JP2003250582A - Aspergillus oryzae dao c gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a new Aspergillus amino acid oxidase in a large amount by culturing a transformant wherein the enzyme has an action to oxidize D-amino acids, the amino acid sequence includes 349 amino acid residues and is a new compound, and the enzyme specifically acts on D-aspartic acid and D-glutamic acid, consequently these amino acids can be readily detected and their racemic resolution can be effectively achieved. <P>SOLUTION: A gene encoding a new D-amino acid oxidase daoC is cloned and the base sequence of the gene is determined (1.1 Kbp). The cloned new D-amino acid oxidase DAOC gene is transduced into a vector and a host (Escherichia coli) is transformed with the resultant expression plasmid. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel D-amino acid oxidase daoC gene and its use. More specifically, the present invention uses a D-amino acid oxidase daoC gene newly isolated from Aspergillus oryzae,
D-amino acid oxidase DAOC, which is a novel enzyme, can be produced in large quantities and used in various industrial fields such as D-amino acid measuring reagents.

[0002]

2. Description of the Related Art Generally, amino acids constituting proteins in vivo are constructed with L-amino acids, but it is known that some special molecules contain D-amino acids. It is known that the D-amino acid and the biopolymer containing the D-amino acid have various special functions, and the detection of the amino acid in the living body and the measurement of the content are very important items as clinical tests. . For example, in recent years, it has been shown that the measured value of D-amino acid in blood may serve as an index of renal failure (see, for example, Non-Patent Documents 1, 2, and 3), and D-amino acid is used as an evaluation item of renal function. Has been added.

For the quantification of this D-amino acid, measurement by an enzymatic method using D-amino acid oxidase has been widely studied (for example, see Non-Patent Document 4). The features of this method are:
Since it is an enzyme that specifically oxidizes D-amino acids, L
-It does not react with amino acids at all, and hydrogen peroxide produced by the oxidase reaction can be easily detected as a color reaction by a coupling reaction with peroxidase. However, like the D-amino acid oxidase of porcine pancreas, there are still problems regarding the stability and production amount of the enzyme, and the technique completed as a reagent for clinical examination has not been reached.

As microorganisms producing D-amino acid oxidase, there are reports of cephalosporium potronii, trigonopsis variabilis, gliocladium, pseudomonas, rhodotorula glutinis and the like. However, the enzymes produced from these microorganisms have not been put to practical use because their productivity and stability have not been solved. In addition, when it is derived from bacteria, there is a possibility that the expression in eukaryote may be impaired.

On the other hand, in recent years, the production of D-amino acid oxidase has also been examined at the gene level, and the present inventors have succeeded in cloning the D-amino acid oxidase gene and determined the base sequence thereof. Has also succeeded, and the expression in Aspergillus has been confirmed (see, for example, Patent Document 1).

[0006]

[Patent Document 1] Japanese Patent Laid-Open No. 2002-253246

[Non-patent document 1] "Clinical Science (Clinical
Science), 73, p.105-108, 1987

[Non-Patent Document 2] "Journal of Chromatography", Volume 614, p.7-17,
1993

[Non-patent document 3] "Kitasato medicine", Volume 23, p.51-62, 1993.
Year

[Non-patent document 4] "Analytical Biochemistry", 150 volumes, p.238-242,
1985

[0007]

DISCLOSURE OF THE INVENTION The problems to be solved by the present inventors are, in view of the usefulness of D-amino acid oxidase, which can be used in various industries such as quantification of D-amino acids, and the like. Succeeded in developing recombinant D-
Amino acid oxidase (Japanese Patent Laid-Open No. 2002-253246)
Is to efficiently produce another novel D-amino acid oxidase.

[0008]

[Means for Solving the Problems] Among the filamentous fungi, koji molds containing Aspergillus oryzae, etc., are especially used in the brewing industry in Japan for producing sake, miso, soy sauce, mirin, etc. It is a fungus that has been used for a long time and has been eaten directly.
GRAS (Generally Recognized) by DA (Food and Drug Administration)
It is a safe gene source listed in ized as Safe). Therefore, in a safety examination such as a chronic toxicity test required when using a gene derived from a normal bacterium in foods, it takes about 1 billion yen for a gene derived from a normal bacterium, whereas the above-mentioned GRAS In case of a gene of a grade, the cost is about one-third of that, and there is an advantage that the time required for the examination is shorter. Thus, filamentous fungi, especially koji mold, can be said to be a treasure trove of genes with extremely high utility value in terms of safety and economy.

