Modified l-amino acid ligase
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- AJINOMOTO CO INC
- Filing Date
- 2026-01-19
- Publication Date
- 2026-07-30
Smart Images
Figure JP2026001366_30072026_PF_FP_ABST
Abstract
Description
Modified L-amino acid ligase
[0008]
[0001] The present invention relates to a modified L-amino acid ligase, a polynucleotide encoding the L-amino acid ligase, a recombinant vector containing the polynucleotide, and a transformant into which the vector has been introduced.
[0002] A peptide is a substance in which a plurality of amino acids are linked by peptide bonds. A substance in which two amino acids are linked is called a dipeptide, and a substance in which three amino acids are linked is called a tripeptide. Dipeptides and tripeptides may have physiological activities that are not possessed by the amino acids that constitute them, and their applications to foods and beverages have been studied.
[0003] For example, carnosine, a dipeptide in which β-alanine and histidine are peptide-bonded, has an antioxidant effect. By adding carnosine to foods and beverages, functions such as fatigue reduction, enhanced motor function, and improved brain function can be imparted.
[0004] As a method for synthesizing carnosine, a method using a microorganism having imidazole dipeptide synthesis activity is known (Patent Documents 1-3). Specifically, a microorganism having the ability to produce a mutant of L-amino acid ligase YwfE is used.
[0005] JP-A-2018-102287 JP-A-2020-022433 JP-A-2013-081405
[0006] Although the modified L-amino acid ligases described in Patent Documents 2 and 3 have an improved production efficiency of carnosine compared to the wild type, there is a need for a modified L-amino acid ligase that can synthesize carnosine with higher efficiency. An object of the present invention is to provide a modified L-amino acid ligase with improved activity.
[0007] As a result of intensive studies to solve the above problems, the present inventors have found that by substituting specific amino acid residues contained in L-amino acid ligase YwfE, the substrate affinity is improved, and the present invention has been completed.
[0008] In other words, the present invention can be illustrated as follows: [1] The protein described in (A), (B), or (C) below. (A) A protein comprising an amino acid sequence in the amino acid sequence shown in Sequence ID No. 1, wherein the substitution of the glutamine residue at position 78 to histidine, the glycine residue at position 90 to glutamic acid, the methionine residue at position 104 to isoleucine, the isoleucine residue at position 112 to valine, the lysine residue at position 285 to glutamic acid, the glutamic acid residue at position 361 to aspartic acid, the methionine residue at position 374 to isoleucine, and the histidine residue at position 378 to arginine; (B) A protein having L-amino acid ligase activity, comprising an amino acid sequence in which the amino acid sequence shown in Sequence ID No. 1 has the substitution of a glutamine residue at position 78 to histidine, a glycine residue at position 90 to glutamic acid, a methionine residue at position 104 to isoleucine, an isoleucine residue at position 112 to valine, a lysine residue at position 285 to glutamic acid, a glutamic acid residue at position 361 to aspartic acid, a methionine residue at position 374 to isoleucine, and a histidine residue at position 378 to arginine, wherein one or more amino acid residues other than those substituted are substituted, deleted, inserted, or added; (C) A protein having 90% or more identity with an amino acid sequence having the substitutions described above, and having L-amino acid ligase activity, in the amino acid sequence shown in Sequence ID No. 1, the substitution of the glutamine residue at position 78 to histidine, the glycine residue at position 90 to glutamic acid, the methionine residue at position 104 to isoleucine, the isoleucine residue at position 112 to valine, the lysine residue at position 285 to glutamic acid, the glutamic acid residue at position 361 to aspartic acid, the methionine residue at position 374 to isoleucine, and the histidine residue at position 378 to arginine.[2] The amino acid sequence is subject to the substitution of glutamic acid from the alanine residue at position 26, the serine residue at position 67 to aspartic acid, the proline residue at position 82 to aspartic acid, the leucine residue at position 92 to isoleucine, the arginine residue at position 94 to lysine, the alanine residue at position 101 to valine, the valine residue at position 153 to isoleucine, the glutamine residue at position 156 to lysine, the serine residue at position 162 to glutamic acid, the aspartic acid residue at position 198 to glutamic acid, the asparagine residue at position 215 to lysine, and the alanine residue at position 232 to glutamic acid. The protein described in [1] further comprising a substitution of alanine at position 243 to serine, a substitution of glutamine at position 287 to lysine, a substitution of arginine at position 295 to lysine, a substitution of glutamine at position 365 to glutamic acid, a substitution of tyrosine at position 372 to histidine, a substitution of valine at position 402 to isoleucine, a substitution of lysine at position 418 to phenylalanine, a substitution of valine at position 420 to alanine, a substitution of serine at position 422 to alanine, a substitution of threonine at position 463 to lysine, a substitution of isoleucine at position 464 to leucine, and a deletion of glycine at position 473. [3] The protein according to [1], wherein the amino acid sequence further comprises a substitution of aspartic acid from the glutamic acid residue at position 239, an insertion of threonine at position 474, an insertion of leucine at position 475, and an insertion of glutamic acid at position 476. [4] The protein according to (A'), (B'), or (C') below. (A') A protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 3; (B') A protein comprising an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted, or added in the amino acid sequence shown in SEQ ID NO: 2 or 3, and having L-amino acid ligase activity; (C') A protein having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 or 3, and having L-amino acid ligase activity. [5] The protein according to [4], which comprises the amino acid sequence shown in SEQ ID NO: 2 or 3. [6] A polynucleotide encoding the protein according to any one of [1] to [5].[7] A recombinant vector containing the polynucleotide described in [6]. [8] A transformant into which the recombinant vector described in [7] has been introduced. [9] A method for producing a dipeptide, comprising acting on one or more amino acids with a protein described in any of [1] to [5].
