JP2003219888A - Aminopeptidase P, aminopeptidase P gene, recombinant DNA and method for producing aminopeptidase P - Google Patents

Abstract

(57) [Summary] (Revised) [Solution] Aminopeptidase P having a specific amino acid sequence, aminopeptidase P gene encoding this protein, aminopeptidase gene having a specific nucleotide sequence, and these genes as vector DNA An inserted recombinant DNA, a transformant or a transductant containing the recombinant DNA, and culturing the transformant or the transductant in a medium, and collecting aminopeptidase P from the culture; aminopeptidase; This is a method for producing P. According to the present invention, a novel aminopeptidase P,
A method for producing aminopeptidase P gene, recombinant DNA and aminopeptidase P was provided. According to the present invention, protein engineering of the aminopeptidase P can be improved. Further, the present invention can be used for production of enzymes for food processing and improvement of microorganisms used for production of brewed foods.

Description

Detailed Description of the Invention

[0001]

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

[0002]

2. Description of the Related Art Aminopeptidase P (X-Pro aminopeptidase, proline aminopeptidase, aminoacylproline aminopeptidase, EC 3.4.11.9, hereinafter referred to as aminopeptidase P) has proline as the second amino acid from the amino terminus. It is an enzyme that releases amino-terminal amino acids from peptide chains containing dipeptides / tripeptides. Aminopeptidase P is known to be involved in metabolism of the vasodilator bradykinin in the body of animals.

Until now, the enzymatic properties of many aminopeptidases have been revealed from mammals, plants and bacteria,
Also, genes have been analyzed. For example, in mammals, porcine kidney origin [Biochem J. 319: 197-201 (199
6)], derived from rat pulmonary blood vessels [J. Pharmacol Exp. Ther., 26.
9: 941-947 (1994)], and bacteria include Escherichia coli [J. Biochem., 104: 93-97 (1988)] and lactic acid bacteria [J. Dairy Res., 64: 399-407]. (1997)]. In addition, in plants, tomato [J. Biol. Che
m., 276: 31732-31737, (2002)] and the like are known. The above aminopeptidase P has also been reported as a gene.

Aspergillus oryzae and Aspergillus soya, which are molds of Aspergillus oryzae, have been used for a long time in the production of brewed foods in Japan such as miso, soy sauce, sake, etc., and have high enzyme productivity and many years of use. It is a microorganism that is particularly important in industry due to its high reliability in safety due to its use. With regard to aminopeptidase P derived from the genus Aspergillus containing these Aspergillus oryzae, no reports have been made on the purified enzyme and the enzyme activity of the gene product.

[0005]

In the production of soy sauce, the raw material is decomposed into a polypeptide by a protease produced by Aspergillus oryzae. Further, it is decomposed from leucine aminopeptidase from the amino terminus and from acidic carboxypeptidase from the carboxy terminus to a low-molecular peptide mainly composed of amino acids and dipeptides. However, when proline is present in the peptide, leucine aminopeptidase ceases to act in the state of Xaa-Pro-peptide in which one amino acid is linked to the imino group side of proline. Therefore, an enzyme that releases Xaa and subsequent proline from Xaa-Pro-peptide in soy sauce moromi is strongly desired.

Aminopeptidase P releases N-terminal Xaa from the Xaa-Pro-peptide on which leucine aminopeptidase does not act. Further, when proline iminopeptidase acts, N-terminal proline is also released and can be used again as a substrate for leucine aminopeptidase. Proline is
Since it is an amino acid having a taste property, the taste can be modified, and the effect of improving the taste can be expected even in the case of an amino acid such as Glu-Pro-peptide or Asp-Pro-peptide whose terminal is related to the taste. Further, as the dipeptide remaining in soy sauce, Ser-Pro, Thr-Pro, Ala-Pro, Gly-Pro have been identified, these terminal amino acids are all amino acids that present sweetness and umami, It can be expected to improve the sweetness and umami of soy sauce. Therefore, it is considered that in the production of soy sauce and enzymatically decomposed seasonings, aminopeptidase P largely contributes to the improvement of amination ratio and the improvement of taste. Therefore, it is desired to obtain aminopeptidase P from a microorganism in which a large amount of enzyme can be prepared relatively easily, and it is further strongly desired to isolate aminopeptidase P from a highly safe microorganism such as koji mold. The present invention provides a method for producing aminopeptidase P, aminopeptidase P gene, recombinant DNA and aminopeptidase P capable of degrading Xaa-Pro and Xaa-Pro-peptide present in soy sauce and enzymatically decomposed seasonings. The purpose is to do.