[0009] Therefore, by clarifying the genomic DNA information of these bacteria and clarifying the functions of the genes and the like encoded by them, it is possible to identify a safe genetic resource in the food industry such as substance production using biotechnology. In addition to providing effective utilization methods, it is possible to provide useful information for screening various genes in the fields of agricultural chemicals and medicine. Further, it can be a useful tool for analyzing genomic information of grain contaminated bacteria and human infectious bacteria such as Aspergillus flavus and Aspergillus fumigatus which are related species.

From the above viewpoints, the present inventors have conducted a genome analysis of Aspergillus oryzae, which is a kind of Aspergillus,
Finally succeeded in it, and also succeeded in determining their nucleotide sequences and various functions, and patented the results of these (first application: Japanese Patent Application No. 2001-403).
261).

Further, this time, the present inventors have actually created a transformant using gene recombination means based on the nucleotide sequence information shown in the above-mentioned prior application and shown in SEQ ID NO. The product was obtained to verify the sequence of the gene and its function in more detail. As a result of the verification, the novel fact that the gene isolated according to the gene sequence information shown in SEQ ID NO: 36897 in the previous application has a specific function and utility of expressing D-amino acid oxidase was confirmed for the first time.

That is, the present invention uses the base information shown in SEQ ID NO: 36897 in the previous application to obtain A
A gene having homology to it was isolated in Spergillus oryzae, and it was confirmed that the host into which this gene was introduced actually produces a protein having D-amino acid oxidase activity shown in the description of SEQ ID NO: 36897. It is based on what you have done. Specifically, the nucleotide sequence information shown in SEQ ID NO: 36897 was analyzed, and as a result, a predicted gene showing homology to D-amino acid oxidase was found. This is dao
It was named C.

Next, in order to investigate the function of this daoC gene, an attempt was made to introduce this gene into E. coli. In order to obtain the cDNA of the daoC gene, the expression pattern of the daoC gene was examined. Specifically, DPY liquid culture, DPY +
RNA was extracted from the cells obtained by D-amino acid-added culture and rice koji culture, and cDNA was amplified by reverse transcription PCR. As a result, amplification of cDNA was confirmed in D-amino acid-added culture and rice koji culture. In particular, cDNA was remarkably amplified in the culture containing D-amino acid.

The daoC cDN thus obtained
A (1.0 kb) is a vector pET23 for highly expressing E. coli
b (manufactured by Novagen), and the transformant was cultured according to a conventional method. As a result, the lysate of the obtained transformant had a significantly higher D-amino acid oxidase activity than the lysate of the transformant transformed with pET23b. Therefore, it was shown that the daoC gene isolated by us encodes a protein having D-amino acid oxidase activity.

The present invention has been made on the basis of these useful new findings, and relates to a protein having D-amino acid oxidase activity and a gene encoding the protein, and its embodiment examples are shown below.

(Aspect 1) The following protein (a) or (b): (a) a protein consisting of the amino acid sequence shown by SEQ ID NO: 1, or (b) a deletion of an amino acid in the amino acid sequence (a), A D-amino acid oxidase protein consisting of a substituted or added amino acid sequence.

(Aspect 2) A gene encoding the protein (a) according to claim 1, which is represented by the nucleotide sequence of SEQ ID NO: 2 or a D-amino acid which hybridizes with the gene under stringent conditions DNA of a gene encoding an oxidase protein (b).

In the present invention, the above-mentioned specific amino acid sequence also includes an amino acid sequence substantially the same as this. An amino acid sequence which is substantially the same as a specific amino acid sequence in the present invention means an average of about 30% or more, preferably about 50% as a whole when the amino acid sequence and the entire amino acid sequence are aligned and compared. Or more, more preferably about 80% or more, and particularly preferably about 9%.
It means an amino acid sequence in which 0% or more of amino acids are identical. Therefore, a protein having an amino acid sequence substantially the same as a certain amino acid sequence is considered to have substantially the same function as a protein having the amino acid sequence.