[10] The method for producing the dipeptide according to [9], wherein the dipeptide is an imidazole peptide.
[11] The method for producing the dipeptide according to [9], wherein the dipeptide is carnosine or alanylglutamine.
[0009] The present invention provides an L-amino acid ligase with high activity.
[0010] This graph shows the amount of carnosine produced when BaYwfE, BaYwfE_I112V / H378R, or BpYwfE_I112V / H378R is used as the L-amino acid ligase. This graph shows the amount of carnosine produced when BaYwfE_I112V / H378R, FcYwfE, or AncYwfE-3 is used as the L-amino acid ligase. This graph shows the amount of alanylglutamine produced when FcYwfE or AncYwfE-3 is used as the L-amino acid ligase.
[0011] <1> One embodiment of a protein is a protein comprising an amino acid sequence in the amino acid sequence shown in Sequence ID No. 1, which includes a substitution of histidine from the glutamine residue at position 78, glutamic acid from the glycine residue at position 90, isoleucine from the methionine residue at position 104, valine from the isoleucine residue at position 112, glutamic acid from the lysine residue at position 285, aspartic acid from the glutamic acid residue at position 361, isoleucine from the methionine residue at position 374, and arginine from the histidine residue at position 378.
[0012] The above protein is an L-amino acid ligase having L-amino acid ligase activity. In this specification, L-amino acid ligase activity is the activity of condensing the α-carboxyl group of one free amino acid molecule with the amino group of another free amino acid molecule to form a peptide bond. In this specification, unless otherwise specified, "activity" refers to activity at 37°C. Furthermore, the protein according to one embodiment of the present invention will also be referred to as "modified L-amino acid ligase" or simply "L-amino acid ligase" below.
[0013] The protein of this embodiment includes an amino acid sequence in the amino acid sequence shown in Sequence ID No. 1, which includes substitutions of the glutamine residue at position 78 to histidine, the glycine residue at position 90 to glutamic acid, the methionine residue at position 104 to isoleucine, the isoleucine residue at position 112 to valine, the lysine residue at position 285 to glutamic acid, the glutamic acid residue at position 361 to aspartic acid, the methionine residue at position 374 to isoleucine, and the histidine residue at position 378 to arginine. In other words, the protein of this embodiment is a specific mutant of Bacillus pumilus-derived L-amino acid ligase (hereinafter also referred to as "BpYwfE") having the amino acid sequence shown in Sequence ID No. 1.
[0014] Furthermore, the protein of this embodiment may be a protein containing the amino acid sequence shown in SEQ ID NO: 2 or 3. The amino acid sequence shown in SEQ ID NO: 2 or 3 has the substitutions shown in Table 1 relative to the amino acid sequence of the Bacillus pumilus-derived L-amino acid ligase shown in SEQ ID NO: 1.
[0015] The protein of this embodiment may be a variant of the L-amino acid ligase exemplified above, as long as its original function is maintained. Such a variant in which the original function is maintained is sometimes called a "conserved variant." Examples of conserved variants include homologs of the L-amino acid ligase or the gene encoding it, as well as artificially modified versions.
[0016] "Maintaining the original function" means that a variant of a gene or protein possesses a function (activity or properties) that corresponds to the function (activity or properties) of the original gene or protein. In other words, "maintaining the original function" in the case of an L-amino acid ligase means that the protein variant possesses L-amino acid ligase activity. Furthermore, "maintaining the original function" in the case of an L-amino acid ligase gene means that the gene variant codes for a protein whose original function is maintained (i.e., a protein that possesses L-amino acid ligase activity).
