JP2018102287A - Proteins having imidazole dipeptide synthesis activity and imidazole dipeptide production method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To address the problem of simply providing a low cost imidazole dipeptide, to thereby provide proteins that produce imidazole dipeptides with high efficiency such as carnosine, anserine and balenine, and a production method using same.SOLUTION: A modified protein comprises an L-amino acid α-ligase illustrated by sequence number 1 in which at least 1-3 amino acid residues of an amino acid sequence of YwfE are substituted wherein the asparagine (N) residue at position 108 from the N terminal is substituted for an alanine (A) residue, glutamic acid (E) residue or glutamine (Q) residue, or additionally, the isoleucine (I) residue at position 112 is substituted for a valine (V) residue, and/or the histidine (H) residue at position 378 is substituted for a lysine (K) residue or an arginine (R) residue.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a protein having synthetic activity for imidazole dipeptides such as carnosine, anserine, and valenin, and a method for producing the imidazole dipeptide.

  Imidazole dipeptide is a generic term for peptides to which amino acid residues containing an imidazole group are bonded, and includes carnosine (β-alanyl-L-histidine), anserine (β-alanyl-3-methyl-L-histidine) and Examples include dipeptides containing histidine residues or derivatives thereof such as valenine (Nα-β-alanyl-1-methyl-L-histidine). These dipeptides are abundant in muscles of marine organisms that travel long distances, such as chicken breasts, tuna, skipjack, and whales that fly over long distances, and have anti-fatigue effects, active oxygen scavenging ability, and blood pressure lowering effects. Anti-inflammatory action, uric acid level effect action, and the like are known, and these imidazole dipeptides extracted from muscles of domestic animals such as chickens are used as supplements (Non-Patent Documents 1 to 3). However, a simple and low-cost production method for carnosine, anserine and valenin has not yet been established.

Song B. Et al., Nutr Res Pract. 2014, 8: 3-10 Bellia F.M. Molecules 2014, 19: 2299-2329. Boldyrev A.R. A. Et al., Physiol Rev. 2013, 93: 1803-45

  The present invention provides a protein capable of producing carnosine, anserine and valenine imidazole dipeptides with high efficiency and a gene encoding the protein for the purpose of providing these imidazole dipeptides simply and at low cost. It is an object of the present invention to provide a method for producing the imidazole peptide using the introduced host cell and the protein.

  The present inventor noted that L-amino acid α-ligase is an enzyme that is coupled to the hydrolysis reaction of ATP and catalyzes peptide synthesis by forming an α peptide bond using an unprotected amino acid as a substrate, A mutant enzyme introduced with a site-specific mutation of protein YwfE derived from Bacillus and having L-amino acid α-ligase activity is evaluated, its imidazole dipeptide synthesis activity is evaluated, and a specific site is substituted with a specific amino acid residue The present inventors have found that the mutated enzyme has strong imidazole peptide synthesis activity, particularly carnosine synthesis activity, anserine synthesis activity and / or valenine synthesis activity, and completed the present invention.

Specifically, the present invention is a mutant protein comprising a substitution of at least 1 to 3 amino acid residues in the amino acid sequence of L-amino acid α-ligase: YwfE represented by SEQ ID NO: 1,
An asparagine (N) residue at position 108 from the N-terminus is substituted with an alanine (A) residue, a glutamic acid (E) residue or a glutamine (Q) residue; or
The isoleucine (I) residue at position 112 is replaced with a valine (V) residue and / or the histidine (H) residue at position 378 is replaced with a lysine (K) residue or an arginine (R) residue A mutant protein is provided.

  The mutant protein of the present invention may have L-amino acid α-ligase activity.

The mutant protein of the present invention is
(I) a protein having the amino acid sequence of SEQ ID NOs: 2 to 16,
(Ii) a protein having 80% or more homology with the amino acid sequences of SEQ ID NOs: 2 to 16 and having L-amino acid α-ligase activity;
(Iii) It consists of an amino acid sequence in which one or more amino acid residues of the amino acid sequences of SEQ ID NOs: 2 to 16 are deleted, substituted, inserted, and / or added, and has L-amino acid α-ligase activity May be a protein.

  In the present invention, the L-amino acid α-ligase activity may be selected from carnosine synthesis activity, anserine synthesis activity and / or valenine synthesis activity.

In the present invention, when the L-amino acid α-ligase activity is carnosine synthesis activity, the mutant protein is:
(I) a protein having an amino acid sequence of SEQ ID NOs: 2 to 4, 6, 7, 9, 10, 12 to 16,
(Ii) a protein having 80% or more homology with the amino acid sequences of SEQ ID NOs: 2 to 4, 6, 7, 9, 10, 12 to 16 and having a carnosine synthesis activity;
(Iii) consisting of an amino acid sequence in which one or more amino acid residues of the amino acid sequences of SEQ ID NOs: 2 to 4, 6, 7, 9, 10, 12 to 16 are deleted, substituted, inserted, and / or added In some cases, the protein has carnosine synthesis activity.

In the present invention, when the L-amino acid α-ligase activity is anserine synthesis activity, the mutant protein is:
(I) a protein having an amino acid sequence of SEQ ID NOs: 2, 3, 5, 6, 8 to 16,
(Ii) a protein having 80% or more homology with the amino acid sequences of SEQ ID NOs: 2, 3, 5, 6, 8 to 16 and having anserine synthesis activity;
(Iii) It consists of an amino acid sequence in which one or more amino acid residues of the amino acid sequences of SEQ ID NOs: 2, 3, 5, 6, 8 to 16 are deleted, substituted, inserted and / or added, and anserine A protein having synthetic activity,
The mutant protein according to claim 4, wherein

In the present invention, when the L-amino acid α-ligase activity is a valenine synthesis activity, the mutant protein is:
(I) a protein having the amino acid sequence of SEQ ID NOs: 6, 7, 11, 14-16,
(Ii) a protein having 80% or more homology with the amino acid sequences of SEQ ID NOs: 6, 7, 11, 14 to 16 and having a valenin synthesis activity,
(Iii) It consists of an amino acid sequence in which one or more amino acid residues of the amino acid sequences of SEQ ID NOs: 6, 7, 11, 14 to 16 are deleted, substituted, inserted and / or added, and has a valenin synthesis activity In some cases.

  The present invention provides a nucleic acid encoding the mutant protein of the present invention.

  The present invention provides a vector into which a polynucleotide containing the nucleic acid of the present invention has been inserted.

  The present invention provides a host cell comprising at least one vector of the present invention.

  The present invention provides a method for producing a dipeptide using the mutant protein of the present invention.

  In the production method of the present invention, the protein of the present invention may be used together with a peptidase activity inhibitor.

  In the production method of the present invention, the host cell of the present invention may be used.

  In the production method of the present invention, the host cell may be a peptidase-deficient cell.

  In the production method of the present invention, the peptidase may be peptidase D.

  In the production method of the present invention, the defective cell may be JW0227 strain.

  In the production method of the present invention, the dipeptide may be an imidazole dipeptide.

  In the production method of the present invention, the imidazole dipeptide may be carnosine, anserine and / or valenin.