Further, an amino acid sequence in which a part of amino acids is deleted, substituted or added in the specific amino acid sequence of the protein of the present invention, as long as it has a function substantially equivalent to that of the protein consisting of the amino acid sequence, It preferably means an amino acid sequence in which about 1 to 20, more preferably about 1 to 10 and still more preferably several amino acids are deleted, substituted or added, or an amino acid sequence combining them. The "function" of a protein means a biological function or activity that the protein exhibits inside and / or outside the cell (bacteria).

The protein consisting of an amino acid sequence substantially the same as the above-mentioned specific amino acid sequence, or the protein consisting of an amino acid sequence in which a part of the amino acids is deleted, substituted or added is, for example, a site-directed mutagenesis method. , A gene homologous recombination method, a primer extension method, a PCR method, and the like, which are well known to those skilled in the art, can be appropriately combined and easily prepared. At this time, in order to have substantially the same function, homologous amino acids (polar / nonpolar amino acids, hydrophobic / hydrophilic amino acids, positive / negatively charged amino acids, aromatic amino acids, etc. Substitution among amino acids) is possible.
Further, in order to maintain substantially the same function, it is desirable that amino acids in the functional domain contained in each protein of the present invention be retained.

In the present specification, the term "stringent conditions" means that the degree of homology between the nucleotide sequences is, for example, about 80% or more, preferably about 90% or more, and more preferably the average of the entire sequence. It means a condition that a hybrid is specifically formed only between base sequences having high homology, such as about 95% or more. Specifically, for example, at a temperature of 60 ° C to 68 ° C, the sodium concentration is 15
0 to 900 mM, preferably 600 to 900 mM, pH
Conditions such as 6 to 8 can be mentioned. Hybridization can be performed, for example, by using Current Protocols in Molecular Biology (Current pr
otocols in molecular biology (edited by Frederick
M. Ausubel et al., 1987)) and the like known in the art or a method similar thereto. When a commercially available library is used, it can be performed according to the method described in the attached instruction manual.

As described above, the daoC gene according to the present invention is novel and has a novel D-amino acid oxidase activity, which is a gene encoding a novel D-amino acid oxidase different from the conventionally known D-amino acid oxidase. It indicates at least one selected from genes encoding proteins and genes containing at least one of these genes (hereinafter, simply da.
Sometimes called the oC gene, its base sequence (2640
bp) is SEQ ID NO: 2 in the sequence listing and FIGS. 3 and 4 in the drawings.
Shown in. )

The DNA of the present invention can be prepared by a method known to those skilled in the art. For example, each DNA represented by a specific nucleotide sequence in the present specification can be prepared, for example, by the shotgun cloning method using the Aspergillus oryzae genome as a starting material. At that time, each fragmented chromosomal DNA is ligated to an appropriate cloning vector such as a plasmid vector or a phage according to its length and the like, and using this, an E. coli etc. by an appropriate method such as electroporation. Suitable host cells of, and the fragmented chromosomal DNA
A clone library can be prepared for cloning. Furthermore, according to known methods such as chemical decomposition method (Maxam-Gilbert method) and dideoxy method,
The base sequence of each fragmented chromosomal DNA obtained from such a clone library can be determined.

Alternatively, the DNA of the present invention described herein.
It can also be prepared by amplification based on the information on the nucleotide sequence or amino acid sequence of the above, by chemical synthesis well known to those skilled in the art, or by PCR using the primer of the present invention.

As an example of the Aspergillus oryzae genome used as the DNA source according to the present invention, the Aspergillus oryzae RIB40 strain genome can be used. The strain has been deposited as FERM P-18273 at the Patent Organism Depositary Center, National Institute of Advanced Industrial Science and Technology.

In the present invention, the daoC gene thus isolated is introduced into a host for expression,
It is intended to produce D-amino acid oxidase or a protein having D-amino acid oxidase enzymatic activity.
In the present invention, the protein having D-amino acid oxidase enzymatic activity also includes D-amino acid oxidase, and its amino acid sequence is shown in SEQ ID NO: 1 (FIGS. 1 and 2).

For example, the coding region of the daoC gene (1.1 kbp) is inserted into a vector (for example, the E. coli high expression vector pET23b) to prepare a recombinant vector. The E. coli expression plasmid thus prepared was designated as pET23b-daoC, and was deposited as FERM P-19108 at the Japan Patent Organism Depositary (FIG. 8).

