JP2005058212A - METHOD FOR PRODUCING alpha-L-ASPARTYL-L-PHENYLALANINE-beta-ESTER AND METHOD FOR PRODUCING alpha-L-ASPARTYL-L-PHENYLALANINE-alpha-METHYL ESTER - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for simply producing an α-L-aspartyl-L-phenylalanine-β-ester which is an intermediate for α-L-aspartyl-L-phenylalanine-α-methyl ester in a high yield at a low cost, and to provide a method for simply producing the α-L-aspartyl-L-phenylalanine-α-methyl ester in a high yield at a low cost. <P>SOLUTION: This α-L-aspartyl-L-phenylalanine-β-ester is formed out of an L-aspartic acid-α,β-diester and L-phenylalanine, by using an enzyme, or a material containing the enzyme, having ability of catalyzing reaction in which the L-phenylalanine does not nucleophilically attack the β-ester position of the L-aspartic acid-α,β-diester but nucleophilically attacks the α-ester position thereof. Further, the α-L-aspartyl-L-phenylalanine-α-methyl ester is formed out of the α-L-aspartyl-L-phenylalanine-β-ester. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for producing α-L-aspartyl-L-phenylalanine-β-ester (or α-L- (β-o-substituted aspartyl) -L-phenylalanine (abbreviation: α-ARP)) and α More specifically, a method for producing L-aspartyl-L-phenylalanine-α-methyl ester (α-L-aspartyl-L-phenylalanine methyl ester (abbreviation: α-APM)) has a large demand as a sweetener. -L-aspartyl-L-phenylalanine-α-methyl ester (product name: aspartame), an intermediate for production of α-L-aspartyl-L-phenylalanine-β-ester, and the α-L The present invention relates to a method for producing α-L-aspartyl-L-phenylalanine-α-methyl ester using a method for producing aspartyl-L-phenylalanine-β-ester.

  Conventionally known methods for producing α-L-aspartyl-L-phenylalanine-α-methyl ester (sometimes abbreviated as “α-APM”) include chemical synthesis methods and enzyme synthesis methods. As a chemical synthesis method, N-protected L-aspartic anhydride and L-phenylalanine methyl ester are condensed to synthesize N-protected APM, and this N-protecting group is eliminated to obtain APM. , N-protected L-aspartic acid and L-phenylalanine methyl ester are condensed to synthesize N-protected APM, and this N-protecting group is eliminated to obtain APM. Introducing and removing the protecting group was necessary, and the process was extremely complicated. On the other hand, an APM production method that does not use an N-protecting group has been studied (Patent Document 1), but the yield is extremely low and it is not suitable for industrial production. Under such circumstances, development of a further inexpensive industrial production method for aspartame has been desired.

Japanese Patent Publication No. 02-015196

  The present invention relates to α-L-aspartyl-L-phenylalanine, which is an intermediate of α-L-aspartyl-L-phenylalanine-α-methyl ester, easily and at a low yield without complicated synthesis methods. It is an object to provide a method capable of producing a -β-ester. Another object of the present invention is to provide a method for producing α-L-aspartyl-L-phenylalanine-α-methyl ester, which is simple, high yield and inexpensive.

  As a result of intensive studies in view of the above-mentioned object, the present inventors have newly found an enzyme or an enzyme-containing substance from L-aspartic acid-α, β-diester and L-phenylalanine, α-L-aspartyl- It has been found that it has the ability to selectively produce L-phenylalanine-β-ester and has led to the completion of the present invention.

That is, the present invention is as follows.
[1] L-aspartic acid-α, β-, using an enzyme or an enzyme-containing product having the ability to selectively bond a peptide to the α-ester site of L-aspartic acid-α, β-diester. Α-L-aspartyl-L-phenylalanine-β-ester (ie, α-L- (), characterized in that α-L-aspartyl-L-phenylalanine-β-ester is produced from diester and L-phenylalanine. β-o-substituted aspartyl) -L-phenylalanine).
[2] A microorganism culture having the ability to selectively peptide bond L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester, which is separated from the enzyme or the enzyme-containing product. The α-L-aspartyl-L-phenylalanine according to the above [1], which is one or more selected from the group consisting of the microbial cells obtained and the processed microbial cells of the microorganisms -Production method of β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine).
[3] The microorganism is an Aeromonas genus, Azotobacter genus, Alkagenes genus, Brevibacterium genus, Corynebacterium genus, Escherichia genus, Empedobacter genus, Flavobacterium genus, Microbacterium genus, Propionibacterium genus, Brevibacillus, Penibacillus, Pseudomonas, Serratia, Stenotrophomonas, Sphingobacteria, Streptomyces, Xanthomonas, Williopsis, Candida, Geotricum, Pichia, Saccharomyces, Torraspola , Cerroferga, Weeksera, Pedobacter, Pericobacter, Flexitrix, Titinophaga, Cyclobacterium, Lunella, Thermonema, Cycloserpense, Gelidibacter Genus, diadbacter, flameovirga, spirosoma, flectobacils, tenashibakuramu, rhodotermas, zoberia, murikauda, salegentibacter, taxiobacter, chitophaga, marinilavillia, levinella, saprospira, and The α-L-aspartyl-L-phenylalanine-β-ester (ie, α-L- () described in the above [2], which is a microorganism belonging to a genus selected from the group consisting of the genus Harriskomenobacter. β-o-substituted aspartyl) -L-phenylalanine).
[4] The α-L-aspartyl-L according to the above [2], wherein the microorganism is a transformed microorganism capable of expressing the protein shown in the following (A) or (B): A method for producing phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine).
(A) Protein having the amino acid sequence of amino acid residues 23 to 616 in the amino acid sequence described in SEQ ID NO: 6 in the sequence listing (B) Amino acid residue number 23 in the amino acid sequence described in SEQ ID NO: 6 in the sequence listing ˜616 having an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, and / or inversions, and L-phenylalanine is converted to L-aspartic acid-α, β -A protein having an activity of selectively binding a peptide to the α-ester site of a diester [5] The microorganism is a transformed microorganism capable of expressing the protein shown in the following (C) or (D): The method for producing α-L-aspartyl-L-phenylalanine-β-ester as described in [2] above.
(C) Protein having the amino acid sequence of amino acid residues 21 to 619 among the amino acid sequences described in SEQ ID NO: 12 of the sequence listing (D) Amino acid residue number 21 of the amino acid sequences described in SEQ ID NO: 12 of the sequence listing In the amino acid sequence of ˜619, having an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, and / or inversions, and L-phenylalanine to L-aspartic acid-α, β -A protein having an activity of selectively binding a peptide to the α-ester site of a diester [6] The microorganism is a transformed microorganism capable of expressing the protein shown in the following (E) or (F): The α-L-aspartyl-L-phenylalanine-β-ester according to claim 2 (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine) Manufacturing method.
(E) a protein having an amino acid sequence of amino acid residues 23 to 625 in the amino acid sequence set forth in SEQ ID NO: 18 in the sequence listing (F) amino acid residue No. 23 in the amino acid sequence set forth in SEQ ID NO: 18 in the sequence listing ˜625 amino acid sequence having an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, and / or inversions, and L-phenylalanine is converted to L-aspartic acid-α, β -A protein having an activity of selectively binding a peptide to the α-ester site of a diester [7] The microorganism is a transformed microorganism capable of expressing the protein shown in (G) or (H) below. Α-L-aspartyl-L-phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine according to [2] above, ) Manufacturing method.
(G) A protein having an amino acid sequence of amino acid residues 23 to 645 in the amino acid sequence described in SEQ ID NO: 23 of the sequence listing (H) Amino acid residue No. 23 of the amino acid sequences described in SEQ ID NO: 23 of the sequence listing In an amino acid sequence of ˜645, having an amino acid sequence including substitution, deletion, insertion, addition, and / or inversion of one or several amino acids, and L-phenylalanine as L-aspartic acid-α, β -A protein having an activity of selectively binding a peptide to the α-ester site of a diester [8] The microorganism is a transformed microorganism capable of expressing the protein shown in (I) or (J) below: Α-L-aspartyl-L-phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine according to [2] above, ) Manufacturing method.
(I) Protein having the amino acid sequence of amino acid residues 26 to 620 in the amino acid sequence described in SEQ ID NO: 25 in the sequence listing (J) Amino acid residue number 26 in the amino acid sequence described in SEQ ID NO: 25 of the sequence listing An amino acid sequence comprising one or several amino acid substitutions, deletions, insertions, additions, and / or inversions, and L-phenylalanine is converted to L-aspartic acid-α, β -A protein having an activity of selectively binding a peptide to the α-ester site of a diester [9] The microorganism is a transformed microorganism capable of expressing the protein shown in the following (K) or (L): Α-L-aspartyl-L-phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine according to [2] above, ) Manufacturing method.
(K) Protein having the amino acid sequence of amino acid residues 18 to 644 among the amino acid sequences described in SEQ ID NO: 27 of the sequence listing (L) Amino acid residue number 18 of the amino acid sequences described in SEQ ID NO: 27 of the sequence listing An amino acid sequence comprising one or several amino acid substitutions, deletions, insertions, additions, and / or inversions, and L-phenylalanine is converted to L-aspartic acid-α, β -A protein having an activity of selectively binding a peptide to the α-ester site of a diester [10] The microorganism is a transformed microorganism capable of expressing the protein shown in (M) or (N) below. The α-L-aspartyl-L-phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine described in [2] above, ine) manufacturing method.
(M) A protein having the amino acid sequence set forth in SEQ ID NO: 6 in the sequence listing (N) Substitution, deletion, insertion, addition of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 6 in the sequence listing And / or a protein having an amino acid sequence containing an inversion and a mature protein region having peptide-forming activity [11] A transformed microorganism capable of expressing the protein shown in (O) or (P) below: The method for producing α-L-aspartyl-L-phenylalanine-β-ester according to [2] above, which is a microorganism.
(O) Protein having the amino acid sequence set forth in SEQ ID NO: 12 in the Sequence Listing (P) Substitution, deletion, insertion, addition of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 12 in the Sequence Listing And / or a protein comprising a mature protein region having an amino acid sequence containing an inversion and having an activity of selectively peptide-binding L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester [ [12] The α-L-aspartyl-L- described in [2] above, wherein the microorganism is a transformed microorganism capable of expressing the protein shown in (Q) or (R) below. A method for producing phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine).
(Q) Protein having the amino acid sequence set forth in SEQ ID NO: 18 in the sequence listing (R) Substitution, deletion, insertion, addition of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 18 in the sequence listing And / or a protein comprising a mature protein region having an amino acid sequence containing an inversion and having an activity of selectively peptide-binding L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester [ [13] The α-L-aspartyl-L- described in [2] above, wherein the microorganism is a transformed microorganism capable of expressing the protein shown in (S) or (T) below. A method for producing phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine).
(S) a protein having the amino acid sequence set forth in SEQ ID NO: 23 in the sequence listing (T) substitution or deletion, insertion, addition of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 23 in the sequence listing And / or a protein comprising a mature protein region having an amino acid sequence containing an inversion and having an activity of selectively peptide-binding L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester [ [14] The α-L-aspartyl-L- described in [2] above, wherein the microorganism is a transformed microorganism capable of expressing the protein shown in the following (U) or (V): A method for producing phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine).
(U) a protein having the amino acid sequence set forth in SEQ ID NO: 25 in the sequence listing (V) one or several amino acid substitutions, deletions, insertions, additions in the amino acid sequence set forth in SEQ ID NO: 25 in the sequence listing; And / or a protein comprising a mature protein region having an amino acid sequence containing an inversion and having an activity of selectively peptide-binding L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester [ [15] The α-L-aspartyl-L- described in [2] above, wherein the microorganism is a transformed microorganism capable of expressing the protein shown in (W) or (X) below. A method for producing phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine).
(W) a protein having the amino acid sequence set forth in SEQ ID NO: 27 in the sequence listing (X) substitution, deletion, insertion, addition of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 27 in the sequence listing And / or a protein comprising a mature protein region having an amino acid sequence containing an inversion and having an activity of selectively peptide-binding L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester [ [16] The α-L-aspartyl-L-phenylalanine-β-ester (ie, α) according to [1], wherein the enzyme is at least one selected from the group consisting of (A) to (X) below: -L- (β-o-substituted aspartyl) -L-phenylalanine)
(A) Protein having the amino acid sequence of amino acid residues 23 to 616 in the amino acid sequence described in SEQ ID NO: 6 in the sequence listing (B) Amino acid residue number 23 in the amino acid sequence described in SEQ ID NO: 6 in the sequence listing ˜616 having an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, and / or inversions, and L-phenylalanine is converted to L-aspartic acid-α, β -Protein having the activity of selectively binding a peptide to the α-ester site of the diester (C) Protein having the amino acid sequence of amino acid residues 21 to 619 among the amino acid sequences shown in SEQ ID NO: 12 of the Sequence Listing (D) In the amino acid sequence of amino acid residues 21 to 619 in the amino acid sequence described in SEQ ID NO: 12 in the sequence listing, 1 or a number Of amino acid substitutions, deletions, insertions, additions, and / or inversions, and L-phenylalanine is selectively used as an α-ester site of L-aspartic acid-α, β-diester A protein having an activity of peptide binding (E) The amino acid sequence described in SEQ ID NO: 18 of the protein (F) sequence table having the amino acid sequence of amino acid residues 23 to 625 among the amino acid sequences described in SEQ ID NO: 18 of the sequence table Among the amino acid sequences of amino acid residues 23 to 625, and having an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or several amino acids, and L-phenylalanine is L A protein (G) sequence having an activity of selectively binding a peptide to the α-ester site of aspartic acid-α, β-diester The amino acid sequence of amino acid residues 23 to 645 in the amino acid sequence of SEQ ID NO: 23 of the protein (H) sequence table having the amino acid sequence of amino acid residues 23 to 645 of the amino acid sequence of SEQ ID NO: 23 Having an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions and / or inversions, and L-phenylalanine is α- of L-aspartic acid-α, β-diester A protein having an activity of selectively binding a peptide to an ester site (I) A protein having an amino acid sequence of amino acid residues 26 to 620 among amino acid sequences set forth in SEQ ID NO: 25 of the sequence listing (J) SEQ ID NO: of the sequence listing In the amino acid sequence of amino acid residues 26 to 620 of the amino acid sequence described in 25, one or several amino acids Having an amino acid sequence containing no acid substitution, deletion, insertion, addition, and / or inversion, and L-phenylalanine selectively at the α-ester site of L-aspartic acid-α, β-diester The amino acid sequence shown in SEQ ID NO: 27 of the protein (L) sequence table having the amino acid sequence of amino acid residues 18 to 644 out of the amino acid sequence shown in SEQ ID NO: 27 of the protein (K) sequence table having the activity to bind the peptide In the amino acid sequence of amino acid residues 18 to 644, and having an amino acid sequence including substitution, deletion, insertion, addition, and / or inversion of one or several amino acids, and L-phenylalanine is L -Sequence of the protein (M) sequence table having the activity of selectively binding a peptide to the α-ester site of aspartic acid-α, β-diester Amino acids comprising one or several amino acid substitutions, deletions, insertions, additions and / or inversions in the amino acid sequence set forth in SEQ ID NO: 6 in the protein (N) sequence listing having the amino acid sequence set forth in No. 6 SEQ ID NO: of the protein (O) sequence listing having a sequence and having a mature protein region that selectively binds L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester Amino acid sequence comprising one or several amino acid substitutions, deletions, insertions, additions, and / or inversions in the amino acid sequence shown in SEQ ID NO: 12 of the protein (P) sequence listing having the amino acid sequence shown in FIG. And L-phenylalanine is selectively bonded to the α-ester site of L-aspartic acid-α, β-diester In the amino acid sequence shown in SEQ ID NO: 18 in the protein (R) sequence list, which has the amino acid sequence shown in SEQ ID NO: 18 in the protein (Q) sequence list, including the mature protein region having the activity of one or several amino acids It has an amino acid sequence including substitution, deletion, insertion, addition and / or inversion, and L-phenylalanine is selectively peptide-bonded to the α-ester site of L-aspartic acid-α, β-diester. Substitution of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 23 of the protein (T) sequence list having the amino acid sequence shown in SEQ ID NO: 23 of the protein (S) sequence list including the mature protein region having activity , Deletions, insertions, additions, and / or inversions, and L-phenylalanine is L- A protein (V) having the amino acid sequence shown in SEQ ID NO: 25 in the protein (U) sequence table, which contains a mature protein region having an activity of selectively binding a peptide to the α-ester site of aspartic acid-α, β-diester It has an amino acid sequence containing substitution, deletion, insertion, addition, and / or inversion of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 25 in the Table, and L-phenylalanine is converted to L-asparagine Protein (X) Sequence Listing having the amino acid sequence set forth in SEQ ID NO: 27 in the Protein (W) Sequence Listing, which contains a mature protein region having an activity of selectively binding a peptide to the α-ester site of the acid-α, β-diester 1 or several amino acid substitutions, deletions, insertions, additions, and And / or a protein comprising a mature protein region having an amino acid sequence containing an inversion and having an activity of selectively peptide-binding L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester [17] α-L-aspartyl-L-phenylalanine-β-methyl by the method for producing α-L-aspartyl-L-phenylalanine-β-ester according to any one of [1] to [16] A reaction step of synthesizing an ester (ie, α-L- (β-o-methyl aspartyl) -L-phenylalanine);
a reaction step of converting α-L-aspartyl-L-phenylalanine-β-methyl ester to α-L-aspartyl-L-phenylalanine-α-methyl ester, α-L-aspartyl-L-phenylalanine-α- A method for producing methyl ester (ie, α-L-aspartyl-L-phenylalanine methyl ester).

  According to the present invention, α-L-aspartyl-L-phenylalanine-β-ester can be easily produced using an enzyme. By the method of the present invention, complicated synthesis methods such as introduction and removal of protecting groups can be reduced, and α-L-aspartyl-L-phenylalanine-β-ester can be produced simply and at a high yield at low cost. .

  Moreover, according to the present invention, α-L-aspartyl-L-phenylalanine-α-methyl ester can be easily produced at a high yield and at a low cost.

Hereinafter, regarding the present invention,
<1> Method for producing α-L-aspartyl-L-phenylalanine-β-ester 1. Method for producing α-L-aspartyl-L-phenylalanine-β-ester 2. Microorganisms used in the present invention A method for producing the enzyme <2> α-L-aspartyl-L-phenylalanine-α-methyl ester used in the present invention;
Will be described in the order.

<1> Method for producing α-L-aspartyl-L-phenylalanine-β-ester Method for Producing α-L-Aspartyl-L-Phenylalanine-β-Ester Method for Producing α-L-Aspartyl-L-Phenylalanine-β-Ester of the Present Invention (hereinafter also referred to as “peptide production method of the present invention”) Reacts L-phenylalanine with L-aspartic acid-α, β-diester in the presence of an enzyme having a predetermined peptide-forming activity. That is, in the method for producing a peptide of the present invention, L-phenylalanine is used with an enzyme or an enzyme-containing substance having an ability to selectively bond a peptide to the α-ester site of L-aspartic acid-α, β-diester. Α-L-aspartyl-L-phenylalanine-β-ester is produced from aspartic acid-α, β-diester and L-phenylalanine. An enzyme or an enzyme-containing substance having the ability to selectively peptide bond L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester is substantially defined as L-phenylalanine is L-aspartic acid- It refers to an enzyme or an enzyme-containing substance that has an ability or activity to catalyze a reaction that does not allow nucleophilic attack on the β-ester site of α, β-diester but only nucleophilic attack on only the α-ester site. In addition, as shown in the following reference examples, contrary to the above-mentioned ability, L-phenylalanine cannot undergo nucleophilic attack on the α-ester site of L-aspartic acid-α, β-diester, and β-ester Having the ability to catalyze a reaction that nucleophilically attacks only the site, and β-L-aspartyl-L-phenylalanine-α-ester (or β-L) from L-aspartic acid-α, β-diester and L-phenylalanine An enzyme or an enzyme-containing product that produces-(α-o-substituted aspartyl) -L-phenylalanine (abbreviation: β-ARP) has also been obtained.