  By providing a protein that produces imidazole dipeptide with high efficiency and a host cell containing the protein, imidazole dipeptides containing these carnosine, anserine, and valenin can be provided simply and at low cost. Further, by using peptidase-deficient cells as host cells, imidazole dipeptides, particularly carnosine, can be produced with high efficiency.

The figure showing the result of having evaluated the carnosine synthesis activity of the mutant type enzyme containing wild type YwfE and site-specific mutant type YwfE. In the figure, * indicates that the p value is less than 0.05, ** indicates that the p value is less than 0.01, and *** indicates that the p value is less than 0.001. It shows that. The figure showing the result of having evaluated the anserine synthetic | combination activity of the mutant enzyme containing wild type YwfE and site-specific mutant YwfE. In the figure, * indicates that the p value is less than 0.05, ** indicates that the p value is less than 0.01, and *** indicates that the p value is less than 0.001. It shows that. The figure showing the result of having evaluated the valenine synthetic activity of the mutant type enzyme containing wild type YwfE and site-specific mutant type YwfE. In the figure, * indicates that the p value is less than 0.05, ** indicates that the p value is less than 0.01, and *** indicates that the p value is less than 0.001. It shows that. The figure showing the result of having evaluated the influence of the peptidase in carnosine synthesis.

1. Protein of the present invention One embodiment of the present invention is a mutant enzyme of protein YwfE derived from Bacillus subtilis, a bacterium belonging to the genus Bacillus, and having L-amino acid α-ligase activity.

  In the present specification, the L-amino acid α-ligase activity is imidazole dipeptide synthesis activity, and examples of the imidazole dipeptide include carnosine, anserine, and valenin.

  In the mutant enzyme, the asparagine (N) residue at position 108 from the N-terminus of wild-type YwfE is substituted with an alanine (A) residue, glutamic acid (E) residue or glutamine (Q) residue, or The leucine (I) residue at position 112 is replaced with a valine (V) residue and / or the histidine (H) residue at position 378 is replaced with a lysine (K) residue or an arginine (R) residue. By doing so, the ligase activity for an amino acid whose N-terminal substrate is β-alanine and whose C-terminal substrate is histidine or a derivative thereof is improved.

  In the present specification, protein sequences are described in the three-letter or one-letter notation of amino acids well known to those skilled in the art. In the present specification, amino acids are in L form unless otherwise specified. In addition, when a mutant protein is represented, a one-letter code of an amino acid into which a wild-type protein mutation is introduced, a number representing the mutation position, and a one-letter code of the mutated amino acid, which are methods commonly used by those skilled in the art, are shown. used. In the present specification, unless otherwise specified, an amino acid represents an L-form.

Specifically, the mutant enzyme which is the protein of the present invention is a mutation comprising substitution of at least 1 to 3 amino acid residues in the amino acid sequence of L-amino acid α-ligase: YwfE represented by SEQ ID NO: 1. Protein,
An asparagine (N) residue at position 108 from the N-terminus is substituted with an alanine (A) residue, a glutamic acid (E) residue or a glutamine (Q) residue; or
The isoleucine (I) residue at position 112 is replaced with a valine (V) residue and / or the histidine (H) residue at position 378 is replaced with a lysine (K) residue or an arginine (R) residue A mutant protein is provided.

That is, the protein of the present invention is
(I) a protein having the amino acid sequence of SEQ ID NOs: 2 to 16,
(Ii) 80% or more, preferably 85% or more, more preferably 90% or more, most preferably 95% or more of homology with the amino acid sequence of SEQ ID NO: 2 to 16, and L-amino acid α-ligase Active protein,
(Iii) It consists of an amino acid sequence in which one or more amino acid residues of the amino acid sequences of SEQ ID NOs: 2 to 16 are deleted, substituted, inserted, and / or added, and has L-amino acid α-ligase activity It may be a protein.

  The L-amino acid α-ligase activity is preferably dipeptide synthesis activity, and more preferably carnosine (L-Carnosine) synthesis activity, anserine (L-Anserine) synthesis activity and / or valenine (L-Balenine). Synthetic activity.

  In the present specification, the “as long as it has L-amino acid α-ligase activity” refers to an activity equivalent to or improved from the L-amino acid α-ligase activity of wild-type YwfE. A protein that exhibits improved L-amino acid α-ligase activity compared to the L-amino acid α-ligase activity of wild-type YwfE when the protein activity is compared under the same conditions except for the difference in the test protein.

  In the present specification, L-amino acid α-ligase activity, in particular, dipeptide synthesis activity is evaluated, for example, in a buffered aqueous solution containing, for example, a test protein, a substrate amino acid, and ATP at a pH of 5 to 11, for example, 20 to 50 ° C. Incubation at a predetermined temperature of, for example, 2 to 150 hours, increasing the amount or concentration of a dipeptide produced by the incubation, decreasing the amount or concentration of a substrate amino acid, decreasing the amount or concentration of ATP, ADP Measured using, for example, high performance liquid chromatography with at least one of an increase in the amount or concentration of inorganic phosphate or an increase in the amount or concentration of inorganic phosphate as an index, and the conditions other than the test protein are measured under the same conditions Can be carried out by comparing with the dipeptide synthesis activity of wild-type YwfE.

In addition, in the present specification, “a protein in which an amino acid residue corresponding to the Y-th Z ₁ residue (Z ₁ ) from the N-terminus of SEQ ID NO: X is substituted with a Z ₂ residue (Z ₂ )” (“X” And “Y” represents an integer of 1 or more, “Z ₁ ” and “Z ₂ ” represent any amino acid, and “(Z ₁ )” and “(Z ₂ )” represent each amino acid in one letter. Is equivalent to the Y-th position from the N-terminal of the amino acid sequence of SEQ ID NO: X when the amino acid sequence of the protein and SEQ ID NO: X are aligned so as to give the highest homology score Z ₁ residues refers to a protein having the amino acid sequence substituted with Z ₂ residues.

  Below, the characteristic of the protein of this invention is demonstrated in detail.

  Although the wild-type protein YwfE, which is derived from Bacillus and has L-amino acid α-ligase activity, has a synthetic activity of a dipeptide whose C-terminal is L-histidine (Tabata K. et al., J. Bacteriol., 2005, 187: 5195-5202), and does not possess strong α-ligase activity for the production of carnosine, which is a dipeptide having β-alanine at the N-terminus and histidine at the C-terminus, and derivatives thereof, anserine and valenin (see FIG. 2). . That is, strong carnosine synthesizing activity, strong anserine synthesizing activity, and valenine synthesizing activity are brought about by making a mutant of the wild type protein of YwfE.

  In the expression of L-amino acid α-ligase activity of the protein of the present invention, Ala and Gly are used as the N-terminal substrate, and His is used as the C-terminal substrate. When His is used, the protein of the present invention shows stronger activity, and the protein of the present invention has a strong selective activity particularly for the synthesis of carnosine or its derivatives, anserine or valenin. Optimal mutant enzymes for the synthesis of carnosine or its derivatives, anserine or valenin, do not necessarily match.

  Based on the characteristics obtained from the single amino acid mutant protein of the present invention, the present invention is designed with a mutant enzyme in which two or three amino acid residues are substituted, and a double mutant enzyme or triple mutant enzyme. A protein is provided by the present invention.

