JP2003250584A - Carboxypeptidase gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a totally new carboxypeptidase. <P>SOLUTION: This carboxypeptidase is (a) a protein consisting of a specific amino acid sequence derived from Aspergillus oryzae or (b) a protein consisting of an amino acid sequence obtained by the deletion, substitution or addition of one or plural number of amino acids from/for/to the specific amino acid sequence derived from the Aspergillus oryzae and having the carboxypeptidase activity. The protein is a new glutaminase. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a carboxypeptidase, a carboxypeptidase gene, a novel recombinant DNA, and a method for producing carboxypeptidase.

[0002]

BACKGROUND OF THE INVENTION Carboxypeptidase is an enzyme that sequentially releases amino acids from the carboxy terminus of proteins. So far, the enzymatic properties of many carboxypeptidases from mammals to microorganisms have been determined, and genes have been analyzed. For example, those derived from human pancreas (see Non-Patent Document 1) and from Saccharomyces cerevisiae (see Non-Patent Document 2) are known.

Aspergillus which is a mold of Aspergillus
Orise and Aspergillus soya have been used for a long time in the production of brewed foods in Japan, such as miso, soy sauce, sake, etc., and are highly industrially productive and highly reliable due to their long-term use. Above all, it is a particularly important microorganism.

By the way, in the production of soy sauce, the raw material begins to be decomposed into a polypeptide by a protease produced by Aspergillus oryzae, and subsequently, a polypeptide is decomposed from leucine aminopeptidase from the amino terminal and acid carboxypeptidase from the carboxyl terminal. , Is decomposed into low molecular weight peptides mainly consisting of amino acids and dipeptides. These enzymes complement each other to decompose many kinds of peptides, and in order to completely solubilize, decompose, and amino acid high-molecular proteins,
Carboxypeptidase is essential. This is because when soybean protein is decomposed only with protease, the solubilization of nitrogen content is remarkable, but there is almost no production of glutamic acid and other amino acids, whereas by adding carboxypeptidase to protease and decomposing,
It can be seen from the fact that the solubilization of nitrogen increases and the elution of glutamic acid and other amino acids is remarkable (see Non-Patent Document 3).

It is also known that undecomposed peptides remain in soy sauce and enzymatically decomposed seasonings. If carboxypeptidase is present, the peptides remaining in the soy sauce and the enzymatically decomposed seasoning can be rapidly decomposed, and thus it is expected to contribute to the improvement of the amination ratio and the improvement of the taste due to the change of the amino acid composition. Therefore, it is strongly desired to obtain carboxypeptidase from Aspergillus oryzae.

[0006]

[Non-Patent Document 1] Eur. J. Biochem, 179 (3), 609-616.
(1989)

[Non-Patent Document 2] Biochemistry, 13 (19), 3871-3877.
(1974)

[Non-patent document 3] Science and technology of soy sauce, edited by Tatsurokuro Tochikura

[0007]

Therefore, the present invention provides a novel carboxypeptidase capable of degrading peptides present in soy sauce, enzymatically decomposed seasonings, etc., a novel gene encoding the carboxypeptidase, and a recombinant containing the gene. It is an object of the present invention to provide a somatic DNA, a transformant or transductant containing the recombinant DNA, and a method for producing carboxypeptidase using the transformant or transductant.

[0008]

Means for Solving the Problems The present inventors, as a result of various studies on the above problems, isolated a carboxypeptidase gene derived from Aspergillus oryzae, determined the structure, confirmed the carboxypeptidase activity, The present invention has been completed.

That is, the first invention is the following carboxypeptidase (a) or (b). (A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and having a carboxypeptidase activity Protein to have

The second invention is a carboxypeptidase gene encoding the following protein (a) or (b). (A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and having a carboxypeptidase activity Protein to have

A third aspect of the present invention is the following (a) or (b):
It is a carboxypeptidase gene consisting of DNA. (A) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 (b) DNA hybridizing with the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 under stringent conditions and encoding a protein having carboxypeptidase activity

