JP2009183255A - Peptide synthetase, gene thereof and application thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for readily, rapidly and inexpensively producing a peptide having a proline residue at the C-terminus without requiring complicated operation. <P>SOLUTION: The method for producing the peptide includes producing the peptide having the proline residue at the C-terminus from an amino acid ester or a peptide ester and the proline by using an enzyme or an enzyme-containing material, having ability for forming the peptide having the proline at the C-terminus from the amino acid ester or the peptide ester and the proline. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  A method of chemically condensing an amino acid protected at the N-terminus and proline protected at the C-terminus (Pro) is disclosed in JP-A-61-134321 (Patent Document 1) and JP-A-62-000098 (Patent Document 1). Document 2).

  In addition, a method in which DNA encoding a repeat of alanylproline (Ala-Pro) or lysylproline (Lys-Pro) is transcribed and translated using a cell system, and then enzymatically hydrolyzed is disclosed in International Publication No. 96 / No. 32468 (Patent Document 3) and US Pat. No. 6,204,008B1 (Patent Document 4).

  A method of extracting from a natural product is described in JP-A-2003-024012 (Patent Document 5).

  Methods for condensing 2,2-dichloropropionyl chloride and L-proline to derive L-alanyl-L-proline are disclosed in US Pat. No. 5,424,454A (Patent Document 6) and US Pat. )It is described in.

JP 61-134321 A JP-A-62-000098 International Publication No. 96/32468 Pamphlet US Pat. No. 6,204,008B1 Japanese Patent Laid-Open No. 2003-024012

  An enzymatic condensation method of amino acid ester or amino acid amide and proline is also known.

However, all of the above-described methods have problems such as the need for introduction / desorption of a protecting group, a multi-step step, or a very low yield.
The present invention solves the above-mentioned problems and provides a means for producing a peptide having a proline residue at the C-terminus in a simple, rapid and inexpensive manner without requiring a complicated operation. Objective.

The present invention provides the following [1] to [13].
[1] From an amino acid ester or peptide ester and proline using an enzyme or an enzyme-containing product having the ability to produce a peptide having a proline residue at the C-terminus from an amino acid ester or peptide ester and proline, A method for producing a peptide, which comprises producing a peptide having C at the C-terminus.
[2] A culture of a microorganism having the ability to produce a peptide having a proline residue at the C-terminus from an amino acid ester or peptide ester and proline, or a microbial cell separated from the culture And the method for producing a peptide according to [1], wherein the peptide is one or more selected from the group consisting of a treated product of the microorganism.
[3] The method for producing a peptide according to [2], wherein the microorganism is a microorganism belonging to the genus Streptomyces or Philobacterium.
[4] The method for producing a peptide according to [2], wherein the microorganism is a transformed microorganism capable of expressing a protein selected from the group consisting of the following (A) to (D): .
(A) a protein having the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing (B) substitution or deletion and / or insertion of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing The amino acid sequence described in SEQ ID NO: 4 in the protein (C) sequence table, which has an amino acid sequence containing and has an activity of generating a peptide having a proline residue at the C-terminus from an amino acid ester or peptide ester and proline. The amino acid sequence shown in SEQ ID NO: 4 in the protein (D) sequence table, having an amino acid sequence containing one or several amino acid substitutions, deletions and / or insertions, and an amino acid ester or peptide ester; A protein having an activity to produce a peptide having a proline residue at the C-terminus from proline [5] The method for producing a peptide according to [2], wherein the microorganism is a transformed microorganism into which a polynucleotide selected from the group consisting of the following (a) to (d) is introduced.
(A) a polynucleotide having a base sequence of base numbers 1197 to 2198 in the base sequence set forth in SEQ ID NO: 1 in the sequence listing (b) a base sequence of base numbers 1197 to 2198 in the base sequence set forth in SEQ ID NO: 1 of the sequence listing A protein that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence and has an activity of generating a peptide having a proline residue at the C-terminus from an amino acid ester or peptide ester and proline (C) a polynucleotide having the nucleotide sequence of nucleotide numbers 410 to 1705 out of the nucleotide sequence set forth in SEQ ID NO: 3 in the sequence listing (d) the nucleotide in the nucleotide sequence set forth in SEQ ID NO: 3 in the sequence listing Polynucleotide and stream consisting of a base sequence complementary to the base sequence of numbers 410 to 1705 A polynucleotide encoding a peptide that hybridizes under a gentle condition and has an amino acid ester or peptide ester and proline, and a proline residue at the C-terminus [6] The enzyme is represented by the following (A) to (D): The method for producing a peptide according to [1], which is at least one selected from the group consisting of:
(A) a protein having the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing (B) substitution or deletion and / or insertion of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing The amino acid sequence described in SEQ ID NO: 4 in the protein (C) sequence table, which has an amino acid sequence containing and has an activity of generating a peptide having a proline residue at the C-terminus from an amino acid ester or peptide ester and proline. The amino acid sequence shown in SEQ ID NO: 4 in the protein (D) sequence table, having an amino acid sequence containing one or several amino acid substitutions, deletions and / or insertions, and an amino acid ester or peptide ester; A protein [7] having the activity of producing a peptide having a proline residue at the C-terminus from proline A protein derived from a microorganism belonging to the genus Leptomyces or Philobacterium, and having an activity of generating a peptide having a proline residue at the C-terminus from an amino acid ester or peptide ester and proline.
[8] A protein selected from the group consisting of the following (A) to (D).
(A) a protein having the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing (B) substitution or deletion and / or insertion of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing The amino acid sequence described in SEQ ID NO: 4 in the protein (C) sequence table, which has an amino acid sequence containing and has an activity of generating a peptide having a proline residue at the C-terminus from an amino acid ester or peptide ester and proline. The amino acid sequence shown in SEQ ID NO: 4 in the protein (D) sequence table, having an amino acid sequence containing one or several amino acid substitutions, deletions and / or insertions, and an amino acid ester or peptide ester; Protein [9] [8] which has an activity to produce a peptide having a proline residue at the C-terminus from proline ] The polynucleotide which codes protein as described in.
[10] A polynucleotide selected from the group consisting of the following (a) to (d).
(A) a polynucleotide having a base sequence of base numbers 1197 to 2198 in the base sequence set forth in SEQ ID NO: 1 in the sequence listing (b) a base sequence of base numbers 1197 to 2198 in the base sequence set forth in SEQ ID NO: 1 of the sequence listing A protein that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence and has an activity of generating a peptide having a proline residue at the C-terminus from an amino acid ester or peptide ester and proline (C) a polynucleotide having the nucleotide sequence of nucleotide numbers 410 to 1705 out of the nucleotide sequence set forth in SEQ ID NO: 3 in the sequence listing (d) the nucleotide in the nucleotide sequence set forth in SEQ ID NO: 3 in the sequence listing Polynucleotide and stream consisting of a base sequence complementary to the base sequence of numbers 410 to 1705 A polynucleotide encoding a protein that has an activity of producing a peptide having a proline residue at the C-terminus from an amino acid ester or peptide ester and proline, and which hybridizes under a gentle condition [11] The polynucleotide according to [10], which is a condition under which washing is performed at a salt concentration corresponding to 1 × SSC and 0.1% SDS at 60 ° C.
[12] A recombinant polynucleotide comprising the polynucleotide according to any one of [9] to [11].
[13] A transformed cell into which the polynucleotide according to [12] has been introduced.

  According to the present invention, a peptide having a proline residue at the C-terminus can be produced simply, rapidly and inexpensively without performing complicated operations such as introduction of a protecting group.

上記反応式中、Ｒ_１はアミノ酸の側鎖、またはペプチドのＣ末端のアミノ酸残基の側鎖を示し、Ｒ_２は置換または無置換の炭化水素基を表す。 1. Method for Producing Peptide The method for producing the peptide of the present invention (hereinafter also referred to as “the peptide production method of the present invention”) comprises an amino acid ester or peptide ester and proline in the presence of an enzyme having a predetermined peptide-forming activity. A peptide having a proline residue at the C-terminus (carboxyl terminus) is generated. That is, the peptide production method of the present invention uses an enzyme or an enzyme-containing product having an ability to produce a peptide having a proline residue at the C-terminus from an amino acid ester or peptide ester and proline, and the amino acid ester or peptide ester. A peptide having a proline residue at the C-terminus is produced from proline. An enzyme or an enzyme-containing substance having the ability to produce a peptide having a proline residue at the C-terminus from an amino acid ester or peptide ester and proline is substantially an amino acid ester or peptide ester represented by the following chemical formula and proline. An enzyme or an enzyme-containing substance having the ability or activity to catalyze the condensation due to the acyl transfer reaction.