[0007]

Therefore, as a result of intensive studies to solve the above problems, the present inventors succeeded in cloning a novel aminopeptidase P gene from Aspergillus oryzae, and completed the present invention. . That is, the present invention is: The following protein (a) or (b). (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (b) an aminopeptidase consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 A protein having P activity 2. A protein comprising an amino acid sequence having 70% or more sequence homology with the amino acid sequence represented by SEQ ID NO: 2 or a partial fragment thereof and having aminopeptidase P activity. 3. An aminopeptidase P gene encoding the following protein (a) or (b). (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (b) an aminopeptidase consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 Protein having P activity 4. An aminopeptidase P gene, which is a protein comprising an amino acid sequence having 70% or more sequence homology with the amino acid sequence represented by SEQ ID NO: 2 or a partial fragment thereof, and which encodes a protein having aminopeptidase P activity. 5. An aminopeptidase P gene consisting of the following DNA (a) or (b): (a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 (b) Hybridizing with DNA consisting of the nucleotide sequence complementary to the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions, And DNA encoding a protein having aminopeptidase P activity 6. A recombinant DNA, characterized in that the gene described in 3, 4, or 5 is inserted into vector DNA. 7. A transformant or transductant containing the recombinant DNA according to 6 above. 8. A method for producing aminopeptidase P, which comprises culturing the transformant or transductant described in 7 above in a medium and collecting aminopeptidase P from the culture.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. 1. Aminopeptidase P of the Present Invention The aminopeptidase P of the present invention is a protein consisting of the amino acid sequence represented by SEQ ID NO: 2. The enzyme is, for example, Aspergillus soja or Aspergillus
It can be purified from a culture of Aspergillus oryzae such as oryzae.
The enzyme can be obtained by expressing the aminopeptidase P gene cloned from Aspergillus oryzae in an appropriate host-vector system.

The aminopeptidase P of the present invention may have one or more amino acids deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 as long as it has an enzymatic activity. Further, as long as it has aminopeptidase P activity, it has 70% or more, preferably 75% or more, more preferably 8% with the amino acid sequence represented by SEQ ID NO: 2.
It may be a protein consisting of an amino acid sequence showing a sequence homology of 0% or more, and most preferably 85% or more, or a partial fragment thereof.

In order to determine the sequence homology between two amino acid sequences or base sequences, the sequences are pre-treated in an optimal state for comparison. For example, by inserting a gap in one sequence, alignment with the other sequence is optimized. Then, the amino acid residues or bases at each site are compared. If a site in the first sequence has the same amino acid residue or base as the corresponding site in the second sequence, then the sequences are identical at that site. Sequence homology between two sequences is shown as a percentage of the number of identical sites between sequences with respect to the total number of sites (all amino acids or all bases).

According to the above-mentioned principle, the sequence homology between two amino acid sequences or base sequences is Karlin and Altschu.
l algorithm (Proc. Natl. Acad. Sci. USA 87: 226
4-2268, 1990 and Proc. Natl. Acad. Sci. USA 90: 5873.
-5877, 1993). The BLAST program using such an algorithm was developed by Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). Furthermore, Gap
ped BLAST is a program for determining sequence homology with higher sensitivity than BLAST (Nucleic Acids Res. 25: 3389-3402,
1997). The above program is mainly used to search a database for a sequence showing high sequence homology with a given sequence. These are, for example, US Nati
It is available on the website of the onal Center for Biotechnology Information on the Internet.

Sequence homology between sequences refers to BLAST 2 developed herein by Tatiana A. Tatusova et al.
Sequences software (FEMS Microbiol Lett., 174:
247-250, 1999). This software is the National Center for Biotechnology
It is available and available on the Information website on the Internet. The programs and parameters used are as follows. In the case of amino acid sequences, the parameters are Open gap: 11 and extension gap: 1 penalties, ga using the blastp program.
p x_dropoff: 50, expect: 10, word size: 3, Filter:
Use ON. In the case of nucleotide sequences, the parameters are Reward for a match: 1, Pen using the blastn program.
alty for amismatch: -2, Strand option: Both strand
s, Open gap: 5 and extension gap: 2 penalties, g
ap x_dropoff: 50, expect: 10, word size: 11, Filte
Use r: ON. Both parameters are used as default values on the website.

However, when a sequence showing significant sequence homology cannot be found by the above BLAST software,
Highly sensitive FASTA software (WR Pearson and DJ Li
pman, Proc. Natl. Acad. Sci., 85: 2444-2448, 1988)
It is also possible to search the database for sequences showing sequence homology. FASTA software is available, for example, on the GenomeNet website. Also in this case,
The parameters use default values. For example, when performing a search for a nucleotide sequence, nr
-nt is used and ktup value is 6.

