JP2961143B2 - Aminopeptidase gene, plasmid vector containing the gene and transformant - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an aminopeptidase gene, a plasmid vector containing the gene, and a transformant. Aminopeptidase is an enzyme that decomposes a peptide from the N-terminus. Since the aminopeptidase of the present invention acts well on a peptide whose N-terminal amino acid is a hydrophobic amino acid, the protein is hydrolyzed by the enzyme. It has the effect of decomposing the bitter peptide rich in hydrophobic amino acids that is produced when it is used. By using this enzyme, bitterness can be reduced or eliminated, so that it is expected to be used in the food field.

[0002]

2. Description of the Related Art In recent years, materials containing protein degradation products such as fermented foods and seasonings have been developed and used in various food fields. However, many of the materials containing these protein hydrolyzates exhibit bitterness, which may cause difficulty in commercialization. This often involves a bitter peptide rich in hydrophobic amino acids, which is generated when a protein is hydrolyzed by an enzyme or the like. If this bitter peptide is decomposed with a protease, bitterness can be reduced or eliminated.

[0003] Proteolytic enzymes are classified into endo-type proteases that degrade proteins at random and exo-type proteases that degrade sequentially from the ends of the proteins. Exo-type proteases are further classified into aminopeptidases and carboxypeptidases. So far, many endo-type proteases have been reported and are commercially available, but exo-type proteases, especially aminopeptidases derived from microorganisms belonging to the genus Streptomyces, have been reported. There are few things that have been done, and their practical use is limited. Further, there is no report that an aminopeptidase gene derived from a microorganism of the genus Aeromonas has been isolated.

[0004]

Means for Solving the Problems In order to utilize aminopeptidase for food processing, the present inventors have planned the cloning of aminopeptidase from microorganisms of the genus Aeromonas, and have designed a precursor gene for aminopeptidase and an aminopeptidase gene. Succeeded in cloning,
The present invention has been completed.

[0005] That is, the invention according to claim 1 is a gene for an aminopeptidase precursor derived from a microorganism of the genus Aeromonas having the nucleotide sequence of SEQ ID NO: 1 in the sequence listing.
The invention according to claim 2 is an aminopeptidase gene derived from a microorganism of the genus Aeromonas having the nucleotide sequence of SEQ ID NO: 2 in the sequence listing. The invention according to claim 3 is a plasmid containing the aminopeptidase precursor gene according to claim 1. According to a fourth aspect of the present invention, there is provided a plasmid containing the aminopeptidase gene of the second aspect.
The invention according to claim 5 is Escherichia coli transformed with the plasmid according to claim 3. The invention according to claim 6 is Escherichia coli transformed with the plasmid according to claim 4.

[0006] The present inventors isolated bacteria of the genus Aeromonas having high productivity of aminopeptidase from soil, highly purified aminopeptidase produced by the bacterium, and determined the N-terminal amino acid sequence thereof. Furthermore, the aminopeptidase is enzymatically decomposed to prepare peptide fragments, and their amino acid sequences are determined.
A was synthesized to produce a probe. On the other hand, genomic DNA was extracted from bacteria belonging to the genus Aeromonas, cut with restriction enzymes, and packaged in vitro into lambda phage to prepare a phage library.

A phage having an aminopeptidase gene was screened from a phage library using the above probe, the gene was extracted from a positive phage, and subcloned into a plasmid. Various deletion mutants were prepared from this plasmid, their base sequences were decoded, and these sequences were joined to construct the aminopeptidase precursor gene and the aminopeptidase gene. Further, the aminopeptidase gene was confirmed from the N-terminal amino acid sequence of the active aminopeptidase and the molecular weight of the aminopeptidase. The aminopeptidase precursor is converted into an aminopeptidase (active aminopeptidase) having enzymatic activity under the action of a protease. Further, a plasmid containing the aminopeptidase precursor gene and the aminopeptidase gene was prepared by a conventional method using the above gene, and Escherichia coli was transformed using the plasmid.