In the above reaction formula, R ₁ represents a side chain of an amino acid or a side chain of an amino acid residue at the C-terminal of a peptide, and R ₂ represents a substituted or unsubstituted hydrocarbon group.

  Examples of a method of allowing an enzyme or an enzyme-containing substance to act on an amino acid ester or peptide ester and proline include a method of mixing the enzyme or the enzyme-containing substance, an amino acid ester or peptide ester and proline. More specifically, a method in which an enzyme or an enzyme-containing substance is added to a solution containing an amino acid ester or peptide and proline and allowed to react can be used. In addition, when a microorganism that produces the enzyme is used as the enzyme-containing material, the microorganism that produces the enzyme is cultured in addition to the method in which the microorganism that produces the enzyme is added and reacted in the solution as described above. Alternatively, a method of generating and accumulating the enzyme in a microorganism or a culture solution of the microorganism and adding an amino acid ester or a peptide ester and proline to the culture solution may be used. The produced peptide having a proline residue at the C-terminus can be recovered by a conventional method and purified as necessary.

  The “enzyme-containing material” is a mixture containing the enzyme. The enzyme-containing material can further contain one or more other substances. Specifically, an enzyme-containing mixture in which a step of producing an enzyme, such as culturing of a microorganism, is performed therein, or a mixture obtained by subjecting it to post-treatment as necessary can be used as the enzyme-containing material. The other substance may include microbial cells and microbial cell debris in addition to the substances constituting the medium. The post-treatment includes recovery of the medium from the culture solution after completion of the culture or recovery of the cells; disruption of the cells, lysis and lyophilization; removal of some other substances by rough purification, etc .; Examples thereof include immobilization of an enzyme or an enzyme-containing structure (for example, a cell) by an adsorption method, an entrapment method, and the like, and combinations thereof. In addition, some microorganisms lyse during culture, and in this case, the culture supernatant can also be used as an enzyme-containing material.

  Moreover, as a microorganism containing the enzyme, a wild strain may be used, or a genetically modified strain expressing the enzyme may be used. The microorganisms are not limited to enzyme microorganisms but may be treated with acetone, lyophilized cells, etc., and these may be fixed by a covalent bond method, adsorption method, inclusion method, etc. Immobilized microbial cells and immobilized microbial cell processed products may be used.

  When using a wild strain capable of producing an enzyme having an activity to produce a peptide having a proline residue at the C-terminus, it is preferable in that peptide production can be performed more easily without the need to prepare a genetically modified strain. is there. On the other hand, a mutant strain modified so as to produce a large amount of an active enzyme that produces a peptide having a proline residue at the C-terminus (for example, a genetically modified strain transformed so that the enzyme gene can be expressed in a large amount) ) Can be used to synthesize a peptide having a proline residue at the C-terminus more rapidly. That is, in the method of the present invention, as a microorganism, an active enzyme that produces a peptide having a proline residue at the C-terminus, for example, a microorganism transformed so as to express the protein of the present invention described later, A microorganism transformed so that the enzyme gene, for example, the polynucleotide of the present invention described later can be expressed can also be used. Wild-type or mutant-type microorganisms are cultured in a medium, and the enzyme is accumulated in the medium and / or in the microorganism, mixed with amino acid ester or peptide ester and proline, and has a proline residue at the C-terminus. Peptides can be generated.

  In addition, in the case of using a cultured product, cultured bacterial cells, washed bacterial cells, or a processed bacterial cell product obtained by disrupting or lysing bacterial cells, the produced peptide is not involved in the production of a peptide having a proline residue at the C-terminus. There are often enzymes that degrade. Therefore, it is possible to add a metal protease inhibitor such as ethylenediaminetetraacetic acid (EDTA). The addition amount can be appropriately determined, for example, in the range of 0.1 mM to 300 mM, preferably in the range of 1 mM to 100 mM.

  The amount of the enzyme or the enzyme-containing material used may be an amount (effective amount) that exhibits the intended effect. This effective amount is easily determined by a person skilled in the art by a simple preliminary experiment. For example, when an enzyme is used, about 0.01 to 100 units (U), and when a washing cell is used, 0.1 to 0.1 unit. It is about 500 g / L. In addition, 1U is 1 minute when it is made to react on 25 degreeC temperature conditions using 100 mM boric-acid-potassium chloride buffer solution (pH 9.0) containing 50 mM amino acid ester or peptide ester, and proline. The amount of enzyme used to generate a peptide having 1 μmole of proline residue at the C-terminus.

  In the production method of the present invention, the first reaction raw material is an amino acid ester or a peptide ester.

The amino acid constituting the amino acid ester may be, for example, either an L-amino acid or a D-amino acid, or an amino acid ester corresponding to a natural amino acid or an amino acid corresponding to a non-natural amino acid or a derivative thereof. May be. Furthermore, as an amino acid, amino acids such as β-, γ-, ω-, etc., which differ in the binding position of the amino group, in addition to α-amino acids are exemplified. Preferred are L-alanine, L-valine, L-leucine, L-isoleucine, L-methionine, L-proline, L-serine, L-threonine, L-lysine, L-phenylalanine, L-tryptophan, L- The respective esters of histidine are mentioned.
Examples of the ester include methyl ester, ethyl ester, n-propyl ester, iso-propyl ester, n-butyl ester, iso-butyl ester, tert-butyl ester and the like of these amino acids. Among these, methyl ester, ethyl Esters are preferred.
Preferred amino acid esters in the present invention include L-alanine methyl ester, L-valine methyl ester, L-leucine methyl ester, L-isoleucine methyl ester, L-methionine methyl ester, L-proline methyl ester, L-serine methyl ester. , L-threonine methyl ester, L-lysine methyl ester, L-phenylalanine methyl ester, L-tryptophan methyl ester, and L-histidine methyl ester.

  The peptide ester means a peptide in which two or more amino acids are peptide-bonded and the carboxyl group of the C-terminal amino acid residue is esterified. The amino acid constituting the peptide may be either L-form or D-form, and may be a natural type or non-natural type, and may be an amino acid derivative having a substituent. The number of amino acids constituting the peptide may be two or more. Examples of the ester include peptide methyl ester, ethyl ester, n-propyl ester, iso-propyl ester, n-butyl ester, iso-butyl ester, tert-butyl ester and the like. Among them, methyl ester and ethyl ester are preferable. Preferable peptide ester in the present invention includes alanylalanine methyl ester.

  In the production method of the present invention, the second reaction raw material is proline. Proline may be either L-form or D-form, but L-proline is preferred. In view of the object of the present invention, it is desirable that proline does not have a protecting group.

  The concentration of amino acid ester or peptide ester as a starting material and proline is 1 mM to 2 M, preferably 20 to 600 mM. Further, when the reaction is inhibited when the concentration of the substrate is high, it can be added successively at a concentration that does not inhibit these during the reaction.

  The reaction temperature is 5 to 60 ° C., and peptide formation is possible, preferably 10 to 40 ° C. Moreover, reaction pH is pH 5-12 and a peptide production | generation is possible, Preferably it is pH 6-11.

2. Microorganism used in the present invention As the microorganism used in the present invention, a microorganism having the ability to produce a peptide having a proline residue at the C-terminus from an amino acid ester or peptide ester and proline is used without particular limitation. it can. Examples of microorganisms having the ability to produce a peptide having a proline residue at the C-terminus from an amino acid ester or peptide ester and proline include microorganisms belonging to each of the genus Streptomyces and Philobacterium. Examples include Philobacterium sp. And Streptomyces sp. Specific examples include Philobacterium sp. S1201 (AJ110317) and Streptomyces sp. AJ9587.
Philobacterium sp. S1201 (AJ110317) and Streptomyces sp. AJ9587 are registered in the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (IPOD) (Tsukuba Center, 1-1-1 Tsukuba City Center, Ibaraki, Japan) Deposited on November 6, 2007, the deposit number of S1201 (AJ110317) is FERM P-21431, and the deposit number of AJ9587 is FERM P-21430. Therefore, they can be sold from IPOD.

  As these microorganisms, either wild strains or mutant strains may be used, and recombinant strains induced by genetic techniques such as cell fusion or genetic manipulation may also be used.

  In order to obtain cells of such microorganisms, the microorganisms are preferably grown and cultured in an appropriate medium. The medium for this is not particularly limited as long as the microorganism can grow, and is a normal medium containing a normal carbon source, nitrogen source, phosphorus source, sulfur source, inorganic ions, and if necessary, an organic nutrient source. Good.