The E. coli expression plasmid pET23b-daoC thus prepared is introduced into a host (eg E. coli) to obtain a transformant. The transformant thus obtained was transformed into Escherichia coli (Escherichia coli).
coli) T7-DAOC. When this transformant was cultured, the expression of this gene was confirmed because the disrupted cell suspension showed extremely high D-amino acid oxidase activity.

The D-amino acid oxidase protein according to the present invention has an action of oxidizing various D-amino acids to D-keto acids, like the previously unknown D-amino acid oxidase. Corresponding to D
-Can be oxidized to keto acids: D-aspartic acid, D-glutamic acid, D-glutamine and the like. However, since its amino acid sequence was different from the conventional one, this enzyme was identified as a novel enzyme and named D-amino acid oxidase DAOC. Hereinafter, the present invention will be described in more detail by the following experimental examples.

[0030]

Example 1 Cloning of daoC Gene and dao
Determination of the nucleotide sequence of C gene As described in the previous application, among the genomic analysis results distributed by the Aspergillus genome analysis consortium, the predicted gene Con1 showing homology to D-amino acid oxidase.
The adequacy of 601_h_1200 was examined. This gene was predicted as a gene with a total length of 3.4 kbp, which is encoded on the reverse strand of genomic contig 1601. 1-1000bp of which is the promoter region, 1
001-1032bp, 1098-1684, 1777
-2183 and 2828-3121 bp are coding regions, 3
It was predicted that 122-3421 bp contained the terminator region and three introns. It was said that three introns were included in the genome prediction.
Sequence analysis revealed the absence of the third intron.

Therefore, the gene contained in Con1601_h_1200 is designated as daoC, and a primer (oligo DNA # 1) containing a predicted initiation codon and oligo are included.
Using odT primer, RNA obtained from rice malt was used as a template to amplify cDNA, and the nucleotide sequence was analyzed. The base sequence of oligo DNA # 1 is SEQ ID NO: 3 (FIG. 5).
Shown in.

As a result, unlike the prediction, the daoC gene contained only two introns, and its coding region encoded 349 amino acids. However, although the intron portion and the CDS region are different from those predicted, the nucleotide sequence of the gene encoding daoC is entirely contained in the nucleotide sequence shown in SEQ ID NO: 36897 of the previous application except for a partially substituted portion. Therefore, it was confirmed that the gene shown in SEQ ID NO: 36897 of the previous application is a D-amino acid oxidase gene.

[0033]

Example 2 Introduction of daoC Gene into E. coli Next, in order to confirm the function of the daoC gene, an attempt was made to introduce this gene into E. coli. The coding region of the daoC gene (1.1 Kbp) was treated with Pyrobest manufactured by Takara Shuzo Co., Ltd. using daoC cDNA as a template, and the fragment amplified with oligo DNA # 2 and oligo DNA # 3 was treated with restriction enzyme NdeI to obtain high expression of E. coli. The vector pET23b (manufactured by Novagen) was inserted into the NdeI site to obtain a new recombinant vector pET23b-dao.
Establish C and use it as a FERM for the Patent Biological Depository Center
Deposited as P-19108. Escherichia coli was transformed with this recombinant vector according to a conventional method, and the obtained novel transformant was transformed into Escherichia coli.
It was named T7-DAOC.

The base sequences of oligo DNA # 2 and oligo DNA # 3 used as PCR primers in the above are shown in SEQ ID NO: 4 (FIG. 6) and SEQ ID NO: 5 (FIG. 7), respectively.

The transformant obtained as described above was cultured. As a result, the lysate of the obtained transformant was pE.
It showed a significantly higher D-amino acid oxidase activity than that of the disrupted cell suspension, which was transformed with T23b.

Therefore, it was revealed that the daoC gene isolated by us encodes a protein having D-amino acid oxidase activity. As described above, the daoC gene according to the present invention is novel and encodes a gene encoding a novel D-amino acid oxidase different from the conventionally known D-amino acid oxidase, and a protein having the novel D-amino acid oxidase activity. It is intended to indicate at least one selected from genes and genes containing at least one of these genes.

[0037]

Example 3 Recombinant D-amino acid oxidase DAOC
The reaction formula of the reaction catalyzed by D-amino acid oxidase is shown below.

