RU2312149C2 - Method for preparing alpha-l-aspartyl-l-phenylalanine-beta-ester and method for preparing alpha-l-aspartyl-l-phenylalanine-alpha-methyl ester - Google Patents

Abstract

FIELD: biotechnology, food processing industry, derivatives of amino acids.

SUBSTANCE: invention proposes a method for enzymatic preparing α-L-aspartyl-L-phenylalanine-β-ester (α-L-(β-o-substituted aspartyl)-L-phenylalanine) from L-aspartic acid α,β-diester and L-phenylalanine at temperature 5-40⁰C and pH 7-10 for period sufficient for formation of α-L-aspartyl-L-phenylalanine-β-ester from L-aspartic acid α,β-diester and L-phenylalanine. Proposed method is used in a method for preparing α-L-aspartyl-L-phenylalanine-α-methyl ester that comprises a step for synthesis of α-L-aspartyl-L-phenylalanine-β-methyl ester and step for conversion of indicates ester to α-L-aspartyl-L-phenylalanine-α-methyl ester. Using this invention provides synthesis of α-L-aspartyl-L-phenylalanine-β-ester followed by preparing α-L-aspartyl-L-phenylalanine-α-methyl ester by simplified and inexpensive scheme and with enhanced yield of the end product.

EFFECT: improved method of synthesis.

5 cl, 1 dwg, 4 tbl, 12 ex

Description

FIELD OF THE INVENTION

The present invention relates to a method for producing α-L-aspartyl-L-phenylalanine-β-ester (also called "α-L- (β-o-substituted aspartyl) -L-phenylalanine" (abbreviation: α-ARP)) and to a method for producing α-L-aspartyl-L-phenylalanine-α-methyl ester (also called "α-L-aspartyl-L-phenylalanine methyl ester (abbreviation: α-APM). More specifically, the present invention relates to a method for producing α- L-aspartyl-L-phenylalanine-β-ester, which is an important intermediate for the production of α-L-aspartyl-L-phenylalanine-α-methyl ester (product name: a Spartam), which is in high demand as a sweetener, and to a method for producing α-L-aspartyl-L-phenylalanine-α-methyl ester using the method for producing α-L-aspartyl-L-phenylalanine-β-ester.

State of the art

Well-known methods for producing α-L-aspartyl-L-phenylalanine-α-methyl ester (hereinafter “α-APM” for abbreviation in some cases) include a chemical synthesis method and an enzymatic synthesis method. As for the chemical synthesis method, a method is known for condensing N-protected L-aspartic anhydride with L-phenylalanine methyl ester to obtain N-protected APM and removing the N-protecting group to obtain APM, and as for the enzymatic synthesis method, the known method condensing the N-protected L-aspartic acid with L-phenylalanine methyl ester to give an N-protected APM and removing the N-protecting group to give APM. In both methods, however, the steps of introducing and removing the protecting group are necessary, and the processes are very difficult. On the other hand, a method has been developed to produce APM without using an N-protecting group (see Japanese Patent Publication No. H02-015196 Gazette). However, this method is not suitable for industrial production due to the very low yield of the product. Thus, in this regard, it is desirable to develop an inexpensive method for the industrial production of aspartame.

Disclosure of invention

The aim of the present invention is a simple inexpensive method for producing α-L-aspartyl-L-phenylalanine-β-ester, which is an intermediate of α-L-aspartyl-L-phenylalanine-α-methyl ether, providing a high yield without using a complex synthesis method . In addition, it is an object of the present invention to provide a simple, inexpensive method for producing α-L-aspartyl-L-phenylalanine-α-methyl ester, providing a high yield.

As a result of research on the above objectives, the inventors of the present invention found that a recently discovered enzyme or substance containing the enzyme is capable of selectively synthesizing α-L-aspartyl-L-phenylalanine-β-ester from α, β-diester of L-aspartic acid and L-phenylalanine, which led to the implementation of the present invention.

Specifically, the present invention is described below.

[1] A method for producing α-L-aspartyl-L-phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine), comprising producing α-L-aspartyl-L -phenylalanine-β-ester from α, β-diester of L-aspartic acid and L-phenylalanine using an enzyme or enzyme-containing substances that are able to selectively attach L-phenylalanine to the α-ester portion of the α, β-diester of L-aspartic acid via peptide bond.

[2] A method for producing α-L-aspartyl-L-phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine) according to [1] above, wherein the enzyme or containing the enzyme substance is one or two or more types selected from the group comprising a culture of a microorganism that has the ability to selectively attach L-phenylalanine to the α-ether portion of the α, β-diester of L-aspartic acid via a peptide bond, a microorganism cell isolated from culture, and the processed product of the microorganism cells of the microorganism.

[3] A method for producing α-L-aspartyl-L-phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine) according to [2] above, wherein the microorganism is is a microorganism belonging to a genus selected from the group consisting of Aeromonas, Azotobacter, Alcaligenes, Brevibacterium, Corynebacterium, Escherichia, Empedobacter, Flavobacterium, Microbacterium, Propionibacterium, Brevibacillus, Paenibacillus, Prophomida, Pseudomata Candida, Geotrichum, Pichia, Saccharomyces, Torulaspora, Cellulophaga, Weeksell, Pedobacter, Persicobacter, Flexithrix, Chitinophaga, Cyclobacterium, Runella, Thermonema, Psychroserpens, Gelidibacter, Dyadobacter, Flammeovirumumobomobomobomobomobomobomobomobomobomobomobomobomomibobomoma auda, Salegentibacter, Taxeobacter, Cytophaga, Marinilabilia, Lewinella, Saprospira, and Haliscomenobacter.

[4] A method for producing α-L-aspartyl-L-phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine) according to [2] above, wherein the microorganism is a transformed microorganism that is able to express protein (A) or (B):

(A) a protein with an amino acid sequence consisting of amino acid residues 23 to 616 of the amino acid sequence shown in SEQ ID NO: 6 of the sequence listing,

(B) a protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or many amino acids in an amino acid sequence consisting of amino acid residues 23 to 616 of the amino acid sequence shown in SEQ ID NO: 6 of the sequence listing, and capable of selectively attaching L-phenylalanine to the α-ether portion of the α, β-diester of L-aspartic acid via a peptide bond.

[5] The method for producing α-L-aspartyl-L-phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine) according to [2], wherein the microorganism is transformed a microorganism that is capable of expressing protein (C) or (D):

(C) a protein with an amino acid sequence consisting of amino acid residues numbered 21 through 619 of the amino acid sequence shown in SEQ ID NO: 12 of the sequence listing,

(D) a protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or many amino acids in an amino acid sequence consisting of amino acid residues 21 to 619 of the amino acid sequence shown in SEQ ID NO: 12 of the sequence listing, and capable of selectively attaching L-phenylalanine to the α-ether portion of the α, β-diester of L-aspartic acid via a peptide bond.

[6] A method for producing α-L-aspartyl-L-phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine) according to [2], wherein the microorganism is a transformed microorganism that is capable of expressing a protein (E) or (F) below:

(E) a protein with an amino acid sequence consisting of amino acid residues numbered 23 to 625 of the amino acid sequence shown in SEQ ID NO: 18 of the sequence listing,

(F) a protein with an amino acid sequence including substitution, deletion, isertia, addition and / or inversion of one or many amino acids in the amino acid sequence consisting of amino acid residues 23 to 625 of the amino acid sequence shown in SEQ ID NO: 18 of the sequence listing, and capable of selectively attaching L-phenylalanine to the α-ether portion of the α, β-diester of L-aspartic acid via a peptide bond.

[7] A method for producing α-L-aspartyl-L-phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine) according to [2] above, wherein the microorganism is is a transformed microorganism that is able to express protein (G) or (H), below:

(G) a protein with an amino acid sequence consisting of amino acid residues numbered 23 to 645 of the amino acid sequence shown in SEQ ID NO: 23 of the sequence listing,

(H) a protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or many amino acids in the amino acid sequence consisting of amino acid residues 23 to 645 of the amino acid sequence shown in SEQ ID NO: 23 of the sequence listing, and capable of selectively attaching L-phenylalanine to the α-ether portion of the α, β-diester of L-aspartic acid via a peptide bond.

[8] The method for producing α-L-aspartyl-L-phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine) according to [2], wherein the microorganism is a transformed microorganism that is able to express protein (I) or (J) below:

(I) a protein with an amino acid sequence consisting of amino acid residues with numbers 26 to 620 of the amino acid sequence shown in SEQ ID NO: 25 of the sequence listing,

(J) a protein with an amino acid sequence including the substitution, deletion, insertion, addition and / or inversion of one or many amino acids in the amino acid sequence consisting of amino acid residues 26 to 620 of the amino acid sequence shown in SEQ ID NO: 25 of the sequence listing, and capable of selectively attaching L-phenylalanine to the α-ether portion of the α, β-diester of L-aspartic acid via a peptide bond.

[9] A method for producing α-L-aspartyl-L-phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine) according to [2] above, wherein the microorganism is a transformed microorganism that is capable of expressing a protein (K) or (L) below:

(K) a protein with an amino acid sequence consisting of amino acid residues 18 to 644 of the amino acid sequence shown in SEQ ID NO: 27 of the sequence listing,

(L) a protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or many amino acids in an amino acid sequence consisting of amino acid residues 18 to 644 of the amino acid sequence shown in SEQ ID NO: 27 of the sequence listing, and capable of selectively attaching L-phenylalanine to the α-ether portion of the α, β-diester of L-aspartic acid via a peptide bond.

[10] The method for producing α-L-aspartyl-L-phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine) according to [2], wherein the microorganism is a transformed microorganism that is capable of expressing a protein (M) or (N) below:

(M) a protein with the amino acid sequence shown in SEQ ID NO: 6 of the sequence listing,

(N) a protein containing a region of a mature protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or multiple amino acids in the amino acid sequence shown in SEQ ID NO: 6 of the sequence listing, and capable of selectively attaching L-phenylalanine to the α-ether site of the α, β-diester of L-aspartic acid via a peptide bond.

[11] The method for producing α-L-aspartyl-L-phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine) according to [2], wherein the microorganism is a transformed microorganism that is capable of expressing a protein (O) or (P) below:

(O) a protein with the amino acid sequence shown in SEQ ID NO: 12 sequence listing,

(P) a protein containing a region of a mature protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or many amino acids in the amino acid sequence shown in SEQ ID NO: 12 of the sequence listing, and capable of selectively attaching L-phenylalanine to the α-ether site of the α, β-diester of L-aspartic acid via a peptide bond.

[12] A method for producing α-L-aspartyl-L-phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine) according to [2], wherein the microorganism is a transformed microorganism that is able to express protein (Q) or (R), below:

(Q) a protein comprising a mature protein region with the amino acid sequence shown in SEQ ID NO: 18 of the sequence listing,

(R) a protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or multiple amino acids in the amino acid sequence shown in SEQ ID NO: 18 of the sequence listing, and capable of selectively attaching L-phenylalanine to the α-ether site α, β-diester of L-aspartic acid via a peptide bond.

[13] The method for producing α-L-aspartyl-L-phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine) according to [3], wherein the microorganism is a transformed microorganism that is capable of expressing a protein (S) or (T) below:

(S) a protein with the amino acid sequence shown in SEQ ID NO: 23 sequence listing,

(T) a protein containing a region of a mature protein with an amino acid sequence including the substitution, deletion, insertion, addition and / or inversion of one or many amino acids in the amino acid sequence shown in SEQ ID NO: 23 of the sequence listing, and capable of selectively attaching L-phenylalanine to the α-ether site of the α, β-diester of L-aspartic acid via a peptide bond.

[14] The method for producing α-L-aspartyl-L-phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine) according to [2], wherein the microorganism is a transformed microorganism that is capable of expressing a protein (U) or (V) below:

(U) a protein with the amino acid sequence shown in SEQ ID NO: 25 sequence listing,

(V) a protein containing a region of a mature protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or many amino acids in the amino acid sequence shown in SEQ ID NO: 25 of the sequence listing, and capable of selectively attaching L-phenylalanine to the α-ether site of the α, β-diester of L-aspartic acid via a peptide bond.

[15] The method for producing α-L-aspartyl-L-phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine) according to [2] above, wherein the microorganism is a transformed microorganism that is capable of expressing a protein (W) or (X) below:

(W) a protein with the amino acid sequence shown in SEQ ID NO: 27 of the sequence listing,

(X) a protein containing a region of a mature protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or many amino acids in the amino acid sequence shown in SEQ ID NO: 27 of the sequence listing, and capable of selectively attaching L-phenylalanine to the α-ether site of the α, β-diester of L-aspartic acid via a peptide bond.

[16] A method for producing α-L-aspartyl-L-phenylalanine-β-ester (ie, α-L- (β-o-substituted aspartyl) -L-phenylalanine) according to [1] above, wherein the enzyme is at least one of the proteins selected from the group comprising (A) - (X) below:

(A) a protein with an amino acid sequence consisting of amino acid residues 23 to 616 of the amino acid sequence shown in SEQ ID NO: 6 of the sequence listing,

(B) a protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or many amino acids in an amino acid sequence consisting of amino acid residues 23 to 616 of the amino acid sequence shown in SEQ ID NO: 6 of the sequence listing and capable of selectively attaching L-phenylalanine to the α-ester portion of the α, β-diester of L-aspartic acid via a peptide bond,

(C) a protein with an amino acid sequence consisting of amino acid residues numbered 21 to 619 of the amino acid sequence shown in SEQ ID NO: 12 of the sequence listing,

(D) a protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or many amino acids in an amino acid sequence consisting of amino acid residues 21 to 619 of the amino acid sequence shown in SEQ ID NO: 12 of the sequence listing and capable of selectively attaching L-phenylalanine to the α-ester portion of the α, β-diester of L-aspartic acid via a peptide bond,

(E) a protein with an amino acid sequence consisting of amino acid residues numbered 23 to 625 of the amino acid sequence shown in SEQ ID NO: 18 of the sequence listing,

(F) a protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or many amino acids in an amino acid sequence consisting of amino acid residues 23 to 625 of the amino acid sequence shown in SEQ ID NO: 18 of the sequence listing and capable of selectively attaching L-phenylalanine to the α-ester portion of the α, β-diester of L-aspartic acid via a peptide bond,

(G) a protein with an amino acid sequence consisting of amino acid residues numbered 23 to 645 of the amino acid sequence shown in SEQ ID NO: 23 of the sequence listing,

(H) a protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or a plurality of amino acids in an amino acid sequence consisting of amino acid residues 23 to 645 of the amino acid sequence shown in SEQ ID NO: 23 of the sequence listing and capable of selectively attaching L-phenylalanine to the α-ester portion of the α, β-diester of L-aspartic acid via a peptide bond,

(I) a protein with an amino acid sequence consisting of amino acid residues with numbers 26 to 620 of the amino acid sequence shown in SEQ ID NO: 25 of the sequence listing,

(J) a protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or many amino acids in an amino acid sequence consisting of amino acid residues 26 to 620 of the amino acid sequence shown in SEQ ID NO: 25 of the sequence listing and capable of selectively attaching L-phenylalanine to the α-ester portion of the α, β-diester of L-aspartic acid via a peptide bond,

(K) a protein with an amino acid sequence consisting of amino acid residues 18 to 644 of the amino acid sequence shown in SEQ ID NO: 27 of the sequence listing,

(L) a protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or many amino acids in an amino acid sequence consisting of amino acid residues 18 to 644 of the amino acid sequence shown in SEQ ID NO: 27 of the sequence listing and capable of selectively attaching L-phenylalanine to the α-ester portion of the α, β-diester of L-aspartic acid via a peptide bond,

(M) a protein with the amino acid sequence shown in SEQ ID NO: 6 of the sequence listing,

(N) a protein containing a region of a mature protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or many amino acids in the amino acid sequence shown in SEQ ID NO: 6 of the sequence listing, and capable of selectively attaching L-phenylalanine to the α-ether site of the α, β-diester of L-aspartic acid via a peptide bond,

(O) a protein with the amino acid sequence shown in SEQ ID NO: 12 of the sequence listing,

(P) a protein containing a region of a mature protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or multiple amino acids in the amino acid sequence shown in SEQ ID NO: 12 of the sequence listing, and capable of selectively attaching L-phenylalanine to the α-ether site of the α, β-diester of L-aspartic acid through a peptide bond,

(Q) a protein with the amino acid sequence shown in SEQ ID NO: 18 of the sequence listing,

(R) a protein containing a region of a mature protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or many amino acids in the amino acid sequence shown in SEQ ID NO: 18 of the sequence listing, and capable of selectively attaching L-phenylalanine to the α-ether site of the α, β-diester of L-aspartic acid via a peptide bond,

(S) a protein with the amino acid sequence shown in SEQ ID NO: 23 sequence listing,

(T) a protein containing a region of a mature protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or many amino acids in the amino acid sequence shown in SEQ ID NO: 23 of the sequence listing, and capable of selectively attaching L-phenylalanine to the α-ether site of the α, β-diester of L-aspartic acid via a peptide bond,

(U) a protein with the amino acid sequence shown in SEQ ID NO: 25 sequence listing,

(V) a protein containing a region of a mature protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or many amino acids in the amino acid sequence shown in SEQ ID NO: 25 of the sequence listing, and capable of selectively attaching L-phenylalanine to the α-ether site of the α, β-diester of L-aspartic acid via a peptide bond,

(W) a protein with the amino acid sequence shown in SEQ ID NO: 27 of the sequence listing, and

(X) a protein containing a region of a mature protein with an amino acid sequence in the amino acid sequence set forth in SEQ ID NO: 27 of the sequence listing and capable of selectively attaching L-phenylalanine to the α-ester portion of the α, β-diester of L-aspartic acid via the peptide communication.

[17] A method for producing α-L-aspartyl-L-phenylalanine-α-methyl ester (ie, α-L-aspartyl-L-phenylalanine methyl ester), comprising: a reaction step for synthesizing α-L-aspartyl-L- phenylalanine-β-methyl ester (also called α-L- (β-o-methylaspartyl) -L-phenylalanine (abbreviated: α-AMP) using the method for producing α-L-aspartyl-L-phenylalanine-β-ester according to any from 1-16; and a reaction step for converting α-L-aspartyl-L-phenylalanine-β-methyl ester (i.e., α-L- (β-o-methylaspartyl) -L-phenylalanine) to α-L- aspartyl-L-phenylalanine-α-methyl ether.

α-L-aspartyl-L-phenylalanine-β-ether can be easily obtained using the present invention. α-L-aspartyl-L-phenylalanine-β-ether can be easily obtained and have a high yield along with reduced use of complex synthesis methods, such as the introduction / removal of protective groups using the method of the present invention.

Moreover, using the present invention, α-L-aspartyl-L-phenylalanine-α-methyl ether can be easily obtained and can have a high yield at a minimum cost.

Other objects, features and advantages of the present invention will be described in detail or will become apparent from the following description, accompanied by figures.

Brief description of the figure

The drawing shows a diagram showing the number of enzymes that exists in the cytoplasmic fraction (Cy) and periplasm fraction (Pe).

Best Mode for Carrying Out the Invention

Further, the present invention will be described in the following order

<1> Method for producing α-L-aspartyl-L-phenylalanine-β-ester

1. The method of obtaining α-L-aspartyl-L-phenylalanine-β-ester

2. Microorganisms used in the present invention

3. Enzymes used in the present invention; and

<2> Method for producing α-L-aspartyl-L-phenylalanine-α-methyl ester.

<1> Method for producing α-L-aspartyl-L-phenylalanine-β-ester

1. The method of obtaining α-L-aspartyl-L-phenylalanine-β-ester

In the method for producing α-L-aspartyl-L-phenylalanine-β-ester of the present invention (also referred to herein as the “method of producing the peptide of the present invention"), L-phenylalanine and α, β-diester of L-aspartic acid are reacted in the presence of an enzyme, with known peptide-forming activity. That is, the method for producing the peptide of the present invention is that α-L-aspartyl-L-phenylalanine-β-ether is formed from the α, β-diester of L-aspartic acid and L-phenylalanine using an enzyme or substances containing the enzyme capable of selectively attach L-phenylalanine to the α-ether portion of the α, β-diester of L-aspartic acid via a peptide bond. An enzyme or enzyme-containing substance capable of selectively attaching L-phenylalanine to the α-ester portion of the α, β-diester of L-aspartic acid, refers to an enzyme or enzyme-containing substance having the ability or activity to catalyze a reaction in which essentially L-phenylalanine does not it is capable of exerting a nucleophilic effect on the β-ether portion of the α, β-diester of L-aspartic acid, but carries out nucleophilic effects only on its α-ether portion. As shown hereinafter in the reference example, however, an enzyme or an enzyme-containing substance is obtained which is capable of catalyzing a reaction in which essentially L-phenylalanine is not capable of influencing the α-ester portion of the α, β-diester of L-aspartic acid, but carries out a nucleophilic effect only on its β-ether site, in contrast to the above ability, and that gives β-L-aspartyl-L-phenylalanine-α-ether (also called β-L- (α-o-substituted aspartyl) - L-phenylalanine (abbreviation: β-ARP) from α, β-diester L- sparaginovoy acid and L-phenylalanine.