However, in any case, if there is no overlap of 30% or more, 50% or more, or 70% or more of the whole, it is not necessarily presumed that they are functionally correlated, and therefore the two sequences It is not used as a value indicating the sequence homology between the two.

2. Cloning of aminopeptidase P gene The aminopeptidase P gene of the present invention can be obtained from Aspergillus oryzae such as Aspergillus sojae or Aspergillus oryzae, other filamentous fungi or other fungi. More specifically, for example, Aspergillus oryzae var. Viridis
Murakami, anamorph; ATCC 42149). Total RNA is recovered from a culture of these cells in a medium that produces aminopeptidase P by a conventional method. Examples of the medium include bran medium (2.78 g of wheat bran
2.22 g of deionized water was added and autoclaved at 121 ° C. for 50 minutes). After culturing in the above medium for an appropriate time, for example, 30 hours, an appropriate amount (for example, 1 g) is transferred to a mortar filled with liquid nitrogen, crushed with a pestle, and the method of Cathala et al. [DNA, 2 (4): 329-335, 1983]
Prepare RNA.

RT-PCR is performed using the thus obtained total RNA as a template. As the primer, any combination may be used as long as it can amplify the aminopeptidase P gene of the present invention. For example, use of oligonucleotides having the sequences of SEQ ID NO: 3 and SEQ ID NO: 4. You can RT-PCR can be performed by a conventional method using a commercially available kit, for example, RNA LA-PCR Kit (manufactured by Takara Shuzo). The obtained DNA containing the aminopeptidase P gene of the present invention can be incorporated into a plasmid by a conventional method, for example. The nucleotide sequence of the DNA thus obtained can be determined by the Sanger method using a commercially available reagent and a DNA sequencer. Examples of the DNA containing the aminopeptidase P gene of the present invention thus obtained and the aminopeptidase P gene encoded thereby are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

The aminopeptidase P gene of the present invention comprises:
In addition to the above, as long as it has aminopeptidase P activity, it encodes a protein containing an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2. May be. Such a gene can be obtained by various known mutagenesis methods in addition to the selection by hybridization described below.

The aminopeptidase P gene of the present invention comprises:
It can also be obtained using a selection method by hybridization as follows. Examples of the gene source include Aspergillus oryzae such as Aspergillus soja or Aspergillus oryzae. From these organisms, RN
A or genomic DNA is prepared and integrated into a plasmid or phage to prepare a library. Then, the nucleic acid used as a probe is labeled by a method suitable for the detection method. The nucleic acid used as a probe may have a length that allows sufficient specificity, for example, at least 100 bases or more, preferably 200 bases or more, and most preferably 450 bases or more of the sequence of SEQ ID NO: 1, or The thing including the whole is mentioned. Then, a clone that hybridizes to the labeled probe under stringent conditions is selected from the above library. Hybridization can be carried out by colony hybridization if it is a plasmid library, and by plaque hybridization if it is a phage library.
The stringent condition is a condition under which a signal of a specific hybrid is clearly distinguished from a signal of a non-specific hybrid, and varies depending on the hybridization system used, the type of probe, the sequence and the length. Such conditions can be determined by varying the temperature of hybridization and the temperature of washing and salt concentration. For example, if the signal of a non-specific hybrid is strongly detected, raise the temperature of hybridization and washing,
If necessary, the specificity can be increased by lowering the salt concentration for washing. When no specific hybrid signal is detected, the hybrid can be stabilized by lowering the temperature of hybridization and washing and, if necessary, increasing the salt concentration of washing. Such optimization can be easily performed by a researcher in this technical field.

Specific examples of stringent conditions include, for example, hybridization of 5 × SSC, 1.
Using 0% (W / V) blocking reagent for nucleic acid hybridization (manufactured by Boehringa Mannheim), 0.1% (W / V) N-lauroylsarcosine, 0.02% (W / V) SDS overnight (8-
About 16 hours), wash 0.5 × SSC, 0.1% (W /
V) SDS, preferably 0.1 x SSC, 0.1% (W / V) SDS, 15
Do twice a minute. The temperature of hybridization and washing is 52 ° C or higher, preferably 57 ° C or higher, more preferably
It is 62 ° C or higher, most preferably 67 ° C or higher.

The nucleotide sequence shown in SEQ ID NO: 1 and 70
% Or more, preferably 75% or more, more preferably 80%
As described above, it is considered that the most preferable nucleotide sequence showing 85% or more sequence homology encodes a protein having substantially the same activity as aminopeptidase P of the present invention. DNA showing the sequence homology of the above base sequence or the sequence homology of the encoded amino acid sequence
Can also be obtained by using hybridization as an index as described above, and is found by a search using the above-mentioned BLAST software, for example, from DNA groups of unknown function obtained by genomic nucleotide sequence analysis or public databases. It's also easy. Such a search is a method usually used by researchers in this technical field.