[0017] The L-amino acid ligase activity of the L-amino acid ligase variant is preferably 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the activity of the original L-amino acid ligase when measured under the same conditions. The L-amino acid ligase activity can be measured, for example, by quantifying the product when reacting an aqueous solution (pH 9.0) containing the substrate amino acid at 37°C for 2 hours.
[0018] L-amino acid ligase may be a protein containing an amino acid sequence that includes the substitution, deletion, insertion, or addition of one or more amino acids at one or more positions other than the substituted residue in the above amino acid sequence (for example, the amino acid sequence having the above substitution in the amino acid sequence shown in Sequence ID No. 1), as long as the original function is maintained. The above "one or more" will vary depending on the position and type of amino acid residue in the three-dimensional structure of the protein, but specifically it means, for example, 1 to 47, 1 to 23, 1 to 10, preferably 1 to 8, more preferably 1 to 5, and particularly preferably 1 to 3.
[0019] The above substitutions, deletions, insertions, or additions of one or more amino acids are conservative mutations that maintain the normal function of the protein. A typical example of a conservative mutation is a conservative substitution. A conservative substitution is a mutation in which the substitution site is between Phe, Trp, and Tyr if the substitution site is an aromatic amino acid; between Leu, Ile, and Val if the substitution site is a hydrophobic amino acid; between Gln and Asn if the substitution site is a polar amino acid; between Lys, Arg, and His if the substitution site is a basic amino acid; between Asp and Glu if the substitution site is an acidic amino acid; and between Ser and Thr if the amino acid has a hydroxyl group. Substitutions considered conservative include, specifically, substitutions from Ala to Ser or Thr, from Arg to Gln, His or Lys, from Asn to Glu, Gln, Lys, His or Asp, from Asp to Asn, Glu or Gln, from Cys to Ser or Ala, from Gln to Asn, Glu, Lys, His, Asp or Arg, from Glu to Gly, Asn, Gln, Lys or Asp, from Gly to Pro, from His to Asn, Lys, Gln, Arg or Tyr, and Il Examples of substitutions include: substitution of e to Leu, Met, Val, or Phe; substitution of Leu to Ile, Met, Val, or Phe; substitution of Lys to Asn, Glu, Gln, His, or Arg; substitution of Met to Ile, Leu, Val, or Phe; substitution of Phe to Trp, Tyr, Met, Ile, or Leu; substitution of Ser to Thr or Ala; substitution of Thr to Ser or Ala; substitution of Trp to Phe or Tyr; substitution of Tyr to His, Phe, or Trp; and substitution of Val to Met, Ile, or Leu.
[0020] Furthermore, the L-amino acid ligase may be a protein having an amino acid sequence that has 90% or more, preferably 95% or more, more preferably 97% or more, and even more preferably 99% or more homology to the entire amino acid sequence, as long as its original function is maintained. In this case, the protein has the above substitution. In this specification, "homology" may refer to "identity".
[0021] L-amino acid ligases may be any of the above-mentioned proteins, excluding those whose amino acid sequence or the base sequence of the gene encoding them is publicly known at the time of filing the application of the present invention.
[0022] L-amino acid ligase may be a fusion protein with other amino acid sequences. The "other amino acid sequence" can be appropriately selected according to various conditions such as its intended use. Examples of "other amino acid sequences" include peptide tags, signal sequences, and protease recognition sequences. The "other amino acid sequence" may be linked to the N-terminus, C-terminus, or both of the L-amino acid ligase, for example. The "other amino acid sequence" may be a single amino acid sequence, or a combination of two or more amino acid sequences may be used.
[0023] Peptide tags can be used, for example, to detect and purify expressed L-amino acid ligases. Specific examples of peptide tags include His tags, FLAG tags, GST tags, Myc tags, MBP (maltose binding protein), CBP (cellulose binding protein), TRX (thioredoxin), GFP (green fluorescent protein), HRP (horseradish peroxidase), ALP (alkaline phosphate), and the Fc region of antibodies. A 6xHis tag is an example of a His tag.