A fourth invention is a novel recombinant DNA characterized by inserting the above-mentioned carboxypeptidase gene into vector DNA. A fifth invention is a transformant or transductant containing the above recombinant DNA. Sixth
The present invention is a method for producing carboxypeptidase, which comprises culturing the above transformant or transductant in a medium and collecting carboxypeptidase from the culture.
In addition, in this specification, the base sequence represented by SEQ ID NO: 1 and the amino acid sequence represented by SEQ ID NO: 2 are the same as those in the previous application.
It is the same as SEQ ID NOs: 22705 and 22706 in the specification of 2001-403261 (filed on Dec. 27, 2001), respectively.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. 1. Carboxypeptidase and Gene Encoding It The carboxypeptidase of the present invention is the following (a) or (b) carboxypeptidase. (A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and having a carboxypeptidase activity Protein to have

The carboxypeptidase shown in (a) is a chromosomal DNA of a filamentous fungus of the genus Aspergillus or
It can be obtained by cloning a natural carboxypeptidase gene derived from cDNA, introducing it into an appropriate vector-host system, and expressing it. In the following explanation,
The carboxypeptidase shown in (a) is referred to as "natural carboxypeptidase".

As shown in (b), the carboxypeptidase of the present invention has one or more amino acids deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 as long as it has carboxypeptidase activity. May be. In the following description, SEQ ID NO: 2
A protein consisting of an amino acid sequence in which one or more amino acids in the amino acid sequence shown in 1 is deleted, substituted or added and having carboxypeptidase activity is referred to as "mutant carboxypeptidase". In the present invention, “plurality” is usually 2 to 300, preferably 2 to 300, though it varies depending on the position or type of the amino acid residue in the three-dimensional structure of the carboxypeptidase protein.
It means 200, more preferably 2 to 50, and most preferably 2 to 10. The carboxypeptidase activity can be measured according to the method described in "2. Method for producing carboxypeptidase" described later.

Substitution with respect to the nucleotide sequence of the mutant carboxypeptidase or natural carboxypeptidase gene,
It can be obtained by introducing a mutation such as deletion, insertion, addition or inversion to prepare a mutant carboxypeptidase gene, and introducing this into an appropriate vector-host system to express it. As a method for introducing a mutation into a gene, for example, a site-directed mutagenesis method, a random mutation introduction method by PCR, further, a gene is selectively cleaved, then, after removing or adding a selected nucleotide, the gene And the like.

The carboxypeptidase gene of the present invention is a DN encoding the protein of (a) or (b) above.
It is a gene containing A. The carboxypeptidase gene of the present invention is a protein that hybridizes under stringent conditions with a DNA consisting of a nucleotide sequence complementary to the DNA encoding the protein of (a) or (b) and has carboxypeptidase activity. It may be a gene encoding In the present invention, “stringent conditions” means, for example, that the NaCl concentration is 50 to 300 mM,
It is preferably 150 mM and the temperature is 42 to 68 ° C., preferably 65 ° C. or higher.

DNA encoding the protein of (a) above
As an example of the gene containing, there is a DNA containing the nucleotide sequence of SEQ ID NO: 1. This DNA is the natural carboxypeptidase gene. The natural carboxypeptidase gene can be obtained by cloning a natural gene derived from chromosomal DNA or cDNA of a filamentous fungus belonging to the genus Aspergillus. As a gene cloning method, for example, carboxypeptidase is purified and a partial amino acid sequence is determined, and then a suitable probe DNA is synthesized, and using this, Aspergillus oryzae chromosomal DNA is used.
The method of screening from is mentioned. In addition, prepare an appropriate primer DNA based on the partial amino acid sequence,
By an appropriate polymerase chain reaction (hereinafter, abbreviated as PCR method) such as 5'-RACE method or 3'-RACE method,
A method of amplifying DNA containing a fragment of the gene and ligating them to obtain a DNA containing the full-length gene is also included.

More specifically, the natural carboxypeptidase gene can be obtained as follows. First, Aspergillus oryzae va
r. viridis Murakami, anamorph ATCC 42149) was cultured, and the obtained bacterial cells were frozen in liquid nitrogen, and then physically ground using a mortar or the like to give a fine powdery bacterial cell piece, A total RNA fraction is extracted from the cell pieces by a usual method. A commercially available RNA extraction kit can be used for the extraction operation. RNA may be recovered from the obtained RNA extract by ethanol precipitation, and RNA having a poly A chain may be fractionated from this RNA by a usual method. For the fractionation operation, commercially available O
ligo dT columns are available.