The fact that the DNA thus obtained encodes a protein having aminopeptidase P activity means that, as will be described later, it is incorporated into an appropriate vector, an appropriate host is transformed, and a transformant is obtained. It can be confirmed by culturing and measuring aminopeptidase P activity.

3. Preparation of Recombinant Vector The recombinant vector of the present invention can be obtained by ligating the aminopeptidase P gene of the present invention on a suitable vector. As the vector, any vector can be used as long as it can produce aminopeptidase P in a host to be transformed. For example, a vector such as a plasmid, cosmid, phage, virus, chromosome-integrated type, or artificial chromosome can be used.

The above-mentioned vector may contain a marker gene for allowing the selection of transformed cells. Examples of marker genes include UR
Examples include genes that complement the auxotrophy of the host, such as A3 and niaD, and resistance genes to drugs such as ampicillin, kanamycin, and oligomycin. Also,
The recombinant vector preferably contains a promoter or other regulatory sequences capable of expressing the gene of the present invention in a host cell (for example, enhancer sequence, terminator sequence, polyadenylation sequence, etc.). Specific examples of the promoter include GAL1 promoter, amyB promoter, lac promoter and the like. Also, a tag for purification can be attached. For example, a linker sequence is appropriately connected downstream of the aminopeptidase P gene, and the base sequence encoding histidine is 6
By connecting more than the codon, it is possible to enable purification using a nickel column.

4. Obtaining Transformant The transformant of the present invention can be obtained by transforming a host with the recombinant vector of the present invention. As a host,
It is not particularly limited as long as it can produce the aminopeptidase P of the present invention, and examples thereof include yeasts such as Saccharomyces cerevisiae and Tigosaccharomyces lucii, and filamentous fungi such as Aspergillus soya, Aspergillus oryzae and Aspergillus niger. , Escherichia coli, Bacillus subtilis, and other bacteria.
Transformation can be performed by a known method depending on the host. In the case of yeast, for example, using lithium acetate, Me
The method of thods Mol. Cell. Biol., 5, 255-269 (1995) can be used. In the case of filamentous fungi, for example, a method using polyethylene glycol and calcium chloride after protoplast formation [Mol. Gen. Genet., 218, 99-104 (1
989)] can be used. When using bacteria, for example, by methods such as electroporation, Methods Enzymo
The method of L., 194, 182-187 (1990) and the like can be used.

5. Production of aminopeptidase P The method for producing aminopeptidase P of the present invention comprises culturing the transformant or transductant of the present invention and collecting the aminopeptidase P protein from the resulting culture. Appropriate medium and culture method may be selected depending on the type of host and the expression control sequence in the recombinant vector. For example, when the host is Saccharomyces cerevisiae and the expression control sequence is the GAL1 promoter, for example, cells pre-cultured in a liquid minimal medium containing raffinose as a carbon source, a liquid minimal medium containing galactose and raffinose as a carbon source. The aminopeptidase P of the present invention can be produced by diluting, inoculating, and culturing. Further, for example, when the host is Aspergillus soja and the expression control sequence is the amyB promoter,
For example, the aminopeptidase P of the present invention can be produced by culturing in a minimal liquid medium containing maltose as a carbon source.

Further, for example, when the host is Escherichia coli and the expression control sequence is the lac promoter, isopropyl-
The aminopeptidase P of the present invention can be produced by culturing in a liquid medium containing β-D (−)-thiogalactopyranoside (IPTG). When the aminopeptidase P of the present invention is produced in or on the surface of cells,
The aminopeptidase P of the present invention can be obtained by separating the cells from the medium and treating the cells appropriately. For example, when produced on the surface of Saccharomyces cerevisiae cells, the cells themselves are used as an enzyme agent, and the cells are crushed, then Triton X-100, Tween-20, Nonidet P-
A nonionic surfactant such as 40 is allowed to act at a low concentration, the mixture is centrifuged, and the supernatant is used to extract the aminopeptidase P of the present invention.
Can be recovered. When the aminopeptidase P of the present invention is produced in the culture medium, the aminopeptidase P of the present invention can be obtained by removing the bacterial cells by centrifugation, filtration or the like. In any case, ammonium sulfate fractionation,
The aminopeptidase P of the present invention can be obtained in higher purity by a conventional method using various chromatography, alcohol precipitation, ultrafiltration and the like.