Hereinafter, the present invention will be described in detail. As described above, the aminopeptidase precursor gene and aminopeptidase gene of the present invention are derived from a microorganism of the genus Aeromonas having a high productivity of aminopeptidase. Examples of aminopeptidase-producing bacteria include Aeromonas hydrophila, Aeromonas sobria, and Aeromonas sobria.
Salmonicida (Aeromonas salmonicida), Aeromonas shigelloides (Aeromonas shigelloides), Aeromonas caviae (Aeromonascaviae) and the like. Aeromonas caviae T-64 is a strain isolated from the soil by the present inventors. In the present invention, after culturing this strain in a nutrient medium by a conventional method, separating cells from the culture, disrupting the cells by a conventional method, and obtaining a fraction containing aminopeptidase, column chromatography , F
A highly purified aminopeptidase can be obtained by a purification means using PLC, HPLC or the like.

The amino-terminal amino acid sequence of this aminopeptidase was determined using a protein sequencer type 477A (manufactured by PerkinElmer). Furthermore, the aminopeptidase is enzymatically decomposed to prepare peptide fragments, and their amino acid sequences are determined.
A was synthesized to produce three types of probes. on the other hand,
Genomic DNA was extracted from bacteria belonging to the genus Aeromonas, partially digested with restriction enzymes, and packaged in vitro with lambda phage to prepare a phage library.

A phage having an aminopeptidase gene was screened from a phage library using the above-mentioned probe, and a positive phage was obtained. The gene was extracted from this positive phage, degraded with restriction enzymes, and the resulting D
The NA fragment was subcloned into a plasmid. Various deletion mutants were prepared from this plasmid, the nucleotide sequences of the genes were decoded, and these sequences were joined to construct the aminopeptidase precursor gene. This gene has the nucleotide sequence of SEQ ID NO: 1 in the sequence listing. Further, from the N-terminal amino acid sequence of the active aminopeptidase and the molecular weight of the aminopeptidase, it was found that the aminopeptidase gene was present in the aminopeptidase precursor gene, and thus the aminopeptidase precursor gene was constructed. The aminopeptidase gene was constructed using the same procedure as described above. However, Southern hybridization was omitted, and a partial base sequence in the plasmid was deleted, followed by ligation. This aminopeptidase gene has the nucleotide sequence of SEQ ID NO: 2 in the sequence listing. The aminopeptidase precursor gene and aminopeptidase gene of the present invention are genes having a novel nucleotide sequence.
No gene with 0% or more homology was found.

Next, using the aminopeptidase precursor gene and the aminopeptidase gene of the present invention, a plasmid containing the gene is prepared by a conventional method ("Cloning and Sequence", supervised by Watanabe, Rural Culture Co., 1989, p. 224). Prepared by Furthermore, using the plasmid, Sambrook,
J., Fritsch, EF and Maniatis, T. "Molecular Clon
ing, A Laboratory Manual 2nd Edition ”Chapter 6.3 Vol.1 (19
E. coli was transformed according to the method described in 89). The transformed Escherichia coli has been deposited with the National Institute of Advanced Industrial Science and Technology, and the accession numbers thereof are FERM P-14715 and FERM P-1 respectively.
4716.

The expression of the aminopeptidase gene was determined by culturing transformed Escherichia coli and determining the aminopeptidase in the Escherichia coli and the culture supernatant by the method described by Hayashi (Journal of Japan).
General Microbiology, 135, 2027-2034, 1990
Year). When aminopeptidase is expressed in a large amount, various enzymes and the like necessary for the growth and survival of cells may be decomposed, but such a problem does not occur in the case of a precursor. The expressed aminopeptidase precursor can be converted into active aminopeptidase by extracting and allowing the protease to act in vitro.

[0013] Further, this transformant was placed in an LB medium for 15 to 15 hours.
After culturing at 40 ° C for 1 to 3 days, usually at 30 ° C for 1 day, the obtained cells are sonicated, and the supernatant obtained by solid-liquid separation is subjected to ion exchange column chromatography to purify the enzyme. can do. This enzyme has the following physicochemical properties.