  For example, any of the above microorganisms can be used as the carbon source. Specifically, sugars such as glucose, fructose, maltose and amylose, alcohols such as sorbitol, ethanol and glycerol, fumaric acid and citric acid In addition, organic acids such as acetic acid and propionic acid and salts thereof, hydrocarbons such as paraffin or mixtures thereof can be used.

  Nitrogen sources include ammonium salts of inorganic acids such as ammonium sulfate and ammonium chloride, ammonium salts of organic acids such as ammonium fumarate and ammonium citrate, phosphates such as monopotassium phosphate and dipotassium phosphate, magnesium sulfate, etc. Sulfates, nitrates such as sodium nitrate and potassium nitrate, organic nitrogen compounds such as peptone, yeast extract, meat extract and corn steep liquor, or mixtures thereof can be used.

  In addition, nutrient sources used in normal media such as inorganic salts, trace metal salts, vitamins, and the like can be appropriately mixed and used.

  The culture conditions are not particularly limited. For example, the culture may be performed for about 12 to 48 hours while appropriately limiting the pH and temperature in the range of pH 5 to 8 and temperature 15 to 40 ° C. under aerobic conditions. .

3. Enzyme used in the present invention In the present invention, an enzyme having the ability to produce a peptide having a proline residue at the C-terminus from an amino acid ester or peptide ester and proline is used. In the present invention, any enzyme having such an activity can be used without limitation on its origin and acquisition method. Hereinafter, the enzyme protein of the present invention, its purification, utilization of genetic engineering techniques, etc. will be described.

(3-1) Protein of the present invention A protein having an activity of producing a peptide having a proline residue at the C-terminus from the amino acid ester or peptide ester of the present invention and proline is obtained from a proline residue of amino acid ester or peptide ester and proline. Can be obtained from a microorganism having the ability to produce a peptide having at the C-terminus. For example, microorganisms belonging to the genus mentioned as examples of the microorganisms used in the present invention, that is, bacteria belonging to the genus Streptomyces or Philobacterium can be used. More specifically, mention may be made of the aforementioned Philobacterium sp. S1201 (AJ110317) and Streptomyces sp. AJ9587.

  A method for isolating and purifying the protein of the present invention from the aforementioned microorganism will be described.

  First, the bacterial cells are disrupted from the microorganisms by a physical method such as ultrasonic disruption, or an enzymatic method using cell wall lysing enzymes, etc., and the insoluble fraction is removed by centrifugation or the like to remove the bacterial cell extract. Prepare. The bacterial cell extract thus obtained is combined with conventional protein purification methods such as ammonium sulfate fractionation, dialysis, anion exchange chromatography, hydrophobic chromatography, cation exchange chromatography, gel filtration chromatography, etc. Thus, the peptide-forming enzyme can be purified.

  Examples of the carrier for anion exchange chromatography include HiLoad-16 / 10-Q-Sepharose HP, Q-Sepharose FF, Mono-Q5 / 5 (manufactured by Amersham Biosciences (GE Healthcare Bioscience)).

  Examples of the carrier for hydrophobic chromatography include Phenyl Sepharose HP, HiLoad-16 / 10-Phenyl Sepharose HP, Resource ETH, and Resource ISO (all manufactured by Amersham Biosciences (GE Healthcare Bioscience)). The extract containing the enzyme is passed through a column packed with these carriers to adsorb the enzyme to the column, the column is washed, and then the enzyme is eluted using a buffer solution with a high salt concentration. At that time, the salt concentration may be increased stepwise, or a concentration gradient may be applied.

  Examples of the carrier for cation chromatography include MonoS HR (manufactured by Pharmacia (GE Healthcare Science)). The extract containing the enzyme is passed through a column packed with these carriers to adsorb the enzyme to the column, the column is washed, and then the enzyme is eluted using a buffer solution with a high salt concentration. At that time, the salt concentration may be increased stepwise, or a concentration gradient may be applied.

  Examples of the carrier for gel filtration chromatography include HiLoad-16 / 60-Superdex 200 pg and Sephadex 200 pg (manufactured by Amersham Biosciences (GE Healthcare Bioscience)).

  In the above purification operation, the fraction containing the present enzyme can be confirmed by measuring the peptide-forming activity of each fraction by the method shown in Examples described later. A partial amino acid sequence of the present enzyme purified as described above from Philobacterium sp. S1201 (AJ110317) is shown in SEQ ID NO: 5 of the sequence listing. A partial amino acid sequence of the present enzyme purified as described above from Streptomyces sp. AJ9587 is shown in SEQ ID NO: 10 of the sequence listing.

  A protein having the amino acid sequence of SEQ ID NO: 5 or 10 as a part thereof as a protein having an activity of generating a peptide having a proline residue at the C-terminus from an amino acid ester or ester of a peptide and proline of the present invention Can be mentioned. More specifically, a protein selected from the group consisting of the following (A) to (D) can be mentioned.

(A) a protein having the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing (B) substitution or deletion and / or insertion of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing The amino acid sequence described in SEQ ID NO: 4 in the protein (C) sequence table, which has an amino acid sequence containing and has an activity of generating a peptide having a proline residue at the C-terminus from an amino acid ester or peptide ester and proline. The amino acid sequence shown in SEQ ID NO: 4 in the protein (D) sequence table, having an amino acid sequence containing one or several amino acid substitutions, deletions and / or insertions, and an amino acid ester or peptide ester; A protein having an activity to produce a peptide having a proline residue at the C-terminus from proline

  (A) A protein having the amino acid sequence shown in SEQ ID NO: 2 was newly isolated from Philobacterium sp. S1201 (AJ110317) by the present inventors and specified as the amino acid sequence of the protein of the above peptide-forming enzyme It is. The protein (A) exhibits acyl transfer activity between an amino acid ester or peptide ester and an amino acid, peptide, or derivative thereof such as amide. In addition, it exhibits activity as an esterase for amino acid esters and peptide esters. In particular, the protein (A) is excellent in the activity of producing a peptide having a proline residue at the C-terminus from an amino acid ester or peptide ester and proline, and this point is proved in Examples described later.

  (C) The protein having the amino acid sequence described in SEQ ID NO: 4 is newly isolated from Streptomyces sp. AJ9587 by the present inventors and specified as the amino acid sequence of the above-mentioned peptide-forming enzyme protein. The protein (C) exhibits an acyl transfer activity between an amino acid ester or peptide ester and an amino acid or peptide or a derivative such as amide thereof. In addition, it exhibits endopeptidase activity against peptides consisting of various amino acids. In particular, the protein (C) is excellent in the activity of producing a peptide having a proline residue at the C-terminus from an amino acid ester or peptide ester and proline, and this point is proved in the examples described later.

  The protein of the present invention also includes a protein that is substantially the same as the protein having the amino acid sequence described in any one of SEQ ID NO: 2 and SEQ ID NO: 4 in the sequence listing. Specific examples include (B) and (D).

  Here, “several” means a range that does not greatly impair the three-dimensional structure and activity of the amino acid residue protein, although it varies depending on the position and type of the three-dimensional structure of the amino acid residue protein. Is 2-50, preferably 2-30, and more preferably 2-10. Further, the sequence having one or several amino acid mutations is 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, relative to the sequence having no mutation. Even more preferably, it may have a homology of 97% or more. However, in the case of an amino acid sequence containing substitution, deletion, and / or insertion of one or several amino acid residues in the amino acid sequence of the proteins of (B) and (D), the conditions are at 50 ° C. and pH 8 It is desirable that the enzyme activity is maintained at about half or more, more preferably 80% or more, and still more preferably 90% or more of the protein in a state containing no mutation. For example, (B) the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing has an amino acid sequence containing one or several amino acid substitutions, deletions, and / or insertions, and an amino acid ester or peptide ester In the case of a protein having an activity to produce a peptide having a proline residue at the C-terminus from proline, half of the protein having the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing under conditions of 50 ° C. and pH 8 It is desirable that the enzyme activity is maintained at a degree or more, more preferably 80% or more, and still more preferably 90% or more. The homology referred to here is obtained by calculating the number of amino acid residues corresponding to the two sequences to be compared as the numerator, using the total number of amino acid residues as the denominator, and multiplying this by 100. The homology analysis can also be obtained by using Genetyx (Genetics Co., Ltd.) and calculating the parameters as initial setting values.

  In the amino acid mutation as shown in (B) above, the amino acid at a specific site in the amino acid sequence shown in (A) above is substituted, deleted, and / or inserted by, for example, site-specific mutagenesis. It is obtained by designing in advance an amino acid sequence modified in such a manner and expressing a base sequence corresponding to this amino acid sequence.