A reaction formula in which L-phenylalanine exerts a nucleophilic effect on the α-ester portion of the α, β-diester of L-aspartic acid to produce α-L-aspartyl-L-phenylalanine-β-ester (abbreviation: α-ARP) is presented in the following formula (I-α) (where “Me” is a methyl group) by citing the case in which α, β-dimethyl ester of L-aspartic acid is used as the α, β-diester of L-aspartic acid. As shown in formula (I-α), in the method for producing the peptide of the present invention, the amino group of L-phenylalanine interacts with the α-methyl ester portion of the α, β-dimethyl ester of L-aspartic acid to form a peptide bond. On the other hand, the following formula (I-β) indicates a reaction in which the β-methyl ester portion of the α, β-dimethyl ester of L-aspartic acid is subjected to nucleophilic action to form β-L-aspartyl-L-phenylalanine-α-methyl ether (also called β-L- (α-o-methyl-aspartyl) -L-phenylalanine (abbreviation: β-AMP)). The peptide bond in β-AMP is formed at the β-methyl ether portion of the α, β-dimethyl ester of L-aspartic acid. The enzyme or enzyme-containing substance used in the present invention substantially only accelerates the reaction as in formula (I-α), but essentially does not cause a reaction as in formula (I-β). α-APM can be obtained from α-AMP via a simple reaction step (formula (II)), but α-APM cannot be obtained directly from β-AMP. Thus, the method of the present invention is extremely effective as a method for producing the α-APM intermediate and can be used for industrial production.

Reaction formula I-α

Reaction formula I-β

Reaction Formula II

A method for allowing an enzyme or an enzyme-containing substance to interact with an α, β-diester of L-aspartic acid and L-phenylalanine can be carried out by mixing the enzyme or an enzyme-containing substance with an α, β-diester of L-aspartic acid and L-phenylalanine. More specifically, a method can be used in which an enzyme or an enzyme-containing substance is added to a solution containing L-aspartic acid diester and L-phenylalanine to influence the reaction. When a microorganism is used as an enzyme-containing substance that produces an enzyme, the reaction can be carried out either as described above, or a method can be used that involves culturing a microorganism that produces an enzyme to produce and accumulate the enzyme in the microorganism or culture medium in which it is cultured. microorganism, and the addition of α, β-diester of L-aspartic acid and L-phenylalanine to the culture fluid, or a similar method. The α-L-aspartyl-L-phenylalanine-β-ether thus obtained is isolated in a conventional manner and, if necessary, can be purified.

An “enzyme-containing substance” can be any substance if it contains an enzyme, and specific types thereof include a microorganism culture that produces the enzyme, a microorganism cell isolated from the culture, and a processed microorganism cell product. Under the culture of a microorganism is meant a substance obtained by culturing a microorganism, and, specifically, a mixture of cells of the microorganism, the medium used to cultivate the microorganism, and the substance produced by the cultured microorganism, and so on. In addition, the microorganism cell can be purified for use as a purified microorganism cell. Moreover, the processed microorganism cell product includes products that are obtained by disrupting, lysing or freezing-thawing the microorganism cell and further recovering the crude enzyme by treating the microorganism cell and purifying the enzyme by further purification. As an enzyme processed for purification, a partially purified enzyme obtained by various purification methods and so on can be used. In addition, immobilized enzymes that are immobilized using the covalent bond formation method, the adsorption method, the capture method, and so on can be used. In addition, in the case of some microorganisms used, part of the cells of the microorganism can be lysed during cultivation, and in this case, the supernatant of the culture fluid can also be used as an enzyme-containing substance.

In addition, a wild-type strain can be used as the microorganism that contains the enzyme, or a recombinant strain in which the enzyme is expressed can be used. Such a microorganism is not limited to a microorganism producing cell of the enzyme, and processed microorganism cell products such as an acetone-treated microorganism cell and a lyophilized microorganism cell can also be used. In addition, immobilized microorganism cells obtained by immobilizing a treated microorganism cell product using a covalent bonding method, an adsorption method, a capture method or the like, or an immobilized processed microorganism cell product can be used.

The use of a wild-type strain that can produce a peptide-forming enzyme and capable of forming α-L-aspartyl-L-phenylalanine-β-ester is preferred since the preparation of the peptide can be carried out more easily without going through the step of creating a recombinant strain. Alternatively, a recombinant strain that has been transformed and expresses a peptide forming enzyme and capable of forming α-L-aspartyl-L-phenylalanine-β-ester can be modified so that the peptide forming enzyme is produced in greater quantity. Thus, it is possible to synthesize α-L-aspartyl-L-phenylalanine-β-ether in greater quantity and with higher intensity. Cultivating a microorganism of a wild-type strain or a recombinant strain in a medium for accumulation of a peptide-forming enzyme and / or microorganism in a medium and mixing the product thus accumulated with the α, β-diester of L-aspartic acid and L-phenylalanine can produce α-L- aspartyl-L-phenylalanine-β-ester.

It should be noted that when using cultured products, cultured microorganism cells, washed microorganism cells and processed microorganism cell products obtained by disrupting or lysing microorganism cells, an enzyme is often present that destroys the formed α-L-aspartyl-L-phenylalanine-β- ether without being involved in the formation of α-L-aspartyl-L-phenylalanine-β-ester. In such a case, in some cases, it is preferable to add a metalloproteinase inhibitor such as ethylenediaminetetraacetic acid (EDTA). The amount added is in the range from 0.1 millimolar (mM) to 300 mM, preferably from 1 mM to 100 mM.

The amount of enzyme or enzyme-containing substance used may be sufficient if that amount is the amount at which the desired effect is observed (effective amount). Although a person skilled in the art can easily determine such an effective amount by a simple preliminary experiment, the amount used is, for example, from about 0.01 to about 100 units ("Units") in the case of an enzyme and from about 0.1 up to approximately 500 g / l in the washed cells of the microorganism. It should be noted that 1 Unit is defined as the amount of enzyme that allows 1 micromole (μmol) of α-L-aspartyl-L-phenylalanine-β-methyl ester to be produced from 100 mM α, L-aspartic acid β-dimethyl ester and 200 mM L -phenylalanine at 25 ° C in one minute.

The α, β-L-aspartic acid diester used in the reaction may be any ester condensed with L-phenylalanine to give α-L-aspartyl-L-phenylalanine-β-ester. Examples of α, β-diester of L-aspartic acid include α, β-dimethyl ester of L-aspartic acid and α, β-diethyl ester of L-aspartic acid. When L-aspartic acid α, β-dimethyl ester and L-phenylalanine react, then α-L-aspartyl-L-phenylalanine-β-methyl ester (α-AMP) is formed, and when α, β-diethyl ester L- Aspartic acid and L-phenylalanine, then α-L-aspartyl-L-phenylalanine-β-ethyl ether (also called α-L- (β-o-ethyl aspartyl) -L-phenylalanine (abbreviation: α-AEP) is formed.

While the concentration of α, β-diester of L-aspartic acid and L-phenylalanine, which are the starting products, in each case are from 1 mM to 10 mM, preferably from 0.05 M to 2 M, there may be cases where it is preferable to add one substrate in equimolar or greater relative to another substrate and, if necessary, make a choice. In addition, in cases where high concentrations of substrates cause inhibition of the reaction, then the substrates are added during the reaction at a concentration that does not cause inhibition.

The reaction temperature at which it is possible to obtain α-L-aspartyl-L-phenylalanine-β-ester is from 0 to 60 ° C, preferably from 5 to 40 ° C. In addition, the pH of the reaction, in which it is possible to obtain α-L-aspartyl-L-phenylalanine-β-ester, is from 6.5 to 10.5, preferably from 7.0 to 10.0.

2. Microorganisms used in the present invention

As microorganisms used in the present invention, those microorganisms which are capable of producing α-L-aspartyl-L-phenylalanine-β-ether from α, β-diester of L-aspartic acid and L-phenylalanine can be used without particular limitation. Microorganisms that are capable of producing α-L-aspartyl-L-phenylalanine-β-ester from α, β-diester of L-aspartic acid and L-phenylalanine include, for example, genus-related microorganisms

Aeromonas, Azotobacter, Alcaligenes, Brevibacterium, Corynebacterium, Escherichia, Empedobacter, Flavobacterium, Microbacterium, Propionibacterium, Brevibacillus, Paenibacillus, Pseudomonasomophisomphisophida, Somphida, Somphida, Somphidae Weeksella, Pedobacter, Persicobacter, Flexithrix, Chitinophaga, Cyclobacterium, Runella, Thermonema, Psychroserpens, Gelidibacter, Dyadobacter, Flammeovirga, Spirosoma, Flectobacillus, Tenacibacilobecobacta biblactaibecomactum, Rhodotermus, Zobelliabilobecomibica Murica

The following specific examples may be given.

Aeromonas hydrophila ATCC 13136

Azotobacter vinelandii IFO 3741

Alcaligenes faecalis FERM P-8460

Brevibacterium minutiferuna FERM BP-8277

Corynebacterium flavescens ATCC 10340

Escherichia coli FERM BP-8276

Empedobacter brevis ATCC 14234

Flavobacterium resinovorum ATCC 14231

Microbacterium arborescens ATCC 4348

Propionibacterium shermanii FERM BP-8100

Brevibacillus parabrevis ATCC 8185

Paenibacillus alvei IFO 14175

Pseudomonas fragi IFO 3458

Serratia grimesii ATCC 14460

Stenotrophomonas maltophilia ATCC 13270

Sphingobacterium sp. PERM BP-8124

Streptomyces griseolus NRRL B-1305

(Streptomyces lavendulae)

Xanthomonas maltophilia FERM BP-5568

Williopsis saturnus IFO 0895

Candida magnoliae IFO 0705

Geotrichum fragrance CBS 152.25

(Geotrichum amycelium)

Geotrichum amycelium IFO 0905

Pichia ciferrii IFO 0905

Saccharomyces unisporus IFO 0724

Torulaspora delbrueckii IFO 0422

Cellulophaga lytica NBRC 14961

Weeksella virosa NBRC 16016

Pedobacter heparinus NBRC 12017

Persicobacter diffluens NBRC 15940

Flexithrix dorotheae NBRC 15987

Chitinophaga pinensis NBRC 15968

Cyclobacterium marinum ATCC 25205

Runella slithyformis ATCC 29530

Thermonema lapsum ATCC 43542

Psychroserpens burtonensis ATCC 700359

Gelidibacter algens ATCC 700364

Dyadobacter fermentans ATCC 700827

Flammeovirga aprica NBRC 15941

Spirosoma linguale DSMZ 74

Flectobacillus major DSMZ 103

Tenacibaculum maritimum ATCC 43398

Rhodotermus marinus DSMZ 4252

Zobellia galactanivorans DSMZ 12802

Muricauda ruestringensis DSMZ 13258

Salegentibacter salegens DSMZ 5424

Taxeobacter gelupurpurascens DSMZ 11116

Cytophaga hutchinsonii NBRC 15051

Marinilabilia salmonicolor NBRC 15948

Lewinella cohaerens ATCC 23123

Saprospira grandis ATCC 23119

Haliscomenobacter hydrossis ATCC 27775

Among the above microorganism strains, those microorganisms designated by the FERM number were deposited by an independent corporation, the National Institute for the Development of Industrial Science and Technology, the international depository of patented organisms (Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan), and can be provided with a link to each number.

Among the above microorganism strains, those microorganisms indicated by ATCC numbers were deposited by the American Type Culture Collection (P.O. Box 1549, Manassas, VA 20110, United States of America) and may be provided by reference to each number.

Among the above-mentioned microorganism strains, those microorganisms designated by IFO numbers were deposited by the Osaka Fermentation Institute (2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan) and can be provided by reference to each number.

Among the above microorganism strains, those microorganisms designated by NBRC numbers were deposited by the NITE Center for Biological Sources of the National Institute of Technology and Computing (5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) and can be provided at link to each number.

Among the above microorganism strains, those microorganisms indicated by DSMZ numbers were deposited with Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures) (Mascheroder Weg 1b, 38124 Braunschweig, Germany) and can be provided by reference to each number.

Like the above strains, those microorganisms that are marked with FERM numbers are microorganisms that have been deposited with an independent corporation, the National Institute for the Development of Industrial Science and Technology, the international depository of patented organisms (Chuo Dai-6, 1-1 Higashi 1 -Chome, Tsukuba-shi, Ibaraki-ken, 305-8566 Japan Alcaligenes faecalis FERM P-8460 is a microorganism that was deposited on September 30, 1985 and is assigned a deposit number of FERM P-8460. Propionibacterium shermanii FERM P-9737 represents a microorganism which was originally deposited on December 4, 1987, and control of this organism was subsequently transferred to the international depository under the terms of the Budapest Treaty of July 1, 2002, and was assigned the deposit number FERM BP-8100. Xanthomonas maltophilia FERM BP-5568 is a microorganism which was originally zadeponirovan June 14, 1995, and control of the body was later transferred to international deposition under the provisions of the Budapest treaty on June 14, 1996 Brevibacterium minutiferuna FERM BP-8277 has been internationally deposited on zadeponirovan n the conditions of the Budapest Treaty on January 20, 2002 Esherichia coli FERM BP-8276 was zadeponirovan an international deposition under the provisions of Budapest Treaty on January 20, 2002

Empedobacter brevis , strain ATCC 14234 (strain FERM P-18545, strain FERM BP-8113), was deposited in the international depository of patented organisms independent in the management of the corporation, the National Institute for the Development of Industrial Science and Technology (Chuo Dai-6, 1-1 Higashi 1 -Chome, Tsukuba-shi, Ibaraki-ken, Japan), October 1, 2001, and has been assigned the deposit number FERM P-18545. The control over this organism was subsequently transferred to the depository under the terms of the Budapest Treaty at the international depositary of patented organisms, an independent corporation, the national institute for the development of industrial science and technology, July 8, 2002, and was assigned the deposit number FERM P-8113 (microorganism index: Empedobacter brevis strain AJ 13933).

Sphingobacterium sp ., Strain AJ 110003, was deposited in the international depository of patented organisms independent in the management of the corporation, the National Institute for the Development of Industrial Science and Technology (address of the depository institution: Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan), July 22, 2002, and was assigned the deposit number FERM BP-8124.

It should be noted that strain AJ 110003 (FERM BP-8124) was identified as Sphingobacterium sp. in the identification experiment described below. Strain FERM BP-8124 is a gram-negative bacillus (from 0.7 to 0.8 x 1.5 to 2.0 microns), which does not form a spore and is stationary. Its colonies are round with completely smooth borders, contain few protrusions and have a brilliant, bright yellow color. The organism grows at 30 ° C and is catalase-positive, oxidase-positive and negative for the OF test (for glucose) and is identified on the basis of its properties as a bacterium belonging to the genus Sphingobacterium. Moreover, due to the fact that it is negative for nitrate reduction, negative for indole production, negative for glucose acid production, negative for arginine dihydrolase, positive for urease, positive for esculin hydrolysis, negative for hydrolysis of gelatin, positive for β-galactosidase, positive for assimilation of glucose, negative for assimilation of L-arabinose, positive for assimilation of D-mannose, negative for and assimilation of D-mannitol, positive for assimilation of N-acetyl-D-glucosamine, positive for assimilation of maltose, negative for assimilation of potassium gluconate, negative for assimilation of n-capric acid, negative for assimilation of adipic acid, negative for assimilation of dl-malic acid, negative for assimilation of sodium citrate, negative for assimilation of phenylacetate and positive for assimilation of cytochrome oxidase, it was found to have Twas that are similar to those of Sphingobacterium multivorum or Sphingobacterium spiritivorum . Moreover, although the results of the 16S rRNA gene base sequence homology analysis indicate that the highest degree of homology is found with Sphingobacterium multivorum (98.8%), there is no strain with which the bacterial strain fully coincides. Accordingly, this bacterial strain was therefore identified as Sphingobacterium sp .

As microorganisms, either wild-type strains or mutant strains can be used, or recombinant strains obtained by cell fusion or genetic methods such as genetic manipulations can be used.

To obtain cells of such microorganisms, microorganisms can be cultured and grown in a suitable medium. There are no particular restrictions on the environment used for this purpose, if it allows the growth of a microorganism. This medium, if necessary, may be a conventional medium containing conventional carbon sources, nitrogen sources, phosphorus sources, sulfur sources, inorganic ions and sources of organic nutrients.

For example, any carbon source can be used if microorganisms can use it. Specific examples of the carbon source that can be used include sugars such as glucose, fructose, maltose and amylose, alcohols such as sorbitol, ethanol and glycerin, organic acids such as fumaric acid, citric acid, acetic acid and propionic acid and their salts, hydrocarbons, such as paraffin, as well as mixtures thereof.

Examples of nitrogen sources that can be used 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, organic compounds containing nitrogen, such as peptones, yeast extract, meat extract and corn extract liquid, and mixtures thereof.

In addition, nitrogen sources used in conventional environments, such as inorganic salts, trace metal salts and vitamins, can also be suitably mixed and applied.

There is no particular limitation on the conditions of cultivation and cultivation can be carried out, for example, from about 12 to about 48 hours at a properly controlled pH and temperature with a pH range of 5 to 8 and a temperature in the range of 15 to 40 ° C. accordingly, under aerobic conditions.

3. Enzymes used in the present invention

In the method for producing the peptide of the present invention described above, an enzyme is used that is capable of selectively attaching L-phenylalanine to the α-ester portion of the α, β-diester of L-aspartic acid via a peptide bond. In the method for producing the peptide of the present invention, the enzyme is not limited by its origin and the proposed method, if it has such activity. Next, the purification of the enzymes used in the present invention and the application of genetic engineering methods will be described.

(3-1) Microorganisms containing the enzyme, which can be used for the method of obtaining the present invention

As microorganisms that produce the enzyme of the present invention, all microorganisms that are capable of producing α-L-aspartyl-L-phenylalanine-β-ester from α, β-diester of L-aspartic acid and L-phenylalanine can be used. Microorganisms include bacteria and the like, which belong to the genera selected from the group consisting of

Aeromonas, Azotobacter, Alcaligenes, Brevibacterium, Corynebacterium, Escherichia, Empedobacter, Flavobacterium, Microbacterium, Propionibacterium, Brevibacillus, Paenibacillus, Pseudomonasomophisomphisophida, Somphida, Somphida, Somphidae Weeksella, Pedobacter, Persicobacter, Flexithrix, Chitinophaga, Cyclobacterium, Runella, Thermonema, Psychmserpens, Gelidibacter, Dyadobacter, Flammeovirga, Spirosoma, Flectobacillus, Tenacibacerobecobacta biblioma bibuta, Bloombacteribactum, Rhodotermus

More specifically, microorganisms include Empedobacter brevis ATCC 14234 (FERM strain P-18545, FERM strain BP-8113 (depository institute: independent management corporation, national institute for the development of industrial science and technology, international patented organisms depository, depository institution address: Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, date of international transfer of deposit: July 8, 2002)), Sphingobacterium sp. FERM strain BP-8124 (depository institute: independent corporation in management, national institute for the development of industrial science and technology, international patented organisms depository, depository institution address: Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international deposit date: July 22, 2002), Pedobacter heparinus IFO 12017 (depository institute: fermentation institute, Osaka; 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan) Taxeobacter gelupurpurascens DSMZ 11116 (depository institute; the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German collection of microorganisms and cell cultures, Depository Institution Address; Mascheroder Weg 1b, 38124 Braunschweig, Germany), Cyclobacterium marinum ATCC 25205 (Depository Institute; American Type Culture Collection, Depository Institute Address; PO Box 1549, Manassas, VA 20110, United States of America) and Psychroserpens burtonensis ATCC 700359 (Depository Institute; American Type Culture Collection, Address of the Depository Institute; PO Box 1549, Manassas, VA 20110, United States of America) and so on. Empedobacter brevis ATCC strain 14234 (FERM strain P-18545, FERM strain BP-8113 (depository institute: independent management corporation, national institute for the development of industrial science and technology, international patented organisms depository, depository institution address: Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, date of international transfer of deposit: July 8, 2002)) and Sphingobacterium sp . FERM strain BP-8124 (depository institute: independent corporation in management, national institute for the development of industrial science and technology, international patented organisms depository, depository institution address: Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, international deposit date: July 22, 2002), Pedobacter heparinus IFO strain 12017 (depository institute: fermentation institute, Osaka; 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan ), Taxeobacter gelupurpurascens DSMZ strain 11116 (depository institute; the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German collection of microorganisms and cells cultures, depository institution address; Mascheroder Weg 1b, 38124 Braunschweig, Germany), Cyclobacterium marinum ATCC strain 25205 (depository institute; American crop type collection, depository institution address; PO Box 1549, Manassas, VA 20110, United States of America ) and Psyclloserpens burtonensis ATCC strain 700359 (depository institute; American Type Culture Collection, Depository Institution Address; PO Box 1549, Manassas, VA 20110, United States of America) and the like are microorganisms selected by the applicants of the present invention as a result of the search for microorganisms producing an enzyme that produces α-L-aspartyl-L-phenylalanine-β-ether from α, β -diester of L-aspartic acid and L-phenylalanine in high yield.

(3-2) Purification of the enzyme

As indicated above, the peptide forming enzyme used in the present invention can be purified from bacteria belonging, for example, to the genus Empedobacter . A method for isolating and purifying a peptide-forming enzyme from Empedobacter brevis is provided as an example of purification of an enzyme.

