JP4631436B2 - Metalloendopeptidase - Google Patents

Description

  The present invention relates to a culture showing a metalloendopeptidase, a metalloendopeptidase, a method for producing the same, a nucleic acid encoding the metalloendopeptidase, a carrier for expression of the metalloendopeptidase, a transformed cell, and a method for producing a recombinant metalloendopeptidase About.

  Metalloendopeptidase plays an important role in the production of optically active α-amino acids by hydrolyzing L-Trp methyl ester.

Such a metalloendopeptidase is obtained by using, for example, a specific substrate specificity of a specific metalloendopeptidase from a polypeptide in which the α-amino group of the amino-terminal amino acid residue is modified with an acyl group or the like. It is used for isolation of peptides, identification of polypeptides in which the α-amino group of the amino terminal amino acid residue is modified, and amino terminal portions thereof (see Patent Document 1).
JP-A-9-224698

  The first aspect of the present invention is that a high specific activity is achieved in that the other proteolytic activities are not substantially detected, have resistance to organic solvents, exhibit high metalloendopeptidase activity, and metalloendopeptidase activity. It is an object of the present invention to provide a culture exhibiting metalloendopeptidase activity having at least one property such as exhibiting a long life as an enzyme. The second aspect of the present invention is a metalloend that enables the second aspect to be easily purified, produced in large quantities, and substantially free from other enzymes having proteolytic activity. It is in providing the manufacturing method of a peptidase. In the third aspect, the present invention provides a means based on proteolytic activity in the presence of an organic solvent, provides a means for analysis such as protein analysis, produces various peptides, An object of the present invention is to provide a metalloendopeptidase or a recombinant metalloendopeptidase that enables at least one of producing intermediate compounds, lead compounds and the like. In the fourth aspect of the present invention, in the fourth aspect, the metalloendopeptidase is produced in a large amount, the metalloendopeptidase is expressed in a heterologous cell, and an isozyme having the same physiological activity as the metalloendopeptidase is screened. It is an object of the present invention to provide a nucleic acid encoding a metalloendopeptidase that enables at least one of, for example, improving the physiological activity of the metalloendopeptidase. The sixth aspect of the present invention relates to providing a metalloendopeptidase expression carrier that enables at least one of expressing the metalloendopeptidase in a heterologous cell. Furthermore, in the seventh aspect of the present invention, the metalloendopeptidase is expressed by genetic engineering, the metalloendopeptidase is expressed in a state where it can be easily purified, and the physiological activity of the metalloendopeptidase is improved. It is to provide a transformed cell that enables at least one of the above. In the eighth aspect, the present invention provides a method for producing a recombinant metalloendopeptidase that enables at least one of easy purification, efficient production, and improvement of the bioactivity of metalloendopeptidase. There is to do. Other objects of the present invention are clearly derived from the following description of the present specification.

That is, the gist of the present invention is as follows.
[1] Metalloendopeptidase activity obtained as a culture supernatant by culturing Streptomyces septatus TH-2 (FERM P-17329) in the presence of 80 to 160 mM glucose and 10 to 50 mM K ₂ HPO ₄ A culture showing,
[2] A culture supernatant is obtained by culturing Streptomyces septatus TH-2 (FERM P-17329) in the presence of 80 to 160 mM glucose and 10 to 50 mM K ₂ HPO ₄ . Method for producing metalloendopeptidase,
[3] The metalloendopeptidase is
(A) the amino acid sequence shown in SEQ ID NO: 2,
(B) In SEQ ID NO: 2, an amino acid sequence having substitution, deletion, insertion or addition of one to several amino acid residues, and (C) the sequence shown in SEQ ID NO: 2, using the FASTA algorithm , Amino acid sequence having a sequence identity of 98% or more calculated under the conditions of KTUP 2, expect value 10.0, Cost to open gap 11, and Cost to extend gap 1;
Having an amino acid sequence selected from the group consisting of:

(In the formula, Xaa is phenylalanine residue, leucine residue, tyrosine residue, methionine residue, threonine residue, alanine residue, histidine residue, arginine residue, serine residue, isoleucine residue, lysine residue. , Glutamine residue, asparagine residue, tryptophan residue, valine residue, glycine residue, glutamic acid residue, proline residue or aspartic acid residue, Yaa is a proline residue, tyrosine residue, lysine residue An isoleucine residue or an aspartic acid residue, and Zaa represents a phenylalanine residue, an alanine residue, a valine residue, a glutamic acid residue, or an arginine residue)
Is a polypeptide that recognizes the sequence shown in (SEQ ID NO: 3) and acts on a peptide bond between the amino acid residue of amino acid number 4 of SEQ ID NO: 3 and the amino acid residue of amino acid number 5 , The production method according to the above [2],
[4] The metalloendopeptidase has an amino acid sequence in which one to several amino acid residues are conservatively substituted with other amino acids in SEQ ID NO: 2, and the conservative substitution is represented by the following amino acid group: (1) to (6):
(1) glycine and alanine,
(2) valine, isoleucine and leucine,
(3) Aspartic acid, glutamic acid, asparagine and glutamine,
(4) serine and threonine,
(5) lysine and arginine,
(6) Performed within an amino acid residue belonging to the amino acid group selected from the group consisting of phenylalanine and tyrosine, the following formula:

(In the formula, Xaa is phenylalanine residue, leucine residue, tyrosine residue, methionine residue, threonine residue, alanine residue, histidine residue, arginine residue, serine residue, isoleucine residue, lysine residue. , Glutamine residue, asparagine residue, tryptophan residue, valine residue, glycine residue, glutamic acid residue, proline residue or aspartic acid residue, Yaa is a proline residue, tyrosine residue, lysine residue An isoleucine residue or an aspartic acid residue, and Zaa represents a phenylalanine residue, an alanine residue, a valine residue, a glutamic acid residue, or an arginine residue)
Is a polypeptide that recognizes the sequence shown in (SEQ ID NO: 3) and acts on a peptide bond between the amino acid residue of amino acid number 4 of SEQ ID NO: 3 and the amino acid residue of amino acid number 5 , The production method according to the above [3],
[5] (A) the amino acid sequence represented by SEQ ID NO: 2,
(B) In SEQ ID NO: 2, an amino acid sequence having substitution, deletion, insertion or addition of one to several amino acid residues, and (C) the sequence shown in SEQ ID NO: 2, using the FASTA algorithm , Amino acid sequence having a sequence identity of 98% or more calculated under the conditions of KTUP 2, expect value 10.0, Cost to open gap 11, and Cost to extend gap 1;
Having an amino acid sequence selected from the group consisting of:

(In the formula, Xaa is phenylalanine residue, leucine residue, tyrosine residue, methionine residue, threonine residue, alanine residue, histidine residue, arginine residue, serine residue, isoleucine residue, lysine residue. , Glutamine residue, asparagine residue, tryptophan residue, valine residue, glycine residue, glutamic acid residue, proline residue or aspartic acid residue, Yaa is a proline residue, tyrosine residue, lysine residue An isoleucine residue or an aspartic acid residue, and Zaa represents a phenylalanine residue, an alanine residue, a valine residue, a glutamic acid residue, or an arginine residue)
Metalloendopeptidase that recognizes the sequence shown in SEQ ID NO: 3 and acts on a peptide bond between the amino acid residue of amino acid number 4 of SEQ ID NO: 3 and the amino acid residue of amino acid number 5 (SEQ ID NO: 3) ,
[6] The metalloendopeptidase according to [5] obtained by the production method according to any one of [2] to [4],
[7] (a) a base sequence encoding the amino acid sequence shown in SEQ ID NO: 2,
(B) a base sequence encoding an amino acid sequence having substitution, deletion, insertion or addition of one to several amino acid residues in SEQ ID NO: 2;
(C) The sequence identity calculated by the FASTA algorithm with respect to the sequence shown in SEQ ID NO: 2 under the conditions of KTUP 2, expect value 10.0, Cost to open gap 11, and Cost to extend gap 1 is 98. A base sequence encoding an amino acid sequence that is at least% , and (d) a base sequence of a nucleic acid that hybridizes under stringent conditions with an antisense strand of a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 2.
A polypeptide encoded by containing a base sequence selected from the group consisting of:

(In the formula, Xaa is phenylalanine residue, leucine residue, tyrosine residue, methionine residue, threonine residue, alanine residue, histidine residue, arginine residue, serine residue, isoleucine residue, lysine residue. , Glutamine residue, asparagine residue, tryptophan residue, valine residue, glycine residue, glutamic acid residue, proline residue or aspartic acid residue, Yaa is a proline residue, tyrosine residue, lysine residue An isoleucine residue or an aspartic acid residue, and Zaa represents a phenylalanine residue, an alanine residue, a valine residue, a glutamic acid residue, or an arginine residue)
And has an activity that acts on a peptide bond between the amino acid residue of amino acid number 4 of SEQ ID NO: 3 and the amino acid residue of amino acid number 5 of SEQ ID NO: 3. A nucleic acid encoding a metalloendopeptidase,
[8] A recombinant metalloendopeptidase encoded by the nucleic acid according to [7],
[9] A metalloendopeptidase expression carrier comprising the nucleic acid of [7] above,
[10] A transformed cell having the nucleic acid according to [7] above,
[11] A method for producing a recombinant metalloendopeptidase, comprising culturing the transformed cell according to [10], and recovering metalloendopeptidase from the obtained culture,