  Moreover, naturally occurring mutations such as differences between microorganism species or strains are included in the above-described base substitution, deletion, and / or insertion. By expressing a polynucleotide encoding an amino acid having a mutation as described above in an appropriate cell and examining the enzyme activity of the expression product, a protein having the amino acid sequence described in SEQ ID NO: 2 or 4 in the sequence listing Substantially identical proteins are obtained.

  Proteins (B) and (D) have an enzyme activity of about half or more, more preferably 80% or more, and still more preferably 90% or more of each protein having the original amino acid sequence under conditions of 50 ° C. and pH 8. It is desirable to hold For example, in the case of the protein (B), about 50% or more, more preferably 80% or more, more preferably 90% or more of the protein having the amino acid sequence shown in SEQ ID NO: 2 under the conditions of 50 ° C. and pH 8. It is desirable to retain the enzyme activity of

This enzyme is a transferase that has the activity of synthesizing a dipeptide by the transfer reaction between an ester of an amino acid ester and an amino acid. However, even if it is generally called a transferase, there are many that perform both a transfer reaction and an esterolysis reaction. It is called transferase if the ratio of the amino acid binding when the ester of the ester is cut is high, and it is called esterase if the ratio of water entering is high.
Confirmation of retention of transferase activity is confirmed by experimenting whether the target protein is allowed to act on an amino acid ester or peptide ester and proline to produce a peptide having a proline residue at the C-terminus. be able to. However, since it is necessary to individually set analysis conditions according to the type of substrate, etc., in order to confirm the substrate specificity at the N-terminus, the target protein is replaced with an amino acid ester or peptide for convenience. A confirmation test may be performed in which the hydrolysis reaction is observed by acting on the liquid.

(3-2) Production of Polynucleotide, Recombinant Polynucleotide, Transformant of the Present Invention and Purification of Peptide Generating Enzyme (3-2-1) Polynucleotide Encoding Peptide Gene Enzyme Protein A polynucleotide encoding the product enzyme protein is also provided. The polynucleotide of the present invention is a polynucleotide that encodes the amino acid sequence that constitutes the above-described peptide-generating enzyme protein, but there may be a plurality of base sequences that define one amino acid sequence due to the degeneracy of codons. Specifically, a polynucleotide having a base sequence encoding a protein selected from the group consisting of the above (A) to (D) is included.

  Preferable specific examples of the polynucleotide of the present invention include a polynucleotide selected from the group consisting of the following (a) to (d).

(A) a polynucleotide having a base sequence of base numbers 1197 to 2198 in the base sequence set forth in SEQ ID NO: 1 in the sequence listing (b) a base sequence of base numbers 1197 to 2198 in the base sequence set forth in SEQ ID NO: 1 of the sequence listing A protein that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence and has an activity of generating a peptide having a proline residue at the C-terminus from an amino acid ester or peptide ester and proline (C) a polynucleotide having the nucleotide sequence of nucleotide numbers 410 to 1705 out of the nucleotide sequence set forth in SEQ ID NO: 3 in the sequence listing (d) the nucleotide in the nucleotide sequence set forth in SEQ ID NO: 3 in the sequence listing Polynucleotide and stream consisting of a base sequence complementary to the base sequence of numbers 410 to 1705 Hybridizes under stringent conditions, and from the amino acid ester or peptide ester and proline, which encodes a protein having the activity to form a peptide having a proline residue at the C-terminal polynucleotide

The polynucleotide having the base sequence set forth in SEQ ID NO: 1 in the sequence listing is isolated from Philobacterium sp. S1201 (AJ110317). The polynucleotide consisting of the nucleotide sequences of SEQ ID NO: 1 and nucleotide numbers 1197 to 2198 is ORF. It has been demonstrated in the Examples that the protein encoded by this ORF exhibits high peptide-forming activity.
The polynucleotide having the base sequence set forth in SEQ ID NO: 3 in the Sequence Listing is isolated from Streptomyces sp. AJ9587. The polynucleotide comprising the nucleotide sequence of base numbers 410 to 1705 of SEQ ID NO: 3 is the ORF. It has been demonstrated in the Examples that the protein encoded by this ORF exhibits high peptide-forming activity.

  The various gene recombination techniques listed below can be performed according to the description of Molecular Cloning, 2nd edition, Cold Spring Harbor press (1989).

  The polynucleotide of the present invention can be obtained from PCR (polymerase chain reaction, White, TJ et al; Trends Genet., 5, 185 (1989) from cDNAs such as Philobacterium sp. And Streptomyces sp. ))) Or hybridization. The primer used for PCR is determined based on the peptide-forming enzyme purified as described in the section (3) above, and can be designed based on the partial amino acid sequence. Moreover, since the base sequences (SEQ ID NO: 1, SEQ ID NO: 3) of the peptide-generating enzyme gene have been clarified by the present invention, primers and probes for hybridization can be designed based on these base sequences. It can also be isolated using When a primer having a sequence corresponding to a 5 ′ untranslated region and a 3 ′ untranslated region is used as a primer for PCR, the entire coding region of the present enzyme can be amplified. Specifically, taking the base sequence of SEQ ID NO: 1 as an example, a primer having a base sequence in a region upstream of base number 1197 in SEQ ID NO: 1 as a 5 ′ primer is a base as a 3 ′ primer. And a primer having a sequence complementary to the base sequence in the region downstream of No. 2198.

  The primer can be synthesized, for example, using a DNA synthesizer model 380B manufactured by Applied Biosystems, using the phosphoamidite method (see Tetrahedron Letters (1981), 22, 1859). The PCR reaction can be performed, for example, using Gene Amp PCR System 9600 (manufactured by PERKIN ELMER) and TaKaRa LA PCR in vitro Cloning Kit (manufactured by Takara Shuzo) according to the method specified by the supplier such as each manufacturer.

The polynucleotide encoding the enzyme that can be used in the method for producing a peptide of the present invention includes a polynucleotide that is substantially the same as the ORF among the polynucleotides shown in SEQ ID NO: 1 in the Sequence Listing. That is, a probe prepared from a polynucleotide or a base sequence complementary to the ORF described in SEQ ID NO: 1 in the Sequence Listing from a polynucleotide encoding the present enzyme having a mutation or a cell retaining the enzyme A polynucleotide substantially identical to the polynucleotide of the present invention can also be obtained by isolating a polynucleotide encoding a protein having a peptide-forming activity and hybridizing under stringent conditions.
The polynucleotide encoding the enzyme that can be used in the method for producing a peptide of the present invention also includes a polynucleotide that is substantially the same as the ORF among the polynucleotides shown in SEQ ID NO: 3 in the Sequence Listing. That is, a probe prepared from a polynucleotide or a base sequence complementary to the ORF described in SEQ ID NO: 3 in the Sequence Listing from a polynucleotide encoding the present enzyme having a mutation or a cell retaining the enzyme A polynucleotide substantially identical to the polynucleotide of the present invention can also be obtained by isolating a polynucleotide encoding a protein having a peptide-forming activity and hybridizing under stringent conditions.

  The probe can be prepared by a conventional method based on the base sequence described in SEQ ID NO: 1, for example. In addition, a method of picking up a polynucleotide that hybridizes with a probe using a probe and isolating the target polynucleotide may be performed according to a conventional method. For example, a DNA probe can be prepared by amplifying a base sequence cloned in a plasmid or a phage vector, cutting out and extracting the base sequence to be used as a probe with a restriction enzyme. The location to be cut out can be adjusted according to the target polynucleotide.

  The “stringent conditions” referred to herein are conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Although it is difficult to quantify this condition clearly, for example, highly homologous polynucleotides, for example, 50% or more, more preferably 80% or more, more preferably 90% or more, and most preferably More than 95%, more preferably more than 97% homologous polynucleotides hybridize, and polynucleotides with lower homology do not hybridize with each other, or under normal Southern hybridization washing conditions. The conditions include hybridization at a salt concentration corresponding to a certain 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS. The genes that hybridize under these conditions include those that have generated stop codons in the middle and those that have lost activity due to mutations in the active center. It can be easily removed by expressing in an appropriate host and measuring the enzyme activity of the expression product by the method described below.

  However, in the case of a polynucleotide comprising a base sequence that hybridizes under stringent conditions as described above, half of the protein having the amino acid sequence encoded by the original base sequence at 50 ° C. and pH 8 It is desirable that the enzyme activity is maintained at a degree or more, more preferably 80% or more, and still more preferably 90% or more. For example, in the case of a base sequence that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence complementary to the base sequence of base numbers 1197 to 2198 of the base sequence described in (b) SEQ ID NO: 1, It is desirable to maintain an enzyme activity of about half or more, more preferably 80% or more, and still more preferably 90% or more of the protein having the amino acid sequence shown in SEQ ID NO: 2 at 50 ° C. and pH 8 .