Primers used for PCR are synthesized with reference to the Aspergillus oryzae genome database. This primer DNA and RNA obtained as described above
The DNA containing the fragment of the gene is amplified by an appropriate RT-PCR reaction such as 5'-RACE method or 3'-RACE method, and these are ligated to obtain a DNA containing the full-length gene. A commercially available kit can be used for the partial cDNA synthesis operation by the 5'-RACE method or the 3'-RACE method. Using the cDNA as a template, a 5'terminal sequence and
DNA is amplified by performing PCR using synthetic primers complementary to the 3'terminal sequence. The amplified DNA can be cloned according to a usual method.

The amplified DNA is inserted into an appropriate vector to obtain a recombinant DNA. For cloning, TA Clonin
Commercial kit such as g Kit (Invitrogen) or pUC1
19 (Takara Shuzo), pBR322 (Takara Shuzo), pBluescrip
Commercially available plasmid vector DNA such as t SK + (manufactured by Stratagene) and commercially available bacteriophage vector DNA such as λEMBL3 (manufactured by Stratagene) can be used.

The obtained recombinant DNA is used to transform or transduce, for example, Escherichia coli K-12, preferably Escherichia coli JM109 (manufactured by Takara Shuzo), XL-Blue (manufactured by Stratagene), etc. A transformant or transductant is obtained. Transformation can be performed, for example, by the method of DM Morrison (Methods in E
nzymology, 68, 326-331, 1979). For the transduction, for example, the method of B. Hohn (Meth
ods in Enzymology, 68, 299-309, 1979). As the host cell, other than Escherichia coli, other bacteria such as yeast, filamentous fungi, actinomycetes, animal cells, plant cells and the like can be used.

The total nucleotide sequence of the DNA amplified above (see SEQ ID NO: 1) is, for example, Li-COR MODEL 4200L sequencer (purchased from Aloka), 370 DNA sequence system (Perkin Elmer), CEQ2000XL DNA analysis system. (Manufactured by Beckman) can be used for analysis.
By comparing the nucleotide sequence with the information on the partial amino acid sequence, it can be confirmed whether or not the natural carboxypeptidase gene was obtained. Then, by analyzing the natural carboxypeptidase gene, the translated polypeptide, that is, the amino acid sequence shown in SEQ ID NO: 2 is determined.

2. Method for Producing Carboxypeptidase When producing the carboxypeptidase of the present invention, first, a recombinant DNA containing the carboxypeptidase gene of the present invention is prepared. Then, a transformant or transductant containing the recombinant DNA may be prepared, cultured, and carboxypeptidase may be collected from the culture.

In order to produce a protein having carboxypeptidase activity using the carboxypeptidase gene of the present invention, it is necessary to select a suitable vector-host system. As such a system, pST14 (Unkle
s et al., 1989, Mol. Gen. Genet., 218, 99-104) and filamentous fungi [Aspergillus soja
e), Aspergillus oryzae,
Aspergillus nidulan
s), Aspergillus niger,
Penicillium chrysogenu
m) etc.], yeast expression vector YEpFLAG-1 (manufactured by SIGMA) and yeast Saccharomyces cerevisiae (Saccharo
myces cerevisiae) system, E. coli expression vector pTE (Str
made by atagene) and E. coli Escherichia coli
richia coli) system and the like. It is preferable to use a filamentous fungus or yeast system because sugar chain addition to a protein occurs.

Recombinant DNA can be obtained by inserting the carboxypeptidase gene into an appropriate vector. As the vector, commercially available products such as yeast expression vector YEp-FLAG-1 (manufactured by SIGMA), pYES2, pYD1 (Invitoro
gen), pUR123 (Takara Shuzo), pYEX-BX, pYEX-S
1, pYEX-4T (CLONTECH), E. coli expression vector pSET
(Invitorogen), pTE (Stratagene), etc. can be used.

Next, the recombinant DNA is transformed or transduced into a host cell. For transduction into yeast, for example, a method such as Becker DM (Methods in Enzymology, 194,
182-187, 1991). As the host cell, in addition to E. coli or yeast, other microorganisms such as bacteria, filamentous fungi, actinomycetes, or animal cells can be used. As described above, a transformant or transductant capable of producing carboxypeptidase can be obtained. For culturing the transformant or transductant, a usual solid culture method or liquid culture method can be adopted.

