JP2005218319A - Aminopeptidase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide aminopeptidase equipped with sufficient properties for application to a catalyst, etc. <P>SOLUTION: The aminopeptidase has optimum reaction pH 7.5-10.5, hydrolysis activity to L-Leu-pNA, L-Phe-pNA, L-Met-pNA, L-Lys-pNA, L-Ala-pNA and L-Pro-pNA, optimum reaction temperature of about 75°C, thermostability of about 78°C as 50% heat inactivation temperature and no substantial influence on catalytic activity by calcium ion. The specific base sequence derived from Streptomyces or its variant nucleic acid is obtained. The recombinant aminopeptidase is obtained. The method for producing the recombinant aminopeptidase is provided. The carrier for expressing the aminopeptidase is obtained. The transformed cell has the nucleic acid. The probe for detecting the nucleic acid is obtained. The primer pair for amplifying the nucleic acid or its part is obtained. The method for searching the aminopeptidase comprises using the probe and/or the primer pair. The antibody or its fragment to the aminopeptidase or the recombinant aminopeptidase is obtained. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to aminopeptidases. More specifically, the present invention relates to an isolated aminopeptidase, a nucleic acid encoding the aminopeptidase, an expression vector containing the nucleic acid, a transformant retaining the nucleic acid, a recombinant aminopeptidase, and an antibody against the aminopeptidase. And a probe for detecting the nucleic acid and a primer for amplifying the nucleic acid.

  Aminopeptidases catalyze the hydrolysis of amino acid residues from the N-terminus of peptide or protein substrates.

  The aminopeptidase is a catalyst for reaction during the production of a seasoning (see Patent Documents 1 to 3), a molecular tool for protein sequencing, and an additive in a two-stage assay for endopeptidase [Non-Patent Document 1] It is used as a curing reagent in the processing of recombinant proteins and fusion products (see Non-Patent Document 2) and optically active amino acid production (see Non-Patent Document 3).

However, conventionally used aminopeptidases have the disadvantage that the use conditions may be limited in terms of stability, reactivity, cofactor requirements, substrate specificity, and the like.
JP 2002-355047 A JP 2000-325090 A Japanese Patent Application Laid-Open No. 2000-232828 Orlowsky, M. et al., Biochemistry, 18, 4942-4950 (1981). Ben-Bassat, A. et al. Bacteriol. , 169, 751-757 (1987) Yamskov et al., Enzyme Microb. Technol. 3,137-140 (1981)

  One aspect of the present invention is that it has sufficiently high thermal stability for application to a catalyst, etc., has an optimum reaction temperature sufficiently high for application to a catalyst, etc., and conditions suitable for application to a catalyst, etc. Reactivity underneath, reactivity not affected by calcium ions, high substrate specificity even in the absence of calcium ions, substrate specificity suitable for application to catalysts, etc. It is to provide an aminopeptidase that can solve at least one of obtaining various products by hydrolytic activity.

  Another aspect of the present invention is to efficiently express the aminopeptidase, to supply a large amount of the aminopeptidase, to modify the properties of the aminopeptidase, and to have other properties equivalent to those of the aminopeptidase. It is an object of the present invention to provide a nucleic acid encoding an aminopeptidase, which can solve at least one of detecting the aminopeptidase of the present invention, obtaining the aminopeptidase of substantially high purity, and the like.

  Furthermore, other aspects of the present invention are substantially high in purity, have sufficiently high thermal stability for application to a catalyst, etc., and have an optimum reaction temperature sufficiently high for application to a catalyst, etc. At least one of exhibiting reactivity under conditions suitable for application to a catalyst, etc., exhibiting substrate specificity suitable for application to a catalyst, etc., obtaining various products by hydrolysis activity, etc. It is to provide a recombinant aminopeptidase that can be solved.

  Further, another aspect of the present invention can solve at least one of detecting another aminopeptidase having properties equivalent to the aminopeptidase, detecting a biological source having the aminopeptidase, and the like. It is to provide a probe and primer pair.

  Furthermore, another aspect of the present invention can solve at least one of detecting another aminopeptidase having properties equivalent to the aminopeptidase, detecting a biological source having the aminopeptidase, and the like. The object is to provide a search method for aminopeptidase.

  Further, another aspect of the present invention is to detect the aminopeptidase or another aminopeptidase having properties equivalent to the aminopeptidase, and to detect the aminopeptidase or other aminopeptidases having properties equivalent to the aminopeptidase. An object of the present invention is to provide an antibody or fragment thereof that can solve at least one of inhibiting function and the like.

  Furthermore, another aspect of the present invention is suitable for efficiently expressing the aminopeptidase, supplying a large amount of the aminopeptidase, obtaining the aminopeptidase of substantially high purity, and producing an aminopeptidase. An object of the present invention is to provide an aminopeptidase expression carrier capable of solving at least one of introducing the nucleic acid into a cell and efficiently introducing the nucleic acid.

  Another object of the present invention is to provide a transformed cell capable of solving at least one of supplying a large amount of the aminopeptidase and obtaining the aminopeptidase having substantially high purity.

  Furthermore, another aspect of the present invention solves at least one of efficiently expressing the aminopeptidase, supplying a large amount of the aminopeptidase, obtaining the aminopeptidase of substantially high purity, and the like. Another object is to provide a method for producing the aminopeptidase.

That is, the present invention
[1] The following properties:
(1) Optimal reaction pH:
pH 7.5-10.5
(2) Substrate specificity:
L-leucine-p-nitroanilide, L-phenylalanine-p-nitroanilide, L-methionine-p-nitroanilide, L-lysine-p-nitroanilide, L-alanine-p-nitroanilide and L-proline-p -Exhibits hydrolytic activity towards nitroanilide,
(3) Optimal reaction temperature:
About 75 ° C,
(4) Thermal stability:
As a 50% heat inactivation temperature, about 78 ° C
(5) There is substantially no influence of catalytic activity by calcium ions,
An aminopeptidase having,
[2] The aminopeptidase according to the above [1], which is produced by Streptomyces septatus TH-2 (FERM P-173295),
[3] (A) a base sequence encoding the amino acid sequence shown in SEQ ID NO: 2,
(B) a base sequence represented by SEQ ID NO: 1,
(C) A nucleotide sequence having substitution, deletion, addition or insertion of at least one nucleotide residue in the nucleotide sequence of (A) or (B), and the encoded polypeptide has aminopeptidase activity. The base sequence that is shown,
(D) a nucleotide sequence of a nucleic acid that hybridizes under stringent conditions with a complementary strand of the nucleic acid comprising the nucleotide sequence of (A) or (B), and the encoded polypeptide exhibits aminopeptidase activity Obtained by aligning the base sequence and (E) the amino acid sequence shown in SEQ ID NO: 2 with the BLAST algorithm under the conditions of Gap Costs (Extension 11, Extension 1), Expect 10, and Word Size 3. A base sequence that encodes an amino acid sequence having a sequence identity of at least 75% and wherein the encoded polypeptide exhibits aminopeptidase activity;
An amino acid comprising a base sequence selected from the group consisting of: wherein the encoded polypeptide exhibits hydrolytic activity against aminoacyl-p-nitroanilide in the absence of calcium ions, A nucleic acid encoding a peptidase,
[4] An aminopeptidase expression carrier comprising the nucleic acid according to [3],
[5] A transformed cell retaining the nucleic acid according to [3],
[6] 1) a step of culturing the transformed cell according to [5] to obtain a culture, and 2) a step of recovering aminopeptidase from the culture obtained in the step 1).
A method for producing a recombinant aminopeptidase,
[7] A recombinant aminopeptidase encoded by the nucleic acid according to [3],
[8] (i) A sequence containing nucleotide residues selected from the group consisting of base numbers: 268 to 274, 325 to 334, and 534 to 558 in the sequence of the nucleic acid encoding the amino acid sequence represented by SEQ ID NO: 2. (Ii) a nucleic acid encoding the nucleotide sequence consisting of at least 100 nucleotide residues or a nucleotide sequence substantially complementary to the nucleotide sequence, or (ii) the amino acid sequence shown in SEQ ID NO: 2. A base sequence of an oligonucleotide showing complementarity equivalent to an oligonucleotide consisting of a base sequence or a base sequence substantially complementary to the base sequence,
A probe for detecting the nucleic acid according to the above [3], comprising:
[9] A. (I) Nucleotide residues selected from the group consisting of base numbers: 268 to 274, 325 to 334, and 534 to 558 in the sequence of the nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 2 A nucleotide sequence consisting of nucleotide residues, or (II) an oligonucleotide having complementarity equivalent to the oligonucleotide consisting of the nucleotide sequence of (I) above with respect to the nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 2. Base sequence,
An oligonucleotide containing
B. In the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2, the nucleotide sequence is at least 100 nucleotide residues away from the 3 ′ terminal nucleotide residue of the nucleotide sequence corresponding to the oligonucleotide A. A primer pair for amplifying the nucleic acid or the part thereof according to the above [3], comprising an oligonucleotide containing a sequence substantially complementary to a base sequence comprising at least 20 nucleotide residues,
[10] (A) a base sequence encoding the amino acid sequence represented by SEQ ID NO: 2 by hybridization using the probe according to [8] and / or PCR using the primer pair according to [9],
(B) a base sequence represented by SEQ ID NO: 1,
(C) A nucleotide sequence having substitution, deletion, addition or insertion of at least one nucleotide residue in the nucleotide sequence of (A) or (B), and the encoded polypeptide has aminopeptidase activity. The base sequence that is shown,
(D) a nucleotide sequence of a nucleic acid that hybridizes under stringent conditions with a complementary strand of the nucleic acid comprising the nucleotide sequence of (A) or (B), and the encoded polypeptide exhibits aminopeptidase activity Obtained by aligning the base sequence and (E) the amino acid sequence shown in SEQ ID NO: 2 with the BLAST algorithm under the conditions of Gap Costs (Extension 11, Extension 1), Expect 10, and Word Size 3. A base sequence that encodes an amino acid sequence having a sequence identity of at least 75% and wherein the encoded polypeptide exhibits aminopeptidase activity;
A region corresponding to the probe and / or a region corresponding to a PCR amplification product obtained using the primer pair in a base sequence selected from the group consisting of aminoacyl- A screening method for aminopeptidase, comprising screening for aminopeptidase having hydrolytic activity against p-nitroanilide, and [11] the aminopeptidase according to [1] or [2] above or [4 Or an antibody against the recombinant aminopeptidase according to [5] or a fragment thereof,
About.