First, an extract of microorganism cells is obtained from the cells of the microorganism Empedobacter brevis , for example, strain FERM BP-8113 (depository institute: independent corporation in management, national institute for the development of industrial science and technology, international patented organisms depository, depository institution address: Chuo Dai-6 , 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, date of international transfer of deposit: July 8, 2002) by cell destruction using a physical method of destruction, such as ultrasonic destruction, or enzymatic Allowances fracture using an enzyme dissolving cell wall and removing the insoluble fraction by centrifugation and so forth. The peptide forming enzyme can then be purified by fractionating the cell extract obtained by the above method using a combination of conventional protein purification methods such as anion exchange chromatography, cation exchange chromatography or gel filtration chromatography.

An example of a carrier for use in anion exchange chromatography is Q-Sepharose HP (manufactured by Amersham). After passing the cell extract containing the enzyme through the column with the carrier, the enzyme is restored in the non-adsorbed fraction at pH 8.5.

An example of a carrier used in cation exchange chromatography is MonoS HR (manufactured by Amersham). After passing the cell extract containing the enzyme through the column with the carrier and adsorbing the enzyme on the column, the column is washed and the enzyme is eluted with a high salt concentration buffer solution. At this stage, the salt concentration may increase sequentially or a concentration gradient may be applied. For example, in the case of using MonoS HR, the enzyme adsorbed on the column is eluted at a NaCl concentration of from about 0.2 to about 0.5 M.

The enzyme purified by the method described above can then be further purified by standard gel filtration chromatography and so on. An example of a carrier used in gel filtration chromatography is Sephadex 200 pg (manufactured by Amersham).

In the above purification procedure, a fraction containing the enzyme can be identified by testing each fraction for peptide-forming activity in accordance with the method described in the examples that will be described later. The internal amino acid sequence of the enzyme purified by the method described above is presented in SEQ ID NO: 1 and SEQ ID NO: 2 of the sequence listing.

(3-3) Isolation of DNA, obtaining a transformant and purification of the enzyme forming the peptide

(3-3-1) DNA Isolation

The inventors of the present invention first managed to isolate one type of DNA of the enzyme that forms the peptide, which can be used in the method for producing the peptide of the present invention from the strain FERM BP-8113 Empedobacter brevis (depository institute: independent corporation in management, national institute for the development of industrial science and technology, international depository of patented organisms, the address of the depository institution: Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, date of international transfer of the deposit: July 8, 2002).

A DNA containing a base sequence consisting of bases 61 to 1908 of the base sequence shown in SEQ ID NO: 5, which is the DNA of the present invention, was isolated from the FERM BP-8113 Empedobacter brevis strain (Depository Institution: independent in management corporation, national institute for the development of industrial science and technology, international depository of patented organisms, depository institution address: Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, date of international transfer of deposit: 8 July 2002) . DNA containing a base sequence consisting of bases numbered 61 through 1908 is the coding part of the sequence (CDS). The base sequence, consisting of bases number 61 to 1908, contains the region of the signal sequence and the region of the mature protein. The region of the signal sequence is the region that consists of bases numbered 61 to 126, while the region of the mature protein is the region which consists of bases numbered 127 to 1908. Thus, the present invention relates to an enzymatic protein gene, forming a peptide that contains a signal sequence, and the gene of the enzymatic protein forming the peptide in the form of a mature protein. The signal sequence contained in the sequence shown in SEQ ID NO: 5 is a type of leader sequence. The main function of the leader peptide encoded by the leader sequence is thought to be excretion from outside the cell. The protein encoded by bases 127 to 1908, namely the ether region, with the exception of the leading peptide, is believed to be a mature protein and exhibits a high degree of activity with respect to the formation of the peptide.

DNA, consisting of a base sequence which consists of bases numbered 61 to 1917, presented in SEQ ID NO: 11, which is also the DNA of the present invention, was isolated from Sphingobacterium sp ., Strain FERM BP-8124 (depository institute : independent corporation in management, national institute for the development of industrial science and technology, international depository of patented organisms, address of depository institution: Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, date of international deposit : July 22, 2002). DNA, consisting of a base sequence, which consists of bases number 61 to 1917, is the coding part of the sequence (CDS). The base sequence, consisting of bases number 61 to 1917, contains the region of the signal sequence and the region of the mature protein. The region of the signal sequence is the region that consists of bases number 61 to 120, while the mature protein region is the region that consists of bases number 121 to 1917. Thus, the present invention relates to an enzymatic protein gene, forming a peptide that contains a signal sequence, and to the gene of the enzymatic protein forming the peptide in the form of a mature protein. The signal sequence contained in the sequence shown in SEQ ID NO: 11 is a type of leader sequence. The main function of the leader peptide encoded by the leader sequence is thought to be excretion from the cell. The protein encoded by bases 121 to 1917, namely the part, with the exception of the leading peptide, is believed to be a mature protein and exhibits a high degree of activity with respect to the formation of the peptide.

DNA, consisting of a base sequence which consists of bases numbered 61 to 1935, presented in SEQ ID NO: 17, which is also the DNA of the present invention, was isolated from Pedobacter heparinus , strain IFO 12017 (depository institute: Osaka fermentation institute ; 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan). DNA, consisting of a base sequence, which consists of bases from numbers 61 to 1935, presented in SEQ ID NO: 17, is the coding part of the sequence (CDS). The base sequence, consisting of bases number 61 to 1935, contains the region of the signal sequence and the region of the mature protein. The region of the signal sequence is the region that consists of bases number 61 to 126, while the mature protein region is the region that consists of bases number 127 to 1935. Thus, the present invention relates to an enzymatic protein gene, forming a peptide that contains a signal sequence, and to the gene of the enzymatic protein forming the peptide in the form of a mature protein. The signal sequence contained in the sequence shown in SEQ ID NO: 17 is a type of leader sequence. The main function of the leader peptide encoded by the leader sequence is thought to be excretion from the inside out through the cell membrane. The protein encoded by bases 127 to 1935, namely the part, with the exception of the leading peptide, is believed to be a mature protein and exhibits a high degree of activity with respect to the formation of the peptide.

DNA consisting of a base sequence which consists of bases numbered 61 to 1995 as set forth in SEQ ID NO: 22, which is also the DNA of the present invention, was isolated from Taxeobacter gelupurpurascens DSMZ 11116 (depository institution; Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of Microorganisms and Cell Culture, Address of the Depository Institute; Mascheroder Weg 1b, 38124 Braunschweig, Germany). DNA consisting of a base sequence which consists of bases number 61 to 1995 described in SEQ ID NO: 22, is a coding cha sequence sequence (CDS). The base sequence consisting of bases number 61 to 1995 contains the signal sequence region and the mature protein region. The signal sequence region is the region that consists of bases 61 to 126, while the region a mature protein is a region that consists of bases from 127 to 1995. Thus, the present invention relates to a gene for an enzymatic protein forming a peptide that contains a signal sequence NOSTA and enzyme protein to a gene forming peptide to form a mature protein. The signal sequence contained in the sequence shown in SEQ ID NO: 22 is a type of leader sequence. The main function of the leader peptide encoded by the leader sequence is thought to be excretion from the cell. The protein encoded by bases 127 to 1995, namely the part, with the exception of the leading peptide, is believed to be a mature protein and exhibits a high degree of activity with respect to peptide formation.

DNA, consisting of a base sequence that consists of bases numbered 29 through 1888, presented in SEQ ID NO: 24, which is also the DNA of the present invention, was isolated from Cyclobacterium marinum ATCC 25205 (Depository Institute; American Type Culture Collection, Depository Institution; PO Box 1549, Manassas, VA 20110, United States of America). DNA, consisting of a base sequence, which consists of bases number 29 through 1888, described in SEQ ID NO: 24, is the coding part of the sequence (CDS). The base sequence, consisting of bases 291 to 1888, contains a region of the signal sequence and a region of mature protein. The region of the signal sequence is the region that consists of bases number 29 to 103, while the mature protein region is the region that consists of bases number 104 to 1888. Thus, the present invention relates to an enzymatic protein gene, forming a peptide that contains a signal sequence, and to the gene of the enzymatic protein forming the peptide in the form of a mature protein. The signal sequence contained in the sequence shown in SEQ ID NO: 24 is a type of leader sequence. The main function of the leader peptide encoded by the leader sequence is thought to be excretion from the cell. The protein encoded by bases number 104 to 1888, namely the part, with the exception of the leading peptide, is supposed to be a mature protein and exhibits a high degree of activity with respect to the formation of the peptide.

DNA, consisting of a base sequence that consists of bases numbered 61 to 1992, presented in SEQ ID NO: 26, which is also the DNA of the present invention, was isolated from Psychroserpens burtonensis ATCC 700359 (Depository Institute; American Type Culture Collection, Depository Institution; PO Box 1549, Manassas, VA 20110, United States of America). DNA, consisting of a base sequence, which consists of bases from numbers 61 to 1992, presented in SEQ ID NO: 26, is the coding part of the sequence (CDS). The base sequence, consisting of bases number 61 to 1992, contains the region of the signal sequence and the region of the mature protein. The region of the signal sequence is the region that consists of bases number 61 to 111, while the mature protein region is the region that consists of bases number 112 to 1992. Thus, the present invention relates to an enzymatic protein gene, forming a peptide that contains a signal sequence, and to the gene of the enzymatic protein forming the peptide in the form of a mature protein. The signal sequence contained in the sequence shown in SEQ ID NO: 26 is a type of leader sequence. The main function of the leader peptide encoded by the leader sequence is thought to be excretion from the cell. The protein encoded by bases 112 to 1992, namely the part, with the exception of the leading peptide, is believed to be a mature protein and exhibits a high degree of activity with respect to peptide formation.

Moreover, the various gene recombination methods described below can be carried out as described in Molecular Cloning, 2nd edition, Cold Spring Harbor Press (1989) and other publications.

DNA encoding an enzyme that can be used in the present invention can be obtained by polymerase chain reaction (PCR, link to White TJ et al., Trends Genet., 5, 185 (1989)) or by hybridization from a chromosomal DNA or library DNA of Empedobacter brevis, Sphingobacterium sp., Pedobacter heparinus, Taxeobacter gelupurpurascens, Cyclobacterium marinum or Psychroserpens burtonensis . The primers used in PCR can be created on the basis of internal amino acid sequences determined on the basis of a purified peptide-forming enzyme, as explained in the previous section (3). In addition, since the base sequences of the peptide-forming enzyme genes were identified (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), primers or probes for hybridization can be created based on these base sequences, and genes can be isolated using these probes. If primers having sequences corresponding to the 5'-untranslated region and the 3'-untranslated region, respectively, are used as primers for PCR, the full-length coding region of the enzyme can be amplified. Using, as an example, the case of amplification of a region containing both a leading sequence and a coding region of a mature protein, as presented in SEQ ID NO: 5, specific examples of primers include a primer with a base sequence of the region above base number 61 in SEQ ID NO: 5 for the primer 5'-end, and a primer having a sequence complementary to the base sequence of the region below base number 1908 for the 3'-end primer.

Primers can be synthesized, for example, in accordance with conventional methods using a phosphoamidite method (reference to Tetrahedron Letters (1981), 22, 1859) using a Model 380B DNA synthesizer manufactured by Applied Biosystems. The PCR reaction can be carried out, for example, using the Gene Amp PCR System 9600 (Perkin-Elmer) and the Takara LA PCR In Vitro Cloning Kit (Takara Shuzo) in accordance with the method described by the supplier, such as the manufacturer.

DNA that encodes an enzyme that can be used in the method for producing the peptide of the present invention, whether the DNA contains the leading sequence or not, includes DNA that is essentially identical to the DNA consisting of CDS shown in SEQ ID NO: 5 of the list sequences. Namely, DNA essentially identical to the DNA of the present invention can be obtained by isolating DNA that hybridizes with DNA with a base sequence complementary to the CDS shown in SEQ ID NO: 5 of the sequence listing, or with a probe obtained from the base sequence in harsh conditions, and encoding a protein with peptide-forming activity, from DNA encoding a mutant enzyme or cells that contain this DNA.

The DNA of the present invention, whether it contains a leader sequence or not, includes DNA that is essentially identical to the DNA consisting of the CDS shown in SEQ ID NO: 11 of the sequence listing. Namely, DNA essentially identical to the DNA of the present invention can be obtained by isolating DNA that hybridizes with a DNA consisting of a base sequence complementary to the CDS shown in SEQ ID NO: 11 of the sequence listing, or with a probe obtained from the sequence bases under severe conditions, and encoding a protein, with peptide-forming activity from DNA encoding a mutant enzyme, or from cells that possess this DNA.

The DNA of the present invention, whether it contains a leader sequence or not, includes DNA which is essentially identical to the DNA consisting of CDS shown in SEQ ID NO: 17 of the sequence listing. Namely, DNA essentially identical to the DNA of the present invention can be obtained by isolating DNA that hybridizes with a DNA consisting of a base sequence complementary to the CDS shown in SEQ ID NO: 17 of the sequence listing, or with a probe obtained from a base sequence under stringent conditions, and encoding a protein, with peptide-forming activity from DNA encoding a mutant enzyme, or from cells that contain this DNA.

The DNA of the present invention, whether it contains a leader sequence or not, includes DNA that is essentially identical to the DNA consisting of the CDS shown in SEQ ID NO: 22 of the sequence listing. Namely, DNA essentially identical to the DNA of the present invention can be obtained by isolating DNA that hybridizes with a DNA consisting of a base sequence complementary to the CDS shown in SEQ ID NO: 22 of the sequence listing, or with a probe obtained from the sequence bases under severe conditions, and encoding a protein, with peptide-forming activity from DNA encoding a mutant enzyme, or from cells that possess this DNA.

The DNA of the present invention, whether it contains a leader sequence or not, includes DNA that is essentially identical to the DNA consisting of the CDS shown in SEQ ID NO: 24 of the sequence listing. Namely, DNA essentially identical to the DNA of the present invention can be obtained by isolating DNA that hybridizes with a DNA consisting of a base sequence complementary to the CDS shown in SEQ ID NO: 24 of the sequence listing, or with a probe obtained from a base sequence under stringent conditions, and encoding a protein, with peptide-forming activity from DNA encoding a mutant enzyme, or from cells that possess this DNA.

The DNA of the present invention, whether it contains a leader sequence or not, includes DNA that is essentially identical to the DNA consisting of the CDS shown in SEQ ID NO: 26 of the sequence listing. Namely, DNA essentially identical to the DNA of the present invention can be obtained by isolating DNA that hybridizes with a DNA consisting of a base sequence complementary to the CDS shown in SEQ ID NO: 26 of the sequence listing, or with a probe obtained from a base sequence under stringent conditions, and encoding a protein, with peptide-forming activity from DNA encoding a mutant enzyme, or from cells that possess this DNA.

The probe can be obtained, for example, in accordance with conventional methods based, for example, on the base sequence shown in SEQ ID NO: 5 of the sequence listing. In addition, you can also use the method of extraction of DNA of interest, using a probe to determine DNA that hybridizes with the probe, in accordance with conventional methods. For example, a DNA probe can be obtained by amplifying a base sequence cloned in a plasmid or phage vector, cleaving the base sequence that is desired to be used as a probe using a restriction enzyme, and then extracting the desired base sequence. The cleavable portion may be adjusted depending on the DNA of interest.

The term “harsh conditions” as used herein refers to conditions under which a non-specific hybrid is not formed, but a so-called specific hybrid. It is difficult to accurately convey these conditions in numerical values. For example, you can specify the conditions under which DNA with high homology, for example 50% or more, preferably 80% or more, more preferably 90% or more, hybridize with each other, and DNA with a lower degree of homology than these do not hybridize each other with another, or the usual conditions for washing for Southern hybridization, in which hybridization is carried out at 60 ° C. at a salt concentration corresponding to 1xSSC and 0.1% SDS, preferably 0.1xSSC and 0.1% SDS. Although genes that hybridize under such conditions include those genes whose sequences have stop codons at certain positions or that have lost activity due to a mutation in the active site, they can be easily removed by ligation with a commercially available expression vector expressing them in a suitable host, and testing the enzymatic activity of the expression product using the method that will be described later.

However, in the case of a base sequence that hybridizes under stringent conditions, as described above, it is preferable that the protein encoded by this base sequence contains about half or more, preferably 80% or more, and more preferably 90% or more, of the enzymatic activity of the protein with amino acid the sequence encoded by the original base sequence, serving as the base, able to be stored at 50 ° C and pH 8. For example, as an example, when a base sequence that hybridizes under stringent conditions with DNA with a base sequence complementary to the base sequence consisting of bases 127 through 1908 of the base sequence shown in SEQ ID NO: 5, it is preferred that the protein encoded by this base sequence retains approximately half or more, preferably 80% or more and more preferably 90% or more, of the enzymatic activity of a protein with an amino acid sequence that consists of minokislotnyh residues numbers 23 to 616 of the amino acid sequence shown in SEQ ID NO: 6 under conditions of 50 ° C and pH 8.

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

The complete amino acid sequence shown in SEQ ID NO: 6 contains the leading peptide and the mature protein region with amino acid residues 1 through 22 constituting the leading peptide and amino acid residues 23 through 616 constituting the mature protein region.

The complete amino acid sequence shown in SEQ ID NO: 11 includes a leading peptide and a mature protein region with amino acid residues 1 through 20 constituting the leading peptide and amino acid residues 21 through 619 constituting the mature protein region.

The complete amino acid sequence shown in SEQ ID NO: 18 contains the leading peptide and the mature protein region with amino acid residues 1 through 22 constituting the leading peptide and amino acid residues 23 through 625 constituting the mature protein region.

The complete amino acid sequence shown in SEQ ID NO: 23 contains a leading peptide and a mature protein region with amino acid residues 1 through 22 constituting the leading peptide and amino acid residues 23 through 645 constituting the mature protein region.

The complete amino acid sequence shown in SEQ ID NO: 25 contains a leading peptide and a mature protein region with amino acid residues 1 through 25 constituting the leading peptide and amino acid residues 26 through 620 constituting the mature protein region.

The complete amino acid sequence shown in SEQ ID NO: 27 contains a leading peptide and a mature protein region with amino acid residues 1 through 17 constituting the leading peptide and amino acid residues 18 through 644 constituting the mature protein region.

The protein encoded by the DNA of the present invention is a protein in which the mature protein has peptide-forming activity, and the DNA that encodes the protein is essentially identical to the protein with the amino acid sequence shown in SEQ ID NO: 6 in SEQ ID NO: 12, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 25 or in SEQ ID NO: 27 of the sequence listing, regardless of whether it contains a leading sequence or not, is also included in the DNA of the present invention . (It should be noted that base sequences are described from amino acid sequences in accordance with universal codon codes). Namely, the present invention relates to DNA that encodes the proteins indicated from (A) to (X) below:

(A) a protein with an amino acid sequence consisting of amino acid residues 23 to 616 of the amino acid sequence shown in SEQ ID NO: 6 of the sequence listing,

(B) a protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or a plurality of amino acids in an amino acid sequence consisting of amino acid residues 23 to 616 of the amino acid sequence shown in SEQ ID NO: 6 of the sequence listing capable of selectively attaching L-phenylalanine to the α-ester portion of the α, β-diester of L-aspartic acid via a peptide bond,

(C) a protein with an amino acid sequence consisting of amino acid residues numbered 21 through 619 of the amino acid sequence shown in SEQ ID NO: 12 of the sequence listing,

(D) a protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or many amino acids in an amino acid sequence consisting of amino acid residues 21 to 619 of the amino acid sequence shown in SEQ ID NO: 12 of the sequence listing capable of selectively attaching L-phenylalanine to the α-ester portion of the α, β-diester of L-aspartic acid via a peptide bond,

(E) a protein with an amino acid sequence consisting of amino acid residues numbered 23 to 625 of the amino acid sequence shown in SEQ ID NO: 18 of the sequence listing,

(F) a protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or a plurality of amino acids in an amino acid sequence consisting of amino acid residues 23 to 625 of the amino acid sequence shown in SEQ ID NO: 18 of the sequence listing capable of selectively attaching L-phenylalanine to the α-ester portion of the α, β-diester of L-aspartic acid via a peptide bond,

(G) a protein with an amino acid sequence consisting of amino acid residues numbered 23 to 645 of the amino acid sequence shown in SEQ ID NO: 23 of the sequence listing,

(H) a protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or many amino acids in an amino acid sequence consisting of amino acid residues 23 to 645 of the amino acid sequence shown in SEQ ID NO: 23 of the sequence listing capable of selectively attaching L-phenylalanine to the α-ester portion of the α, β-diester of L-aspartic acid via a peptide bond,

(I) a protein with an amino acid sequence consisting of amino acid residues with numbers 26 to 620 of the amino acid sequence shown in SEQ ID NO: 25 of the sequence listing,

(J) a protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or many amino acids in an amino acid sequence consisting of amino acid residues 26 to 620 of the amino acid sequence shown in SEQ ID NO: 25 of the sequence listing capable of selectively attaching L-phenylalanine to the α-ester portion of the α, β-diester of L-aspartic acid via a peptide bond,

(K) a protein with an amino acid sequence consisting of amino acid residues 18 to 644 of the amino acid sequence shown in SEQ ID NO: 27 of the sequence listing,

(L) a protein with an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or a plurality of amino acids in an amino acid sequence consisting of amino acid residues 18 to 644 of the amino acid sequence shown in SEQ ID NO: 27 of the sequence listing capable of selectively attaching L-phenylalanine to the α-ester portion of the α, β-diester of L-aspartic acid via a peptide bond,

(M) a protein with the amino acid sequence shown in SEQ ID NO: 6 of the sequence listing,

(N) a protein containing a region of a mature protein having an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or many amino acids in the amino acid sequence shown in SEQ ID NO: 6 of the sequence listing, and capable of selectively attaching L- phenylalanine to the α-ether site of the α, β-diester of L-aspartic acid via a peptide bond,

(O) a protein with the amino acid sequence shown in SEQ ID NO: 12 sequence listing,

(P) a protein containing a region of a mature protein having an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or many amino acids in the amino acid sequence shown in SEQ ID NO: 12 of the sequence listing, and capable of selectively attaching L- phenylalanine to the α-ether site of the α, β-diester of L-aspartic acid via a peptide bond,

(Q) a protein with the amino acid sequence shown in SEQ ID NO: 18 sequence listing,

(R) a protein containing a region of a mature protein having an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or many amino acids in the amino acid sequence shown in SEQ ID NO: 18 of the sequence listing, and capable of selectively attaching L- phenylalanine to the α-ether site of the α, β-diester of L-aspartic acid via a peptide bond,

(S) a protein with the amino acid sequence shown in SEQ ID NO: 23 sequence listing,

(T) a protein containing a region of a mature protein having an amino acid sequence including the substitution, deletion, insertion, addition and / or inversion of one or many amino acids in the amino acid sequence shown in SEQ ID NO: 23 of the sequence listing, and capable of selectively attaching L- phenylalanine to the α-ether site of the α, β-diester of L-aspartic acid via a peptide bond,

(U) a protein with the amino acid sequence shown in SEQ ID NO: 25 sequence listing,

(V) a protein containing a region of a mature protein having an amino acid sequence including substitution, deletion, insertion, addition and / or inversion of one or multiple amino acids in the amino acid sequence shown in SEQ ID NO: 25 of the sequence listing, and capable of selectively attaching L- phenylalanine to the α-ether site of the α, β-diester of L-aspartic acid via a peptide bond,

(W) a protein with the amino acid sequence shown in SEQ ID NO: 27 of the sequence listing, and

(X) a protein containing a mature protein region having an amino acid sequence in the amino acid sequence shown in SEQ ID NO: 27 of the sequence listing and capable of selectively attaching L-phenylalanine to the α-ester portion of the α, β-diester of L-aspartic acid via the peptide communication.