[0024] Signal sequences can be used, for example, in the secretion and production of L-amino acid ligases. Examples of signal sequences include those recognized by the Sec secretory pathway and those recognized by the Tat secretory pathway. Specifically, examples of signal sequences recognized by the Sec secretory pathway include signal sequences of cell surface proteins of Corynebacteria. Examples of cell surface proteins of Corynebacteria include PS1 (CspA) and PS2 (CspB) of C. glutamicum (Japanese Patent Publication No. 6-502548), and SlpA (CspA) of C. ammoniagenes (C. stationis) (Japanese Patent Publication No. 10-108675). Specifically, signal sequences recognized by the Tat secretory pathway include the TorA signal sequence of E. coli, the SufI signal sequence of E. coli, the PhoD signal sequence of Bacillus subtilis, the LipA signal sequence of Bacillus subtilis, and the IMD signal sequence of Arthrobacter globiformis (WO2013 / 118544). Signal sequences can be used, for example, by being added to the N-terminus of the protein to be produced. Generally, signal sequences are cleaved by signal peptidases when the translation product is secreted outside the bacterial cell. Therefore, when L-amino acid ligase is secreted using a signal sequence, L-amino acid ligase without a signal sequence may be secreted outside the bacterial cell.
[0025] The protease recognition sequence can be used, for example, to cleave expressed L-amino acid ligase. Preferably, the protease recognition sequence is a protease recognition sequence with high substrate specificity. Specifically, examples of protease recognition sequences with high substrate specificity include the recognition sequences of Factor Xa proteases and proTEV proteases. Factor Xa proteases recognize the amino acid sequence Ile-Glu-Gly-Arg (=IEGR) (SEQ ID NO: 4) in proteins, and proTEV proteases recognize the amino acid sequence Glu-Asn-Leu-Tyr-Phe-Gln (=ENLYFQ) (SEQ ID NO: 5) in proteins, and specifically cleave the C-terminal side of each sequence. For example, when expressing an L-amino acid ligase as a fusion protein with other amino acid sequences such as peptide tags or pro-sequences, by introducing a protease recognition sequence between the L-amino acid ligase and the other amino acid sequence, the other amino acid sequence can be removed from the expressed L-amino acid ligase using the protease, thereby obtaining an L-amino acid ligase that does not contain the other amino acid sequence.
[0026] <2> Polynucleotide Another embodiment of the present invention may be a polynucleotide encoding the above-mentioned protein. Hereinafter, the polynucleotide of this embodiment will also be referred to as the L-amino acid ligase gene.
[0027] The polynucleotide of this embodiment may include, for example, a gene having a missense mutation in the codons corresponding to the amino acid residues at positions 78, 90, 104, 112, 285, 361, 374, and 378 of the L-amino acid ligase protein in the base sequence shown in Sequence ID No. 6. A missense mutation refers to a mutation in which the amino acid to which the codon corresponds changes due to a mutation in the bases constituting the codon. Furthermore, the L-amino acid ligase gene may be a conserved variant of the L-amino acid ligase gene exemplified above, as long as the original function is maintained. In addition, the L-amino acid ligase gene may be one in which any codon in the base sequence of the L-amino acid ligase gene exemplified above or its conserved variant is replaced with an equivalent codon. For example, the L-amino acid ligase gene may be modified to have the optimal codons depending on the codon usage frequency of the host being used.
[0028] In this specification, the term "gene" is not limited to DNA, but may include any polynucleotide, as long as it codes for the target protein. That is, "L-amino acid ligase gene" may mean any polynucleotide that codes for L-amino acid ligase. The L-amino acid ligase gene may be DNA, RNA, or a combination thereof. The L-amino acid ligase gene may be single-stranded or double-stranded. The L-amino acid ligase gene may be single-stranded DNA or single-stranded RNA. The L-amino acid ligase gene may be double-stranded DNA, double-stranded RNA, or a hybrid strand consisting of a DNA strand and an RNA strand. The L-amino acid ligase gene may contain both DNA residues and RNA residues in a single polynucleotide chain. When the L-amino acid ligase gene contains RNA, the descriptions of DNA such as the base sequence exemplified above may be appropriately interpreted to correspond to RNA. The form of the L-amino acid ligase gene can be appropriately selected according to various conditions such as its intended use.
[0029] <3> Production of L-amino acid ligase L-amino acid ligase can be produced using organisms capable of producing L-amino acid ligase. That is, another embodiment of the present invention is a method for producing L-amino acid ligase, which includes culturing an organism capable of producing L-amino acid ligase in a culture medium to generate L-amino acid ligase, and recovering the L-amino acid ligase from the culture. This method is also referred to as "the method for producing L-amino acid ligase of this embodiment." L-amino acid ligase can also be produced by expressing the L-amino acid ligase gene in a cell-free protein synthesis system.
[0030] Organisms capable of producing L-amino acid ligases include hosts into which the L-amino acid ligase gene has been introduced.