When yeast is used as a host, a general rich medium such as YPD medium or YM medium can be used as the medium. When a selective medium is used due to the genetic characteristics of the host, SD medium, which is the minimum medium, can be used. When a selection medium is used, the selection pressure varies depending on the host vector system used, and thus amino acids or nucleic acids other than the selection pressure are appropriately added to the minimum medium according to the genetic requirements of the host.

In addition, if necessary, inorganic salts, sugar raw materials, vitamins and the like may be appropriately added to the medium. The initial pH of the medium is appropriately adjusted to pH 6-9. Furthermore, depending on the vector used, the expression of a protein may be regulated. When using these vectors, a carboxypeptidase can be induced by adding an inducer suitable for the vector, for example, galactose or copper ion.

In the case of culturing yeast, it is preferable to cultivate at 25 to 35 ° C., preferably around 30 ° C. for 24 to 48 hours by aeration and agitation deep culture, shaking culture, static culture and the like. Expressed carboxypeptidase was analyzed by JPTW van
It can be purified by a partially modified method of the method of den Homvergh et al. (Gene, 151 (1994) 73-79). In the case of yeast,
After culturing the transformed yeast by the above-mentioned appropriate method, the culture solution is centrifuged to obtain cells. A cell wall lysing enzyme is added to the cells to sufficiently dissolve the cell wall, followed by centrifugation to obtain a supernatant. Ammonium sulfate is added to this supernatant for salting out, followed by centrifugation to remove insoluble proteins and obtain a crude enzyme solution containing carboxypeptidase.

From the crude enzyme solution to phenyl sepharose column, DEAE-sepharose column, gel filtration column, HPLC
Can be used to purify the carboxypeptidase active fraction to obtain a purified carboxypeptidase. The genetic engineering method in the present invention includes, for example, “Molecular Cloning: A Laboratory Manu
al2nd edition ”(1989), Cold Spring Harbor La
boratory Press, ISBN 0-8769-309-6, `` Current Proto
cols in Molecular Biology "(1989), John Wiely
& Sons, Inc. ISBN 0-471-50338-X and the like.

The activity of the carboxypeptidase contained in the crude enzyme solution or the purified carboxypeptidase is measured by, for example, phenylalanine released from peptides or proteins such as Z-glycyl-L-phenylalanine by the ninhydrin method, or hydrolyzed. It can be evaluated by measuring the decrease in absorbance at 224 nm associated with. The above-mentioned activity can also be evaluated using a commercially available reagent for measuring carboxypeptidase activity. Examples of commercially available reagents for measuring carboxypeptidase activity include "Acid Carboxypeptidase Assay Kit" manufactured by Kikkoman Corporation.

Since the carboxypeptidase of the present invention is derived from a filamentous fungus belonging to the genus Aspergillus, the use of the carboxypeptidase of the present invention allows rapid degradation of undegraded peptides contained in soy sauce and enzymatically decomposed seasonings, for example. . Therefore, if the carboxypeptidase of the present invention is used, it is possible to improve the amination ratio and change the amino acid composition in soy sauce and enzyme-decomposed seasonings, which can greatly contribute to the improvement of the taste.

[0034]

The present invention will be described in more detail with reference to the following examples, but the technical scope of the present invention is not limited to these examples. Example 1 Acquisition of carboxypeptidase cDNA (1) Search for carboxypeptidase homologous gene in Aspergillus genus Aspergillus niger-derived gene encoding a protein having homology to the amino acid sequence of serine-type carboxypeptidase F -A search from the Orize genome database yielded a genomic clone Con1393_h_791 consisting of 1602 bases. The clone was presumed to be a fragment of the carboxypeptidase gene, and the cDNA of the gene was cloned. Extraction of total RNA from Aspergillus oryzae RIB40 (Aspergillus oryzae)
var. viridis Murakami, anamorph ATCC 42149) 3 spores in 50 ml of soybean flour medium (3% soybean flour, 1% KH ₂ PO ₄ , pH 6.0)
The cells were inoculated at a density of × 10 ⁵ / ml, and cultured in a 150 ml Erlenmeyer flask at 30 ° C for 48 hours with shaking at 150 rpm.