Here, although the meaning of the term “multitude” varies depending on the positions and types of amino acid residues in the three-dimensional structure of the protein, nevertheless, it is in the range in which there is no significant violation of the three-dimensional structure and the activity of the protein with amino acid residues is specifically from 2 to 50, preferably from 2 to 30, and more preferably from 2 to 10. However, in the case of amino acid sequences including substitution, deletion, insertion, addition and / or inversion of one or many amino acids in amino acid sequences of proteins (B), (D), (F), (H), (J), (L), (N), (P), (R), (T), (V) or (X) , preferably, the proteins retained approximately half or more, more preferably 80% or more, and even more preferably 90% or more, the enzymatic activity of the proteins in a state where there is no mutation, at 50 ° C. and pH 8. As an example, a description is given. case (B); in the case of the amino acid sequence (B), including the substitution, deletion, insertion, addition and / or inversion of one or many amino acids in the amino acid sequence shown in SEQ ID NO: 6 of the sequence listing, it is preferable that this protein retains approximately half or more, more preferably 80% or more and even more preferably 90% or more, the enzymatic activity of a protein with the amino acid sequence shown in SEQ ID NO: 6 of the sequence listing under conditions of 50 ° C. and pH 8.

An amino acid mutation, such as that indicated in the sequence (B) above, and so on, is obtained by modifying the base sequence so that, with respect to the amino acid of a specific ester region in the enzyme gene of the present invention, replacement, deletion, insertion, addition by, for example, mutagenesis directed to the ether region. In addition, modified DNA, similar to that described above, can also be obtained using mutagenic effects known in this field. Mutagenicity refers, for example, to a method in which a DNA encoding an enzyme of the present invention is treated in vitro with hydroxylamine and so on, as well as a method in which Escherichia bacteria that contain a DNA encoding an enzyme of the present invention are treated with a mutagen, usually used for artificial mutagenesis, such as ultraviolet radiation, N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or nitrous acid.

In addition, the substitution, deletion, insertion, addition and / or inversion described above can be species-specific natural mutations or mutations that differ in different strains of microorganisms. By expressing the DNA containing such a mutation in suitable cells and examining the enzymatic activity of the expression product, DNA can be obtained that encodes a protein that is substantially identical to the protein described in SEQ ID NO: 6, 12, 18, 23, 25 or 27 of the sequence listing .

(3-3-2) Preparation of Transformants and Production of Peptide- Forming Enzymes

Peptide-forming enzymes that can be used in the method for producing the peptide of the present invention can be obtained by introducing the DNA described in (3-3-1) above into a suitable host and expressing the DNA in that host.

In relation to hosts for expression of a DNA-detectable protein, examples of hosts that can be used include various prokaryotic cells, including Escherichia bacteria, such as Escherichia coli , Empedobacter bacteria, Sphingobacterium bacteria, Flavobacterium and Bacillus subtilis bacteria, as well as various eukaryotic cells, including Saccharomyces cerevisiae, Pichia stipitis, and Aspergillus oryzae .

Recombinant DNA used to introduce DNA into the host can be obtained by inserting the introduced DNA into a vector corresponding to the type of host in which the DNA is to be expressed, in such a way that the protein encoded by that DNA can be expressed. In the case where the promoter is unique to the gene of the peptide-forming enzyme Empedobacter brevis and further functions in host cells, the promoter can be used as a promoter for the expression of DNA of the present invention. In addition, another promoter that acts in host cells can be ligated with the DNA of the present invention, and DNA can be expressed, if necessary, under the control of the promoter.

Examples of transformation methods for introducing recombinant DNA into host cells include method D.M. Morrison (see Methods in Enzymology, 68, 326 (1979)) or a method in which DNA permeability is increased by treating the receiving cells of the microorganism with calcium chloride (see Mandel, H. and Higa, A., J. Mol. Biol., 53, 159 (1970)).

In the case of large-scale protein production using the recombinant DNA method, conjugation of a protein in a transformant that produces a protein to form protein inclusion bodies is also a preferred embodiment of the present invention. The advantages of this method of expression and production are the protection of the target protein from hydrolysis by proteases located in the cells of the microorganism, and the simple and easy purification of the target protein by destroying the cells of the microorganism, followed by separation by centrifugation and so on.

Protein inclusion bodies obtained by this method are solubilized with a protein denaturing agent, and the protein is converted to a properly laid, physiologically active protein through a recovery procedure, which consists primarily of removing the denaturing agent. There are many such examples, including the restoration of human interleukin-2 activity (see Japanese Patent Application, Attached Publication No. S61-257931).

To obtain the active protein from inclusion bodies, a number of procedures are required, including solubilization and restoration of activity, and the procedure is more complicated than in the case of direct preparation of the active protein. However, in the case of obtaining a protein that has a negative effect on the growth of microorganisms in large volumes in the cells of the microorganism, this effect can be suppressed by the accumulation of proteins in the form of Taurus inclusion inactive protein in the cells of the microorganism.

Examples of large-scale production methods for producing a protein of interest in the form of inclusion bodies include a method in which a protein of interest is expressed independently under the control of a strong promoter, and a method in which a protein of interest is expressed in the form of a fusion protein with a protein that is known to be expressed in large quantities.

The present invention will now be described more specifically with reference to an example of a method for producing a transformed Escherichia coli and the use of such a transformed microorganism to produce a peptide-forming enzyme. Moreover, in the case of producing a peptide-forming enzyme, DNA that encodes a precursor protein containing a leader sequence can be introduced into a microorganism such as Escherichia coli , or DNA that consists only of a region of a mature protein that is not contains the leading sequence, and DNA may be suitably selected for the protein coding sequence, depending on the production conditions, form, conditions of use, and so on for the enzyme produced.

The promoters commonly used to produce heterologous proteins in Escherichia coli can be used as a promoter for the expression of DNA encoding a peptide-forming enzyme. Examples of such promoters include the T7 promoter, the lac promoter, the trp promoter, the trc promoter, the tac promoter, the lambda phage PR promoter, the PL promoter, and other strong promoters. In addition, examples of vectors that can be used include pUC19, pUC18, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219 and pMW218. In addition, vectors based on phage DNA can be used. Moreover, expression vectors that contain promoters and are capable of expressing the introduced DNA sequence can be used.

To obtain a peptide-forming enzyme in the form of fusion protein inclusion bodies, a gene encoding another protein and preferably a hydrophilic peptide is ligated above or below the peptide-forming enzyme gene to produce a fusion protein gene. A gene that encodes another protein in this method can be any gene that increases the amount of accumulated fusion protein and increases the solubility of the fusion protein after denaturation and reduction steps. Examples of possible genes include the 10 T7 gene, the β-galactosidase gene, the dehydrofolate reductase gene, the γ-interferon gene, the interleukin-2 gene, and the prochymosin gene.

If genes are ligated with genes that encode peptide-forming enzymes, the genes are ligated so that the reading frames of the codons match. It is recommended that genes be ligated at a suitable restriction site of the ester enzyme or DNA having a suitable sequence is used.

In addition, to increase the amount of the produced peptide-forming enzyme, in some cases it is preferable that the terminator, which is a transcription termination sequence, be ligated below the fusion protein gene. The terminator includes, for example, the T7 terminator, the phage fd terminator, the T4 terminator, the tetracycline resistance gene terminator, and the Escherichia coli trpA gene terminator.

As vectors for introducing a gene that encodes a protein-forming enzyme, or a fusion protein of a peptide-forming enzyme, and another protein, so-called multicopy types of vectors are preferred in Escherichia coli, examples of which include a plasmid having a replicator derived from ColE1, for example a plasmid based on pUC and a plasmid based on pBR322 or their derivatives. The term “derivatives” as used herein refers to those plasmids which undergo modification by substitution, deletion, insertion, addition and / or inversion of bases. It should be noted that the modification used here includes modifications by mutagenicity with a mutagen or UV radiation or modifications by spontaneous mutation.

For screening transformants, it is preferred that the vectors have markers, such as the ampicillin resistance gene. Such plasmids are commercially available expression vectors with strong promoters (pUC-based vector (manufactured by Takara Shuzo, Co., Ltd.), pRROK-based vector (manufactured by Clonetech Laboratories, Inc.), pKK233-2 (manufactured by Clonetech Laboratories, Inc. ) and so on.

Recombinant DNA is obtained by ligation of a DNA fragment with a DNA vector. In this case, a promoter, a gene encoding a hydrolase of L-amino acid amides, or a hybrid protein consisting of a hydrolase of L-amino acid amides and another protein, and, depending on the case, the terminator, are ligated in this order.

If Escherichia coli is transformed using recombinant DNA and the resulting Escherichia coli is cultured, a peptide-forming enzyme or a hybrid protein consisting of a peptide-forming enzyme and another protein is expressed and produced. Although a strain that is commonly used to express a heterogeneous gene is used as the host to be transformed, Escherichia coli strain JM109, for example, is preferred. Transformation methods and transformant screening methods are described in Molecular Cloning, 2nd Edition, Cold Spring Harbor Press (1989) and other publications.

In the case of expression of a peptide forming enzyme in the form of a fusion protein, the peptide forming enzyme can be cleaved using a restriction protease such as coagulation factor Xa or kallikrein, which uses a sequence not present in the peptide forming enzyme as a recognizable sequence.

As the production medium, a medium commonly used for culturing Escherichia coli , such as M9 casamino acid medium or LB medium, can be used. In addition, cultivation conditions and conditions for product induction are suitably selected in accordance with the marker of the vector used, the promoter, the type of host microorganism, and so on.

To obtain a peptide forming enzyme or a fusion protein consisting of a peptide forming enzyme and another protein, the following method can be used. If the enzyme forming the peptide or its fusion protein is soluble in the cells of the microorganism, after receiving the cells of the microorganism, the cells of the microorganism are destroyed or lysed so that they can be used as a crude liquid containing the enzyme. Moreover, the peptide-forming enzyme or its fusion protein can be purified before use by conventional methods, such as precipitation, filtration or column chromatography, as necessary. In this case, a purification method may also be used in which an anti-peptide-forming antibody or a fusion protein thereof is used.

In the case where protein inclusion bodies are formed, the inclusion bodies are solubilized with a denaturing agent. They can be solubilized with the protein of the microorganism cell. However, when considering the following purification procedure, inclusion bodies are preferably prepared and then solubilized. To obtain inclusion bodies from microorganism cells, well-known conventional methods can be applied. For example, inclusion bodies can be obtained by disrupting microorganism cells, followed by separation by centrifugation. Examples of denaturing agents capable of solubilizing inclusion bodies include guanidine hydrochloride (e.g., 6 M, pH 5 to 8) and urea (e.g., 8 M).

An active protein is recovered by deleting these denaturing agents by dialysis. During dialysis, a solution of Tris-HCl buffer or a solution of phosphate buffer and so on can be used as a solution for dialysis and the concentration can be, for example, from 20 mm to 0.5 M, while the pH can be, for example, from 5 up to 8.

The protein concentration during the recovery step is preferably approximately 500 μg / ml or less. The dialysis temperature is preferably 5 ° C. or lower to inhibit the intrinsic crosslinking of the reduced peptide-forming enzyme. Moreover, in addition to dialysis, dilution or ultrafiltration can be used to remove denaturing agents, and it is expected that activity can be restored regardless of which denaturing agent was used.

<2> Method for producing α-L-aspartyl-L-phenylalanine-α-methyl ester

The method of producing α-APM in accordance with the present invention includes a first step in the synthesis of α-L-aspartyl-L-phenylalanine-β-methyl ester in accordance with "<1> a method for producing α-L-aspartyl-L-phenylalanine-β-methyl ester ”and a second step for converting α-L-aspartyl-L-phenylalanine-β-methyl ether to α-L-aspartyl-L-phenylalanine-α-methyl ether.

Preferred conditions in the first step and the like are described in “<1> Method for the Preparation of α-L-Aspartyl-L-Phenylalanine-β-Methyl Ester”. In addition, the second stage can be carried out in accordance with a known method and reference can be made to the method and preferred conditions described, for example, in Japanese patent application No. H4-41155 and so on. Using the method for producing α-APM according to the present invention, α-APM, which is important as a sweetener and the like, can be obtained inexpensively and in high yield.

Examples

Hereinafter, the present invention will be described using examples. However, the present invention is not limited to these examples. In addition to confirming with ninhydrin staining of thin-layer chromatograms (qualitative), quantitative determinations were carried out using the following high-resolution liquid chromatography for product analysis. Column: InertsiL ODS-2 (manufactured by GL Science, Inc.), eluate: aqueous phosphate solution containing a mixture of 5.0 mm sodium 1-octanesulfonate (pH 2.1): methanol = 100: 15 to 50, flow rate 1, 0 ml / min, measurement of 210 nanometers (nm).

Example 1

Microorganisms that produce α-L-aspartyl-L-phenylalanine-β-methyl ester

To cultivate the bacteria and actinomycetes shown in Table 1-1, 50 milliliters ("ml") of medium (pH 7.0) containing 20 grams ("g") of glycerol, 5 g of ammonium sulfate, 1 g of monopotassium hydrogen phosphate, 3 were used. g of dipotassium hydrogen phosphate, 0.5 g of magnesium sulfate, 10 g of yeast extract and 10 g of peptone in 1 liter (l), which was transferred to a 500 ml Sakagushi vessel and sterilized at 115 ° C for 15 minutes (medium 1). Obtained beveled agar (pH 7.0) containing 5 g / L glucose, 10 g / L yeast extract, 10 g / L peptone, 5 g / L NaCl and 20 g / L agar, and the microorganisms shown in Table 1 were cultured in a mowed agar strain at 30 ° C for 24 hours. Then, the microorganisms collected with one loop were cultured in medium 1 at 30 ° C for 24 hours and then cultivated by shaking at 30 ° C and 120 shakes / min for 17 hours. After cultivation, the cells of the microorganism were separated from the culture fluid by centrifugation and suspended in 0.1 M borate buffer (pH 9.0) containing 10 mM EDTA, up to 100 g / l of crude microorganism cells.

To cultivate the yeast indicated in Table 1-1, 50 ml of medium (pH 6.0) containing 10 g / l glucose, 10 g glycerol, 5 g ammonium sulfate, 1 g monopotassium hydrogen phosphate, 3 g dipotassium hydrogen phosphate, 0.5 g of magnesium sulfate, 5 g of yeast extract 5 g of malt extract and 10 g of peptone in 1 l transferred to a 500 ml Sakagushi vessel and sterilized at 115 ° C for 15 minutes (medium 2). An adapted agar medium (pH 6.0) was obtained containing 5 g / L glucose, 5 g / L yeast extract, 5 g / L malt extract, 10 g / L peptone, 5 g / L NaCl and 20 g / L agar in medium 2, and the microorganisms shown in table 1, were cultured in this adapted agar medium at 30 ° C for 24 hours. Then, the yeast collected with one loop was cultured by shaking at 30 ° C for 24 hours in medium 2 at 25 ° C and 120 shaking / min for 17 hours. After cultivation, the cells of the microorganism were separated from the liquids of this culture by centrifugation and suspended in 0.1 M borate buffer (pH 9.0) containing 10 mM EDTA, up to 100 g / l of crude microorganism cells.

The microorganisms shown in table 1-2, were cultured as follows. For cultivation of Cellulophaga lytica NBRC 14961 (depository institution; the NITE Biological Resource Center of the National Institute of Technology and Evaluation, address of the depository institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) or Flexithrix dorotheae NBRC 15987 (depository institution; the NITE Biological Resource Center of the National Institute of Technology and Evaluation, address of the depository institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) were used solid agar medium (pH 7.2, 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 SP artificial sea water. The seeded cells of the microorganism Cellulophaga lytica NBRC 14961 (depository institution; the NITE Biological Resource Center of the National Institute of Technology and Evaluation, address of the depository institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan ) or Flexithrix dorotheae NBRC 15987 (depository institution; the NITE Biological Resource Center of the National Institute of Technology and Evaluation, address of the depository institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) cultivated in this medium at 30 ° C for 48 hours to obtain seed material, after which they were applied to the same medium, followed by main cultivation at 30 ° C for 48 hours.

For cultivation Weeksella virosa NBRC 16016 (depository institution; the NITE Biological Resource Center of the National Institute of Technology and Evaluation, address of the depository institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) Sheep agar medium (Nissui Plate, Nissui Pharmaceutical) was used. Cells of the microorganism Weeksella virosa NBRC 16016 (depository institution; the NITE Biological Resource Center of the National Institute of Technology and Evaluation, address of the depository institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) which were cultured in this medium at 30 ° C for 48 hours to obtain seed material was applied to the same medium, followed by main cultivation at 30 ° C for 48 hours.

For cultivation of Pedobacter heparinus NBRC 12017 (depository institution; the NITE Biological Resource Center of the National Institute of Technology and Evaluation, address of the depository institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) an agar solid medium (pH 7.0 sterilized at 120 ° C. for 15 minutes) containing 10 g peptone, 2 g yeast extract, 1 g MgSO ₄ · 7H ₂ O and 15 g agar in 1 liter of distilled water was used. Cells of the microorganism Pedobacter heparinus NBRC 12017 (depository institution; the NITE Biological Resource Center of the National Institute of Technology and Evaluation, address of the depository institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) which were cultured in this medium at 30 ° C for 48 hours to obtain seed material was applied to the same medium, followed by main cultivation at 30 ° C for 48 hours.

For cultivation of Persicobacter diffluens NBRC 15940 (depository institution; the NITE Biological Resource Center of the National Institute of Technology and Evaluation, address of the depository institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) used agar solid medium (pH 7.0, sterilized at 120 ° C for 15 minutes) containing 0.5 g KNO ₃ , 0.1 g sodium glycerophosphate, 1 g trishydroxymethylaminomethane, 5 g tryptone, 5 g yeast extract, 15 g of agar and 1 ml of a solution of trace elements in 1 l of artificial sea water Daigo SP. It should be noted that the trace element solution contained 2.85 g of H ₃ BO ₄ , 1.8 g of MnCl ₂ · 4H ₂ O, 1.36 g of FeSO ₄ · 7H ₂ O, 26.9 mg of CuCl ₂ · 2H ₂ O, 20 , 8 mg of ZnCl ₂ , 40.4 mg of CoCl ₂ · 6H ₂ O, 25.2 mg of Na ₂ MoO ₄ · 2H ₂ O and 1.77 g of sodium tartrate. Cells of the microorganism Persicobacter diffluens NBRC 15940 (depository institution; the NITE Biological Resource Center of the National Institute of Technology and Evaluation, address of the depository institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) which were cultured in this medium at 25 ° C for 48 hours to obtain seed, was applied to the same medium, followed by main cultivation at 25 ° C for 48 hours.

For cultivation of Chitinophaga pinensis NBRC 15968 (depository institution; the NITE Biological Resource Center of the National Institute of Technology and Evaluation, address of the depository institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) used agar solid medium (pH 7.0, sterilized at 120 ° C for 15 minutes) containing 3 g of bactocasitone, 1 g of yeast extract, 1.36 g of CaCl ₂ · H ₂ O and 15 g of agar in 1 l of distilled water . Cells of the microorganism Chitinophaga pinensis NBRC 15968 (depository institution; the NITE Biological Resource Center of the National Institute of Technology and Evaluation, address of the depository institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) which were cultured in this medium at 25 ° C for 48 hours to obtain seed, was applied to the same medium, followed by main cultivation at 25 ° C for 48 hours.