  Reaction formula in which L-phenylalanine undergoes nucleophilic attack on the α-ester site of L-aspartic acid-α, β-diester to produce α-L-aspartyl-L-phenylalanine-β-ester (α-ARP) Is represented by the following formula (I-α) as an example when L-aspartic acid-α, β-dimethyl ester is used as L-aspartic acid-α, β-diester (where “Me” is methyl As shown in the formula (I-α), in the peptide production method of the present invention, the amino group of L-phenylalanine and the α-methyl ester moiety of L-aspartic acid-α, β-dimethyl ester Reacts to form a peptide bond. On the other hand, in the following formula (I-β), the β-methyl ester site of L-aspartic acid-α, β-dimethyl ester is subjected to nucleophilic attack to produce β-L-aspartyl-L-phenylalanine-α-methyl. It shows a reaction in which an ester (or β-L- (α-o-methyl aspartyl) -L-phenylalanine (abbreviation: β-AMP) is formed. The peptide bond in β-AMP is L-aspartic acid-α, β- It is formed at the β-methyl ester site of dimethyl ester, and the enzyme or enzyme-containing substance used in the present invention substantially promotes only the reaction of the formula (I-α), and has the formula (I-β) From α-AMP, α-APM can be produced from α-AMP by a simple reaction step (formula (II)), but α-APM can be directly produced from β-AMP. Cannot be manufactured, ie, the present invention The method is very efficient as an intermediate production method of α-APM and is useful for industrial production.

  As a method of allowing an enzyme or an enzyme-containing substance to act on L-aspartic acid-α, β-diester and L-phenylalanine, the enzyme or the enzyme-containing material, L-aspartic acid-α, β-diester, L- What is necessary is just to mix with phenylalanine. More specifically, a method may be used in which an enzyme or an enzyme-containing material is added to a solution containing L-aspartic acid diester and L-phenylalanine and reacted, or a microorganism that produces the enzyme as the enzyme-containing material. May be reacted as described above, or the microorganism producing the enzyme may be cultured, the enzyme may be produced and accumulated in the microorganism or in the culture medium of the microorganism, and L- A method of adding aspartic acid-α, β-diester and L-phenylalanine may be used. The produced α-L-aspartyl-L-phenylalanine-β-ester can be recovered by a conventional method and purified as necessary.

  The “enzyme-containing product” may be any material that contains the enzyme. Specific examples of the enzyme-containing material include a culture of a microorganism that produces the enzyme, a microbial cell separated from the culture, and a treated product of the cell. Etc. are included. A microorganism culture is a product obtained by culturing a microorganism, and more specifically, a microbial cell, a medium used for culturing the microorganism, and a mixture of substances produced by the cultured microorganism. And so on. Further, the microbial cells may be washed and used as washed cells. In addition, the processed microbial cells include those obtained by crushing, lysing, lyophilizing the microbial cells, and further include crude enzymes recovered by treating the microbial cells, and further purified enzymes. As the purified enzyme, partially purified enzymes obtained by various purification methods may be used. Further, an immobilized enzyme obtained by immobilizing these enzyme-containing materials by a covalent bond method, an adsorption method, a comprehensive method, or the like may be used. 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 active peptide-forming enzyme that produces α-L-aspartyl-L-phenylalanine-β-ester, peptide production can be carried out more easily without the need to prepare a genetically modified strain. It is preferable in that it can be performed. On the other hand, a genetically modified strain transformed so as to be able to express an active peptide-forming enzyme that produces α-L-aspartyl-L-phenylalanine-β-ester should be modified to produce a larger amount of peptide-forming enzyme. Therefore, it may be possible to synthesize α-L-aspartyl-L-phenylalanine-β-ester more quickly and in large quantities. A microorganism of a wild strain or a genetically modified strain is cultured in a medium, and the peptide-forming enzyme is accumulated in the medium and / or in the microorganism and mixed with L-aspartic acid-α, β-diester and L-phenylalanine. Thus, α-L-aspartyl-L-phenylalanine-β-ester can be produced.

  In the case of using a culture, cultured cells, washed cells, or a cell-treated product obtained by crushing or lysing cells, it is not involved in the production of α-L-aspartyl-L-phenylalanine-β-ester. In many cases, there is an enzyme that degrades the produced α-L-aspartyl-L-phenylalanine-β-ester. In this case, it is preferable to add a metal protease inhibitor such as ethylenediaminetetraacetic acid (EDTA). There is. The addition amount is in the range of 0.1 mM to 300 mM, preferably 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 can be 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. 1 U is 1 μmole of α-L-aspartyl-L-phenylalanine-β- from 100 mM L-aspartic acid-α, β-dimethyl ester and 200 mM L-phenylalanine per minute at a temperature of 25 ° C. The amount of enzyme that produces methyl ester (or α-L- (β-o-methyl aspartyl) -L-phenylalanine (abbreviation: α-AMP)).

  Any L-aspartic acid-α, β-diester used in the reaction may be used as long as it can be condensed with L-phenylalanine to produce α-L-aspartyl-L-phenylalanine-β-ester. It's okay. Examples of L-aspartic acid-α, β-diester include L-aspartic acid-α, β-dimethyl ester, L-aspartic acid-α, β-diethyl ester, and the like. When L-aspartic acid-α, β-dimethyl ester is reacted with L-phenylalanine, α-L-aspartyl-L-phenylalanine-β-methyl ester (α-AMP) is converted to L-aspartic acid-α. , Β-diethyl ester and L-phenylalanine are reacted with α-L-aspartyl-L-phenylalanine-β-ethyl ester (or α-L- (β-o-ethyl aspartyl) -L-phenylalanine ( (Abbreviation: α-AEP)) is produced.

  The concentration of the starting materials L-aspartic acid-α, β-diester and L-phenylalanine is 1 mM to 10 M, preferably 0.05 M to 2 M, respectively, but it is preferable to add an equal amount or more of either substrate There are also choices as needed. 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.

  Reaction temperature is 0-60 degreeC, (alpha) -L-aspartyl-L-phenylalanine-beta-ester production | generation is possible, Preferably it is 5-40 degreeC. The reaction pH is pH 6.5 to 10.5, and α-L-aspartyl-L-phenylalanine-β-ester can be produced, preferably pH 7.0 to 10.0.

2. Microorganism used in the present invention The microorganism used in the present invention has the ability to produce α-L-aspartyl-L-phenylalanine-β-ester from L-aspartic acid-α, β-diester and L-phenylalanine. The microorganisms can be used without any particular limitation. Examples of microorganisms having the ability to produce α-L-aspartyl-L-phenylalanine-β-ester from L-aspartic acid-α, β-diester and L-phenylalanine include, for example, Aeromonas, Azotobacter, Alkagenes, Brevibacterium, Corynebacterium, Escherichia, Empedobacter, Flavobacterium, Microbacteria, Propionibacterium, Brevibacillus, Penibacillus, Pseudomonas, Serratia, Stenotrohomonanas Genus, Sphingobacterium, Streptomyces, Xanthomonas, Williopsis, Candida, Geotricum, Pichia, Saccharomyces, Torlaspola, Cellulophaga, Weekera, Pedobacter, -Shikobacter, Flexitrix, Titinophaga, Cyclobacteria, Lunella, Thermonema, Cycloserpence, Gelidibacter, Diadacter, Flameovirga, Spirosoma, Frectobacilli, Tenasibacrum, Rhodothermas The microorganisms belonging to the genus, Zoberia, Murikauda, Salegentibacter, Taxebacter, Chitophaga, Marinillaviria, Levinella, Saprospira, and Harris Comobacter can be mentioned. Things can be illustrated.

Aeromonas Hydrophila ATCC 13136
(Aeromonas hydrophila)
Azotobacter Vinelandi IFO 3741
(Azotobacter vinelandii)
Alkali Genes Fecaris FERM P-8460
(Alcaligenes faecalis)
Brevibacterium Minutifera FERM BP-8277
(Brevibacterium minutiferuna)
Corynebacterium flavesens ATCC 10340
(Corynebacterium flavescens)
Escherichia coli FERM BP-8276
(Escherichia coli)
Empedobacter Brevis ATCC 14234
(Empedobacter brevis)
Flavobacterium Resinovoram ATCC 14231
(Flavobacterium resinovorum)
Microbacterium Arborescens ATCC 4348
(Microbacterium arborescens)
Propionibacterium Shermani FERM BP-8100
(Propionibacterium shermanii)
Brevibacillus Parabrevis ATCC 8185
(Brevibacillus parabrevis)
Penibacillus albey ifo 14175
(Paenibacillus alvei)
Pseudomonas Frazi IFO 3458
(Pseudomonas fragi)
Serratia Grimesi ATCC 14460
(Serratia grimesii)
Stenotrophomonas Martofilia ATCC 13270
(Stenotrophomonas maltophilia)
Sphingobacterium sp FERM BP-8124
(Sphingobacterium sp.)
Streptomyces griseorus NRRL B-1305
(Streptomyces griseolus, aka Streptomyces lavendulae)
Xanthomonas maltophilia FERM BP-5568
(Xanthomonas maltophilia)
Williopsis Saturnas IFO 0895
(Williopsis saturnus)
Candida Magnoliae IFO 0705
(Candida magnoliae)
Geotricum Fragrance CBS 152.25
(Geotrichum flagrance, also known as Geotrichum amycelium)
Geotricum Amicerium IFO 0905
(Geotrichum amycelium)
Pichia Ciferilly IFO 0905
(Pichia ciferrii)
Saccharomyces Unisporous IFO 0724
(Saccharomyces unisporus)
Torlas Polar Del Brucchi IFO 0422
(Torulaspora delbrueckii)
Cellulofagaritica NBRC 14961
(Cellulophaga lytica)
Week Sera Bilosa NBRC 16016
(Weeksella virosa)
Pedobacter Heparinas NBRC 12017
(Pedobacter heparinus)
Persicobacter fluence NBRC 15940
(Persicobacter diffluens)
Flexitrix Dorothe NBRC 15987
(Flexithrix dorotheae)
Chitinofaga Pinensis NBRC 15968
(Chitinophaga pinensis)
Cyclobacterium marinum ATCC 25205
(Cyclobacterium marinum)
Renella Sriciformis ATCC 29530
(Runella slithyformis)
Thermonema Lapsum ATCC 43542
(Thermonema lapsum)
Cycloserpence Brutonensis ATCC 700359
(Psychroserpens burtonensis)
Geridi Bacter Argens ATCC 700364
(Gelidibacter algens)
Dead Bacter Fermentance ATCC 700827
(Dyadobacter fermentans)
Flameo Bilga Aprica NBRC 15941
(Flammeovirga aprica)
Spirosoma ring array DSMZ 74
(Spirosoma linguale)
Frecto Bacillus Major DSMZ 103
(Flectobacillus major)
Tenashi Bacram Maritimum ATCC43398
(Tenacibaculum maritimum)
Rhodotamus Marinas DSMZ 4252
(Rhodothermus marinus)
Zoberia Galactanibolans DSMZ 12802
(Zobellia galactanivorans)
Murikauda Ruestringensis DSMZ 13258
(Muricauda ruestringensis)
Salegentibacter Salegens DSMZ 5424
(Salegentibacter salegens)
Taxiobacter gel pull pull essence DSMZ 11116
(Taxeobacter gelupurpurascens)
Chitofaga Fucchin Sonny NBRC 15051
(Cytophaga hutchinsonii)
Marinillaville Salmoni Color NBRC 15948
(Marinilabilia salmonicolor)
REVINELLA COHAERENCE ATCC 23123
(Lewinella cohaerens)
Saprospira Grandis ATCC 23119
(Saprospira grandis)
Harris Comenobacter Hydrosis ATCC 27775
(Haliscomenobacter hydrossis)

Among the above strains, those with a FERM number are deposited at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (1st, 1st East, 1st Street, Tsukuba City, Ibaraki, Japan). You can get a lot by referring to the number.
Among the above strains, those with ATCC numbers are deposited with the American Type Culture Collection (POBox 1549 Manassas, VA 20110, the United States of America), and will be sold with reference to each number. be able to.
Among the above strains, those with IFO numbers are deposited with the Fermentation Research Institute (2-17-85 Juzahoncho, Yodogawa-ku, Osaka, Japan). Can receive.
Among the above strains, those with NBRC numbers are deposited in the Biological Genetic Resource Department of the National Institute for Product Evaluation (2-5-8, Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan). You can get a lot by referring.
Among the above strains, those with DSMZ numbers are deposited in the Dutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures) (Mascheroder Weg 1b, 38124 Braunschweig, Germany). You can get a lot by referring to.

  Like the above strains, those with a FERM number are deposited at the National Institute of Advanced Industrial Science and Technology Patent Microorganism Depositary Center (Central 1-1, Higashi 1-1-1 Tsukuba, Ibaraki, Japan 305-8586) It is a microorganism that has been assigned a trust number. Alkaligenes faecalis FERM P-8460 is a microorganism that was deposited on September 30, 1985 and was assigned the deposit number FERM P-8460. Propionibacterium Shermani FERM P-9737 was originally deposited on December 4, 1987, transferred to an international deposit based on the Budapest Treaty on July 1, 2002, and assigned the accession number FERM BP-8100. Microorganisms. Xanthomonas maltophilia FERM BP-5568 was originally deposited on June 14, 1995 and transferred to an international deposit on June 14, 1996 under the Budapest Treaty. Brevibacterium Minutifera FERM BP-8277 is entrusted to the international deposit under the Budapest Treaty on January 20, 2002. Escherichia coli FERM BP-8276 was commissioned to an international organization based on the Budapest Treaty on January 20, 2002.

  Empedobacter Brevis ATCC 14234 strain (FERM P-18545 strain, FERM BP-8113 strain) was established on October 1, 2001 by the National Institute of Advanced Industrial Science and Technology Patent Microbiology Depositary Center (Ibaraki, 305-8586, Japan) Deposited at Tsukuba City East 1-chome, 1st Central 6) and given the FERM P-18545 deposit number, and on July 8, 2002, at the Patent Deposit Center of the National Institute of Advanced Industrial Science and Technology, the Budapest Treaty The microorganism was transferred to the deposit based on the above and given FERM BP-8113 (indication of microorganism: Empedobacter brevis AJ 13933 strain).

  Sphingobacterium sp. AJ 110003 strain was deposited on July 22, 2002 at the National Institute of Advanced Industrial Science and Technology Patent Microorganism Depositary, and has been assigned a deposit number of FERM BP-8124.

  The AJ 110003 (FERM BP-8124) strain was identified as the sphingobacterium sp. By the following classification experiment. FERM BP-8124 strain is gonococcus (0.7-0.8 × 1.5-2.0 μm), gram negative, no sporulation, no motility, colony shape is circular, smooth all edges, low convex, It was identified as a bacterium belonging to sphingobacterium because of its glossy, pale yellow color, growth at 30 ° C., catalase positive, oxidase positive, and OF test (glucose) negative. Furthermore, nitrate reduction negative, indole production negative, acid generation from glucose negative, arginine dihydrolase negative, urease positive, esculin hydrolysis positive, gelatin hydrolysis negative, β-galactosidase positive, glucose utilization positive, L-arabinose utilization Negative, D-mannose utilization positive, D-mannitol utilization negative, N-acetyl-D-glucosamine utilization positive, maltose utilization positive, potassium gluconate utilization negative, n-capric acid utilization negative, adipic acid utilization It is similar to the properties of sphingobacterium multiborum or sphingobacterum spiritibolum due to its negative properties, negative dl-malate utilization, negative use of sodium citrate, negative use of phenyl acetate, and positive cytochrome oxidase. There was found. Furthermore, as a result of homology analysis of the base sequence of the 16S rRNA gene, it showed the highest homology (98.8%) with the sphingobacterium multiborum. It was identified as SP (Sphingobacterium sp.).

  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, nitrates such as sodium nitrate and potassium nitrate, peptone, yeast extract, meat extract, corn steep Organic nitrogen compounds such as 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 peptide production method of the present invention, an enzyme having the ability to selectively peptide bond L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester is used. In the method for producing a peptide of the present invention, as long as the enzyme has such activity, it is not limited to its origin and acquisition method. Hereinafter, purification of the enzyme used in the present invention, utilization of genetic engineering techniques, and the like will be described.

(3-1) Microorganism having an enzyme that can be used in the production method of the present invention The microorganism producing the enzyme of the present invention includes α-L-aspartyl-from L-aspartic acid-α, β-diester and L-phenylalanine. Any microorganism having an ability to produce L-phenylalanine-β-ester can be used. For example, Aeromonas, Azotobacter, Alkagenes, Brevibacterium, Corynebacterium, Escherichia, Empedobacter, Flavobacterium, Microbacterium, Propionibacterium, Brevibacillus, Penibacillus Pseudomonas sp. Weeksera, Pedobacter, Pericobacter, Flexitrix, Titinophaga, Cyclobacterium, Lunella, Thermonema, Cycloserpense, Gelidibacter, Dia Bacter, Flameovirga, Spirosoma, Flectobacillus, Tenacibacrum, Rhodotermas, Zoberia, Murikauda, Salegentibacter, Taxobacter, Chitophaga, Marinillaviria, Levinella, Saprospira Examples include bacteria belonging to a genus selected from the group consisting of a genus, and more specifically, Empedobacter brevis ATCC 14234 strain (FERM P-18545 strain, FERM BP-8113 strain), Sphingobacterium sp. (Sphingobacterium sp.) FERM BP-8124 strain, Pedobacter heparinus IFO 12017 strain, Taxeobacter gelupurpurascens DSMZ 11116 strain, Examples thereof include Cyclobacterium marinum ATCC 25205 strain, Cycloserpens burtonensis ATCC 70000359 strain, and the like. Empedobacter brevis ATCC 14234 strain (FERM P-18545 strain, FERM BP-8113 strain) and Sphingobacterium sp. FERM BP-8124 strain, Pedobacter heparinus IFO 12017 strain, Taxeobacter gel Sense (Taxeobacter gelupurpurascens) DSMZ 11116 strain, Cyclobacterium marinum ATCC 25205 strain, Cycloserpens burtonensis ATCC 70000359 strain, etc. are obtained by the present inventors as L-aspartic acid-α, β- Selected as a result of searching for bacteria producing enzymes that produce α-L-aspartyl-L-phenylalanine-β-ester in high yield from diester and L-phenylalanine It is biological.

(3-2) Purification of enzyme As described above, the peptide-forming enzyme used in the present invention can be purified from bacteria belonging to the genus Empedobacter, for example. As an example of purifying the enzyme, a method for isolating and purifying a peptide-forming enzyme from Empedobacter brevis is described.