  Carnosine synthesizing activity, anserine synthesizing activity in many of the double mutant enzyme or triple mutant enzyme by mutation of N108A, N108E or N108Q, mutation of I112V, mutation of H378K or H378R, and combinations of these mutations, The activity of synthesizing valenine is improved over the activities of wild type YwfE, but not all activities are improved as well.

  That is, when the example of the L-amino acid α-ligase activity of the protein of the present invention is carnosine synthesis activity, the protein is alanine (of the 108th asparagine (N) residue from the N-terminus of SEQ ID NO: 1. A) Substitution with an amino acid residue selected from a residue, glutamic acid (E) residue or glutamine (Q) residue; valine (V) of isoleucine (I) residue at position 112 from the N-terminus of SEQ ID NO: 1 And / or substitution of the 378 th histidine (H) residue of SEQ ID NO: 1 with a lysine (K) residue or an arginine (R) residue.

  In addition, when the example of the L-amino acid α-ligase activity of the protein of the present invention is anserine synthesis activity, the protein is an alanine (N) residue at the 108th asparagine (N) residue from the N-terminus of SEQ ID NO: 1. A) Substitution with an amino acid residue selected from a residue, glutamic acid (E) residue or glutamine (Q) residue; valine (V) of isoleucine (I) residue at position 112 from the N-terminus of SEQ ID NO: 1 And / or substitution of the 378 th histidine (H) residue of SEQ ID NO: 1 with a lysine (K) residue or an arginine (R) residue.

  Furthermore, when the example of the L-amino acid α-ligase activity of the protein of the present invention is a valenine synthesis activity, the protein is an alanine (A) of the 108th asparagine (N) from the N-terminus of SEQ ID NO: 1. Substitution with an amino acid residue selected from a residue or glutamine (Q) residue; substitution of an isoleucine (I) residue at position 112 from the N-terminus of SEQ ID NO: 1 with a valine (V) residue; and / or Alternatively, it is preferable to include substitution of the 378th histidine (H) residue of SEQ ID NO: 1 with a lysine (K) residue or an arginine (R) residue.

  The mutant protein of YwfE is, for example, site-directed mutagenesis by conventional PCR well-known to those skilled in the art, using a DNA having the nucleic acid sequence shown in SEQ ID NOs: 33 to 44 using the DNA shown in SEQ ID NO: 17 as a template. And a vector into which a polynucleotide containing a nucleic acid encoding the protein of SEQ ID NOs: 2 to 16 of the present invention described below is inserted (hereinafter referred to as “nucleic acid insertion vector”), and the nucleic acid insertion vector further introduced below A host cell containing the nucleic acid insertion vector of the present invention (hereinafter sometimes referred to as “transformed cell”), culturing the host cell containing the transformed cell in an appropriate medium, and transformant By using ordinary enzyme isolation and purification methods such as salt precipitation, gel chromatography, and gel electrophoresis, the protein produced by Away purified, it is possible to produce a mutant enzyme.

  The DNA used as the template is, for example, a probe designed from a partial sequence of the polynucleotide sequence of SEQ ID NO: 17, and Southern hybridization to a chromosomal DNA of a microorganism encoding a YwfE protein such as Bacillus subtilis 168 or a similar protein. A full-length polynucleotide of SEQ ID NO: 17 can be obtained by a method or the like.

  In addition, the double mutant enzyme uses the nucleic acid insertion vector into which the one-mutated site-specific mutation used in the production of the one-mutated enzyme is used as a template, and further the nucleic acid sequence shown in SEQ ID NOs: 33 to 44. After site-directed mutagenesis by conventional PCR well known to those skilled in the art, and after culturing a host cell containing a double-mutated nucleic acid vector in an appropriate medium, similar to the production of a single mutant enzyme It can be produced by isolating and purifying the double mutant enzyme.

  Further, the triple mutant enzyme has a nucleic acid sequence shown in SEQ ID NOs: 33 to 44, using as a template a nucleic acid insertion vector into which a two-mutated site-specific mutation used in the production of the two-mutated enzyme is introduced. Using a primer, site-directed mutagenesis is carried out by conventional PCR well-known to those skilled in the art, and in the same manner as in the production of the two-mutant enzyme, host cells containing a nucleic acid vector into which a triple mutation has been introduced are cultured in an appropriate medium, It can be produced by isolating and purifying the mutant enzyme.

  The protein of the present invention is used as an enzyme for producing carnosine, anserine, or valenin, and further, a transformed cell into which a nucleic acid encoding the amino acid sequence of the protein is introduced is prepared, and the cell of the transformed cell is produced. It is used for the production of carnosine, anserine and / or valenin by culturing.

2. Nucleic acids of the invention Another embodiment of the invention is a nucleic acid,
(I) a protein having the amino acid sequence of SEQ ID NOs: 2 to 16,
(Ii) 80% or more, preferably 85% or more, more preferably 90% or more, most preferably 95% or more of homology with the amino acid sequence of SEQ ID NO: 2 to 16, and L-amino acid α-ligase Active protein,
(Iii) It consists of an amino acid sequence in which one or more amino acid residues of the amino acid sequences of SEQ ID NOs: 2 to 16 are deleted, substituted, inserted, and / or added, and has L-amino acid α-ligase activity Mention may be made of nucleic acids encoding proteins.

  The nucleic acid is, for example, a primer designed from the polynucleotide sequence of SEQ ID NO: 17, and a site-directed mutagenesis method using a chromosomal DNA of a microorganism encoding a YwfE protein such as Bacillus subtilis 168 or a similar protein as a template. Obtained by introducing site-specific mutations.

  More specifically, the targeted mutation introduction into the template gene is basically based on PCR amplification using the polynucleotide of SEQ ID NO: 17 as the template DNA and replication reactions with various DNA polymerases. Can be performed using any site-directed mutagenesis method. The site-directed mutagenesis method can be performed by any method such as PCR method or annealing method (edited by Muramatsu et al., “Revised 4th edition, New Genetic Engineering Handbook”, Yodosha, p. 82-88). it can. Use various commercially available site-specific mutagenesis kits such as QuickChange II Site-Directed Mutagenesis Kit (Stratagene, USA) and QuickChange Multi Site-Directed Mutagenesis Kit (Agilent Technologies, USA) as necessary You can also.

  The template DNA containing the YwfE gene can be prepared by extracting genomic DNA from bacteria producing the YwfE protein by a conventional method, or extracting RNA and synthesizing cDNA by reverse transcription. Bacteria that produce YwfE protein have been reported in plants and animals in addition to bacteria including Bacillus subtilis, Bacillus subtilis, Clostridium bacteria, Acid Thermus bacteria, but Bacillus subtilis, etc. The Bacillus bacterium is preferred and can be easily obtained by those skilled in the art. For example, KSM-S237 strain (Accession No. FERM BP-7875), KSM-64 strain (Accession No. FERM BP-2886), and KSM-635 strain (Accession No. FERM BP-1485) of Bacillus Sp. Deposited at the Research Center for Biological Biology (1st, 1st, 1st, 1st East, Tsukuba City, Ibaraki Prefecture, Japan) based on the accession number written together.