[0031] The host into which the L-amino acid ligase gene is introduced is not particularly limited as long as it can express a functional L-amino acid ligase. Examples of hosts include bacteria, actinomycetes, yeasts, fungi, plant cells, insect cells, and animal cells. Preferred hosts include microorganisms such as bacteria and yeasts. More preferred hosts include bacteria. Examples of bacteria include Gram-negative bacteria and Gram-positive bacteria. Examples of Gram-negative bacteria include bacteria belonging to the Enterobacteriaceae family, such as Escherichia, Enterobacter, and Pantoea. Examples of Gram-positive bacteria include Corynebacteria, such as Bacillus and Corynebacterium. Among these, Escherichia coli can be preferably used as a host. Furthermore, when L-amino acid ligase is secreted and produced outside the bacterial cell, Corynebacterium-type bacteria such as Corynebacterium glutamicum and Corynebacterium stationis can be suitably used as hosts (WO2013 / 065869, WO2013 / 065772, WO2013 / 118544, WO2013 / 062029).
[0032] The L-amino acid ligase gene having the nucleotide sequence shown in Sequence ID No. 6 can be obtained by cloning from an organism that possesses the L-amino acid ligase gene. Bacillus pumilus is an example of an organism that possesses the L-amino acid ligase gene. Nucleic acids such as genomic DNA or cDNA containing the gene can be used for cloning. The L-amino acid ligase gene can also be obtained by chemical synthesis (Gene, 60(1), 115-127 (1987)).
[0033] Variants can be obtained by modifying the L-amino acid ligase gene shown in Sequence ID No. 6, which was obtained as described above. Gene modification can be carried out by known methods. For example, a target mutation can be introduced into the target site of the gene by site-directed mutagenesis. That is, for example, by site-directed mutagenesis, the coding region of the gene can be modified to include substitutions of amino acid residues at specific sites such as positions 78, 90, 104, 112, 285, 361, 374, and 378 of the encoded protein, or to include substitutions, deletions, insertions, or additions of other amino acid residues. Site-directed mutagenesis methods include those using PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and those using phages (Kramer, W. and Frits, HJ, Meth. in Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. in Enzymol., 154, 367 (1987)). In addition, variants of L-amino acid ligase genes can also be obtained, for example, by mutational treatment. Mutation methods include in vitro treatment of the gene itself with hydroxylamine, etc., treatment of microorganisms that possess the L-amino acid ligase gene, such as bacteria belonging to the Actinobacteria class, with X-rays, ultraviolet light, or mutagens such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), etc., and methods such as ella-prone PCR (Cadwell, RC PCR Meth. Appl. 2, 28 (1992)), DNA shuffling (Stemmer, WP Nature 370, 389 (1994)), and StEP-PCR (Zhao, H. Nature Biotechnol. 16, 258 (1998)).
[0034] The method for introducing the L-amino acid ligase gene into the host is not particularly limited. In the host, the L-amino acid ligase gene only needs to be expressible under the control of a promoter that functions in the host. In the host, the L-amino acid ligase gene may be located on a vector that autonomously replicates outside the chromosome, such as a plasmid, or it may be introduced onto the chromosome. The host may have only one copy of the L-amino acid ligase gene, or it may have two or more copies. The host may have only one type of L-amino acid ligase gene, or it may have two or more types of L-amino acid ligase genes.
[0035] The promoter for expressing the L-amino acid ligase gene is not particularly limited as long as it functions in the host. "Host-functioning promoter" means a promoter that has promoter activity in the host. The promoter may be of host origin or heterologous origin. The promoter may be the intrinsic promoter of the L-amino acid ligase gene or a promoter of another gene. The promoter may be a potent promoter to achieve high expression levels. Specific examples of potent promoters that function in Enterobacteriaceae bacteria such as Escherichia coli include the T7 promoter, trp promoter, trc promoter, lac promoter, tac promoter, tet promoter, araBAD promoter, rpoH promoter, PR promoter, and PL promoter. Furthermore, potent promoters that function in Corynebacteria include the artificially modified P54-6 promoter (Appl. Microbiol. Biotechnol., 53, 674-679(2000)), the pta, aceA, aceB, adh, and amyE promoters that can be induced in Corynebacteria with acetic acid, ethanol, pyruvate, etc., and the cspB, SOD, and tuf((EF-Tu)) promoters, which are potent promoters with high expression levels in Corynebacteria (Journal of Biotechnology 104 (2003) 311-323, Appl Environ Microbiol. 2005 Dec;71(12):8587-96.), the lac promoter, the tac promoter, and the trc promoter. In addition, highly active versions of conventional promoters may be obtained and used by using various reporter genes. For example, the activity of the promoter can be increased by bringing the -35 and -10 regions within the promoter region closer to the consensus sequence (International Publication No. 00 / 18935).Examples of highly active promoters include various tac-like promoters (Katashkina JI et al. Russian Federation Patent application 2006134574) and the pnlp8 promoter (WO2010 / 027045). Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al.'s paper (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)), among others.