  According to the aminopeptidase of the present invention, there is an excellent effect that it can be used at a sufficiently high thermal stability and optimum reaction temperature for application to a catalyst or the like. In addition, the aminopeptidase of the present invention exhibits reactivity under conditions suitable for application to a catalyst and the like, exhibits high substrate affinity even in the absence of calcium ions, and is substantially affected by catalytic activity due to calcium ions. In addition, it has an excellent effect of exhibiting substrate specificity suitable for application to a catalyst or the like. Furthermore, according to the aminopeptidase of the present invention, there is an excellent effect that various products can be obtained by hydrolysis activity.

  In addition, according to the nucleic acid of the present invention, the aminopeptidase can be efficiently expressed and supplied in a large amount, and the aminopeptidase having substantially high purity can be obtained. Furthermore, according to the nucleic acid of the present invention, there is an excellent effect that the properties of the aminopeptidase can be modified. Moreover, according to the nucleic acid of this invention, there exists an outstanding effect that the other aminopeptidase which has the property equivalent to the said aminopeptidase can be detected.

  Furthermore, the recombinant aminopeptidase of the present invention exhibits the excellent property of being substantially highly pure. The recombinant aminopeptidase of the present invention exhibits excellent properties such as high thermal stability sufficient for application to a catalyst and the like and high optimum reaction temperature. In addition, the recombinant aminopeptidase of the present invention exhibits reactivity under conditions suitable for application to a catalyst and the like, exhibits high substrate affinity even in the absence of calcium ions, and exhibits catalytic activity due to calcium ions. It exhibits an excellent property that there is substantially no influence. Furthermore, the recombinant aminopeptidase of the present invention exhibits an excellent property of exhibiting substrate specificity suitable for application to a catalyst or the like. Therefore, according to the recombinant aminopeptidase of the present invention, it is possible to perform analysis under suitable conditions, and to obtain an excellent effect that various products can be obtained by hydrolysis activity.

  In addition, according to the probe and primer pair of the present invention, other aminopeptidases having properties equivalent to those of the aminopeptidase can be detected, and an excellent effect that a biological source having the aminopeptidase can be detected. .

  Furthermore, according to the aminopeptidase search method of the present invention, it is possible to detect other aminopeptidases having the same properties as the aminopeptidase, and to detect a biological source having the aminopeptidase. Play.

  Moreover, according to the antibody of the present invention or a fragment thereof, the aminopeptidase or another aminopeptidase having the same properties as the aminopeptidase can be detected. Furthermore, according to the antibody or fragment thereof of the present invention, there is an excellent effect that the function of the aminopeptidase or another aminopeptidase having the same properties as the aminopeptidase can be inhibited.

  Furthermore, according to the aminopeptidase expression carrier of the present invention, there is an excellent effect that a large amount of the aminopeptidase having substantially high purity can be supplied. In addition, according to the aminopeptidase expression carrier of the present invention, the nucleic acid can be efficiently introduced into cells suitable for aminopeptidase production, and the aminopeptidase can be efficiently expressed. Moreover, according to the aminopeptidase expression carrier of the present invention, there is an excellent effect that the nucleic acid can be efficiently introduced.

  Moreover, according to the transformed cell of the present invention, there is an excellent effect that the aminopeptidase can be supplied in large quantities with substantially high purity.

  Furthermore, according to the method for producing aminopeptidase of the present invention, the aminopeptidase can be supplied in a large amount with substantially high purity. Furthermore, according to the method for producing an aminopeptidase of the present invention, there is an excellent effect that the aminopeptidase can be efficiently expressed.

  One aspect of the invention is an aminopeptidase.

  The aminopeptidase of the present invention has one major characteristic in that it has a property of having a high affinity for a substrate even in the absence of calcium ions and substantially not affecting the catalytic activity of calcium ions. . Therefore, according to the aminopeptidase of the present invention, an excellent effect of suppressing the generation of precipitation derived from calcium ions, white turbidity and the like during the reaction is exhibited. Therefore, it is suitable for application to analysis, catalyst, and the like.

  The activity measurement of the aminopeptidase of the present invention, specifically the measurement of the hydrolysis activity, is carried out, for example, in a reaction solution containing the enzyme to be measured [composition: 80 mM Tris-HCl, pH 8.0], with a final concentration of 3.2 mM. Thus, aminoacyl-p-nitroanilide is added as a substrate, and continuous optical measurement at 405 nm is performed at 37 ° C. to detect the release of p-nitroaniline. The initial rate of the enzymatic reaction can be determined from the linear portion of the optical density profile. In the present specification, one unit of aminopeptidase activity is defined as the amount of enzyme that liberates 1 μmol of p-nitroaniline per minute.

  As a substrate used for the activity measurement of the aminopeptidase of the present invention, for example, L-leucine-p-nitroanilide (L-Leu-pNA), L-phanylalanine-p-nitroanilide (L-Phe-pNA), L-lysine-p-nitroanilide (L-Lys-pNA), L-methionine-p-nitroanilide (L-Met-pNA), etc. are mentioned.

  The aminopeptidase of the present invention has one major feature in that it has an optimum reaction pH of pH 6 to 10.5, preferably pH 7 to 10, more preferably pH 8 to 9, and more preferably about pH 8.0. is there. The aminopeptidase of the present invention exhibits reactivity under conditions suitable for application to a catalyst or the like. Further, since the aminopeptidase of the present invention can be hydrolyzed under a wide range of pH conditions, it exhibits excellent properties that it is useful for detection of proteases using synthetic substrates, synthesis of optically active compounds, and the like. . The optimum reaction pH is determined by measuring the activity of the aminopeptidase activity by appropriately using a buffer solution corresponding to the buffer capacity instead of 80 mM Tris-HCl used for the reaction solution. Can be evaluated.

  The aminopeptidase of the present invention includes L-leucine-p-nitroanilide, L-phenylalanine-p-nitroanilide, L-methionine-p-nitroanilide, L-lysine-p-nitroanilide, L-alanine-p-nitro. One major feature is that it exhibits substrate specificity that exhibits hydrolytic activity against anilide and L-proline-p-nitroanilide. In addition, the aminopeptidase of the present invention can be used for, for example, 5,5-dimethyl-1,3-4 azolidine, 4-carboxyl acid-methyl ester (DMTA methyl ester), ε-polylysine, and derivatives thereof. Shows hydrolytic activity. Therefore, the aminopeptidase of the present invention is suitable for application to a catalyst or the like. Furthermore, since the aminopeptidase of the present invention exhibits the substrate specificity, it can be used to obtain various products by hydrolysis activity. Since the aminopeptidase of the present invention can be hydrolyzed to methyl esters in addition to amides, it exhibits excellent properties that it is useful for the synthesis of pharmaceutical intermediate compounds, the synthesis of high-value-added compounds, etc. To do. Furthermore, the aminopeptidase of the present invention has one major feature in that it has a wide substrate specificity, and in particular, it can efficiently perform a catalytic reaction with a basic amino acid. The substrate specificity can be evaluated by measuring the activity in the same manner as the activity measurement of the aminopeptidase.

  The aminopeptidase of the present invention has an optimal reaction temperature of 10 ° C. or higher, preferably 25 ° C. or higher, more preferably 37 ° C. or higher, more preferably 70 to 75 ° C., and still more preferably about 75 ° C. There is one major feature. The aminopeptidase of the present invention can be used at a high reaction temperature suitable for application to analysis, catalyst and the like. Since the aminopeptidase of the present invention retains its catalytic activity even under low temperature conditions (for example, 10 ° C. or higher), hydrolysis of a heat labile compound and a synthetic substrate for a heat labile protease were used. It exhibits an excellent property that it is useful for detection and the like. The optimum reaction pH can be evaluated by measuring the activity by appropriately changing the reaction temperature of 37 ° C. in the activity measurement of the aminopeptidase.

  The aminopeptidase of the present invention has a 50% heat inactivation temperature of about 78 ° C., a 100% heat inactivation temperature of about 90 ° C., and an untreated range from low to about 70 ° C. One major feature is that it exhibits thermal stability that it can retain 100% of its activity. Therefore, since the aminopeptidase of the present invention has high thermal stability, the catalyst and the like can be stably performed. The thermostability can be evaluated by incubating aminopeptidase at various temperatures for 30 minutes prior to measurement of the aminopeptidase activity, and measuring the activity using the resulting aminopeptidase. .

  In addition, the aminopeptidase of the present invention exhibits the same activity as that of untreated when incubated for 30 minutes under each pH condition, even under a wide range of pH conditions of pH 5-10. It exhibits excellent properties such as being useful for hydrolysis and the like.

  The aminopeptidase of the present invention includes aminopeptidase produced from Streptomyces septatus TH-2.

  The Streptomyces septatus TH-2 is a product of Streptomyces sp. Named and displayed as TH-2, deposit number: FERM P-173295 (deposit date: March 24, 1999), National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (Higashi 1-chome, Tsukuba, Ibaraki, Japan) 1-1).

  In one aspect, the aminopeptidase of the present invention has a trade name of Superdex 200 gel filtration AKTA fast protein liquid chromatography system (Pharmacia Biotech) as a molecular weight of about 30 kDa, 15% by weight. On a gel containing polyacrylamide, the enzyme has a molecular weight of about 33 kDa as determined by SDS-PAGE under denaturing conditions.

  In another aspect, the aminopeptidase of the present invention has, for example, an amino terminal (N-terminal) amino acid sequence: AGAPAIPVANVKAHLNQL (corresponding to amino acid numbers: 65 to 80 in SEQ ID NO: 2; SEQ ID NO: 3), a tryptic fragment peptide N-terminal amino acid sequence: AGPGINDN (corresponding to amino acid numbers 155 to 161 in SEQ ID NO: 2; SEQ ID NO: 4).