(1) Action: Acts on a peptide to release amino acids from the N-terminus. (2) Substrate specificity: acts well on the N-terminal hydrophobic amino acid of the peptide. (3) Optimal pH: 8.5 (4) pH stability: 8 to 10 (5) Optimal reaction temperature: 50 ° C. (6) Temperature stability: 55 ° C. Regarding substrate specificity, the affinity for amino acids is The Km value of a compound of leucine (leucine paranitroanilide), which is a typical hydrophobic amino acid, is 0.1.
082 mM, Vmax 74.1 (μmole / min
Mg), but the Km value was 1.87 mM and the Vmax was 0.128 (μmole / m) for a compound of lysine (lysine paranitroanilide) which is a hydrophilic amino acid.
in.mg).

This aminopeptidase is an enzyme that degrades a peptide rich in hydrophobic amino acids from the N-terminus. Utilizing this property, when aminopeptidase acts on a bitter peptide rich in hydrophobic amino acids, the bitter peptide is decomposed and bitterness can be reduced or eliminated. When this is applied to the field of food processing, especially when aminopeptidase is added in the cheese manufacturing process, it is possible to shorten the ripening period of cheese while suppressing an increase in bitterness. Furthermore, it can be used for the production of EMC (enzyme-modified cheese) having a cheese-like flavor, or used for decomposing protein-containing materials such as various fermented foods and seasonings.

[0016]

Next, the present invention will be described in detail with reference to examples. Example 1 After Aeromonas caviae T-64 isolated from soil was cultured in a nutrient medium, cells were separated from the culture.
Next, the cells were disrupted by a conventional method, and a disrupted solution was obtained by centrifugation, subjected to ion exchange column chromatography, and further highly purified aminopeptidase using FPLC, HPLC and the like.

The aminopeptidase was converted to a protein sequencer 477A (Perkin Elmer) using
The N-terminal amino acid sequence (described in SEQ ID NO: 3 in the sequence listing) was determined. Further, the aminopeptidase was digested with lysyl endopeptidase (manufactured by Merck) to prepare peptide fragments, and the amino acid sequences of two peptide fragments (described in SEQ ID NOs: 4 and 5 in the sequence listing) were determined. To produce three types of probes (described in SEQ ID NOs: 6, 7 and 8 in the sequence listing), and a megalabel kit (manufactured by Takara Shuzo Co., Ltd.)
Was labeled with ³² P radioisotope.

On the other hand, genomic DNA is extracted from the Aeromonas caviae T-64 strain by the method of Saito (Protein nucleic acid enzyme, Vol. 11, p. 446), digested with the restriction enzyme SauIII AI, partially digested, and subjected to ultracentrifugation. The obtained fraction of about 20 Kb was packaged in vitro into lambda phage using Gigapack II Gold (manufactured by Stratagene) to prepare a phage library. Using the above probe, the usual method ("Cloning and sequencing", supervised by Watanabe,
A phage having the aminopeptidase gene was screened from the phage library by Rural Culture Company, 1989, p. 134), and five positive phages were obtained.

Further, a gene is extracted from the phage, digested with the restriction enzyme SacI, and the obtained digest of the restriction enzyme is separated by agarose gel electrophoresis. Rural Culture Company, 1989, p. 157) and 2.9 Kb
It was confirmed that the desired aminopeptidase gene was present in the DNA fragment of p. Note that this 2.9 Kbp D
The NA fragment contains a gene for the aminopeptidase precursor, which contains an active gene.

Next, Sambrook, J., Fritsch, EF and
Maniatis, T. "Molecular Cloning, A Laboratory Manua
l This 2.9 Kbp fragment was prepared by agarose gel electrophoresis according to the method described in the second edition, Chapter 6.3, Vol. 1 (1989).
8 was digested with the restriction enzyme SacI and dephosphorylated using alkaline phosphatase. Using a DNA ligation kit (manufactured by Takara Shuzo Co., Ltd.), the above 2.9 Kbp fragment was cloned and sequenced into this dephosphorylated plasmid by “Cloning and Sequence”, supervised by Watanabe, Rural Bunkasha, 1989
Year, p. 134, the plasmid P
NI2R was prepared (see FIG. 1).