  Preparation of genomic DNA from these Bacillus bacteria is described, for example, in Pitcher et al. Lett. Appl. Microbiol. 1989, 8: p. 151-156. The template DNA containing the YwfE gene may be prepared by inserting a DNA fragment containing the YwfE gene excised from the prepared cDNA or genomic DNA into an arbitrary vector.

  Introduction of a site-specific mutation into the YwfE gene can be most commonly performed using a mutation primer containing a nucleotide mutation to be introduced. Such a mutation primer is annealed to a region containing a nucleotide sequence encoding the amino acid residue to be replaced in the YwfE gene, and after substitution in place of the nucleotide sequence (codon) encoding the amino acid residue to be replaced. What is necessary is just to design so that the base sequence which has a nucleotide sequence (codon) which codes amino acid residue may be included.

  Using the nucleic acid obtained by these methods, preparation of a vector for expressing the following protein of the present invention in a host cell, preparation of a host cell into which the vector is introduced, that is, a transformed cell, and the above-described book It can be used to produce the protein of the invention.

3. A vector into which a polynucleotide containing a nucleic acid encoding the protein of the present invention is inserted (nucleic acid insertion vector)
Another embodiment of the present invention is a nucleic acid insertion vector comprising a gene encoding the above-described protein of the present invention and an appropriate expression sequence. Expression vectors for producing these recombinant DNAs are commercially available. By obtaining and utilizing these vectors, the nucleic acid insertion vector of the present invention can be prepared and used for the preparation of a host cell containing the following nucleic acid insertion vector, that is, a transformed cell.

  As a vector, for example, when Escherichia coli is used as a host cell, pColdI (manufactured by Takara Bio Inc.), pCDF-1b, pRSF-1b (all manufactured by Novagen), pMAL-c2x (manufactured by New England Biolabs) PGEX-4T-1 (manufactured by GE Healthcare Bioscience), pTrcHis (manufactured by Invitrogen), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-30 (manufactured by Qiagen), pET- 3 (manufactured by Novagen), pBluescript II SK (+), pBluescript II KS (−) (manufactured by Stratagene), pTrS30 [prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], and the like. Yes.

  As the promoter in the case of using the vector, any promoter can be used as long as it functions in a host cell such as Escherichia coli. For example, trp promoter (Ptrp), lac promoter (Plac), PL promoter, PR promoter A promoter derived from Escherichia coli, phage, or the like, such as a PSE promoter, can be used. When a microorganism belonging to the genus Bacillus is used as a parent strain, an SPO1 promoter, an SPO2 promoter, a penP promoter, etc. that function in Bacillus subtilis can also be used. In addition, artificially designed and modified promoters such as a promoter in which two Ptrps are connected in series, a tac promoter, a lacT7 promoter, and a let I promoter can be used.

  For the purpose of producing the protein of the present invention, an expression vector is particularly useful when a vector is used. The expression vector is not particularly limited as long as it is a vector that expresses a protein in vitro, in E. coli, in cultured cells, or in an individual organism. For example, in the case of in vitro expression, pBEST vector (manufactured by Promega), E. coli If present, pET vector (manufactured by Invitrogen), pME18S-FL3 vector (GenBank Accession No. AB009864) for cultured cells, pME18S vector (Mol Cell Biol. 8: 466-472 (1988)) for individual organisms, and the like. preferable. The DNA of the present invention can be inserted into a vector by a conventional method, for example, by a ligase reaction using a restriction enzyme site (Current protocol in Molecular Biology Edited by Ausubel et al. (1987) Publish. John Wiley &. Sons.Section 11.4-11.11).

4). Host cells of the invention Another embodiment of the invention is a transformed host cell that introduces the nucleic acid insertion vector into a host cell and expresses the protein of the invention. As a reagent for introducing the nucleic acid insertion vector into the host cell, various reagents are commercially available. These can be obtained and used for the production of transformed cells.

  The host cell is selected from yeast cells, fungal cells, algae cells, animal cells, insect cells, bacterial cells, plant cells and archaeal cells, preferably bacterial cells. Examples of bacterial cells include, but are not limited to, Escherichia cells, Bacillus cells, Lactobacillus cells, Rhodococcus cells, Pseudomonas cells, Aspergillus cells, and the like. From the viewpoint of producing an imidazole dipeptide product (for example, carnosine, anserine, and valenine) with high efficiency, the host cell is preferably a cell lacking imidazole dipeptide degrading enzyme, specifically peptidase D.

  Specifically, examples of host cells into which the vector of the present invention is introduced include Escherichia bacteria such as Escherichia coli, Actinomycetes bacteria, Bacillus bacteria, Serratia genera. Bacteria, Pseudomonas genus, Corynebacterium genus, Brevibacterium genus, Rhodococcus genus, Lactobacillus genus Lactobacillus genus Streptomyces genus Streptomyces , Thermus genus, Streptococcus genus, Saccharomyces (Sa) yeasts of the genus charomyces, yeasts of the genus Pichia, yeasts of the genus Kluyveromyces, yeasts of the genus Candida, yeasts of the genus Schizosaccharomyces, yeasts of the genus Debaryomyros Genus yeast, Cryptococcus genus, Xanthophyllomyces genus yeast, Mortierella genus fungus, Fusarium genus fungus, Schizochytrium striatum Can be suitably used. That. Preferably, the host cell is Escherichia coli, actinomycetes, bacteria belonging to the genus Pseudomonas, yeast belonging to the genus Saccharomyces.

  More specifically, the host cell of the present invention is Escherichia coli, Bacillus subtilis, Bacillus brevis, Bacillus stearothermophilus, Bacillus stearotherm.・ Mercense (Serratia marcescens), Pseudomonas putida, Pseudomonas aeruginosa, Corynebacterium umba Levibacterium lactofermentum, Rhodococcus erythropolis (Rhodococcus erythropolis), Thermus thermophilus (Thermus certocilia lactis, Streptococcus lactos) Streptomyces lividans, Saccharomyces cerevisiae, Saccharomyces bayanus, Pichia pastoris (Pichia pasto) is), Kluyveromyces lactis, Candida utilis, Candida glabrata, Schizosaccharomyces pombe D Yarrowia lipolytica, Cryptococcus curvatus, Xanthophyllomyces dendrorous, Aspergillus niger lu, Aspergillus niger Aspergillus oryzae, Mortierella ramaniana, Mortierella baineri, Mortierella alpina ganeruna Schizochytrium limacium, Thraustochytrium aureum, etc. can be used.