The aminopeptidase of the present invention is not particularly limited. For example, at 4 ° C., the following steps:
- the Streptomyces Seputatasu TH-2, medium [composition: 0.5 wt% of glucose, 0.5 wt% citric acid, 0.2 wt% K ₂ HPO _4, 0.05 wt% MgSO ₄ · 7H ₂ O, 0.5 wt% polypeptone and 0.5 wt% yeast extract) at 28 ° C. for 72 hours, aerobically until stationary phase,
-The obtained culture is centrifuged at 40000 xg for 10 minutes or filtered to obtain a medium filtrate (supernatant),
The obtained medium filtrate is heated with stirring at 65 ° C. for 30 minutes to remove the resulting precipitate;
The resulting solution is dialyzed against buffer A [composition: 10 mM Tris-HCl buffer, pH 8.0]
-Anion exchange column chromatography in which the solution after dialysis was equilibrated with buffer A, specifically, a DEAE-Sepharose (Amersham) column (inner diameter 1.6 × 10 cm, 20 ml) was loaded and bound. The protein is eluted with a linear gradient of buffer 0-0.5 M NaCl at an average flow rate of 2 ml / ml,
-Of the obtained elution fractions, the fractions showing high specific activity are collected, and the protein is precipitated by salting out with 80% saturated ammonium sulfate,
The obtained precipitate is dissolved in buffer A containing 200 mM NaCl, the resulting solution is dialyzed against buffer A containing 200 mM NaCl,
-The solution obtained after dialysis was loaded onto a gel filtration column chromatography, specifically a trade name Superdex 200 gel filtration column (Pharmacia Biotech, ID 1.6 x 60 cm, 60 ml) The protein can be obtained by eluting the protein with buffer A containing 200 mM NaCl at an average flow rate of 1 ml / min.

  It should be noted that the aminopeptidase of the present invention has substantially no influence on activation and stabilization by calcium ions, although the sequence identity with Streptomyces griseus aminopeptidase is about 70%. It has the unexpected property that it has high substrate affinity and is stable to heat.

  The aminopeptidase of the present invention has one major feature in that it has a low affinity for a conventional inhibitor for aminopeptidase, that is, the influence of the inhibitor is small.

  Another aspect of the present invention is a nucleic acid encoding the aminopeptidase, that is, an aminopeptidase having the above properties.

  Examples of the nucleic acid encoding the aminopeptidase of the present invention include (A) a base sequence encoding the amino acid sequence shown in SEQ ID NO: 2.

  In (A) above, the amino acid sequence shown in SEQ ID NO: 2 is the aminopeptidase amino acid sequence produced from Streptomyces septatus TH-2.

  Specific examples of the nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 2 include (B) the base sequence shown in SEQ ID NO: 1. In the present invention, a base sequence that differs from the base sequence shown in SEQ ID NO: 1 through degeneracy is also included.

  In the present invention, the encoded polypeptide exhibits an activity equivalent to that of the aminopeptidase encoded by the nucleic acid (A) or (B), specifically, the aminopeptidase activity. As long as it is substantially unaffected by calcium ions and exhibits hydrolysis activity with respect to p-nitroanilide, it is a variant of a nucleic acid containing the base sequence (A) or (B) Also good.

  The aminopeptidase activity of the polypeptide encoded by the variant nucleic acid, specifically the hydrolysis activity in the absence of calcium ions, can be evaluated according to the activity measurement of the aminopeptidase.

  In the present specification, the “activity equivalent to aminopeptidase” refers to aminopeptidase encoded by the nucleic acid (A) or (B) when L-leucine-p-nitroanilide is used as a substrate. The same activity or at least 10 to 100% activity, preferably the same activity or at least 50 to 100% activity as the aminopeptidase encoded by the nucleic acid (A) or (B). The “activity equivalent to aminopeptidase” also includes having at least the property of exhibiting hydrolytic activity with respect to aminoacyl-p-nitroanilide in the absence of calcium ions.

As one aspect of the base sequence of the variant,
(C) A nucleotide sequence having substitution, deletion, addition or insertion of at least one nucleotide residue in the nucleotide sequence of (A) or (B), and the encoded polypeptide has aminopeptidase activity. The base sequence that is shown,
Is mentioned.

  In the present specification, the “base sequence having substitution, deletion, addition or insertion” may be a base sequence having a naturally-occurring mutation or a base sequence into which an artificial mutation has been introduced. Good.

  In this specification, “at least one” means one or more, or more, preferably one or several.

  In the above (C), “substitution, deletion, addition or insertion of nucleotide residue” occurs for a nucleic acid comprising the base sequence (A) or (B) by a conventional site-directed mutagenesis method or the like. It can be made. The obtained nucleic acid containing the base sequence is one in which the polypeptide encoded by the activity measurement of the aminopeptidase shows the same activity as the aminopeptidase encoded by the nucleic acid (A) or (B). I just need it.

The nucleic acid containing the base sequence (C) may be a nucleic acid encoding a polypeptide having a conservative substitution in the amino acid sequence shown in SEQ ID NO: 2. The above-mentioned “conservative substitution” means substitution with an amino acid having similar properties (that is, hydrophobicity, charge, pK, steric characteristics); It is meant to include substitution with an amino acid that cannot change the three-dimensional structure or folding structure of the peptide. As a conservative substitution, for example, one specific amino acid is replaced with the following groups I to VI:
I. Glycine, alanine;
II. Valine, isoleucine, leucine;
III. Asparagine, glutamic acid, aspartic acid, glutamine;
IV. Serine, threonine;
V. Lysine, arginine; and VI. Substitution with other amino acids within each group of phenylalanine and tyrosine.

  Specific examples of the nucleic acid (C) are not particularly limited. For example, the amino acid sequence of amino acid number 81 shown in SEQ ID NO: 2 is an amino acid sequence in which serine is threonine, and tyrosine amino acid number 234 is phenylalanine. The amino acid sequence etc. which are are mentioned.

In addition, as a base sequence of the variant, in another aspect,
(D) a nucleotide sequence of a nucleic acid that hybridizes under stringent conditions with a complementary strand of the nucleic acid comprising the nucleotide sequence of (A) or (B), and the encoded polypeptide exhibits aminopeptidase activity The base sequence which is a thing is mentioned.

  In the above (D), “stringent conditions” refers to medium stringency conditions, preferably high stringency conditions. Specifically, Cold Spring Harbor Laboratories, Sambrook Et al., Molecular Cloning: A Laboratory Manual 3rd Edition and 2nd Edition (Molecular Cloning: A Laboratory Manual 3rd ed. (Issued in 2001), 2nd ed. (Issued in 1989)) and the like. Specifically, for example, 6 × SSC (composition of 1 × SSC: 0.15M NaCl, 0.015M sodium citrate, pH 7.0), 0.5% SDS, 5 × Denhardt and 100 mg / ml herring sperm DNA And a probe is incubated at 65 ° C. overnight. In the hybridization under the “stringent conditions”, from the viewpoint of improving accuracy, the ionic strength is lower, for example, 5 × SSC, and more stringent is 2 × SSC and / or higher temperature. For example, the Tm value (80 to 90) of the nucleic acid containing the base sequence (A) or (B) is 25 ° C., more stringent at 22 ° C., and further stringent at 20 ° C. For further stringent conditions, hybridization under conditions such as 10 ° C., particularly for stringent conditions such as 5 ° C .; more stringent washing conditions, specifically low ionic strength buffers such as 6 × SSC, more stringent, 5 × SSC, more stringent, 2 × SSC, etc., and higher temperature, eg, Tm value By washing under conditions of 55 ° C, more stringent, 50 ° C, further stringent, 30 ° C, more stringent, 25 ° C, etc. At least 75%, more stringent, 80% or more, more stringent, 85%, more stringent, 90% or more, more stringent than the amino acid sequence of SEQ ID NO: 2. It is possible to obtain a nucleic acid encoding an amino acid sequence having a sequence identity of 95% or more.

  That is, the nucleic acid containing the base sequence (D) is hybridized by performing hybridization under the “stringent conditions” using the nucleic acid comprising the base sequence (A) or (B). And the nucleic acid containing the obtained base sequence has an activity equivalent to that of the aminopeptidase encoded by the nucleic acid (A) or (B), as determined by measuring the activity of the aminopeptidase. Anything is acceptable.

As another aspect of the base sequence of the variant,
(E) The sequence identity obtained by aligning the amino acid sequence shown in SEQ ID NO: 2 with the BLAST algorithm under the conditions of Gap Costs (Extension 11, Extension 1), Expect 10, and Word Size 3 A base sequence that encodes an amino acid sequence that is 75% and wherein the encoded polypeptide exhibits aminopeptidase activity;
Is mentioned.

  In the present specification, “sequence identity” refers to the sequence similarity of residues between two molecules. The “sequence identity” can be determined by comparing two sequences that are optimally aligned with the BLAST algorithm over the region of the sequence to be compared. Here, the molecule to be compared may have an addition or a deletion (for example, a gap) compared to a reference sequence (for example, a consensus sequence) for optimal alignment of the two sequences. The sequence identity number (percentage) determines the number of matching sites by determining the identical residues present in both sequences, and then the total number of residues in the sequence region to be compared. It can be calculated by dividing the number of sites and multiplying the resulting value by 100. The BLAST algorithm is a BLASTN or BLASTP web page address: http: // www. ncbi. nlm. nih. Generally available in gov / BLAST.

  The sequence identity may be at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%.

  Since the nucleic acid of the present invention encodes the aminopeptidase of the present invention, according to the nucleic acid of the present invention, the aminopeptidase can be efficiently expressed and supplied in large quantities, and the substantially high purity of the aminopeptidase can be supplied. An aminopeptidase can be obtained.

  Moreover, according to the nucleic acid of the present invention, the properties of the aminopeptidase can be modified. Moreover, according to the nucleic acid of the present invention, other aminopeptidases having properties equivalent to those of the aminopeptidases can be detected.

  The nucleic acid of the present invention is, for example, based on the base sequence shown in SEQ ID NO: 1, from the genomic DNA of Streptomyces septatus TH-2 by conventional means such as hybridization using an appropriate probe, appropriate primer It can be isolated by PCR using a pair.