Furthermore, using this plasmid, Sambroo
k, J., Fritsch, EF and Maniatis, T. "Molecular C
loning, A Laboratory Manual 2nd edition “Chapter 1.74 Vol.1
(1989) according to the method described in (1989). The transformed Escherichia coli has been deposited with the National Institute of Advanced Industrial Science and Technology, and the accession number is FERM.
P-14715. In addition, plasmid PNI2R
Has an aminopeptidase precursor gene incorporated therein, and this gene includes an active gene.

A large amount of plasmid PNI2 was obtained from the transformant.
R was prepared, and was released using a deletion kit for kilosequence (manufactured by Takara Shuzo Co., Ltd.).
The blunt ends were obtained using an NA branding kit (manufactured by Takara Shuzo Co., Ltd.), and ligated using a DNA ligation kit (manufactured by Takara Shuzo Co., Ltd.) to prepare 20 types of deletion mutants. The nucleotide sequences of these deletion mutant inserted gene portions were decoded using a tack die primer cycle sequencing kit (Perkin Elmer).

[0023] The information of the decoded base sequence was joined to construct the aminopeptidase precursor gene, which is shown in SEQ ID NO: 1 in the sequence listing. A ribosome binding site and a transcription initiation codon were confirmed in the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing. On the other hand, the amino acid sequence at the N-terminus of the active aminopeptidase (see SEQ ID NO: 3 in the sequence listing) has been found, and the amino acid sequence was identical to the sequence at positions 121 to 130 in the amino acid sequence shown in SEQ ID NO: 1. .
Furthermore, the sequence of the aminopeptidase-derived peptide fragment (see SEQ ID NOs: 4 and 5 in the Sequence Listing) was identical to the 187-195 and 359-367 sequences in the amino acid sequence shown in SEQ ID NO: 1, respectively. The molecular weight of the activated aminopeptidase was determined by using a laser ionized TOF-MS KOMPACT MLD manufactured by Shimadzu Corporation.
It was 29,800 daltons as measured by type IIII. From the above results, the aminopeptidase gene was found in the base sequence of the precursor gene. It was confirmed that the aminopeptidase gene was present after the 361st position in the nucleotide sequence of the precursor gene. The active aminopeptidase gene is composed of an aminopeptidase precursor gene based on the amino terminal amino acid sequence of aminopeptidase, and is shown in SEQ ID NO: 2 in the sequence listing.

Example 2 Genes were extracted from the positive phage prepared in Example 1,
After digestion with the restriction enzyme Pst I, the resulting restriction enzyme digest was separated by agarose gel electrophoresis, and then analyzed by Sambrook, J., Fritsch,
EF and Maniatis, T. "Molecular Cloning, A Labora
A 2.1 kbp fragment was prepared by agarose electrophoresis according to the method described in “Tory Manual, 2nd edition”, Chapter 6.3, Vol. 1 (1989), in which the aminopeptidase gene is present.
On the other hand, the plasmid puc18 was digested with the restriction enzyme PstI and dephosphorylated using alkaline phosphatase. Using a DNA ligation kit (manufactured by Takara Shuzo Co., Ltd.), the 2.1k
The bp DNA fragment was subcloned according to “Cloning and Sequence”, supervised by Watanabe, Rural Culture Company, 1989, p. 163 to prepare a plasmid PNI3R (4.8 kbp) containing the aminopeptidase gene (see FIG. 2). .

Further, using this plasmid, Sambroo
k, J., Fritsch, EF and Maniatis, T. "Molecular C
loning, A Laboratory Manual 2nd edition “Chapter 1.74 Vol.1
(1989) according to the method described in (1989).