After completion of the culture, the obtained culture solution was added to Miracloth
The cells were collected by filtration (manufactured by CALBIOCHEM). The collected bacterial cells were washed with sterilized water, frozen in liquid nitrogen, and physically ground using a mortar to give fine powdery bacterial cell pieces.
Total RN from the bacterial cells using ISOGEN (Nippon Gene)
A was extracted. All the operations followed the attached protocol.

(2) Acquisition of carboxypeptidase cDNA using RACE method From about 200 μg of total RNA obtained above, Oligotex-dT30 <Super
> Using mRNA Purification Kit (Takara Bio)
4 μg of mRNA was obtained. Marathon c from 1 μg of mRNA
DNA Amplification Kit (Clontech), Advantage c
5'-RACE and 3'-R using DNA PCR kit (Clontech)
ACE was done. Oligo DNAs represented by SEQ ID NOS: 3 to 6 were synthesized as primers used in the RACE method. That is, to perform 5'-RACE, genomic clone Con1393_
Antisense primer for h_791 (SEQ ID NO: 3
And 4) and 3'-RACE sense primers (SEQ ID NOs: 5 and 6). All the operations followed the attached protocol. GeneAmp 5700 DNA as a PCR device
A detection system (Perkin Elmer) was used.
As a result, a DNA fragment of about 0.3 kb corresponding to the 5'region of carboxypeptidase cDNA and a D of about 0.4 kb corresponding to the 3'region.
The amplification of the NA fragment was confirmed.

The amplified DNA fragments were separated in 0.7% agarose gel and extracted using QIAquick Gel Extraction Kit (manufactured by QIAGEN). The operation followed the attached protocol. The extracted DNA fragment is the TOPO TA Cloning Kit (In
Vitrogen) was used to incorporate the pCR TOPO vector. The resulting recombinant plasmid is Thermo Sequena
seCycle Sequencing Kit (Amersham Pharmacia Biotec
(manufactured by h company) is used for sequence reaction, and LI-COR MO
The nucleotide sequence was determined with a DEL4200L sequencer (purchased from Aloka). As a result, the DNA sequence of the open reading frame (ORF) of about 1.6 kb shown in SEQ ID NO: 1 was revealed, and the genomic clone Con1393_h_791 was found to be a partial fragment thereof. This DNA encoded a protein consisting of 533 amino acids. This amino acid sequence is shown in SEQ ID NO: 2.

PCR was performed using the cDNA prepared during 5'-RACE as a template to obtain the full-length cD of carboxypeptidase.
NA was cloned. The oligo DNAs shown in SEQ ID NOS: 7 and 8 were used as primers. Amplified DN of about 1.6 kb obtained
The A fragment was extracted by the method already described and pYES2.1 TOPO TA Clo
pYES2.1 / V5-His-T using ning Kit (Invitrogen)
Carboxypeptidase full-length cDN integrated into OPO vector
A recombinant plasmid pAOcp containing A was obtained. The base sequence of the recombinant plasmid pAOcp was analyzed again to confirm the base sequence of the carboxypeptidase cDNA (SEQ ID NO: 1).

A full-length carboxypeptidase cDNA, that is, a plasmid containing the nucleotide sequence represented by nucleotide numbers 1-1602 of SEQ ID NO: 1 is named "pAOcp", December 2002.
On the 2nd, the National Institute of Advanced Industrial Science and Technology became the Patent Biological Depository Center (1-1, Higashi, Tsukuba, Ibaraki, Japan)
1 Central No. 6) Deposited as FERM P-19150.

According to the protocol attached to pYES2.1 TOPO TA Cloning Kit, pAOcp was transformed into yeast host INVSc (Invitrogen
(Manufactured by the company) to induce the expression of the target protein. Non-induction medium (2% raffinose, 0.67% yeast nitrogen
base, 0.192% Yeast Synthetic Drop-out Medium Supp
transformants grown in lement without uracil)
Non-induction medium and induction medium (2% galactose so that 00 becomes 0.4)
ose, 0.67% yeast nitrogen base, 0.192% Yeast Synth
The carboxypeptidase activity was measured using the culture supernatant after inoculating each of the cells to etic Drop-out Medium Supplement without uracil) and culturing for 40 hours. An acid carboxypeptidase measurement kit (manufactured by Kikkoman) was used for the measurement. The results are shown in Table 1. The numerical values in Table 1 show carboxypeptidase activity (unit: mU / ml).