  The nucleic acid source of the present invention is not particularly limited. For example, Streptomyces septatus TH-2, other microorganisms, specifically, although not particularly limited, for example, Streptomyces griseus, Streptomyces sericolor Streptomyces avermitilis, plants, animals and the like.

  Another aspect of the invention is a recombinant aminopeptidase.

  The recombinant aminopeptidase of the present invention is an enzyme encoded by the nucleic acid of the present invention.

More specifically, the recombinant aminopeptidase of the present invention includes, for example,
(A) the amino acid sequence shown in SEQ ID NO: 2,
(B) the amino acid sequence of a polypeptide exhibiting aminopeptidase activity having a substitution, deletion, addition or insertion of at least one amino acid residue in SEQ ID NO: 2, and (c) the amino acid sequence shown in SEQ ID NO: 2 Sequence identity obtained by aligning amino acid sequences under the conditions of Gap Costs (Existence 11, Extension 1), Expect 10, and Word Size 3 with the BLAST algorithm is at least 75%, preferably 80% or more, and more Preferably, it contains an amino acid sequence selected from the group consisting of 85% or more, more preferably 90% or more, and even more preferably 95% or more, in the absence of calcium ions. Hydrolysis of aminoacyl-p-nitroanilide It includes polypeptides exhibiting sex.

  The recombinant aminopeptidase of the present invention has a high purity substantially free from other aminopeptidases. In addition, the recombinant aminopeptidase of the present invention, like the aminopeptidase of the present invention, exhibits high thermal stability sufficient for application to a catalyst, etc., and a high optimum reaction temperature, and is suitable for application to analysis, catalyst, etc. It reacts under certain conditions and reacts in the absence of calcium ions. Furthermore, the recombinant aminopeptidase of the present invention exhibits substrate specificity suitable for application to analysis, catalysis and the like. Therefore, according to the recombinant aminopeptidase of the present invention, analysis can be performed under suitable conditions, and various products can be obtained by hydrolysis activity.

The recombinant aminopeptidase of the present invention is, for example,
The nucleic acid of the invention is held in a carrier suitable for introduction into the cells to be expressed with the aminopeptidase,
-Introducing the obtained carrier (hereinafter referred to as aminopeptidase expression carrier) into the cells,
-Culturing the resulting transformed cells;
-It can be produced by recovering aminopeptidase from the obtained culture by an appropriate means.

  That is, another aspect of the present invention is an aminopeptidase expression carrier, a transformed cell, and a method for producing a recombinant aminopeptidase.

  The expression carrier for aminopeptidase of the present invention contains the nucleic acid of the present invention. Therefore, according to the expression carrier for aminopeptidase of the present invention, a large amount of the aminopeptidase having substantially high purity can be supplied, and the nucleic acid can be efficiently introduced into cells suitable for production of aminopeptidase, The aminopeptidase can be expressed efficiently. Further, according to the aminopeptidase expression carrier of the present invention, the nucleic acid can be efficiently introduced.

  Specifically, the carrier for expression of the present invention is one in which the nucleic acid of the present invention is held on a carrier suitable for introduction into cells to be expressed with aminopeptidase.

  Examples of the cells include conventional host cells, and specific examples include bacterial cells such as Escherichia coli, Bacillus subtilis, actinomycetes, yeast cells, insect cells, animal cells, plant cells, and the like. More specifically, examples of the E. coli include BL21 (DE3) and JM109. Among these cells, Escherichia coli BL21 (DE3) and JM109 are preferable from the viewpoints of easy purification, mass preparation, safety and the like.

  Examples of the carrier include biological carriers, specifically, vectors suitable for the cells, non-biological carriers such as gold particles and dextran.

  As the “vector suitable for cells”, an appropriate vector may be selected according to the purpose of use of the recombinant DNA, and examples thereof include a plasmid vector and a phage vector. When the cell to be introduced is an E. coli cell, examples of the “vector suitable for the cell” include pET22b, pTrc99A, pQE32, pETKmS2 [Mishima, N. et al., Biotechnol Prog. 13, 846-868 (1997)] and the like. Such a vector may appropriately have a transcription element such as a promoter (specifically, for example, an inducible promoter, a constitutive promoter, etc.), a marker gene for selection, and a terminator.

  The inducible promoter is not particularly limited, and examples thereof include a T7 promoter, a T5 promoter, a Lac promoter, and a Tac promoter.

  Examples of the marker gene for selection include ampicillin, kanamycin, chloramphenicol, tetracycline, streptomycin and the like.

  Such a vector may contain factors for facilitating purification of aminopeptidase, such as an extracellular secretion signal, a fusion partner (His tag, pelB signal, etc.) and the like.

  When the carrier is the “vector suitable for cells”, the carrier for expression of the present invention operably links the nucleic acid of the present invention to a restriction enzyme site (eg, cloning site) on the vector. Can be produced.

  When the carrier is the non-biological carrier, if necessary, the nucleic acid construct obtained by operably linking the nucleic acid of the present invention under the control of the promoter is prepared by carrying the non-biological carrier on the non-biological carrier. Can be done. The nucleic acid construct may further contain a replication origin, a terminator, and the like.

  In the present specification, “operably linked” means that the expression of a polypeptide encoded by a nucleic acid is linked so that it is expressed in a state exhibiting biological activity under the control of a transcription element. Means.

  The transformed cell of the present invention is a cell that holds the nucleic acid of the present invention. According to the transformed cell of the present invention, since the nucleic acid of the present invention is retained and the recombinant aminopeptidase is expressed from the nucleic acid, the aminopeptidase can be supplied in large quantities with substantially high purity.

  The transformed cell of the present invention can be obtained by introducing the expression carrier of the present invention into the cell and transforming the cell.

  The method for introducing the expression carrier into the cell is not particularly limited, and examples thereof include an electroporation method, a calcium phosphate method, and a DEAE-dextran method.

The method for producing the recombinant aminopeptidase of the present invention includes 1) a step of culturing the transformed cell of the present invention to obtain a culture, and 2) recovery of the aminopeptidase from the culture obtained in the above step 1). Process,
It is a method including. According to the method for producing aminopeptidase of the present invention, since the transformed cell of the present invention is used, according to the method for producing aminopeptidase of the present invention, the aminopeptidase can be efficiently expressed. Peptidases can be supplied in large quantities with substantially high purity.

  In the above step 1), the transformed cells may be cultured under culture conditions depending on the cells, carriers, transcription elements, etc. used, and if necessary, under expression-inducing conditions.

  In the step 2), when the transformed cell of the present invention accumulates recombinant aminopeptidase in the cell, the culture obtained in the step 1) is subjected to centrifugation or the like to recover the transformed cell. That's fine. In this case, from the obtained transformed cells, conventional protein purification methods such as ultrasonic disruption, lysis, freeze disruption, centrifugation, ultrafiltration, salting out, dialysis, ion exchange column chromatography, adsorption Recombinant aminopeptidase is substantially isolated by appropriately combining column chromatography, affinity chromatography, gel filtration column chromatography, etc., and performing aminopeptidase activity measurement, protein quantification, SDS-PAGE, etc. be able to.

  On the other hand, when the transformed cell of the present invention secretes recombinant aminopeptidase outside the cell in the step 2), the culture supernatant is removed from the culture obtained in the step 1) by centrifugation, filtration or the like. Collect it. In this case, although depending on the cells used, the obtained culture supernatant is appropriately combined with heat treatment, the protein purification method, etc., and aminopeptidase activity measurement, protein quantification, purity test by SDS-PAGE, etc. Recombinant aminopeptidase can be substantially isolated. For example, when mesophilic bacteria such as Escherichia coli and Bacillus subtilis are used, the recombinant aminopeptidase can be substantially isolated by heat-treating the obtained culture supernatant.

In the preparation of the transformed cell of the present invention, E. coli BL21 DE3 was used as the cell, and pETKmS2 [Mishima, N., et al., Biotechnol Prog. , 13, 864-868 (1997)], since the recombinant aminopeptidase of the present invention is secreted into the culture supernatant of the transformed cell, the culture supernatant is heat-treated. This is advantageous in that a substantially isolated recombinant aminopeptidase can be easily obtained. In this case, for example,
- the transformed cells, _{0.5% KH 2 PO 4, 0.5%} K 2 HPO 4, 0.44 wt% Na ₂ HPO _4, 0.3 _{wt% (NH 4) 2 SO 4} , 0 0.5 wt% glucose, 0.3 wt% MgSO ₄ .7H ₂ O, 0.004 wt% FeSO ₄ .7H ₂ O, 0.004 wt% CaCl ₂ , 0.00029 wt% CoCl ₂ .6H ₂ O, 0.0003 wt% CuSO ₄ .5H ₂ O, 0.000036 wt% Na ₂ MoO.2H ₂ O, 0.001 wt% H ₃ BO ₃ , 0.001 wt% ZnSO ₄ .7H ₂ O, 0.2 Incubate at 26 ° C. for 12 hours in a synthetic medium containing wt% glycerol and 50 μg / ml kanamycin,
-Add IPTG to the resulting culture to a final concentration of 0.5 mM,
The recombinant aminopeptidase can be expressed by culturing the culture for 36 hours at 18 ° C. with shaking.

  Another aspect of the present invention is a probe for detecting the nucleic acid of the present invention and a primer pair for amplifying the nucleic acid of the present invention or a part thereof.

One embodiment of the probe of the present invention is selected from the group consisting of (i) base numbers: 268 to 274, 325 to 334, and 534 to 558 in the sequence of the nucleic acid encoding the amino acid sequence represented by SEQ ID NO: 2. A probe comprising a base sequence comprising at least 20 consecutive nucleotide residues including a nucleotide residue or a base sequence substantially complementary to the base sequence (referred to as probe (i)). According to such a probe, other aminopeptidases having properties equivalent to those of the aminopeptidase can be detected. Further, according to the probe of the present invention, a biological source having the aminopeptidase can be detected.

  In the probe (i) of the present invention, a nucleotide residue selected from the group consisting of base numbers: 268 to 274, 325 to 334, and 534 to 558 in the nucleic acid sequence encoding the amino acid sequence represented by SEQ ID NO: 2 One major feature is the inclusion of groups. Therefore, according to the probe of the present invention, other aminopeptidases having the same properties as the aminopeptidases and biological sources having the aminopeptidases can be detected.