  Confirmation that the enzyme activity is retained is confirmed by experimenting whether the target protein is allowed to act on an amino acid ester or peptide ester and proline to produce a peptide having a proline residue at the C-terminus. be able to. However, it is necessary to individually set analysis conditions according to the type of substrate. Therefore, for convenience, instead of the above-described experiment, a confirmation test may be performed in which the target protein is allowed to act on an amino acid ester or peptide and the hydrolysis reaction is observed.

  Such a modified polynucleotide can be obtained by a conventionally known mutation treatment. As the mutation treatment, a polynucleotide encoding the enzyme, for example, a method of treating one of the polynucleotides (a) and (c) in vitro with hydroxylamine and the like, and retaining the polynucleotide encoding the enzyme Examples include treatment of Escherichia bacteria with ultraviolet light irradiation or a mutation agent that is commonly used for artificial mutation such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrous acid.

  The amino acid sequence encoded by the ORF in SEQ ID NO: 1 in the sequence listing is shown in SEQ ID NO: 2 in the sequence listing. The amino acid sequence encoded by the ORF in SEQ ID NO: 3 is shown in SEQ ID NO: 4 in the sequence listing.

(3-2-2) Production of recombinant polynucleotide and transformant, and production of peptide-forming enzyme The polynucleotide described in (3-2-1) above is introduced into an appropriate host to obtain a recombinant polynucleotide, The peptide-forming enzyme that can be used in the present invention can also be produced by expressing the protein specified by the polynucleotide in the resulting transformed cell (transformant).

  The host for expressing the protein specified by the polynucleotide includes Escherichia coli, such as Escherichia coli, and various prokaryotic cells including Saccharomyces cerevisiae, including Bacillus subtilis. Various eukaryotic cells such as (Saccharomyces cerevisiae), Pichia stipitis, Aspergillus oryzae can be used.

  A recombinant polynucleotide used for introducing a polynucleotide into a host is in a form in which a protein encoded by the polynucleotide can be expressed in a vector corresponding to the type of host to be expressed. It can be prepared by inserting in As a promoter for expressing the polynucleotide of the present invention, when a promoter specific to a peptide-forming enzyme gene such as Philobacterium sp. Or Streptomyces sp. Functions in a host cell, the promoter can be used. Further, if necessary, another promoter that works in the host cell may be linked to the polynucleotide of the present invention and expressed under the control of the promoter.

  Examples of transformation methods for introducing a recombinant polynucleotide into a host cell include D.I. M.M. Morrison's method (Methods in Enzymology 68, 326 (1979)) or a method in which recipient cells are treated with calcium chloride to increase polynucleotide permeability (Mandel, M. and Higa, A., J. Mol. Biol. 53, 159 (1970)).

  In the case where a protein is mass-produced using recombinant polynucleotide technology, a form in which the protein associates in a transformed cell that produces the protein to form an inclusion body is also mentioned as a preferred embodiment. It is done. Advantages of this expression production method are that the target protein is protected from digestion by proteases present in the microbial cells, and that the target protein can be easily purified by centrifugation following cell disruption.

  The protein inclusion body obtained in this way is solubilized by a protein denaturing agent, and after being subjected to an activity regeneration operation mainly by removing the denaturing agent, it is converted into a correctly folded physiologically active protein. . For example, there are many examples such as activity regeneration of human interleukin-2 (Japanese Patent Laid-Open No. 61-257931).

  In order to obtain an active protein from a protein inclusion body, a series of operations such as solubilization and activity regeneration are necessary, and the operation becomes more complicated than when directly producing an active protein. However, when a large amount of a protein that affects the growth of bacterial cells is produced in the bacterial cells, the effect can be suppressed by accumulating them in the bacterial cells as inactive protein inclusion bodies.

  As a method for mass production of the target protein as inclusion bodies, there is a method of expressing the target protein alone under the control of a strong promoter, or a method of expressing it as a fusion protein with a protein known to be expressed in large quantities. is there.

  Hereinafter, a method for producing transformed Escherichia coli and producing a peptide-producing enzyme using the transformed Escherichia coli will be described more specifically.

  As a promoter for expressing a polynucleotide encoding a peptide-forming enzyme, a promoter usually used for heterologous protein production in Escherichia coli can be used. For example, T7 promoter, lac promoter, trp promoter, trc promoter, tac promoter, lambda Examples include strong promoters such as phage PR promoter and PL promoter. Moreover, pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219, pMW218 etc. can be used as a vector. In addition, phage polynucleotide vectors can be used. Furthermore, an expression vector containing a promoter and capable of expressing the inserted polynucleotide sequence can also be used.

  In order to produce a peptide-forming enzyme as a fusion protein inclusion body, a gene encoding another protein, preferably a hydrophilic peptide, is linked upstream or downstream of the peptide-forming enzyme gene to form a fusion protein gene. . Such a gene encoding another protein may be any gene that increases the accumulation amount of the fusion protein and enhances the solubility of the fusion protein after the denaturation / regeneration process. For example, T7gene 10, β-galactosidase gene, Dehydrofolate reductase gene, γ interferon gene, interleukin-2 gene, prochymosin gene and the like are listed as candidates.

  When these genes and a gene encoding a peptide-forming enzyme are linked, the codon reading frames are made to coincide. They may be linked at an appropriate restriction enzyme site, or a synthetic polynucleotide having an appropriate sequence may be used.

  In order to increase the production amount, it may be preferable to link a terminator, which is a transcription termination sequence, downstream of the fusion protein gene. Examples of the terminator include rrnB terminator, T7 terminator, fd phage terminator, T4 terminator, tetracycline resistance gene terminator, E. coli trpA gene terminator, and the like.

  As a vector for introducing a gene encoding a peptide-forming enzyme or a gene encoding a peptide-forming enzyme and another protein into E. coli, a so-called multi-copy type is preferable, and a plasmid having a replication origin derived from ColE1, for example, Examples include pUC-type plasmids, pBR322-type plasmids, pQE-type plasmids, and derivatives thereof. Here, the “derivative” means one obtained by modifying a plasmid by base substitution, deletion, and / or insertion. In addition, the modification here includes modification by mutation treatment with a mutation agent, UV irradiation, or natural mutation.

  In order to select transformants, the vector preferably has a marker such as an ampicillin resistance gene. As such plasmids, expression vectors having a strong promoter are commercially available (pUC system (Takara Shuzo Co., Ltd.), pPROK system (Clontech), pKK233-2 (Clontech), etc.).

  A recombinant polynucleotide is obtained by ligating a polynucleotide encoding a gene encoding a promoter, a peptide-forming enzyme or a fusion protein of a peptide-forming enzyme and another protein, or a terminator in some cases, and a vector polynucleotide.

  When the recombinant polynucleotide is used to transform Escherichia coli, and the Escherichia coli is cultured, a peptide-forming enzyme or a fusion protein of the peptide-forming enzyme and another protein is expressed and produced. As a host to be transformed, a strain usually used for expression of a heterologous gene can be used, but Escherichia coli JM109 strain is preferable. Methods for performing transformation and methods for selecting transformants are described in Molecular Cloning, 2nd edition, Cold Spring Harbor press (1989) and the like.

  When expressed as a fusion protein, the peptide-forming enzyme may be excised using a restriction protease such as blood coagulation factor Xa, kallikrein, etc., which has a sequence not present in the peptide-forming enzyme as a recognition sequence.

  As the production medium, a medium usually used for culturing Escherichia coli such as M9-casamino acid medium and LB medium may be used. The culture conditions and production induction conditions are appropriately selected according to the type of the marker, promoter, host fungus and the like used.

  There are the following methods for recovering a peptide-forming enzyme or a fusion protein of a peptide-forming enzyme and another protein. If the peptide-forming enzyme or its fusion protein is solubilized in the microbial cells, the microbial cells can be recovered and then disrupted or lysed to be used as a crude enzyme solution. Furthermore, if necessary, the peptide-forming enzyme or its fusion protein can be purified and used by a conventional method such as precipitation, filtration or column chromatography. In this case, a purification method using a peptide-forming enzyme or a fusion protein antibody can also be used.

  When protein inclusion bodies are formed, they are solubilized with a denaturing agent. Although it may be solubilized together with the bacterial protein, it is preferable to take out the inclusion body and solubilize it in consideration of the subsequent purification operation. In order to recover the inclusion body from the microbial cell, a conventionally known method may be used. For example, the microbial cells are destroyed, and the inclusion bodies are collected by centrifugation operation or the like. Examples of denaturing agents that solubilize protein inclusion bodies include guanidine hydrochloride (eg, 6M, pH 5-8), urea (eg, 8M), and the like.