Agar solid medium (pH 7.0 sterilized at 120 ° C.) was used to cultivate Cyclobacterium marinum ATCC 25205 (depository institution; American collection of crop types; depository institution address; PO Box 1549, Manassas, VA 20110, United States of America) for 15 minutes) containing 5 g of peptone, 1 g of yeast extract, 0.2 g of FeSO ₄ · 7H ₂ O and 15 g of agar in 1 l of artificial sea water Daigo SP. Cells of the microorganism Cyclobacterium marinum ATCC 25205 (depository institution; American collection of crop types, depository institution address; PO Box 1549, Manassas, VA 20110, United States of America), which were cultured in this medium at 25 ° C. for 48 hours to obtain seed material was applied to the same medium, followed by main cultivation at 25 ° C for 48 hours.

An agar solid medium (pH 7.0 sterilized at 120 ° C.) was used to cultivate Runella slithyformis ATCC 29530 (depository institution; American collection of crop types; depository institution address; PO Box 1549, Manassas, VA 20110, United States of America). 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. Cells of the microorganism Runella slithyformis ATCC 29530 (depository institution; American collection of crop types, address of depository institution; PO Box 1549, Manassas, VA 20110, United States of America), which were cultured in this medium at 25 ° C. for 48 hours to obtain seed material was applied to the same medium, followed by main cultivation at 25 ° C for 48 hours.

For cultivation of Thermonema lapsum ATCC 43542 (depository institution; American collection of crop types, address of depository institution; PO Box 1549, Manassas, VA 20110, United States of America), an agar solid medium (pH 8.2, sterilized at 120 ° C. for 15 minutes) containing 0.2 g of nitrile triacetic acid, 2 ml of a 0.03% solution of FeCl ₃ , 0.12 g of CaSO ₄ · 2H ₂ O, 0.2 g of MgSO ₄ · 7H ₂ O, 0.016 g of NaCl, 0 , 21 g of KNO ₃ , 1.4 g of NaNO ₃ , 0.22 g of Na ₂ HPO ₄ , 2 ml of a trace element solution and 15 g of agar in 1 liter of distilled water. It should be noted that the trace element solution contained 0.5 ml of H ₂ SO ₄ , 2.2 g of MnSO ₄ , 0.5 g of ZnSO ₄ , 0.5 g of H ₃ BO ₃ , 0.016 g of CuSO ₄ , 0.025 g of Na ₂ MoO ₄ and 0.046 g of CoCl ₂ . Cells of the Thermonema lapsum microorganism ATCC 43542 (depository institution; American collection of crop types, depository institution address; PO Box 1549, Manassas, VA 20110, United States of America), which were cultured in this medium at 60 ° C. for 48 hours to obtain seed material was applied to the same medium, followed by main cultivation at 25 ° C for 48 hours.

For cultivation, Gelidibacter algens ATCC 700364 (depository institution; American collection of crop types, address of depository institution; PO Box 1549, Manassas, VA 20110, United States of America), Lewinella cohaerens ATCC 23123 (depository institution; American collection of crop types, address depository institutions; PO Box 1549, Manassas, VA 20110, United States of America), Psychroserpens burtonensis ATCC 700359 (depository institution; American collection of crop types, address of depository institution; PO Box 1549, Manassas, VA 20110, United States of America) or Salegentibacter salegens DSMZ 5424 (custodian institution; th e Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German collection of microorganisms and cell cultures, address of depository institution; Mascheroder Weg 1b, 38124 Braunschweig, Germany) used marine agar (manufactured by Difco). In the case of Gelidibacter algens ATCC 700364 (American depository institution) collection of crop types, address of the depository institution; PO Box 1549, Manassas, VA 20110, United States of America) or Psychroserpens burtonensis ATCC 700359 (depository institution; American collection of crop types, address of depository institution; PO Box 1549, Manassas, VA 20110, United States of America) used Gelidibacter cells algens ATCC 700364 or Psychroserpens burtonensis ATCC 700359 (depository institution; American collection of crop types; depository institution address; PO Box 1549, Manassas, VA 20110, United States of America) that were cultured on this medium at 10 ° C for 72 hours to obtain seed material, followed by nym culturing at 10 ° C for 72 hours. In the case of Lewinella cohaerens ATCC 23123 (depository institution; American collection of crop types; address of depository institution; PO Box 1549, Manassas, VA 20110, United States of America) cells of the microorganism Lewinella cohaerens ATCC 23123 (depository institution; American collection of crop types, address of depository institution; PO Box 1549, Manassas, VA 20110, United States of America), which were cultivated in this medium at 30 ° C for 48 hours to obtain seed, was applied to the same medium, followed by main cultivation at 30 ° C within 48 hours. In the case of Salegentibacter salegens DSMZ 5424 (depository institution; the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German collection of microorganisms and cell cultures, address of the depository institution; Mascheroder Weg 1b, 38124 Braunschweig, Germany) microorganism cells SalegentibZergentz 54 depository; the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German collection of microorganisms and cell cultures, address of the depository institution; Mascheroder Weg 1b, 38124 Braunschweig, Germany), which were cultivated in this medium at 25 ° C for 48 hours to obtain seed applied to in same medium followed by main culturing at 25 ° C for 48 hours.

An agar solid medium (pH 7.0 sterilized at 120 ° C.) was used to cultivate Dyadobacter fermentans ATCC 700827 (depository institution; American collection of crop types; depository institution address; PO Box 1549, Manassas, VA 20110, United States of America) for 15 minutes) containing 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 , 15 mg CaCl ₂ · 2H ₂ O, 20 mg MgCl ₂ · 6H ₂ O, 7 mg FeSO ₄ · 7H ₂ O, 5 mg Na ₂ SO ₄ , 5 mg MnCl ₂ · 4H ₂ O, 0.5 mg H ₃ BO ₃ , 0.5 mg ZnCl ₂ , 0.5 mg CoCl ₂ · 6H ₂ O, 0.5 mg NiSO ₄ · 6H ₂ O, 0.3 mg CuCl ₂ · 2H ₂ O, 10 mg Na ₂ MoO ₄ · 2H ₂ O, 0.5 g of yeast extract, 0.5 g peptone, 0.5 g casamic acid, 0.5 g dextrose, 0.5 g solution starch, 0.5 g of sodium pyruvate and 15 g of agar in 1 liter of distilled water. Cells of the microorganism Dyadobacter fermentans ATCC 700827 (depository institution; American collection of crop types, depository institution address; PO Box 1549, Manassas, VA 20110, United States of America) that were cultured in this medium at 25 ° C. for 48 hours to obtain seed material, then subjected to basic cultivation at 25 ° C for 48 hours.

For cultivation Flammeovirga aprica NBRC 15941 (depository institution; the NITE Biological Resource Center of the National Institute of Technology and Evaluation, address of the depository institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) used agar solid medium (pH 7.2, sterilized at 120 ° C for 15 minutes) containing 2 g of tryptone, 0.5 g of meat extract, 0.5 g of yeast extract, 0.2 g of sodium acetate and 15 g of agar in 1 liter of artificial sea water Daigo SP. Cells of the microorganism Flammeovirga aprica NBRC 15941 (depository institution; the NITE Biological Resource Center of the National Institute of Technology and Evaluation, address of the depository institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) which were cultured in this medium at 25 ° C for 48 hours to obtain seed, then subjected to the main cultivation at 25 ° C for 48 hours.

For cultivation of Spirosoma linguale DSMZ 74 (depository institution; the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German collection of microorganisms and cell cultures, address of depository institution; Mascheroder Weg 1b, 38124 Braunschweig, Germany) or Flectobacillus major DSMZ 103 ; the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German collection of microorganisms and cell cultures, address of the depository institution; Mascheroder Weg 1b, 38124 Braunschweig, Germany) (pH 7.0, sterilized at 120 ° C for 15 minutes), containing 1 g glucose, 1 g peptone, 1 g yeast extract and 15 g agar in 1 liter of dis illirovannoy water Cells microorganism Spirosoma linguale DSMZ 74 (institution depositary;. the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH ( German Collection of Microorganisms and Cell Cultures, Address of the Depositary institution; Mascheroder Weg 1b, 38124 Braunschweig, Germany) or Flectobacillus major DSMZ 103 ( depository institution; the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German collection of microorganisms and cell cultures, address of depository institution; Mascheroder Weg 1b, 38124 Braunschweig, Germany), which were cultivated in this medium at 25 ° C for 48 hours to obtain seed, then subjected to basic cultivation at 25 ° C for 48 hours.

Agar solid medium (pH 7.0 sterilized at 120 ° C.) was used to cultivate Tenacibaculum maritimum ATCC 43398 (depository institution; American collection of crop types; depository institution address; PO Box 1549, Manassas, VA 20110, United States of America). for 15 minutes) containing 0.5 g of tryptone, 0.5 g of yeast extract, 0.2 g of meat extract, 0.2 g of sodium acetate and 15 g of agar in 300 ml of distilled water and 700 ml of artificial sea water Daigo SP. Cells of the microorganism Tenacibaculum maritimum ATCC 43398 (depository institution; American collection of crop types, depository institution address; PO Box 1549, Manassas, VA 20110, United States of America) that were cultured in this medium at 25 ° C. for 48 hours to obtain seed material, then subjected to basic cultivation at 25 ° C for 48 hours.

For cultivation of Rhodothermus marinus DSMZ 4252 (depository institution; the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German collection of microorganisms and cell cultures, address of depository institution; Mascheroder Weg 1b, 38124 Braunschweig, Germany), agar solid medium 7 was used ( sterilized at 120 ° C for 15 minutes) containing 2.5 g of yeast extract, 2.5 g of tryptone, 100 mg of nitrile triacetic acid, 40 mg of CaSO ₄ · H ₂ O, 200 mg of MgCl ₂ · 6H ₂ O, 0 , 5 ml of 0.01 M Fe citrate, 0.5 ml of a trace element solution, 100 ml of phosphate buffer, 900 ml of distilled water, 28 g of agar in 1 liter. That the trace element solution contained 12.8 g of nitrilotriacetic acid, 1 g of FeCl ₂ · 4H ₂ O, 0.5 g MnCl ₂ · 4H ₂ O, 0.3 g CoCl ₂ · 4H ₂ O, 50 mg CuCl ₂ · 2H ₂ O, 50 mg Na ₂ MoO ₄ · 2H ₂ O, 20 mg H ₃ BO ₃ and 20 mg NiCl ₂ · 6H ₂ O. Cells of the microorganism Rhodothermus marinus DSMZ 4252 (depository institution; the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German collection of microorganisms and cell cultures, address of the depository institution; Mascheroder Weg 1b, 38124 Braunschweig, Germany), which were cultured in this medium at 60 ° C for 48 hours to obtain seed, was applied to the same medium, followed by main cultivation at 60 ° C for 48 hours.

For cultivation of Zobellia galactanivorans DSMZ 12802 (depository institution; the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German collection of microorganisms and cell cultures, depository institution address; Mascheroder Weg 1b, 38124 Braunschweig, Germany), 1.5% agar solid medium was used agar, pH 7.6, sterilized at 120 ° C. for 15 minutes) containing Bacto Marine BROTH broth (Difco 2216). This medium was used with cells of the microorganism Zobellia galactanivorans DSMZ 12802 (depository institution; the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German collection of microorganisms and cell cultures, address at depository institutions; Mascheroder Weg 1b, 38124 Braunschweig, Germany), which were cultured in this medium at 30 ° C for 48 hours to obtain seed and applied to the same medium, followed by main cultivation at 30 ° C for 48 hours.

For cultivation of Muricauda ruestringenesis DSMZ 13258 (depository institution; the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German collection of microorganisms and cell cultures, address of the depository institution; Mascheroder Weg 1b, 38124 Braunschweig, Germany), pH 7 agar was used sterilized at 120 ° C for 15 minutes) containing 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 O, 4.46 g MgSO ₄ · 7H ₂ O, 0.98 g CaCl ₂ and 15 g agar in 1 liter of distilled water Cells of the microorganism Muricauda ruestringenesis DSMZ 13258 (depository institution; the Deutche Sammlung von Mikroorg anismen und Zellkulturen GmbH (German collection of microorganisms and cell cultures, address of the depository institution; Mascheroder Weg 1b, 38124 Braunschweig, Germany), which were cultivated in this medium at 30 ° C for 48 hours to obtain seed, was applied to the same medium followed by main cultivation at 30 ° C for 48 hours.

For the cultivation of Taxeobacter gelupurpurascens DSMZ 11116 (depository institution; the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German collection of microorganisms and cell cultures, address of the depository institution; Mascheroder Weg 1b, 38124 Braunschweig, solid media 7) (Ag 7) sterilized at 120 ° C for 15 minutes) containing 3 g casiton, 1 g yeast extract, 1.36 g CaCl ₂ · 2H ₂ O and 15 g agar in 1 liter of distilled water Taxeobacter gelupurpurascens DSMZ 11116 microorganism cells (institution - depository; the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German microorgan collection serum and cell cultures, address of the depository institution; Mascheroder Weg 1b, 38124 Braunschweig, Germany), which were cultured in this medium at 30 ° C for 48 hours to obtain seed, was applied to the same medium, followed by main cultivation at 30 ° C for 48 hours.

For cultivation of Cytophaga hutchinsonii NBRC 15051 (depository institution; the NITE Biological Resource Center of the National Institute of Technology and Evaluation, address of the depository institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) used agar solid medium (pH 7.2, sterilized at 120 ° C for 15 minutes) containing 3 g of casitone, 1 g of yeast extract, 1.36 g of CaCl ₂ · 2H ₂ O, 5 g of cellobiose and 15 g of agar in 1 liter of distilled water. Cells of the microorganism Cytophaga hutchinsonii NBRC 15051 (depository institution; the NITE Biological Resource Center of the National Institute of Technology and Evaluation, address of the depository institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) which were cultured in this medium at 30 ° C for 48 hours to obtain seed, was applied to the same medium, followed by main cultivation at 30 ° C for 48 hours.

For cultivation of Marinilabilia salmonicolor NBRC 15948 (depository institution; the NITE Biological Resource Center of the National Institute of Technology and Evaluation, address of the depository institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) used agar solid medium (pH 7.2, sterilized at 120 ° C for 15 minutes) containing 10 g of peptone, 2 g of yeast extract, 0.5 g of MgSO ₄ · 7H ₂ O and 15 g of agar in 200 ml of distilled water and 750 ml of artificial sea water Daigo SP. Cells of the microorganism Marinilabilia salmonicolor NBRC 15948 (depository institution; the NITE Biological Resource Center of the National Institute of Technology and Evaluation, address of the depository institution; 5-8 Kazusa-Kamaashi 2-Chome, Kisarazu-shi, Chiba-ken, Japan) which were cultured in this medium at 30 ° C for 48 hours to obtain seed material was applied to the same medium, followed by main cultivation at 30 ° C for 48 hours.

For cultivation of Saprospira grandis ATCC 23119 (depository institution; American collection of crop types, depository institution address; PO Box 1549, Manassas, VA 20110, United States of America), an agar solid medium (pH 7.0 sterilized at 120 ° C. for 15 minutes) containing 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 and 1 ml of a trace element in 1 l of artificial sea water Daigo SP. It should be noted that the trace element solution contained 2.85 g of H ₃ BO ₄ , 1.8 g of MnCl ₂ · 4H ₂ O, 1.36 g of FeSO ₄ · 7H ₂ O, 26.9 mg of CuCl ₂ · 2H ₂ O, 20 , 8 mg of ZnCl ₂ , 40.4 mg of CoCl ₂ · 6H ₂ O, 25.2 mg of Na ₂ MoO ₄ · 2H ₂ O and 1.77 g of sodium tartrate. Cells of the microorganism Saprospira grandis ATCC 23119 (depository institution; American collection of crop types, depository institution address; PO Box 1549, Manassas, VA 20110, United States of America) that were cultured in this medium at 30 ° C. for 48 hours to obtain seed material was applied to the same medium, followed by main cultivation at 30 ° C for 48 hours.

For cultivation of Haliscomenobacter hydrossis ATCC 27775 (depository institution; American collection of crop types, depository institution address; PO Box 1549, Manassas, VA 20110, United States of America), an agar solid medium (pH 7.5 sterilized at 120 ° C. for 15 minutes) containing 27 mg KH ₂ PO ₄ , 40 mg K ₂ HPO ₄ , 40 mg Na ₂ HPO ₄ · 2H ₂ O, 50 mg CaCl ₂ · 2H ₂ O, 75 mg MgCl ₂ · 7H ₂ O, 5 mg FeCl ₃ · 6H ₂ O, 3 mg MnSO ₄ · H ₂ O, 1.31 g glutamic acid, 2.5 mg trypticase soy broth without glucose, 0.4 mg thiamine, 0.01 mg vitamin B12, 2 g glucose and 1 ml of a solution of trace elements in 1 liter of distilled water. It should be noted that the trace element solution contained 0.1 g of ZnSO ₄ · 7H ₂ O, 0.03 g of MnCl ₂ · 4H ₂ O, 0.3 g of H ₃ BO ₃ , 0.2 g of CoCl ₂ · 6H ₂ O, 0 , 01 g CuCl ₂ · 2H ₂ O, 0.02 g NiCl ₂ · 6H ₂ O and 0.03 g Na ₂ MoO ₄ · H ₂ O. Cells of the microorganism Haliscomenobacter hydrossis ATCC 27775 (depository institution; American collection of crop types, address of depository institution; PO Box 1549, Manassas, VA 20110, United States of America), which were cultivated in this medium at 25 ° C for 48 hours to obtain seed, was applied to the same medium, followed by main cultivation at 25 ° C within 48 hours.

The microorganism cells thus obtained in each case were collected from agar medium and suspended in 0.1 M borate buffer (pH 9.0) containing 10 mM EDTA in an amount of 100 g / l in the form of non-dried microorganism cells.

0.1 ml of 100 mM borate buffer (pH 9.0) containing 10 mM EDTA, 100 mM α-hydrochloride, L-aspartic acid β-dimethyl ester and 200 mM L-phenylalanine was added to 0.1 ml of each cell suspension of these microorganisms. bringing the total volume to 0.2 ml. Then the reaction was carried out at 20 ° C for 3 hours when the microorganism indicated in Table 1-1 was used, or within 1 hour when the microorganism indicated in Table 1-2 was used. The obtained amount (mM) of α-L-aspartyl-L-phenylalanine β-methyl ester (α-AMP) is shown in Tables 1-1 and 1-2, was determined in all cases when microorganisms were used. Moreover, in all cases of the use of microorganisms, β-AMP was not recorded.

Table 1-1

Table 1-2

Reference example 1

A microorganism that produces β-L-aspartyl-L-phenylalanine α-methyl ester

The microorganisms shown in Table 2 were cultured similarly to the bacteria procedure of Table 1 of Example 1. After cultivation, the cells of the microorganism were isolated from these culture broths by centrifugation and suspended in 0.1 M borate buffer (pH 9.0) containing 10 mM EDTA up to 100 g / l crude microorganism cells. To 0.1 ml of each of the microorganism suspensions of these microorganisms was added 0.1 ml of 100 mM borate buffer (pH 9.0) containing 10 mM EDTA, 100 mM α hydrochloride, β-dimethyl ether L-aspartic acid and 200 mm L- phenylalanine to obtain a total volume of 0.2 ml, followed by reaction at 30 ° C for 2 hours. The amount (mM) of β-L-aspartyl-L-phenylalanine α-methyl ester (β-AMP) produced in this case is shown in Table 2. It should be noted that α-AMP was detected in all microorganisms.

table 2 Microorganism β-AMP (mm) Hafnia alvei ATCC 9760 0.30 Klebsiella pneumoniae ATCC 8308 0.26

Example 2

Purification of the enzyme from Empedobacter brevis

50 ml of medium (pH 6.2) containing 5 grams (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 and 10 g of peptone in 1 l, transferred to a 500 ml Sakagushi vessel and sterilized at 115 ° C for 15 minutes. This medium was then inoculated with 2 milliliters (ml) of the FERM BP-8113 Empedobacter brevis strain (depository institute: independent management corporation, national institute for the development of industrial science and technology, international patented organisms depository, depository institution address: Chuo Dai-6, 1 -1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, date of international transfer of deposit: July 8, 2002), which was cultivated at 30 ° C for 16 hours in the same medium and then cultured with shaking at 30 ° C and 120 shakes / min for 16 hours.

