloride 1.0%, ampicillin 0.010%, deionized water, pH 7. 0, except that ampicillin is added after sterilization) and then aerobically cultured by shaking at 37 ° C for 24 hours. The bacterial cells are collected from the obtained culture broth by centrifugation, suspended in 0.1 M Tris-HCl buffer (pH 8.0), disrupted by ultrasonic waves, and then centrifuged to obtain the cells derived from the bacterial cells. Insoluble matters were removed, and a transformant D-amino acid oxidase enzyme solution was obtained. Using the obtained enzyme solution, D-amino acid oxidase activity for D-phenylalanine, D-alanine and D-methionine was measured by the method shown below. The results are shown in Table 1.

  The activity of D-amino acid oxidase was measured by quantifying hydrogen peroxide produced by the reaction by an enzymatic method using peroxidase. The quantitative determination of hydrogen peroxide by the enzymatic method was performed as follows. 25 mM The above D-amino acid, 0.80 mM 4-aminoantipyrine, 1.31 mM N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine, 6 u / ml peroxidase (manufactured by TOYOBO Inc.) , And 250 μl of coloring solution consisting of 50 mM Tris-hydrochloric acid buffer (pH 8.0) and 50 μl of appropriately diluted enzyme solution, reacted at 30 ° C. for 30 minutes, measured for absorbance at 555 nm, and prepared a calibration curve prepared in advance. Based on this, the activity was measured by quantifying the amount of hydrogen peroxide produced in the reaction solution.

Example 6 Preparation of Transformant Using Recombinant DNA Containing D-Amino Acid Oxidase 2 Gene D-amino acid oxidase was prepared in the same manner as in Example 5 using plasmid pCDA006 obtained in Example 4. Transformant Escherichia coli JM109 (pCDA006) containing recombinant DNA containing the gene was obtained as a colony.

  The obtained transformant colony was dissolved in a sterilized medium (tryptone 1.0%, yeast extract 0.5%, sodium chloride 1.0%, ampicillin 0.010%, deionized water, pH 7. 0, but ampicillin is added after sterilization), and after aerobic culture at 37 ° C. for 5 hours, isopropyl-1-thio-β-D-galactoside (IPTG) is added to the culture solution. Was added to a final concentration of 1 mM, and further cultured for 19 hours with shaking. The bacterial cells are collected from the obtained culture broth by centrifugation, suspended in 0.1 M Tris-HCl buffer (pH 8.0), disrupted by ultrasonic waves, and then centrifuged to obtain the cells derived from the bacterial cells. Insoluble matters were removed, and a transformant D-amino acid oxidase enzyme solution was obtained. Using the obtained enzyme solution, D-amino acid oxidase activity for D-phenylalanine, D-alanine and D-methionine was measured by the method shown in Example 5. The results are shown in Table 2.

FIG. 1 is a diagram showing the structure of a recombinant plasmid pCDA001 containing a gene for D-amino acid oxidase 1 as an embodiment of the present invention. FIG. 2 is a diagram showing the structure of a recombinant plasmid pCDA006 containing the gene for D-amino acid oxidase 2 as an embodiment of the invention.

Claims (28)

The following polypeptide (a) or (b):
(A) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing;
(B) a polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, inserted, deleted and / or added in the amino acid sequence shown in SEQ ID NO: 1 and having D-amino acid oxidase activity; 2. A polypeptide according to claim 1 derived from Candida intermedia. The polypeptide according to claim 1, which is derived from Candida intermedia NBRC0761 strain. DNA encoding the polypeptide according to any one of claims 1 to 3. The following DNA (g) or (h):
(G) DNA consisting of the base sequence shown in SEQ ID NO: 3 in the Sequence Listing;
(H) A polypeptide having a base sequence in which one or several bases are substituted, inserted, deleted and / or added in the base sequence shown in SEQ ID NO: 3 and having D-amino acid oxidase activity DNA to encode . A recombinant plasmid obtained by inserting the DNA according to claim 4 or 5 into a vector. The recombinant plasmid according to claim 6, wherein the vector is pUC18, pUC19, pBR322, pACYC184, pSTV28, pSTV29, pSC101, pT7Blue, or pUCNT. The recombinant plasmid according to claim 6 or 7, wherein the recombinant plasmid is pCDA001 identified by the restriction enzyme map of FIG. A transformant obtained by transforming a host microorganism with the recombinant plasmid according to any one of claims 6 to 8 . The transformant according to claim 9 , wherein the host microorganism is Escherichia coli. The transformant according to claim 9 , wherein the transformant is Escherichia coli HB101 (pCDA001). A method for producing D-amino acid oxidase, comprising culturing the transformant according to any one of claims 9 to 11 , accumulating the polypeptide in the culture, and collecting the polypeptide. The polypeptide according to any one of claims 1 to 3 or the transformant according to claim 9 is represented by the general formula (1).