D-amino acid + O ₂ + H ₂ O → keto acid + H ₂ O ₂ + NH ₃

As is clear from the reaction formula, the D-amino acid can be quantified by detecting any of the reaction products pyruvic acid, hydrogen peroxide or ammonia. For example, a colorimetric method, a fluorescent method, a chemiluminescent method, an electrode method, and the like are known for detecting the generated hydrogen peroxide, and any of them can be used. In the colorimetric method, a substrate of peroxidase is oxidatively developed with hydrogen peroxide using a catalyst such as peroxidase, and the color density is measured with a spectrophotometer. Substrates for peroxidase include o-phenylenediamine, 5-aminosalicylic acid, 4-aminoantipyrine and phenol, 2,
2'-azinobis (3-ethylbenzothiazoline-6-
Sulfonic acid), bis [3-bis (4-chlorophenyl) methyl-4-dimethyl-aminophenyl] amine and the like can be used.

In the fluorescence method, the substrate is oxidized with hydrogen peroxide by a catalyst such as peroxidase to produce a fluorescent substance, and the fluorescence intensity thereof is measured with a fluorometer. As a substrate for peroxidase, p-hydroxyphenylacetic acid,
For example, p-hydroxyphenylpropionic acid can be used. In the chemiluminescence method, the substrate is oxidized with hydrogen peroxide by a catalyst such as peroxidase to emit light, and the emission intensity is measured with a luminometer. Luminol compounds, lucigenin, compounds of allyl oxalates and the like can be used as the chemiluminescent substrate. A reagent containing the D-amino acid oxidase and the above-mentioned reagent components necessary for detecting D-amino acid can be prepared and assembled into a diagnostic agent or the like as a D-amino acid measurement kit.

Further, this D-amino acid oxidase DA
When OC is used, DL-amino acid can be racem-divided to efficiently separate, purify, and produce L-amino acid, and for example, only L-amino acid can be efficiently obtained from chemically synthesized racemic amino acid. .

The results of examining the substrate specificity of dao CD-amino acid oxidase are shown below. The composition of the reaction solution is shown below.

[0043] 50 mM D-amino acid 50 mM potassium phosphate buffer (pH 8.0) 0.86 mM o-dianisidine 3000U peroxidase 0.5 μg / ml D-amino acid oxidase solution

After reacting at room temperature for 5 minutes, 0.5 ml of a 30% sulfuric acid solution was added to 1 ml of the above reaction mixture, and 540 nm.
The activity was measured by measuring the absorbance at. The obtained results are shown in Table 1 below.

(Table 1) ──────────────────── Substrate relative activity (%) D-aspartic acid 100 D-glutamic acid 71 D-glutamine 16 Other D-amino acids 5-10 ────────────────────

As a result of examining the substrate specificity, it was found that the present D-amino acid oxidase specifically reacts with D-aspartic acid and D-glutamic acid. Therefore, it is found that the detection of these D-amino acids and the racemic resolution are very effective.

[0047]

INDUSTRIAL APPLICABILITY According to the present invention, it becomes possible for the first time to produce a large amount of a novel D-amino acid oxidase DAOC of Aspergillus oryzae, and the D-amino acid oxidase is industrially applied to research reagents and test agents for D-amino acid measurement. It enables to be widely used. Further, the present invention is an epoch-making technology in which the enzyme of koji mold is produced by koji mold, and thus the produced enzyme protein is very safe and can be applied to the food, pharmaceutical and cosmetic industries.

[0048]