  The introduction of the nucleic acid insertion vector into the host cell includes calcium phosphate transfection, DEAE-dextran mediated transfection, polybrene mediated transfection, protoplast fusion, liposome mediated transfection (lipofection), conjugation, natural transformation, electroporation and It can be implemented by various methods including other methods known to those skilled in the art. Moreover, the expression vector can be introduced into a host cell by obtaining commercially available transfection reagents and using them. [Reference: Current protocols in molecular biology. 3 vols. Edited by Ausubel F. M.M. et al. , John Wiley & Son, Inc. , Current Protocols. ]

5. Method for Producing Dipeptide of the Present Invention Using the protein or transformed cell of the present invention described above, an amino acid that is a production raw material serving as a substrate for the protein of the present invention is incubated in a buffer in the presence of ATP, or a medium for cell culture These dipeptides can be produced by isolating the desired dipeptides produced by culturing the cells in a buffer or medium. As amino acids used as raw materials, L-alanine (L-Ala), L-glutamine (L-Gln), L-glutamic acid (L-Glu), L-valine (L-Val), L-leucine (L-Leu) ), L-isoleucine (L-Ile), L-proline (L-Pro), L-phenylalanine (L-Phe), L-tryptophan (L-Trp), L-methionine (L-Met), L-serine (L-Ser), L-threonine (L-Thr), L-cysteine (L-Cys), L-asparagine (L-Asn), L-tyrosine (L-Tyr), L-lysine (L-Lys) L-arginine (L-Arg), L-histidine (L-His), L-aspartic acid (L-Asp), L-α-Aminobutyric acid, L-Azaserine, L The group consisting of Theanine, L-4-Hydroxyproline, L-3-Hydroxyproline, L-Ornitine, L-Citrulline, L-6-Diazo-5-oxo-norleucine, glycine (Gly) and β-alanine (β-Ala) The combination of 1 type or 2 types of amino acids chosen from more can be mentioned.

  Examples of the buffer include a phosphate buffer, a borate buffer, a citrate buffer, an acetate buffer, and a Tris-HCl buffer that are commonly used by those skilled in the art.

  As a component contained in the cell culture medium, any carbon source may be used as long as it can be assimilated by the host cell and the transformed cell, and carbohydrates such as glucose, fructose, sucrose, molasses containing these, starch or starch hydrolysate, etc. Further, organic acids such as acetic acid and propionic acid, alcohols such as ethanol and propanol, and the like can be used.

  Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of organic acids such as ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, casein Hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells, digested products thereof, and the like can be used.

  As the inorganic salt, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used. In addition, nutrients such as various vitamins such as peptone, meat extract, yeast extract, corn steep liquor, casamino acid, and biotin can be added to the medium.

  The culture is usually carried out under aerobic conditions such as aeration stirring and shaking. The culture temperature is not particularly limited as long as it is a temperature at which a host cell or transformed cell can grow, and the pH during the culture is not particularly limited as long as the host cell or transformed cell can grow. The pH adjustment during the culture can be performed by adding an acid or an alkali.

  In the above culture, when using the protein of the present invention in order to prevent degradation of imidazole dipeptide products (eg, carnosine, anserine, valenine) by peptidase, an activity inhibitor of peptidase, particularly peptidase D is used, or a host When cells are used, carnosine, anserine, and / or valenin can be produced with high efficiency by using peptidase-deficient cells, particularly JW0227 strain, as host cells.

  The desired enzyme can be collected from the culture by a known collection method using the activity of the desired enzyme as an index. The desired enzyme does not necessarily have to be purified to homogeneity, and may be purified to a degree of purification according to the application.

  Hereinafter, the case where the dipeptide is carcinone, anserine, and valenin will be described more specifically.

(1) Method of producing carnosine by incubating the protein of the present invention in a buffer containing an amino acid as a substrate Carnosine is a condensation reaction using β-alanine and L-histidine as substrates and the protein of the present invention as a catalyst. It is produced by doing. Therefore, the desired carnosine can be produced by adding the protein of the present invention to a buffer containing β-alanine and L-histidine, and incubating the produced carnosine after isolation for a predetermined time.

  In the production method, the protein of the present invention is added in an amount of 0.01 to 100 mg, preferably 0.1 to 10 mg, per 1 mg of amino acid used as a substrate. In the production method, the amino acid used as a substrate is added to the aqueous medium at the beginning or in the middle of the reaction so as to have a concentration of 0.1 to 100 g / L, preferably 0.2 to 40 g / L. In the production method, ATP can be used as an energy source, and ATP is used at a concentration of 0.5 to 1 mol / L.

  The production reaction by incubation is carried out in an aqueous medium at pH 5-11, preferably pH 6-10, 20-50 ° C, preferably 25-45 ° C, for 2-72 hours, preferably 6-36 hours.

  Carnosine produced and accumulated in the buffer can be isolated and purified by methods using activated carbon or ion exchange resins commonly used by those skilled in the art, extraction with organic solvents, crystallization, thin layer chromatography, high performance liquid chromatography, etc. Can be performed.

(2) Method for producing carnosine using the present invention Carnosine is produced by subjecting β-alanine and L-histidine as substrates to the condensation reaction of the protein of the present invention as a catalyst. Therefore, the desired carnosine can be produced and obtained by isolating and purifying the produced carnosine after culturing the transformed cell of the present invention in a cell culture medium containing β-alanine and L-histidine.

  In the production method, the protein of the present invention is added in an amount of 0.01 to 100 mg, preferably 0.1 to 10 mg, per 1 mg of amino acid used as a substrate. In the said manufacturing method, the quantity of (beta) -alanine and L-histidine to add is 0.5-100 g / L normally, respectively, Preferably it is 2-50 g / L. In a cell culture medium containing these amino acids, the transformed cell of the present invention is cultured according to a method well known to those skilled in the art.

  The culture is usually carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is preferably 15 to 40 ° C., and the culture time is usually 5 hours to 7 days. During the culture, the pH is maintained at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.

  Carnosine produced and accumulated in the buffer can be isolated and purified by methods using activated carbon or ion exchange resins commonly used by those skilled in the art, extraction with organic solvents, crystallization, thin layer chromatography, high performance liquid chromatography, etc. Can be performed.

(3) Method for producing anserine by incubating the protein of the present invention in a buffer containing an amino acid as a substrate Anserine is a dipeptide in which β-alanine and 3-methyl-L-histidine are peptide-bonded. Therefore, when producing anserine using the protein of the present invention, it is described in “(1) Method for producing carnosine by incubating the protein of the present invention in a buffer containing an amino acid as a substrate”. In this method, incubation is carried out under the same conditions as in “(1) Carnosine production method” except that 3-methyl-L-histidine is used instead of L-histidine, which is one of the substrates, and isolation and purification are performed. Thus, anserine can be produced and obtained.

(4) Method for producing anserine using the transformed cell of the present invention Anserine is a dipeptide in which β-alanine and 3-methyl-L-histidine are peptide-bonded. Therefore, in the case of producing anserine using the protein of the present invention, in the above-mentioned “(2) Method for producing carnosine using the transformed cell of the present invention”, L- Except for using 3-methyl-L-histidine instead of histidine, anserine was produced by carrying out the same conditions as in “(2) Carnosine production method using transformed cells of the present invention”. Can get.

(5) Method for producing valerin by incubating the protein of the present invention in a buffer containing an amino acid as a substrate Anserine is a dipeptide in which β-alanine and 1-methyl-L-histidine are peptide-bonded. Therefore, when producing valenin using the protein of the present invention, it is described in “(1) Method for producing carnosine by incubating the protein of the present invention in a buffer containing an amino acid as a substrate”. 1) Incubate, isolate and purify under the same conditions as in “(1) Method for producing carnosine” except that 1-methyl-L-histidine is used instead of L-histidine which is one of the substrates. Thus, it is possible to produce and obtain barenin.