  The culture of the present invention is excellent in that no other proteolytic activities are substantially detected, it has a resistance to organic solvents, exhibits a high metalloendopeptidase activity and a high specific activity, and has a long life as an enzyme. Expresses properties. Therefore, according to the culture of the present invention, it is possible to perform analysis such as protein analysis, and to produce an excellent effect that various peptides, intermediate compounds for pharmaceuticals, lead compounds and the like can be produced. Play. Further, according to the method for producing a metalloendopeptidase of the present invention, a metalloendopeptidase having resistance to organic solvents, recognizing and cleaving a specific amino acid sequence can be easily purified, and produced in large quantities. There is an excellent effect of being able to. According to the metalloendopeptidase or recombinant metalloendopeptidase of the present invention, a reaction based on proteolytic activity in the presence of an organic solvent can be performed. Analysis such as protein analysis, various peptides, for example, functional peptides The production of an intermediate compound, a lead compound, and the like for production and medicine has an excellent effect. Furthermore, according to the nucleic acid of the present invention, the metalloendopeptidase can be produced by genetic engineering, and the metalloendopeptidase can be expressed in heterologous cells. Furthermore, according to the nucleic acid of the present invention, it is possible to screen for isozymes exhibiting physiological activity equivalent to that of the metalloendopeptidase. According to the expression carrier for metalloendopeptidase of the present invention, there is an excellent effect that the metalloendopeptidase can be expressed in heterologous cells. Therefore, it is possible to provide a means for industrial production of the metalloendopeptidase. Furthermore, according to the transformed cell of the present invention, the metalloendopeptidase can be expressed in a state that can be easily purified by genetic engineering, and the metalloendopeptidase can be produced industrially. There is an effect. In addition, according to the method for producing a recombinant metalloendopeptidase of the present invention, the metallopeptidase can be easily purified and can be produced efficiently.

  In this specification, an amino acid may be represented by three letters or one letter. That is, alanine is Ala or A, arginine is Arg or R, asparagine is Asn or N, aspartic acid is Asp or D, cysteine is Cys or C, glutamic acid is Glu or E, glutamine is Gln or Q, glycine is Gly or G, histidine is His or H, isoleucine is Ile or I, leucine is Leu or L, lysine is Lys or K, methionine is Met or M, phenylalanine is Phe or F , Proline is Pro or P, serine is Ser or S, threonine is Thr or T, tryptophan is Trp or W, tyrosine is Tyr or Y, and valine is Val or V.

In one aspect, the present invention relates to S. septatus TH-2 [Streptomyces sp. In the presence of 80-160 mM glucose and 10-50 mM K ₂ HPO ₄ . Named and displayed as TH-2, deposit number: FERM P-17329 (deposit date: March 24, 1999), National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (Higashi 1-chome, Tsukuba, Ibaraki, Japan) It is related with the culture which shows metalloendopeptidase activity obtained as a culture supernatant by culture | cultivating].

The cultures of the present invention are prepared from S. cerevisiae in the presence of 80-160 mM glucose and 10-50 mM K ₂ HPO ₄ . One major characteristic is that the culture supernatant is obtained by culturing septus TH-2.

  Therefore, the culture of the present invention expresses an excellent property that no proteolytic activity other than metalloendopeptidase is substantially detected. For this reason, the culture of the present invention recognizes as a substrate, and there is substantially no variation in the products generated by the decomposition of the cleaved compound. Therefore, analysis such as protein analysis, various peptides such as functional peptides, pharmaceuticals, etc. Suitable for the production of intermediate compounds, lead compounds and the like. Further, according to such a culture, the metalloendopeptidase described later can be easily purified.

Moreover, according to the culture of the present invention, S. cerevisiae is present in the presence of 80 to 160 mM glucose and 10 to 50 mM K ₂ HPO ₄ . Since it is obtained by culturing septus TH-2, it exhibits excellent properties such as high metalloendopeptidase activity and specific activity. Therefore, it can be used for analysis such as protein analysis, production of various peptides such as functional peptides, intermediate compounds for pharmaceuticals, lead compounds, etc. without further purification.

Examples of the medium used for producing the culture of the present invention include S. It is a medium suitable for growth of septus TH-2, and may be any medium containing glucose as a carbon source and inorganic phosphate. For example, 80 to 160 mM glucose and 10 to 50 mM K ₂ HPO ₄ And a medium containing 0.05% by weight MgSO ₄ .7H ₂ O, 0.5% by weight polypeptone and 0.5% by weight yeast extract.

  The content of glucose in the medium is preferably 80 mM or more, more preferably 100 mM or more, from the viewpoint of sufficiently inducing the expression of metalloendopeptidase activity, and the expression of metalloendopeptidase activity by the glucose. From the viewpoint of sufficiently exhibiting the inducing ability, it is preferably 160 mM or less, and more preferably 120 mM or less.

  In the present invention, instead of glucose, it may be used as a derivative compound of glucose, for example, a product produced by reacting with other components in the medium. Examples of such a glucose derivative compound include compounds produced by autoclaving with a medium. In this case, the final concentration is preferably 80 mM or more, more preferably 100 mM or more, depending on the glucose, from the viewpoint of sufficiently inducing the expression of metalloendopeptidase activity as the amount of glucose added to the medium. From the viewpoint of sufficiently exhibiting the ability to induce the expression of the metalloendopeptidase activity, it is preferably formulated to be 160 mM or less, more preferably 120 mM or less.

In the case of K ₂ HPO ₄ , for example, in the case of K ₂ HPO ₄ , the content of inorganic phosphate is preferably 10 mM or more, more preferably 30 mM or more, from the viewpoint of sufficiently inducing the expression of metalloendopeptidase activity. From the viewpoint of sufficiently exhibiting the ability to induce the expression of the metalloendopeptidase activity by K ₂ HPO ₄ , it is preferably 50 mM or less, more preferably 50 mM or less.

  The S. As the culture conditions for septus TH-2, Although it will not specifically limit if it is a range which septus TH-2 grows, The conditions which culture | cultivate at about 30 degreeC, for example, 25-34 degreeC, for about 5 days, for example, 3-5 days are mentioned. Note that the S.P. In the culture of septus TH-2, it is desirable that aeration be sufficient from the viewpoint of growing the cells well, for example, it is desirable to rotate and shake at 125 rpm.

  The culture of the present invention comprises the aforementioned S.P. The culture obtained by culturing septus TH-2 can be obtained as a medium supernatant by subjecting it to means capable of separating cells and medium components, for example, centrifugation, ultrafiltration and the like. In the present invention, the obtained culture supernatant may be dialyzed with an appropriate buffer or the like.

The metalloendopeptidase activity by the culture of the present invention is, for example, 100 μM FRETS-GRYFA [D-A2pr (Nma) -Gly-Arg-Tyr-Phe-Ala-Phe-Pro-Lys (Dnp) -D-Arg-D -Arg] [D-A2pr (Nma)-represents a D-2,3-diaminopropionic acid (D-A2pr) group having a 2- (N-methylamino) benzoyl (Nma) group in the side chain, and Lys (Dnp) represents a lysine residue having a 2,4-dinitrophenyl (Dnp) group in the ε-amino group; a solution containing SEQ ID NO: 4] was added to a 50 mM acetate buffer (pH 6) containing 2 mM CaCl _2. In 1), the sample to be measured can be evaluated by incubating at 37 ° C. for 1 minute and measuring λex 340 nm and λem 440 nm with a spectrofluorometer. One unit of metalloendopeptidase activity can be calculated as the amount of enzyme that digests 1 μmol of FRETS-GRYFA per minute. The metalloendopeptidase activity can be measured by, for example, incubating lysorufin-labeled casein and the sample to be measured for 15 minutes at 37 ° C. in an incubation buffer (0.2 M acetic acid buffer (pH 6.1) containing 8 mM CaCl ₂ ). Thereafter, 5 w / v% trichloroacetic acid is added to the obtained product to stop the reaction, and the supernatant can also be evaluated as proteolytic activity by measuring absorption at 574 nm. Here, one unit of proteolytic activity is defined as the amount of liberated 1 μmol of lysorphine [molecular attenuation coefficient of 524 nm of lysorfin (ε mM = 6.6)] per hour.

  In addition, the culture of the present invention exhibits an excellent property of exhibiting resistance to organic solvents. Therefore, according to the culture of the present invention, a reaction based on metalloendopeptidase activity can be performed in a reaction system using an organic solvent. Therefore, for example, it can be used for the production of a compound that requires a series of reactions in an organic solvent.

In another aspect, the present invention relates to S. cerevisiae in the presence of 80-160 mM glucose and 10-50 mM K ₂ HPO ₄ . The present invention relates to a method for producing a metalloendopeptidase, wherein culture supernatant is obtained by culturing septus TH-2.

  The process for producing the metalloendopeptidase of the present invention is carried out under the same culture conditions as the culture of the present invention. There is one major feature in culturing septus TH-2 to obtain a culture supernatant. Therefore, according to the production method of the present invention, since proteolytic activity other than metalloendopeptidase is not substantially detected in the culture supernatant, the metalloendopeptidase can be easily purified.

  In addition, according to the production method of the present invention, the metalloendopeptidase in the culture supernatant is increased by culturing under the culturing conditions. Therefore, the metalloendopeptidase can be obtained easily and efficiently in large quantities. Exhibits an excellent effect.