[0036] Furthermore, a terminator for transcription termination can be placed downstream of the L-amino acid ligase gene. The terminator is not particularly limited as long as it functions in the host. The terminator may be of host origin or of heterologous origin. The terminator may be a terminator specific to the L-amino acid ligase gene or a terminator of another gene. Specific examples of terminators include, for example, the T7 terminator, T4 terminator, fd phage terminator, tet terminator, and trpA terminator.
[0037] The L-amino acid ligase gene can be introduced into a host, for example, using a vector containing the gene. A vector containing the L-amino acid ligase gene is also called an L-amino acid ligase gene expression vector or a recombinant vector. An L-amino acid ligase gene expression vector can be constructed, for example, by ligating a DNA fragment containing the L-amino acid ligase gene with a vector that functions in the host. By transforming the host with the L-amino acid ligase gene expression vector, a transformant into which the vector has been introduced can be obtained, that is, the gene can be introduced into the host. As the vector, a vector capable of autonomous replication within the host cell can be used. The vector is preferably a multicopy vector. Furthermore, the vector is preferably equipped with markers such as antibiotic resistance genes for selecting transformants. The vector may also be equipped with a promoter or terminator for expressing the inserted gene. The vector may be, for example, a bacterial plasmid-derived vector, a yeast plasmid-derived vector, a bacteriophage-derived vector, a cosmid, or a phagemid. Examples of vectors capable of autonomous replication in Enterobacteriaceae bacteria such as Escherichia coli include pUC19, pUC18, pHSG299, pHSG399, pHSG398, pBR322, pSTV29 (all available from Takara Bio), pACYC184, pMW219 (Nippon Gene), pTrc99A (Pharmacia), pPROK vector (Clontech), pKK233-2 (Clontech), pET vector (Novagen), pQE vector (Qiagen), pACYC, and the broad-host-range vector RSF1010.Specifically, examples of vectors capable of autonomous replication in Corynebacteria include pHM1519 (Agric, Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol. Chem., 48, Examples include plasmids having drug resistance genes that are improved versions of these (2901-2903 (1984)); plasmid pCRY30 described in Japanese Patent Publication No. 3-210184; plasmids pCRY21, pCRY2KE, pCRY2KX, pCRY31, pCRY3KE, and pCRY3KX described in Japanese Patent Publication No. 2-72876 and U.S. Patent No. 5,185,262; plasmids pCRY2 and pCRY3 described in Japanese Patent Publication No. 1-191686; pAJ655, pAJ611, and pAJ1844 described in Japanese Patent Publication No. 58-192900; pCG1 described in Japanese Patent Publication No. 57-134500; pCG2 described in Japanese Patent Publication No. 58-35197; and pCG4 and pCG11 described in Japanese Patent Publication No. 57-183799. When constructing an expression vector, for example, the L-amino acid ligase gene containing a specific promoter region may be directly incorporated into the vector, the coding region of the L-amino acid ligase may be bound downstream of the promoter as described above before being incorporated into the vector, or the coding region of the L-amino acid ligase may be incorporated downstream of a promoter already present on the vector.
[0038] Vectors, promoters, and terminators usable in various organisms are described in detail in, for example, "Basic Microbiology Course 8: Genetic Engineering," Kyoritsu Shuppan, 1987, and these can be utilized.
[0039] Furthermore, L-amino acid ligase genes can be introduced, for example, onto host chromosomes. Gene introduction onto chromosomes can be carried out, for example, using homologous recombination. Examples of gene introduction methods using homologous recombination include Red-driven integration (WO2005 / 010175), transduction using phages such as P1 phage, methods using conjugate vectors, and methods using suicide vectors that do not have replication origins and function within the host. Only one copy of the gene may be introduced, or two or more copies may be introduced. For example, multiple copies of the gene can be introduced into a chromosome by performing homologous recombination targeting a sequence that has multiple copies in the chromosome. Examples of sequences that have multiple copies in the chromosome include repetitive DNA sequences and inverted repeats located at both ends of transposons. Furthermore, genes can be randomly introduced onto chromosomes using methods such as transposons or Mini-Mu (Japanese Patent Publication No. 2-109985, US5,882,888, EP805867B1). When introducing genes into chromosomes, for example, an L-amino acid ligase gene containing a specific promoter region may be directly incorporated into the chromosome, the coding region of the L-amino acid ligase may be bound downstream of such a promoter before being incorporated into the chromosome, or the coding region of the L-amino acid ligase may be incorporated downstream of a promoter that is already present on the chromosome.