The subsequent centrifugation separation was carried out either on ice or at 4 ° C. The resulting culture broth was centrifuged to collect microorganism cells. After washing 16 g of the microorganism cells with 50 mM Tris-HCl buffer (pH 8.0), they were suspended in 40 milliliters (ml) of the same buffer and subjected to sonication for 45 minutes at 195 W. The ultrasound-disrupted fluid was then centrifuged (10,000 rpm, 30 minutes) to remove fragments of the disrupted cells and the supernatant of the ultrasonic disrupted fluid was obtained. This supernatant of sonicated fluid was dialyzed overnight against 50 mM Tris-HCl buffer (pH 8.0), followed by removal of the insoluble fraction by ultracentrifugation (50,000 rpm, 30 minutes) to give the soluble fraction in the form of a supernatant. The resulting soluble fraction was applied to an HP Q-Sepharose column (manufactured by Amersham), previously equilibrated with Tris-HCl buffer (pH 8.0), and the active fraction was collected from the non-adsorbed fraction. This active fraction was dialyzed overnight against 50 mM acetate buffer (pH 4.5), followed by removal of the insoluble fraction by centrifugal separation (10,000 rpm, 30 minutes) to give the dialyzed fraction in the form of a supernatant. This dialyzed fraction was then applied to a mono S column (manufactured by Amersham), previously equilibrated with 50 mM acetate buffer (pH 4.5) to elute the enzyme with a linear concentration gradient of the same buffer containing 0 to 1 M NaCl. Of the active fractions, the fraction containing the lowest level of contaminating protein was applied to a Superdex 200 pg column (manufactured by Amersham), previously equilibrated with 50 mM acetate buffer (pH 4.5) containing 1 M NaCl and a gel fraction, was carried out with the same buffer ( pH 4.5) containing 1M NaCl, which was passed for free passage through the column to obtain a solution of the active fraction. As a result of these procedures using experimental electrophoresis data, it was confirmed that the enzyme involved in the formation of the peptide used in the present invention was purified to a homogeneous state. The degree of enzyme yield in the above purification process was 12.2%, and the degree of purification was 707-fold.

Example 3

Obtaining β-methyl ester of α-L-aspartyl-L-phenylalanine using the enzymatic fraction Empedobacter brevis

10 microliters (μl) of the Mono S enzyme fraction (approximately 20 U / ml) obtained in Example 2 was added to 190 μl of borate buffer (pH 9.0) containing 105.3 mM L-aspartic α-β-dimethyl ether hydrochloride acid, 210.5 mm L-phenylalanine and 10.51 mm EDTA, and the reaction was carried out at 20 ° C. The preparation of α-L-aspartyl-L-phenylalanine β-methyl ester (α-AMP) is shown in Table 3. It should be noted that the almost complete absence of the formation of β-methyl ester of α-L-aspartyl-L-phenylalanine was confirmed with a batch without adding an enzyme.

Moreover, 10 μl of the Mono S enzyme fraction (approximately 20 U / ml) obtained in Example 2 was added to 190 μl of borate buffer (pH 9.0) containing 105.3 mM L-aspartic acid α-methyl ester hydrochloride, 210.5 mm L-phenylalanine and 10.51 mm EDTA, and the reaction was carried out at 20 ° C. As a result, the formation of the corresponding peptides was not observed.

Table 3 Reaction time (minutes) Formed α-AMP (mm) thirty 23.0 60 42.1 120 61.7

Example 4

Purification of the enzyme from Sphingobacterium sp.

Strain FERM BP-8124 Sphingobacterium sp. (depository institute: independent corporation in management, national institute for the development of industrial science and technology, international patented organisms depository, depository institution address: Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan , date of international transfer of the deposit: July 22, 2002) were cultivated in the same way as in example 2, using the medium specified in example 2. The subsequent procedure after separation by centrifugation was carried out either on ice or at 4 ° C. The resulting culture broth was centrifuged (10,000 rpm, 15 minutes) to collect microorganism cells. After washing 2 g of the microorganism cells with 20 mM Tris-HCl buffer (pH 7.6), they were suspended in 8 ml of the same buffer and subjected to ultrasonic disruption treatment for 45 minutes at 195 W. The ultrasound-disrupted fluid was then centrifuged (10,000 rpm, 30 minutes) to remove fragments of the disrupted cells and the supernatant of the ultrasonic disrupted fluid was obtained. This supernatant of sonicated fluid was dialyzed overnight against 20 mM Tris-HCl buffer (pH 7.6), followed by removal of the insoluble fraction by ultracentrifugation (50,000 rpm, 30 minutes) to give the soluble fraction in the form of a supernatant. The resulting soluble fraction was applied to a HP Q-Sepharose column (manufactured by Amersham) pre-equilibrated with Tris-HCl buffer (pH 7.6), and the active fraction was collected from the non-adsorbed fraction. This active fraction was dialyzed overnight against 20 mM acetate buffer (pH 5.0), followed by removal of the insoluble fraction by centrifugal separation (10,000 rpm, 30 minutes) to give the dialyzed fraction in the form of a supernatant. This dialyzed fraction was then applied to an HP SP-Sepharose column (manufactured by Amersham), previously equilibrated with 20 mM acetate buffer (pH 5.0) to give an active fraction in which the enzyme was eluted with a linear concentration gradient of the same buffer containing from 0 to 1 M NaCl.

Example 5

Preparation of α-L-aspartyl-L-phenylalanine β-methyl ester and α-L-aspartyl-L-phenylalanine β-ethyl ester using the enzymatic fraction of Sphingobacterium sp.

In the case of α-L-aspartyl-L-phenylalanine β-methyl ester (α-AMP), 15 μl of the concentrated solution of the HP SP-Sepharose fraction (approximately 15 U / ml) obtained in Example 4 was added to 185 μl of borate buffer ( pH 9.0) containing 108.1 mM hydrochloride of α, L-aspartic acid β-dimethyl ester, 216.2 mM L-phenylalanine and 10.8 mM EDTA, and the reaction was carried out at 20 ° C. Similarly, in the case of α-L-aspartyl-L-phenylalanine β-ethyl ester (α-AEP), 10 μl of the concentrated HP SP-Sepharose fraction solution (approximately 15 U / ml) obtained in Example 4 was added to 190 μl borate buffer (pH 9.0) containing 52.6 mM hydrochloride α, L-aspartic acid β-diethyl ester, 105.2 mM L-phenylalanine and 10.8 mM EDTA, and the reaction was carried out at 20 ° C. The progress of AMP or AEP is shown in Table 4. It should be noted that the almost complete absence of AMP or AEP formation was confirmed with the batch without the addition of the enzyme. For the formation of AEP, numerical values are described using a standard AMP product.

Table 4 Reaction time (minutes) Received α-AMP (mm) Received α-AEP (mm) thirty 25.8 7.5 60 40.7 13.3 120 56.0 20.6 180 61.8 -

Example 6

Isolation of a peptide-forming enzyme genetically derived from Empedobacter brevis

Next, the isolation of the gene of the peptide-forming enzyme will be explained here. Strain FERM BP-8113 Empedobacter brevis (depository institution: independent corporation in management, national institute for the development of industrial science and technology, international depository of patented organisms, depository institution address: Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba- shi, Ibaraki-ken, Japan, date of international transfer of deposit: July 8, 2002) was used as a microorganism. When the gene was isolated, Escherichia coli JM-109 was used as the host, and pUC118 was used as the vector.

(1) Preparation of a PCR Primer Based on a Specific Internal Amino Acid Sequence

A mixed primer having the base sequences indicated in SEQ ID NO .: 3 and SEQ ID NO: 4, respectively, was obtained based on the amino acid sequences (SEQ ID NOs: 1 and 2) determined according to the Edman decomposition method of the hydrolysis product of lysyl endopeptidase Peptide-forming enzyme originating from the FERM BP-8113 Empedobacter brevis strain (depository institute: independent corporation in management, national institute for the development of industrial science and technology, international depository of patented organisms, institu uta depositary: Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, date of international transfer of deposit: July 8, 2002).

(2) Obtaining microorganism cells

Strain FERM BP-8113 Empedobacter brevis (depository institution: independent corporation in management, national institute for the development of industrial science and technology, international depository of patented organisms, address of depository institution: Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba- shi, Ibaraki-ken, Japan, date of international transfer of deposit: July 8, 2002) were cultivated at 30 ° C for 24 hours on CM2G agar medium (containing 50 g / l glucose, 10 g / l yeast extract, 10 g / l peptone 5 g / l sodium chloride and 20 g / l agar, pH 7.0). One loop of the obtained microorganism cells was inoculated into a 500 ml Sakagushi vessel containing 50 ml of CM2G liquid medium (the above medium excluding agar), followed by cultivation with shaking at 30 ° C.

(3) Obtaining chromosomal DNA from microorganism cells

50 ml of culture broth was centrifuged (12000 rpm, 4 ° C, 15 minutes) to collect microorganism cells. Then, chromosomal DNA was obtained from the cells of the microorganism using the QIAGEN Genomic-Tip System (Qiagen) based on the procedure described by its manufacturer.

(4) Obtaining a DNA fragment containing a portion of a gene forming a peptide enzyme using PCR

DNA fragment containing a portion of the gene of the peptide-forming enzyme originating from the FERM BP-8113 Empedobacter brevis strain -6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, date of international transfer of the deposit: July 8, 2002), was obtained using the PCR method using LA-Taq (manufactured by Takara Shuzo). PCR was performed on chromosomal DNA obtained from FERM BP-8113 Empedobacter brevis strain (depository institute: independent corporation in management, national institute for the development of industrial science and technology, international patented organisms depository, depository institution address: Chuo Dai-6, 1 -1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, date of international transfer of deposit: July 8, 2002), using primers having the sequences SEQ ID NOs: 3 and 4.

The PCR reaction was carried out for 30 cycles under the following conditions using Tekara PCR Thermal Cycler PERSONAL (manufactured by Takara Shuzo).

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

After completion of the reaction, 3 μl of the reaction liquid was applied to 0.8% agarose for electrophoresis. As a result, it was confirmed that a DNA fragment of approximately 1,500 bases is amplified (the so-called)

(5) Cloning the gene of the peptide-forming enzyme from the gene library

To obtain the full-sized gene of the peptide-forming enzyme, Southern hybridization was performed using a DNA fragment amplified by PCR as a probe. The Southern hybridization procedure is explained in Molecular Cloning, 2nd edition, Cold Spring Harbor Press (1989).

A DNA fragment of approximately 1.5 kb amplified by PCR was isolated by electrophoresis in 0.8% agarose. The desired strip was then cut and cleaned. This DNA fragment was labeled with a digoxinigen probe using DIG High Prime (manufactured by Boehringer-Mannheim) based on the procedure described in the guidelines for the use of DIG High Prime (manufactured by Boehringer-Mannheim).

After the hydrolysis of the chromosomal DNA of Empedobacter brevis obtained in step (3) of the present Example 6 was completed by reaction at 37 ° C. for 16 hours with the HindIII restriction enzyme, the resulting product was electrophoresed on a 0.8% agarose gel. Electrophoresis-separated chromosomal DNA was transferred after electrophoresis from an agarose gel to a positively charged nylon membrane filter (manufactured by Roche Diagnostics), followed by treatment consisting of alkaline denaturation, neutralization and immobilization. Hybridization was performed using EASY HYB (manufactured by Boehringer-Mannheim). After pre-hybridizing the filter at 50 ° C. for 1 hour, a digoxinigen labeled probe, prepared as described above, was added and hybridization was carried out at 50 ° C. for 16 hours. The filter was then washed for 20 minutes at room temperature with 2 x SSC containing 0.1% SDS. Moreover, the filter was further washed twice at 65 ° C. for 15 minutes with 0.1 x SSC containing 0.1% SDS.

Detection of bands that hybridized with the probe was performed using the DIG Nucleotide Detection Kit (manufactured by Boehringer-Mannheim). As a result, a band of approximately 4 so-called capable of hybridizing with the probe was detected.

Thereafter, 5 μg of the chromosomal DNA obtained in step (3) of the present Example 6 was completely hydrolyzed with HindIII. DNA approximately 4 so-called was separated by electrophoresis in 0.8% agarose gel and then the DNA was purified using the Gene Clean II Kit (manufactured by Funakoshi) and dissolved in 10 μl of TE. 4 μl of this product was then mixed with pUC118 HindIII / BAP (manufactured by Takara Shuzo) and a ligation reaction was performed using DNA Ligation Kit Ver. 2 (manufactured by Takara Shuzo). 5 μl of the ligation reaction mixture and 100 μl of competent Escherichia coli JM109 cells (manufactured by Toyobo) were mixed to transform Escherichia coli . The contents were then applied to a suitable solid medium to obtain a chromosomal DNA library.

To obtain a whole, full-length gene of a peptide-forming enzyme, the chromosomal DNA library was screened by colony hybridization, as explained in Molecular Cloning, 2nd edition, Cold Spring Harbor Press (1989).

Colonies of the chromosomal DNA library were transferred to a Nylon Membrane for Colony and Plaque Hybridization filter (manufactured by Roche Diagnostics) followed by alkaline denaturation, neutralization and immobilization treatment. Hybridization was performed using EASY HYB (manufactured by Boehringer-Mannheim). filter hybridization at 37 ° C. for 1 hour, the above digoxinigen labeled probe was added, followed by hybridization at 50 ° C. for 16 hours, then the filter was washed for 20 minutes at room temperature with 2 x SSC containing 0.1% SDS. Furthermore, the filter was further washed twice at 65 ° C. for 15 minutes with 0.1 x SSC containing 0.1% SDS.

Colonies that hybridized with a labeled probe were detected using the DIG Nucleotide Detection Kit (manufactured by Boehringer-Mannheim) based on the explanation described in the DIG Nucleotide Detection Kit (manufactured by Boehringer-Mannheim). As a result, the coloration of two colonies capable of hybridizing with a labeled probe was revealed.

(6) The base sequence of the gene forming the peptide enzyme derived from Empedobacter brevis

The plasmids contained in Escherichia coli JM109 were obtained from the above two clones of microorganism cells, which, as confirmed by the Wizard Plus Minipreps DNA Purification System (manufactured by Promega), hybridized with a labeled probe, and the base sequence of the part where the hybridization occurred was determined , and close to her. The sequencing reaction was carried out using the CEQ DTCS-Quick Start Kit (manufactured by Beckman-Coulter) based on the procedure described in its manual. In addition, electrophoresis was performed using CEQ 2000-XL (manufactured by Beckman-Coulter).

As a result, it was confirmed that the open reading frame, which encodes a protein containing the internal amino acid sequences of the peptide forming enzyme (SEQ ID NOs: 1 and 2), does exist, thereby confirming that the open reading frame is a gene, peptide-forming enzyme. The base sequence of the full-length genes of the peptide-forming enzyme, along with the corresponding amino acid sequences, is shown in SEQ ID NO: 5 of the sequence listing. According to the results of the homology analysis of the obtained open reading frame carried out using the BLASTP program, homology between the two enzymes was detected; he showed a homology of 34% at the level of the amino acid sequence exhibited by the hydrolase of α-amino acid esters of Acetobacter pasteurianus (see Appl. Environ. Microbiol., 68 (1), 211-218 (2002), and 26% homology at the level of the amino acid sequence exhibited glutaryl-7ACA acylase Brevibacillus laterosporum (see J. Bacteriol., 173 (24), 7848-7855 (1991).

(7) Expression of the gene of the peptide-forming protein originating from Empedobacter brevis in Escherichia coli

The region of the target gene in the promoter region of the trp operon of the Escherichia coli W3110 chromosomal DNA was amplified by PCR using the oligonucleotides indicated in SEQ ID NOs: 7 and 8 as primers and the resulting DNA fragments were ligated with the pGEM-Teasy vector (manufactured by Promega). Then, E. coli JM109 was transformed in this ligation solution, and clones containing the desired plasmid were selected from ampicillin-resistant strains, in which the inserted trp promoter was in the opposite direction to the lac promoter. Then, the DNA fragment containing the trp promoter obtained by processing this EcoO109 / EcoRI plasmid was ligated with EcoO109 / EcoRI, the pUC19 treatment product (manufactured by Takara). Then, the DNA fragment obtained by processing this HindIII / PvuII plasmid was ligated with the DNA fragment containing the rrnB terminator obtained by processing pKK223-3 (manufactured by Amersham Pharmacia) HindIII / HincII. Then, E. coli JM109 was transformed with this ligation solution, strains containing the desired plasmid were selected from ampicillin-resistant strains, and the plasmid was designated as pTrpT.

The desired gene was amplified by PCR using the chromosomal DNA of the strain FERM BP-8113 Empedobacter brevis (depository institute: independent corporation in management, national institute for the development of industrial science and technology, international patented organisms depository, depository institution address: Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, date of international transfer of the deposit: July 8, 2002) as the matrix and oligonucleotides indicated in SEQ ID NOs: 9 and 10, as primers. This DNA fragment was then treated with NdeI / PstI and the resulting DNA fragment was ligated with the NdeI / PstI pTrpT treatment product. Then, Escherichia coli JM109 was transformed with this ligation solution, strains containing the desired plasmid were selected from ampicillin-resistant strains, and the plasmid was designated as pTrpT_Gtg2.

Escherichia coli JM109 containing pTrpT_Gtg2 was cultured to obtain seed at 30 ° C. for 24 hours in LB medium containing 100 mg / L ampicillin. 1 ml of the resulting culture broth was sown in a 500 ml Sakagushi vessel containing 50 ml of medium (2 g / l D glucose, 10 g / l yeast extract, 10 g / l casamino acids, 5 g / l ammonium sulfate, 3 g / l monopotassium hydrogen phosphate , 1 g / l of dipotassium hydrogen phosphate, 0.5 g / l of magnesium sulfate heptahydrate and 100 mg / l of ampicillin), followed by cultivation at 25 ° C for 24 hours. The culture broth had the activity of producing α-L-aspartyl-L-phenylalanine 0.11 U β-methyl ester per 1 ml of culture broth, and it was confirmed that the cloned gene is expressed by E. coli. Moreover, 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: 6 described in the sequence listing was analyzed using Signal P v 1.1 (see Protein Engineering, Vol. 12, No. 1, pp. 3-9, 1999), it was predicted that amino acids from numbers 1 to 22 act as a signal that is secreted into the periplasm, and the mature protein has been found to be located below amino acid number 23.

Secretion confirmation

Escherichia coli JM109 containing pTrpT_Gtg2 was cultured to obtain seed at 30 ° C. for 24 hours in LB medium containing 100 mg / L ampicillin. 1 ml of the resulting culture broth was sown in a 500 ml Sakagushi vessel containing 50 ml of medium (2 g / l D glucose, 10 g / l yeast extract, 10 g / l casamino acids, 5 g / l ammonium sulfate, 3 g / l monopotassium hydrogen phosphate , 1 g / l of dipotassium hydrogen phosphate, 0.5 g / l of magnesium sulfate heptahydrate and 100 mg / l of ampicillin), followed by cultivation at 25 ° C for 24 hours to obtain a microorganism cell culture.

The cultured cells of the microorganism were fractionated into the periplasm fraction and the cytoplasmic fraction using the shock osmotic pressure method using a sucrose solution of 20 grams / deciliter (g / dl). Microorganism cells immersed in a 20 g / dl sucrose solution were immersed in a 5 mM aqueous MgSO ₄ solution. The supernatant after centrifugation was called the periplasmic fraction. ("Pe"). In addition, the precipitate after centrifugation was resuspended and subjected to ultrasonic destruction. The resulting was called the cytoplasmic fraction ("Cy"). The activity of glucose-6-phosphate dehydrogenase, which is known to be in the cytoplasm, was used as an indicator to confirm the separation of the cytoplasm. This measurement was carried out by adding a suitable amount of enzyme to the reaction solution at 30 ° C containing 1 mM glucose-6-phosphate, 0.4 mM NADP, 10 mM MgSO ₄ and 50 mM Tris-HCl (pH 8), followed by measurement of absorption at 340 nm to measure the formation of NADPH.

The number of enzymes in the periplasmic fraction and cytoplasmic fraction, when the activity of the obtained cell-free extract was taken as 100%, is shown in the drawing. The fact that glucose-6-phosphate dehydrogenase activity did not mix with the periplasmic fraction indicates that the periplasmic fraction was not mixed with the cytoplasmic fraction. Approximately 60% of the activity forming α-L-aspartyl-L-phenylalanine β-methyl ester (α-AMP) was detected in the periplasmic fraction, and it was confirmed that the Ala-Gln-forming enzyme is secreted into periplasm, as was predicted by based amino acid sequence using the program Signal P v 1.1.

Example 7

Isolation of a gene forming a peptide enzyme derived from Sphingobacterium sp.

The following describes the isolation of a gene forming a peptide enzyme. The microorganism used was the strain FERM BP-8124 Sphingobacterium sp . (depository institution: independent corporation in management, national institute for the development of industrial science and technology, international depository of patented organisms, address of depository institution: Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan , date of international transfer of deposit: July 22, 2002). To isolate the gene, Escherichia coli DH5α was used as the host, and pUC118 was used as the vector.

(1) Obtaining cells of a microorganism

Strain FERM BP-8124 Sphingobacterium sp . (depository institution: independent corporation in management, national institute for the development of industrial science and technology, international depository of patented organisms, address of depository institution: Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan , date of international transfer of the deposit: July 22, 2002) were cultured for 24 hours at 25 ° C on CM2G agar medium (containing 50 g / l glucose, 10 g / l yeast extract, 10 g / l peptone, 5 g / l sodium chloride and 20 g / l agar, pH 7.0). One loop of the obtained microorganism cells was inoculated into a 500 ml Sakagushi vessel containing 50 ml of CM2G liquid medium (the above medium, except for agar), followed by cultivation with shaking at 25 ° C.

(2) Obtaining chromosomal DNA from microorganism cells

50 ml of culture broth was centrifuged (12000 rpm, 4 ° C, 15 minutes) to collect microorganism cells. Then, chromosomal DNA was obtained from the cells of the microorganism using the Qiagen Genomic-Tip System (Qiagen) based on the procedure described by its manufacturer.