[0041]

[Table 1]

From the results shown in Table 1, the control strain pYES
No carboxypeptidase activity was observed in the culture supernatant of the strain into which only 2 was introduced. The transduced strain also showed no activity in the non-induction medium (raffinose medium), but a carboxypeptidase activity was specifically found in the induction medium (galactose medium). From the above results, it was revealed that the base sequence of SEQ ID NO: 1 isolated in this example is a gene encoding carboxypeptidase.

[0043]

As described above, according to the present invention, a completely novel carboxypeptidase, the gene, a recombinant DNA containing the gene, a transformant or transductant containing the recombinant DNA, and the transformant or transductant. It is possible to provide a method for producing carboxypeptidase using In particular,
According to the carboxypeptidase gene of the present invention, carboxypeptidase can be efficiently produced, which is extremely useful industrially.

[0044]

[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> A carboxypeptidase and a carboxypeptidase gene <130> P02-0979 <150> JP 2001-403261 <151> 2001-12-27 <160> 8 <170> PatentIn Ver. 2.0 <210> 1 <211> 1602 <212> DNA <213> Aspergillus oryzae <400> 1 atgctgttca aaagcatcgt gtcaacagct atcttagccg ctgccctgtg cacggacaat 60 gtatctgctg ccaaacatgg ccggttcggc caaaaggccc gcgactcttt gaatttggcc 120 aagcgagctg cagaacagca gaagtcgtcg ttcaagacgc cgcttgacga cttccggttc 180 ctaaccaata aaacaaaatc ctacagagtc gaccacctcc cagatgtccc ctttgatgtc 240 ggcgagatgt actcgggatt ggttcctatc gacaaggacg acaagtctag ggctcttttc 300 ttcgtcttcc agcctacact tggcgacccc gttgacgaga tcacaatctg gctcaacggt 360 ggaccgggat gtagctctct cgagggtttc ttccaggaaa acggccggtt tacatggcag 420 cctggcacgt ttgcgcctgt tgagaatccg tactcgtggg tgaacttgac taatgtgctt 480 tgggtcgagc agccagttgg tacaggtttc gcgatcggca agccgaatgc cacaacccag 540 gaagagactg ccgaggactt tgtgagattc ttcaagaatt tccaggagct ctttggcatc 600 aagaacttca agatttacgt tactggtgaa agttacgctg ggcgttatgt tccgtacatc 660 tccgctgcta tgttggaccg gaacgacacg gagcactatg acctcaaagg tgcccttgta 720 tacgacccct gtatcggcca acacgactac atccaggaag aagtgcccgc cgtgcccttc 780 gtccagcaaa atgccaacct cttcaacttc aactccagct tcatgtcgga actggagaaa 840 ctccacgact catgcggtta caaggattac ctagacgagt acctcgtctt ccctcccgcc 900 ggtgtgcaac cccagaaatc cttcaactac accagcgacg ccgactgcga cgtcttcgac 960 ctgatcagca acgaagccct ggtcgccaac tcctgcttcg acatctacga gatcaacctc 1020 atgtgcccgc tcgcctggga cgttctcgcc atgcccacag cattcaacta ccagcccgcc 1080 ggtgcgacgg tgtacttcga ccgcccggac gtcaaacgtg ccatgcacgc ccccctgaac 1140 gtcacttggt caggttgttc cagcgagaac gtctacgttg gcggcgacgc cggcccggag 1200 caagagggag acttgtccgc caacccgatc gaacacgtcc tgcctcaggt tattgaggga 1260 acgaaccgcg ttttggtcag caacggcgac tatgacatga tcatcctcac caatggaacc 1320 ctgctggcta tccagaatat gacctggaac gggcagctcg gattccagtc tgcgcctgct 1380 acgcctatca ccattgatct gcctgatcta gcgtggggcg aggtgtttga ggaaaatgga 1440 caagagatgt tgcagagcca gggcgtcatg ggtgtgcagc attatgagcg tgggcttatg 1500 tgggcggaga cctaccaatc tggacatatg cagccgcagt accagccgcg ggtgtcgtat 1560 cgtcatttgc aatggctttt gggtcgggtc gataagcttt aa 1602 <210> 2 <211> 533 <212> PRT <213> Aspergillus oryzae <400> 2 Met Leu Phe Lys Ser Ile Val Ser Thr Ala Ile Leu Ala Ala Ala Leu   1 5 10 15 Cys Thr Asp Asn Val Ser Ala Ala Lys His Gly Arg