  First, from the cells of Empedobacter brevis, for example, FERM BP-8113 strain, the cells are destroyed by a physical method such as ultrasonic disruption or an enzyme method using cell wall lysing enzyme, etc., and insoluble by centrifugation or the like. A bacterial cell extract is prepared by removing the fraction. Peptide-producing enzyme is purified by fractionating the bacterial cell extract obtained in this way by combining conventional protein purification methods, anion exchange chromatography, cation exchange chromatography, gel filtration chromatography, etc. can do.

As a carrier for anion exchange chromatography, Q-Sepharose is used.
HP (made by Amersham) is mentioned. When the extract containing the present enzyme is passed through a column packed with these carriers, the enzyme is recovered in the non-adsorbed fraction under the condition of pH 8.5.

  As the carrier for cation chromatography, MonoS HR (manufactured by Amersham) can be mentioned. 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. For example, when MonoS HR is used, the present enzyme adsorbed on the column is eluted with about 0.2 to 0.5 M NaCl.

  The enzyme purified as described above can be further purified uniformly by gel filtration chromatography or the like. Examples of the carrier for gel filtration chromatography include Sephadex 200 pg (manufactured by Amersham).

  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. The internal amino acid sequences of the present enzyme purified as described above are shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing.

(3-3) Isolation of DNA, preparation of transformant and purification of peptide-forming enzyme (3-3-1) Isolation of DNA The present inventors firstly employed Empedobacter brevis FERM BP-8113 strain and the like. The present inventors succeeded in isolating one kind of peptide-forming enzyme DNA that can be used in the peptide production method of the present invention.

  The DNA comprising the nucleotide sequence of nucleotide numbers 61 to 1908 set forth in SEQ ID NO: 5, which is the DNA of the present invention, is Empedobacter brevis FERM BP-8113 strain (depositing institution; 1) Higashi 1-chome, 1-chome Tsukuba, Ibaraki, Japan, Chuo No. 6, International deposit transfer date; July 8, 2002). The DNA consisting of the base sequences of base numbers 61 to 1908 is a code sequence (CDS) part. The base sequence of base numbers 61 to 1908 includes a signal sequence region and a mature protein region. The signal sequence region is a region of base numbers 61 to 126, and the mature protein region is a region of base numbers 127 to 1908. That is, the present invention provides both a peptide enzyme protein gene containing a signal sequence and a peptide enzyme protein gene as a mature protein. The signal sequence contained in the sequence described in SEQ ID NO: 5 is a kind of leader sequence, and it is presumed that the main function of the leader peptide encoded by the leader sequence is to secrete it from the cell membrane to the outside of the cell membrane. The protein encoded by base numbers 127 to 1908, that is, the site excluding the leader peptide is a mature protein, and is presumed to exhibit high peptide production activity.

  The DNA of the present invention comprising the nucleotide sequence of nucleotide numbers 61 to 1917 described in SEQ ID NO: 11 is sphingobacterium sp. FERM BP-8124 strain (deposited organization; National Institute of Advanced Industrial Science and Technology) Deposit center, depository institution address; 1-1-1 Higashi 1-chome Tsukuba-shi, Ibaraki, Japan Chuo 6th, international deposit date; July 22, 2002). DNA consisting of the base sequences of base numbers 61 to 1917 described in SEQ ID NO: 11 is a code sequence (CDS) portion. The base sequences of base numbers 61 to 1917 include a signal sequence region and a mature protein region. The signal sequence region is a region of base numbers 61 to 120, and the mature protein region is a region of base numbers 121 to 1917. That is, the present invention provides both a peptide enzyme protein gene containing a signal sequence and a peptide enzyme protein gene as a mature protein. The signal sequence contained in the sequence described in SEQ ID NO: 11 is a kind of leader sequence, and it is presumed that the main function of the leader peptide encoded in the leader sequence region is to secrete it from the cell membrane to the outside of the cell membrane. The The protein encoded by base numbers 121 to 1917, that is, the site excluding the leader peptide is a mature protein, and is presumed to exhibit high peptide production activity.

  In addition, the DNA of the present invention, which consists of the nucleotide sequence of base numbers 61 to 1935 set forth in SEQ ID NO: 17, is Pedobacter heparinus IFO 12017 strain (deposited organization; Foundation for Fermentation Research Institute, deposited address: Osaka, Japan) 2-17-85 Jusohoncho, Yodogawa-ku). DNA consisting of the base sequence of base numbers 61 to 1935 described in SEQ ID NO: 17 is a coding sequence (CDS) part. The base sequence of base numbers 61 to 1935 includes a signal sequence region and a mature protein region. The signal sequence region is a region of base numbers 61 to 126, and the mature protein region is a region of base numbers 127 to 1935. That is, the present invention provides both a peptide enzyme protein gene containing a signal sequence and a peptide enzyme protein gene as a mature protein. The signal sequence contained in the sequence described in SEQ ID NO: 17 is a kind of leader sequence, and it is presumed that the main function of the leader peptide encoded in the leader sequence region is to secrete it from the cell membrane to the outside of the cell membrane. The The protein encoded by base numbers 127 to 1935, that is, the site excluding the leader peptide is a mature protein, and is presumed to exhibit high peptide production activity.

  In addition, the DNA of the present invention, which has the base sequence of base numbers 61 to 1995 set forth in SEQ ID NO: 22, is a Taxobacter gelpurpurense DSMZ 11116 strain (deposited institution; Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures, deposited address: Mascheroder Weg 1b, 38124 Braunschweig, Germany) DNA consisting of the base sequence of base numbers 61 to 1995 described in SEQ ID NO: 22 is a coding sequence (CDS). The signal sequence region and the mature protein region are included in the base sequence of base numbers 61 to 1995. The signal sequence region is the region of base numbers 61 to 126, and the mature protein region is the base number. 127 to 1995. That is, the present invention includes a signal sequence. It provides both a peptide enzyme protein gene and a peptide enzyme protein gene as a mature protein The signal sequence contained in the sequence of SEQ ID NO: 22 is a kind of leader sequence and is encoded by the leader sequence region. The main function of the leader peptide is presumed to be secreted from inside the cell membrane to the outside of the cell membrane, the protein encoded by base numbers 127 to 1995, that is, the site excluding the leader peptide is a mature protein, and has high peptide production activity. It is estimated that

  The DNA comprising the nucleotide sequence of nucleotide numbers 29 to 1888 described in SEQ ID NO: 24, which is the DNA of the present invention, is Cyclobacterium marinum ATCC 25205 strain (depositing institution; American Type Culture Collection, depositing address; POBox 1549 Manassas, VA 20110, the United States of America). DNA consisting of the nucleotide sequences of nucleotide numbers 29 to 1888 described in SEQ ID NO: 24 is a coding sequence (CDS) portion. The base sequence of base numbers 29 to 1888 includes a signal sequence region and a mature protein region. The signal sequence region is a region of base numbers 29 to 103, and the mature protein region is a region of base numbers 104 to 1888. That is, the present invention provides both a peptide enzyme protein gene containing a signal sequence and a peptide enzyme protein gene as a mature protein. The signal sequence contained in the sequence described in SEQ ID NO: 24 is a kind of leader sequence, and it is presumed that the main function of the leader peptide encoded in the leader sequence region is to secrete it from the cell membrane to the outside of the cell membrane. The The protein encoded by base numbers 104 to 1888, that is, the site excluding the leader peptide is a mature protein, and is presumed to exhibit high peptide production activity.

  The DNA comprising the nucleotide sequence of nucleotide numbers 61 to 1992 set forth in SEQ ID NO: 26, which is the DNA of the present invention, is Cycloserpens brutonensis ATCC 7000035 strain (depositing organization; American Type Culture Collection, depositing address; POBox 1549 Manassas, VA 20110, the United States of America). The DNA consisting of the base sequence of base numbers 61 to 1992 described in SEQ ID NO: 26 is a code sequence (CDS) part. The base sequence of base numbers 61 to 1992 includes a signal sequence region and a mature protein region. The signal sequence region is a region of base numbers 61 to 111, and the mature protein region is a region of base numbers 112 to 1992. That is, the present invention provides both a peptide enzyme protein gene containing a signal sequence and a peptide enzyme protein gene as a mature protein. The signal sequence contained in the sequence shown in SEQ ID NO: 26 is a kind of leader sequence, and it is presumed that the main function of the leader peptide encoded in the leader sequence region is to secrete it from the cell membrane to the outside of the cell membrane. The It is presumed that the protein encoded by base numbers 112 to 1992, that is, the site excluding the leader peptide is a mature protein and exhibits high peptide production activity.

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

  The DNA encoding the enzyme that can be used in the present invention is chromosomal DNA such as Empedobacter brevis, Sphingobacterium sp, Pedobacter heparinus, Taxobacter gelpurpurense, Cyclobacterium marinum, or Cycloserpens brutenensis, or DNA From the library, it can be obtained by PCR (polymerase chain reacion, White, TJ et al; Trends Genet., 5, 185 (1989)) or hybridization. Primers used for PCR can be designed based on the internal amino acid sequence determined based on the peptide-forming enzyme purified as described in the section (3) above. In addition, since the base sequences of the peptide-generating enzyme genes (SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 17, SEQ ID NO: 22, SEQ ID NO: 24 and SEQ ID NO: 26) have been clarified by the present invention, based on these base sequences Primers or probes for hybridization can be designed and isolated using probes. 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. Taking the case of amplifying the region containing both the leader sequence and the mature protein coding region described in SEQ ID NO: 5 as an example, specifically, as a 5′-side primer, SEQ ID NO: 5 is more than base number 61 Examples of the primer having the base sequence in the upstream region include a primer having a sequence complementary to the base sequence in the region downstream from the base number 1908 as the 3′-side primer.

  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) according to a conventional method. 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 DNA encoding the enzyme that can be used in the method for producing a peptide of the present invention is substantially the same as the DNA consisting of CDS described in SEQ ID NO: 5 in the Sequence Listing, whether or not it contains a leader sequence. Identical DNA is also included. That is, from a DNA encoding the present enzyme having a mutation or a cell retaining the same, a DNA comprising a nucleotide sequence complementary to the CDS described in SEQ ID NO: 5 in the sequence listing or a probe and a string prepared from the nucleotide sequence By isolating a DNA that hybridizes under a gentle condition and encodes a protein having a peptide-forming activity, a DNA substantially identical to the DNA of the present invention can be obtained.

  The DNA of the present invention includes substantially the same DNA as the DNA consisting of CDS described in SEQ ID NO: 11 in the sequence listing, whether or not the leader sequence is included. That is, from a DNA encoding the present enzyme having a mutation or a cell holding the same, a DNA comprising a nucleotide sequence complementary to the CDS described in SEQ ID NO: 11 of the Sequence Listing or a probe and a string prepared from the nucleotide sequence By isolating a DNA that hybridizes under a gentle condition and encodes a protein having a peptide-forming activity, a DNA substantially identical to the DNA of the present invention can be obtained.

  The DNA of the present invention includes substantially the same DNA as the DNA consisting of CDS described in SEQ ID NO: 17 in the sequence listing, whether or not it contains a leader sequence. That is, from a DNA encoding the present enzyme having a mutation or a cell retaining the enzyme, a DNA comprising a nucleotide sequence complementary to CDS described in SEQ ID NO: 17 of the sequence listing or a probe and a string prepared from the nucleotide sequence By isolating a DNA that hybridizes under a gentle condition and encodes a protein having a peptide-forming activity, a DNA substantially identical to the DNA of the present invention can be obtained.

  The DNA of the present invention includes DNA substantially the same as the DNA consisting of CDS described in SEQ ID NO: 22 in the sequence listing, whether or not the leader sequence is included. That is, from a DNA encoding the present enzyme having a mutation or a cell holding the same, a DNA comprising a nucleotide sequence complementary to the CDS described in SEQ ID NO: 22 in the sequence listing or a probe and a string prepared from the nucleotide sequence By isolating a DNA that hybridizes under a gentle condition and encodes a protein having a peptide-forming activity, a DNA substantially identical to the DNA of the present invention can be obtained.

  The DNA of the present invention includes DNA substantially identical to the DNA consisting of CDS described in SEQ ID NO: 24 in the Sequence Listing, whether or not it contains a leader sequence. That is, from a DNA encoding the present enzyme having a mutation or a cell retaining the same, a DNA comprising a nucleotide sequence complementary to the CDS described in SEQ ID NO: 24 of the Sequence Listing or a probe and a string prepared from the nucleotide sequence By isolating a DNA that hybridizes under a gentle condition and encodes a protein having a peptide-forming activity, a DNA substantially identical to the DNA of the present invention can be obtained.

  The DNA of the present invention includes DNA substantially the same as the DNA consisting of CDS described in SEQ ID NO: 26 in the sequence listing, whether or not it contains a leader sequence. That is, from a DNA encoding the present enzyme having a mutation or a cell holding the same, a DNA comprising a nucleotide sequence complementary to CDS described in SEQ ID NO: 26 of the Sequence Listing or a probe and a string prepared from the nucleotide sequence By isolating a DNA that hybridizes under a gentle condition and encodes a protein having a peptide-forming activity, a DNA substantially identical to the DNA of the present invention can be obtained.

  The probe can be prepared by a conventional method based on the base sequence described in SEQ ID NO: 5, for example. A method for picking up DNA that hybridizes with a probe using a probe and isolating the target DNA 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, and cutting out and extracting the base sequence to be used as a probe with a restriction enzyme. The part to be cut out can be adjusted according to the target DNA.

  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 clearly quantify this condition, for example, DNAs having high homology, for example, 50% or more, more preferably 80% or more, and more preferably 90% or more have high homology. 60 ° C., 1 × SSC, 0.1% SDS, preferably 0, which is a condition in which DNAs hybridize and DNAs having lower homology do not hybridize, or washing conditions of normal Southern hybridization Conditions for hybridizing at a salt concentration corresponding to 0.1 × SSC and 0.1% SDS are included. 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 base sequence that hybridizes under stringent conditions as described above, about 50% or more of a protein having an amino acid sequence encoded by the original base sequence under conditions of 50 ° C. and pH 8, It is desirable that the enzyme activity is maintained at 80% or more, more preferably 90% or more. For example, in the case of a base sequence that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence of base numbers 127 to 1908 of the base sequence set forth in SEQ ID NO: 5, Enzyme activity of about 8% or more, more preferably 80% or more, more preferably 90% or more of the protein having the amino acid sequence of amino acid residues 23 to 616 in the amino acid sequence shown in SEQ ID NO: 6 under the condition of pH 8 It is desirable to hold

  The amino acid sequence encoded by the CDS described in SEQ ID NO: 5 in the sequence listing is shown in SEQ ID NO: 6 in the sequence listing. The amino acid sequence encoded by the CDS described in SEQ ID NO: 11 in the sequence listing is shown in SEQ ID NO: 12 in the sequence listing. The amino acid sequence encoded by the CDS described in SEQ ID NO: 17 in the sequence listing is shown in SEQ ID NO: 18 in the sequence listing. The amino acid sequence encoded by the CDS described in SEQ ID NO: 22 in the sequence listing is shown in SEQ ID NO: 23 in the sequence listing. The amino acid sequence encoded by the CDS described in SEQ ID NO: 24 in the sequence listing is shown in SEQ ID NO: 25 in the sequence listing. The amino acid sequence encoded by the CDS described in SEQ ID NO: 26 in the sequence listing is shown in SEQ ID NO: 27 in the sequence listing.

  The entire amino acid sequence described in SEQ ID NO: 6 includes a leader peptide and a mature protein region, amino acid residues 1 to 22 are leader peptides, and 23 to 616 are mature protein regions.

  The entire amino acid sequence described in SEQ ID NO: 11 includes a leader peptide and a mature protein region, amino acid residues 1 to 20 are leader peptides, and 21 to 619 are mature protein regions.

  The entire amino acid sequence described in SEQ ID NO: 18 includes a leader peptide and a mature protein region, amino acid residues 1 to 22 are leader peptides, and 23 to 625 are mature protein regions.

  The entire amino acid sequence described in SEQ ID NO: 23 includes a leader peptide and a mature protein region, amino acid residues 1 to 22 are leader peptides, and 23 to 645 are mature protein regions.

  The entire amino acid sequence described in SEQ ID NO: 25 includes a leader peptide and a mature protein region, amino acid residues 1 to 25 are leader peptides, and 26 to 620 are mature protein regions.

  The entire amino acid sequence described in SEQ ID NO: 27 includes a leader peptide and a mature protein region, amino acid residues 1 to 17 are leader peptides, and 18 to 644 are mature protein regions.

  The protein encoded by the DNA of the present invention is a protein whose mature protein has peptide-forming activity, and whether or not it contains a leader peptide, SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 18, DNA encoding a protein that is substantially the same as the protein having the amino acid sequence set forth in SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27 is also included in the DNA that can be used in the present invention (note that the universal codon The base sequence is specified from the amino acid sequence according to the code). That is, according to the present invention, DNAs encoding the proteins shown in the following (A) to (X) are provided.