A large amount of a plasmid was prepared from the transformant and cut with restriction enzymes KpnI and SalI. The cut plasmid was subjected to deletion by about 90 bp using a release kit for kilosequence (manufactured by Takara Shuzo Co., Ltd.), and then blunt-ended using a DNA blunting kit (manufactured by Takara Shuzo Co., Ltd.). Ligation using
Ten kinds of deletion mutants were created. The nucleotide sequences of these deletion mutant-inserted gene portions were read using a Tack Dye Primer Cycle Sequencing Kit (Perkin Elmer) to construct the aminopeptidase gene. The sequence of the aminopeptidase gene thus obtained was identical to that of the precursor gene obtained in Example 1 and the molecular weight of the aminopeptidase. The nucleotide sequence of the aminopeptidase gene was described in SEQ ID NO: 2 in the sequence listing. Among these deletion mutants, the plasmid PNI4R containing the aminopeptidase (active type) gene, which lacks the specific site of the aminopeptidase precursor,
(4.7 kbp) was obtained (see FIG. 2).

Further, using this plasmid, Sambroo
k, J., Fritsch, EF and Maniatis, T. "Molecular C
loning, A Laboratory Manual 2nd edition “Chapter 1.74 Vol.1
(1989) according to the method described in (1989). The transformed Escherichia coli has been deposited with the National Institute of Advanced Industrial Science and Technology, and the accession number is FERM.
P-14716.

[0028]

According to the present invention, there is provided a gene for aminopeptidase which degrades a bitter peptide rich in hydrophobic amino acids produced by proteolysis and has a debittering effect,
An enzyme obtained by expressing this is useful in the field of food processing.