As a manufacturing method of the present invention, specifically, for example,
I) S. cerevisiae in the presence of 80-160 mM glucose and 10-50 mM K ₂ HPO ₄ . culturing septus TH-2 to obtain a culture supernatant;
II) The culture supernatant obtained in the above step II) is dialyzed, for example, against 25 mM Tris-HCl buffer (pH 8.0), and the obtained product is equilibrated with the appropriate buffer. The product was subjected to ion exchange chromatography, for example, Vivapure-Q spin column (manufactured by Vivascience, product number: VS-IX20QHGP) equilibrated with 25 mM Tris-HCl buffer (pH 8.0). Elution with 25 mM Tris-HCl buffer (pH 8.0) containing 5 M NaCl;
III) The fraction obtained in the above step II) is dialyzed against 10 mM potassium phosphate buffer (pH 7.0), and the obtained product is subjected to a hydroxyapatite column to give 10 to 400 mM potassium phosphate. A step of obtaining a fraction containing metalloendopeptidase as an elution fraction of 200 mM potassium phosphate, and IV) the fraction obtained in step III) above with respect to a 20 mM Tris-HCl buffer. Dialysis and equilibration with an appropriate ion exchange chromatography equilibrated with the above buffer, for example, trade name: Mono Q HR5 / 5 column equilibrated with 25 mM Tris-HCl buffer (pH 8.0) [Amersham Biotech And then eluting with a linear gradient of 0 to 0.5 M NaCl. As a 115 mM elution fraction, Obtaining a fraction containing taro endopeptidase,
And the like. In each step, the activity of metalloendopeptidase can be measured by the same method as described above. Moreover, you may use for centrifugation for 10 minutes at 4000xg about the fraction after a dialysis as needed between each process.

  Metalloendopeptidases obtained by such production methods are also included in the present invention.

  In another aspect, the present invention relates to the metalloendopeptidase.

  More specifically, the metalloendopeptidase of the present invention includes the following 1. ~ 6. The nature of

1. Molecular Weight The metalloendopeptidase of the present invention has a molecular weight calculated by 12.5 wt% SDS-PAGE of about 35 kD and is equilibrated with phosphate buffered saline. Superdex 200 10/300 GL The molecular weight calculated by a gel filtration method (flow rate 0.5 ml / min, column size: 1 cm (inner diameter) × 30 cm) using a column (Amersham Biotech) is about 40 kD, and is a monomer.

2. Presence of metal ions in the molecule Each metalloendopeptidase molecule of the present invention has 0.9 zinc molecules.

3. Inhibitor effects Inhibited by EDTA and phosphoramidon.

4). Optimum pH for reaction
pH 5-8, preferably pH 5.5-7, especially at around pH 6.0

5. Effect of metal ions 1) In the presence of 2 mM calcium, the upper limit of the range of the optimum reaction temperature is increased by about 10 ° C., and the relative activity is about 100% relative to the activity when incubated at 15 ° C. in the absence of calcium. The upper limit of the range showing the thermal stability of the film increases by about 10 ° C.
2) In the presence of 2 mM MgCl ₂ , 2 mM CoCl ₂ , 2 mM NiSO ₄ , 2 mM FeSO ₄ and 2 mM ZnSO ₄ , the activity does not increase, especially with the addition of 2 mM NiSO ₄ , 2 mM FeSO ₄ and 2 mM ZnSO ₄ Activity is reduced to less than 20% of activity.

6). Substrate specificity 1) Under the conditions of the method for measuring metallopeptidase activity, the Phe residue, Leu residue, Tyr residue, Met residue, Thr residue at the P1 position for the substrate shown in SEQ ID NO: 3 Selectable in order of group and Ala residue, selective for hydrophobic or basic residue at P2 position and basic or small residue at P3 position, selected for acidic residues at P2 and P3 positions Not right. Specifically, selectivity for Ala, Arg, Val, Phe and Lys residues at position P2, selectivity for Arg, Val and Ala residues at position P3 Indicates.
2) For the FRETS-GRYFA, Km value: 5.0 ± 1.6 μM, kcat value: 5.0 ± 1.0 (1 / second), kcat / Km: 1.0 ± 0.30 (1 / Second 1 / μM).

  In addition, the metalloendopeptidase of the present invention exhibits proteolytic activity and metalloendopeptidase activity even in the presence of an organic solvent such as dimethylformamide, dimethylsulfoxide, ethanol, methanol, ethylene glycol, etc., and is resistant to organic solvents. Show. Therefore, according to the metalloendopeptidase of the present invention, a reaction based on proteolytic activity in the presence of an organic solvent can be carried out. Analysis such as protein analysis, production of various peptides such as functional peptides, Intermediate compounds, lead compounds and the like can be produced.

As the metalloendopeptidase of the present invention, more specifically,
(A) the amino acid sequence shown in SEQ ID NO: 2,
(B) an amino acid sequence having a substitution, deletion, insertion or addition of at least one amino acid residue in SEQ ID NO: 2; and (C) the sequence shown in SEQ ID NO: 2, using the FASTA algorithm, 2, an amino acid sequence having a sequence identity of at least 75% calculated under the conditions of expect value 10.0, cost to open gap 11, cost to extend gap 1;
Having an amino acid sequence selected from the group consisting of:

(In the formula, Xaa is phenylalanine residue, leucine residue, tyrosine residue, methionine residue, threonine residue, alanine residue, histidine residue, arginine residue, serine residue, isoleucine residue, lysine residue. , Glutamine residue, asparagine residue, tryptophan residue, valine residue, glycine residue, glutamic acid residue, proline residue or aspartic acid residue, Yaa is a proline residue, tyrosine residue, lysine residue An isoleucine residue or an aspartic acid residue, and Zaa represents a phenylalanine residue, an alanine residue, a valine residue, a glutamic acid residue, or an arginine residue)
And a polypeptide that acts on a peptide bond between the amino acid residue of amino acid number 4 of SEQ ID NO: 3 and the amino acid residue of amino acid number 5 (SEQ ID NO: 3). Can be mentioned.

  In (A) above, the amino acid sequence shown in SEQ ID NO: 2 is S. It is the amino acid sequence of the metalloendopeptidase of septus TH-2.

In the present invention, the amino acid sequence (recognition sequence) shown in SEQ ID NO: 3 is recognized under the conditions for the measurement method of the metalloendopeptidase activity, and the amino acid sequence of SEQ ID NO: 3 Any metalloendopeptidase of the present invention can be used as long as it exhibits at least an activity that acts on a peptide bond between an amino acid residue and the amino acid residue of amino acid number: 5, specifically, an activity that cleaves the peptide bond. Includes a polypeptide having a variant sequence of the amino acid sequence shown in SEQ ID NO: 2. Examples of the variant of the amino acid sequence shown in SEQ ID NO: 2 include the amino acid sequence of (B) or (C). In addition, a polypeptide encoded by a nucleic acid described later, which shows the activity of recognizing and cleaving the recognition sequence under the conditions for the measurement method of the metalloendopeptidase activity, can be used. Included in peptidase.

  The organism that is the natural source of the amino acid sequence variants is not particularly limited. Examples include other strains classified as septatus.

  Regarding the amino acid sequence of (B) above, the number of amino acid residue substitutions, deletions, insertions or additions (hereinafter also referred to as “mutation”) in the sequence shown in SEQ ID NO: 2 is the measurement of the metalloendopeptidase activity. It may be in a range showing the activity of recognizing and cleaving the recognition sequence under the conditions of the method, and is at least one, preferably one to plural, more preferably one to several. The amino acid sequence having the mutation may be a naturally occurring amino acid sequence, that is, an amino acid sequence having a naturally occurring mutation, and is an amino acid sequence artificially introduced with a mutation at a desired site or amino acid residue. There may be. When a mutation is artificially introduced into a desired site or amino acid residue, the mutation may be artificially introduced according to a variant of the sequence shown in SEQ ID NO: 2 that exists in nature. Artificial mutation can be introduced, for example, by a site-specific mutagenesis method using conventional PCR.

The amino acid sequence of (B) is not particularly limited. For example, in SEQ ID NO: 2, at least one amino acid residue is substituted with another amino acid residue having similar physical properties (conservative). Substituted) amino acid sequences. The conservative substitution is, for example, substitution to an amino acid residue that exhibits a function similar to hydrophobicity, charge, pK, steric features, etc., and only to the extent that the physiological activity of the original polypeptide is maintained. Examples include substitution with an amino acid residue that cannot change the three-dimensional structure or folding structure of a polypeptide. More specifically, the amino acid sequence of (B) has an amino acid sequence in which at least one amino acid residue is conservatively substituted with another amino acid in SEQ ID NO: 2, and the conservative substitution Are the following amino acid groups (1) to (6):
(1) glycine and alanine,
(2) valine, isoleucine and leucine,
(3) Aspartic acid, glutamic acid, asparagine and glutamine,
(4) serine and threonine,
(5) lysine and arginine,
(6) An amino acid sequence that is performed within an amino acid residue belonging to an amino acid group selected from the group consisting of phenylalanine and tyrosine.

  In (C), the sequence identity is a value calculated by appropriately aligning a query sequence (sequence to be evaluated) with respect to a reference sequence (for example, the amino acid sequence shown in SEQ ID NO: 2). It is. Specifically, in the present specification, the sequence identity is a default value of an algorithm that can be used in multiple alignment according to the trade name: GENETYX-MAC (Software Development Co., Ltd.), more specifically, Pearson and It is a value calculated by using the FASTA algorithm by Lipman under the conditions of KTUP 1-2 (amino acid sequence base), expect value 10.0, Cost to open gap 11, and Cost to extend gap 1.

  In the present invention, the sequence identity is at least 75%, preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, and particularly preferably 98% or more.

  In addition, the metalloendopeptidase of the present invention has an excellent property that it is substantially less susceptible to self-digestion. For example, the metalloendopeptidase exhibits an excellent property that its lifetime as an enzyme is longer than that of thermolysin. Therefore, it can be stably stored during use.