(A) Protein having the amino acid sequence of amino acid residues 23 to 616 in the amino acid sequence described in SEQ ID NO: 6 in the sequence listing (B) Amino acid residue number 23 in the amino acid sequence described in SEQ ID NO: 6 in the sequence listing ˜616 having an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, and / or inversions, and L-phenylalanine is converted to L-aspartic acid-α, β -Protein having the activity of selectively binding a peptide to the α-ester site of the diester (C) Protein having the amino acid sequence of amino acid residues 21 to 619 among the amino acid sequences shown in SEQ ID NO: 12 of the Sequence Listing (D) In the amino acid sequence of amino acid residues 21 to 619 in the amino acid sequence described in SEQ ID NO: 12 in the sequence listing, 1 or a number Of amino acid substitutions, deletions, insertions, additions, and / or inversions, and L-phenylalanine is selectively used as an α-ester site of L-aspartic acid-α, β-diester A protein having an activity of peptide binding (E) The amino acid sequence described in SEQ ID NO: 18 of the protein (F) sequence table having the amino acid sequence of amino acid residues 23 to 625 among the amino acid sequences described in SEQ ID NO: 18 of the sequence table Among the amino acid sequences of amino acid residues 23 to 625, and having an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or several amino acids, and L-phenylalanine is L A protein (G) sequence having an activity of selectively binding a peptide to the α-ester site of aspartic acid-α, β-diester The amino acid sequence of amino acid residues 23 to 645 in the amino acid sequence of SEQ ID NO: 23 of the protein (H) sequence table having the amino acid sequence of amino acid residues 23 to 645 of the amino acid sequence of SEQ ID NO: 23 Having an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions and / or inversions, and L-phenylalanine is α- of L-aspartic acid-α, β-diester A protein having an activity of selectively binding a peptide to an ester site (I) A protein having an amino acid sequence of amino acid residues 26 to 620 among amino acid sequences set forth in SEQ ID NO: 25 of the sequence listing (J) SEQ ID NO: of the sequence listing In the amino acid sequence of amino acid residues 26 to 620 of the amino acid sequence described in 25, one or several amino acids Having an amino acid sequence containing no acid substitution, deletion, insertion, addition, and / or inversion, and L-phenylalanine selectively at the α-ester site of L-aspartic acid-α, β-diester The amino acid sequence shown in SEQ ID NO: 27 of the protein (L) sequence table having the amino acid sequence of amino acid residues 18 to 644 out of the amino acid sequence shown in SEQ ID NO: 27 of the protein (K) sequence table having the activity to bind the peptide In the amino acid sequence of amino acid residues 18 to 644, and having an amino acid sequence including substitution, deletion, insertion, addition, and / or inversion of one or several amino acids, and L-phenylalanine is L -Sequence of the protein (M) sequence table having the activity of selectively binding a peptide to the α-ester site of aspartic acid-α, β-diester Amino acids comprising one or several amino acid substitutions, deletions, insertions, additions and / or inversions in the amino acid sequence set forth in SEQ ID NO: 6 in the protein (N) sequence listing having the amino acid sequence set forth in No. 6 SEQ ID NO: of the protein (O) sequence listing having a sequence and having a mature protein region that selectively binds L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester Amino acid sequence comprising one or several amino acid substitutions, deletions, insertions, additions, and / or inversions in the amino acid sequence shown in SEQ ID NO: 12 of the protein (P) sequence listing having the amino acid sequence shown in FIG. And L-phenylalanine is selectively bonded to the α-ester site of L-aspartic acid-α, β-diester In the amino acid sequence shown in SEQ ID NO: 18 in the protein (R) sequence list, which has the amino acid sequence shown in SEQ ID NO: 18 in the protein (Q) sequence list, including the mature protein region having the activity of one or several amino acids It has an amino acid sequence including substitution, deletion, insertion, addition and / or inversion, and L-phenylalanine is selectively peptide-bonded to the α-ester site of L-aspartic acid-α, β-diester. Substitution of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 23 of the protein (T) sequence list having the amino acid sequence shown in SEQ ID NO: 23 of the protein (S) sequence list including the mature protein region having activity , Deletions, insertions, additions, and / or inversions, and L-phenylalanine is L- A protein (V) having the amino acid sequence shown in SEQ ID NO: 25 in the protein (U) sequence table, which contains a mature protein region having an activity of selectively binding a peptide to the α-ester site of aspartic acid-α, β-diester It has an amino acid sequence containing substitution, deletion, insertion, addition, and / or inversion of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 25 in the Table, and L-phenylalanine is converted to L-asparagine Protein (X) Sequence Listing having the amino acid sequence set forth in SEQ ID NO: 27 in the Protein (W) Sequence Listing, which contains a mature protein region having an activity of selectively binding a peptide to the α-ester site of the acid-α, β-diester 1 or several amino acid substitutions, deletions, insertions, additions, and Has an amino acid sequence comprising a beauty / or inversions, and, L- phenylalanine L- aspartic acid-.alpha., proteins comprising the mature protein region having a selectively active for peptide bond α- ester moiety β- diester

  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. However, (B), (D), (F), (H), (J), (L), (N), (P), (R), (T), (V), (X) In the case of an amino acid sequence containing substitution, deletion, insertion, addition and / or inversion of one or several amino acid residues in the amino acid sequence of the protein, a state containing no mutation at 50 ° C. and pH 8 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. For example, in the case of (B), (B) including substitution, deletion, insertion, addition and / or inversion of one or several amino acid residues in the amino acid sequence set forth in SEQ ID NO: 6 in the sequence listing In the case of an amino acid sequence, enzyme activity of about half 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: 6 in the sequence listing under conditions of 50 ° C. and pH 8 It is desirable to hold

  For amino acid mutations as shown in (B) above, the nucleotide sequence is modified by, for example, site-specific mutagenesis so that the amino acid at a specific site of this enzyme gene is substituted, deleted, inserted, or added. Can be obtained. The modified DNA as described above can also be obtained by a conventionally known mutation treatment. Mutation treatment includes a method of treating DNA encoding the enzyme in vitro with hydroxylamine or the like, and an Escherichia bacterium carrying the DNA encoding the enzyme by ultraviolet irradiation or N-methyl-N′-nitro-N -A method of treating with a mutating agent usually used for artificial mutation such as nitrosoguanidine (NTG) or nitrous acid.

  Moreover, naturally occurring mutations such as differences between microorganism species or strains are included in the above-described base substitution, deletion, insertion, addition, and / or inversion. By substantially expressing the DNA having the mutation as described above in an appropriate cell and examining the enzyme activity of the expression product, it is substantially the same as the protein shown in SEQ ID NO: 6, 12, 18, 23, 25 or 27 in the sequence listing. DNA encoding the same protein is obtained.

(3-3-2) Production of transformant and production of peptide-forming enzyme In the method for producing a peptide of the present invention, the DNA described in (3-3-1) above is introduced into a suitable host and expressed. Peptide producing enzymes that can be used can be produced.

  As a host for expressing a protein specified by DNA, Escherichia coli such as Escherichia coli, Empedobacter bacterium, Sphingobacteria bacterium, Flavobacterium bacterium and Bacillus subtilis Various prokaryotic cells, including (Bacillus subtilis), Saccharomyces cerevisiae, Pichia stipitis, Aspergillus oryzae, and other cells such as Aspergillus oryzae

  Recombinant DNA used to introduce DNA into a host is inserted into a vector corresponding to the type of host to be expressed in a form in which the protein encoded by the DNA can be expressed. Can be prepared. As a promoter for expressing the DNA of the present invention, when a promoter specific to a peptide-generating enzyme gene such as Empedobacter brevis 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 DNA of the present invention and expressed under the control of the promoter.

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

  When a protein is mass-produced using recombinant DNA technology, a form in which the protein associates in a transformant producing the protein to form an inclusion body of the protein is also mentioned as a preferred embodiment. 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. When preparing a peptide-generating enzyme in a microorganism such as Escherichia coli, only the DNA of the mature protein region that does not contain the leader sequence is incorporated even if the DNA encoding the precursor protein containing the leader sequence is incorporated as the protein coding sequence. However, it can be appropriately selected depending on the production conditions, form, use conditions, etc. of the enzyme to be produced.

  As a promoter for expressing DNA encoding a peptide-forming enzyme, a promoter usually used for heterologous protein production in E. coli can be used. For example, T7 promoter, lac promoter, trp promoter, trc promoter, tac promoter, lambda phage Powerful promoters such as PR promoter and PL promoter. Moreover, as a vector, pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219, pMW218, etc. can be used. In addition, phage DNA vectors can also be used. Furthermore, an expression vector containing a promoter and capable of expressing the inserted DNA 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. Ligation may be performed at an appropriate restriction enzyme site, or synthetic DNA 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 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 plasmids, pBR322 plasmids, and derivatives thereof. Here, the “derivative” means one obtained by modifying a plasmid by base substitution, deletion, insertion, addition and / or inversion. 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 DNA encoding a promoter, a peptide-forming enzyme or a gene encoding a fusion protein of a peptide-forming enzyme and another protein, or in some cases a terminator-linked DNA fragment, and vector DNA are ligated to obtain recombinant DNA.

  When E. coli is transformed with the recombinant DNA and the E. coli is cultured, a peptide-forming enzyme or a fusion protein of a 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.

<2> Method for Producing α-L-Aspartyl-L-Phenylalanine-α-Methyl Ester The method for producing α-APM of the present invention is the above-mentioned “<1> α-L-aspartyl-L-phenylalanine-β-ester”. First step of synthesizing α-L-aspartyl-L-phenylalanine-β-methyl ester in accordance with the “Production Method” and α-L-aspartyl-L-phenylalanine-β-methyl ester as α-L-aspartyl A second step of converting to -L-phenylalanine-α-methyl ester.

  Preferred conditions and the like in the first step are as described in the column of “<1> Production method of α-L-aspartyl-L-phenylalanine-β-ester” above. Moreover, about a 2nd process, it can carry out by a well-known method, for example, refer to the method described in patent publication 4-41155 etc., and suitable conditions. According to the method for producing α-APM of the present invention, α-APM important as a sweetener or the like can be produced simply and at a high yield at a low cost.

  EXAMPLES Hereinafter, although an Example is given and it demonstrates further in detail, this invention is not limited to these. In the measurement of the product, in addition to the confirmation (qualitative) of the thin-layer chromatogram by ninhydrin coloration, the product was quantitatively determined by high performance liquid chromatography shown below. Column: InertsiL ODS-2 (manufactured by GL Science), eluent: phosphoric acid aqueous solution (pH 2.1) containing 5.0 mM sodium 1-octanesulfonate: methanol = 100: 15-50, flow rate: 1.0 mL / min, detection 210 nm.

(Example 1) Microorganism producing α-L-aspartyl-L-phenylalanine-β-methyl ester For culturing bacteria and actinomycetes shown in Table 1-1, 20 g of glycerol, 5 g of ammonium sulfate, and monophosphate 50 mL of a medium (pH 7.0) containing 1 g of potassium, 3 g of dipotassium phosphate, 0.5 g of magnesium sulfate, 10 g of yeast extract, and 10 g of peptone was dispensed into a 500 mL Sakaguchi flask and sterilized at 115 ° C. for 15 minutes. (Medium 1). 1 platinum of the microorganisms shown in Table 1 cultured for 24 hours at 30 ° C. on a slope agar medium (pH 7.0) containing 5 g glucose, 10 g yeast extract, 10 g peptone, 5 g NaCl, 20 g agar in 1 L. Inoculated by ear and cultured with shaking at 30 ° C. and 120 reciprocations / minute for 17 hours. After cultivation, the cells were centrifuged and suspended in 0.1 M borate buffer (pH 9.0) containing 10 mM EDTA so as to be 100 g / L as wet cells.

For yeast culture shown in Table 1-1, 10 g glucose, 10 g glycerol, 5 g ammonium sulfate, 1 g monopotassium phosphate, 3 g dipotassium phosphate, 0.5 g magnesium sulfate, 5 g yeast extract, 5 g malt extract, 1 peptone per liter. A medium (pH 6.0) containing 10 g (50 mL) was dispensed into a 500 mL Sakaguchi flask and sterilized at 115 ° C. for 15 minutes (Medium 2). This medium 2 was cultured in a slope agar medium (pH 6.0) containing 5 g of glucose, 5 g of yeast extract, 5 g of yeast extract, 5 g of agar extract, 5 g of malt extract, 10 g of peptone, and 5 g of NaCl in 1 L, as shown in the table below. 1 platinum ear was inoculated and cultured with shaking at 25 ° C. and 120 reciprocations / min for 17 hours. After completion of the culture, the cells were centrifuged from these cultures and suspended in 0.1 M borate buffer (pH 9.0) containing 10 mM EDTA so as to be 100 g / L as wet cells.

  The microorganisms shown in Table 1-2 were cultured as follows. Cellulophaga lytica NBRC 14961 or Flexithrix dorotheae NBRC 15987 is cultured using an agar solid medium (pH7.2, sterilized at 120 ° C for 15 minutes) containing 1 g of tryptone, 1 g of yeast extract, and 15 g of agar in 1 L of Daigo artificial seawater SP It was. Cellulophaga lytica NBRC 14961 or Flexithrix dorotheae NBRC 15987 cells seed-cultured at 30 ° C. for 48 hours in this medium were applied to the same medium, and main culture was performed at 30 ° C. for 48 hours.

  For the culture of Weeksella virosa NBRC 16016, sheep blood agar medium (Nissui Plate, Nissui Pharmaceutical) was used. Weeksella virosa NBRC 16016 seed-cultured at 30 ° C. for 48 hours in this medium was applied to the same medium and cultured at 30 ° C. for 48 hours.

For the culture of Pedobacter heparinus NBRC 12017, an agar solid medium (pH 7.0p, sterilized at 120 ° C for 15 minutes) containing 10 g of peptone, 2 g of yeast extract, 1 g of MgSO ₄ · 7H ₂ O and 15 g of agar in 1 L of distilled water Using. Cells of Pedobacter heparinus NBRC 12017 seed-cultured in this medium at 30 ° C. for 48 hours were applied to the same medium, and main culture was performed at 30 ° C. for 48 hours.

For cultivation of Persicobacter diffluens NBRC 15940, 1 g of Daigo artificial seawater SP, 0.5 g of KNO ₃ , 0.1 g of sodium glycerophosphate, 1 g of trishydroxymethylaminomethane, 5 g of tryptone, 5 g of yeast extract, 15 g of agar, trace element solution agar solid medium containing 1ml was used (pH 7.0, 15 minutes sterilized at 120 ° C.) (Note that trace elements solution in distilled water _{1L, H 3 BO 4 2.85g,} MnCl 2 · 4H 2 0 1.8g, FeSO ₄ · 7H ₂ 0 1.36 g, CuCl ₂ · 2H ₂ 0 26.9 mg, ZnCl ₂ 20.8 mg, CoCl ₂ · 6H ₂ 0 40.4 mg, Na ₂ MoO ₄ · 2H ₂ 0 25.2 mg, sodium tartarate 1.77 g). The cells of Persicobacter diffluens NBRC 15940 seed-cultured at 25 ° C. for 48 hours in this medium were applied to the same medium, and cultured at 25 ° C. for 48 hours.

Chitinophaga pinensis NBRC 15968 is cultured in an agar solid medium (pH 7.0, sterilized at 120 ° C for 15 minutes) containing 3 g of bactocaditone, 1 g of yeast extract, 1.36 g of CaCl ₂ · 2H ₂ 0, and 15 g of agar in 1 L of distilled water. Was used. Cells of Chitinophaga pinensis NBRC 15968 seed-cultured at 25 ° C. for 48 hours in this medium were applied to the same medium, and main culture was performed at 25 ° C. for 48 hours.

Cyclobacterium marinum ATCC 25205 is cultured in an agar solid medium (pH 7.0, 15 minutes at 120 ° C.) containing 5 g of peptone, 1 g of yeast extract, FeSO ₄ · 7H ₂ 0.2 g, and 15 g of agar in 1 L of Daigo artificial seawater SP. Sterilization) was used. Cells of Cyclobacterium marinum ATCC 25205 seed-cultured in this medium at 25 ° C. for 48 hours were applied to the same medium, and main culture was performed at 25 ° C. for 48 hours.

For culturing Runella slithyformis ATCC 29530, an agar solid medium (pH 7.0, sterilized at 120 ° C. for 15 minutes) containing 1 g of peptone, 1 g of yeast extract, 1 g of glucose, and 15 g of agar in 1 L of distilled water was used. The cells of Runella slithyformis ATCC 29530 seed-cultured in this medium at 25 ° C. for 48 hours were applied to the same medium and cultured at 25 ° C. for 48 hours.

For cultivation of Thermonema lapsum ATCC 43542, 1 g of distilled water, 0.2 g of Nitrilotriacetic acid, 2 ml of 0.03% FeCl ₃ solution, 0.12 g of CaSO ₄ · 2H ₂ 0, 0.2 g of MgSO ₄ · 7H ₂ 0, NaCl 0 .016 g, KNO ₃ 0.21 g, NaNO ₃ 1.4 g, Na ₂ HPO ₄ 0.22 g, trace element solution 2 ml, agar 15 g agar (pH 8.2, sterilized at 120 ° C. for 15 minutes) was used ( In addition, the trace element solution is 1 ml of distilled water, H ₂ SO ₄ 0.5 ml, MnSO ₄ 2.2 g, ZnSO ₄ 0.5 g, H ₃ BO ₃ 0.5 g, CuSO ₄ 0.016 g, Na ₂ MoO ₄ 0.025 g, CoCl ₂ Including 0.046g). The cells of Thermonema lapsum ATCC 43542 seed-cultured at 60 ° C. for 48 hours in this medium were applied to the same medium, and main culture was performed at 25 ° C. for 48 hours.

  Marine Agar 2216 (Difco) was used for culturing Gelidibacter algens ATCC 700364, Lewinella cohaerens ATCC 23123, Psychroserpens burtonensis ATCC 700359 or Salegentibacter salegens DSMZ 5424. In this medium, in the case of Gelidibacter algens ATCC 700364 or Psychroserpens burtonensis ATCC 700359, the seed cells cultured at 10 ° C. for 72 hours were applied to the same medium and cultured at 10 ° C. for 72 hours. In the case of Lewinella cohaerensATCC 23123, the seed cells cultured at 30 ° C. for 48 hours were applied to the same medium and cultured at 30 ° C. for 48 hours. In the case of Salegentibacter salegens DSMZ 5424, the seed cells cultured at 25 ° C. for 48 hours were applied to the same medium and cultured at 25 ° C. for 48 hours.

For the culture of Dyadobacter fermentans ATCC 700827, 1 g of distilled water was charged with 0.8 g of NH ₄ Cl, 0.25 g of KH ₂ PO ₄ , 0.4 g of K ₂ HPO ₄ , 0.505 g of KNO ₃ , CaCl ₂ · 2H ₂ 0 15 mg , MgCl ₂ · 6H ₂ 0 20 mg, FeSO ₄ · 7H ₂ 0 7 mg, Na ₂ SO ₄ 5 mg, MnCl ₂ · 4H ₂ 0 5 mg, H ₃ BO ₃ 0.5 mg, ZnCl ₂ 0.5 mg, CoCl ₂ · 6H ₂ 0 0.5 mg, NiSO ₄ · 6H ₂ 0.5 mg, CuCl ₂ · 2H ₂ 0 0.3 mg, Agar containing Na ₂ MoO ₄ · 2H ₂ 10 mg, yeast extract 0.5 g, peptone 0.5 g, casamino acid 0.5 g, dextrose 0.5 g, soluble starch 0.5 g, sodium pyruvate 0.5 g, agar 15 g The cells of Dyadobacter fermentans ATCC 700827 seed-cultured in this medium using solid medium (pH 7.0, sterilized at 120 ° C. for 15 minutes) at 25 ° C. for 48 hours were applied to the same medium. Cultured.

  Flammeovirga aprica NBRC 15941 was cultured in an agar solid medium (pH 7.2, 120) containing 2 g of tryptone, 0.5 g of beef extract, 0.5 g of beef extract, 0.2 g of sodium acetate and 15 g of agar in 1 L of Daigo artificial seawater SP. Sterilized at 15 ° C. for 15 minutes). The cells of Flammeovirga aprica NBRC 15941 seed-cultured at 25 ° C. for 48 hours in this medium were applied to the same medium, and main culture was performed at 25 ° C. for 48 hours.

  For culturing Spirosoma linguale DSMZ 74 or Flectobacillus major DSMZ 103, use an agar solid medium (pH 7.0, sterilized at 120 ° C for 15 minutes) containing 1 g of glucose, 1 g of peptone, 1 g of yeast extract and 15 g of agar in 1 L of distilled water. It was. Cells of Spirosoma linguale DSMZ 74 or Flectobacillus major DSMZ 103 seed-cultured at 25 ° C. for 48 hours in this medium were applied to the same medium, and main culture was performed at 25 ° C. for 48 hours.

  Tenacibaculum maritimum ATCC43398 was cultured in 300 ml of distilled water and 700 ml of Daigo artificial seawater SP with an agar solid medium containing 0.5 g of tryptone, 0.5 g of yeast extract, 0.2 g of beef extract, 0.2 g of sodium acetate, and 15 g of agar ( Sterilized at pH 7.0, 120 ° C. for 15 minutes). The cells of Tenacibaculum maritimum ATCC43398 seed-cultured at 25 ° C. for 48 hours in this medium were applied to the same medium and cultured at 25 ° C. for 48 hours.