[0029]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1179 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin: Organism name: Aeromonas caviae Strain name: T-64 Direct origin Plasmid Name: Characteristics of PNI2R sequence Symbol indicating characteristic: mat peptide Location: 1..1179 Method for determining characteristics: E sequence ATG AAA CCC TTG TTG ACC GCC CTC TTG CTC CCC CTG TCG CCT CTC GCC 48 Met Lys Pro Leu Leu Thr Ala Leu Leu Leu Pro Leu Ser Pro Leu Ala 5 10 15 GCA CAA GCC GCC GAG CCC GTC TGG ATC ACG GTC GGT GCC GAC AGC GGC 96 Ala Gln Ala Ala Ala Glu Pro Val Trp Ile Thr Val Gly Ala Asp Ser Gly 20 25 30 GTC GAA CTC AAG CAG GTC AAG GCC AGG CTG GCC CCC CTG TTC AGT GCC 144 Val Glu Leu Lys Gln Val Lys Ala Arg Leu Ala Pro Leu Phe Ser Ala 35 40 45 AGC GGC GCC CCC ATC CAG CTG GCC CAG GTC GAG GCG AGC GAA CTC GGC 192 Ser Gly Ala Pro Ile Gln Leu Ala Gln Val Glu Ala Ser Glu Leu Gly 50 55 60 ACC CTC TCG CAC CTG ATG CAC GAA GGG CAC CAG CGC TGC GGC GGT TAC 240 Thr Leu Ser His Leu Met His Glu Gly His Gln Arg Cys Gly Gly Tyr 65 70 75 80 GTG GTG CAC AGC ACC CTG GCC GAC GCC CTG CAG AGC ATG GCC CAG CCC 288 Val Val His Ser Thr Leu Ala Asp Ala Leu Gln Ser Met Ala Gln Pro 85 90 95 ATC AGC CAG AAC CTG TTC AGC GCC CCG CCG CTG ACT CAA GGG GCC AGC 336 Ile Ser Gln Asn Leu Phe Ser Ala Pro Pro Leu Thr Gln Gly Ala Ser 100 105 110 GTC AAC CGG CTG CTG CCC TAT CTG GAT CAG GGC AAC ATA GTC GGC ACC 384 Val Asn Arg Leu Leu Pro Tyr Leu Asp Gln Gly Asn Ile Val Gly Thr 115 120 125 ATC AGC CAG CTC GCC AGC TGG CGC AAC CGC TAC TAC TAC ACC ACC ACC ACA 432 Ile Ser Gln Leu Ala Ser Trp Arg Asn Arg Tyr Tyr Thr Thr Thr Thr Thr 130 135 140 GGG GTG CAG TCG GCA GAC TGG GTG GCC GGC CAG TGG CAG AGT CTG AGC 480 Gly Val Gln Ser Ala Asp Trp Val Ala Gly Gln Trp Gln Ser Leu Ser 145 150 155 160 GCG ACC CTG CCC TGG GCC AGC GTC AGC CGG GTC AAG CAC AGC GGC TAT 528 Ala Thr Leu Pro Trp Ala Ser Val Ser Arg Val Lys His Ser Gly Tyr 165 170 175 CCG CAG CAA TCC GTG GTG CTG ACC CTC AAG GGC AGT CGC TAC CCG GAC 576 Pro Gln Gl n Ser Val Val Leu Thr Leu Lys Gly Ser Arg Tyr Pro Asp 180 185 190 GAA GTG GTG GTG CTC GGC GGC CAC CTC GAC TCC ACC GCG GGC TCG GCT 624 Glu Val Val Val Leu Gly Gly His Leu Asp Ser Thr Ala Gly Ser Ala 195 200 205 CCC AAC AGC CGT ACC CTG GCT CCG GGC GCC GAC GAC GAT GCC TCC GGC 672 Pro Asn Ser Arg Thr Leu Ala Pro Gly Ala Asp Asp Asp Ala Ser Gly 210 215 220 ATC GCC ACC CTG ACC GAG GTG CTG CGG GTC ATC GCC GAG CAG GGC CGC 720 Ile Ala Thr Leu Thr Glu Val Leu Arg Val Ile Ala Glu Gln Gly Arg 225 230 235 240 CAG CCA GAG CGC ACC CTG CAA TTT ATC GGC TAT GCG GCG GAA GAG GTG 768 Gln Pro Glu Arg Thr Leu Gln Phe Ile Gly Tyr Ala Ala Glu Glu Val 245 250 255 GGG CTG CGG GGC TCC AAG GAC ATC GCC ACC CGC TAC AAG GCA GCC AAT 816 Gly Leu Arg Gly Ser Lys Asp Ile Ala Thr Arg Tyr Lys Ala Ala Asn 260 265 270 270 ACC AAG GTG CTG GCG GCG TTG CAG CTG GAC ATG ACC AAC TAT CAG GGA 864 Thr Lys Val Leu Ala Ala Leu Gln Leu Asp Met Thr Asn Tyr Gln Gly 275 280 285 TCG GCG GAA GAC ATC GTC TTC ATG ACC GAC TAC ACC GAC CAG GGG CTG 91 2 Ser Ala Glu Asp Ile Val Phe Met Thr Asp Tyr Thr Asp Gln Gly Leu 290 295 300 ACC GGC TAT CTG GCC CAG CTG CTC GAC GCC TAT CTG CCC CAG ATC CGC 960 Thr Gly Tyr Leu Ala Gln Leu Leu Asp Ala Tyr Leu Pro Gln Ile Arg 305 310 315 320 TAT GGC TAC GAC AGC TGC GGC TAC GGC TGC TCG GAT CAC GCC TCC TGG 1008 Tyr Gly Tyr Asp Ser Cys Gly Tyr Gly Cys Ser Asp His Ala Ser Trp 325 330 335 CAC AAT CAG GGC TAC CCA GCA GCC ATG CCG TTC GAG TCG CGC TTC AAC 1056 His Asn Gln Gly Tyr Pro Ala Ala Met Pro Phe Glu Ser Arg Phe Asn 340 345 350 GAC TAC AAC CCC AAG ATC CAC ACC GCC CAG GAC ACC TTG CAG AAC TCT 1104 Asp Tyr Asn Pro Lys Ile His Thr Ala Gln Asp Thr Leu Gln Asn Ser 355 360 365 GAC CCC TCG GCC GCC CAC GCC CTC AAG TTT GCC CAG CTG GCC ACC AGC 1152 Asp Pro Ser Ala Ala His Ala Leu Lys Phe Ala Gln Leu Ala Thr Ser 370 375 380 TTC GCC ATC GAG ATG GGC AAG ATA AAC 1179 Phe Ala Ile Glu Met Gly Lys Ile Asn 385 390