  When these denaturing agents are removed by dialysis or the like, they are regenerated as active proteins. As a dialysis solution used for dialysis, a Tris-HCl buffer or a phosphate buffer may be used, and the concentration may be 20 mM to 0.5 M, and the pH may be 5 to 8.

  The protein concentration during the regeneration step is preferably suppressed to about 500 μg / ml or less. In order to suppress the regenerated peptide-forming enzyme from self-crosslinking, the dialysis temperature is preferably 5 ° C. or lower. Further, as a method for removing the denaturant, there are a dialysis method, a dilution method, an ultrafiltration method, and the like, and the regeneration of activity can be expected by using any of them.

Example 1 Purification of esterase [Preparation of cell-free extract]
Phyllobacterium sp. 50 ml of medium (0.5% yeast extract, 0.5% tryptone, 0.3% KH ₂ PO ₄ , 0.05%) in which S1201 (AJ110317) (FERM P-21431) is put in a 500 ml Sakaguchi flask MgSO ₄ · 7H ₂ O (pH 7.2)) and cultured with shaking at 30 ° C. for 24 hours. The cells were collected by centrifugation (8,000 g, 10 min, 4 ° C.), washed with 50 mM Tris-HCl buffer (pH 8.0), suspended in the same buffer, and subjected to ultrasonic disruption (200 W, 20 min, The cells were disrupted by 4 ° C). Next, undisrupted cells were removed by centrifugation (18,000 g, 15 min, 4 ° C.), and the resulting supernatant was used as a cell-free extract.

[Step I]
According to the above method, a cell-free extract containing 2110 mg of protein prepared from 3 liters of culture solution was applied to Q-Sepharose FF (Amersham Bioscience) previously equilibrated with 50 mM Tris-HCl buffer (pH 8.0). Protein was eluted with a 0-1 M NaCl gradient.

[Step II]
The active fraction obtained in Step I is collected, dialyzed against 50 mM Tris-HCl buffer (pH 8.0) containing 1 M ammonium sulfate, and then subjected to Phenyl-Sepharose HP (Amersham Bioscience) pre-equilibrated with the same buffer. Apply and elute the protein with a 1-0 M ammonium sulfate gradient.

[Step III]
The active fraction obtained in Step II was concentrated with Centricon YM100 (Millipore), applied to Superdex 200 pg (Amersham Bioscience) equilibrated with 50 mM Tris-HCl buffer (pH 8.0), and eluted at 1 ml / min. .

[Step IV]
The active fraction obtained in Step III was applied to MonoQ5 / 5 (Amersham Bioscience) equilibrated with 50 mM Tris-HCl buffer (pH 8.0), and the protein was eluted with a 0-1 M NaCl concentration gradient.

[Process V]
The active fraction obtained in Step IV is collected, dialyzed against 50 mM Tris-HCl buffer (pH 8.0) containing 1 M ammonium sulfate, and applied to Resource ISO (Amersham Bioscience) pre-equilibrated with the same buffer. The protein was eluted with a 1-0 M ammonium sulfate gradient.

[Process VI]
The active fractions obtained in Step V were collected, dialyzed against 50 mM potassium phosphate buffer (pH 7.0) containing 1 M NaCl, and then equilibrated with Benzamidine-Sepharose HP (Amersham Bioscience) equilibrated with the same buffer. After applying, 50 mM monopotassium dihydrogen phosphate-50 mM citrate buffer (pH 4.0) was eluted every 20% by the stepwise method.

  In all steps, L-alanyl-L-proline production activity was measured at 100 mM L-alanine methyl ester, 100 mM L-proline, 100 mM borate-potassium chloride buffer (pH 9.0), 25 ° C. 1 U was the amount of enzyme that produced 1 μmol of L-alanyl-L-proline per minute, and was purified 491 times by purification (Table 1).

  The obtained enzyme fraction was dialyzed against 50 mM Tris-HCl buffer (pH 8.0) and subjected to SDS-polyacrylamide electrophoresis. Thereafter, it was confirmed by Coomassie Brilliant Blue staining that it was electrophoretically single and had a molecular size of about 37 kDa (FIG. 1).

  The prepared enzyme was transferred to a PVDF membrane after SDS-polyacrylamide electrophoresis, and a 30-residue amino acid sequence was obtained by a protein sequencer Model 491 Procise cLC (manufactured by Applied Biosystems) (SEQ ID NO: 5).

Example 2 Determination of base sequence After 5 μg of S1201 (AJ110317) chromosomal DNA was digested with BglII (50 U), it was ligated with the Sau3AI cassette according to the method described in the manual of TaKaRaLA PCR in vitro Cloning Kit. PCR (94 ° C .: 30 seconds, 42 ° C .: 2 minutes, 72 ° C .: 1 minute, 30 cycles) using the combination of cassette primer C1 and primer S1 (1) (SEQ ID NO: 6) attached to the kit using the ligated mix as a template. ) Next, using this PCR reaction solution as a template, the second PCR (94 ° C .: 30 seconds, 47 ° C .: 2 minutes, 72 ° C .: 1 minute, 30 cycles) was performed with the cassette primer C2 and primer S2 (1) ( This was performed using SEQ ID NO: 7).

  A fragment of about 1.1 kb long confirmed to be amplified was ligated to pGEM-Teasy (Promega), and Escherichia coli JM109 was transformed. The nucleotide sequence of the plasmid having the target fragment was confirmed by a DNA sequencer (CEQ2000, manufactured by Beckman Coulter). A digoxigenin label probe was prepared by DIG High Prime (Roche) from a gene fragment of about 1.1 kb length obtained by treating this plasmid with SphI / PstI.

  The S1201 (AJ110317) chromosomal DNA was subjected to EcoRI, BglII, and BglII / EcoRI, then subjected to agarose electrophoresis, and subjected to Southern analysis using the above probe according to the method described in the manual. Positive signals were confirmed at .2 kb and BglII at about 1.7 kb, and BglII / EcoRI at about 1.2 kb and about 0.8 kb.

  Subsequently, S1201 (AJ110317) chromosomal DNA was treated with EcoRI and subjected to agarose electrophoresis, and a fragment of about 2.5 kb was purified and ligated to pUC118. Using this reaction solution, Escherichia coli JM109 was transformed to prepare a library. In the same manner, a library was prepared using an approximately 1.7 kb fragment obtained by BglII treatment.

  Colony hybridization was performed using the above probe according to the method described in the manual to obtain a positive colony. A plasmid having an EcoRI fragment was designated as pUC1201E, and a plasmid having a BglII fragment was designated as pUC1201Bg. When the nucleotide sequence of the fragment of each plasmid was determined, the ORF consisting of 334 amino acids was present in pUC1201E, and the target gene was found to have the same sequence as the amino acid sequence obtained from the N-terminal analysis result. Confirmed the acquisition. Further, pUC1201Bg contained 1.2 kb upstream of the ORF and a part of the ORF. From the above analysis, a base sequence of 3016 bp including ORF was determined (SEQ ID NO: 1).

Example 3 Heterologous Expression and Purification by Escherichia coli S1201 (AJ110317) Using chromosomal DNA as a template, a base sequence encoding a histidine tag sequence at the C-terminus using primers 1 (1) and 2 (2) (SEQ ID NOs: 8 and 9) The added fragment was amplified, treated with EcoRI / HindIII, inserted into pKK223-3 (Amersham Biosciences), and Escherichia coli JM109 was transformed. A transformant having the desired plasmid was selected and designated JM109 / pKK_est_his.

  This strain was cultured in LB medium (100 mg / l ampicillin sodium, 0.1 mM IPTG added) at 30 ° C. for 16 hours, and Mag-Extractor His-tag (TOYOBO) was used as a starting material. Then, affinity purification was performed, and it was confirmed that a uniform enzyme was prepared at about 37 kDa by SDS-PAGE.

  This enzyme preparation was dialyzed against 50 mM potassium phosphate buffer (pH 7.5), and the Ala-Pro synthetic activity was measured and found to be 23 U / mg.

Example 4 Effect of Reaction pH on Esterase Activity Using the enzyme prepared in Example 3, 100 mM L-alanine methyl ester hydrochloride, 200 mM of each buffer solution at 25 ° C. (potassium phosphate, Tris-HCl, boron) The esterase activity was measured in the range of starting pH 6.0-9.2 using (acid-potassium chloride). In addition, the activity value computed the production | generation of L-alanine in the aqueous solution in each pH of alanine methyl ester from the difference of enzyme addition conditions and enzyme non-addition conditions. The esterase activity in this study was maximum when potassium phosphate buffer (pH 7.5) was used, and was 1240 U / mg (FIG. 2).