(6) Method for Producing Valenin Using the Transformed Cell of the Present Invention Valenin is a dipeptide in which β-alanine and 1-methyl-L-histidine are peptide-bonded. Therefore, when producing valenin using the protein of the present invention, in the above-mentioned “(2) Method for producing carnosine using the transformed cell of the present invention”, L- By carrying out under the same conditions as in “(2) Method for producing carnosine using the transformed cell of the present invention” except that 1-methyl-L-histidine is used instead of histidine, valenin is produced. Can get.

  All documents mentioned herein are hereby incorporated by reference in their entirety. The examples described herein are illustrative of embodiments of the invention and should not be construed as limiting the scope of the invention.

  In the following examples, polynucleotide (DNA, mRNA) preparation, PCR, sequencing, transformation, protein expression, protein purification, HPLC analysis, etc. should be performed using methods well known to those skilled in the art. Can do. For example, Sambrook, J. et al. And Russell, D .; W. See, Molecular Cloning A Laboratory Manual 4rd Edition, Cold Spring Harbor Laboratory Press (2012), and the like.

Introduction of site-specific mutation into YwfE Using pET vector incorporating YwfE gene (SEQ ID NO: 11) as a template, the target mutation was introduced using Quick Change Site-Directed Mutagenesis (Strategene, USA) according to the manufacturer's instructions. . The PCR reaction was performed under the reaction conditions shown in Table 1 (composition) and Table 2 (PCR cycle). In the PCR reaction, KOD-Plus-Neo-DNA polymerase (Toyobo Co., Ltd., Osaka) was used. Using a primer (SEQ ID NO: 33-44), N1
A vector into which a site-specific mutation of 08A, N108E or N108Q was introduced was obtained. A vector for N108A is a polynucleotide (SEQ ID NO: 18) encoding a YwfE protein in which the 108th asparagine (N) residue from the N-terminus of the YwfE protein is substituted with an alanine (A) residue, and a vector for N108E is YwfE. A polynucleotide (SEQ ID NO: 19) encoding a YwfE protein in which the 108th asparagine (N) residue of the protein is replaced with a glutamic acid (E) residue, and the vector for N108Q is the 108th asparagine (N) residue of the YwfE protein. This represents a polynucleotide (SEQ ID NO: 20) encoding a YwfE protein having a group substituted with a glutamine (Q) residue.

  After the PCR reaction, the obtained PCR product was analyzed with a DNA sequencer (Applied Biosystems, Life Technologies Japan Co., Ltd., Tokyo) to confirm whether a site-specific mutation was introduced.

  In the vector extracted from E. coli, the Dpn I site is methylated by Dam methylase, whereas the PCR product is not methylated in the Dpn I site. Distinguish from products. Briefly, in order to remove the template vector contained in the reaction solution after PCR, the purified PCR product was treated with Dpn I at 37 ° C. for 2 hours. The restriction enzyme reaction was performed under the reaction conditions shown in Table 3.

  After digestion with Dpn I, the site-directed mutagenesis vector purified by phenol / chloroform treatment and ethanol precipitation was dissolved in TE buffer pH 8.0.

Transformation of E. coli JM109 with site-directed mutagenesis vector Both the competent cell of E. coli JM109 and the site-specific mutagenesis vector were heat-treated at 42 ° C. Thereafter, an SOC medium was added for cultivation, inoculated into an LB agar medium containing 50 μg / mL kanamycin, and cultured at 37 ° C. overnight.

  One colony was selected from the grown colonies, suspended in 3 mL of LB medium containing kanamycin, and cultured at 37 ° C. for 5 hours. After culture, a site-specific mutagenesis vector was extracted from the transformed cells by alkaline SDS method. The extracted site-directed mutagenesis vector was purified by phenol / chloroform treatment and ethanol precipitation, and dissolved in TE buffer (pH 8.0).

Preparation of purified enzyme
Transformation of E. coli BL21 with site-directed mutagenesis vector Escherichia coli BL21 was transformed by the heat shock method using the purified site-specific mutagenesis vector. Both the competent cell of E. coli BL21 and the site-directed mutagenesis vector were heat-treated at 42 ° C. Thereafter, an SOC medium was added for cultivation, inoculated into an LB agar medium containing 50 μg / mL kanamycin, and cultured at 37 ° C. overnight. Here, E. coli that formed colonies was used as YwfE-expressing transformed cells.

Expression induction by IPTG One colony was selected from the colonies of YwfE-expressing transformed cells grown and suspended in 3 mL of LB medium containing kanamycin in a test tube, and precultured at 37 ° C. for 5 hours. Next, 2 mL of the preculture solution was added to 200 mL of the LB medium containing kanamycin in the Erlenmeyer flask with a 500 mL baffle. After main culture at 37 ° C. and 120 rpm for 2 hours, 200 μL of 100 mM IPTG was added. The culture solution after addition of IPTG was cultured overnight at 25 ° C. and 120 rpm.

  After overnight culture, the cells were collected by centrifugation at 5000 × g for 10 minutes, and the cell pellet was suspended in 100 mM Tris-HCl buffer (pH 8.0) and washed. This operation was repeated twice to remove the medium components. After washing, the cell pellet was stored frozen at -80 ° C.

BugBuster (BugBuster ^™ Protein Extraction Reagent, Merck KGaA, Germany) and lysozyme were added to the enzyme-purified and thawed cell pellet to extract the protein. The cell disruption solution was separated into a precipitate (insoluble fraction) and a supernatant (cell-free extract) by centrifugation. The cell-free extract was applied using His GraviTrap (GE Healthcare, USA) according to the manufacturer's instructions. The eluted solution was desalted using a PD-10 column (GE Healthcare) according to the manufacturer's instructions. The purified enzyme was stored at −80 ° C. until used in the following experiments.

Concentration measurement of purified enzyme The concentration of the purified enzyme was measured with a microplate reader MODEL550 (Bio-Rad, USA) after a color reaction using Coomassie Brilliant Blue. The purified enzyme concentration was determined from the absorbance measurement at 595 nm by the calibration curve method.

Double mutant enzymes N108A / I112V (SEQ ID NO: 5), N108A / H378K (SEQ ID NO: 6), N108A / N108A / N108Q (SEQ ID NO: 6), which are a combination of double mutant enzyme N108A, N108E or N108Q mutation and I112V, H378K or H378R mutation H378R (SEQ ID NO: 7), N108E / I112V (SEQ ID NO: 8), N108E / H378K (SEQ ID NO: 9), N108E / H378R (SEQ ID NO: 10), N108Q / I112V (SEQ ID NO: 11), N108Q / H378K (SEQ ID NO: 12) ) And N108Q / H378R (SEQ ID NO: 13) were prepared, and carnosine synthesis activity was evaluated. Briefly, similar to the introduction of the site-specific mutation described above, a vector having a site-specific mutation of N108A, N108E or N108Q is used as a template, the primer of I112V (SEQ ID NOs: 39 and 40), the primer of H378K (sequence No. 41 and 42) or a H378R primer (SEQ ID No. 43 and 44) was used to further introduce a site-specific mutation (I112V, H378K or H378R) to obtain a vector having a double site-specific mutation. Thereafter, in the same manner as described above, purified double-site-specific mutant YwfE (SEQ ID NOs: 5 to 13) was obtained.