[Method for measuring aminopeptidase P activity] The activity of aminopeptidase P of the present invention is determined by Hooper and Turne.
r, etc. (1988, FEBS Lett. 229, 340-344). That is, using Gly-Pro-HyPro as a substrate, Pro-HyPro released by enzymatic action is separated by reverse layer high performance liquid chromatography to separate the product, and Pro-Hy produced per unit time from the absorbance of ultraviolet light at 214 nm.
Pro can be quantified.

[0028]

EXAMPLES The present invention will be described more concretely with reference to the following examples.

Example 1. Aspergillus oryzae aminopeptidase P
0 Gene Cloning Aspergillus oryzae RIB40
Strain (Aspergillus oryzae var.viridis Murakami, anamor
Approximately 100,000 conidia of ph; ATCC42149) were inoculated into 5 g of bran medium (described above) in a 150 ml Erlenmeyer flask, and the temperature was 30 ° C.
Then, static culture was carried out for 30 hours. The culture was put into a mortar cooled by pouring liquid nitrogen, liquid nitrogen was added, and then the culture was carefully crushed using a pestle cooled with liquid nitrogen. From the crushed cells, the method of Cathala et al. [DNA, 2 (4): 329-335,
1983], total RNA was extracted. In addition, the DNA-free kit (Ami
DNase I treatment was performed using bion) to decompose the contaminated DNA. RT-PCR was carried out using 1.0 μg of the obtained total RNA as a template and a Marathon cDNA Amplification Kit (Clontech).

Oligo dT was used as a primer for the reverse transcription reaction.
The reverse transcription reaction was carried out at 42 ° C. for 60 minutes using the one attached to the kit, which had an adapter sequence on the 3 ′ side of. Next, a cocktail containing RNaseH, DNA polymerase, DNA ligase, etc. was added to the reverse transcription reaction product according to the instructions attached to the kit, and the mixture was reacted at 16 ° C for 90 minutes, and then T4 was further added.
DNA polymerase was added and reacted at 16 ° C. for 50 minutes to synthesize a double-stranded cDNA library. Next, adapter DNA, DNA ligase, etc. were added to the above reaction product to synthesize a double-stranded cDNA library to which an adapter was bound.
The composition of each reaction solution and the reaction conditions were in accordance with the attached instructions.

Next, PCR is carried out using the double-stranded cDNA library obtained above as a template and the primers of SEQ ID NO: 3 and SEQ ID NO: 4. The'side was amplified immediately after the stop codon.
A restriction enzyme site was added to the 5'side of each primer to facilitate cloning (for example, KpnI was introduced into SEQ ID NO: 3 and XbaI into SEQ ID NO: 4). As a thermostable DNA polymerase, Takara EX Taq DNA Pol
ymerase (Takara Shuzo) was used, and the composition of the reaction solution was in accordance with the instructions attached to the polymerase.

The PCR reaction was carried out at 94 ° C. for 2 minutes, then at 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, 72 ° C. for 2 minutes for 30 cycles, and
It was performed at 2 ° C for 5 minutes. When a part of the amplified product was electrophoresed on 0.7% agarose gel, a band of about 2.0 kb was confirmed. As a thermal cycler, GeneAmp
5700 Sequence detection system (PE Applied Biosyst
(manufactured by ems) was used, and the temperature control method was a calculate control.

Next, TOPO TA Cloning Kit (Invitroge
(manufactured by N. Co., Ltd.) was used to incorporate the above amplification product into the pCR2.1TOPO vector, and E. coli TOP10F ′ strain (manufactured by Invitrogen) was transformed to obtain a transformant. QIAprep sp from transformants
Plasmids were extracted using in Miniprep Kit (Qiagen), and Thermo Sequenase Cycle Sequencing was used.
Kit) (manufactured by Amersham Pharmacia Biotech) was used to carry out a sequence reaction, and the nucleotide sequence was determined using a LI-COR MODEL4200L sequencer (manufactured by LI-COR).

As a result, the DNA sequence of the approximately 2.0 kb open reading frame (ORF) shown in SEQ ID NO: 1 was revealed. This plasmid pAoAPP1354 was transferred to FERM at the Patent Biological Deposit Center, National Institute of Advanced Industrial Science and Technology.
Deposited as BP-7868. Also, pAoAPP13
The nucleotide sequence of the clone contained in 54 from the start codon to immediately before the stop codon is shown in SEQ ID NO: 1. Analysis of the above nucleotide sequence revealed that this DNA encodes a protein consisting of 654 amino acids. This amino acid sequence is shown in SEQ ID NO: 2.