[0040] The introduction of a gene onto a chromosome can be confirmed, for example, by Southern hybridization using a probe with a nucleotide sequence complementary to all or part of the gene, or by PCR using primers created based on the nucleotide sequence of the gene.
[0041] The transformation method is not particularly limited, and conventionally known methods can be used. Examples of transformation methods include the method of treating receptor bacterial cells with calcium chloride to increase DNA permeability, as reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol. Biol. 1970, 53, 159-162), and the method of preparing competent cells from cells in the growth stage and introducing DNA, as reported for Bacillus subtilis (Duncan, CH, Wilson, GA and Young, FE, 1997. Gene 1: 153-167). Furthermore, as a transformation method, a method is also applicable in which the cells of DNA-receiving bacteria are made into protoplasts or spheroplasts that readily incorporate recombinant DNA, as is known for Bacillus subtilis, actinomycetes, and yeasts, and recombinant DNA is introduced into the DNA-receiving bacteria (Chang, S. and Choen, SN, 1979. Mol. Gen. Genet. 168: 111-115; Bibb, MJ, Ward, JM and Hopwood, OA 1978. Nature 274: 398-400; Hinnen, A., Hicks, JB and Fink, GR 1978. Proc. Natl. Acad. Sci. USA 75: 1929-1933). In addition, as a transformation method, the electropulse method (Japanese Patent Publication No. 2-207791), as reported for Corynebacteria, can also be used.
[0042] By culturing an organism having the ability to produce the above-described L-amino acid ligase in a medium, L-amino acid ligase can be expressed. At that time, gene expression induction may be carried out as necessary. The culturing conditions of the organism and the conditions for gene expression induction may be appropriately selected according to various conditions such as the type of marker, the type of promoter, and the type of the organism. The medium used for culturing is not particularly limited as long as the organism can grow and L-amino acid ligase can be expressed. As the medium, for example, a normal medium containing a carbon source, a nitrogen source, a sulfur source, inorganic ions, and other organic components as necessary can be used.
[0043] Examples of the carbon source include sugars such as glucose, fructose, sucrose, molasses, and hydrolyzates of starch; alcohols such as glycerol and ethanol; and organic acids such as fumaric acid, citric acid, and succinic acid.
[0044] Examples of the nitrogen source include inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate; organic nitrogen such as soybean hydrolyzate; ammonia gas; and aqueous ammonia.
[0045] Examples of the sulfur source include inorganic sulfur compounds such as sulfates, sulfites, sulfides, hyposulfites, and thiosulfates.
[0046] Examples of the inorganic ions include calcium ions, magnesium ions, manganese ions, potassium ions, iron ions, and phosphate ions.
[0047] Examples of the other organic components include organic micronutrient sources. Examples of the organic micronutrient sources include required substances such as vitamin B1 and yeast extracts containing them.
[0048] The culture method may be liquid culture or solid culture, but liquid culture is preferred. Culture is preferably carried out aerobically. Examples of aerobic culture methods include shaking culture and aerobic deep culture using a jar fermenter. The oxygen concentration in this case may be adjusted to, for example, 5 to 50% of the saturation concentration, preferably about 10%. The culture temperature may be, for example, 10 to 50°C, preferably 20 to 45°C, more preferably 25 to 40°C. The pH of the culture medium may be adjusted to, for example, 3 to 9, preferably 5 to 8. Inorganic or organic acidic or alkaline substances, such as calcium carbonate, ammonia gas, or ammonia water, can be used to adjust the pH. The culture period may be, for example, 12 hours to 20 days, preferably 1 to 7 days.
[0049] By culturing under the conditions described above, a culture containing L-amino acid ligase can be obtained. The L-amino acid ligase accumulates, for example, within the host bacterial cells and / or in the culture medium. "Bacterial cells" may be replaced with "cells" as appropriate, depending on the type of host. Depending on the host used and the design of the L-amino acid ligase gene, it is also possible to accumulate L-amino acid ligase in the periplasm or to secrete and produce L-amino acid ligase outside the bacterial cells.
[0050] L-amino acid ligase may be used as is contained in the culture, or it may be recovered from the culture by known methods and used. Alternatively, it may be isolated and purified from bacterial cells, etc., and used as a crude enzyme fraction or purified enzyme.