In another aspect, the present invention provides (a) a base sequence encoding the amino acid sequence represented by SEQ ID NO: 2,
(B) a base sequence encoding an amino acid sequence having a substitution, deletion, insertion or addition of at least one amino acid residue in SEQ ID NO: 2;
(C) The sequence identity calculated by the FASTA algorithm with respect to the sequence shown in SEQ ID NO: 2 under the conditions of KTUP 2, expect value 10.0, Cost to open gap 11, and Cost to extend gap 1 is at least A base sequence encoding an amino acid sequence that is 75%, and (d) a base sequence of a nucleic acid that hybridizes under stringent conditions with an antisense strand of a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 2.
The encoded polypeptide recognizes SEQ ID NO: 3 and comprises the amino acid residue of amino acid number 4 and SEQ ID NO: 5 of SEQ ID NO: 3. The present invention relates to a nucleic acid encoding a metalloendopeptidase, which has an activity that acts on a peptide bond between the amino acid residues.

  According to the nucleic acid of the present invention, the metalloendopeptidase can be produced by genetic engineering. In addition, according to the nucleic acid of the present invention, the metalloendopeptidase can be expressed in a heterologous cell different from S. septus TH-2.

  The base sequences (a) to (b) are base sequences encoding the amino acid sequences (A) to (C), respectively.

  The “stringent conditions” in the above (c) refers to moderately stringent conditions, preferably highly stringent conditions. More specifically, the “stringent conditions” are, for example, 6 × SSC (composition of 1 × SSC: 0.15M NaCl, 0.015M sodium citrate, pH 7.0) and 0.5% by weight. Incubate at room temperature, more stringent at 45 ° C., more stringent at 60 ° C. for 12 hours in a solution containing SDS, 5 × Denhardt, 100 μg / ml denatured salmon sperm DNA and 50% by volume formamide, and more Low ionic strength, for example 0.5 × SSC, preferably 0.2 × SSC, more preferably 0.1 × SSC, etc. and / or higher temperatures, for example depending on the Tm value of the nucleic acid used. 50 ° C. or higher, preferably 60 ° C. or higher, more preferably 65 ° C. or higher. That. In the present invention, the nucleic acid that hybridizes with the antisense strand of the nucleic acid encoding the amino acid sequence of SEQ ID NO: 2 under stringent conditions is related to the amino acid sequence encoded by the nucleic acid, SEQ ID NO: 2 Sequence identity with the amino acid sequence of at least 75%, preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, particularly preferably 98% or more. Regarding the sequence, the sequence identity with the base sequence encoding the amino acid sequence of SEQ ID NO: 2 is at least 70%, preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, particularly The conditions are preferably such that specific hybridization with a nucleic acid of 95% or more can be achieved.

  In the present specification, the sequence identity of the base sequence is a value calculated by the FASTA algorithm under the conditions of KTUP 6, expect value 2.0, Cost to open gap 14, and Cost to extend gap 2.

  In addition, based on the nucleic acid of the present invention, a probe and primer pair for screening for an isozyme exhibiting a physiological activity equivalent to that of the metalloendopeptidase can be provided.

  Examples of the probe include all or part of the nucleic acid of the present invention or an antisense strand thereof. As a part of the nucleic acid of the present invention or a part of the antisense strand, for example, the nucleic acid encoding the amino acid sequence of SEQ ID NO: 2 or the base sequence of the antisense strand (for example, the base sequence of SEQ ID NO: 1 and the like) ) And a base sequence of a known database, for example, in a region showing 30% or less, preferably 20% or less, more preferably 10% or less, and particularly preferably 0% sequence identity. And a nucleic acid comprising a base sequence that is at least 12 nucleotides in length, preferably 18 nucleotides or more in length and shorter than the base sequence shown in SEQ ID NO: 1.

  The primer pair includes an oligonucleotide consisting of at least 9 nucleotides, preferably 9 to 40 nucleotides, more preferably 12 to 30 nucleotides in the base sequence of the nucleic acid of the present invention, and the nucleic acid of the present invention. Of the nucleotide sequence corresponding to the oligonucleotide, the nucleotide sequence corresponding to the nucleotide sequence comprising at least 100 nucleotide residues away from the 3 ′ terminal nucleotide residue of the nucleotide sequence, and at least 9 consecutive nucleotides, preferably Examples thereof include a primer pair composed of a continuous 9 to 40 nucleotides, and more preferably a continuous 12 to 30 nucleotide oligonucleotide. In addition, the primer pair takes into account the region containing the amino acid sequence unique to the metalloendopeptidase of the present invention and the nucleic acid of the present invention using conventional primer design software, etc. in consideration of secondary structure formation, Tm value, etc. It can be designed as a primer pair suitable for amplifying a region containing a unique base sequence.

  Furthermore, according to the nucleic acid of the present invention, an isozyme having a physiological activity equivalent to that of the metalloendopeptidase can be screened from organisms other than S. septus TH-2. The isozyme screening uses the nucleic acid of the present invention, the probe or the primer pair, and uses the nucleic acid of the present invention in a nucleic acid derived from an organism other than S. septus TH-2, the region corresponding to the probe, and the primer pair. The region corresponding to the PCR amplification product obtained in this manner can be detected, and then the nucleic acid encoding the polypeptide capable of detecting the metalloendopeptidase activity can be screened by the method for measuring the metalloendopeptidase activity.

  In particular, when the nucleic acid of the present invention and the probe are used, as a test sample, for example, a sample such as a biological cell or a microbial colony, a nucleic acid extracted from the sample fixed on a membrane, or extracted from the sample Nucleic acids and the like can be used. When the nucleic acid of the present invention or the probe is used, a nucleic acid encoding an isozyme can be screened by performing hybridization under the above stringent conditions. On the other hand, when using a primer, a sample such as a cell or an extract derived from the cell can be used as a test sample. In addition, you may perform operation which removes the other component in the said sample in the range which does not impair the nucleic acid used as the template in the case of PCR. For such screening, various reagents for hybridization typified by a membrane for immobilizing nucleic acids, hybridization buffers, etc., PCR reagents typified by heat-resistant DNA polymerase, dNTP mixed solution, PCR buffer, etc. A reagent for detecting a probe or amplified DNA, a culture medium for microbial growth, a reagent for extracting nucleic acid from a sample, or the like can be used as appropriate.

  The invention in another aspect relates to a recombinant metalloendopeptidase encoded by a nucleic acid of the invention.

  According to the recombinant metalloendopeptidase of the present invention, a reaction based on proteolytic activity in the presence of an organic solvent can be performed. Analysis such as protein analysis, production of various peptides such as functional peptides, Therefore, it is possible to produce an intermediate compound, a lead compound, and the like.

  The recombinant metalloendopeptidase of the present invention can be obtained by the following production method.

  In another aspect, the present invention relates to a metalloendopeptidase expression carrier containing the nucleic acid of the present invention. In the present specification, the “expression carrier” means a nucleic acid construct containing a conventional biological vector or the like as a basic skeleton and operably linked to the nucleic acid of the present invention at an appropriate position; gold particle, liposome, dextran It means a construct in which a nucleic acid of the present invention having an element suitable for expression in a cell is supported on a carrier such as calcium phosphate.

  According to the carrier for expression of metalloendopeptidase of the present invention, the metalloendopeptidase is S. aureus. It can be introduced into cells other than septus TH-2, whereby the metalloendopeptidase of the present invention can be expressed in heterologous cells. Therefore, a means for industrial production of the metalloendopeptidase of the present invention can be provided.

  Examples of the biological vector include pUC118, pUC119, pBR322, pCR3, pCMVSPORT, pETkmS2 [Mishima N. et al., Biotechnol. Prog. Vol. 13, 864-868 (1997)], pOMal (New England Biolab) and other plasmid vectors for E. coli; λZAPII, λgt11 and other E. coli phage vectors; Yeast vectors; insect cell vectors such as pAcSGHisNT-A; animal cell vectors such as pKCR, pEFBOS, cDM8, and pCEV4. Such a vector may appropriately include elements such as a suitable promoter (eg, lac promoter, tac promoter, trc promoter, trp promoter, CMV promoter, SV40 early promoter, etc.), a marker gene for selection, and a terminator.

  The vector may be a vector capable of expressing the metalloendopeptidase of the present invention in a fused state with a conventional tag (eg, His tag) or a fusion partner (eg, glutathione S-transferase). .

  In still another aspect, the present invention relates to a transformed cell retaining the nucleic acid of the present invention. The transformed cell of the present invention can be obtained by introducing the expression carrier for the metalloendopeptidase of the present invention into a suitable host.

  According to the transformed cell of the present invention, the metalloendopeptidase of the present invention can be expressed in a state that can be easily purified by genetic engineering, whereby the metalloendopeptidase can be produced industrially. it can

  Examples of the host include Escherichia coli, yeast (for example, methanol-utilizing yeast Pichia, etc.), Bacillus subtilis and the like.

  Examples of the method for introducing the expression carrier of the present invention into the host include a calcium phosphate method, a DEAE-dextran method, an electroporation method, and a bombardment method using a particle gun.

  A cell obtained by introducing the expression carrier of the present invention into a host is a target transformed cell. The above-mentioned metalloendopeptidase activity measurement method is used for the cell or the culture supernatant of the cell. This can be confirmed using the detection of endopeptidase activity as an indicator.

  In still another aspect, the present invention relates to a method for producing a recombinant metalloendopeptidase, comprising culturing the transformed cell of the present invention and recovering metalloendopeptidase from the obtained culture.

  According to the method for producing a recombinant metalloendopeptidase of the present invention, since the transformed cell of the present invention is used, the metallopeptidase can be easily purified and can be produced efficiently. Play.

  In the method for producing a recombinant metalloendopeptidase of the present invention, the optimum conditions for the expression of metalloendopeptidase are determined in terms of medium composition, medium pH, culture temperature, culture time, the amount of inducer used, the time of use, etc. By determining, recombinant metalloendopeptidase can be produced efficiently.