For cultivation of Rhodothermus marinus DSMZ 4252, 1 g of yeast extract 2.5 g, tryptone 2.5 g, Nitrilotriacetic acid 100 mg, CaSO ₄ · 2H ₂ 0 40 mg, MgCl ₂ · 6H ₂ 0 200 mg, 0.01 M Fe citrate 0. 5 ml, 0.5 ml of trace element solution, 100 ml of phosphate buffer, 900 ml of distilled water, and agar solid medium (pH 7.2, sterilized at 120 ° C. for 15 minutes) were used (the trace element solution was in 1 L of distilled water. Nitrilotriacetic acid 12.8 g, FeCl ₂・ 4H ₂ 0 1g, MnCl ₂・ 4H ₂ 0 0.5g, CoCl ₂・ 4H ₂ 0 0.3g, CuCl ₂・ 2H ₂ 0 50mg, Na ₂ MoO ₄・ 2H ₂ 0 50mg , including _{H 3 BO 3 20mg, KH 2} PO 4 5.44g, K 2 HPO 4 43g is. phosphate buffer containing NiCl ₂ · 6H ₂ 0 20mg of distilled water 1L). Cells of Rhodothermus marinus DSMZ 4252 seed-cultured at 60 ° C. for 48 hours in this medium were applied to the same medium, and main culture was performed at 60 ° C. for 48 hours.

  For the cultivation of Zobellia galactanivorans DSMZ 12802, an agar solid medium (Agar 1.5%, pH 7.6, sterilized at 120 ° C. for 15 minutes) containing BACTO MARINE BROTH (DIFCO 2216) was used. Bacterial cells of Zobellia galactanivorans DSMZ 12802 seed-cultured at 30 ° C. for 48 hours in this medium were applied to the same medium, and main culture was performed at 30 ° C. for 48 hours.

For the cultivation of Muricauda ruestringensis DSMZ 13258, 1 g of distilled water, 1.5 g of yeast extract, 2.5 g of peptone, 2 g of hexadecane, 17.7 g of NaCl, 0.48 g of KCl, 3.4 g of MgCl ₂ · 6H ₂ , MgSO _{4 · 7H 2 0 4.46g, CaCl} 2 0.98g, agar solid medium containing agar 15 g (pH 7.2, 15 minutes sterilized at 120 ° C.) 30 ° C. at the medium with, 48 hours, Muricauda were seeded culture The cells of ruestringensis DSMZ 13258 were applied to the same medium and cultured at 30 ° C. for 48 hours.

For cultivation of Taxeobacter gelupurpurascens DSMZ 11116, an agar solid medium (pH 7.2, sterilized at 120 ° C. for 15 minutes) containing 3 g of kaditon, 1 g of yeast extract, 1.36 g of CaCl ₂ .2H ₂ 0, 15 g of agar in 1 L of distilled water The cells of Taxeobacter gelupurpurascens DSMZ 11116 seed-cultured at 30 ° C. for 48 hours in this medium were applied to the same medium and cultured at 30 ° C. for 48 hours.

Cytophaga hutchinsonii NBRC 15051 was cultured in an agar solid medium (pH 7.2, 120 ° C.) containing 3 g of kaditon, 1 g of yeast extract, 1.36 g of CaCl ₂ .2H ₂ 0, 5 g of cellobiose and 15 g of agar in 1 L of distilled water. The cells of Cytophaga hutchinsonii NBRC 15051 seed-cultured at 30 ° C. for 48 hours in this medium using sterilization were applied to the same medium and cultured at 30 ° C. for 48 hours.

For the cultivation of Marinilabilia salmonicolor NBRC 15948, an agar solid medium (pH 7.2, 120) containing 250 g of distilled water, 750 ml of Daigo artificial seawater SP, 10 g of peptone, 2 g of yeast extract, 0.5 g of MgSO ₄ .7H ₂ 0 0.5 g, and 15 g of agar. The cells of Marinilabilia salmonicolor NBRC 15948 seed-cultured at 30 ° C. for 48 hours in this medium using sterilization at 15 ° C. for 15 minutes were applied to the same medium and cultured at 30 ° C. for 48 hours.

For culturing Saprospira grandis ATCC 23119, 1 g of Daigo artificial seawater SP, 0.5 g of KNO ₃ , 0.1 g of sodium glycerophosphate, 1 g of trishydroxymethylaminomethane, 2 g of tryptone, 2 g of yeast extract, 15 g of agar, trace element solution 1 ml of agar solid medium (pH 7.0, sterilized at 120 ° C. for 15 minutes) was used. (The trace element solution was H ₃ BO ₄ 2.85 g, MnCl ₂ · 4H ₂ 0 1.8 g, FeSO in 1 L of distilled water. ₄ · 7H ₂ 0 1.36 g, CuCl ₂ · 2H ₂ 0 26.9 mg, ZnCl ₂ 20.8 mg, CoCl ₂ · 6H ₂ 0 40.4 mg, Na ₂ MoO ₄ · 2H ₂ 0 25.2 mg, sodium tartrate 1.77 g). The cells of Saprospira grandis ATCC 23119 seed-cultured in this medium at 30 ° C. for 48 hours were applied to the same medium and cultured at 30 ° C. for 48 hours.

Haliscomenobacter hydrossis ATCC 27775 was cultured in 1 L of distilled water with 27 mg KH ₂ PO ₄ , 40 mg K ₂ HPO _4, 40 mg Na ₂ HPO ₄ · 2H ₂ 0 40 mg, CaCl ₂ · 2H ₂ 0 50 mg, MgSO ₄ · 7H ₂ 0 75 mg, FeCl ₃ · 6H ₂ 0 5 mg, MnSO ₄ · H ₂ 0 3 mg, glutamic acid 1.31 g, Trypticase Soy Broth without glucose 2.5 mg, thiamine 0.4 mg, vitamin B12 0.01 mg, glucose 2 g, trace element solution Agar solid medium (pH 7.5, sterilized at 120 ° C for 15 minutes) containing 1 ml was used. (Note that the trace element solution was ZnSO ₄ · 7H ₂ 0.1g, MnCl ₂ · 4H ₂ 0 0.03g in 1L of distilled water. , H ₃ BO ₃ 0.3g, CoCl ₂ · 6H ₂ 0 0.2g, CuCl ₂ · 2H ₂ 0 0.01g, NiCl ₂ · 6H ₂ 0 0.02g, Na ₂ MoO ₄ · H ₂ 0 0.03g included). The cells of Haliscomenobacter hydrossis ATCC 27775 seed-cultured at 25 ° C. for 48 hours in this medium were applied to the same medium, and main culture was performed at 25 ° C. for 48 hours.

  Each bacterial cell thus obtained is collected from an agar medium and suspended in 0.1 M borate buffer (pH 9.0) containing 10 mM EDTA so as to be 100 g / L as a wet bacterial cell. It became cloudy.

  100 mL borate buffer (pH 9.0) containing EDTA 10 mM, L-aspartic acid-α, β-dimethyl ester hydrochloride 100 mM, and L-phenylalanine 200 mM in 0.1 mL of a cell suspension of these microorganisms. 1 mL each was added to make a total volume of 0.2 mL, and at 20 ° C., when the microorganisms shown in Table 1-1 were used, 3 hours, when the microorganisms shown in Table 1-2 were used Reacted for 1 hour. The production amount (mM) of α-L-aspartyl-L-phenylalanine-β-methyl ester (α-AMP) at this time is shown in Table 1-1 and Table 1-2. Note that β-AMP was not detected from any microorganism.

(Reference Example 1) Microorganism producing β-L-aspartyl-L-phenylalanine-α-methyl ester The microorganisms shown in Table 2 were cultured in the same manner as in the case of the bacteria in Table 1 of Example 1. After cultivation, the cells were centrifuged and suspended in 0.1 M borate buffer (pH 9.0) containing 10 mM EDTA so as to be 100 g / L as wet cells. 100 mL borate buffer (pH 9.0) containing EDTA 10 mM, L-aspartic acid-α, β-dimethyl ester hydrochloride 100 mM, and L-phenylalanine 200 mM in 0.1 mL of a cell suspension of these microorganisms. 1 mL of each was added to make the total amount 0.2 mL, and then reacted at 30 ° C. for 2 hours. Table 2 shows the production amount (mM) of β-L-aspartyl-L-phenylalanine-α-methyl ester (β-AMP) at this time. In addition, α-AMP was not detected in any microorganism.

(Example 2) Purification of enzyme from Empedobacter brevis Medium containing 1 g of glucose, 5 g of glucose, 5 g of ammonium sulfate, 1 g of monopotassium phosphate, 3 g of dipotassium phosphate, 0.5 g of magnesium sulfate, 10 g of yeast extract, 10 g of peptone (PH 6.2) 50 mL was dispensed into a 500 mL Sakaguchi flask and sterilized at 115 ° C. for 15 minutes (medium 3). Empedobacter brevis FERM BP-8113 strain cultured in medium 3 at 30 ° C. for 16 hours in the same medium (deposited organization; National Institute of Advanced Industrial Science and Technology, Patent Organism Depositary, Depositary Organization Address; East Tsukuba City, Ibaraki, Japan 1-chome, 1-address 1 Chuo No. 6, International Deposit Transfer Date; July 8, 2002) 2 ml of inoculation, followed by shaking culture at 30 ° C. and 120 reciprocations / min for 16 hours.

  Hereinafter, the operations after centrifugation were performed on ice or at 4 ° C. The obtained culture solution was centrifuged (10,000 rpm, 15 minutes), and the cells were collected. 16 g of cells were washed with 50 mM Tris-HCl buffer (pH 8.0), suspended in 40 ml of the same buffer, and subjected to ultrasonic crushing at 195 W for 45 minutes. This ultrasonic disruption liquid was centrifuged (10,000 rpm, 30 minutes), and the ultrasonic disruption liquid supernatant was obtained by removing the fragment of disrupted cells. The ultrasonic disrupted supernatant was dialyzed overnight against 50 mM Tris-HCl buffer (pH 8.0), and the insoluble fraction was removed by ultracentrifugation (50,000 rpm, 30 minutes). A soluble fraction was obtained as a liquid. The obtained soluble fraction was applied to a Q-Sepharose HP column (manufactured by Amersham) equilibrated in advance with Tris-HCl buffer (pH 8.0), and the active fraction was collected from the non-adsorbed fraction. This active fraction is dialyzed overnight against 50 mM acetate buffer (pH 4.5), and the insoluble fraction is removed by centrifugation (10,000 rpm, 30 minutes), whereby the dialysis fraction is obtained as a supernatant. Obtained. This dialysis fraction is applied to a Mono S column (manufactured by Amersham) previously equilibrated with 50 mM acetate buffer (pH 4.5), and the enzyme is eluted with a linear concentration gradient of the same buffer containing 0 to 1 M NaCl. It was. Among the active fractions, the fraction with the least contaminating protein was applied to a Superdex 200 pg column (manufactured by Amersham) pre-equilibrated with 50 mM acetate buffer (pH 4.5) containing 1 M NaCl, and the same buffer containing 1 M NaCl. Gel filtration was performed by flowing a liquid (pH 4.5) to obtain an active fraction solution. By these operations, it was confirmed that the peptide-forming enzyme obtained in Example 2 was uniformly purified from the results of electrophoresis experiments. The activity recovery rate in the above purification step was 12.2%, and the degree of purification was 707 times.

(Example 3) Production of α-L-aspartyl-L-phenylalanine-β-methyl ester using Empedobacter brevis enzyme fraction 10 μl of MonoS fraction enzyme (about 20 U / ml) obtained in Example 2 Is added to 190 μl borate buffer (pH 9.0) containing 105.3 mM L-aspartic acid-α, β-dimethyl ester hydrochloride, 210.5 mM L-phenylalanine, 10.51 mM EDTA, and 20 Reacted at ° C. The production process of α-L-aspartyl-L-phenylalanine-β-methyl ester (α-AMP) is shown in Table 3. In addition, almost no α-L-aspartyl-L-phenylalanine-β-methyl ester was formed in the enzyme-free group.

  Furthermore, 10 μl of the MonoS fraction enzyme (about 20 U / ml) obtained in Example 2 was added to 105.3 mM L-aspartic acid-α-methyl ester hydrochloride or 105.3 mM L-aspartic acid-β-methyl. Each ester hydrochloride was reacted with 190 μl borate buffer (pH 9.0) containing 210.5 mM L-phenylalanine and 10.51 mM EDTA and reacted at 20 ° C. to produce the corresponding peptide. Was not seen.

(Example 4) Purification of enzyme from Sphingobacterium sp. Using the medium shown in Example 2, the same method as in Example 2 was carried out in the same manner as in Example 2, but the strain Sphingobacterium sp. FERM BP-8124 (deposited institution; Research Center for Patent Biological Deposits, Address of Depositary Institution; 1-1-1 Higashi 1-chome, Tsukuba City, Ibaraki, Japan 6th, International Deposit Date; July 22, 2002). Hereinafter, the operations after centrifugation were performed on ice or at 4 ° C. The obtained culture solution was centrifuged (10,000 rpm, 15 minutes), and the cells were collected. 2 g of cells were washed with 20 mM Tris-HCl buffer (pH 7.6), suspended in 8 ml of the same buffer, and subjected to ultrasonic crushing at 195 W for 45 minutes. This ultrasonic disruption liquid was centrifuged (10,000 rpm, 30 minutes), and the ultrasonic disruption liquid supernatant was obtained by removing the fragment of disrupted cells. The supernatant of this ultrasonic disruption solution was dialyzed overnight against 20 mM Tris-HCl buffer (pH 7.6), and the insoluble fraction was removed by ultracentrifugation (50,000 rpm, 30 minutes). A soluble fraction was obtained as a liquid. The obtained soluble fraction was applied to a Q-Sepharose HP column (manufactured by Amersham) previously equilibrated with Tris-HCl buffer (pH 7.6), and the active fraction was collected from the non-adsorbed fraction. This active fraction is dialyzed overnight against 20 mM acetate buffer (pH 5.0), and the insoluble fraction is removed by centrifugation (10,000 rpm, 30 minutes). Obtained. This dialysis fraction is applied to an SP-Sepharose HP column (manufactured by Amersham) previously equilibrated with 20 mM acetate buffer (pH 5.0), and the enzyme is extracted with a linear concentration gradient of the same buffer containing 0 to 1 M NaCl. The eluted active fraction was obtained.

(Example 5) Production of α-L-aspartyl-L-phenylalanine-β-methyl ester and α-L-aspartyl-L-phenylalanine-β-ethyl ester using an enzyme fraction of Sphingobacteria sp. In the case of α-L-aspartyl-L-phenylalanine-β-methyl ester (α-AMP) production, 108.1 mM of the concentrated solution (about 15 U / ml) of the SP-Sepharose HP fraction obtained in step 1 L-aspartic acid-α, β-dimethyl ester hydrochloride was added to 185 μl borate buffer (pH 9.0) containing 216.2 mM L-phenylalanine, 10.8 mM EDTA, and reacted at 20 ° C. . Similarly, in the case of α-L-aspartyl-L-phenylalanine-β-ethyl ester (α-AEP) production, a concentrated solution (about 15 U / ml) of the SP-Sepharose HP fraction obtained in Example 4 10 μl is added to 190 μl borate buffer (pH 9.0) containing 52.6 mM L-aspartic acid-α, β-diethyl ester hydrochloride, 105.2 mM L-phenylalanine, 10.8 mM EDTA, The reaction was performed at 20 ° C. Table 4 shows the production process of AMP or AEP. In addition, almost no AMP or AEP production was observed in the enzyme-free group. In addition, the production | generation of AEP described the value using the standard product of AMP.

(Example 6) Isolation of peptide-forming enzyme gene derived from Empedobacter brevis Hereinafter, the isolation of the peptide-forming enzyme gene will be described, but the microorganism used was Empedobacter brevis FERM BP-8113 strain. For isolation of the gene, Escherichia coli JM-109 was used as the host, and pUC118 was used as the vector.

(1) Preparation of PCR Primer Based on Determined Internal Amino Acid Sequence Amino acid sequence (SEQ ID NO: 1) determined by Edman degradation method of digestion by lysyl endopeptidase of peptide producing enzyme derived from the aforementioned Empedobacter brevis FERM BP-8113 strain And 2), mixed primers having the base sequences shown in SEQ ID NOs: 3 and 4 were prepared.

(2) Acquisition of bacterial cells Empedobacter brevis FERM BP-8113 strain was added to CM2G agar medium (50 g / l glucose, 10 g / l yeast extract, 10 g / l peptone, 5 g / l sodium chloride, 20 g / l agar, pH 7. 0)) and cultured at 30 ° C. for 24 hours. One microbial ear of this bacterial cell was inoculated into a 500 ml Sakaguchi flask in which 50 ml of CM2G liquid medium (medium obtained by removing agar from the above medium) was placed, and cultured at 30 ° C. with shaking.

(3) Acquisition of chromosomal DNA from microbial cells 50 ml of the culture solution was centrifuged (12,000 rpm, 4 ° C., 15 minutes) and collected. Using QIAGEN Genomic-tip System (Qiagen), chromosomal DNA was obtained from the cells based on the method described in the manual.

  (4) Obtaining a DNA fragment containing a part of a peptide-forming enzyme gene by PCR method A DNA fragment containing a part of a peptide-forming enzyme gene derived from Empedobacter brevis FERM BP-8113 strain was obtained by LA-Taq (manufactured by Takara Shuzo). ). PCR reaction was performed on the chromosomal DNA obtained from Empedobacter brevis FERM BP-8113 using primers having the nucleotide sequences shown in SEQ ID NOs: 3 and 4.

  The PCR reaction was performed using Takara PCR Thermal Cycler PERSONAL (Takara Shuzo), and the reaction under the following conditions was performed for 30 cycles.

94 ° C 30 seconds 52 ° C 1 minute 72 ° C 1 minute

  After the reaction, 3 μl of the reaction solution was subjected to 0.8% agarose electrophoresis. As a result, it was confirmed that a DNA fragment of about 1.5 kb was amplified.

(5) Cloning of the peptide-forming enzyme gene from the gene library In order to obtain the full-length peptide-forming enzyme gene, first, Southern hybridization was performed using the DNA fragment amplified in the PCR as a probe. Southern hybridization of operation, Molecular Cloning, 2 ^nd edition, is described in Cold Spring Harbor press (1989).

  The approximately 1.5 kb DNA fragment amplified by the PCR was separated by 0.8% agarose electrophoresis. The target band was cut out and purified. Using this DNA fragment, DIG High Prime (manufactured by Boehringer Mannheim) was used to label the probe with digoxinigen based on the instructions.

  The chromosomal DNA of Empedobacter brevis obtained in Example 6 (3) was reacted with the restriction enzyme HindIII at 37 ° C. for 16 hours for complete digestion, and then electrophoresed on a 0.8% agarose gel. The agarose gel after electrophoresis was blotted onto a nylon membrane filter Nylon membranes positively charged (manufactured by Roche Diagnostics), and subjected to alkali denaturation, neutralization, and immobilization. Hybridization was performed using EASY HYB (Boehringer Mannheim). The filter was prehybridized at 50 ° C. for 1 hour, and then the digoxinigen-labeled probe prepared above was added, and hybridization was carried out at 50 ° C. for 16 hours. Thereafter, the filter was washed with 2 × SSC containing 0.1% SDS at room temperature for 20 minutes. Further, washing was performed twice at 65 ° C. for 15 minutes with 0.1 × SSC containing 0.1% SDS.

  Detection of the band hybridizing with the probe was performed based on the instructions using a DIG Nucleotide Detection Kit (manufactured by Boehringer Mannheim). As a result, a band of about 4 kb that hybridized with the probe could be detected.