(3) Obtaining a DNA fragment of a probe by PCR

DNA fragment containing a portion of the gene of the peptide-forming enzyme originating from the FERM BP-8113 Empedobacter brevis strain -6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, date of international transfer of the deposit: July 8, 2002), was obtained using the PCR method using LA-Taq (manufactured by Takara Shuzo). Then, a PCR reaction was performed on chromosomal DNA obtained from the FERM BP-8113 Empedobacter brevis strain (depository institute: independent corporation in management, national institute for the development of industrial science and technology, international patented organisms depository, depository institute address: Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, date of international transfer of deposit: July 8, 2002), using primers having base sequences of SEQ ID NOs: 3 and 4.

The PCR reaction was carried out using Takara PCR Thermal Cycler - PERSONAL (Takara Shuzo) for 30 cycles under the following conditions.

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

After completion of the reaction, 3 μl of the reaction liquid was applied to 0.8% agarose for electrophoresis. As a result, it was confirmed that a DNA fragment of approximately 1.5 kb was amplified.

(4) Cloning a gene of a peptide-forming enzyme from a gene library

To obtain the full-sized gene of the peptide-forming enzyme, Southern hybridization was performed using a DNA fragment amplified in the above PCR procedure as a probe. Southern hybridization operations are explained in Molecular Cloning, 2nd edition, Cold Spring Harbor Press (1989).

A DNA fragment of approximately 1.5 kb amplified by the above PCR procedure was isolated by electrophoresis in 0.8% agarose. The desired strip was then cut and cleaned. This DNA fragment was labeled with a digoxinigen probe using DIG High Prime (manufactured by Boehringer-Mannheim) based on the procedure described in the manual.

After the interaction of the chromosomal DNA of Sphingobacterium sp ., Obtained in stage (2) of the present example 7, with the SacI restriction enzyme at 37 ° C for 16 hours until the complete hydrolysis of the DNA, the obtained one was subjected to electrophoresis on a 0.8% agarose gel. After electrophoresis, electrophoresis-separated DNA from an agarose gel was transferred to a positively charged nylon membrane filter (manufactured by Roche Diagnostics), followed by treatment consisting of alkaline denaturation, neutralization and immobilization. Hybridization was performed using EASY HYB (manufactured by Boehringer-Mannheim). After pre-hybridizing the filter at 37 ° C for 1 hour, a digoxinigen-labeled probe, prepared as described above, was added and hybridization was carried out at 37 ° C for 16 hours. The filter was then washed twice at 60 ° C with 1 x SSC containing 0.1% SDS.

The bands that hybridized with the probe were detected using the DIG Nucleotide Detection Kit (manufactured by Boehringer-Mannheim) based on the procedure described in his manual. As a result, a band of approximately 3 so-called, which hybridized with the probe, was successfully detected.

5 μg of the chromosomal DNA obtained in step (2) of the present Example 7 was completely hydrolyzed using SacI. DNA approximately 3 so-called was separated by electrophoresis in 0.8% agarose gel, DNA was purified using Gene Clean II Kit (manufactured by Funakoshi) and dissolved in 10 μl of TE. 4 μl of the resulting solution were mixed with alkaline phosphatase-treated pUC118 (E. coli C75) at 37 ° C for 30 minutes and 50 ° C for 30 minutes after complete hydrolysis with SacI at 37 ° C for 16 hours, and was performed ligation reaction using DNA Ligation Kit Ver. 2 (manufactured by Takara Shuzo). 5 μl of ligation reaction fluid and 100 μl of competent Escherichia coli DH5α cells (manufactured by Takara Shuzo) were mixed to transform Escherichia coli . The contents were then applied to a suitable solid medium to obtain a chromosomal DNA library.

To obtain the full-length gene of the peptide-forming enzyme, the chromosomal DNA library was screened by colony hybridization using the probe indicated above. The procedure for colony hybridization is explained in Molecular Cloning, 2nd edition, Cold Spring Harbor Press (1989).

Colonies of the chromosomal DNA library were transferred to a nylon membrane filter (Nylon Membrane for Colony and Plaque Hybridization, manufactured by Roche Diagnostics), followed by alkaline denaturation, neutralization and immobilization. Hybridization was performed using EASY HYB (manufactured by Boehringer-Mannheim). After pre-hybridizing the filter at 37 ° C for 1 hour, the above digoxinigen-labeled probe was added, followed by hybridization at 37 ° C for 16 hours. The filter was then washed twice at 60 ° C with 1 x SSC containing 0.1% SDS.

Colonies that hybridized with a labeled probe were detected using the DIG Nucleotide Detection Kit (manufactured by Boehringer-Mannheim) based on the explanation described in his manual. As a result, six strains of colonies capable of hybridizing with a labeled probe were identified.

(5) The base sequence of the gene forming the peptide of the enzyme derived from Sphingobacterium sp .

The plasmids contained in Escherichia coli DH5α were obtained from six clones of microorganism cells, which, as confirmed by the Wizard Plus Minipreps DNA Purification System (manufactured by Promega), hybridized with a labeled probe to determine the base sequence of the part where the probe hybridized and close to her. The sequencing reaction was carried out using the CEQ DTCS-Quick Start Kit (manufactured by Beckman-Coulter) based on the procedure described in its manual. In addition, electrophoresis was performed using CEQ 2000-XL (manufactured by Beckman-Coulter).

As a result, it was confirmed that an open-reading frame that encodes a peptide-forming enzyme does exist. The full length base sequence of the gene for the peptide-forming enzyme from Sphingobacterium sp., Along with the corresponding amino acid sequence, is shown in SEQ ID NO: 11. The peptide-forming enzyme derived from Sphingobacterium sp. Showed a 63.5% homology at the amino acid sequence level derived from Empedobacter brevis peptide-forming enzyme (as determined using the BLASTP program).

(6) Expression of the gene of the peptide-forming enzyme originating from Sphingobacterium sp. , in Escherichia coli

The desired gene was amplified by PCR using chromosomal DNA of Sphingobacterium sp . FERM BP-8124 (depository institute: independent corporation in management, national institute for the development of industrial science and technology, international patented organisms depository, depository institute address: Chuo Dai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki -ken, Japan, date of international transfer of deposit: July 22, 2002) as the template and oligonucleotides indicated in SEQ ID NOs: 13 and 14, as primers. This DNA fragment was treated with NdeI / XbaI and the obtained DNA fragment was ligated with the NdeI / XbaI pTrpT treatment product. Then Escherichia coli JM109 was transformed with this ligation solution, and strains containing the desired plasmid were selected from ampicillin-resistant strains. The plasmid was designated as pTrpT_Sm_aet.

Escherichia coli JM109 containing pTrpT_Sm_aet was cultured at 25 ° C for 20 hours by inoculating one of its loops in a standard test tube containing 3 ml of medium (2 g / l D glucose, 10 g / l yeast extract, 10 g / l casamino acids, 5 g / l of ammonium sulfate, 3 g / l of monopotassium hydrogen phosphate, 1 g / l of dipotassium hydrogen phosphate, 0.5 g / l of magnesium sulfate heptahydrate and 100 mg / l of ampicillin). It has been confirmed that a cloned gene having an activity of α-AMP formation of 0.53 E per ml of culture broth is expressed by Escherichia. coli. Moreover, activity was not detected in the transformant containing only pTrpT as a control.

Signal sequence prediction

When the amino acid sequence of SEQ ID NO: 12 described in the sequence listing was analyzed using Signal P v 1.1 (see Protein Engineering, Vol. 12, No. 1, pp. 3-9, 1999), it was predicted that amino acids from numbers 1 to 20 act as a signal that is secreted into the periplasm, and the mature protein has been found to be located below amino acid number 21.

Signal sequence confirmation

One loop of Escherichia coli JM109 containing pTrpT_Sm_aet was inoculated into ordinary test tubes containing 50 ml of medium (2 g / l glucose, 10 g / l yeast extract, 10 g / l casamino acids, 5 g / l ammonium sulfate, 3 g / l of monopotassium hydrogen phosphate, 1 g / l of dipotassium hydrogen phosphate, 0.5 g / l of magnesium sulfate heptahydrate and 100 mg / l of ampicillin), and the main cultivation was carried out at 25 ° C for 20 hours.

Further, here, the procedures after separation by centrifugation were carried out either on ice or at 4 ° C. After cultivation, the cells of the microorganism were separated from the culture broth by centrifugation, washed with 100 mM phosphate buffer (pH 7) and then suspended in the same buffer. The cells of the microorganism were then subjected to ultrasonic disruption treatment for 20 minutes at 195 W, and the liquid destroyed by ultrasound was centrifuged (12000 rpm, 30 minutes) to remove fragments of the destroyed cells and obtain a soluble fraction. The resulting soluble fraction was loaded onto a CHT-II column (manufactured by Biorad), previously equilibrated with 100 mM phosphate buffer (pH 7), and the enzyme was eluted with a linear concentration gradient of 500 mM phosphate buffer. The solution obtained by mixing the active fraction with 5-fold volumes of 2 M ammonium sulfate and 100 mM phosphate buffer was applied to a Resource-PHE column (manufactured by Amersham), previously equilibrated with 2 M ammonium sulfate and 100 mM phosphate buffer, and the enzyme was eluted with a linear concentration gradient from 2 to 0 M ammonium sulfate to obtain a solution of the active fraction. As a result of these procedures, it was confirmed that the peptide-forming enzyme was purified to electrophoretic uniformity.

When the amino acid sequence of the aforementioned peptide-forming enzyme was determined using the Edman decomposition method, the amino acid sequence of SEQ ID NO: 15 was obtained and it was confirmed that the mature protein was located after amino acid number 21, as was predicted using the Signal P v 1.1 program .

Example 8

Isolation of the gene forming the peptide enzyme from Pedobacter heparinus IFO 12017

The following describes the isolation of a gene forming a peptide enzyme. The microorganism used was Pedobacter heparinus IFO 12017 (depository institute: Institute of Fermentation, Osaka, depository institute address: 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan). To isolate the gene, Escherichia coli JM109 was used as the host, and pUC118 was used as the vector.

(1) Obtaining cells of a microorganism

Pedobacter heparinus IFO 12017 (depository institute: Institute of Fermentation, Osaka, depository institution address: 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan) was cultivated for 24 hours at 25 ° C on agar CM2G medium (containing 50 g / l glucose, 10 g / l yeast extract, 10 g / l peptone, 5 g / l sodium chloride and 20 g / l agar, pH 7.0). One loop of the obtained microorganism cells was inoculated into a 500 ml Sakagushi vessel containing 50 ml of CM2G liquid medium (the above medium, except for agar), followed by cultivation with shaking at 25 ° C.

(2) Obtaining chromosomal DNA from microorganism cells

50 ml of culture broth was centrifuged (12000 rpm, 4 ° C, 15 minutes) to collect microorganism cells. Then, chromosomal DNA was obtained from the cells of the microorganism using the Qiagen Genomic-Tip System (Qiagen) based on the procedure described by its manufacturer.

(3) Obtaining a DNA fragment of a probe by PCR

DNA fragment containing a portion of the gene of the peptide-forming enzyme, originating from Pedobacter heparinus IFO 12017 (depository institute: Institute of Fermentation, Osaka, depository institute address: 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan), obtained using the PCR method using LA-Taq (manufactured by Takara Shuzo). Then, a PCR reaction was performed on chromosomal DNA obtained from Pedobacter heparinus IFO 12017 (depository institute: Institute of Fermentation, Osaka, depository institute address: 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan), using primers having a base sequence of SEQ ID NOs: 15 and 16. Using electrophoresis in 0.8% agarose, a DNA fragment of approximately 1 so-called amplified by PCR was isolated. Then the desired strip was cut out and cleaned. The DNA fragment was labeled with a digoxinigen probe using DIG High Prime (manufactured by Boehringer-Mannheim) based on the procedure described in his manual.

(4) Cloning the gene of the peptide-forming enzyme from the gene library

To obtain the full-sized gene of the peptide-forming enzyme, Southern hybridization was performed using a DNA fragment amplified in the above PCR procedure as a probe. Southern hybridization operations are explained in Molecular Cloning, 2nd edition, Cold Spring Harbor Press (1989).

After the interaction of the pedobacter heparinus IFO 12017 chromosomal DNA (depository institute: Institute of Fermentation, Osaka, depository institute address: 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan) with the HindIII restriction enzyme at 37 ° C for 16 hours until complete hydrolysis of the DNA obtained was subjected to electrophoresis in 0.8% agarose gel. After electrophoresis, electrophoresis-separated DNA from an agarose gel was transferred to a positively charged nylon membrane filter (manufactured by Roche Diagnostics), followed by treatment consisting of alkaline denaturation, neutralization and immobilization. Hybridization was performed using EASY HYB (manufactured by Boehringer-Mannheim). After pre-hybridizing the filter at 50 ° C. for 1 hour, a digoxinigen labeled probe, prepared as described above, was added and hybridization was carried out at 50 ° C. for 16 hours. The filter was then washed twice at 60 ° C with 1 x SSC containing 0.1% SDS.

The bands that hybridized with the probe were detected using the DIG Nucleotide Detection Kit (Boehringer-Mannheim) based on the procedure described in its manual. As a result, a band of approximately 5 so-called, which hybridized with the probe, was successfully detected.

5 μg of Pedobacter heparinus IFO 12017 chromosomal DNA was completely hydrolyzed using HindIII. DNA approximately 5 so-called was separated by electrophoresis in 0.8% agarose gel, DNA was purified using Gene Clean II Kit (manufactured by Funakoshi) and dissolved in 10 μl of TE. 4 μl of the resulting solution and pUC118 HindIII / BAP were mixed and the ligation reaction was performed using DNA Ligation Kit Ver. 2 (manufactured by Takara Shuzo). 5 μl of a mixture of this ligation reaction fluid and 100 μl of competent Escherichia coli JM109 cells (manufactured by Takara Shuzo) were mixed to transform Escherichia coli. The contents were then applied to a suitable solid medium to obtain a chromosomal DNA library.

To obtain the full-length gene of the peptide-forming enzyme, the chromosomal DNA library was screened using the probe indicated above. Colony hybridization procedure is explained in Molecular Cloning, 2nd edition, Cold Spring Harbor Press (1989).

Colonies of the chromosomal DNA library were transferred to a Nylon Membrane Filter for Colony and Plaque Hybridization (manufactured by Roche Diagnostics), followed by alkaline denaturation, neutralization, and immobilization. Hybridization was performed using EASY HYB (manufactured by Boehringer-Mannheim). After pre-hybridizing the filter at 37 ° C for 1 hour, the above digoxinigen-labeled probe was added, followed by hybridization at 37 ° C for 16 hours. The filter was then washed twice at 60 ° C with 1 x SSC containing 0.1% SDS.

Colonies that hybridized with the labeled probe were detected using the DIG Nucleotide Detection Kit (manufactured by Boehringer-Mannheim) based on the explanation described in its manual. As a result, one strain was detected whose colony hybridized with a labeled probe.

(5) The base sequence of the gene forming the peptide of the enzyme derived from Pedobacter heparinus IFO 12017

The plasmids contained in Escherichia coli JM109 were obtained from a strain that was confirmed to hybridize with a labeled probe, and the base sequence of the part where hybridization with the probe and in the vicinity thereof was determined was determined. The sequencing reaction was carried out using the CEQ DTCS-Quick Start Kit (manufactured by Beckman-Coulter) based on the procedure described in its manual. In addition, electrophoresis was performed using CEQ 2000-XL (manufactured by Beckman-Coulter).

As a result, it was found that an open-reading frame that encodes a peptide-forming enzyme does exist. Full-length base sequence of the gene of the peptide-forming enzyme derived from Pedobacter heparinus IFO 12017 (depository institute: Institute of Fermentation, Osaka, address of depository institute: 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan) along with the corresponding amino acid sequence shown in SEQ ID NO: 17.

Example 9

Expression of the gene of the peptide-forming enzyme derived from Pedobacter heparinus IFO 12017 in Escherichia coli

The desired gene was amplified by PCR using the pedobacter heparinus IFO 12017 chromosomal DNA (depository institute: Institute of Fermentation, Osaka, depository institute address: 2-17-85 Jusanbon-cho, Yodogawa-ku, Osaka-shi, Japan) in as the matrix and oligonucleotides shown in SEQ ID NOs: 19 and 20, as primers. The DNA fragment was treated with NdeI / HindIII and the resulting DNA fragment and the NdeI / HindIII processing product pTrpT were ligated. Then Escherichia coli JM109 was transformed with this ligation solution, and strains containing the desired plasmid were selected from ampicillin-resistant strains. The plasmid was designated as pTrpT_Ph_aet.

One loop of Escherichia coli JM109 containing pTrpT_Ph_aet was inoculated into a standard test tube containing 3 ml of medium (2 g / l glucose, 10 g / l yeast extract, 10 g / l casamino acids, 5 g / l ammonium sulfate, 3 g / l of monopotassium hydrogen phosphate, 1 g / l of dipotassium hydrogen phosphate, 0.5 g / l of magnesium sulfate heptahydrate and 100 mg / l of ampicillin), and the main cultivation was carried out at 25 ° C for 20 hours. It was confirmed that the culture broth had an α-AMP producing activity of 0.01 U per ml of culture broth, which confirmed the expression of the cloned gene in Escherichia coli. Moreover, activity was not detected in the transformant containing only pTrpT, used as a control.

Example 10

Isolation of a gene forming a peptide enzyme derived from Taxeobacter gelupurpurascens DSMZ 11116

The following describes the isolation of a gene forming a peptide enzyme. The microorganism used was Taxeobacter gelupurpurascens DSMZ 11116 (depository institution; the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German collection of microorganisms and cell cultures); address of the depository institution; Mascheroder Weg 1b, 38124 Braunschweig. To isolate the gene, Escherichia coli JM109 was used as the host, and pUC118 was used as the vector.

(1) Obtaining cells of a microorganism

Taxeobacter gelupurpurascens DSMZ 11116 (depository institution; the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German collection of microorganisms and cell cultures, address of the depository institution; Mascheroder Weg 1b, 38124 Braunschweig, Germany) was cultivated for 24 hours at 25 ° C at 24 hours CM2G medium (containing 50 g / l glucose, 10 g / l yeast extract, 10 g / l peptone, 5 g / l sodium chloride and 20 g / l agar, pH 7.0). One loop of the obtained microorganism cells was inoculated in 500 ml Sakagushi vessel containing 50 ml of CM2G liquid medium (the above medium, excluding agar), followed by cool tivirovaniem with shaking at 25 ° C.

(2) Obtaining chromosomal DNA from microorganism cells

50 ml of culture broth was centrifuged (12000 rpm, 4 ° C, 15 minutes) to collect microorganism cells. Then, chromosomal DNA was obtained from the cells of the microorganism using the Qiagen Genomic-Tip System (Qiagen) based on the procedure described by its manufacturer.

(3) Obtaining a DNA fragment of a probe by PCR

DNA fragment containing a portion of the gene of the peptide-forming enzyme originating from Taxeobacter gelupurpurascens DSMZ 11116 (depository institution; the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German collection of microorganisms and cell cultures, address of the depository institution; Mascheroder Weg1 Brabe 1 Germany) was obtained using a PCR method using LA-Taq (manufactured by Takara Shuzo), and then a PCR reaction was performed on chromosomal DNA obtained from Taxeobacter gelupurpurascens DSMZ 11116 (depository institution; the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German collection of microorganisms) and to cultures, address of custodian institution; Mascheroder Weg 1b, 38124 Braunschweig, Germany) using primers having base sequences of SEQ ID NOs: 21 and 16. A DNA fragment of approximately 1 kb amplified by PCR was isolated with by electrophoresis in 0.8% agarose, the desired strip was then cut and cleaned. The DNA fragment was labeled with a digoxinigen probe using DIG High Prime (manufactured by Boehringer-Mannheim) based on the procedure described in his manual.

(4) Cloning a gene of a peptide-forming enzyme from a gene library

To obtain the full-sized gene of the peptide-forming enzyme, Southern hybridization was performed using a DNA fragment amplified in the above PCR procedure as a probe. Southern hybridization operations are explained in Molecular Cloning, 2nd edition, Cold Spring Harbor Press (1989).

After chromosomal DNA interaction Taxeobacter gelupurpurascens DSMZ 11116 (depository institution; the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German collection of microorganisms and cell cultures, address of the depository institution; Mascheroder Weg 1b, 38124 Braun enzyme restriction, Germany) C for 16 hours until complete hydrolysis of the DNA obtained was subjected to electrophoresis in 0.8% agarose gel.After electrophoresis, the chromosomal DNA separated by electrophoresis from the agarose gel was transferred to a positively charged nylon membrane filter (manufactured by Roche D iagnostics) followed by treatment consisting of alkaline denaturation, neutralization and immobilization Hybridization was carried out using EASY HYB (manufactured by Boehringer-Mannheim). After preliminary hybridization of the filter at 50 ° C for 1 hour, a probe labeled with digoxinigen obtained as described above, and hybridization was carried out at 50 ° C. for 16 hours, then the filter was washed twice at 60 ° C. with 1 x SSC containing 0.1% SDS.

The bands that hybridized with the probe were detected using the DIG Nucleotide Detection Kit (Boehringer-Mannheim) based on the procedure described in his manual. As a result, a band of approximately 5 so-called, which hybridized with the probe, was successfully detected.