  Conventional methods are used to purify polypeptides having metalloendopeptidase activity from transformed cell cultures. If the transformed cells accumulate a polypeptide having metalloendopeptidase activity in the cells like Escherichia coli, the transformed cells are collected by centrifugation after culturing, and the obtained cells are disrupted by sonication or the like. After that, a cell-free extract is obtained by centrifugation or the like. Using this as a starting material, in addition to the salting-out method, it can be purified by general protein purification methods such as various types of chromatography such as ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography and affinity chromatography. Depending on the transformant used, the expression product may be secreted extracellularly. In such a case, purification may be similarly performed from the culture supernatant.

  Recombinant metalloendopeptidase produced by a transformed cell coexists with other proteins, other polypeptides, etc. in the cell when it is produced in the cell, but this is the amount of metalloendopeptidase expressed. Since it is only a very small amount compared to the above, there is an excellent advantage that its purification is extremely easy. Further, when a cell exocrine secretion type vector is used as a vector, metalloendopeptidase is secreted outside the cell.

Using the metalloendopeptidase of the present invention or the recombinant metalloendopeptidase of the present invention, for example, Current Protocols in Immunology [John E. et al. The metalloend of the present invention can be obtained by immunizing an appropriate animal according to the method described in, for example, edited by John E. Coligan, 1992, published by John Weilly & Sons, Inc. A monoclonal antibody or an antibody fragment thereof against peptidase or the recombinant metalloendopeptidase of the present invention can be obtained. The antibody is not particularly limited as long as it has an ability to specifically bind to the metalloendopeptidase of the present invention or the recombinant metalloendopeptidase of the present invention, and examples thereof include polyclonal antibodies and monoclonal antibodies. According to such an antibody or an antibody fragment thereof, affinity purification, detection, function inhibition, etc. of the metalloendopeptidase of the present invention or the recombinant metalloendopeptidase of the present invention are possible. Examples of the antibody fragment include an Fab fragment obtained by digesting a monoclonal antibody with papain, for example, and an F (ab ′) ₂ fragment obtained by digesting a monoclonal antibody with pepsin, for example.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these Examples.

Streptomyces septatus (S. septatus) A proteolytic activity was found in the culture supernatant of TH-2. For proteolytic activity found in such media supernatants, see Septatus TH-2 in a 500 mL baffle flask in a 50 mL medium containing a carbon source, a nitrogen source, 0.05 wt% MgSO ₄ .7H ₂ O, 0.5 wt% polypeptone, and 0.5 wt% yeast extract The cells were cultured at 30 ° C. for 5 days while being rotated and shaken at 125 rpm, and the effects of the carbon source and inorganic phosphoric acid were examined.

The proteolytic activity was determined as follows. In a 1.5 mL sample tube, 50 μL of a substrate solution [universal protease substrate (lysorphine-labeled casein, manufactured by Roche Molecular Biochemicals), 4 mg / mL water] and incubation buffer (0 containing 8 mM CaCl ₂₎ .2M Acetate Buffer (pH 6.1)] 50 μL and 100 μL of the sample solution to be measured were mixed, and the resulting reaction mixture was incubated at 37 ° C. for 15 minutes. Thereafter, 480 μL of 5 w / v% trichloroacetic acid was added to the mixture to stop the reaction. The mixture was then incubated at 37 ° C. for 10 minutes and then centrifuged at 20000 × g for 5 minutes. 400 μL of the obtained supernatant and 600 μL of 0.5 M Tris-HCl buffer (pH 8.8) were pipetted and placed in a 1.5 mL cuvette and mixed. Thereafter, the absorption at 574 nm was immediately measured against a blank in which distilled water was added to the reaction mixture instead of the sample. One unit of proteolytic activity was defined as the amount of liberated 1 μmol of lysorphine per hour under assay conditions. In addition, the molecular attenuation coefficient (ε mM = 6.6) of 524 nm lysorufin was used to calculate proteolytic activity. The amount of protein was measured using a protein assay kit (manufactured by Bio-Rad) according to the manufacturer's instructions.

  As a result, among the carbon sources, S. It was found that when septatus TH-2 was cultured, the proteolytic activity found in the culture supernatant was higher than when cultured in a medium containing other carbon sources such as fructose.

Then S. The effects of glucose and K ₂ HPO _{4 on} the proteolytic activity in the culture supernatant of septus TH-2 were examined. The result is shown in FIG.

As shown in FIG. The proteolytic activity found in the culture supernatant of septus TH-2 was increased. In addition, when a medium containing 2.0 wt% glucose and 0.8 wt% K ₂ HPO ₄ was used, the specific activity and SSMP content of the medium supernatant were the highest. Therefore, in the following examples, S.M. Septatus TH-2 was cultured using a medium [composition: 2.0 wt% (110 mM) glucose, 0.8 wt% (45 mM) K ₂ HPO ₄ , 0.05 wt% MgSO ₄ .7H ₂ O, 0. 5 wt% polypeptone, 0.5 wt% yeast extract, balance water].

  Further, the lysorphine casein degrading activity was examined for the culture supernatant. As a result, only one enzyme showing proteolytic activity was found in the culture supernatant. Therefore, S.M. It was suggested that an enzyme exhibiting proteolytic activity can be produced industrially by septus TH-2.

S. septus TH-2 was added to a medium [composition: 2.0 wt% (110 mM) glucose, 0.8 wt% (45 mM) K ₂ HPO ₄ , 0.05 wt% MgSO ₄ .7H ₂ O in a 500 mL baffle flask. 0.5 wt% polypeptone, 0.5 wt% yeast extract, remaining water] In 50 mL, the cells were cultured at 30 ° C for 5 days while rotating and shaking at 125 rpm to obtain a culture.

  In addition, about the following operation, all the experiments were performed at 4 degreeC unless there was particular mention. The proteolytic activity was measured in the same manner as in the above example.

  20 mL of the culture was subjected to an ultrafiltration device (Millipore, 0.45 μm pore size) to obtain a culture filtrate. The obtained filtrate was dialyzed against 25 mM Tris-HCl buffer (pH 8.0).

  The product obtained after dialysis was centrifuged at 7400 × g for 10 minutes. The obtained sample was subjected to a trade name: Vivapure-Q spin column (manufactured by Vivascience) equilibrated with the 25 mM Tris-HCl buffer (pH 8.0), and then the protein bound to the column was treated with 0. Elution was performed with 25 mM Tris-HCl (pH 8.0) buffer containing 5 M NaCl. The obtained eluate was dialyzed against 10 mM potassium phosphate buffer (pH 7.0).

  The product obtained after dialysis was centrifuged at 7400 × g for 10 minutes. The obtained supernatant was pre-equilibrated with 10 mM potassium phosphate buffer (pH 7.0). Product name: Bioscale CHT2-I Hydroxyapatite column [manufactured by Bio-Rad; 0.7 cm (inner diameter) ) X 5.2 cm, flow rate: 1 ml / min], and elution with a linear gradient with 10 mM to 400 mM potassium phosphate (pH 7.0) gives high proteolytic activity as an elution fraction of 200 mM potassium phosphate. The fraction having was obtained. The obtained fraction was dialyzed against 20 mM Tris-HCl (pH 8.0) buffer.

  The product obtained after dialysis was centrifuged at 7400 × g for 10 minutes. The obtained supernatant was pre-equilibrated with 20 mM Tris-HCl (pH 8.0) buffer. Product name: Mono Q HR5 / 5 column (Amersham Biotech; 0.5 cm (inner diameter) × 5 cm, flow rate: 1 ml / min] and eluted with a linear gradient of 0 to 0.5 M NaCl to obtain a fraction having high proteolytic activity as a 115 mM elution fraction.

  S. A purification table of enzymes having proteolytic activity from the culture supernatant of septus TH-2 is shown in Table 1.

  Moreover, about the fraction obtained after using for a brand name: Mono Q HR5 / 5 column, the gel [Bio-Rad (Bio-) (12.5 weight% sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (PAGE)). Rad)]. The result is shown in FIG.

  As shown in Table 1 and FIG. 2, the enzyme having proteolytic activity was uniformly applied with a purity of 2.2 times and a yield of 16.6% from 20 mL of culture filtrate (medium supernatant) in a three-step procedure. Could be purified. Further, from the results of SDS-PAGE, it was found that the enzyme having proteolytic activity has a molecular weight of about 35 kD.

  Further, the fraction obtained after being subjected to the trade name: Mono Q HR5 / 5 column was equilibrated with phosphate buffered saline, trade name: Superdex 200 10/300 GL column [Amersham Biotech (Amersham Biotech). In addition, the protein molecular marker for gel filtration (made by Oriental Yeast Co., Ltd.) was used as a molecular weight marker.

  As a result, it was found that the enzyme having proteolytic activity is a monomer having a molecular weight of about 40 kD.

(1) Effect of inhibitor on enzyme having proteolytic activity The enzyme obtained in Example 2 was adjusted to a protein concentration of 24 μg / mL to obtain an enzyme solution. 20 μL of the obtained enzyme solution, 5 μL of inhibitor solution [protease inhibitor set [Roche Molecular Biochemicals]], and 12.5 μL of 0.2 M acetic acid buffer (pH 6.1) containing 8 mM CaCl ₂ Mix and incubate at 37 ° C. for 10 minutes. Next, 12.5 μL of universal substrate solution (4 mg / mL in H ₂ O) was added to the resulting mixture, and the resulting mixture was incubated at 37 ° C. for 10 minutes. Thereafter, the obtained product was centrifuged at 3000 × g for 5 minutes to obtain a supernatant. 100 μL of the obtained supernatant and 150 μL of 0.5 M Tris-HCl buffer (pH 8.8) were added to the wells of a 96-well microplate and mixed.