  5 μg of chromosomal DNA prepared in Example 6 (3) was completely digested with HindIII. About 4 kb of DNA was separated by 0.8% agarose gel electrophoresis, and the DNA was purified using Gene Clean II Kit (manufactured by Funakoshi) and dissolved in 10 μl of TE. 4 μl of this was mixed with pUC118 HindIII / BAP (Takara Shuzo), and DNA Ligation Kit Ver. The ligation reaction was performed using 2 (Takara Shuzo). Escherichia coli was transformed by mixing 5 μl of this ligation reaction solution and 100 μl of Escherichia coli JM109 competent cell (manufactured by Toyobo). This was applied to an appropriate solid medium to prepare a chromosomal DNA library.

In order to obtain the full length of the peptide-forming enzyme gene, a chromosomal DNA library was screened by colony hybridization using the probe. The procedure for colony hybridization, Molecular Cloning, 2 ^nd edition, is described in Cold Spring Harbor press (1989).

  The colonies of the chromosomal DNA library were transferred to a nylon membrane filter Nylon Membranes for Colony and Place Hybridization (Roche Diagnostics), and subjected to alkali denaturation, neutralization, and immobilization. Hybridization was performed using EASY HYB (Boehringer Mannheim). The filter was prehybridized at 37 ° C. for 1 hour, and then the above labeled probe with digoxinigen was added, followed by hybridization at 50 ° C. for 16 hours. Thereafter, the filter was washed with 2 × SSC containing 0.1% SDS at room temperature for 20 minutes. Further, washing was performed twice at 65 ° C. for 15 minutes with 0.1 × SSC containing 0.1% SDS.

  Detection of colonies that hybridized with the labeled probe was performed based on the instructions using DIG Nucleotide Detection Kit (Boehringer Mannheim). As a result, two colonies that hybridize with the labeled probe were confirmed.

(6) Base sequence of Empedobacter brevis-derived peptide-forming enzyme gene From the two strains confirmed to hybridize with the labeled probe, plasmids possessed by Escherichia coli JM109 were obtained from Wizard Plus Minipreps DNA Purification System (manufactured by Promega). ) And the base sequence in the vicinity of the hybridized probe was determined. The sequence reaction was performed using CEQ DTCS-Quick Start Kit (manufactured by Beckman Coulter) based on the instructions. Electrophoresis was performed using CEQ 2000-XL (Beckman Coulter, Inc.).

  As a result, it was confirmed that there was an open reading frame encoding a protein containing the internal amino acid sequence (SEQ ID NOs: 1 and 2) of the peptide generating enzyme, and the gene was encoding the peptide generating enzyme. The full-length base sequence of the peptide-forming enzyme gene and the corresponding amino acid sequence are shown in SEQ ID NO: 5 in the sequence listing. The resulting open reading frame was BLASTP. As a result of the homology analysis by the program, homology was found between the two enzymes. Acetobacter pasteurianus α-amino acid ester hydrolase (Appl. Environ. Microbiol., 68 (1), 211-218 (2002) The amino acid sequence showed 34% homology with the glutaryl-7ACA acylase of Brevibacillus laterosporum (J. Bacteriol., 173 (24), 7848-7855 (1991)).

(7) Expression of peptide-forming enzyme gene derived from Empedobacter genus in Escherichia coli The target gene by PCR using the trp operon promoter region on Escherichia coli W3110 chromosomal DNA as the primers shown in SEQ ID NOs: 7 and 8 The region was amplified, and the obtained DNA fragment was ligated into a pGEM-Teasy vector (Promega). With this ligation solution E. coli JM109 was transformed, and a strain having the desired plasmid in which the direction of the trp promoter was inserted in the opposite direction to the lac promoter was selected from ampicillin resistant strains. Next, this plasmid was ligated with a DNA fragment containing the trp promoter obtained by treating with EcoO109I / EcoRI and an EcoO109I / EcoRI-treated product of pUC19 (manufactured by Takara). Escherichia coli JM109 was transformed with this ligation solution, and a strain having the target plasmid was selected from ampicillin resistant strains. Next, the DNA fragment obtained by treating this plasmid with HindIII / PvuII and the DNA fragment containing the rrnB terminator obtained by treating pKK223-3 (manufactured by Amersham Pharmacia) with HindIII / HincII were ligated. With this ligation solution E. coli JM109 was transformed, a strain having the target plasmid was selected from ampicillin resistant strains, and this plasmid was named pTrpT.

  The target gene was amplified by PCR using the chromosomal DNA of Empedobacter brevis FERM BP-8113 as a template and the oligonucleotides shown in SEQ ID NOs: 9 and 10 as primers. This DNA fragment was treated with NdeI / PstI, and the obtained DNA fragment was ligated with the NdeI / PstI-treated product of pTrpT. Escherichia coli JM109 was transformed with this ligation solution, a strain having the target plasmid was selected from ampicillin resistant strains, and this plasmid was named pTrpT_Gtg2.

  Escherichia coli JM109 having pTrpT_Gtg2 was seed-cultured at 30 ° C. for 24 hours in an LB medium containing 100 mg / l ampicillin. 1 ml of the obtained culture solution was added to 50 ml of a medium (2 g / l D-glucose, 10 g / l yeast extract, 10 g / l casamino acid, 5 g / l ammonium sulfate, 3 g / l potassium dihydrogen phosphate, 1 g / l phosphate. A 500 ml Sakaguchi flask filled with dipotassium hydrogen, 0.5 g / l magnesium sulfate heptahydrate, 100 mg / l ampicillin) was seeded, and main culture was performed at 25 ° C. for 24 hours. It has 0.11 U of α-L-aspartyl-L-phenylalanine-β-ester producing activity per 1 ml of culture solution. It was confirmed that it was expressed in E. coli. In addition, the activity was not detected in the transformant into which only pTrpT was introduced as a control.

(Signal sequence prediction)
The amino acid sequence of SEQ ID NO: 6 described in the sequence listing was analyzed with the SignalP v1.1 program (Protein Engineering, vol12, no.1, pp.3-9, 1999). Was predicted to function as a signal and secreted into the periplasm, and the mature protein was assumed to be downstream from the 23rd.

(Confirmation of secretion)
Escherichia coli JM109 having pTrpT_Gtg2 was seed-cultured at 30 ° C. for 24 hours in an LB medium containing 100 mg / l ampicillin. 1 ml of the obtained culture broth was added to 50 ml of medium (2 g / l glucose, 10 g / l yeast extract, 10 g / l casamino acid, 5 g / l ammonium sulfate, 3 g / l potassium dihydrogen phosphate, 1 g / l dihydrogen phosphate. A 500 ml Sakaguchi flask filled with potassium, 0.5 g / l magnesium sulfate heptahydrate, 100 mg / l ampicillin) was seeded, and main culture was performed at 25 ° C. for 24 hours to obtain cultured cells.

The cultured cells were fractionated into a periplasm fraction and a cytoplasm fraction by an osmotic shock method using a 20 g / dl sucrose solution. The bacterial cells immersed in a 20 g / dl sucrose solution were immersed in 5 mM MgSO ₄ aqueous solution, and this centrifugal supernatant was used as a periplasm fraction (Pe). Further, the centrifugal precipitate was resuspended and subjected to ultrasonic disruption to obtain a cytoplasm fraction (Cy). In order to confirm that the cytoplasm was separated, the activity of glucose 6-phosphate dehydrogenase known to exist in the cytoplasm was used as an index. The measurement method is 1 mM glucose 6 phosphate, 0.4 mM NADP, 10 mM MgSO ₄ , 50 mM Tris-Cl (pH 8), an appropriate amount of enzyme is added to the reaction solution at 30 ° C., and NADPH is generated by measuring absorbance at 340 nm. It was performed by measuring.

  FIG. 1 shows the enzyme amounts of the cytoplasmic fraction and the periplasmic fraction when the activity of the cell-free extract prepared separately is 100%. The absence of glucose 6-phosphate dehydrodrogenase activity in the periplasm fraction indicates that the periplasm fraction is not mixed in the cytoplasm fraction. About 60% of the α-L-aspartyl-L-phenylalanine-β-ester (α-AMP) producing activity was recovered in the periplasm fraction, and the above SignalP vl. As predicted from the amino acid sequence using one program, it was confirmed that α-AMP producing enzyme was secreted into the periplasm.

(Example 7) Isolation of peptide-forming enzyme gene derived from sphingobacterium sp. Hereinafter, isolation of the peptide-forming enzyme gene will be described. For isolation of the gene, Escherichia coli DH5α was used as a host, and pUC118 was used as a vector.

(1) Acquisition of bacterial cells Sphingobacterium sp. FERM BP-8124 strain was obtained from CM2G agar medium (50 g / l glucose, 10 g / l yeast extract, 10 g / l peptone, 5 g / l sodium chloride, 20 g / l agar, pH 7. 0)) and cultured at 25 ° C. for 24 hours. This microbial cell was inoculated with 1 platinum ear in a 500 ml Sakaguchi flask in which 50 ml of CM2G liquid medium (medium obtained by removing agar from the above medium) was spread, and cultured at 25 ° C. with shaking.

(2) Acquisition of chromosomal DNA from microbial cells 50 ml of the culture solution was centrifuged (12,000 rpm, 4 ° C., 15 minutes) and collected. Using QIAGEN Genomic-tip System (Qiagen), chromosomal DNA was obtained from the cells based on the method described in the manual.

(3) Acquisition of DNA fragment for probe by PCR method DNA fragment containing a part of peptide-forming enzyme gene derived from Empedobacter brevis FERM BP-8113 strain was obtained by PCR method using LA-Taq (Takara Shuzo). I got it. PCR reaction was performed on the chromosomal DNA obtained from Empedobacter brevis FERMBP-8113 using primers having the nucleotide sequences shown in SEQ ID NOs: 3 and 4.

  The PCR reaction was performed using Takara PCR Thermal Cycler PERSONAL (Takara Shuzo), and the reaction under the following conditions was performed for 30 cycles.

94 ° C 30 seconds 52 ° C 1 minute 72 ° C 1 minute

  After the reaction, 3 μl of the reaction solution was subjected to 0.8% agarose electrophoresis. As a result, it was confirmed that a DNA fragment of about 1.5 kb was amplified.

(4) Cloning of peptide-forming enzyme gene from gene library In order to obtain the full-length peptide-forming enzyme gene, Southern hybridization was first performed using the DNA fragment amplified in the PCR as a probe. Southern hybridization of operation, Molecular Cloning, 2 ^nd edition, is described in Cold Spring Harbor press (1989).

  The approximately 1.5 kb DNA fragment amplified by the PCR was separated by 0.8% agarose electrophoresis. The target band was cut out and purified. Using this DNA fragment, DIG High Prime (manufactured by Boehringer Mannheim) was used to label the probe with digoxinigen based on the instructions.

  The sphingobium sp. Chromosomal DNA obtained in Example 7 (2) was reacted with the restriction enzyme SacI at 37 ° C. for 16 hours to complete digestion, and then electrophoresed on a 0.8% agarose gel. The agarose gel after electrophoresis was blotted onto a nylon membrane filter Nylon membranes positively charged (manufactured by Roche Diagnostics), and subjected to alkali denaturation, neutralization, and immobilization. Hybridization was performed using EASY HYB (Boehringer Mannheim). The filter was prehybridized at 37 ° C. for 1 hour, and then the digoxinigen-labeled probe prepared above was added, and hybridization was carried out at 37 ° C. for 16 hours. Thereafter, the filter was washed twice at 60 ° C. with 1 × SSC containing 0.1% SDS.

  Detection of the band hybridizing with the probe was performed based on the instructions using a DIG Nucleotide Detection Kit (manufactured by Boehringer Mannheim). As a result, a band of about 3 kb that hybridized with the probe could be detected.

  5 μg of the chromosomal DNA prepared in Example 7 (2) was completely digested with SacI. About 3 kb of DNA was separated by 0.8% agarose gel electrophoresis, and the DNA was purified using Gene Clean II Kit (manufactured by Funakoshi) and dissolved in 10 μl of TE. 4 μl of this was reacted with SacI at 37 ° C. for 16 hours and completely digested, and then mixed with pUC118 treated with Alkaline Phosphatase (E. coli C75) at 37 ° C. for 30 minutes, 50 ° C. for 30 minutes, and DNA was mixed. Ligation Kit Ver. The ligation reaction was performed using 2 (Takara Shuzo). Escherichia coli was transformed by mixing 5 μl of this ligation reaction solution and 100 μl of Escherichia coli DH5α competent cell (Takara Shuzo). This was applied to an appropriate solid medium to prepare a chromosomal DNA library.

In order to obtain the full length of the peptide-forming enzyme gene, a chromosomal DNA library was screened by colony hybridization using the probe. The procedure for colony hybridization, Molecular Cloning, 2 ^nd edition, is described in Cold Spring Harbor press (1989).

  The colonies of the chromosomal DNA library were transferred to a nylon membrane filter Nylon Membranes for Colony and Place Hybridization (Roche Diagnostics), and subjected to alkali denaturation, neutralization, and immobilization. Hybridization was performed using EASY HYB (Boehringer Mannheim). The filter was prehybridized at 37 ° C. for 1 hour, and then the above labeled probe with digoxinigen was added, and hybridization was carried out at 37 ° C. for 16 hours. Thereafter, the filter was washed twice at 60 ° C. with 1 × SSC containing 0.1% SDS.

  Detection of colonies that hybridized with the labeled probe was performed based on the instructions using DIG Nucleotide Detection Kit (Boehringer Mannheim). As a result, 6 colonies that hybridize with the labeled probe were confirmed.

(5) Sphingobacterium sp.-derived peptide-forming enzyme gene base sequence From the six strains confirmed to hybridize with the labeled probe, plasmids possessed by Escherichia coli DH5α were obtained from Wizard Plus Minipreps DNA Purification System (manufactured by Promega). ) And the base sequence in the vicinity of the hybridized probe was determined. The sequence reaction was performed using CEQ DTCS-Quick Start Kit (manufactured by Beckman Coulter) based on the instructions. Electrophoresis was performed using CEQ 2000-XL (Beckman Coulter, Inc.).

  As a result, there was an open reading frame encoding a peptide generating enzyme. The full-length nucleotide sequence of the sphingobacterium sp.-derived peptide-forming enzyme gene and the amino acid sequence corresponding thereto are shown in SEQ ID NO: 11 in the Sequence Listing. Sphingobacterium sp.-derived peptide-forming enzyme showed 63.5% homology in amino acid sequence with Empedobacter brevis-derived peptide-forming enzyme (using the BLASTP program).

(6) Expression in Escherichia coli of a peptide-forming enzyme gene derived from the genus Sphingobacteria The target gene was amplified by PCR using the chromosomal DNA of Sphingobacterium sp. . This DNA fragment was treated with NdeI / XbaI, and the obtained DNA fragment was ligated with the NdeI / XbaI-treated product of pTrpT. Escherichia coli JM109 was transformed with this ligation solution, a strain having the target plasmid was selected from ampicillin resistant strains, and this plasmid was named pTrpT_Sm_aet.

  Escherichia coli JM109 containing pTrpT_Sm_aet in 3 ml of medium (2 g / l glucose, 10 g / l yeast extract, 10 g / l casamino acid, 5 g / l ammonium sulfate, 3 g / l potassium dihydrogen phosphate, 1 g / l dipotassium hydrogen phosphate , 0.5 g / l magnesium sulfate heptahydrate, 100 mg / l ampicillin) was inoculated with one platinum ear in a normal test tube, followed by main culture at 25 ° C. for 20 hours. It has confirmed that the cloned gene was expressed in Escherichia coli having 0.53 U of α-AMP generating activity per 1 ml of the culture solution. In addition, the activity was not detected in the transformant into which only pTrpT was introduced as a control.

(Signal sequence prediction)
When the amino acid sequence of SEQ ID NO: 12 described in the sequence listing was analyzed by the SignalP v1.1 program (Protein Engineering, vol12, no.1, pp.3-9, 1999), the amino acid sequence up to the 1-20th position Was predicted to function as a signal and secreted into the periplasm, and the mature protein was assumed to be downstream from the 21st.

(Confirmation of signal sequence)
Escherichia coli JM109 with pTrpT_Sm_aet in 50 ml medium (2 g / l glucose, 10 g / l yeast extract, 10 g / l casamino acid, 5 g / l ammonium sulfate, 3 g / l potassium dihydrogen phosphate, 1 g / l dipotassium hydrogen phosphate , 0.5 g / l magnesium sulfate heptahydrate, 100 mg / l ampicillin) was inoculated with a platinum ear in a normal test tube, followed by main culture at 25 ° C. for 20 hours.

  Hereinafter, the operations after centrifugation were performed on ice or at 4 ° C. After completion of the culture, the cells were centrifuged from these cultures, washed with 100 mM phosphate buffer (pH 7), and suspended in the same buffer. An ultrasonic disruption treatment was performed at 195 W for 20 minutes, the ultrasonic disruption solution was centrifuged (12,000 rpm, 30 minutes), and the disrupted cell fragments were removed to obtain a soluble fraction. The obtained soluble fraction was applied to a CHT-II column (manufactured by Bio-Rad) pre-equilibrated with 100 mM phosphate buffer (pH 7), and the enzyme was eluted with a linear concentration gradient with 500 mM phosphate buffer. . A solution obtained by mixing the active fraction with 5 volumes of 2M ammonium sulfate and 100 mM phosphate buffer was applied to a Resource-PHE column (Amersham) previously equilibrated with 2M ammonium sulfate and 100 mM phosphate buffer. The enzyme was eluted with a linear concentration gradient with ˜0 M ammonium sulfate to obtain an active fraction solution. By these operations, it was confirmed that the peptide-forming enzyme was purified by electrophoresis.

  When the amino acid sequence of the above peptide-forming enzyme was determined by the Edman degradation method, the amino acid sequence of SEQ ID NO: 15 was obtained, and it was confirmed that the mature protein was downstream from the 21st position as predicted by the SignalP v1.1 program. .

(Example 8) Isolation of Peptobacter Heparinas IFO 12017-derived peptide-forming enzyme gene The following describes the isolation of peptide-forming enzyme genes. Address; 2-17-85 Jusohoncho, Yodogawa-ku, Osaka, Japan. For gene isolation, Escherichia coli JM109 was used as a host, and pUC118 was used as a vector.

(1) Acquisition of bacterial cells Pedobacter heparinus IFO 12017 strain on CM2G agar medium (50 g / l glucose, 10 g / l yeast extract, 10 g / l peptone, 5 g / l sodium chloride, 20 g / l agar, pH 7.0)) And cultured at 25 ° C. for 24 hours. This microbial cell was inoculated with 1 platinum ear in a 500 ml Sakaguchi flask in which 50 ml of CM2G liquid medium (medium obtained by removing agar from the above medium) was spread, and cultured at 25 ° C. with shaking.

(2) Acquisition of chromosomal DNA from microbial cells 50 ml of the culture solution was centrifuged (12,000 rpm, 4 ° C., 15 minutes) and collected. Chromosomal DNA was obtained from the cells using QIAGEN Genomic-tip System (Qiagen) based on the method described in the manual.