5 μg of Taxeobacter gelupurpurascens DSMZ chromosomal DNA DSMZ 11116 (depository institution; the Deutche Sammlung von Mikroorganismen und Zellkulturen GmbH (German collection of microorganisms and cell cultures, address of the depository institution; Mascheroder Weg 1b, 38124 Brauns using DNA, Germany). approximately 5 so-called 0.8% agarose gel electrophoresis was separated, DNA was purified using the Gene Clean II Kit (manufactured by Funakoshi) and dissolved in 10 μl TE. 4 μl of the resulting solution was mixed with pUC118 PstI / BAP (manufactured by Takara Shuzo) and conducted a ligation reaction using DNA Ligation Kit Ver. 2 (industrial by Takara Shuzo) 5 μl of this ligation reaction liquid and 100 μl of competent Escherichia coli JM109 cells (manufactured by Toyobo) were mixed to transform Escherichia coli. The contents were then applied to a suitable solid medium to obtain a chromosomal DNA library.

To obtain the full-length gene of the peptide-forming enzyme, the chromosomal DNA library was screened by colony hybridization using the probe indicated above. The procedure for colony hybridization is explained in Molecular Cloning, 2nd edition, Cold Spring Harbor Press (1989).

Colonies of the chromosomal DNA library were transferred onto a Nylon Membrane Filter for Colony and Plaque Hybridization (manufactured by Roche Diagnostics), followed by alkaline denaturation, neutralization and immobilization. Hybridization was performed using EASY HYB (manufactured by Boehringer-Mannheim). After pre-hybridizing the filter at 37 ° C for 1 hour, the above digoxinigen-labeled probe was added, followed by hybridization at 37 ° C for 16 hours. The filter was then washed twice at 60 ° C with 1 x SSC containing 0.1% SDS.

Colonies that hybridized with the labeled probe were detected using the DIG Nucleotide Detection Kit (manufactured by Boehringer-Mannheim) based on the explanation described in his manual. As a result, one strain was identified whose colony hybridized with a labeled probe.

(5) The base sequence of the gene forming the peptide enzyme derived from Taxeobacter gelupurpurascens DSMZ 11116

The plasmids contained in Escherichia coli JM109 were obtained from a strain that was confirmed to hybridize with a labeled probe, and the base sequence of the part where hybridization with the probe and in the vicinity thereof was determined was determined. The sequencing reaction was carried out using the CEQ DTCS-Quick Start Kit (manufactured by Beckman-Coulter) based on the procedure described in its manual. In addition, electrophoresis was performed using CEQ 2000-XL (manufactured by Beckman-Coulter).

As a result, it was found that an open-reading frame that encodes a peptide-forming enzyme does exist. The full length base sequence of the gene for the peptide forming enzyme from Taxeobacter gelupurpurascens DSMZ 11116, along with the corresponding amino acid sequence, is shown in SEQ ID NO: 22.

Example 11

Isolation of the gene forming the peptide enzyme derived from Cyclobacterium marinum ATCC 25205

The following describes the isolation of a gene forming a peptide enzyme. The microorganism used was Cyclobacterium marinum ATCC 25205 (depository institution; American collection of crop types, address of depository institution; PO Box 1549, Manassas, VA 20110, United States of America). To isolate the gene, Escherichia coli JM109 was used as the host, and pUC118 was used as the vector.

(1) Obtaining cells of a microorganism

Cyclobacterium marinum ATCC 25205 (depository institution; American collection of crop types, depository institution address; PO Box 1549, Manassas, VA 20110, United States of America) was cultured for 24 hours at 25 ° C. on CM2G agar medium (containing 50 g / l of glucose, 10 g / l of yeast extract, 10 g / l of peptone, 5 g / l of sodium chloride and 20 g / l of agar, pH 7.0). One loop of the obtained microorganism cells was inoculated into a 500 ml Sakagushi vessel containing 50 ml of CM2G liquid medium (the above medium, except for agar), followed by cultivation with shaking at 25 ° C.

(2) Obtaining chromosomal DNA from microorganism cells

50 ml of culture broth was centrifuged (12000 rpm, 4 ° C, 15 minutes) to collect microorganism cells. Then, chromosomal DNA was obtained from microorganism cells using the Qiagen Genomic-Tip System (Qiagen) based on the procedure described in its manual.

(3) Obtaining a DNA fragment of a probe by PCR

A DNA fragment containing a portion of the gene of the peptide forming enzyme originating from Cyclobacterium marinum ATCC 25205 was obtained by PCR using LA-Taq (manufactured by Takara Shuzo). Then, a PCR reaction was performed on chromosomal DNA obtained from Cyclobacterium marinum ATCC 25205 (depository institution; American collection of culture types, depository institution address; PO Box 1549, Manassas, VA 20110, United States of America) using primers having base sequences SEQ ID NOs: 15 and 16. A DNA fragment of approximately 1 kb amplified by PCR was isolated by electrophoresis in 0.8% agarose. The desired strip was then cut and cleaned. The DNA fragment was labeled with a digoxinigen probe using DIG High Prime (manufactured by Boehringer-Mannheim) based on the procedure described in his manual.

(4) Cloning a gene of a peptide-forming enzyme from a gene library

To obtain the full-sized gene of the peptide-forming enzyme, Southern hybridization was performed using a DNA fragment amplified in the above PCR procedure as a probe. Southern hybridization operations are explained in Molecular Cloning, 2nd edition, Cold Spring Harbor Press (1989).

After the interaction of the chromosomal DNA of Cyclobacterium marinum ATCC 25205 (depository institution; American collection of crop types, depository institution address; PO Box 1549, Manassas, VA 20110, United States of America) with the restriction enzyme PstI or HincII at 37 ° C for 16 hours until complete hydrolysis of the DNA obtained was subjected to electrophoresis in 0.8% agarose gel. After electrophoresis, the chromosomal DNA separated by electrophoresis from the agarose gel was transferred to a positively charged nylon membrane filter (manufactured by Roche Diagnostics), followed by treatment consisting of alkaline denaturation, neutralization and immobilization. Hybridization was performed using EASY HYB (manufactured by Boehringer-Mannheim). After pre-hybridizing the filter at 50 ° C. for 1 hour, a digoxinigen-labeled probe, prepared as described above, was added, and hybridization was carried out at 50 ° C. for 16 hours. The filter was then washed twice at 60 ° C with 1 x SSC containing 0.1% SDS.

The bands that hybridized with the probe were detected using the DIG Nucleotide Detection Kit (Boehringer-Mannheim) based on the procedure described in his manual. As a result, a band of 7 so-called which hybridized with the probe was successfully detected for the hydrolyzed PstI product and a band of 2 so-called which hybridized with the probe was successfully detected for the hydrolyzed HincII product.

5 μg of Cyclobacterium marinum ATCC 25205 chromosomal DNA (depository institution; American collection of crop types, depository institution address; PO Box 1549, Manassas, VA 20110, United States of America) was completely hydrolyzed using PstI or HincII. DNA approximately 7 so-called or 2 so-called separated by electrophoresis in 0.8% agarose gel. DNA was purified using the Gene Clean II Kit (manufactured by Funakoshi) and dissolved in 10 μl of TE. 4 μl of the resulting solution was mixed with pUC118 PstI / BAP (manufactured by Takara Shuzo) or pUC118 HincII / BAP (manufactured by Takara Shuzo) and a ligation reaction was performed using DNA Ligation Kit Ver. 2 (manufactured by Takara Shuzo). 5 μl of this ligation reaction fluid and 100 μl of competent Escherichia coli JM109 cells (manufactured by Takara Shuzo) were mixed to transform Escherichia coli . The contents were then applied to a suitable solid medium to obtain a chromosomal DNA library.

To obtain the full-length gene of the peptide-forming enzyme, the chromosomal DNA library was screened by colony hybridization using the probe indicated above. The procedure for colony hybridization is explained in Molecular Cloning, 2nd edition, Cold Spring Harbor Press (1989).

Colonies of the chromosomal DNA library were transferred onto a Nylon Membrane Filter for Colony and Plaque Hybridization (manufactured by Roche Diagnostics), followed by alkaline denaturation, neutralization and immobilization. Hybridization was performed using EASY HYB (manufactured by Boehringer-Mannheim). After pre-hybridizing the filter at 37 ° C for 1 hour, the above digoxinigen-labeled probe was added, followed by hybridization at 37 ° C for 16 hours. The filter was then washed twice at 60 ° C with 1 x SSC containing 0.1% SDS.

Colonies that hybridized with a labeled probe were detected using the DIG Nucleotide Detection Kit (manufactured by Boehringer-Mannheim) based on the explanation described in his manual. As a result, one strain was identified whose colony hybridized with a labeled probe.

(5) The base sequence of the gene forming the peptide of the enzyme derived from Cyclobacterium marinum ATCC 25205

Plasmids contained in Escherichia coli JM109 were obtained from each strain, which was confirmed to hybridize with a labeled probe, and the base sequence of the part where hybridization with the probe took place and in the vicinity thereof was determined. The sequencing reaction was carried out using the CEQ DTCS-Quick Start Kit (manufactured by Beckman-Coulter) based on the procedure described in its manual. In addition, electrophoresis was performed using CEQ 2000-XL (manufactured by Beckman-Coulter).

As a result, it was found that an open-reading frame that encodes a peptide-forming enzyme does exist. The full-length base sequence of the gene of the peptide-forming enzyme derived from Cyclobacterium marinum ATCC 25205 (depository institution; American collection of crop types, depository institution address; PO Box 1549, Manassas, VA 20110, United States of America), along with the corresponding amino acid sequence, is shown in SEQ ID NO: 24.

Example 12

Isolation of a gene forming a peptide enzyme derived from Psychroserpens burtonensis ATCC 700359

The following describes the isolation of a gene forming a peptide enzyme. The microorganism used was Psychroserpens burtonensis ATCC 700359 (Depository Institution; American Type Culture Collection, Depository Institution Address; PO Box 1549, Manassas, VA 20110, United States of America). To isolate the gene, Escherichia coli JM109 was used as the host, and pUC118 was used as the vector.

(1) Obtaining cells of a microorganism

Psychroserpens burtonensis ATCC 700359 (depository institution; American collection of crop types, depository institution address; PO Box 1549, Manassas, VA 20110, United States of America) was cultured for 24 hours at 25 ° C. on CM2G agar medium (containing 50 g / l of glucose, 10 g / l of yeast extract, 10 g / l of peptone, 5 g / l of sodium chloride and 20 g / l of agar, pH 7.0). One loop of the obtained microorganism cells was inoculated into a 500 ml Sakagushi vessel containing 50 ml of CM2G liquid medium (the above medium, except for agar), followed by cultivation with shaking at 10 ° C.

(2) Obtaining chromosomal DNA from microorganism cells

50 ml of culture broth was centrifuged (12000 rpm, 4 ° C, 15 minutes) to collect microorganism cells. Then, chromosomal DNA was obtained from microorganism cells using the Qiagen Genomic-Tip System (Qiagen) based on the procedure described in its manual.

(3) Obtaining a DNA fragment of a probe by PCR

A DNA fragment containing a portion of the gene of the peptide-forming enzyme originating from Psychroserpens burtonensis ATCC 700359 (depository institution; American collection of crop types, depository institution address; PO Box 1549, Manassas, VA 20110, United States of America) was obtained using the method PCR using LA-Taq (manufactured by Takara Shuzo). Then, a PCR reaction was performed on chromosomal DNA obtained from Psychroserpens burtonensis ATCC 700359 (depository institution; American collection of culture types, depository institution address; PO Box 1549, Manassas, VA 20110, United States of America) using primers having base sequences SEQ ID NOs: 15 and 16. A DNA fragment of approximately 1 kb amplified by PCR was isolated by electrophoresis in 0.8% agarose. The desired strip was then cut and cleaned. The DNA fragment was labeled with a digoxinigen probe using DIG High Prime (manufactured by Boehringer-Mannheim) based on the procedure described in his manual.

(4) Cloning a gene of a peptide-forming enzyme from a gene library

To obtain the full-sized gene of the peptide-forming enzyme, Southern hybridization was performed using a DNA fragment amplified in the above PCR procedure as a probe. Southern hybridization operations are explained in Molecular Cloning, 2nd edition, Cold Spring Harbor Press (1989).

After the interaction of the Psychoserpens burtonensis ATCC 700359 chromosomal DNA with the EcoRI restriction enzyme at 37 ° C for 16 hours until the complete hydrolysis of the DNA, the obtained one was subjected to electrophoresis on a 0.8% agarose gel. After electrophoresis, the chromosomal DNA separated by electrophoresis from the agarose gel was transferred to a positively charged nylon membrane filter (manufactured by Roche Diagnostics), followed by treatment consisting of alkaline denaturation, neutralization and immobilization. Hybridization was performed using EASY HYB (manufactured by Boehringer-Mannheim). After pre-hybridizing the filter at 50 ° C. for 1 hour, a digoxinigen-labeled probe, prepared as described above, was added, and hybridization was carried out at 50 ° C. for 16 hours. The filter was then washed twice at 60 ° C with 1 x SSC containing 0.1% SDS.

The bands that hybridized with the probe were detected using the DIG Nucleotide Detection Kit (Boehringer-Mannheim) based on the procedure described in his manual. As a result, a band of approximately 7 so-called, which hybridized with the probe, was successfully detected.

5 μg of Psychroserpens burtonensis ATCC 700359 chromosomal DNA was completely hydrolyzed using EcoRI. DNA approximately 7 so-called was separated by electrophoresis in 0.8% agarose gel, DNA was purified using Gene Clean II Kit (manufactured by Funakoshi) and dissolved in 10 μl of TE. 4 μl of the resulting solution was mixed with pUC118 EcoRI / BAP (manufactured by Takara Shuzo) and a ligation reaction was performed using DNA Ligation Kit Ver. 2 (manufactured by Takara Shuzo). 5 μl of this ligation reaction fluid and 100 μl of competent Escherichia coli JM109 cells (manufactured by Takara Shuzo) were mixed to transform Escherichia coli. The contents were then applied to a suitable solid medium to obtain a chromosomal DNA library.

To obtain the full-length gene of the peptide-forming enzyme, the chromosomal DNA library was screened by colony hybridization using the probe indicated above. The procedure for colony hybridization is explained in Molecular Cloning, 2nd edition, Cold Spring Harbor Press (1989).

Colonies of the chromosomal DNA library were transferred onto a Nylon Membrane Filter for Colony and Plaque Hybridization (manufactured by Roche Diagnostics), followed by alkaline denaturation, neutralization and immobilization. Hybridization was performed using EASY HYB (manufactured by Boehringer-Mannheim). After pre-hybridizing the filter at 37 ° C for 1 hour, the above digoxinigen-labeled probe was added, followed by hybridization at 37 ° C for 16 hours. The filter was then washed twice at 60 ° C with 1 x SSC containing 0.1% SDS.

Colonies that hybridized with a labeled probe were detected using the DIG Nucleotide Detection Kit (manufactured by Boehringer-Mannheim) based on the explanation described in his manual. As a result, one strain was identified whose colony hybridized with a labeled probe.

(5) The base sequence of the gene forming the peptide enzyme derived from Psychroserpens burtonensis ATCC 700359

The plasmids contained in Escherichia coli JM109 were obtained from a strain that was confirmed to hybridize with a labeled probe, and the base sequence of the part where hybridization with the probe and in the vicinity thereof was determined was determined. The sequencing reaction was carried out using the CEQ DTCS-Quick Start Kit (manufactured by Beckman-Coulter) based on the procedure described in its manual. In addition, electrophoresis was performed using CEQ 2000-XL (manufactured by Beckman-Coulter).

As a result, it was found that an open-reading frame that encodes a peptide-forming enzyme does exist. The full-length base sequence of the gene for the peptide-forming enzyme derived from Psychroserpens burtonensis ATCC 700359 (depository institution; American collection of crop types, depository institution address; PO Box 1549, Manassas, VA 20110, United States of America), along with the corresponding amino acid sequence, is shown in SEQ ID NO: 26.

Although the invention has been described with respect to a specific implementation for a complete and clear disclosure, the appended claims are not limited thereto, and should be construed as including all modifications and alternative designs that can be made by a person skilled in the art and which clearly fall within the basic doctrine.

The list of sequences in the public domain

SEQ ID NO: 3: Synthetic Primer 1

SEQ ID NO: 4: Synthetic Primer 2

SEQ ID NO: 5: Gene Encoding a Peptide-Forming Enzyme

SEQ ID NO: 7: Synthetic primer for pTrpT

SEQ ID NO: 8: Synthetic primer for pTrpT

SEQ ID NO: 9: Synthetic primer to obtain pTrpT_Gtg2

SEQ ID NO: 10: Synthetic Primer for pTrpT_Gtg2

SEQ ID NO: 11: Gene Encoding a Peptide-Forming Enzyme

SEQ ID NO: 13: Synthetic primer to obtain pTrpT_Sm_aet

SEQ ID NO: 14: Synthetic primer to obtain pTrpT_Sm_aet

SEQ ID NO: 15: Mixed Primer 1 for Aet

SEQ ID NO: 16: Mixed Primer 2 for Aet

SEQ ID NO: 19: Primer 1 for the construction of expression vectors originating from pPedobacter

SEQ ID NO: 20: Primer 2 for the construction of expression vectors originating from pedobacter Pedobacter

SEQ ID NO: 21: Mixed Primer 3 for Aet

Claims (5)

1. The method of producing α-L-aspartyl-L-phenylalanine-β-ester, comprising contacting the α, β-diester of L-aspartic acid and L-phenylalanine in the presence of an enzyme, the amino acid sequence of which is selected from the group of sequences which consists of: a) the sequence formed by residues 23 to 616 of the amino acid sequence shown in SEQ ID NO: 6; b) the sequence formed by residues 21 to 619 of the amino acid sequence shown in SEQ ID NO: 12; C) the sequence formed by residues 23 to 625 of the amino acid sequence shown in SEQ ID NO: 18; g) the sequence formed by residues 23 to 645 of the amino acid sequence shown in SEQ ID NO: 23; d) the sequence formed by residues 26 to 620 of the amino acid sequence shown in SEQ ID NO: 25; e) the sequence formed by residues 18 to 644 of the amino acid sequence shown in SEQ ID NO: 27; g) the sequence shown in SEQ ID NO: 6; h) the sequence shown in SEQ ID NO: 12; i) the sequence shown in SEQ ID NO: 18; k) the sequence shown in SEQ ID NO: 23; k) the sequence shown in SEQ ID NO: 25; m) the sequence shown in SEQ ID NO: 27; m) sequences (a) through (m), including a substitution, deletion, insertion, addition and / or inversion of one or a number of amino acids, in which the enzyme is able to selectively attach L-phenylalanine to the α-ether portion of the α, β-diester L aspartic acid via a peptide bond, or a product containing the specified enzyme, where contacting is carried out at 5-40 ° C and pH 7-10 for a time sufficient to form α-L-aspartyl-L-phenylalanine-β-ester from α, β-diester of L-aspartic acids and L-phenylalanine. 2. The method according to claim 1, where the agent containing the enzyme is selected from the group comprising culture, a microorganism cell and a processed cell product of the specified microorganism. 3. The method according to claim 2, where the microorganism is a microorganism belonging to the genus selected from the group consisting of Aeromonas, Azotobacter, Alcaligenes, Brevibacterium, Corynebacterium, Escherichia, Empedobacter, Flavobacterium, Microbacterium, Propionibacterium, Brevibacillus, Paenibacillus, Pseudomonasomphisomphisomphisomphisomphisomphida, Somphida syomphida Weeksella, Pedobacter, Persicobacter, Flexithrix, Chitinophaga, Cyclobacterium, Runella, Thermonema, Psychroserpens, Gelidibacter, Dyadobacter, Flammeovirga, Spirosoma, Flectobacillus, Tenacibaciloberibecobacta, Jerome, Bloomegibacteribactum, Rhodotermus, Zobelliabilobecomibica, Murcia . 4. The method according to claim 2, where the microorganism is a microorganism transformed with a vector that provides the expression of the specified enzyme in the microorganism. 5. A method of producing α-L-aspartyl-L-gphenylalanine-α-methyl ester, comprising the step of synthesizing α-L-aspartyl-L-phenylalanine-β-methyl ester using the method according to any one of claims 1 to 4 and a conversion step α-L-aspartyl-L-phenylalanine-β-methyl ether to α-L-aspartyl-L-phenylalanine-α-methyl ether. Priority according to claims 1-5 in part (a), (b), (g), (h), (n) in part SEQ ID NO: 6 and 12, and also in item 3 in part Aeromonas, Azotobacter, Alcaligenes, Brevibacterium, Corynebacterium, Escherichia, Empedobacter, Flavobacterium, Microbacterium, Propionibacterium, Brevibacillus, Paenibacillus, Pseudomonas, Serratia, Stenotrophomonas, Sphingobacterium, Strehomomochas, Xphomida, Xphomida, Xippomaces, Xpomochida, Xippomaces, filing date JP 2003-016764). Priority according to claims 1-5 in part (c), (d), (e), (e), (i), (k), (l), (m), (n) in part SEQ ID NO: 18, 23, 25 and 27, and also according to claim 3, in terms of Cellulophaga, Weeksella, Pedobacter, Persicobacter, Flexithrix, Chitinophaga, Cyclobacterium, Runella, Thermonema, Psychroserpens, Gelidibacter, Dyadobacter, Flammeovirga, Spirosoma, Flectobaculus Ten Zobellia, Muricauda, Salegentibacter, Taxeobacter, Cytophaga, Marinilabilia, Lewinella, Saprospira, and Haliscomenobacter was established on July 25, 2003 (according to the filing date of JP 2003-201819).

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