  Here, “continuous (base sequence) including the nucleotide residue” means the nucleotide residue and its adjacent sequence on the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2.

  The length of the probe (i) of the present invention is at least 20 nucleotides, preferably 20 to 200 nucleotides, preferably 50 to 200 nucleotides, more preferably 100 to 200 nucleotides.

Another aspect of the probe of the present invention is:
(Ii) a base sequence of an oligonucleotide having the same complementarity as the oligonucleotide consisting of the base sequence of (i) above or substantially equivalent to the base sequence of the nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 2 Base sequence complementary to
Containing a probe (referred to as probe (ii)).

  As used herein, “equivalent complementarity” means that the binding force with the nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 2 is equivalent to that of the oligonucleotide to be compared. The binding force can be evaluated by, for example, hybridization, resonance plasmon interaction analysis, or the like. In the case of evaluation by hybridization, the binding force is, for example, 6 × SSC, 0.5% SDS, 5 × Denhardt, 100 mg / ml herring sperm DNA together with the nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 2. It is only necessary that the hybrid with the nucleic acid can be detected even after incubation with a probe at 55 ° C. for 12 hours in a solution containing and then washing with 5 × SSC for 10 minutes.

  In the present specification, “substantially complementary” means completely complementary.

  The length of the probe (ii) may be the same as that of the probe (i).

  Specific examples of the probe of the present invention include the full length of the nucleic acid of the present invention, the base numbers of nucleic acids encoding the amino acid sequence shown in SEQ ID NO: 2, 259 to 303, 534 to 558, and the like.

The primer pair of the present invention comprises
A. (I) Nucleotide residues selected from the group consisting of base numbers: 268 to 274, 325 to 334, and 534 to 558 in the sequence of the nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 2 A nucleotide sequence consisting of nucleotide residues, or (II) an oligonucleotide having complementarity equivalent to the oligonucleotide consisting of the nucleotide sequence of (I) above with respect to the nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 2. Base sequence,
An oligonucleotide containing
B. In the sequence of the nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 2, the nucleotide sequence is at least 100 nucleotide residues away from the 3 'terminal nucleotide residue of the nucleotide sequence corresponding to the oligonucleotide A. A primer pair consisting of an oligonucleotide containing a sequence substantially complementary to a base sequence consisting of at least 20 nucleotide residues. According to the primer pair of the present invention, other aminopeptidases having the same properties as the aminopeptidase and biological sources having the aminopeptidase can be detected.

  The length of each primer of the primer pair of the present invention is at least 20 nucleotides, preferably 20 to 30 nucleotides, preferably 25 to 30 nucleotides, and more preferably 28 to 30 nucleotides.

  In the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 2, the distance between the oligonucleotide A and the oligonucleotide B is determined from the nucleotide residue at the 3 ′ end of the base sequence corresponding to the oligonucleotide A. It is at least 100 nucleotides long, preferably 200 to 800 nucleotides long, preferably 500 to 800 nucleotides long, more preferably 700 to 800 nucleotides long.

  Specific examples of the primer pair of the present invention include, for example, the base represented by 5′-CC (GC) GC (GC) ATCCC (GC) GT (GC) GC (GC) AACGT-3 ′ (SEQ ID NO: 5). A primer pair consisting of a primer containing the sequence and a primer containing 5'-CCGTTGTCGTTGAT (GC) CC (GC) GG (GC) CC-3 '(SEQ ID NO: 6), 5'-CAAGGGCCACGTCAGGACCATGGCCGGCGC-3' (sequence And a primer pair comprising a primer containing the base sequence shown in No. 7) and a primer containing the base sequence shown in 5′-CTTGAGGATCCGAGGTCAAACCCCGCAG-3 ′ (SEQ ID No. 8).

  Another aspect of the present invention is an aminopeptidase search method.

  The aminopeptidase search method of the present invention comprises a nucleotide sequence selected from the group consisting of the above (A) to (E) by hybridization using the probe of the present invention and / or PCR using the primer pair of the present invention. The region corresponding to the probe and / or the region corresponding to the PCR amplification product obtained using the primer pair is detected, and aminopeptidase showing hydrolytic activity is screened against aminoacyl-p-nitroanilide One major feature is to do this. Therefore, according to the aminopeptidase search method of the present invention, it is possible to detect other aminopeptidases having the same properties as the aminopeptidases and biological sources having the aminopeptidases.

  In the search method of the present invention, examples of the test sample include microorganisms, plant cells, animal cells, and nucleic acid samples thereof.

  In the search method of the present invention, examples of hybridization conditions include hybridization conditions for the evaluation of the “binding force”. In addition, in the search method of the present invention, from the viewpoint of improving accuracy, a lower ionic strength, for example, 6 × SSC, and more stringent, 5 × SSC, and / or a higher temperature as necessary. For example, hybridization under conditions of 10 ° C. or more stringent of the Tm value (80 to 90) of the probe used; conditions such as under 5 ° C .; more severe washing conditions, specifically, low ionic strength A buffer solution such as 6 × SSC, 5 × SSC for more stringent, 2 × SSC or the like for more stringent is used, and is more stringent at a higher temperature, for example, 55 ° C. of the Tm value. For example, washing under conditions such as 50 ° C., further stringent, 30 ° C., and even more stringent, 25 ° C. may be performed.

  The hybridization conditions may be set using, for example, a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 2 as a control.

When the probe length is 18 nucleotides or more, the Tm value is, for example, the following formula:
Tm = 81.5-16.6 (log ₁₀ [Na ⁺ ] ⁺ 0.41 (% G + C)-(600 / N)
(Where N is the chain length of the probe and% G + C is the content of guanine and cytosine residues in the probe)
When the probe length is shorter than 18 nucleotides, for example, the product of the content of A + T (adenine + thymine) residue and 2 ° C., the product of the content of G + C residue and 4 ° C. The sum of [(A + T) × 2 + (G + C) × 4] can be obtained.

  The PCR conditions can be set as appropriate according to the Tm value of the primer used. The PCR conditions may be set using, for example, a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 2 as a control.

  When the region detected by hybridization and PCR is a nucleic acid fragment encoding a partial fragment of aminopeptidase, the full-length amino acid sequence is encoded by a conventional method (for example, colony hybridization) using such a nucleic acid fragment. The nucleic acid to be isolated can be isolated.

  If hybridization and PCR indicate the presence of aminopeptidase, it may be assessed whether the source of the test sample contains the aminopeptidase of the present invention. For this evaluation, the aminopeptidase activity may be measured using aminoacyl-p-nitroanilide as a substrate. When the activity measurement shows hydrolytic activity, it is an indicator that the source of the test sample contains the aminopeptidase of the present invention.

  Yet another aspect of the present invention is an antibody or fragment thereof against the aminopeptidase of the present invention or the recombinant aminopeptidase of the present invention. According to the antibody or fragment thereof of the present invention, the aminopeptidase or other aminopeptidases having properties equivalent to those of the aminopeptidase can be detected. In addition, according to the antibody of the present invention or a fragment thereof, the function of the aminopeptidase or other aminopeptidases having properties equivalent to those of the aminopeptidases can be inhibited.

  The antibody of the present invention may be either a polyclonal antibody or a monoclonal antibody as long as it has an ability to specifically bind to the aminopeptidase of the present invention or the recombinant aminopeptidase of the present invention.

  The antibody of the present invention can be easily produced by a conventional method, for example, by immunizing a rabbit, mouse or the like using all or part of the aminopeptidase of the present invention or the recombinant aminopeptidase of the present invention. In addition, antibodies can be produced by genetic engineering. The antibodies of the present invention also include antibodies that can specifically bind to the aminopeptidase of the present invention or a partial fragment of the recombinant aminopeptidase of the present invention, antibodies modified by known techniques, antibody derivatives, and the like.

The antibody fragment of the present invention can be obtained by treating with a peptidase or the like. Specific examples of the antibody fragment of the present invention include Fab fragment, Fc ′ fragment, F (ab ′) ₂ fragment, Fab ′ fragment and the like.

  EXAMPLES Hereinafter, although an Example etc. demonstrate this invention in detail, this invention is not limited by this Example. Unless otherwise specified, in this specification, various operations were performed according to the Molecular Cloning Laboratory Manual Second Edition [Sambrook, J. et al., Molecular Cloning A Laboratory Manual 2nd Edition, (1989)].

Streptomyces septatus TH-2 [Streptomyces sp. Named and displayed as TH-2, deposit number: FERM P-173295 (deposit date: March 24, 1999), National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (Higashi 1-chome, Tsukuba, Ibaraki, Japan) 1-1)] [Hagishita, T., et al., Biotechnol. Lett. 22, 1587-1590 (2000); Hatanaka, T. et al., Enzyme Microb. Tech. , 31,233-241 (2002)] in a medium [composition: 0.5 wt% glucose, 0.5 wt% citric acid, 0.2 wt% K ₂ HPO ₄ , 0.05 wt% MgSO _4. 7H ₂ O, 0.5 wt% polypeptone and 0.5 wt% yeast extract] was aerobically cultured at 34 ° C. for 26 hours. During the culture, 1 ml of the culture was collected over time, and the obtained culture was centrifuged to obtain a culture supernatant. The obtained medium supernatant was subjected to a phosphatidyl-para-nitrophenol hydrolysis reaction to detect extracellular phospholipase D in the medium supernatant. Thereafter, extracellular phospholipase D in the culture supernatant was analyzed by Western blot hybridization using SDS-PAGE and an anti-PLD antibody.

As a result, it was found that phospholipase D was decomposed after 20 hours (OD ₆₆₀ = 6) from the start of culture. Therefore, it was suggested that the enzyme which decomposes | disassembles protein exists in this culture medium filtrate.

  Therefore, 0.1 ml of the medium filtrate obtained after 72 hours of culture was used with L-leucine-p-nitroanilide (L-Leu-pNA) as a substrate, and a reaction solution [composition: 80 mM Tris-HCl, pH 8.0]. The aminopeptidase activity was measured by detecting the release of p-nitroaniline by continuous optical measurement at 405 nm at 37 ° C.