Furthermore, the amino acid sequence database of this amino acid sequence was searched for a sequence having high sequence homology. Search for NCBI blastp (http://www.ncbi.nlm.ni
h.gov/BLAST/) was used and nr was designated as the database. As a result, there was no matching sequence, and the highest sequence homology was found to be due to human aminopeptidase P (Ge
nBank: AF195530) and showed 46% sequence homology.
Further, the sequence homology of the nucleotide sequence of SEQ ID NO: 1 was examined. When the sequence homology of the entire coding region was examined with the analysis software GENETYX-MAC Ver.10.1, the sequence identity was 53.8% over 1855 bases.

[0036]

INDUSTRIAL APPLICABILITY According to the present invention, a novel aminopeptidase P, aminopeptidase P gene, recombinant DNA
And a method for producing aminopeptidase P. According to the present invention, the above aminopeptidase P can be improved in protein engineering. The present invention can also be used for enzyme production for food processing and improvement of microorganisms used for production of brewed food.

[0037]

[Sequence list]                     SEQUENCE LISTING <110> KIKKOMAN CORPORATION <120> A AMINOPEPTIDASE P, A AMINOPEPTIDASE P GENE,       A RECOMBINANT DNA, AND A PROCESS FOR       PRODUCING AMINOPEPTIDASE P <130> P2263 <160> 4 <210> 1 <211> 1962 <212> DNA <213> Aspergillus oryzae RIB40 <400> 1 atg ctt ttt tct cgc cct cca att cgt tct ccg tgg ata tca gcg ttt cga tcg gct agc 60 cag cta ccc ctc tct cgc ccc agg ttc ttt tct atc tct tta tct cgc tat tca gtc gac 120 atg gag acc gtg aac acc tcc gaa cgt ctt tcg aga ctc aga gag ttg atg cag gaa cac 180 aag gtc gat gta tac att gtc cca tct gaa gat agt cat cag tcg gag tat att gct cca 240 tgc gac ggc cgc cga gaa ttc atc tct ggt ttc tcc ggt tcc gct gga act gcc att gtt 300 tct ctg agc aaa gcc gct ctg tcc act gat ggg cga tat ttc aac cag gct tca aaa cag 360 ctt gac aac aac tgg cag ctc cta aag cgt ggt gtc gaa ggc ttt cct aca tgg cag gaa 420 tgg act acc gaa cag gcc gaa ggc ggt aag gtc gtt ggt gtc gac cca gct ttg atc aca 480 gcg tct ggt gcc cgt agc ctg tcg gaa acc ctc aag aaa aat ggc tcg aca ctg gtg ggt 540 gtt cag cag aat ttg gtt gat cta gtt tgg ggc aag gac cgg cct gct ccc ccg agg gag 600 aag gta agg gtt cat ccg gaa aag tac gct gga aag agc ttc cag gaa aag att agc gaa 660 ctc cgc aaa gag ctg gag tcg aga aaa tcg gcc gga ttc atc gtt tca atg ctc gat gaa 720 att gcg tgg ctg ttc aac tta aga ggc agc gac att cct tat aat ccg gtg ttc ttc tct 780 ttt gcc acc atc aca cct aca act acg gag ctt tat gtc gac gct gac aag ctg acg ccg 840 gag gta aca gct cat ctg ggt caa gac gtt gtg atc aag cca tat gat gcc atc tat gcc 900 gac gcg aag gcc ctc agt gag aca aga aag caa gaa gcg gga gaa acg gcc tct aag ttt 960 ttg ttg tcc aat aaa gct tca tgg gca ctt agc ctt agc ctt gga ggc gag gga cag gtc 1020 gag gaa gtc cgt agc ccc att ggc gat gct aaa gca gtg aag aat gat gtg gaa ctc gcg 1080 ggc atg cgg gca tgc cac atc cgc gac ggt gcc gcg cta aca gaa tac ttc gcc tgg ctc 1140 gag aac gaa cta gtc aac aag aaa tcg act ctt gac gaa gtg gac gcc gca gac aaa cta 1200 gag cag atc aga tct aag cac gac ctc ttt gtc ggt ctt tcg ttt gac act atc tcc tct 1260 aca ggc cct aac gga gcg gtt atc cac tat aaa ccg gaa aag gga agt tgc tct atc att 1320 gat cca aat gcc ata tac ctc tgc gat tct ggt gct caa tac ctg gat gga acc acg gat 1380 gtt acc aga acg ttc cat ttc gga caa cct acc gag ctc gag aag aag gcc ttc aca ttg 1440 gtt ctc aag ggc gtg att ggt ctg gat acc gct gtg ttc cct aag ggt acg agt ggg ttt 1500 gca ctt gat gtg ctc gcc aga cag tac ctc tgg aag gag gga ctt gat tac ttg cat gga 1560 acc ggg cac gga atc ggc tcc tat ttg aat gta cac gag gga cca att ggt gtc ggc acc 1620 cgt gtt cag tac acc gag gtc cct ata gca cct gga aac gtc atc tct gat gag cct ggt 1680 ttc tac gag gac ggc aag ttt ggt att cgg ata gag aat gtt atc atg gcg cgt gag gtg 1740 caa acc acg cac aag ttt ggg gat aaa ccg tgg cta ggc ttc gag cat gtc aca atg gcc 1800 cct att ggt cgc aat ttg atc gag cca tct ctt ctc agt gat gct gag ctc aaa tgg gtg 1860 aat gac tac cac cga gag atc tgg gag aag acc cac cac ttc ttc gag aac gat gaa tgc 1920 aca cgc agc tgg ctc cag cgg gaa aca cag ccc atc tct aaa                 1962 <210> 2 <211> 654 <212> PRT <213> Aspergillus oryzae RIB 40 <400> 2     Met Leu Phe Ser Arg Pro Pro Ile Arg Ser Pro Trp Ile Ser Ala                         5 10 15     Phe Arg Ser