Example 5 Substrate Specificity of Esterase Activity Using the enzyme prepared in Example 3, measurement was performed over time at a substrate concentration of 50 mM, 200 mM potassium phosphate buffer (pH 7.5), 25 ° C., up to 15 minutes of reaction. In addition, when there existed decomposition | disassembly in the aqueous solution of a substrate, enzyme non-addition conditions were set and activity was computed from the difference with an enzyme addition group. The activity against L-alanine methyl ester is shown in Table 2 with 1190 U / mg as 100%.

Example 6 Measurement of acyl transfer activity Using the enzyme prepared in Example 3, acyl transfer activity to L-alanine methyl ester was measured at a substrate concentration of 100 mM, 200 mM boric acid-potassium chloride buffer (pH 9.0), 25 ° C. And are shown in Table 3.

Example 7 Enzymatic synthesis of L-alanyl-L-proline 100 mM L-alanine methyl ester, 100-300 mM L-proline, 100 mM boric acid-potassium chloride buffer (pH 9.0) using the enzyme prepared in Example 1. ), 16 μg of the prepared enzyme was reacted in a 1 ml reaction solution at 25 ° C. for 7.5 hours. As a result, as shown in Table 4, the production of L-alanyl-L-proline was confirmed.

Example 8 Preparation of enzyme [Preparation of cell-free extract]
Streptomyces. Sp AJ9587 (FERM P-21430) in a 500 ml baffled Erlenmeyer flask in 100 ml liquid medium (0.5% sodium aspartate monohydrate, 0.5% yeast extract, 0.5% peptone, 0.5 % Malt extract, 0.3% potassium dihydrogen phosphate, 0.05% magnesium sulfate heptahydrate (pH 7.2)), and cultured with stirring at 32 ° C. for 48 hours. The cells were collected by centrifugation, washed with 50 mM potassium phosphate buffer (pH 7.0), suspended in the same buffer, and disrupted by ultrasonic disruption (200 W, 20 min, 4 ° C.). Next, undisrupted cells were removed by centrifugation (18,000 g, 15 min, 4 ° C.), and the resulting supernatant was used as a cell-free extract.

[Step I]
According to the above method, solid ammonium sulfate was added to a cell-free extract containing 1470 mg of protein prepared from 2 liters of culture broth and slowly stirred to 40% saturation until dissolved, and then dissolved. Left at 4 ° C. overnight. The precipitate was recovered by centrifugation (18,000 g, 20 min, 4 ° C.), dissolved in a small amount of 50 mM potassium phosphate buffer (pH 7.0), and dialyzed against the same buffer.

[Step II]
The sample obtained in Step I was collected, dialyzed against 50 mM potassium phosphate buffer (pH 7.0), and then loaded onto HiLoad-16 / 10-Q-Sepharose HP (Amersham Bioscience) pre-equilibrated with the same buffer. Apply and elute protein with 0-1 M sodium chloride gradient. Protein was eluted with a 1-0 M ammonium sulfate gradient.

[Step III]
The active fraction obtained in Step II was dialyzed against 50 mM potassium phosphate buffer (pH 7.0) containing 1 M ammonium sulfate, and HiLoad-16 / 10-Phenyl-Sepharose HP (Amersham) pre-equilibrated with the same buffer. The protein was eluted with a 0-1 M sodium chloride gradient. Protein was eluted with a 1-0 M ammonium sulfate gradient.

[Step IV]
The active fraction obtained in Step III was concentrated with Centricon YM100 (Millipore), applied to HiLoad-16 / 60-Superdex 200 pg (Amersham Biosciences) equilibrated with 50 mM Tris-HCl buffer (pH 8.0), and 1 ml / Elute at min.

[Process V]
The active fraction obtained in Step IV is dialyzed against 50 mM potassium phosphate buffer (pH 7.0) containing 1 M ammonium sulfate, and applied to Resource-ETH (Amersham Bioscience) pre-equilibrated with the same buffer. The protein was eluted with a 1-0 M ammonium sulfate gradient.

  In all steps, L-alanyl-L-proline production activity was measured at 100 mM L-alanine methyl ester, 100 mM L-proline, 100 mM boric acid-potassium chloride buffer (pH 9.0), 25 ° C. 1 U was the amount of enzyme that produced 1 μmol of L-alanyl-L-proline per minute, and was purified 509 times by purification (Table 5).

  The obtained enzyme fraction was dialyzed against 50 mM potassium phosphate buffer (pH 7.0), subjected to SDS-polyacrylamide electrophoresis, and then electrophoretically single by Coomassie brilliant blue staining. The molecular size was confirmed to be about 44 kDa (FIG. 3).

  The prepared enzyme was transferred to a PVDF membrane after SDS-polyacrylamide electrophoresis, and an amino acid sequence of 30 residues was obtained by protein sequencer Model491 Procise cLC (Applied Biosystems) (sequence 7).

Example 9 Determination of base sequence After 5 μg of AJ9587 chromosomal DNA was digested with PstI (50 U), it was ligated with a PstI cassette according to the method described in the manual of TaKaRaLA PCR in vitro Cloning Kit. PCR (94 ° C .: 30 seconds, 42 ° C .: 2 minutes, 72 ° C .: 1 minute, 30 cycles) using the combination of cassette primer C1 and primer S1 (2) (SEQ ID NO: 10) attached to the kit using the ligated mix as a template Went. Next, using this PCR reaction solution as a template, the second PCR (94 ° C .: 30 seconds, 47 ° C .: 2 minutes, 72 ° C .: 1 minute, 30 cycles) was performed with the cassette primer C2 and primer S2 (2) ( This was carried out using SEQ ID NO: 11).

  A fragment of about 0.5 kb long confirmed to be amplified was ligated to pGEM-Teasy (Promega), and Escherichia coli JM109 was transformed. The nucleotide sequence of the plasmid having the target fragment was confirmed by a DNA sequencer (CEQ2000, manufactured by Beckman Coulter). A digoxigenin-labeled probe was prepared by DIG High Prime (Roche) from a gene fragment of about 1.1 kb obtained by treating this plasmid with EcoRI / PstI.

  S1201 (AJ110317) chromosomal DNA was treated with PstI / KpnI, subjected to agarose electrophoresis, and subjected to Southern analysis according to the method described in the manual. As a result, a positive signal was confirmed at 5 kb.

  Subsequently, AJ9587 chromosomal DNA was treated with PstI / KpnI and subjected to agarose electrophoresis, and an approximately 5 kb fragment was purified and ligated to pUC19. Using this reaction solution, Escherichia coli JM109 was transformed to prepare a library.

  Colony hybridization was performed using the probe according to the method described in the manual, positive colonies were obtained, and plasmids were extracted. The obtained plasmid was designated p95887-1, and the nucleotide sequence of 1959 bp was determined. As a result, an ORF consisting of 441 amino acids was present and had the same sequence as the amino acid sequence obtained from the N-terminal analysis result. The acquisition of the gene was confirmed (SEQ ID NO: 3).

Example 10 Measurement of Amino Acid Paranitroanilide Hydrolysis Activity with Purified Enzyme Using the purified enzyme of Example 8, various amino acid paranitroanilide hydrolysis activities were measured for the reaction solution composition (50 mM Tris-HCl buffer (pH 8.0), 0 .1 mM amino acid pNA), measured by following the absorbance at 405 nm at 25 ° C. (Table 6).

  As is clear from Table 6, it was revealed that the purified enzyme can degrade Pro-pNA.

Example 11 Determination of Amino Acid Ester and Amino Acid Amide Hydrolysis Activity with Purified Enzyme Using the enzyme of Example 8, hydrolysis activity was obtained at 100 mM borate-potassium chloride buffer (pH 9.0) and substrate concentration of 100 mM. Was determined by quantification by HPLC. When spontaneous degradation of the substrate was observed, an enzyme-free group was set and the activity was calculated from the difference (Table 7).

Example 12 Measurement of Acyl Transfer Activity Using Purified Enzyme Using the enzyme prepared in Example 8, acyl transfer activity was measured at 100 mM borate-potassium chloride buffer (pH 9.0) and each substrate concentration of 100 mM. Measured by quantification by HPLC (Table 8).

Example 13 Enzymatic synthesis of L-alanyl-L-proline Using the enzyme prepared in Example 8, 100 mM L-alanine methyl ester, 100-300 mM L-proline, 100 mM boric acid-potassium chloride buffer (pH 9.0) ) When 16 μg of the prepared enzyme was reacted in a 1 ml reaction solution at 25 ° C. for 7.5 hours, the production of L-alanyl-L-proline was confirmed as shown in Table 9.