Phe Gly Gln Lys              20 25 30 Ala Arg Asp Ser Leu Asn Leu Ala Lys Arg Ala Ala Glu Gln Gln Lys          35 40 45 Ser Ser Phe Lys Thr Pro Leu Asp Asp Phe Arg Phe Leu Thr Asn Lys      50 55 60 Thr Lys Ser Tyr Arg Val Asp His Leu Pro Asp Val Pro Phe Asp Val  65 70 75 80 Gly Glu Met Tyr Ser Gly Leu Val Pro Ile Asp Lys Asp Asp Lys Ser                  85 90 95 Arg Ala Leu Phe Phe Val Phe Gln Pro Thr Leu Gly Asp Pro Val Asp             100 105 110 Glu Ile Thr Ile Trp Leu Asn Gly Gly Pro Gly Cys Ser Ser Leu Glu         115 120 125 Gly Phe Phe Gln Glu Asn Gly Arg Phe Thr Trp Gln Pro Gly Thr Phe     130 135 140 Ala Pro Val Glu Asn Pro Tyr Ser Trp Val Asn Leu Thr Asn Val Leu 145 150 155 160 Trp Val Glu Gln Pro Val Gly Thr Gly Phe Ala Ile Gly Lys Pro Asn                 165 170 175 Ala Thr Thr Gln Glu Glu Thr Ala Glu Asp Phe Val Arg Phe Phe Lys             180 185 190 Asn Phe Gln Glu Leu Phe Gly Ile Lys Asn Phe Lys Ile Tyr Val Thr         195 200 205 Gly Glu Ser Tyr Ala Gly Arg Tyr Val Pro Tyr Ile Ser Ala Ala Met     210 215 220 Leu Asp Arg Asn Asp Thr Glu His Tyr Asp Leu Lys Gly Ala Leu Val 225 230 235 240 Tyr Asp Pro Cys Ile Gly Gln His Asp Tyr Ile Gln Glu Glu Val Pro                 245 250 255 Ala Val Pro Phe Val Gln Gln Asn Ala Asn Leu Phe Asn Phe Asn Ser             260 265 270 Ser Phe Met Ser Glu Leu Glu Lys Leu His Asp Ser Cys Gly Tyr Lys         275 280 285 Asp Tyr Leu Asp Glu Tyr Leu Val Phe Pro Pro Ala Gly Val Gln Pro     290 295 300 Gln Lys Ser Phe Asn Tyr Thr Ser Asp Ala Asp Cys Asp Val Phe Asp 305 310 315 320 Leu Ile Ser Asn Glu Ala Leu Val Ala Asn Ser Cys Phe Asp Ile Tyr                 325 330 335 Glu Ile Asn Leu Met Cys Pro Leu Ala Trp Asp Val Leu Ala Met Pro             340 345 350 Thr Ala Phe Asn Tyr Gln Pro Ala Gly Ala Thr Val Tyr Phe Asp Arg         355 360 365 Pro Asp Val Lys Arg Ala Met His Ala Pro Leu Asn Val Thr Trp Ser     370 375 380 Gly Cys Ser Ser Glu Asn Val Tyr Val Gly Gly Asp Ala Gly Pro Glu 385 390 395 400 Gln Glu Gly Asp Leu Ser Ala Asn Pro Ile Glu His Val Leu Pro Gln                 405 410 415 Val Ile Glu Gly Thr Asn Arg Val Leu Val Ser Asn Gly Asp Tyr Asp             420 425 430 Met Ile Ile Leu Thr Asn Gly Thr Leu Leu Ala Ile Gln Asn Met Thr         435 440 445 Trp Asn Gly Gln Leu Gly Phe Gln Ser Ala Pro Ala Thr Pro Ile Thr     450 455 460 Ile Asp Leu Pro Asp Leu Ala Trp Gly Glu Val Phe Glu Glu Asn Gly 465 470 475 480 Gln Glu Met Leu Gln Ser Gln Gly Val Met Gly Val Gln His Tyr Glu                 485 490 495 Arg Gly Leu Met Trp Ala Glu Thr Tyr Gln Ser Gly His Met Gln Pro             500 505 510 Gln Tyr Gln Pro Arg Val Ser Tyr Arg His Leu Gln Trp Leu Leu Gly         515 520 525 Arg Val Asp Lys Leu     530 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer DNA <400> 3 tcgtccttgt cgataggaac caatcccgag 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer DNA <400> 4 gtgtaggctg gaagacgaag aaaagagccc 30 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer DNA <400> 5 accctgctgg ctatccagaa tatgacctgg 30 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer DNA <400> 6 cctatcacca ttgatctgcc tgatctagcg 30 <210> 7 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer DNA <400> 7 gcatcgtaaa aatggtgttc aaaagcatcg tgtcaacagc 40 <210> 8 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer DNA <400> 8 aagcttatcg acccgaccca aaagccattg ccaaatg 37