SEQ ID NO: 2 Sequence length: 819 Sequence type: number of nucleic acid strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin: Organism: Aeromonas caviae Strain: T-64 Direct Origin of plasmid Plasmid name: PNI4R Sequence characteristics Characteristic symbol: mat peptide Location: 1..819 Method for determining characteristics: E sequence GAT CAG GGC AAC ATA GTC GGC ACC ATC AGC CAG CTC GCC AGC TGG CGC 48 Asp Gln Gly Asn Ile Val Gly Thr Ile Ser Gln Leu Ala Ser Trp Arg 1 5 10 15 AAC CGC TAC TAC ACC ACC ACC ACA GGG GTG CAG TCG GCA GAC TGG GTG 96 Asn Arg Tyr Tyr Thr Thr Thr Thr Gly Val Gln Ser Ala Asp Trp Val 20 25 30 GCC GGC CAG TGG CAG AGT CTG AGC GCG ACC CTG CCC TGG GCC AGC GTC 144 Ala Gly Gln Trp Gln Ser Leu Ser Ala Thr Leu Pro Trp Ala Ser Val 35 40 45 AGC CGG GTC AAG CAC AGC GGC TAT CCG CAG CAA TCC GTG GTG CTG ACC 192 Ser Arg Val Lys His Ser Gly Tyr Pro Gln Gln Ser Val Val Leu Thr 50 55 60 CTC AAG GGC AGT CGC TAC CCG GAC GAA GTG GTG GTG CTC GGC GGC CAC 240 Le u Lys Gly Ser Arg Tyr Pro Asp Glu Val Val Val Leu Gly Gly His 65 70 75 80 CTC GAC TCC ACC GCG GGC TCG GCT CCC AAC AGC CGT ACC CTG GCT CCG 288 Leu Asp Ser Thr Ala Gly Ser Ala Pro Asn Ser Arg Thr Leu Ala Pro 85 90 95 GGC GCC GAC GAC GAT GCC TCC GGC ATC GCC ACC CTG ACC GAG GTG CTG 336 Gly Ala Asp Asp Asp Ala Ser Gly Ile Ala Thr Leu Thr Glu Val Leu 100 105 110 CGG GTC ATC GCC GAG CAG GGC CGC CAG CCA GAG CGC ACC CTG CAA TTT 384 Arg Val Ile Ala Glu Gln Gly Arg Gln Pro Glu Arg Thr Leu Gln Phe 115 120 125 ATC GGC TAT GCG GCG GAA GAG GTG GGG CTG CGG GGC TCC AAG GAC ATC 432 Ile Gly Tyr Ala Ala Glu Glu Val Gly Leu Arg Gly Ser Lys Asp Ile 130 135 140 GCC ACC CGC TAC AAG GCA GCC AAT ACC AAG GTG CTG GCG GCG TTG CAG 480 Ala Thr Arg Tyr Lys Ala Ala Asn Thr Lys Val Leu Ala Ala Leu Gln 145 150 155 160 CTG GAC ATG ACC AAC TAT CAG GGA TCG GCG GAA GAC ATC GTC TTC ATG 528 Leu Asp Met Thr Asn Tyr Gln Gly Ser Ala Glu Asp Ile Val Phe Met 165 170 175 ACC GAC TAC ACC GAC CAG GGG CTG ACC GGC TAT CTG GCC CAG CTG C TC 576 Thr Asp Tyr Thr Asp Gln Gly Leu Thr Gly Tyr Leu Ala Gln Leu Leu 180 185 190 GAC GCC TAT CTG CCC CAG ATC CGC TAT GGC TAC GAC AGC TGC GGC TAC 624 Asp Ala Tyr Leu Pro Gln Ile Arg Tyr Gly Tyr Asp Ser Cys Gly Tyr 195 200 205 GGC TGC TCG GAT CAC GCC TCC TGG CAC AAT CAG GGC TAC CCA GCA GCC 672 Gly Cys Ser Asp His Ala Ser Trp His Asn Gln Gly Tyr Pro Ala Ala 210 215 220 ATG CCG TTC GAG TCG CGC TTC AAC GAC TAC AAC CCC AAG ATC CAC ACC 720 Met Pro Phe Glu Ser Arg Phe Asn Asp Tyr Asn Pro Lys Ile His Thr 225 230 235 240 GCC CAG GAC ACC TTG CAG AAC TCT GAC CCC TCG GCC GCC CAC GCC CTC 768 Ala Gln Asp Thr Leu Gln Asn Ser Asp Pro Ser Ala Ala His Ala Leu 245 250 255 AAG TTT GCC CAG CTG GCC ACC AGC TTC GCC ATC GAG ATG GGC AAG ATA 816 Lys Phe Ala Gln Leu Ala Thr Ser Phe Ala Ile Glu Met Gly Lys Ile 260 265 270 AAC 819 Asn