[Sequence list]                   SEQUENCE LISTING <110> National Institute of Advanced Industrial Science and            Technology; National Institute of Technology and Evaluation;            National Research Institute of Brewing <120> Koji Mold daoC Gene <130> 6638 <141> 2002-11-29 <160> 5 <210> 1 <211> 349 <212> PRT <213> Aspergillus oryzae <400> 1 Met Pro Glu Lys Glu Thr Ile Val Val Ile Gly Ala Gly Val Ile Gly   1 5 10 15 Leu Thr Thr Ala Leu Arg Ile Gln Glu Pro Leu His Arg Asn Gln Thr              20 25 30 Ile Gln Leu Ile Ala Arg Asp Phe Pro Asn Thr Thr Ser Leu Asn Tyr          35 40 45 Ala Ser Pro Trp Ala Gly Ala His Tyr Arg Pro Val Pro Gly Ser Thr      50 55 60 Pro Gln Ala Leu Arg Glu Glu Ala Gln Ala Lys Gln Thr Tyr Ser Tyr  65 70 75 80 Leu Lys Gln Leu Ala Lys Ser Asp Pro Ser Ser Gly Val Ala Ile Ile           85 90 95 Glu Gly Ile Glu His Leu Glu Asn Pro Pro Gur Tyr Leu Asp Glu             100 105 110 Arg Ser Ile Lys Glu Cys Tyr Gly His Leu Asp Gly Phe Arg Ile Leu         115 120 125 Gly Lys Asp Glu Cys Pro Glu Gly Val Lys Trp Gly Ala Arg Tyr Glu     130 135 140 Thr Ala Val Ile Asn Ser Pro Val Tyr Cys Ala Tyr Leu Leu Arg Lys 145 150 155 160 Phe Val Leu Gly Gly Gly Val Thr Arg Glu Tyr Thr Val Thr Asp Pro                 165 170 175 Arg Glu Ala Phe Tyr Leu Ala Pro Asn Val Arg Thr Val Val Asn Cys             180 185 190 Ser Ala Leu Gly Phe Gly Asp Glu Arg Ser Phe Ile Ile Arg Gly Gln         195 200 205 Thr Cys Leu Val Arg Asn Pro Cys Ser Gln Thr Val Thr Arg Gln Asn     210 215 220 Ser Asp Gly Ser Trp Ser Phe Cys Ile Pro Arg Pro Leu Ser Gly Gly 225 230 235 240 Thr Ile Ile Gly Gly Thr Lys Gln Pro Arg Asp Tyr Asp Pro Asn Pro                 245 250 255 Ser Val Glu Thr Arg Glu Lys Leu Leu Ala Asn Ala Ala Lys Trp Phe             260 265 270 Pro Phe Ala Pro Gly Ser Glu Gly Lys Phe Asp Val Ile Arg Asp Ile         275 280 285 Val Gly Arg Arg Pro Ala Arg Glu Gly Gly Met Arg Ile Glu Ala Glu     290 295 300 Lys Val Gly Glu Gly Arg Phe Val Val His Ala Tyr Gly Ala Gly Gly 305 310 315 320 Arg Gly Phe Glu Leu Ser Tyr Gly Val Ala Gly Asp Val Val Lys Leu                 325 330 335 Met Val Gly Asn Lys Leu Val Gly Glu Arg Ala Ser Leu             340 345 <210> 2 <211> 2640 <212> DNA <213> Aspergillus oryzae <400> 2 atatgttctt cgccgccaac acggccctcg gacctcctta ccatgtgctg gtggatacca 60 actttgtctc ccactctatt cgcgcgaaga cggatatgct gaagtctatg atggacctgt 120 gagtggatat gctattgggt ggtggggtgg cgatggctga ttgtcaacag gttatatgcg 180 aagtgtatcc cgacgtttac ggattgtact attgcggaat tggaaaagtt gggtgataag 240 ttccggttgg cgttgagggt ggctaaggat ccgagatggg cgcgcgtgcg gtgtgatcac 300 cccggtacct atgctgatga ctgtttagtt gatcgggtga gtttttgaga gttcgaaagc 360 tatgggatga catggtctaa tttggtcgaa gattacaaaa cacagaatct acattgtggc 420 gacgaacgat aaggacctgg ttcgccgtat tcgcaagatt cccggtgtgc cgatcatgaa 480 ggtcgcaaga