Example 14 Enzymatic synthesis of Ala-Ala-Pro To a composition solution of 100 mM boric acid-potassium chloride buffer (pH 9.0), L-alanine methyl ester hydrochloride, 100 mM L-alanyl-L-proline in Example 8 20 μg of the prepared purified enzyme was added to make a total volume of 1 ml. When Ala-Ala-Pro was quantified after 2.5 hours of reaction at 25 ° C., accumulation of 56.3 mM was confirmed.

Example 15 Heterologous expression and purification by Escherichia coli E. coli carrying plasmid p95887-1. Using E. coli chromosomal DNA as a template, a gene fragment is amplified by a combination of primer 1 (sequence 11) and primer 2 (sequence 12), or a combination of primer 1 and primer 3 (sequence 13). After BamHI / HindIII treatment, pQE30 ( Was inserted into the BamHI / HindIII site of Qiagen) and Escherichia coli JM109 was transformed. A transformant having the target plasmid was selected and designated as pQE-PIP1 and pQE-PIP2.

  The transformant was cultured in LB medium (100 mg / l ampicillin sodium, 0.02 mM IPTG added) at 30 ° C. for 16 hr, and Mag-Extractor His-tag (TOYOBO) was used as a starting material. It was used for affinity purification, and it was confirmed by SDS-PAGE that a uniform enzyme was prepared at about 44 kDa.

  This enzyme preparation was dialyzed against 50 mM potassium phosphate buffer (pH 7.5), and then the hydrolysis activity of Pro-pNA was determined according to the reaction solution composition (50 mM Tris-HCl buffer (pH 8.0), 0.1 mM Pro- pNA), measured by following the absorption at 405 nm at 25 ° C., which were 37.5 U / mg and 79.1 U / mg, respectively.

FIG. 1 shows Phyllobacterium sp. It is a figure which shows the molecular weight of the enzyme extracted and refined from. FIG. 2 shows reaction pH and Phyllobacterium sp. It is a graph which shows the relationship with the esterase activity of the enzyme derived from. FIG. 3 shows Streptomyces sp. It is a figure which shows the molecular weight of the enzyme extracted and refined from.

SEQ ID NO: 1: Phyllobacterium sp. Base sequence of derived enzyme SEQ ID NO: 2: Phyllobacterium sp. Amino acid sequence of the enzyme derived from SEQ ID NO: 3: Streptomyces sp. Base sequence of enzyme derived from SEQ ID NO: 4: Streptomyces sp. Amino acid sequence of derived enzyme SEQ ID NO: 5: Phyllobacterium sp. Partial amino acid sequence of the derived enzyme SEQ ID NO: 6: nucleotide sequence of primer S1 (1) SEQ ID NO: 7: nucleotide sequence of primer S2 (1) SEQ ID NO: 8: nucleotide sequence of primer 1 (1) SEQ ID NO: 9: primer 2 (1 ) Base sequence SEQ ID NO: 10: Streptomyces sp. Partial amino acid sequence of the derived enzyme SEQ ID NO: 11: base sequence of primer S1 (2) SEQ ID NO: 12: base sequence of primer S2 (2) SEQ ID NO: 13: base sequence of primer 1 (2) SEQ ID NO: 14: primer 2 (2 ) Base sequence SEQ ID NO: 15: base sequence of primer 3

Claims (13)

  An amino acid ester or peptide ester and proline are used to produce a peptide having a proline residue at the C-terminus. A method for producing a peptide, comprising:   A culture of a microorganism having the ability to produce a peptide having a proline residue at the C-terminus from an amino acid ester or a peptide ester and proline, a microorganism cell separated from the culture, and The method for producing a peptide according to claim 1, wherein the peptide is one or more selected from the group consisting of processed microbial cells.   The method for producing a peptide according to claim 2, wherein the microorganism is a microorganism belonging to the genus Streptomyces or Philobacterium. The method for producing a peptide according to claim 2, wherein the microorganism is a transformed microorganism capable of expressing a protein selected from the group consisting of the following (A) to (D).
(A) a protein having the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing (B) substitution or deletion and / or insertion of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing The amino acid sequence described in SEQ ID NO: 4 in the protein (C) sequence table, which has an amino acid sequence containing and has an activity of generating a peptide having a proline residue at the C-terminus from an amino acid ester or peptide ester and proline. The amino acid sequence shown in SEQ ID NO: 4 in the protein (D) sequence table, having an amino acid sequence containing one or several amino acid substitutions, deletions and / or insertions, and an amino acid ester or peptide ester; A protein having an activity to produce a peptide having a proline residue at the C-terminus from proline The method for producing a peptide according to claim 2, wherein the microorganism is a transformed microorganism into which a polynucleotide selected from the group consisting of the following (a) to (d) is introduced.
(A) a polynucleotide having a base sequence of base numbers 1197 to 2198 in the base sequence set forth in SEQ ID NO: 1 in the sequence listing (b) a base sequence of base numbers 1197 to 2198 in the base sequence set forth in SEQ ID NO: 1 of the sequence listing A protein that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence and has an activity of generating a peptide having a proline residue at the C-terminus from an amino acid ester or peptide ester and proline (C) a polynucleotide having the nucleotide sequence of nucleotide numbers 410 to 1705 out of the nucleotide sequence set forth in SEQ ID NO: 3 in the sequence listing (d) the nucleotide in the nucleotide sequence set forth in SEQ ID NO: 3 in the sequence listing Polynucleotide and stream consisting of a base sequence complementary to the base sequence of numbers 410 to 1705 Hybridizes under stringent conditions, and from the amino acid ester or peptide ester and proline, encoding a peptide having a proline residue at the C-terminal polynucleotide The method for producing a peptide according to claim 1, wherein the enzyme is at least one selected from the group consisting of the following (A) to (D).
(A) a protein having the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing (B) substitution or deletion and / or insertion of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing The amino acid sequence described in SEQ ID NO: 4 in the protein (C) sequence table, which has an amino acid sequence containing and has an activity of generating a peptide having a proline residue at the C-terminus from an amino acid ester or peptide ester and proline. The amino acid sequence shown in SEQ ID NO: 4 in the protein (D) sequence table, having an amino acid sequence containing one or several amino acid substitutions, deletions and / or insertions, and an amino acid ester or peptide ester; A protein having an activity to produce a peptide having a proline residue at the C-terminus from proline   A protein derived from a microorganism belonging to the genus Streptomyces or Philobacterium and having an activity of generating a peptide having a proline residue at the C-terminus from an amino acid ester or peptide ester and proline. A protein selected from the group consisting of the following (A) to (D).
(A) a protein having the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing (B) substitution or deletion and / or insertion of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 2 in the sequence listing The amino acid sequence described in SEQ ID NO: 4 in the protein (C) sequence table, which has an amino acid sequence containing and has an activity of generating a peptide having a proline residue at the C-terminus from an amino acid ester or peptide ester and proline. The amino acid sequence shown in SEQ ID NO: 4 in the protein (D) sequence table, having an amino acid sequence containing one or several amino acid substitutions, deletions and / or insertions, and an amino acid ester or peptide ester; A protein having an activity to produce a peptide having a proline residue at the C-terminus from proline   A polynucleotide encoding the protein according to claim 8. A polynucleotide selected from the group consisting of the following (a) to (d).
(A) a polynucleotide having a base sequence of base numbers 1197 to 2198 in the base sequence set forth in SEQ ID NO: 1 in the sequence listing (b) a base sequence of base numbers 1197 to 2198 in the base sequence set forth in SEQ ID NO: 1 of the sequence listing A protein that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence and has an activity of generating a peptide having a proline residue at the C-terminus from an amino acid ester or peptide ester and proline (C) a polynucleotide having the nucleotide sequence of nucleotide numbers 410 to 1705 out of the nucleotide sequence set forth in SEQ ID NO: 3 in the sequence listing (d) the nucleotide in the nucleotide sequence set forth in SEQ ID NO: 3 in the sequence listing Polynucleotide and stream consisting of a base sequence complementary to the base sequence of numbers 410 to 1705 Hybridizes under stringent conditions, and from the amino acid ester or peptide ester and proline, which encodes a protein having the activity to form a peptide having a proline residue at the C-terminal polynucleotide   The polynucleotide according to claim 10, wherein the stringent condition is a condition in which washing is performed at a salt concentration corresponding to 1 × SSC and 0.1% SDS at 60 ° C.   A recombinant polynucleotide comprising the polynucleotide according to any one of claims 9 to 11.   A transformed cell into which the polynucleotide according to claim 12 has been introduced.

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