Ala Ser Gln Leu Pro Leu Ser Arg Pro Arg Phe Phe                        20 25 30     Ser Ile Ser Leu Ser Arg Tyr Ser Val Asp Met Glu Thr Val Asn                        35 40 45     Thr Ser Glu Arg Leu Ser Arg Leu Arg Glu Leu Met Gln Glu His                        50 55 60     Lys Val Asp Val Tyr Ile Val Pro Ser Glu Asp Ser His Gln Ser                        65 70 75     Glu Tyr Ile Ala Pro Cys Asp Gly Arg Arg Glu Phe Ile Ser Gly                        80 85 90     Thr Ala Ile Val Ser Leu Ser Lys Ala Ala Leu Ser Thr Asp Gly                        95 100 105     Arg Phe Ser Gly Ser Ala Gly Tyr Phe Asn Gln Ala Ser Lys Gln                       110 115 120     Leu Asp Asn Asn Trp Gln Leu Leu Lys Arg Gly Val Glu Gly Phe                       125 130 135     Pro Thr Trp Gln Glu Trp Thr Thr Glu Gln Ala Glu Gly Gly Lys                       140 145 150     Val Val Gly Val Asp Pro Ala Leu Ile Thr Ala Ser Gly Ala Arg                       155 160 165     Ser Leu Ser Glu Thr Leu Lys Lys Asn Gly Ser Thr Leu Val Gly                       170 175 180     Val Gln Gln Asn Leu Val Asp Leu Val Trp Gly Lys Asp Arg Pro                       185 190 195     Ala Pro Pro Arg Glu Lys Val Arg Val His Pro Glu Lys Tyr Ala                       200 205 210     Gly Lys Ser Phe Gln Glu Lys Ile Ser Glu Leu Arg Lys Glu Leu                       215 220 225     Glu Ser Arg Lys Ser Ala Gly Phe Ile Val Ser Met Leu Asp Glu                       230 235 240     Ile Ala Trp Leu Phe Asn Leu Arg Gly Ser Asp Ile Pro Tyr Asn                       245 250 255     Pro Val Phe Phe Ser Phe Ala Thr Ile Thr Pro Thr Thr Thr Glu                       260 265 270     Leu Tyr Val Asp Ala Asp Lys Leu Thr Pro Glu Val Thr Ala His                       275 280 285     Leu Gly Gln Asp Val Val Ile Lys Pro Tyr Asp Ala Ile Tyr Ala                       290 295 300     Asp Ala Lys Ala Leu Ser Glu Thr Arg Lys Gln Glu Ala Gly Glu                       305 310 315     Thr Ala Ser Lys Phe Leu Leu Ser Asn Lys Ala Ser Trp Ala Leu                       320 325 330     Ser Leu Ser Leu Gly Gly Glu Gly Gln Val Glu Glu Val Arg Ser                       335 340 345     Pro Ile Gly Asp Ala Lys Ala Val Lys Asn Asp Val Glu Leu Ala                       350 355 360     Gly Met Arg Ala Cys His Ile Arg Asp Gly Ala Ala Leu Thr Glu                       365 370 375     Tyr Phe Ala Trp Leu Glu Asn Glu Leu Val Asn Lys Lys Ser Thr                       380 385 390     Leu Asp Glu Val Asp Ala Ala Asp Lys Leu Glu Gln Ile Arg Ser                       395 400 405     Lys His Asp Leu Phe Val Gly Leu Ser Phe Asp Thr Ile Ser Ser                       410 415 420     Thr Gly Pro Asn Gly Ala Val Ile His Tyr Lys Pro Glu Lys Gly                       425 430 435     Ser Cys Ser Ile Ile Asp Pro Asn Ala Ile Tyr Leu Cys Asp Ser                       440 445 450     Gly Ala Gln Tyr Leu Asp Gly Thr Thr Asp Val Thr Arg Thr Phe                       455 460 465     His Phe Gly Gln Pro Thr Glu Leu Glu Lys Lys Ala Phe Thr Leu                       470 475 480     Val Leu Lys Gly Val Ile Gly Leu Asp Thr Ala Val Phe Pro Lys                       485 490 495     Gly Thr Ser Gly Phe Ala Leu Asp Val Leu Ala Arg Gln Tyr Leu                       500 505 510     Trp Lys Glu Gly Leu Asp Tyr Leu His Gly Thr Gly His Gly Ile                       515 520 525     Gly Ser Tyr Leu Asn Val His Glu Gly Pro Ile Gly Val Gly Thr                       530 535 540     Arg Val Gln Tyr Thr Glu Val Pro Ile Ala Pro Gly Asn Val Ile                       545 550 555     Ser Asp Glu Pro Gly Phe Tyr Glu Asp Gly Lys Phe Gly Ile Arg                       560 565 570     Ile Glu Asn Val Ile Met Ala Arg Glu Val Gln Thr Thr His Lys                       575 580 585     Phe Gly Asp Lys Pro Trp Leu Gly Phe Glu His Val Thr Met Ala                       590 595 600     Pro Ile Gly Arg Asn Leu Ile Glu Pro Ser Leu Leu Ser Asp Ala                       605 610 615     Glu Leu Lys Trp Val Asn Asp Tyr His Arg Glu Ile Trp Glu Lys                       620 625 630     Thr His His Phe Phe Glu Asn Asp Glu Cys Thr Arg Ser Trp Leu                       635 640 645     Gln Arg Glu Thr Gln Pro Ile Ser Lys                       650 654 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence: Primer DNA <400> 3 cgggtacctg catccttctg ttcag 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence: Primer DNA <400> 4 aacaattcta gattactgcc cgccg 25