  The enzymatic reaction was initiated by the addition of substrate. The initial velocity was determined from the linear portion of the optical density profile. One unit of aminopeptidase activity was defined as the amount of enzyme that liberates 1 μmol of p-nitroaniline per minute.

  As a result, aminopeptidase activity was found.

  The following operations were performed at 4 ° C. unless otherwise specified.

Streptomyces septatus TH-2 is added to the medium [composition: 0.5 wt% glucose, 0.5 wt% citric acid, 0.2 wt% K ₂ HPO ₄ , 0.05 wt% MgSO ₄ .7H ₂ O, The culture was aerobically cultured at 28 ° C. for 72 hours in 0.5 wt% polypeptone and 0.5 wt% yeast extract. During the cultivation, 0.1 ml of the culture was collected over time, and the obtained culture was filtered with filter paper to obtain a medium filtrate (supernatant).

  The culture filtrate (10 mg / ml protein solution) was heated at 65 ° C. for 30 minutes with occasional stirring. After removing the precipitate, the resulting solution was dialyzed against buffer A [composition: 10 mM Tris-HCl buffer, pH 8.0]. The dialyzed solution was loaded onto a DEAE-Sepharose (Amersham) column (inner diameter 1.6 × 10 cm, 20 ml) equilibrated with buffer A. The bound protein was eluted with a linear gradient of buffer 0-0.5 M NaCl at an average flow rate of 2 ml / ml. Among the obtained elution fractions, fractions showing high specific activity were collected. To the obtained fraction, ammonium sulfate was added so as to be 80% saturated ammonium sulfate to precipitate proteins. The obtained precipitate was dissolved in buffer A containing 200 mM NaCl. The resulting solution was dialyzed against buffer A containing 200 mM NaCl. The solution obtained after dialysis was loaded onto a trade name: Superdex 200 gel filtration column (Pharmacia Biotech, inner diameter 1.6 × 60 cm, 60 ml) and averaged with buffer A containing 200 mM NaCl. Protein was eluted at a flow rate of 1 ml / min. Protein elution was monitored at 280 nm.

  The aminopeptidase activity was determined by continuous optical measurement at 37 ° C. at 405 nm in a reaction solution [composition: 80 mM Tris-HCl, pH 8.0] by using p-nitro in 3.2 mM L-Leu-pNA as a substrate. It was measured by detecting the release of aniline.

  The enzymatic reaction was initiated by the addition of substrate. The initial velocity was determined from the linear portion of the optical density profile. One unit of aminopeptidase activity was defined as the amount of enzyme that liberates 1 μmol of p-nitroaniline per minute.

  Table 1 shows the purification table.

  The molecular weight (native) of the purified protein was determined using a trade name: Superdex 200 gel filtration AKTA fast protein liquid chromatography system (Pharmacia Biotech).

  As a result, the molecular weight of the purified protein in the native state was about 30 kDa.

  Samples obtained at each purification step were subjected to SDS-PAGE under denaturing conditions on a gel containing 15% by weight polyacrylamide [Laemmli, U., K., Nature, 15, 680-685 (1970)]. ] Was analyzed. The resulting gel was stained with Coomassie Brilliant Blue. FIG. 1 shows the results of SDS-PAGE of the samples obtained in each purification step.

  As a result, as shown in FIG. 1, it can be seen that the protein obtained after gel filtration (hereinafter referred to as aminopeptidase preparation) is substantially uniform. Further, as shown in FIG. 1 panel B, it can be seen that the molecular weight of the purified protein by SDS-PAGE is about 33 kDa. Thus, Streptomyces septatus TH-2 aminopeptidase is suggested to be a monomer.

  Subsequently, trypsin was added to an amount corresponding to 10 μg of the aminopeptidase preparation and incubated under conditions of 1% SDS, pH 8.0, and 37 ° C. The obtained product was blotted on a membrane to obtain a trypsin-degrading partial peptide.

  The N-terminal amino acid sequences of the aminopeptidase preparation and trypsin-degrading partial peptide were analyzed by Edman degradation. For the analysis of the N-terminal amino acid sequence, trade name: sequanaters manufactured by Applied Biosystems was used.

  As a result, the N-terminal amino acid sequence of aminopeptidase was AGAPAIPVANVKAHLNQL (SEQ ID NO: 3), and the N-terminal amino acid sequence of the trypsin-degrading partial peptide was AGPGINDN (SEQ ID NO: 4). Hereinafter, the aminopeptidase of Streptomyces septatus TH-2 is also referred to as SSAP. A gene encoding such aminopeptidase is also expressed as Ssap.

Streptomyces septatus TH-2 is added to the medium [composition: 0.5 wt% glucose, 0.5 wt% citric acid, 0.2 wt% K ₂ HPO ₄ , 0.05 wt% MgSO ₄ .7H ₂ O, The culture was aerobically cultured at 28 ° C. for 72 hours in 0.5 wt% polypeptone and 0.5 wt% yeast extract. The obtained culture was centrifuged at 40000 × g for 10 minutes to obtain bacterial cells.

  From the obtained bacterial cells, Streptomyces septatus TH-2 genomic DNA was obtained by the Saito Miura method [Saito, H. et al., Biochem. Biophys. Acta. 72, 619-629 (1963)].

  Using the N-terminal amino acid sequence shown in SEQ ID NO: 3 and the N-terminal amino acid sequence of the trypsin-degrading partial peptide shown in SEQ ID NO: 4, the forward primer (5′-CC (GC) GC (GC) ATCCC (GC ) Design GT (GC) GC (GC) AACGT-3 '; SEQ ID NO: 5) and reverse primer (5'-CCGTTGTCGTTGAT (GC) CC (GC) GG (GC) CC-3'; SEQ ID NO: 6) And synthesized.

  PCR amplification was performed using the Streptomyces septatus TH-2 genomic DNA as a template and the primers. In addition, PCR uses GC-RICH PCR System (Roche), 95 degreeC 3 minutes 1 cycle, 95 degreeC 30 seconds, 58 degreeC 30 seconds, 72 degreeC 1 minute 1 cycle, 10 cycles, 95 degreeC 30 seconds It was performed under the conditions of 20 cycles of 58 ° C. for 30 seconds and 72 ° C. (starting from 20 seconds and increasing by 5 seconds for each cycle), 72 ° C. for 7 minutes, 1 cycle, and 4 ° C. The obtained PCR product was ligated to a trade name: pGEM-T Easy (manufactured by Promega). Escherichia coli JM109 was transformed with the obtained plasmid.

  The Escherichia coli transformant was cultured at 37 ° C. in an LB medium [1% NaCl, 1% polypeptone, 0.5% yeast extract] containing 50 μg / ml ampicillin.

  From the obtained transformant, an alkaline extraction method [Birnboim, HC, et al., Nucleic Acids Res. , 24, 1513-1523 (1979)].

  (DIG) -labeled PCR probe was synthesized using PCR DIG Probe synthesis kit (Roche Molecular Biochemicals) using the obtained plasmid insert.

  The genomic DNA was partially digested with SacI, and then a 4-6 kb SacI restriction fragment was recovered.

  A genomic library was constructed using the 4-6 kb SacI restriction fragment and SacI-digested pUC19.

  The library was then screened by colony hybridization using a (DIG) -labeled PCR probe.

The genome library colony was transferred to a membrane (manufactured by Amersham, trade name: Nylon membrane Hybond-N ⁺ ), and then an alkaline denaturing solution (composition: 0.5 M NaOH, 1.5 M NaCl) was used. The nucleic acid on the membrane was denatured. Thereafter, the membrane was neutralized using a neutralizing solution [composition: 0.5 M Tris-HCl (pH 7.5), 1.5 M NaCl].

  The membrane was incubated with the (DIG) -labeled PCR probe for 24 hours at 55 ° C. in the trade name: DIG Easy Hyb (Roche), and then the membrane was washed with 2 × SSC; 0.1% SDS. The membrane after washing and washing, the trade name: DIG Wash and Block Buffer Set (Roche), trade name: anti-digoxigenin AP labeled antibody (Roche), trade name: NBT-BCIP solution (Roche) Detection was performed by color development.

  As a result, a positive colony carrying a plasmid having the full-length Ssap gene (pUC-SSAP) [hereinafter referred to as pUC-SSAP1] was obtained.

  From the obtained positive colony, pUC-SSAP1 was extracted, and the base sequence of the inserted fragment of pUC-SSAP1 was analyzed. The nucleotide sequence was determined by the dideoxy chain termination method [Sanger, F. et al., Proc. Natl. Acad. Sci. USA, 74, 5463-5467 (1977)].

  As a result, the determined nucleotide sequence of the Ssap gene in pUC-SSAP contained one open reading frame (FIG. 2). The amino acid sequence obtained by N-terminal analysis of purified SSAP and tryptic peptide is shown in the deduced amino acid sequence (bold in FIG. 2).

  In addition, regarding the deduced amino acid sequence, the BLAST program [Altschul, SF, et al., Nucleic Acid Res. , 25, 3389-3402 (1997)], and database search was performed under the conditions of Gap Costs (Extension 11, Extension 1), Expect 10, and Word Size 3.

  As a result, as shown in FIG. 3, the sequence identity between SSAP and Streptomyces griseus aminopeptidase (hereinafter also referred to as SGAP) was 71%, and SSAP and other streptomyces. The sequence identity with Myces aminopeptidase was shown to be 65-70%.

  Also, as shown in FIG. 3, five amino acid residues of SGAP marked with an asterisk involved in the coordination of two zinc atoms were found in SSAP and other Streptomyces aminopeptidases. .

  As shown in FIG. 1 panel B and FIG. 3, the amino acid sequence analysis and SDS-PAGE analysis of SSAP showed similarity to SGAP. Surprisingly, however, SSAP was not activated at all by calcium ions.