  Subsequently, the remaining activity of the resulting mixture was determined by measuring the absorption of the solution in the plate at 570 nm with a microplate reader. The results are shown in Table 2.

  As shown in Table 2, the enzyme obtained in Example 2 was particularly inhibited by EDTA and phosphoramidon. This indicates that such an enzyme belongs to the metalloendopeptidase family. Hereinafter, such S.P. A metalloendopeptidase derived from septatus is also referred to as SSMP.

(2) Analysis of the amount of zinc in SSMP The zinc present in SSMP was analyzed by atomic absorption spectrometry. As a result, 0.9 zinc molecule was measured per enzyme molecule.

(3) Effect of pH on proteolytic activity of SSMP In the measurement of the proteolytic activity in Example 1, as buffer solutions used for the incubation buffer, pH 5.3, pH 5.5, pH 5.8, pH 6.1 and pH 6.5 acetate buffer, pH 6.0, pH 6.3, pH 6.8, pH 7.1 and pH 7.4, respectively, potassium phosphate buffer and pH 7.0, pH 7.2, pH 7.6, pH 7. The proteolytic activity of SSMP was measured using Tris-HCl buffer of 9 and pH 8.2. The results are shown in FIG. In FIG. 3, the relative activity is indicated by a value calculated with the activity at the pH showing the highest activity as 100.

  As a result, as shown in FIG. 3, the activity was optimized around pH 6.0 and was maximum under mildly acidic conditions.

(4) Effect of calcium ion on proteolytic activity of SSMP The effect of calcium ion on SSMP activity and stability was examined.

  20 mL of SSMP solution (24 μg / mL) was preincubated for 5 minutes at 37 ° C. in the presence of 0 to 10 mM calcium. Subsequently, using the obtained mixture, the activity was measured in the same manner as the measurement of the proteolytic activity in Example 1. The results are shown in panel (A) of FIG. The relative activity is a value calculated with the activity in the absence of calcium as 100.

  As shown in FIG. 4 panel (A), SSMP activity was inhibited when the final concentration of calcium exceeded 2 mM.

  Further, 20 mL of SSMP solution (24 μg / mL) was preincubated for 5 minutes at 37 ° C. in the presence of a final concentration of 2 mM calcium. Subsequently, using the obtained mixture, the activity was measured in the same manner as the measurement of the proteolytic activity in Example 1. The results are shown in panel (B) of FIG. The relative activity is a value calculated with the activity at the optimum temperature as 100.

  Furthermore, the concentration of the SSMP solution was adjusted to a concentration of 0.1 mg protein / mL. The SSMP solution was incubated at 15 to 80 ° C. for 10 minutes in the presence or absence of a final concentration of 2 mM calcium using a thermal cycler (trade name: TaKaRa PCR thermal cycler MP, manufactured by Takara Bio Inc.). After incubation, residual activity was determined from proteolytic activity. The results are shown in panel (C) of FIG. The residual activity is indicated by a value calculated with the activity in the case of incubation at 15 ° C. being 100.

As a result, as shown in panel (B) and panel (C) in FIG. 4, the presence of calcium causes the optimum temperature and thermal stability to be lower than the optimum temperature and thermal stability in the absence of calcium. Shifted by about 10 ° C. Under the same conditions, no increase in activity was observed using ₂ mM MgCl ₂ , 2 mM CoCl ₂ , 2 mM NiSO ₄ , 2 mM FeSO ₄ and 2 mM ZnSO ₄ . In particular, the addition of 2 mM NiSO ₄ , 2 mM FeSO ₄ and 2 mM ZnSO ₄ reduced the activity to less than 20% of maximum activity.

(1) Analysis of substrate specificity by fluorescence energy transfer (FRETS) combinatorial library The FRETS-25Xaa library (manufactured by Peptide Institute) has the structure shown in FIG. 5 and has an amino terminal D-2,3-diamino. 2,4-dinitrophenyl (Dnp) containing a highly fluorescent 2- (N-methylamino) benzoyl (Nma) group attached to the side chain of a propionic acid (D-A2pr) residue and linked to the ε-amino group of Lys ) A substrate library having the property of quenching efficiently by a group.

The FRETS-25Xaa library shown in FIG. 5 (all of the 475 peptide substrate), 50 mM acetate buffer (pH 6.1) containing ₂ mM CaCl ₂ and 50 ng of the purified SSMP obtained in Example 2 were mixed. After incubation at 37 ° C., the increase in fluorescence intensity at λex 355 nm and λem 460 nm was monitored using a trade name: ARVO 1420 multi-label counter (manufactured by Perkin Elmer). In addition, a calibration curve was prepared using two kinds of degradation products of FRETS-25Gly whose Xaa is Gly, and based on the calibration curve, the substrate specificity of SSMP for FRETS-25Xaa was evaluated. The results are shown in FIG.

  As a result, as shown in FIG. 6, with respect to the amino acid residue at the P1 position (Xaa in FIG. 5) in the substrate, SSMP is in descending order at the P1 position, Phe residue, Leu residue, Tyr residue. There was selectivity for the Met, Thr and Ala residues.

Next, as the best substrate for the secondary screening, Xaa mixed PRET residue FRETS-25Phe, 50 mM acetate buffer (pH 6.1) containing ₂ mM CaCl ₂ , and 200 ng of purified SSMP 200 ng. Incubate at 37 ° C. for 5 minutes, and then add EDTA to the reaction mixture to stop the reaction and measure the substrate cleavage rate with a trade name: ARVO 1420 multilabel counter (Perkin Elmer). It was measured. This examined the selectivity by SSMP for the P2 position (Yaa in FIG. 5) and the P3 position (Zaa in FIG. 5) in the substrate. In addition, a calibration curve was prepared using two kinds of degradation products of FRETS-25Gly whose Xaa is Gly, and based on the calibration curve, the substrate specificity of SSMP for FRETS-25Xaa was evaluated.

  As a result, the obtained product was digested to about 15% compared to the sample before the reaction. Moreover, the obtained product was used for liquid chromatography (LC) -mass spectrometry (MS), and the sequence derived from FRETS-25Phe digested by SSMP was identified.

  As shown in FIG. 7, SSMP exhibited selectivity for Tyr, Ala, Arg, Val, Phe and Lys at the P2 position and selectivity for Arg, Val and Ala at the P3 position. That is, SSMP is selective for hydrophobic or basic residues at the P2 position and basic or small residues at the P3 position, and SSMP is not selective for acidic residues at the P2 and P3 positions. .

(2) Kinetic analysis Based on the result of (1) above, a consensus substrate:
D-A2pr (Nma) -Gly-Arg-Tyr-Phe-Ala-Phe-Pro-Lys (Dnp) -D-Arg-D-Arg
(FRETS-GRYFA; SEQ ID NO: 4) was designed and synthesized. The consensus substrate was diluted with 1.6 mM 50 mM acetate buffer (pH 6.1) containing ₂ mM CaCl ₂ to obtain 490 μL of each substrate solution. 490 μL of the resulting diluted solution was preincubated at 37 ° C. for 5 minutes. 10 μL of purified SSMP (2.5 ng / μL) was added to the substrate solution, and the resulting mixture was then incubated at 37 ° C. for 1 minute. In addition, about the said mixture installed in the thermal cell holder, (lambda) ex 340nm and (lambda) em 440nm were measured using Hitachi spectrofluorometer F-4500. The reaction rate was estimated from a standard curve plotted using Nma solution.

  As a result, Km, kcat and kcat / Km for the consensus substrate are 5.0 ± 1.6 μM, 5.0 ± 1.0 (1 / second) and 1.0 ± 0.30 (1 / second), respectively. 1 / μM).

(1) Determination of N-terminal amino acid sequence and internal amino acid sequence of SSMP The purified SSMP obtained in Example 2 was subjected to 12.5 wt% SDS-PAGE. Subsequently, the protein in the obtained gel was subjected to the trade name: Sequlot Blot PVDF [BioRad (BioRad) using the trade name: Transblot SD cell (manufactured by BioRad) according to the manufacturer's instructions. Electroblotting). The blotted SSMP was subjected to a protein sequencer to identify the N-terminal amino acid sequence. SSMP was treated with trypsin to obtain a trypsin-treated peptide fragment. The obtained trypsin-treated peptide fragment was analyzed by LC-MS to identify the internal amino acid sequence. In the analysis of amino acid sequences, Ile and Leu could not be distinguished from each other because of the same molecular weight. Gln and Lys could not be distinguished from each other because their molecular weights were approximately equal.

  As a result, the N-terminal amino acid sequence of SSMP was GTGTSTYSGNVPLTT (SEQ ID NO: 5). A sense primer was prepared using the underlined sequence. The internal amino acid sequence of SSMP is TGSGF (Q / K) (I / L) EDGAR (SEQ ID NO: 6), TYNSPTYDNSK (SEQ ID NO: 7), TTYFTSTTNYK (SEQ ID NO: 8), and AATWTA (I / L) NVK. (SEQ ID NO: 9). An antisense primer was prepared using the underlined sequence.

(2) Cloning of the ssmp gene by inverse PCR Genomic DNA was prepared according to the method of D. A Hopwood et al. [A Laboratory Manual, The John Ines Foundation, Norwich, 1985, pp. 70-84]. S. Prepared from septus TH-2.