[0045]

[Sequence Listing Free Text] SEQ ID NOs: 3 to 8 are primers.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 9/62 C12N 5/00 A (72) Inventor Masayuki Machida 1-1-1 Higashi, Tsukuba, Ibaraki Prefecture Inside Hoken AIST (72) Inventor Keietsu Abe 1-1-1 Higashi, Tsukuba, Ibaraki Prefecture Independent Administrative Law Inside AIST (72) Inventor Katsuya Gomi 1-1-1 Higashi, Tsukuba, Ibaraki Independent Administrative Law Inside the National Institute of Advanced Industrial Science and Technology (72) Inventor Kiyoshi Asai 2-41-6 Aomi, Koto-ku, Tokyo Independent Administrative Law Inside the National Institute of Advanced Industrial Science and Technology (72) Motoaki Sano 1-1-Higashi, Tsukuba, Ibaraki Prefecture 1 Independent Administrative Law, National Institute of Advanced Industrial Science and Technology (72) Inventor Kim Taishin 2-41-6 Aomi, Koto-ku, Tokyo Independent Administrative Law, National Institute of Advanced Industrial Science and Technology (72) Invention Person Hideki Nagasaki 2-41-6 Aomi, Koto-ku, Tokyo Independent Administrative Law, National Institute of Advanced Industrial Science and Technology (72) Inventor Satoshi Hosoyama 2-49-10 Nishihara, Shibuya-ku, Tokyo Independent administrative law, Human Product Evaluation Technology Infrastructure ( 72) Inventor Osamu Akita 3-7-1 Kagamiyama, Higashihiroshima City, Hiroshima Prefecture Independent Administrative Institution Liquor Research Institute (72) Naoki Ogasawara 8916-5 Takayama-cho, Ikoma-shi, Nara Nara Institute of Science and Technology Graduate School of Bioscience Graduate School (72) Inventor Satoshi Kuhara 6-10-1 Hakozaki, Higashi-ku, Fukuoka City, Fukuoka Prefecture Kyushu University Faculty of Agriculture, Department of Genetic Resource Engineering (72) Inventor Toshifumi Matsuda 250 Noda, Noda, Chiba Prefecture Kikkoman Corporation (72) Kenichiro Matsushima, Noda City, Chiba Prefecture, Noda 250, Kikkoman Corporation (72) Inventor, Taiji Koyama 250, Noda City, Chiba Prefecture, Noda City, Kikkoman Corporation, F Term (Reference) 4B024 AA05 BA14 CA04 DA12 GA11 HA12 4B050 CC01 CC03 DD03 LL02 LL03 4B065 A A63Y AA72X AB01 AC14 CA31 CA41 CA46

Claims (6)

[Claims] 1. A carboxypeptidase of the following (a) or (b): (A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and having a carboxypeptidase activity Protein to have 2. A carboxypeptidase gene encoding the following protein (a) or (b). (A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (b) consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and having a carboxypeptidase activity Protein to have 3. A carboxypeptidase gene consisting of the following DNA (a) or (b): (A) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 (b) DNA hybridizing with the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 under stringent conditions and encoding a protein having carboxypeptidase activity 4. A novel recombinant DNA comprising the carboxypeptidase gene according to claim 2 or 3 inserted into vector DNA. 5. A transformant or transductant containing the recombinant DNA according to claim 4. 6. A method for producing carboxypeptidase, which comprises culturing the transformant or transductant according to claim 5 in a medium and collecting carboxypeptidase from the culture.