(3) Acquisition of DNA fragment for probe by PCR method A DNA fragment containing a part of the peptide-forming enzyme gene derived from Pedobacter heparinus IFO 12017 was obtained by PCR method using LA-Taq (Takara Shuzo). PCR reaction was performed on the chromosomal DNA obtained from Pedobacter heparinus IFO 12017 using primers having the nucleotide sequences shown in SEQ ID NOs: 15 and 16. The approximately 1 kb DNA fragment amplified by PCR was separated by 0.8% agarose electrophoresis. The target band was cut out and purified. The DNA fragment was labeled with digoxinigen on the probe based on the instructions using DIG High Prime (Boehringer Mannheim).

(4) Cloning of peptide-forming enzyme gene from gene library In order to obtain the full-length peptide-forming enzyme gene, Southern hybridization was first performed using the DNA fragment amplified in the PCR as a probe. Southern hybridization of operation, Molecular Cloning, 2 ^nd edition, is described in Cold Spring Harbor press (1989).

  The chromosomal DNA of Pedobacter heparinus IFO 12017 was reacted with the restriction enzyme HindIII at 37 ° C. for 16 hours for complete digestion, and then electrophoresed on a 0.8% agarose gel. The agarose gel after electrophoresis was blotted onto a nylon membrane filter Nylon memebranes positively charged (Roche Diagnostics) and subjected to alkali denaturation, neutralization and immobilization. Hybridization was performed using EASY HYB (Boehringer Mannheim). The filter was prehybridized at 50 ° C. for 1 hour, and then the digoxinigen-labeled probe prepared above was added, and hybridization was carried out at 50 ° C. for 16 hours. Thereafter, the filter was washed twice at 60 ° C. with 1 × SSC containing 0.1% SDS.

  Detection of the band hybridizing with the probe was performed based on the instructions using the DIG Nucleotide Detection Kit (Boehringer Mannheim). As a result, a band of about 5 kb that hybridized with the probe could be detected.

  5 μg of chromosomal DNA of Pedobacter heparinus IFO 12017 strain was completely digested with HindIII. About 5 kb of DNA was separated by 0.8% agarose gel electrophoresis, the DNA was purified using Gene Clean II Kit (manufactured by Funakoshi), and dissolved in 10 μl of TE. Of these, 4 μl was mixed with pUC118 HindIII / BAP (Takara Shuzo), and a ligation reaction was performed using DNA Ligation Kit Ver.2 (Takara Shuzo). Escherichia coli was transformed by mixing 5 μl of this ligation reaction solution and 100 μl of Escherichia coli JM109 competent cell (Takara Shuzo). This was applied to an appropriate solid medium to prepare a chromosomal DNA library.

In order to obtain the full length of the peptide-forming enzyme gene, a chromosomal DNA library was screened by colony hybridization using the probe. The procedure for colony hybridization, Molecular Cloning, 2 ^nd edition, is described in Cold Spring Harbor press (1989).

  The colonies of the chromosomal DNA library were transferred to a nylon membrane filter NylonMembranes for Colony and Plaque Hybridization (Roche Diagnostics) and subjected to alkali denaturation, neutralization, and immobilization. Hybridization was performed using EASY HYB (Boehringer Mannheim). The filter was prehybridized at 37 ° C. for 1 hour, and then the above labeled probe with digoxinigen was added, and hybridization was carried out at 37 ° C. for 16 hours. Thereafter, the filter was washed twice at 60 ° C. with 1 × SSC containing 0.1% SDS.

  Colonies that hybridize with the labeled probe were detected based on the instructions using the DIG Nucleotide Detection Kit (Boehringer Mannheim). As a result, one strain of colonies that hybridized with the labeled probe could be confirmed.

(5) Pedobacter Heparinus IFO 12017-derived peptide-forming enzyme gene base sequence From the above-mentioned one strain confirmed to hybridize with the labeled probe, a plasmid possessed by Escherichia coli JM109 was prepared, and the vicinity of the hybridized with the probe The base sequence was determined. The sequencing reaction was performed using CEQ DTCS-Quick Start Kit (manufactured by Beckman Coulter) based on the instructions. Electrophoresis was performed using CEQ 2000-XL (Beckman Coulter).

  As a result, there was an open reading frame encoding a peptide generating enzyme. The base sequence of the full-length peptide-forming enzyme gene derived from Pedobacter heparinus IFO 12017 and the amino acid sequence corresponding thereto are shown in SEQ ID NO: 17 in the Sequence Listing.

(Example 9) Expression in Escherichia coli of a peptide-forming enzyme gene derived from Pedobacter heparinus IFO 12017 strain The target gene was amplified by PCR using the chromosomal DNA of Pedobacter heparinus IFO 12017 strain as a template and the oligonucleotides shown in SEQ ID NOs: 19 and 20 as primers. . This DNA fragment was treated with NdeI / HindIII, and the resulting DNA fragment was ligated with the NdeI / HindIII-treated product of pTrpT. Escherichia with this ligation solution

E. coli JM109 was transformed, a strain having the target plasmid was selected from ampicillin resistant strains, and this plasmid was named pTrpT_Ph_aet.
Escherichia coli JM109 containing pTrpT_Ph_aet in 3 ml of medium (2 g / l glucose, 10 g / l yeast extract, 10 g / l casamino acid, 5 g / l ammonium sulfate, 3 g / l potassium dihydrogen phosphate, 1 g / l dipotassium hydrogen phosphate , 0.5 g / l magnesium sulfate heptahydrate, 100 mg / l ampicillin) was inoculated with a platinum ear in a normal test tube, followed by main culture at 25 ° C. for 20 hours. It has confirmed that the cloned gene was expressed in Escherichia coli with 0.01 U α-AMP producing activity per 1 ml of the culture solution. As a control, no activity was detected in the transformant into which only pTrpT was introduced.

(Example 10) Taxeobacter gelupurpurascens Isolation of peptide-forming enzyme gene derived from DSMZ 11116 strain Hereinafter, isolation of peptide-forming enzyme gene will be described. Depositary organization: Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures, deposit address: Mascheroder Weg 1b, 38124 Braunschweig, Germany), using Escherichia coli JM109 The vector used was pUC118.

(1) Acquisition of bacterial cells Taxeobacter gelpurpurense DSMZ 11116 strain was treated with CM2G agar medium (50 g / l glucose, 10 g / l yeast extract, 10 g / l peptone, 5 g / l sodium chloride, 20 g / l agar, pH 7.0). ) At 25 ° C. for 24 hours. This microbial cell was inoculated with 1 platinum ear in a 500 ml Sakaguchi flask in which 50 ml of CM2G liquid medium (medium obtained by removing agar from the above medium) was spread, and cultured at 25 ° C. with shaking.

(2) Acquisition of chromosomal DNA from microbial cells 50 ml of the culture solution was centrifuged (12,000 rpm, 4 ° C., 15 minutes) and collected. Chromosomal DNA was obtained from the cells using QIAGEN Genomic-tip System (Qiagen) based on the method described in the manual.

(3) Acquisition of DNA fragment for probe by PCR method DNA fragment containing a part of the peptide-forming enzyme gene derived from Taxobacter gel pullulase DSMZ 11116 strain is acquired by PCR method using LA-Taq (Takara Shuzo). did. A PCR reaction was performed on the chromosomal DNA obtained from Taxeobacter gelpurpurense DSMZ 11116 strain using primers having the nucleotide sequences shown in SEQ ID NOs: 21 and 16. The approximately 1 kb DNA fragment amplified by PCR was separated by 0.8% agarose electrophoresis. The target band was cut out and purified. The DNA fragment was labeled with digoxinigen on the probe based on the instructions using DIG High Prime (Boehringer Mannheim).

(4) Cloning of peptide-forming enzyme gene from gene library In order to obtain the full-length peptide-forming enzyme gene, Southern hybridization was first performed using the DNA fragment amplified in the PCR as a probe. Southern hybridization of operation, Molecular Cloning, 2 ^nd edition, is described in Cold Spring Harbor press (1989).

  Texeobacter gel pull pullense DSMZ 11116 chromosomal DNA was reacted with the restriction enzyme PstI at 37 ° C. for 16 hours for complete digestion, and then electrophoresed on a 0.8% agarose gel. The agarose gel after electrophoresis was blotted onto a nylon membrane filter Nylon memebranes positively charged (Roche Diagnostics) and subjected to alkali denaturation, neutralization and immobilization. Hybridization was performed using EASY HYB (Boehringer Mannheim). The filter was prehybridized at 50 ° C. for 1 hour, and then the digoxinigen-labeled probe prepared above was added, and hybridization was carried out at 50 ° C. for 16 hours. Thereafter, the filter was washed twice at 60 ° C. with 1 × SSC containing 0.1% SDS.

  Detection of the band hybridizing with the probe was performed based on the instructions using the DIG Nucleotide Detection Kit (Boehringer Mannheim). As a result, a band of about 5 kb that hybridized with the probe could be detected.

  Taxeobacter gel pull pullense DSMZ 11116 strain chromosomal DNA 5 μg was completely digested with PstI. About 5 kb of DNA was separated by 0.8% agarose gel electrophoresis, the DNA was purified using Gene Clean II Kit (manufactured by Funakoshi), and dissolved in 10 μl of TE. Of these, 4 μl and pUC118 PstI / BAP (Takara Shuzo) were mixed, and a ligation reaction was performed using DNA Ligation Kit Ver.2 (Takara Shuzo). Escherichia coli was transformed by mixing 5 μl of this ligation reaction solution and 100 μl of Escherichia coli JM109 competent cell (Takara Shuzo). This was applied to an appropriate solid medium to prepare a chromosomal DNA library.

In order to obtain the full length of the peptide-forming enzyme gene, a chromosomal DNA library was screened by colony hybridization using the probe. The procedure for colony hybridization, Molecular Cloning, 2 ^nd edition, is described in Cold Spring Harbor press (1989).

  The colonies of the chromosomal DNA library were transferred to a nylon membrane filter NylonMembranes for Colony and Plaque Hybridization (Roche Diagnostics) and subjected to alkali denaturation, neutralization, and immobilization. Hybridization was performed using EASY HYB (Boehringer Mannheim). The filter was prehybridized at 37 ° C. for 1 hour, and then the above labeled probe with digoxinigen was added, and hybridization was carried out at 37 ° C. for 16 hours. Thereafter, the filter was washed twice at 60 ° C. with 1 × SSC containing 0.1% SDS.

  Colonies that hybridize with the labeled probe were detected based on the instructions using the DIG Nucleotide Detection Kit (Boehringer Mannheim). As a result, one strain of colonies that hybridized with the labeled probe could be confirmed.

(5) Taxobabacter gel pullulase DSMZ 11116 strain-derived peptide-forming enzyme gene base sequence From the above-mentioned one strain confirmed to hybridize with the labeled probe, a plasmid possessed by Escherichia coli JM109 is prepared and hybridized with the probe. The base sequence in the vicinity was determined. The sequencing reaction was performed using CEQ DTCS-Quick Start Kit (manufactured by Beckman Coulter) based on the instructions. Electrophoresis was performed using CEQ 2000-XL (Beckman Coulter).

  As a result, there was an open reading frame encoding a peptide generating enzyme. The base sequence of the full-length peptide-forming enzyme gene derived from Taxeobacter gelpurpurense DSMZ 11116 strain and the amino acid sequence corresponding thereto are shown in SEQ ID NO: 22 in Sequence Listing.

(Example 11) Isolation of a peptide-forming enzyme gene from Cyclobacterium marinum ATCC 25205 strain Hereinafter, isolation of a peptide-forming enzyme gene will be described. American Type Culture Collection, deposit address; POBox 1549 Manassas, VA 20110, the United States of America). For gene isolation, Escherichia coli JM109 was used as a host, and pUC118 was used as a vector.

(1) Acquisition of bacterial cells Cyclobacterium marinum ATCC 25205 strain was obtained from CM2G agar medium (50 g / l glucose, 10 g / l yeast extract, 10 g / l peptone, 5 g / l sodium chloride, 20 g / l agar, pH 7.0). ) At 25 ° C. for 24 hours. This microbial cell was inoculated with 1 platinum ear in a 500 ml Sakaguchi flask in which 50 ml of CM2G liquid medium (medium obtained by removing agar from the above medium) was spread, and cultured at 25 ° C. with shaking.

(2) Acquisition of chromosomal DNA from microbial cells 50 ml of the culture solution was centrifuged (12,000 rpm, 4 ° C., 15 minutes) and collected. Chromosomal DNA was obtained from the cells using QIAGEN Genomic-tip System (Qiagen) based on the method described in the manual.

(3) Acquisition of DNA fragment for probe by PCR method A DNA fragment containing a part of the peptide-forming enzyme gene derived from Cyclobacterium marinum ATCC 25205 strain was acquired by PCR method using LA-Taq (Takara Shuzo). . PCR reaction was performed on the chromosomal DNA obtained from Cyclobacterium marinum ATCC 25205 strain using primers having the nucleotide sequences shown in SEQ ID NOs: 15 and 16. The approximately 1 kb DNA fragment amplified by PCR was separated by 0.8% agarose electrophoresis. The target band was cut out and purified. The DNA fragment was labeled with digoxinigen on the probe based on the instructions using DIG High Prime (Boehringer Mannheim).

(4) Cloning of peptide-forming enzyme gene from gene library In order to obtain the full-length peptide-forming enzyme gene, Southern hybridization was first performed using the DNA fragment amplified in the PCR as a probe. Southern hybridization of operation, Molecular Cloning, 2 ^nd edition, is described in Cold Spring Harbor press (1989).

  The chromosomal DNA of Cyclobacterium marinum ATCC 25205 strain was reacted with restriction enzymes PstI or HincII at 37 ° C. for 16 hours for complete digestion, and each was electrophoresed on a 0.8% agarose gel. The agarose gel after electrophoresis was blotted onto a nylon membrane filter Nylon memebranes positively charged (Roche Diagnostics) and subjected to alkali denaturation, neutralization and immobilization. Hybridization was performed using EASY HYB (Boehringer Mannheim). The filter was prehybridized at 50 ° C. for 1 hour, and then the digoxinigen-labeled probe prepared above was added, and hybridization was carried out at 50 ° C. for 16 hours. Thereafter, the filter was washed twice at 60 ° C. with 1 × SSC containing 0.1% SDS.

  Detection of the band hybridizing with the probe was performed based on the instructions using the DIG Nucleotide Detection Kit (Boehringer Mannheim). As a result, a 7k band was detected for the PstI digested product hybridized with the probe, and a 2k band was detected for the HincII digested product.

5 μg of chromosomal DNA of Cyclobacterium marinum ATCC 25205 strain was completely digested with PstI or HincII. About 7 kb or 2 kb of DNA was separated by 0.8% agarose gel electrophoresis, and the DNA was purified using Gene Clean II Kit (manufactured by Funakoshi) and dissolved in 10 μl of TE. Of these, 4 μl and pUC118 PstI / BAP (Takara Shuzo) or pUC118 HincII / BAP (Takara Shuzo) were mixed, and ligation reaction was performed using DNA Ligation Kit Ver.2 (Takara Shuzo). Escherichia coli was transformed by mixing 5 μl of this ligation reaction solution and 100 μl of Escherichia coli JM109 competent cell (Takara Shuzo). This was applied to an appropriate solid medium to prepare a chromosomal DNA library.

In order to obtain the full length of the peptide-forming enzyme gene, a chromosomal DNA library was screened by colony hybridization using the probe. The procedure for colony hybridization, Molecular Cloning, 2 ^nd edition, is described in Cold Spring Harbor press (1989).

  The colonies of the chromosomal DNA library were transferred to a nylon membrane filter NylonMembranes for Colony and Plaque Hybridization (Roche Diagnostics) and subjected to alkali denaturation, neutralization, and immobilization. Hybridization was performed using EASY HYB (Boehringer Mannheim). The filter was prehybridized at 37 ° C. for 1 hour, and then the above labeled probe with digoxinigen was added, and hybridization was carried out at 37 ° C. for 16 hours. Thereafter, the filter was washed twice at 60 ° C. with 1 × SSC containing 0.1% SDS.

  Colonies that hybridize with the labeled probe were detected based on the instructions using the DIG Nucleotide Detection Kit (Boehringer Mannheim). As a result, one strain of each colony hybridizing with the labeled probe was confirmed.

(5) Nucleotide sequence of the peptide-forming enzyme gene derived from Cyclobacterium marinum ATCC25205 strain From each of the strains confirmed to hybridize with the labeled probe, plasmids possessed by Escherichia coli JM109 were prepared and hybridized with the probe. The base sequence in the vicinity was determined. The sequencing reaction was performed using CEQ DTCS-Quick Start Kit (manufactured by Beckman Coulter) based on the instructions. Electrophoresis was performed using CEQ 2000-XL (Beckman Coulter).

  As a result, there was an open reading frame encoding a peptide generating enzyme. The base sequence of the full length peptide-forming enzyme gene derived from Cyclobacterium marinum ATCC 25205 and the amino acid sequence corresponding thereto are shown in SEQ ID NO: 24.

(Example 12) Isolation of a peptide-forming enzyme gene derived from Psycloserpens burtonensis ATCC 70000359 Hereinafter, the isolation of a peptide-forming enzyme gene will be described. American Type Culture Collection, deposit address: POBox 1549 Manassas, VA 20110, the United States of America). For gene isolation, Escherichia coli JM109 was used as a host, and pUC118 was used as a vector.

(1) Acquisition of bacterial cells Cycloserpens bluetonensis ATCC 70000359 strain was obtained from CM2G agar medium (50 g / l glucose, 10 g / l yeast extract, 10 g / l peptone, 5 g / l sodium chloride, 20 g / l agar, pH 7.0). ) At 25 ° C. for 24 hours. This cell was inoculated with 1 platinum ear in a 500 ml Sakaguchi flask in which 50 ml of CM2G liquid medium (medium obtained by removing agar from the above medium) was spread, and cultured at 10 ° C. with shaking.

(2) Acquisition of chromosomal DNA from microbial cells 50 ml of the culture solution was centrifuged (12,000 rpm, 4 ° C., 15 minutes) and collected. Chromosomal DNA was obtained from the cells using QIAGEN Genomic-tip System (Qiagen) based on the method described in the manual.

(3) Acquisition of DNA fragment for probe by PCR method A DNA fragment containing a part of the peptide-forming enzyme gene derived from Cycloserpens brtonensis ATCC 70000359 was acquired by PCR method using LA-Taq (Takara Shuzo). . PCR reaction was carried out on the chromosomal DNA obtained from Cyclocerpense brtonensis ATCC 70000359 using primers having the nucleotide sequences shown in SEQ ID NOs: 15 and 16. The approximately 1 kb DNA fragment amplified by PCR was separated by 0.8% agarose electrophoresis. The target band was cut out and purified. The DNA fragment was labeled with digoxinigen on the probe based on the instructions using DIG High Prime (Boehringer Mannheim).

(4) Cloning of peptide-forming enzyme gene from gene library In order to obtain the full-length peptide-forming enzyme gene, Southern hybridization was first performed using the DNA fragment amplified in the PCR as a probe. Southern hybridization of operation, Molecular Cloning, 2 ^nd edition, is described in Cold Spring Harbor press (1989).

  The chromosomal DNA of Cycloserpens bluetonensis ATCC 70000359 strain was reacted with the restriction enzyme EcoRI at 37 ° C. for 16 hours for complete digestion, and then electrophoresed on a 0.8% agarose gel. The agarose gel after electrophoresis was blotted onto a nylon membrane filter Nylon memebranes positively charged (Roche Diagnostics) and subjected to alkali denaturation, neutralization and immobilization. Hybridization was performed using EASY HYB (Boehringer Mannheim). The filter was prehybridized at 50 ° C. for 1 hour, and then the digoxinigen-labeled probe prepared above was added, and hybridization was carried out at 50 ° C. for 16 hours. Thereafter, the filter was washed twice at 60 ° C. with 1 × SSC containing 0.1% SDS.