SEQ ID NO: 3 Sequence length: 10 Sequence type: amino acid Topology: linear Sequence type: peptide Fragment type: N-terminal fragment Origin: Organism name: Aeromonas caviae Strain name: T-64 Direct origin Characteristic of enzyme sequence produced by Aeromonas caviae Symbol indicating characteristics: Location: 1..10 Method for determining characteristics: E

SEQ ID NO: 4 Sequence length: 9 Sequence type: amino acid Topology: linear Sequence type: peptide Fragment type: middle fragment Origin: Organism name: Aeromonas caviae Strain name: T-64 Direct origin Degradation product of the enzyme produced by Aeromonas caviae Sequence characteristics Symbol indicating characteristics: Location: 1..9 Method for determining characteristics: E

SEQ ID NO: 5 Sequence length: 9 Sequence type: amino acid Topology: linear Sequence type: peptide Fragment type: middle fragment Origin: Organism name: Aeromonas caviae Strain name: T-64 Direct origin Degradation product of the enzyme produced by Aeromonas caviae Sequence characteristics Symbol indicating characteristics: Location: 1..9 Method for determining characteristics: E

SEQ ID NO: 6 Sequence length: 26 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid (created from amino acid sequence) Origin: Organism name: Aeromonas caviae Strain name: T-64 Sequence characteristics Characteristic symbols: Location: 1..26 Characteristic determination method: E sequence GAYCARGGIA AYATHGTIGG IACIAT 26

SEQ ID NO: 7 Sequence length: 17 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid (created from amino acid sequence) Origin: Organism name: Aeromonas caviae Strain name: T-64 Sequence characteristics Symbols indicating characteristics: Location: 1..17 Method for determining characteristics: E sequence TAYCCNGAYG ARGTNGT 17

SEQ ID NO: 8 Sequence length: 26 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid (created from amino acid sequence) Origin: Organism name: Aeromonas caviae Strain name: T-64 Sequence characteristics Characteristic symbols: Location: 1..26 Characteristic determination method: E sequence CAYACIGCIC ARGAYACIYT ICARAA 26

[Brief description of the drawings]

FIG. 1 is a construction diagram of a plasmid containing an aminopeptidase precursor gene.

FIG. 2 is a construction diagram of a plasmid containing an aminopeptidase gene.

Continuation of the front page (51) Int.Cl. ⁶ Identification symbol FI (C12N 9/52 C12R 1:19) (C12N 15/09 ZNA C12R 1:01) (56) References Biochimica et Biophysica Acta, 1131, 337 -340, 1992 The Journal of Biological Chemistry, 267, 8390-8395, 1992 (58) Fields investigated (Int. Cl. ⁶ , DB name) C12N 15/00-15/90 BIOSIS (DIALOG) WPI (DIALOG) ) DDBJ GENESEQ

Claims (6)

(57) [Claims] 1. A gene for an aminopeptidase precursor derived from a microorganism of the genus Aeromonas having the nucleotide sequence of SEQ ID NO: 1 in the sequence listing. 2. An aminopeptidase gene derived from a microorganism of the genus Aeromonas having the nucleotide sequence of SEQ ID NO: 2 in the sequence listing. 3. A plasmid containing the gene for the aminopeptidase precursor according to claim 1. 4. A plasmid containing the aminopeptidase gene according to claim 2. 5. Escherichia coli transformed with the plasmid of claim 3. 6. Escherichia coli transformed with the plasmid of claim 4.