  In order to construct an expression plasmid for the Ssap gene product, an Nco I site and a Bam HI site were introduced into the putative transcription initiation codon on the Ssap gene by PCR. An Ssap gene lacking a putative signal peptide region was subjected to PCR using a sense primer (5′-CAAGGGCCACGTCAGGACCATGGCCGGCGC-3 ′, SEQ ID NO: 7) and an antisense primer (5′-CTTGAGGATCCGAGGTCAAACCCCGCAG-3 ′, SEQ ID NO: 8). Amplified by In addition, PCR uses GC-RICH PCR System (Roche), 95 degreeC 3 minutes 1 cycle, 95 degreeC 30 seconds, 58 degreeC 30 seconds, 72 degreeC 1 minute 1 cycle, 10 cycles, 95 degreeC 30 seconds The test was carried out under the conditions of 20 cycles of 58 ° C. for 30 seconds and 72 ° C. (starting from 20 seconds and increasing by 5 seconds for each cycle), one cycle of 72 ° C. for 7 minutes, and incubation at 4 ° C.

  The obtained PCR product was ligated to a trade name: pGEM-T Easy (manufactured by Promega). The sequence of the obtained plasmid was confirmed. Subsequently, the obtained plasmid insert was ligated to pETKmS2 [Mishima, N. et al., Biotechnol Prog. 13, 864-868 (1997)]. The resulting plasmid was used to transform E. coli BL21 DE3.

E. coli BL21 DE3 cells retaining the pET-SSAP1 were cultured at 37 ° C. for 12 hours in 3 ml of LB medium containing 50 μg / ml kanamycin. The cultured cells were washed with sterilized water. The cells after washing were treated with 0.5 wt% KH ₂ PO ₄ , 0.5% K ₂ HPO ₄ , 0.44 wt% Na ₂ HPO ₄ , 0.3 wt% (NH ₄ ) ₂ SO ₄ ,. 5 wt% glucose, 0.3 wt% MgSO ₄ .7H ₂ O, 0.004 wt% FeSO ₄ .7H ₂ O, 0.004 wt% CaCl ₂ , 0.00029 wt% CoCl ₂ .6H ₂ O, 0 .0003 wt% CuSO ₄ .5H ₂ O, 0.000036 wt% Na ₂ MoO.2H ₂ O, 0.001 wt% H ₃ BO ₃ , 0.001 wt% ZnSO ₄ .7H ₂ O, 0.2 wt Seed in 100 ml of synthetic medium containing% glycerol and 50 μg / ml kanamycin. Thereafter, the cells were cultured at 26 ° C. for 12 hours, and IPTG was added to the resulting culture to a final concentration of 0.5 mM. The culture was then cultured at 18 ° C. for 36 hours with shaking. As a result, recombinant SSAP was secreted into the extracellular fraction to a concentration corresponding to up to about 50% of the total extracellular protein.

  The obtained culture was centrifuged to remove the cells, and the obtained supernatant was cultured at 65 ° C. for 30 minutes, with stirring as necessary, at 65 ° C. for 30 minutes. After removing the precipitate by centrifugation, the solution was concentrated by ultrafiltration and dialyzed against buffer A. The solution after dialysis was used as a recombinant SSAP preparation in the following examples.

  As shown in panel B of FIG. 1, the recombinant SSAP had a molecular weight of approximately 33 kDa, just like purified wild-type SSAP. Moreover, one protein peak with a molecular weight of about 30 kDa was observed by gel filtration column chromatography using the trade name: Superdex 200, suggesting that SSAP is a monomeric enzyme.

  The effect of pH on recombinant aminopeptidase activity was investigated.

  L-Leu-pNA was prepared using 80 mM sodium acetate buffer at pH 4 to 6.5, 80 mM Tris-HCl buffer at pH 6.5 to 9.0, and 80 mM carbonate buffer at pH 9.0 to 11.0, respectively. The aminopeptidase activity was measured by detecting the release of p-nitroaniline by substrate as a substrate and continuous optical measurement at 405 nm at 37 ° C. SGAP was used as a comparative control. The enzymatic reaction was initiated by the addition of substrate. The initial velocity was determined from the linear portion of the optical density profile. One unit of aminopeptidase activity was defined as the amount of enzyme that liberates 1 μmol of p-nitroaniline per minute. The results are shown in FIG.

  As shown in FIG. 4, it can be seen that the recombinant SSAP has an optimum reaction pH of around pH 7.0, while SGAP has an optimum reaction pH of around pH 8-9. Also, from the V vs. pH plot and V / Km vs. pH (Dixon plot) of Panel A and Panel C of FIG. 4, as shown in Table 2, four pKa were determined. Such results suggest that the binding and reaction of the substrate to the enzyme is controlled by at least two ionic residues.

  The hydrolytic activity of recombinant SSAP against pNA derivatives was examined. Continuous optics at 405 nm at 37 ° C. in a reaction solution containing 50 μl of 1-2000 μg / ml recombinant SSAP, 50 μl of 0.2-3.2 mM aminoacyl-pNA and 400 μl of 100 mM Tris-HCl buffer (pH 8.0) It was measured by detecting the release of p-nitroaniline by measurement. The enzymatic reaction was initiated by the addition of substrate. The initial velocity was determined from the linear portion of the optical density profile. One unit of aminopeptidase activity was defined as the amount of enzyme that liberates 1 μmol of p-nitroaniline per minute.

  As the aminoacyl-pNA, L-leucine p-nitroanilide (Leu-pNA) (manufactured by Sigma Chemical Co.), L-phenylalanine p-nitroanilide (Phe-pNA) [Sigma Chemical Co. L-methionine p-nitroanilide (Met-pNA) (Sigma Chemical Co.), L-lysine p-nitroanilide (Lys-pNA) [Sigma Chemical Co. )], L-proline p-nitroanilide (pro-pNA) [Sigma Chemical Co.], L-alanine p-nitroanilide (Ala-pNA) [Sigma Chemical Co., Ltd.] With Sigma Chemical Co.) Co.] and L- glutamic acid p- nitroanilide (Glu-pNA) Peptides Laboratory Ltd.].

  SGAP was used as a comparative control.

  Table 3 shows the results of examining the catalytic ability of various aminoacyl-pNA hydrolysiss for each of the recombinant SSAP and SGAP.

As a result, SSAP showed the highest hydrolysis activity with respect to L-Leu-pNA. Kcat / Km value of the SSAP is a ^{125mM -1 · s -1, kcat /} Km value of SGAP is 345 mM ^-1 · ^s-1, between the SSAP and SGAP, observed significant difference It was.

  The thermal stability of the recombinant SSAP was examined. 100 μl of enzyme sample (5 μg / ml protein) was incubated in a 10 mM Tris-HCl buffer (pH 8.0) for 30 minutes at a temperature of 30 ° C. to 85 ° C. Thereafter, for the obtained enzyme sample, p-nitroaniline was obtained by continuous optical measurement at 405 nm in a reaction solution [composition: 80 mM Tris-HCl, pH 8.0] at 37 ° C. using L-Leu-pNA as a substrate. The aminopeptidase activity was measured by detecting the release of. The enzymatic reaction was initiated by the addition of substrate. The initial velocity was determined from the linear portion of the optical density profile. One unit of aminopeptidase activity was defined as the amount of enzyme that liberates 1 μmol of p-nitroaniline per minute. In addition, it investigated similarly using SGAP as a comparison control. The result is shown in FIG. In FIG. 5, the numerical value is shown as a percentage (residual activity) with respect to the maximum value.

As a result, as shown in FIG. 5, the 50% heat inactivation temperature of recombinant SSAP is about 78 ° C., while the 50% heat inactivation temperature of SGAP is in the absence of 1 mM CaCl _2. About 80 ° C. in the presence of 1 mM CaCl ₂ .

  Next, the effect of temperature on the recombinant SSAP was examined. By detecting the release of p-nitroaniline by continuous optical measurement at 405 nm in a temperature range of 30 to 85 ° C. in 80 mM Tris-HCl buffer (pH 8.0) using L-Leu-pNA as a substrate. Aminopeptidase activity was measured. The enzymatic reaction was initiated by the addition of substrate. The initial velocity was determined from the linear portion of the optical density profile. One unit of aminopeptidase activity was defined as the amount of enzyme that liberates 1 μmol of p-nitroaniline per minute. As a comparative control, SGAP was used, and the activity was measured in the same manner in 80 mM Tris-HCl buffer (pH 8.0) to examine the influence of temperature. The activation energy and the ΔH value were measured using Segel [Segel, I. et al. H. Biochemical Calculations, John Wiley and Sons, New York, 277-281 (1976)]. The results are shown in FIG.

  As a result, as shown in panel A of FIG. 6, the recombinant SSAP showed a bell-shaped curve at an optimal reaction temperature of 75 ° C. Further, as shown in FIG. 6 panel B, the activation energy was calculated to be 46.6 kJ / mol from the Arrhenius diagram of the hydrolysis of L-Leu-pNA in the temperature range of 30 to 70 ° C. It was. As shown in FIG. 6 panel D, from a Van't Hoff diagram of Km values for hydrolysis of the substrate in the temperature range of 25-50 ° C. (panel C in FIG. 6), ΔH is −61.9 kJ / mol. It was calculated that there was.

On the other hand, the optimum reaction temperature of SGAP was around 65 ° C. as shown in panel A of FIG. In addition, as shown in FIG. 6 panel B, from the Arrhenius diagram of the hydrolysis of L-Leu-pNA in the temperature range of 30 to 50 ° C., the activation energy is 38.6 kJ / in the presence of 1 mM CaCl _2. It was calculated to be 27.7 kJ / mol in the absence of 1 mM CaCl ₂ . As shown in panel D of FIG. 6, a Van't Hoff diagram of the Km value of substrate hydrolysis in the temperature range of 25-50 ° C. (panel C of FIG. 6) shows that ΔH is in the presence of 1 mM CaCl _2. -34.6 kJ / mol, and in the absence of 1 mM CaCl ₂ , it was calculated to be -46.6 kJ / mol.

Each enzyme (1 μg / ml, 37 ° C.), SSAP for 100 mM Tris-HCl buffer (pH 8.0) dissolved inhibitor, SGAP for 100 mM Tris-HCl buffer (pH 7.0), 1 mM CaCl ₂ By mixing with the dissolved inhibitor, preincubating for 5 minutes, and adding the substrate to the resulting mixture, bestatin (bestatin hydrochloride, Sigma Chemical Co.), amastatin (peptide research) The inhibition of recombinant SSAP and SGAP by D-Leu and L-Leu was determined. All assays were performed as described above with varying concentrations of L-Leu-pNA as substrate. The results are shown in Table 4.