gcgaaatacg tcattgagag atgcccgacc actttgagtg agcgtgtgca 540 aggatataga atatctcgtg ttgacaacta cggaatccac atctgattgg gacggttgga 600 caaggatatt gcaaatccga ccccaatact gcgataccgg cagatcttta tcggaggaat 660 ccatcatcaa tccaataccg ccgcggactc gcttggattt ctgcggagga gcggtttcag 720 gacacagcta cggagaacac tagatgtaac tagcagggct gtatacgact tcaatgataa 780 tactttgata ccattggtat acgaagagtg gagatggtat agataagcaa gcaagatgac 840 tcaactgcgg cattttctcc aagtactaga ctacattaaa catgaaataa ataccccata 900 aacctacatg cggggtcagc cttacccttc atccatgacc catttatata catctctcaa 960 ttgaattgac aacacaatct gaaccaaccc aaaaacaaaa tgcccgagaa agaaacaatc 1020 gtagtaatcg ggtacgtcat gccaccctta tatccccaat catcaaattc aacctccaaa 1080 actaactcca aaaaagcgcc ggagtaatcg gcctcacaac cgccctccgc atccaagaac 1140 ccctccaccg caaccaaacc atccagctca tagcccgcga cttccccaac accacatccc 1200 taaactacgc ctccccagtg ggccggcgcc cattaccgcc ctgtccccgg ttcaacacca 1260 caagccctcc gcgaagaggc ccaagccaaa caaacctaca gctacctcaa acaattggcc 1320 aaaagcgacc cctcctcggg cgtagcaatc atagaaggaa tcgaacacct cgagaaccca 1380 ccttccgaat atctcgacga aaggagtatc aaagaatgct acggtcatct tgatgggttc 1440 aggatcctgg ggaaagacga atgtcccgag ggggtgaaat ggggagctcg atatgaaact 1500 gcggtgatta attcgccggt gtattgtgcg tatcttctaa ggaagtttgt gcttggtggt 1560 ggggttacga gggaatatac agtcacggat ccgagggagg cgttttattt agcgccgaat 1620 gtgaggacgg tggttaattg ttcggcattg gggtttgggg atgagaggtc gtttattatt 1680 cggggtttgt tttcactctc tctttttctt cactatcctg gtcttttgtt atctgggttt 1740 acatgttgtt atgtggtgtg ttaacgatgt gtctagggca aacgtgtctc gtgcggaatc 1800 cttgcagtca gactgtgacg aggcagaatt ccgatgggtc ttggtcgttt tgtatcccgc 1860 gtcccttgtc cgggggcacc attattgggg gtacgaagca gccgcgcgac tatgatccca 1920 acccgtcggt ggagacgagg gagaagttgc ttgccaatgc ggccaagtgg ttcccgtttg 1980 cgccggggag tgaggggaag tttgatgtga ttcgggacat tgtaggtagg agaccggcta 2040 gagagggtgg gatgaggatt gaggcggaga aggttggaga aggtcggttt gtggtccatg 2100 cttatggggc tggagggaga ggctttgagt tgtcgtacgg agtggcgggg gatgttgtca 2160 agttgatggt gggtaataag ttggttggcg agagagcttc gttgtagatg tcatggatgt 2220 gtttaagttg attgtaaata tgtttggttt ggataggact tttgctgtat acaacatcta 2280 aagatcccaa ctctatagga ccgcagggac actttccagt ctaacatcaa gaagtgcata 2340 aatcattgta cctcaaagga cagaaaaaga aaagaaaaaa cgctaagttc attatagcaa 2400 cgaacgttct tttttacgta caaaatctgg cataacaaat ggtgtccttc actccgccac 2460 gaatgcacga cactcgatct atctcaatct tgtgtacgcg gctgttgtct tctagaacct 2520 ttggacatga atccaactgg gccggagttc acgatcaaaa tgacacacaa aacgtcacac 2580 gttaaccgca gccctcagtc gaggagacca agacaggtga ttactcggta ataaacaaca 2640 <210> 3 <211> 21 <212> DNA <213> Artificial sequence <400> 3 atgcccgaga aagaaacaat c 21 <210> 4 <211> 30 <212> DNA <213> Artificial sequence <400> 4 ggaattccat atgcccgaga aagaaacaat 30 <210> 5 <211> 30 <212> DNA <213> Artificial sequence <400> 5 ggaattccat atgcaacgaa gctctctcgc 30