(In the formula, R has an optionally substituted alkyl group having 1 to 20 carbon atoms, an optionally substituted aralkyl group having 7 to 20 carbon atoms, or a substituent. Or a racemic amino acid represented by the general formula (2):

(Wherein, R is the same as described above). The polypeptide according to any one of claims 1 to 3 or the transformant according to claim 9 is represented by the general formula (1).

(In the formula, R has an optionally substituted alkyl group having 1 to 20 carbon atoms, an optionally substituted aralkyl group having 7 to 20 carbon atoms, or a substituent. Or a racemic amino acid represented by the general formula (3):

(Wherein, R is the same as described above). The polypeptide according to any one of claims 1 to 3 or the transformant according to claim 9 is represented by the general formula (4).

(Wherein R ′ is an optionally substituted alkyl chain having 2 to 7 carbon atoms) and is allowed to act on the cyclic amino acid represented by the general formula (5)

(Wherein R ′ is the same as above), a method for producing a cyclic L-amino acid. The polypeptide according to any one of claims 1 to 3 or the transformant according to claim 9 is represented by the general formula (4).

(Wherein R ′ is an optionally substituted alkyl chain having 2 to 7 carbon atoms) and is allowed to act on the cyclic amino acid represented by the general formula (6)

(Wherein R ′ is the same as described above). The polypeptide according to any one of claims 1 to 3 or the transformant according to claim 9 is represented by the general formula (1) in the presence of an amino acid dehydrogenase and an enzyme having a coenzyme regeneration ability.

(In the formula, R has an optionally substituted alkyl group having 1 to 20 carbon atoms, an optionally substituted aralkyl group having 7 to 20 carbon atoms, or a substituent. Or a racemic amino acid represented by the general formula (2):

(Wherein, R is the same as described above). The polypeptide according to any one of claims 1 to 3 or the transformant according to claim 9 is represented by the general formula (4) in the presence of an amino acid dehydrogenase and an enzyme having a coenzyme regeneration ability.

Wherein R ′ is a cyclic amino acid represented by the general formula (5), wherein R ′ is an optionally substituted alkyl chain having 2 to 7 carbon atoms.

(Wherein R ′ is the same as above), a method for producing a cyclic L-amino acid. The method for producing an L-amino acid according to claim 13 or 17, wherein the polypeptide is a D-amino acid oxidase produced by the transformant according to any one of claims 9 to 11 . The method for producing 2-oxo acid according to claim 14, wherein the polypeptide is D-amino acid oxidase produced by the transformant according to any one of claims 9 to 11 . The method for producing a cyclic L-amino acid according to claim 15 or 18, wherein the polypeptide is a D-amino acid oxidase produced by the transformant according to any one of claims 9 to 11 . The method for producing a cyclic imine according to claim 16, wherein the polypeptide is a D-amino acid oxidase produced by the transformant according to any one of claims 9 to 11 . The method for producing an L-amino acid according to claim 17 , wherein the amino acid dehydrogenase is phenylalanine dehydrogenase or leucine dehydrogenase. The method for producing an L-amino acid according to claim 17 , wherein the enzyme having a coenzyme regeneration ability is formate dehydrogenase or glucose dehydrogenase. The method for producing a cyclic L-amino acid according to claim 18 , wherein the enzyme having the ability to regenerate coenzyme is formate dehydrogenase or glucose dehydrogenase. The method for producing an L-amino acid according to claim 13, 17 or 19, which is carried out in the presence of catalase. The method for producing 2-oxo acid according to claim 14 or 20, which is carried out in the presence of catalase. The method for producing a cyclic imine according to claim 16 or 21, which is carried out in the presence of catalase.

Images