Triple mutant enzymes N108A / I112V / H378R (SEQ ID NO: 14), N108Q / I112V / H378K (SEQ ID NO: 15), which are combinations of triple mutant enzymes N108A / I112V and N108Q / I112V, and mutations of H378K and H378R N108Q / I112V / H378R (SEQ ID NO: 16) was prepared, and carnosine synthesis activity was evaluated. Briefly, in the same manner as the introduction of the above site-specific mutation, H378K primer (SEQ ID NO: 41 and 42) or H378R primer using vectors having N108A / I112V and N108Q / I112V site-specific mutations as templates. A site-specific mutation (H378K or H378R) was further introduced using (SEQ ID NOs: 43 and 44) to obtain a vector having a triple site-specific mutation. Thereafter, in the same manner as described above, purified triple site-specific mutant YwfE (SEQ ID NOs: 14 to 16) was obtained.

Carnosine synthesis activity evaluation (HPLC analysis)
A commercial carnosine (Sigma-Aldrich, USA) was used as a standard carnosine. The amount of peptide synthesized was subjected to HPLC analysis using N ^α- (5-fluoro-2,4-dinitrophenyl) -L-alanineamide (FDAA) derivatization method, and quantified by a calibration curve method. The HPLC analysis was performed according to a conventional method under the conditions shown in Table 4 (eluent composition) and Table 5 (gradient program).
<Analysis conditions>
Equipment used: HITACHI L-7000 series (Hitachi, Ltd., Tokyo)
Column used: WH-C18A (4 × 150 mm) (Hitachi High-Technologies Corporation, Tokyo)
Sample injection volume: 10 μL
Flow rate: 0.5 mL / min Column temperature: 40 ° C
UV detection wavelength: 340 nm

  Carnosine synthesis was performed in a reaction solution having the composition shown in Table 6 at 30 ° C. for 20 hours. As an enzyme, a wild type enzyme (SEQ ID NO: 1), a single mutant enzyme N108A (SEQ ID NO: 2), N108E (SEQ ID NO: 3), N108Q (SEQ ID NO: 4), and a double mutant enzyme N108A / I112V (SEQ ID NO: 5), N108A / H378K (SEQ ID NO: 6), N108A / H378R (SEQ ID NO: 7), N108E / I112V (SEQ ID NO: 8), N108E / H378K (SEQ ID NO: 9), N108E / H378R (SEQ ID NO: 10), N108Q / I112V (SEQ ID NO: 11), N108Q / H378K (SEQ ID NO: 12), N108Q / H378R (SEQ ID NO: 13), triple mutant enzyme N108A / I112V / H378R (SEQ ID NO: 14), N108Q / I112V / H378K (SEQ ID NO: 15) ), N108Q / I112V / H378R (SEQ ID NO: 16) It was used. After the reaction was completed, HPLC analysis was performed in the same manner as described above in order to evaluate carnosine synthesis.

  The statistical test is performed using statistical test software: EZR (http://www.jichi.ac.jp/saitama-sct/SaitamaHP.files/statmed.htm) and the concentration of carnosine produced by the wild-type enzyme. As a control group, post hoc analysis was performed with respect to the concentration of carnosine produced by each mutant enzyme using a one-way analysis of variance (ANOVA) and Dunnett's test with a p-value of 0.05 as a significance level (n for each group). = 3). In addition, the following statistical analysis of anserine synthesis activity evaluation and valenine synthesis activity evaluation was performed in the same manner.

Results FIG. 1 shows a wild type enzyme (SEQ ID NO: 1), a single mutant enzyme N108A (SEQ ID NO: 2), N108E (SEQ ID NO: 3), N108Q (SEQ ID NO: 4), and a double mutant enzyme N108A / I112V ( SEQ ID NO: 5), N108A / H378K (SEQ ID NO: 6), N108A / H378R (SEQ ID NO: 7), N108E / I112V (SEQ ID NO: 8), N108E / H378K (SEQ ID NO: 9), N108E / H378R (SEQ ID NO: 10), N108Q / I112V (SEQ ID NO: 11), N108Q / H378K (SEQ ID NO: 12), N108Q / H378R (SEQ ID NO: 13), triple mutant enzyme N108A / I112V / H378R (SEQ ID NO: 14), N108Q / I112V / H378K (sequence) No. 15), N108Q / I112V / H378R (SEQ ID NO: 16) The carnosine concentration by carnosine synthesis is shown. Except for N108A / I112V (SEQ ID NO: 5), N108E / I112V (SEQ ID NO: 8), and N108Q / I112V (SEQ ID NO: 11), the statistically significant calcinone in all mutant enzymes compared to the wild type An increase in synthetic activity was observed. The calcinone synthesis activity was highest for N108E / H378K (SEQ ID NO: 9), and the yield was 91.4%.

Evaluation of Anserine Synthesis Activity As in the evaluation of carnosine synthesis activity in Example 1, 15 mutant enzymes (N108A, N108E, N108Q, N108A / I112V, N108A / H378K, N108A / H378R, N108E / I112V, N108E / H378K) N108E / H378R, N108Q / I112V, N108Q / H378K, N108Q / H378R, N108A / I112V / H378R, N108Q / I112V / H378K and N108Q / I112V / H378R) were evaluated by comparing with the wild-type activity. . Briefly, anserine synthesis was carried out in a reaction solution having the composition shown in Table 7 at 30 ° C. for 20 hours. After completion of the reaction, the anserine concentration of each mutant enzyme was evaluated by quantifying the anserine concentration by HPLC in the same manner as in Example 1. As an anserine preparation, commercially available anserine (Wako Pure Chemical Industries, Ltd., Osaka) was used.

Results FIG. 2 shows the measurement results. With the exception of N108Q (SEQ ID NO: 4) and N108A / H378R (SEQ ID NO: 7), a statistically significant increase in anserine synthesis activity was observed in all mutant enzymes compared to the wild type. Moreover, N108Q / I112V / H378K (sequence number 15) had the highest anserine synthetic activity, and the yield was 94.7%.

Evaluation of Valenin Synthesis Activity Similar to the evaluation of carnosine synthesis activity in Example 1, 15 mutant enzymes (N108A, N108E, N108Q, N108A / I112V, N108A / H378K, N108A / H378R, N108E / I112V, N108E / H378K) , N108E / H378R, N108Q / I112V, N108Q / H378K, N108Q / H378R, N108A / I112V / H378R, N108Q / I112V / H378K and N108Q / I112V / H378R) were evaluated by comparing with the wild-type activity. . Briefly, valenin synthesis was performed in a reaction solution having the composition shown in Table 8 at 30 ° C. for 20 hours. After completion of the reaction, the valenin concentration of each mutant enzyme was evaluated by quantifying the valenin concentration by HPLC in the same manner as in Example 1. As a standard product of valenin, commercially available valenin (Hamari Pharmaceutical Co., Ltd., Osaka) was used.