[Brief description of drawings]

FIG. 1 shows the amino acid sequence of D-amino acid oxidase DAOC.

FIG. 2 shows the continuation of the above.

FIG. 3 shows the nucleotide sequence of D-amino acid oxidase daoC gene.

FIG. 4 shows the continuation of the above.

FIG. 5 shows oligo DNA # 1 primer.

FIG. 6 shows oligo DNA # 2 primer.

FIG. 7 shows oligo DNA # 3 primer.

FIG. 8: E. coli expression plasmid pET23b-daoC
Shows the construction of and the restriction map thereof.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/68 C12N 15/00 ZNAA 4B065 (72) Inventor Masayuki Machida 1-1-1 Higashi, Tsukuba-shi, Ibaraki Independent Administrative Law, National Institute of Advanced Industrial Science and Technology, Tsukuba Center (72) Inventor, Keietsu Abe 1-1-1, Higashi, Tsukuba, Ibaraki Prefecture Independent Administrative Law, National Institute of Advanced Industrial Science and Technology, Tsukuba Center (72) Inventor, Katsuya Gomi, Tsukuba, Ibaraki Prefecture 1-1-1 Higashi City, Independent Administrative Law, National Institute of Advanced Industrial Science and Technology Tsukuba Center (72) Inventor, Kiyoshi Asai 2-41-6 Ome, Koto-ku, Tokyo Independent Administrative Law, National Institute of Advanced Industrial Science and Technology, Seaside Fukutoshin Center ( 72) Inventor Motoaki Sano 1-1-1 East, Tsukuba City, Ibaraki Prefecture In-center (72) Inventor Kim Taishin 2-41-6 Ome, Koto-ku, Tokyo Independent Administrative Law Institute of Industrial Science and Technology Waterfront Fukutoshin Center (72) Inventor Hideki Nagasaki 2-41, Ome, Koto-ku, Tokyo 6 Independent Administrative Law, National Institute of Advanced Industrial Science and Technology Research Center, Seaside Fukutoshin Center (72) Inventor Satoshi Hosoyama 2-49-10 Nishihara, Shibuya-ku, Tokyo Independent Administrative Law Human Product Evaluation Technology Foundation (72) Inventor Osamu Akita Hiroshima 3-7-1 Kagamiyama, Higashihiroshima City, Higashi-Hiroshima, Incorporated Administrative Agency Alcoholic Beverage Research Institute (72) Inventor Naoki Ogasawara 8916-5 Takayama-cho, Ikoma-shi, Nara Nara Institute of Science and Technology Graduate School of Bioscience (72) Invention Satoshi Kuhara 6-10-1 Hakozaki, Higashi-ku, Fukuoka-shi, Fukuoka Prefecture Department of Genetic Resource Engineering, Faculty of Agriculture, Kyushu University (72) Inventor Hiroki Ishida 300 Katahara-cho, Fushimi-ku, Kyoto City Gekkokappu Co., Ltd. (72) Inventor Hiroshi Obata 300 Katahara-cho, Fushimi-ku, Kyoto City Laurel Crown Research Institute (72) Inventor Yoji Hata 300 Katahara-cho, Fushimi-ku, Kyoto City Gekkeikan Co., Ltd., Research Institute (72) Inventor, Shoji Kawado 24 Shimotoba Koyanagi-cho, Fushimi-ku, Kyoto City Keikyu Corporation Research Institute (72) Inventor, Yasuhisa Abe 300 Katahara-cho, Fushimi-ku, Ichi F-term (Reference) 2G045 BB03 BB20 CA25 CB21 DA12 DA13 DA14 DA20 DA35 FB01 FB04 4B024 AA03 AA11 BA08 BA71 CA01 DA06 GA11 HA20 4B050 CC01 DD03 QS10 QR03 QL05 4B06Q26QA01 QAQ AE03 CA21 CB11 CC24 CD13 DA01 DA13 4B065 AA26X AA63Y AB01 AC14 BA02 BB12 CA17 CA28 CA44 CA46 CA60

Claims (10)

[Claims] 1. A protein of the following (a) or (b): (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1, or (b) a deletion, substitution, or substitution of amino acids in the amino acid sequence (a) A protein comprising an added amino acid sequence and having D-amino acid oxidase activity. 2. A D containing the nucleotide sequence shown in SEQ ID NO: 2.
NA, which encodes the gene encoding the protein (a) according to claim 1, or the protein (b) which hybridizes with the gene under stringent conditions and has D-amino acid oxidase activity. DNA of a gene. 3. A recombinant vector comprising at least a coding region of the DNA according to claim 2. 4. A recombinant vector pET23b-daoC.
(FERM P-19108). 5. A transformant obtained by introducing the recombinant vector according to claim 3 or 4. 6. A transformant, Escherichia coli (Es)
cherichia coli) T7-DAOC. 7. A method for producing a D-amino acid oxidase protein, which comprises using the transformant according to claim 5 or 6. 8. A method for measuring D-amino acid, which comprises using the protein according to claim 1. 9. A reagent for measuring D-amino acids, which comprises the protein according to claim 1. 10. A method for racemic resolution of DL-amino acids, which comprises using the protein according to claim 1.