Two degenerate primers designed from SSMP N-terminal amino acid sequence and internal amino acid sequence (4 μM each):
5′-GG (GC) AACGT (GC) CC (GC) CT (GC) AC (GC) AC-3 ′ (SEQ ID NO: 10), and
5'-TCGTA (GC) GT (GC) GG (GC) (GC) (AT) GTTGTA-3 '(SEQ ID NO: 11)
And GC-RICH PCR system (Roche Molecular Biochemicals) and 0.5M GC-RICH resolution solution (trade name). PCR was performed using the genomic DNA of septus TH-2 as a template, and a DNA fragment consisting of a part of the ssmp gene was amplified. The PCR thermal profile was incubated at 95 ° C for 3 minutes, 10 cycles of reactions consisting of 95 ° C for 30 seconds, 54 ° C for 30 seconds and 72 ° C for 45 seconds, 95 ° C for 30 seconds and 54 ° C. A reaction consisting of 30 seconds and 45 ° C. at 72 ° C. (however, 5 seconds extended for each cycle) was set as a condition for 20 cycles of extension at 72 ° C. for 7 minutes.

  The PCR product was cloned into a trade name: pGEM-T Easy (manufactured by Promega) and sequenced.

Then the internal DNA sequence primer of the cloned PCR product:
5′-ACCAACTCCACCGACACCTGG-3 ′ (SEQ ID NO: 12, corresponding to nucleotide number 2083-2103 of SEQ ID NO: 1), and
5′-CTTGTCGGTGCCGTTGTTGGC-3 ′ (corresponding to the complementary strand of SEQ ID NO: 13, nucleotide number of SEQ ID NO: 1: nucleotide number: 2455-2475)
And product name: PCR DIG probe synthesis kit (Roche Molecular Biochemicals), after incubation at 95 ° C. for 2 minutes, 95 ° C. for 10 seconds, 60 ° C. for 30 seconds and 72 ° C. for 2 minutes. 10 cycles of reaction are performed, and then 20 cycles of reaction are performed with 95 ° C. for 10 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 2 minutes (increased by 20 seconds for each cycle). A digoxigenin (DIG) -labeled probe was synthesized by incubating at 7 ° C. for 7 minutes.

  The S. The genomic DNA of septus TH-2 was digested with various restriction enzymes and electrophoresed on a 1.0 wt% agarose gel. The obtained gel was blotted to a trade name: HybondN (manufactured by Amersham Bioscience). The obtained membrane, the DIG-labeled probe, and the trade name: Easy Hyb (Roche) were hybridized overnight at 42 ° C. The membrane was then washed twice for 5 minutes at 25 ° C. with a solution containing 2 × SSC and 0.1 wt% SDS, and a solution containing 0.1 × SSC and 0.1 wt% DSD was used. And washed twice at 68 ° C. for 15 minutes. Then, the signal was detected using brand name: Anti-Digoxgenin AP Fab fragment and NBT / BCIP Stock solution (Roche).

  As a result, an approximately 1.6 kb Van91I digested fragment, an approximately 1.1 kb SalI digested fragment, and a 3.3 kb ApaI digested fragment hybridized with the probe.

These fragments were then recovered by agarose electrophoresis and self-ligated. A set of primers (1 μM each) designed from a partial DNA fragment of the ssmp gene using the ligation product as a template:
For amplification of the Van91I digested fragment,
5′-TACCTGCTGTCCGAGGGCAGC-3 ′ (SEQ ID NO: 14, corresponding to nucleotide number: 2638-2658 of SEQ ID NO: 1), and
5′-CGCCCCGTCTTCGAGCTGGAAGCC-3 ′ (SEQ ID NO: 15, corresponding to the complementary strand of nucleotide number: 1999-2022 of SEQ ID NO: 1);
For amplification of the SalI digested fragment,
5′-TACCTGCTGTCCGAGGGCAGC-3 ′ (SEQ ID NO: 16, corresponding to nucleotide numbers: 2638-2658 of SEQ ID NO: 1), and
5′-GTAGAAGTCCCAGGTGACGGC-3 ′ (SEQ ID NO: 17, corresponding to the complementary strand of nucleotide numbers: 2149-2169 of SEQ ID NO: 1);
For amplification of ApaI digested fragments,
5′-TGCGCTGTCGGAGGCGGTGG-3 ′ (SEQ ID NO: 18, corresponding to nucleotide numbers 2989-3008 of SEQ ID NO: 1), and
5'-CGCCGAGCCGCTCTGCACTCC-3 '(SEQ ID NO: 19, corresponding to the complementary strand of nucleotide number: 1369-1389 of SEQ ID NO: 1)
And 0.5M product name: GC-RICH resolution solution and GC-RICH PCR system (manufactured by Roche Molecular Biochemicals).

  The PCR thermal profile was as described above except that the annealing temperature was 58 ° C. and the extension time was 45 seconds for the Van91I digested fragment, 25 seconds for the SalI digested fragment, and 60 seconds for the ApaI digested fragment. It is the same. The obtained PCR product was cloned into a trade name: pGEM-T Easy (manufactured by Promega) and sequenced. The results are shown in FIG.

  As shown in FIG. 8, the obtained base sequence (SEQ ID NO: 1, DDBJ database accession number: AB18036) includes the base sequence of all ssmp genes.

  From the results of the nucleotide sequence shown in SEQ ID NO: 1, the N-terminal amino acid sequence analysis and the internal amino acid sequence analysis, it can be seen that an in-frame stop codon TGA exists at nucleotide number: 2902-2904 of SEQ ID NO: 1.

  From the deduced amino acid sequence, it appears to have an N-terminal presequence (33 amino acid residues) and an N-terminal prosequence (184 amino acid residues). In the base sequence shown in SEQ ID NO: 1, a putative ribosome binding site (“SD” in FIG. 8) was found upstream of the base sequence corresponding to the N-terminal pre-sequence. There was no ATG initiation codon in the region of the base sequence corresponding to the N-presequence from the putative ribosome binding site, but it is presumed that the TTG codon found at nucleotides 1288-1290 acts similarly as the initiation codon. It was.

  The open reading frame of the ssmp gene was 1617 nucleotides long and was estimated to encode a 538 amino acid SSMP with a predicted molecular weight of 56232. The calculated molecular weight of the mature SSMP enzyme was 34062, which coincided with the molecular weight (about 35 kD) calculated based on the SDS-PAGE analysis shown in FIG.

Culture supernatant 50 μL, 50 mM acetate buffer (pH 6.0), 2 mM CaCl ₂ 2950 μL, deionized water (control), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), ethanol (EtOH), ethylene glycol ( EG) or 1000 μL of methanol (MeOH). The resulting mixture was incubated at 30 ° C. with rotary shaking at 160 rpm / min. As a control, the mixture using the demineralized water was incubated at 4 ° C. In addition, 10 μL from the mixture at 0, 20.1, 68.1, 115.9, 187.4, 287.0, and 355.0 hours after the start of incubation. The solution was sampled.

10 μL of the above solution was added to 490 μL of 50 mM acetate buffer (pH 6.0) containing 50 μM FRETS-GRYFA and 2 mM CaCl ₂ , incubated at 37 ° C., and the reaction rate of 5-10 seconds was λEx / Em = 340 / Measured by 440 nm (excitation at 340 nm, emission at 440 nm). The results are shown in FIG.

  As shown in FIG. 9, the proteolytic activity found in the culture supernatant was found stably even in the presence of an organic solvent. Thus, it is suggested that SSMP is resistant to organic solvents.

Comparative Example 1

  Commercially available thermolysin was dialyzed overnight against 20 mM Tris-HCl (pH 8.0), and 10 μg of thermolysin before and after dialysis was subjected to 15 wt% SDS-PAGE. The results are shown in FIG.

  As a result, as shown in FIG. 10, it can be seen that thermolysin is self-digested by dialysis. On the other hand, as shown in FIG. 2, the metalloendopeptidase of the present invention does not detect an autolyzed product even after dialysis, indicating that the lifetime as an enzyme is longer than that of thermolysin.

  According to the present invention, there are provided means for analysis such as protein analysis, production of various peptides, intermediate compounds for drugs, lead compounds and the like.

FIG. shows the effects of glucose and K ₂ HPO ₄ in the protease production by septatus TH-2. Medium A-E include glucose, K ₂ HPO _4, a _{0.05% MgSO 4 · 7H 2 O} , 0.5 wt% polypeptone and 0.5% by weight yeast extract. The glucose content and K ₂ HPO ₄ content are shown in the figure. Black bars indicate the proteolytic activity of the culture supernatant, and bars indicate the specific activity.

FIG. It is a figure which shows the result of SDS-PAGE of purified SSMP derived from septus TH-2. Lane 1 shows low molecular weight protein markers (molecular weight, 94,000, 67,000, 43,000, 30,000, 20,100 and 14,400), and lane 2 shows 2 μg of purified SSMP.

FIG. 3 is a diagram showing the influence of pH on the proteolytic activity of SSMP. The activity is shown as the relative activity of the highest pH [acetate buffer (pH 6.1)]. Black circles indicate acetate buffer, black triangles indicate potassium phosphate buffer, and black squares indicate Tris-HCl buffer.

FIG. 4 is a diagram showing the influence of calcium on the activity and thermal stability of the proteolytic activity of SSMP. Panel (A) shows the effect of calcium concentration on SSMP activity. Activity is shown as relative activity at the highest optimum temperature. Panel (B) shows the effect of calcium at the optimum temperature for SSMP activity. Activity is shown as relative activity at the highest optimum temperature. Panel (C) shows the effect of calcium on the thermal stability of SSMP activity. Shown as relative activity in incubation at 15 ° C. White circles indicate absence of calcium, and black circles indicate presence of 2 mM calcium.

FIG. 5 shows the structure of the FRETS-25Xaa combinatorial library. Xaa is a fixed position incorporating each of the 19 natural amino acids except cysteine. For each determined Xaa, incorporate a mixture of 5 amino acid residues at the Yaa position (P, Y, K, I and D) with a mixture of 5 amino acid residues at the Zaa position (F, A, V, E and R) It is.

FIG. 6 is a diagram showing primary screening at the P1 position using the FRETS-25 Xaa combinatorial library.