Results FIG. 3 shows the measurement results. In 6 mutant enzymes (N108A / H378K, N108A / H378R, N108Q / I112V, N108A / I112V / H378R, N108Q / I112V / H378K and N108Q / I112V / H378R), statistically significant compared to the wild type Increase in the activity of synthesizing valenin was observed. In addition, the activity of synthesizing valerin was highest for N108A / H378K (SEQ ID NO: 6), and the yield was 38.8%.

  From the above results, four types of mutant enzymes (N108A / H378K (SEQ ID NO: 6), N108A / I112V / H378R (SEQ ID NO: 14), N108Q / I112V / H378K (SEQ ID NO: 15) and N108Q / I112V / H378R (sequence) Regarding No. 16)), statistically significant increases in carnosine synthesis activity, anserine synthesis activity, and valenin synthesis activity were observed compared to the wild type.

Evaluation of the effect of peptidase on carnosine synthesis Peptidase D (pepD) recognizes and decomposes a dipeptide (Xaa-His) consisting of any amino acid (Xaa) on the N-terminal side and histidine (His) on the C-terminal side. (J. Gen. Microbiol. 172, 2337-2343 (1986)). Therefore, it was considered that in the production method using cells, degradation of carnosine can be suppressed by using bacterial cells lacking pepD.
Therefore, the degradation activity of carnosine was comparatively evaluated using Escherichia coli JM101 strain and JW0227 strain. The JW0227 strain is obtained by deleting the gene of pepD, which is a kind of peptidase, from the JM101 strain. Table 9 shows the Genotype of JM101 strain.

Carnosine degradation by the bacterial cell reaction was performed in a reaction solution having the composition shown in Table 10 at 30 ° C. for 0 or 20 hours. The solution after completion of the reaction was treated at 90 ° C. for 10 minutes to stop the reaction. After centrifugation for 20 minutes, the supernatant was analyzed by HPLC. The carnosine residual rate after reaction for 20 hours when the carnosine concentration at 0 hour was taken as 100% was calculated.

Results FIG. 4 shows the carnosine survival rate when using JM101 strain and JW0227 strain as host cells, respectively. The carnosine residual rate was 64.2% for the JM101 strain and 74.7% for the JW0227 strain. From this, it was clarified that by using a peptidase-deficient strain, degradation of the produced carnosine can be suppressed and carnosine can be produced with high efficiency. It was also suggested that anserine and valenin can be produced with high efficiency by using a peptidase-deficient strain.

  By providing a protein that efficiently produces imidazole dipeptides, particularly carnosine, anserine and / or valenin, a vector containing a nucleic acid encoding the protein, and a host cell containing the protein, these can be conveniently and at low cost. Of imidazole dipeptides can be provided. Further, by using peptidase-deficient cells as host cells, imidazole dipeptides, particularly carnosine, can be produced with high efficiency.

Claims (17)

L-amino acid α-ligase represented by SEQ ID NO: 1 is a mutant protein comprising a substitution of at least 1 to 3 amino acid residues in the amino acid sequence of YwfE;
An asparagine (N) residue at position 108 from the N-terminus is substituted with an alanine (A) residue, a glutamic acid (E) residue or a glutamine (Q) residue; or
The isoleucine (I) residue at position 112 is replaced with a valine (V) residue and / or the histidine (H) residue at position 378 is replaced with a lysine (K) residue or an arginine (R) residue A mutated protein characterized by   The mutant protein according to claim 1, wherein the mutant protein has L-amino acid α-ligase activity. The mutant protein is
(I) a protein having the amino acid sequence of SEQ ID NOs: 2 to 16,
(Ii) a protein having 80% or more homology with the amino acid sequences of SEQ ID NOs: 2 to 16 and having L-amino acid α-ligase activity;
(Iii) It consists of an amino acid sequence in which one or more amino acid residues of the amino acid sequences of SEQ ID NOs: 2 to 16 are deleted, substituted, inserted, and / or added, and has L-amino acid α-ligase activity protein,
The mutant protein according to claim 1 or 2, characterized in that   The mutant protein according to claim 2 or 3, wherein the L-amino acid α-ligase activity is selected from carnosine synthesis activity, anserine synthesis activity and / or valenine synthesis activity. When the L-amino acid α-ligase activity is carnosine synthesis activity, the mutant protein is
(I) a protein having an amino acid sequence of SEQ ID NOs: 2 to 4, 6, 7, 9, 10, 12 to 16,
(Ii) a protein having 80% or more homology with the amino acid sequences of SEQ ID NOs: 2 to 4, 6, 7, 9, 10, 12 to 16 and having a carnosine synthesis activity;
(Iii) consisting of an amino acid sequence in which one or more amino acid residues of the amino acid sequences of SEQ ID NOs: 2 to 4, 6, 7, 9, 10, 12 to 16 are deleted, substituted, inserted, and / or added And a protein having carnosine synthesis activity,
The mutant protein according to claim 4, wherein When the L-amino acid α-ligase activity is anserine synthesis activity, the mutant protein is
(I) a protein having an amino acid sequence of SEQ ID NOs: 2, 3, 5, 6, 8 to 16,
(Ii) a protein having 80% or more homology with the amino acid sequences of SEQ ID NOs: 2, 3, 5, 6, 8 to 16 and having anserine synthesis activity;
(Iii) It consists of an amino acid sequence in which one or more amino acid residues of the amino acid sequences of SEQ ID NOs: 2, 3, 5, 6, 8 to 16 are deleted, substituted, inserted and / or added, and anserine A protein having synthetic activity,
The mutant protein according to claim 4, wherein When the L-amino acid α-ligase activity is valenine synthesis activity, the mutant protein is
(I) a protein having the amino acid sequence of SEQ ID NOs: 6, 7, 11, 14-16,
(Ii) a protein having 80% or more homology with the amino acid sequences of SEQ ID NOs: 6, 7, 11, 14 to 16 and having a valenin synthesis activity,
(Iii) It consists of an amino acid sequence in which one or more amino acid residues of the amino acid sequences of SEQ ID NOs: 6, 7, 11, 14 to 16 are deleted, substituted, inserted and / or added, and has a valenin synthesis activity A protein having
The mutant protein according to claim 4, wherein   A nucleic acid encoding the protein according to any one of claims 1 to 7.   A vector comprising a polynucleotide comprising the nucleic acid according to claim 8 inserted therein.   A host cell comprising at least one vector according to claim 9.   A method for producing a dipeptide, wherein the mutant protein according to any one of claims 1 to 7 is used.   A method for producing a dipeptide, comprising using the protein according to any one of claims 1 to 3 together with a peptidase activity inhibitor.   A method for producing a dipeptide, comprising using the host cell according to claim 10.   The method for producing a dipeptide according to claim 13, wherein the host cell is a peptidase-deficient cell.   The method for producing a dipeptide according to claim 12 or 14, wherein the peptidase is peptidase D.   The method for producing a dipeptide according to any one of claims 11 to 15, wherein the dipeptide is an imidazole dipeptide.   The method for producing a dipeptide according to claim 16, wherein the imidazole dipeptide is carnosine, anserine and / or valenin.