  Detection of the band hybridizing with the probe was performed based on the instructions using the DIG Nucleotide Detection Kit (Boehringer Mannheim). As a result, a band of about 7 kb that hybridized with the probe could be detected.

5 μg of chromosomal DNA of Cycloserpens brtonensis ATCC 70000359 strain was completely digested with EcoRI. About 7 kb of DNA was separated by 0.8% agarose gel electrophoresis, DNA was purified using Gene Clean II Kit (manufactured by Funakoshi), and dissolved in 10 μl of TE. 4 μl of this, pUC118 EcoRI / BAP
(Manufactured by Takara Shuzo) was mixed, and a ligation reaction was performed using DNA Ligation Kit Ver.2 (Takara Shuzo). Escherichia coli was transformed by mixing 5 μl of this ligation reaction solution and 100 μl of Escherichia coli JM109 competent cell (Takara Shuzo). This was applied to an appropriate solid medium to prepare a chromosomal DNA library.

In order to obtain the full length of the peptide-forming enzyme gene, a chromosomal DNA library was screened by colony hybridization using the probe. The procedure for colony hybridization, Molecular Cloning, 2 ^nd edition, is described in Cold Spring Harbor press (1989).

  The colonies of the chromosomal DNA library were transferred to a nylon membrane filter NylonMembranes for Colony and Plaque Hybridization (Roche Diagnostics) and subjected to alkali denaturation, neutralization, and immobilization. Hybridization was performed using EASY HYB (Boehringer Mannheim). The filter was prehybridized at 37 ° C. for 1 hour, and then the above labeled probe with digoxinigen was added, and hybridization was carried out at 37 ° C. for 16 hours. Thereafter, the filter was washed twice at 60 ° C. with 1 × SSC containing 0.1% SDS.

  Colonies that hybridize with the labeled probe were detected based on the instructions using the DIG Nucleotide Detection Kit (Boehringer Mannheim). As a result, one strain of colonies that hybridized with the labeled probe could be confirmed.

(5) Cycloserpens Brutonensis A base sequence of peptide-forming enzyme gene derived from ATCC 7003009 strain From the above-mentioned one strain confirmed to hybridize with the labeled probe, a plasmid possessed by Escherichia coli JM109 was prepared and hybridized with the probe. The nearby nucleotide sequence was determined. The sequencing reaction was performed using CEQ DTCS-Quick Start Kit (manufactured by Beckman Coulter) based on the instructions. Electrophoresis was performed using CEQ 2000-XL (Beckman Coulter).

  As a result, there was an open reading frame encoding a peptide generating enzyme. The base sequence of the full length peptide-forming enzyme gene derived from Cycloserpens brtonensis ATCC 70000359 and the amino acid sequence corresponding thereto are shown in SEQ ID NO: 26 in the Sequence Listing.

It is a figure which shows the relative activity (%) about a cytoplasm fraction and a periplasm fraction.

SEQ ID NO: 1; partial peptide-forming enzyme derived from Empedobacter brevis SEQ ID NO: 2; partial peptide-forming enzyme derived from Empedobacter brevis SEQ ID NO: 3; synthetic primer 1
SEQ ID NO: 4; synthetic primer 2
SEQ ID NO: 5; SEQ ID NO: 6 encoding a peptide-forming enzyme; amino acid sequence SEQ ID NO: 7 of peptide-forming enzyme; synthetic primer SEQ ID NO: 8 for pTrpT preparation; synthetic primer SEQ ID NO: 9 for pTrpT preparation; synthetic primer SEQ ID NO: pTrpT_Gtg2 preparation 10; synthetic primer SEQ ID NO: 11 for preparing pTrpT_Gtg2; gene SEQ ID NO: 12 encoding the peptide-forming enzyme; amino acid sequence SEQ ID NO: 13 of the peptide-forming enzyme; synthetic primer SEQ ID NO: 14 for preparing pTrpT_Sm_aet; synthetic primer SEQ ID NO: 15 for preparing pTrpT_Sm_aet; Mix primer for Aet 1
Sequence number 16; Mix primer 2 for Aet
SEQ ID NO: 17; gene SEQ ID NO: 18 encoding a peptide-forming enzyme; amino acid sequence SEQ ID NO: 19 of a peptide-forming enzyme; primer 1 for constructing an aet expression vector derived from Pedobacter
SEQ ID NO: 20: Primer 2 for constructing an aet expression vector derived from Pedobacter
Sequence number 21; Mix primer 3 for Aet
SEQ ID NO: 22; gene SEQ ID NO: 23 encoding peptide-forming enzyme; amino acid sequence SEQ ID NO: 24 of peptide-generating enzyme; gene SEQ ID NO: 25 encoding peptide-generating enzyme; amino acid sequence SEQ ID NO: 26 of peptide-generating enzyme; Encoding gene SEQ ID NO: 27; gene encoding peptide-forming enzyme

Claims (17)

  L-aspartic acid-α, β-diester and L can be prepared using an enzyme or an enzyme-containing substance having the ability to selectively peptide bond L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester. A method for producing α-L-aspartyl-L-phenylalanine-β-ester, wherein α-L-aspartyl-L-phenylalanine-β-ester is produced from phenylalanine.   A culture of a microorganism having the ability of the enzyme or enzyme-containing substance to selectively bind L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester, and a microbial strain isolated from the culture The α-L-aspartyl-L-phenylalanine-β-ester according to claim 1, wherein the α-L-aspartyl-L-phenylalanine-β-ester is selected from the group consisting of a body and a treated product of the microorganism. Manufacturing method.   The microorganism is Aeromonas, Azotobacter, Alkagenes, Brevibacterium, Corynebacterium, Escherichia, Empedobacter, Flavobacterium, Microbacterium, Propionibacterium, Brevibacillus , Penibacillus genus, Pseudomonas genus, Serratia genus, Stenotrophomonas genus, Sphingobacteria genus, Streptomyces genus, Xanthomonas genus, Williopsis genus, Candida genus, Geotrichum genus, Pichia genus, Saccharomyces genus, Trussola genus, Cellulofarga genus , Weeksera, Pedobacter, Persicobacter, Flexitrix, Titinophaga, Cyclobacterium, Lunella, Thermonema, Cycloserpense, Gelidibacter, De Adbacter, Flameovirga, Spirosoma, Flectobacillus, Tenacibacrum, Rhodtermas, Zoberia, Murikauda, Salegentibacter, Taxebacter, Chitophaga, Marinillaviria, Levinella, Saprospira The method for producing α-L-aspartyl-L-phenylalanine-β-ester according to claim 2, which is a microorganism belonging to a genus selected from the group consisting of a genus. The α-L-aspartyl-L-phenylalanine-β according to claim 2, wherein the microorganism is a transformed microorganism capable of expressing the protein shown in (A) or (B) below. -Manufacturing method of ester.
(A) A protein having an amino acid sequence of amino acid residues 23 to 616 in the amino acid sequence set forth in SEQ ID NO: 6 in the sequence listing (B) Amino acid residue No. 23 in the amino acid sequence set forth in SEQ ID NO: 6 in the sequence listing ˜616 having an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, and / or inversions, and L-phenylalanine is converted to L-aspartic acid-α, β A protein having an activity of selectively binding a peptide to an α-ester site of a diester The α-L-aspartyl-L-phenylalanine-β according to claim 2, wherein the microorganism is a transformed microorganism capable of expressing the protein shown in (C) or (D) below. -Manufacturing method of ester.
(C) Protein having the amino acid sequence of amino acid residues 21 to 619 in the amino acid sequence described in SEQ ID NO: 12 of the sequence listing (D) Amino acid residue number 21 of the amino acid sequence described in SEQ ID NO: 12 of the sequence listing In the amino acid sequence of ˜619, having an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, and / or inversions, and L-phenylalanine to L-aspartic acid-α, β A protein having an activity of selectively binding a peptide to the α-ester site of a diester The α-L-aspartyl-L-phenylalanine-β according to claim 2, wherein the microorganism is a transformed microorganism capable of expressing the protein shown in (E) or (F) below. -Manufacturing method of ester.
(E) a protein having an amino acid sequence of amino acid residues 23 to 625 in the amino acid sequence set forth in SEQ ID NO: 18 in the sequence listing (F) amino acid residue No. 23 in the amino acid sequence set forth in SEQ ID NO: 18 in the sequence listing ˜625 amino acid sequence having an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, and / or inversions, and L-phenylalanine is converted to L-aspartic acid-α, β A protein having an activity of selectively binding a peptide to the α-ester site of a diester The α-L-aspartyl-L-phenylalanine-β according to claim 2, wherein the microorganism is a transformed microorganism capable of expressing the protein shown in (G) or (H) below. -Manufacturing method of ester.
(G) A protein having an amino acid sequence of amino acid residues 23 to 645 in the amino acid sequence described in SEQ ID NO: 23 of the sequence listing (H) Amino acid residue No. 23 of the amino acid sequences described in SEQ ID NO: 23 of the sequence listing In the amino acid sequence of ˜645, which has an amino acid sequence including substitution, deletion, insertion, addition, and / or inversion of one or several amino acids, and L-phenylalanine is converted to L-aspartic acid-α, β A protein having an activity of selectively binding a peptide to an α-ester site of a diester The α-L-aspartyl-L-phenylalanine-β according to claim 2, wherein the microorganism is a transformed microorganism capable of expressing the protein shown in the following (I) or (J). -Manufacturing method of ester.
(I) Protein having the amino acid sequence of amino acid residues 26 to 620 in the amino acid sequence described in SEQ ID NO: 25 of the sequence listing (J) Amino acid residue # 26 of the amino acid sequence described in SEQ ID NO: 25 of the sequence listing In the amino acid sequence of ˜620, having an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, and / or inversions, and L-phenylalanine as L-aspartic acid-α, β A protein having an activity of selectively binding a peptide to an α-ester site of a diester The α-L-aspartyl-L-phenylalanine-β according to claim 2, wherein the microorganism is a transformed microorganism capable of expressing the protein shown in the following (K) or (L). -Manufacturing method of ester.
(K) Protein having the amino acid sequence of amino acid residues 18 to 644 among the amino acid sequences described in SEQ ID NO: 27 of the sequence listing (L) Amino acid residue number 18 of the amino acid sequences described in SEQ ID NO: 27 of the sequence listing An amino acid sequence comprising one or several amino acid substitutions, deletions, insertions, additions, and / or inversions, and L-phenylalanine is converted to L-aspartic acid-α, β A protein having an activity of selectively binding a peptide to the α-ester site of a diester The α-L-aspartyl-L-phenylalanine-β according to claim 2, wherein the microorganism is a transformed microorganism capable of expressing the protein shown in the following (M) or (N). -Manufacturing method of ester.
(M) A protein having the amino acid sequence set forth in SEQ ID NO: 6 in the sequence listing (N) Substitution, deletion, insertion, addition of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 6 in the sequence listing And / or a protein comprising a mature protein region having an amino acid sequence containing an inversion and having an activity of selectively peptide-binding L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester The α-L-aspartyl-L-phenylalanine-β according to claim 2, wherein the microorganism is a transformed microorganism capable of expressing a protein represented by the following (O) or (P): -Manufacturing method of ester.
(O) Protein having the amino acid sequence set forth in SEQ ID NO: 12 in the Sequence Listing (P) Substitution, deletion, insertion, addition of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 12 in the Sequence Listing And / or a protein comprising an mature protein region having an amino acid sequence containing an inversion and having an activity of selectively peptide-binding L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester The α-L-aspartyl-L-phenylalanine-β according to claim 2, wherein the microorganism is a transformed microorganism capable of expressing the protein shown in (Q) or (R) below. -Manufacturing method of ester.
(Q) Protein having the amino acid sequence set forth in SEQ ID NO: 18 in the sequence listing (R) Substitution, deletion, insertion, addition of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 18 in the sequence listing And / or a protein comprising an mature protein region having an amino acid sequence containing an inversion and having an activity of selectively peptide-binding L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester The α-L-aspartyl-L-phenylalanine-β according to claim 2, wherein the microorganism is a transformed microorganism capable of expressing the protein shown in (S) or (T) below. -Manufacturing method of ester.
(S) Protein having the amino acid sequence set forth in SEQ ID NO: 23 in the Sequence Listing (T) Substitution, deletion, insertion, addition of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 23 in the Sequence Listing And / or a protein comprising an mature protein region having an amino acid sequence containing an inversion and having an activity of selectively peptide-binding L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester The α-L-aspartyl-L-phenylalanine-β according to claim 2, wherein the microorganism is a transformed microorganism capable of expressing the protein shown in the following (U) or (V). -Manufacturing method of ester.
(U) a protein having the amino acid sequence set forth in SEQ ID NO: 25 in the sequence listing (V) substitution, deletion, insertion, addition of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 25 in the sequence listing And / or a protein comprising an mature protein region having an amino acid sequence containing an inversion and having an activity of selectively peptide-binding L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester The α-L-aspartyl-L-phenylalanine-β according to claim 2, wherein the microorganism is a transformed microorganism capable of expressing the protein shown in the following (W) or (X). -Manufacturing method of ester.
(W) a protein having the amino acid sequence set forth in SEQ ID NO: 27 in the sequence listing (X) substitution, deletion, insertion, addition of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 27 in the sequence listing And / or a protein comprising an mature protein region having an amino acid sequence containing an inversion and having an activity of selectively peptide-binding L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester The method for producing α-L-aspartyl-L-phenylalanine-β-ester according to claim 1, wherein the enzyme is at least one selected from the group consisting of (A) to (X) below.
(A) Protein having the amino acid sequence of amino acid residues 23 to 616 in the amino acid sequence described in SEQ ID NO: 6 in the sequence listing (B) Amino acid residue number 23 in the amino acid sequence described in SEQ ID NO: 6 in the sequence listing ˜616 having an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, and / or inversions, and L-phenylalanine is converted to L-aspartic acid-α, β -Protein having the activity of selectively binding a peptide to the α-ester site of the diester (C) Protein having the amino acid sequence of amino acid residues 21 to 619 among the amino acid sequences shown in SEQ ID NO: 12 of the Sequence Listing (D) In the amino acid sequence of amino acid residues 21 to 619 in the amino acid sequence described in SEQ ID NO: 12 in the sequence listing, 1 or a number Of amino acid substitutions, deletions, insertions, additions, and / or inversions, and L-phenylalanine is selectively used as an α-ester site of L-aspartic acid-α, β-diester A protein having an activity of peptide binding (E) The amino acid sequence described in SEQ ID NO: 18 of the protein (F) sequence table having the amino acid sequence of amino acid residues 23 to 625 among the amino acid sequences described in SEQ ID NO: 18 of the sequence table Among the amino acid sequences of amino acid residues 23 to 625, and having an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or several amino acids, and L-phenylalanine is L A protein (G) sequence having an activity of selectively binding a peptide to the α-ester site of aspartic acid-α, β-diester The amino acid sequence of amino acid residues 23 to 645 in the amino acid sequence of SEQ ID NO: 23 of the protein (H) sequence table having the amino acid sequence of amino acid residues 23 to 645 of the amino acid sequence of SEQ ID NO: 23 Having an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions and / or inversions, and L-phenylalanine is α- of L-aspartic acid-α, β-diester A protein having an activity of selectively binding a peptide to an ester site (I) A protein having an amino acid sequence of amino acid residues 26 to 620 among amino acid sequences set forth in SEQ ID NO: 25 of the sequence listing (J) SEQ ID NO: of the sequence listing In the amino acid sequence of amino acid residues 26 to 620 of the amino acid sequence described in 25, one or several amino acids Having an amino acid sequence containing no acid substitution, deletion, insertion, addition, and / or inversion, and L-phenylalanine selectively at the α-ester site of L-aspartic acid-α, β-diester The amino acid sequence shown in SEQ ID NO: 27 of the protein (L) sequence table having the amino acid sequence of amino acid residues 18 to 644 out of the amino acid sequence shown in SEQ ID NO: 27 of the protein (K) sequence table having the activity to bind the peptide In the amino acid sequence of amino acid residues 18 to 644, and having an amino acid sequence including substitution, deletion, insertion, addition, and / or inversion of one or several amino acids, and L-phenylalanine is L -Sequence of the protein (M) sequence table having the activity of selectively binding a peptide to the α-ester site of aspartic acid-α, β-diester Amino acids comprising one or several amino acid substitutions, deletions, insertions, additions and / or inversions in the amino acid sequence set forth in SEQ ID NO: 6 in the protein (N) sequence listing having the amino acid sequence set forth in No. 6 SEQ ID NO: of the protein (O) sequence listing having a sequence and having a mature protein region that selectively binds L-phenylalanine to the α-ester site of L-aspartic acid-α, β-diester Amino acid sequence comprising one or several amino acid substitutions, deletions, insertions, additions, and / or inversions in the amino acid sequence shown in SEQ ID NO: 12 of the protein (P) sequence listing having the amino acid sequence shown in FIG. And L-phenylalanine is selectively bonded to the α-ester site of L-aspartic acid-α, β-diester In the amino acid sequence shown in SEQ ID NO: 18 in the protein (R) sequence list, which has the amino acid sequence shown in SEQ ID NO: 18 in the protein (Q) sequence list, including the mature protein region having the activity of one or several amino acids It has an amino acid sequence including substitution, deletion, insertion, addition and / or inversion, and L-phenylalanine is selectively peptide-bonded to the α-ester site of L-aspartic acid-α, β-diester. Substitution of one or several amino acids in the amino acid sequence shown in SEQ ID NO: 23 of the protein (T) sequence list having the amino acid sequence shown in SEQ ID NO: 23 of the protein (S) sequence list including the mature protein region having activity , Deletions, insertions, additions, and / or inversions, and L-phenylalanine is L- A protein (V) having the amino acid sequence shown in SEQ ID NO: 25 in the protein (U) sequence table, which contains a mature protein region having an activity of selectively binding a peptide to the α-ester site of aspartic acid-α, β-diester It has an amino acid sequence containing substitution, deletion, insertion, addition, and / or inversion of one or several amino acids in the amino acid sequence set forth in SEQ ID NO: 25 in the Table, and L-phenylalanine is converted to L-asparagine Protein (X) Sequence Listing having the amino acid sequence set forth in SEQ ID NO: 27 in the Protein (W) Sequence Listing, which contains a mature protein region having an activity of selectively binding a peptide to the α-ester site of the acid-α, β-diester 1 or several amino acid substitutions, deletions, insertions, additions, and Has an amino acid sequence comprising a beauty / or inversions, and, L- phenylalanine L- aspartic acid-.alpha., proteins comprising the mature protein region having a selectively active for peptide bond α- ester moiety β- diester   A reaction step of synthesizing α-L-aspartyl-L-phenylalanine-β-methyl ester by the method for producing α-L-aspartyl-L-phenylalanine-β-ester according to any one of claims 1 to 16. And a reaction step of converting α-L-aspartyl-L-phenylalanine-β-methyl ester to α-L-aspartyl-L-phenylalanine-α-methyl ester, α-L-aspartyl-L-phenylalanine- A method for producing α-methyl ester.

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