FIG. 7 is a diagram showing secondary screening at positions P2 and P3 using FRETS-25Phe. Panel (A) shows the structure of FRETS-25Phe. Panel (B) shows P2 and P3 selectivity determined using FRETS-25Phe. Relative fluorescence intensity was determined from the peak area by LC-MS. The cleavage site is indicated by an arrow.

FIG. 8 shows the base sequence of the ssmp gene. The putative -35 and -10 sequences of the ssmp gene are underlined. The high purine sequence, AGGAGA, 5 nucleotides upstream of the start codon serves as a ribosome binding (SD) sequence. A zinc binding consensus sequence (HEXXH) was enclosed. The signal sequence of the ssmp gene could be divided into an Arg cluster region (Arg residues are shown in bold) and a hydrophobic region (hatched). The putative cleavage site (white triangle) for the signal peptide is shown. During SSMP maturation, secondary cleavage (sites are indicated by black triangles) probably occurs after secretion. In the primary structure deduced from the amino acid sequence, an N-terminal sequence (underlined) and an internal sequence (double line) were found.

FIG. 9 is a diagram showing organic solvent resistance.

FIG. 10 is a diagram showing autolysis of thermolysin. In the figure, panel (A) shows the results of SDS-PAGE of thermolysin before dialysis, and panel (B) shows the results of SDS-PAGE of thermolysin after dialysis overnight with 20 mM Tris-HCl (pH 8.0). It is a result.

  SEQ ID NO: 3 is the amino acid sequence of the consensus sequence for the substrate of SSMP. Xaa at the second position is F, A, V, E or R. Xaa at the third position is P, Y, K, I or D. Xaa at the fourth position is F, L, Y, M, T, A, H, R, S, I, K, Q, N, W, V, G, E, P, or D.

  SEQ ID NO: 4 is the amino acid sequence of the consensus sequence for the substrate of SSMP. Gly at the 1-position has a D-2,3-diaminopropionic acid group having a 2- (N-methylamino) benzoyl group in the side chain. Lys at the 8th position has a 2,4-dinitrophenyl group in the ε-amino group.

  In SEQ ID NO: 6, Xaa at position 6 is Q or K. Xaa at the seventh position is I or L.

  In SEQ ID NO: 9, Xaa at position 7 is I or L.

  SEQ ID NO: 10 is a sequence of a primer for amplifying a partial sequence of SSMP.

  SEQ ID NO: 11 is the sequence of a primer for amplifying a partial sequence of SSMP.

  SEQ ID NO: 12 is the sequence of the SSMP internal sequence primer.

  SEQ ID NO: 13 is the sequence of the SSMP internal sequence primer.

  SEQ ID NO: 14 is the sequence of a primer for amplifying the Van91I fragment.

  SEQ ID NO: 15 is the sequence of a primer for amplifying the Van91I fragment.

  SEQ ID NO: 16 is a sequence of a primer for amplifying SalI fragment.

  SEQ ID NO: 17 is a primer sequence for amplifying SalI fragment.

  SEQ ID NO: 18 is a sequence of a primer for amplifying an ApaI fragment.

  SEQ ID NO: 19 is a sequence of a primer for amplifying an ApaI fragment.

Claims (11)

Culture showing metalloendopeptidase activity obtained as a culture supernatant by culturing Streptomyces septatus TH-2 (FERM P-17329) in the presence of 80-160 mM glucose and 10-50 mM K ₂ HPO ₄ object. Metalloendopeptidase obtained by culturing Streptomyces septatus TH-2 (FERM P-17329) in the presence of 80 to 160 mM glucose and 10 to 50 mM K ₂ HPO ₄ to obtain a culture supernatant Manufacturing method. The metalloendopeptidase is
(A) the amino acid sequence shown in SEQ ID NO: 2,
(B) In SEQ ID NO: 2, an amino acid sequence having substitution, deletion, insertion or addition of one to several amino acid residues, and (C) the sequence shown in SEQ ID NO: 2, using the FASTA algorithm , Amino acid sequence having a sequence identity of 98% or more calculated under the conditions of KTUP 2, expect value 10.0, Cost to open gap 11, and Cost to extend gap 1;
Having an amino acid sequence selected from the group consisting of:

(In the formula, Xaa is phenylalanine residue, leucine residue, tyrosine residue, methionine residue, threonine residue, alanine residue, histidine residue, arginine residue, serine residue, isoleucine residue, lysine residue. , Glutamine residue, asparagine residue, tryptophan residue, valine residue, glycine residue, glutamic acid residue, proline residue or aspartic acid residue, Yaa is a proline residue, tyrosine residue, lysine residue An isoleucine residue or an aspartic acid residue, and Zaa represents a phenylalanine residue, an alanine residue, a valine residue, a glutamic acid residue, or an arginine residue)
Is a polypeptide that recognizes the sequence shown in (SEQ ID NO: 3) and acts on a peptide bond between the amino acid residue of amino acid number 4 of SEQ ID NO: 3 and the amino acid residue of amino acid number 5 The manufacturing method according to claim 2. The metalloendopeptidase has an amino acid sequence in which one to several amino acid residues are conservatively substituted with other amino acids in SEQ ID NO: 2, and the conservative substitution is represented by the following amino acid group (1): To (6):
(1) glycine and alanine,
(2) valine, isoleucine and leucine,
(3) Aspartic acid, glutamic acid, asparagine and glutamine,
(4) serine and threonine,
(5) lysine and arginine,
(6) Performed within an amino acid residue belonging to the amino acid group selected from the group consisting of phenylalanine and tyrosine, the following formula:

(In the formula, Xaa is phenylalanine residue, leucine residue, tyrosine residue, methionine residue, threonine residue, alanine residue, histidine residue, arginine residue, serine residue, isoleucine residue, lysine residue. , Glutamine residue, asparagine residue, tryptophan residue, valine residue, glycine residue, glutamic acid residue, proline residue or aspartic acid residue, Yaa is a proline residue, tyrosine residue, lysine residue An isoleucine residue or an aspartic acid residue, and Zaa represents a phenylalanine residue, an alanine residue, a valine residue, a glutamic acid residue, or an arginine residue)
Is a polypeptide that recognizes the sequence shown in (SEQ ID NO: 3) and acts on a peptide bond between the amino acid residue of amino acid number 4 of SEQ ID NO: 3 and the amino acid residue of amino acid number 5 The manufacturing method according to claim 3. (A) the amino acid sequence shown in SEQ ID NO: 2,
(B) In SEQ ID NO: 2, an amino acid sequence having substitution, deletion, insertion or addition of one to several amino acid residues, and (C) the sequence shown in SEQ ID NO: 2, using the FASTA algorithm , Amino acid sequence having a sequence identity of 98% or more calculated under the conditions of KTUP 2, expect value 10.0, Cost to open gap 11, and Cost to extend gap 1;
Having an amino acid sequence selected from the group consisting of:

(In the formula, Xaa is phenylalanine residue, leucine residue, tyrosine residue, methionine residue, threonine residue, alanine residue, histidine residue, arginine residue, serine residue, isoleucine residue, lysine residue. , Glutamine residue, asparagine residue, tryptophan residue, valine residue, glycine residue, glutamic acid residue, proline residue or aspartic acid residue, Yaa is a proline residue, tyrosine residue, lysine residue An isoleucine residue or an aspartic acid residue, and Zaa represents a phenylalanine residue, an alanine residue, a valine residue, a glutamic acid residue, or an arginine residue)
Metalloendopeptidase that recognizes the sequence shown in SEQ ID NO: 3 and acts on a peptide bond between the amino acid residue of amino acid number 4 of SEQ ID NO: 3 and the amino acid residue of amino acid number 5 (SEQ ID NO: 3) .   The metalloendopeptidase of Claim 5 obtained by the manufacturing method of any one of Claims 2-4. (A) a base sequence encoding the amino acid sequence represented by SEQ ID NO: 2,
(B) a base sequence encoding an amino acid sequence having substitution, deletion, insertion or addition of one to several amino acid residues in SEQ ID NO: 2;
(C) The sequence identity calculated by the FASTA algorithm with respect to the sequence shown in SEQ ID NO: 2 under the conditions of KTUP 2, expect value 10.0, Cost to open gap 11, and Cost to extend gap 1 is 98. A base sequence encoding an amino acid sequence that is at least% , and (d) a base sequence of a nucleic acid that hybridizes under stringent conditions with an antisense strand of a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 2.
A polypeptide encoded by containing a base sequence selected from the group consisting of:

(In the formula, Xaa is phenylalanine residue, leucine residue, tyrosine residue, methionine residue, threonine residue, alanine residue, histidine residue, arginine residue, serine residue, isoleucine residue, lysine residue. , Glutamine residue, asparagine residue, tryptophan residue, valine residue, glycine residue, glutamic acid residue, proline residue or aspartic acid residue, Yaa is a proline residue, tyrosine residue, lysine residue An isoleucine residue or an aspartic acid residue, and Zaa represents a phenylalanine residue, an alanine residue, a valine residue, a glutamic acid residue, or an arginine residue)
And has an activity that acts on a peptide bond between the amino acid residue of amino acid number 4 of SEQ ID NO: 3 and the amino acid residue of amino acid number 5 of SEQ ID NO: 3. A nucleic acid encoding a metalloendopeptidase.   A recombinant metalloendopeptidase encoded by the nucleic acid of claim 7.   A metalloendopeptidase expression carrier comprising the nucleic acid according to claim 7.   A transformed cell comprising the nucleic acid according to claim 7.   A method for producing a recombinant metalloendopeptidase, comprising culturing the transformed cell according to claim 10 and recovering the metalloendopeptidase from the obtained culture.

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