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 9/64 C12N 5/00 A (72) Inventor Osamu Takahashi 250 Noda, Chiba Prefecture Kikkoman Co., Ltd. (72) Inventor Toshifumi Matsuda 250 Noda, Noda, Chiba Prefecture Kikkoman Co., Ltd. (72) Inventor Kotaro Ito 250 Noda, Noda, Chiba Prefecture Kikkoman Co., Ltd. (72) Inventor Takeharu Nakahara Noda, Chiba Prefecture Ichida Noda 250 Kikkoman Co., Ltd. (72) Inventor Riki Masuda 399 Noda City, Noda City, Chiba Prefecture Noda Institute of Industrial Science (72) Inventor Genryu 399 Noda City, Noda City, Chiba Prefecture Noda Foundation F-Term in the Institute of Industrial Science (reference) 4B024 AA05 AA20 BA14 CA01 GA11 HA01 HA20 4B050 CC03 DD03 EE10 LL02 LL10 4B065 AA60Y AA63Y AB01 A C14 BA02 CA33 CA41 CA42 CA60

Claims (8)

[Claims] 1. The following protein (a) or (b): (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (b) an aminopeptidase consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 Protein with P activity 2. The amino acid sequence represented by SEQ ID NO: 2 and 70
A protein comprising an amino acid sequence having a sequence homology of not less than%, or a partial fragment thereof, and having aminopeptidase P activity. 3. An aminopeptidase P gene encoding the following protein (a) or (b). (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 (b) an aminopeptidase consisting of an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2 Protein with P activity 4. The amino acid sequence represented by SEQ ID NO: 2 and 70
An aminopeptidase P gene, which is a protein consisting of an amino acid sequence having a sequence homology of not less than%, or a partial fragment thereof, and which encodes a protein having aminopeptidase P activity. 5. An aminopeptidase P gene consisting of the following DNA (a) or (b): (a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 (b) Hybridizing with DNA consisting of the nucleotide sequence complementary to the DNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions, And a DNA encoding a protein having aminopeptidase P activity 6. A recombinant DNA, wherein the gene according to claim 3, 4 or 5 is inserted into vector DNA. 7. A transformant or transductant containing the recombinant DNA according to claim 6. 8. A method for producing aminopeptidase P, which comprises culturing the transformant or transductant according to claim 7 in a medium and collecting aminopeptidase P from the culture.