As shown in Table 4, it can be seen that Amastatin is a weaker inhibitor against SGAP with K _i = 0.011 μM and Bestatin is K _i = 3.9 μM. Recombinant SSAP was 0.79 μM and 65 μM for inhibition by amastatin and bestatin, respectively. The inhibition constant of free L-Leu is 100 mM or more and 29 mM for recombinant SSAP and SGAP, respectively. Furthermore, the inhibition constant of D-Leu was K _i = 40 mM for SGAP. In contrast, for recombinant SSAP, the inhibition constant of D-Leu was 100 mM or more.

  According to the present invention, it is possible to perform a catalyst for amino acid analysis, synthesis reaction of various lead compounds, and the like under suitable conditions.

FIG. 1 is an electrophoretic diagram of SSAP by SDS-PAGE. Panel A shows the degree of purification of Streptomyces septatus TH-2 wild-type aminopeptidase at each purification step. Panel B is an electropherogram of purified wild type SSAP, heat treated recombinant SSAP and SGAP. The marker is a low molecular weight marker (molecular weight: 94,000, 67,000, 43,000, 30,000, 20,100 and 14,400).

FIG. 2 is a diagram showing the base sequence and deduced amino acid sequence of the SSAP gene. In the figure, the underlined portion of the base sequence is the putative ribosome binding site ("sd" in the figure), -35 sequence ("-35" in the figure) and -10 sequence ("-10" in the figure). ), The asterisk indicates a stop codon, and the arrow indicates a transcription termination factor. In addition, the amino acid sequence determined using purified aminopeptidase and trypsin-degrading partial peptide is shown in bold.

FIG. 3 is a diagram showing the results of analyzing the amino acid sequence of Streptomyces septatus TH-2 aminopeptidase (SSAP). In the figure, “SSAP” is an aminopeptidase (SEQ ID NO: 2) of Streptomyces aptemitus (SEQ ID NO: 2), “SAAP” is an aminopeptidase (SEQ ID NO: 9) of Streptomyces avermitilis, “SGAP” represents an aminopeptidase (SEQ ID NO: 10) of Streptomyces grieseus, and “SCAP” represents an aminopeptidase (SEQ ID NO: 11) of Streptomyces coelicolor. In the figure, an asterisk indicates a zinc coordination site, and “C” indicates a calcium binding site. For the alignment, the trade name: GENETX program was used with default values.

FIG. 4 shows the influence of pH on the activity of recombinant SSAP. SGAP was used as a comparative control. Panel A shows the effect of pH on substrate hydrolysis rate [V (μmol / min / mg)], Panel B shows the effect of pH on Km (mM), Panel C shows V / Km (μmol / min / mg / mg / m). The effect of pH in mM) is shown. In the figure, white circles, SSAP, open squares, CaCl ₂ in the absence SGAP, black squares represent SGAP at 1 mM CaCl ₂ presence. The reaction was carried out at 80 mM sodium acetate buffer, pH 6.5, 7.0, 7.5, 8. at pH 4.0, 4.5, 5.0, 5.5, 6.0 and 6.5, respectively. For each of 0, 8.5, and 9.0, 80 mM Tris-HCl buffer, and for each of pH 9.0, 9.5, 10.0, 10.5, and 11.0, use 80 mM carbonate buffer, Performed at 37 ° C. In the case of SGAP reaction, the reaction was performed even in the presence of 1 mM CaCl ₂ . The substrate hydrolysis rate (V) was performed at a final concentration of 3.2 mM L-Leu-pNA.

FIG. 5 shows the results of examining the thermal stability of recombinant SSAP. SGAP was used as a comparative control. In the figure, numerical values are shown as a percentage of the maximum value (100% of SGAP in the presence of recombinant SSAP, 1 mM CaCl ₂ and SGAP in the absence of CaCl ₂ = 0.23, 1.60 and 0. 0, respectively. 53 μM / min). In the figure, white circles, SSAP, open squares, CaCl ₂ in the absence SGAP, black squares represent SGAP at 1 mM CaCl ₂ presence. Each enzyme was used at 1 μg / ml. Enzyme activity was measured at pH 8.0 after incubating each enzyme at each temperature for 30 minutes.

FIG. 6 is a diagram showing the results of examining the temperature dependence of L-Leu-pNA hydrolysis by recombinant SSAP. SGAP was used as a comparative control. Panel A is the temperature effect on kcat (s ⁻¹ ), Panel B is the Arrrhenius plot, Panel C is the van't Hoff plot of K _m (mM), Panel D is the L-Leu-pNA water addition by SSAP. The energy diagram of decomposition is shown. In the figure, white circles, SSAP, open squares, CaCl ₂ in the absence SGAP, black squares represent SGAP at 1 mM CaCl ₂ presence.

  SEQ ID NO: 5 is the sequence of the primer.

  SEQ ID NO: 6 is the sequence of the primer.

  SEQ ID NO: 7 is the primer sequence.

  SEQ ID NO: 8 is the sequence of a primer.

Claims (11)

The following properties:
(1) Optimal reaction pH:
pH 7.5-10.5
(2) Substrate specificity:
L-leucine-p-nitroanilide, L-phenylalanine-p-nitroanilide, L-methionine-p-nitroanilide, L-lysine-p-nitroanilide, L-alanine-p-nitroanilide and L-proline-p -Exhibits hydrolytic activity towards nitroanilide,
(3) Optimal reaction temperature:
About 75 ° C,
(4) Thermal stability:
As a 50% heat inactivation temperature, about 78 ° C
(5) There is substantially no influence of catalytic activity by calcium ions,
An aminopeptidase having   The aminopeptidase according to claim 1, which is produced by Streptomyces septatus TH-2 (FERM P-173295). (A) a base sequence encoding the amino acid sequence represented by SEQ ID NO: 2,
(B) a base sequence represented by SEQ ID NO: 1,
(C) A nucleotide sequence having substitution, deletion, addition or insertion of at least one nucleotide residue in the nucleotide sequence of (A) or (B), and the encoded polypeptide has aminopeptidase activity. The base sequence that is shown,
(D) a nucleotide sequence of a nucleic acid that hybridizes under stringent conditions with a complementary strand of the nucleic acid comprising the nucleotide sequence of (A) or (B), and the encoded polypeptide exhibits aminopeptidase activity Obtained by aligning the base sequence and (E) the amino acid sequence shown in SEQ ID NO: 2 with the BLAST algorithm under the conditions of Gap Costs (Extension 11, Extension 1), Expect 10, and Word Size 3. A base sequence that encodes an amino acid sequence having a sequence identity of at least 75% and wherein the encoded polypeptide exhibits aminopeptidase activity;
An amino acid comprising a base sequence selected from the group consisting of: wherein the encoded polypeptide exhibits hydrolytic activity against aminoacyl-p-nitroanilide in the absence of calcium ions, Nucleic acid encoding peptidase.   A carrier for expression of aminopeptidase comprising the nucleic acid according to claim 3.   A transformed cell comprising the nucleic acid according to claim 3. 1) a step of culturing the transformed cell according to claim 5 to obtain a culture; and 2) a step of recovering aminopeptidase from the culture obtained in the step 1).
A method for producing a recombinant aminopeptidase, comprising:   A recombinant aminopeptidase encoded by the nucleic acid of claim 3. (I) at least 20 consecutive nucleotide residues selected from the group consisting of base numbers: 268 to 274, 325 to 334, and 534 to 558 in the sequence of the nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 2 A nucleotide sequence consisting of nucleotide residues or a nucleotide sequence substantially complementary to the nucleotide sequence, or (ii) a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 2, from the nucleotide sequence of the above (i) A base sequence of an oligonucleotide showing complementarity equivalent to that of the oligonucleotide or a base sequence substantially complementary to the base sequence,
The probe for detecting a nucleic acid according to claim 3, comprising: A. (I) Nucleotide residues selected from the group consisting of base numbers: 268 to 274, 325 to 334, and 534 to 558 in the sequence of the nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 2 A nucleotide sequence consisting of nucleotide residues, or (II) an oligonucleotide having complementarity equivalent to the oligonucleotide consisting of the nucleotide sequence of (I) above with respect to the nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 2. Base sequence,
An oligonucleotide containing
B. In the sequence of the nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 2, the nucleotide sequence is at least 100 nucleotide residues away from the 3 ′ terminal nucleotide residue of the nucleotide sequence corresponding to the oligonucleotide A. The primer pair for amplifying a nucleic acid or a part thereof according to claim 3, comprising an oligonucleotide containing a sequence substantially complementary to a base sequence comprising at least 20 nucleotide residues. (A) a base sequence encoding the amino acid sequence represented by SEQ ID NO: 2 by hybridization using the probe according to claim 8 and / or PCR using the primer pair according to claim 9;
(B) a base sequence represented by SEQ ID NO: 1,
(C) A nucleotide sequence having substitution, deletion, addition or insertion of at least one nucleotide residue in the nucleotide sequence of (A) or (B), and the encoded polypeptide has aminopeptidase activity. The base sequence that is shown,
(D) a nucleotide sequence of a nucleic acid that hybridizes under stringent conditions with a complementary strand of the nucleic acid comprising the nucleotide sequence of (A) or (B), and the encoded polypeptide exhibits aminopeptidase activity Obtained by aligning the base sequence and (E) the amino acid sequence shown in SEQ ID NO: 2 with the BLAST algorithm under the conditions of Gap Costs (Extension 11, Extension 1), Expect 10, and Word Size 3. A base sequence that encodes an amino acid sequence having a sequence identity of at least 75% and wherein the encoded polypeptide exhibits aminopeptidase activity;
A region corresponding to the probe and / or a region corresponding to a PCR amplification product obtained using the primer pair in a base sequence selected from the group consisting of aminoacyl- A method for searching for an aminopeptidase, comprising screening an aminopeptidase exhibiting hydrolytic activity against p-nitroanilide.   An antibody or fragment thereof against the aminopeptidase according to claim 1 or 2, or the recombinant aminopeptidase according to claim 4 or 5.

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