[0051] In other words, for example, if L-amino acid ligase accumulates in the host bacterial cells, the L-amino acid ligase can be recovered by appropriately disrupting, lysing, or extracting the bacterial cells. The bacterial cells can be recovered from the culture by centrifugation or the like. Disruption, lysing, or extraction of cells can be carried out by known methods. Examples of such methods include sonication, dynomylation, bead disruption, French press disruption, and lysozyme treatment. These methods may be used individually, or two or more may be used in appropriate combinations. Also, for example, if L-amino acid ligase accumulates in the culture medium, the culture supernatant can be obtained by centrifugation or the like, and the L-amino acid ligase can be recovered from the culture supernatant.
[0052] L-amino acid ligases can be purified by known methods used for enzyme purification. Examples of such methods include ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration chromatography, and isoelectric point precipitation. These methods may be used individually or in combination of two or more methods as appropriate. The purification of L-amino acid ligases can be achieved to the desired degree.
[0053] Purified L-amino acid ligase can be used as a catalyst for the dehydration condensation of two free amino acid molecules to synthesize a dipeptide. L-amino acid ligase may be used in its free state or as an immobilized enzyme attached to a solid phase such as a resin.
[0054] Furthermore, the fraction is not limited to purified L-amino acid ligase, but any fraction containing L-amino acid ligase may be used as "L-amino acid ligase" in the condensation reaction of free amino acids. The fraction containing L-amino acid ligase is not particularly limited as long as it is contained in such a way that the L-amino acid ligase can act on the substrate amino acid. Examples of such fractions include cultures of organisms capable of producing L-amino acid ligase, bacterial cells recovered from the culture (cultured bacterial cells), lysates of the bacterial cells, extracts of the bacterial cells (cell-free extracts), immobilized bacterial cells obtained by immobilizing the bacterial cells on carriers such as acrylamide and carrageenan, culture supernatants recovered from the culture, partially purified products thereof (crude products), and combinations thereof. Any of these fractions may be used alone or together with purified L-amino acid ligase.
[0055] The recovered L-amino acid ligase may be formulated as appropriate. The dosage form is not particularly limited and can be set as appropriate according to various conditions such as the intended use of the L-amino acid ligase. Examples of dosage forms include liquids, suspensions, powders, tablets, pills, and capsules. In formulation, pharmacologically acceptable additives such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, odoring agents, fragrances, diluents, and surfactants may be used.
[0056] <4> Production of Dipeptides Another embodiment of the present invention is a method for producing dipeptides, comprising reacting the L-amino acid ligase with one or more amino acids. The dipeptide is not particularly limited, but may be an imidazole peptide, for example. An imidazole peptide is a dipeptide having an imidazole group in its structure, and examples include carnosine, anserine, balenine, homocarnosine, etc. The dipeptide may also be alanylglutamine. Of these, carnosine or alanylglutamine are particularly preferred as the dipeptide.
[0057] The one or more amino acids used as substrates include one or more amino acids selected from the group consisting of L-amino acids, glycine, and β-alanine. Examples of L-amino acids include L-alanine, L-glutamine, L-glutamic acid, L-valine, L-leucine, L-isoleucine, L-proline, L-phenylalanine, L-tryptophan, L-methionine, L-serine, L-threonine, L-cysteine, L-asparagine, L-tyrosine, L-lysine, L-arginine, L-histidine, L-aspartic acid, L-α-aminobutyric acid, L-azaserine, L-theanine, L-4-hydroxyproline, L-3-hydroxyproline, L-ornithine, L-citrulline, and L-6-diazo-5-oxonolleucine. It is particularly preferable to act the L-amino acid ligase on β-alanine and L-histidine. In this case, carnosine is synthesized as a dipeptide. Alternatively, it is also preferable to act on L-amino acid ligase with L-alanine and L-glutamine. In this case, alanylglutamine is synthesized as a dipeptide.
[0058] To allow L-amino acid ligase to act on one or more amino acids, for example, the L-amino acid ligase, one or more amino acids, and ATP can be added to an aqueous solution and reacted. The amino acid used as a substrate can be added in such a way that its content in the aqueous solution is 0.1 to 100 mM, preferably 1 to 30 mM. The L-amino acid ligase is preferably added in an amount of 0.01 g to 100 g, and more preferably 0.1 g to 10 g, per mol of the amino acid used as a substrate. When acting on two or more amino acids, the amount added per mol of the total volume of those amino acids should be within the above range. ATP can be added in such a way that its content in the aqueous solution is 1 to 1000 mM.
[0059] The aqueous solution may contain cofactors and stabilizers. Examples of cofactors include divalent metal salts (e.g., magnesium salts such as magnesium sulfate and magnesium chloride). Examples of stabilizers include antioxidants (e.g., reducing agents such as dithiothreitol and mercaptoethanol). The aqueous solution may also contain surfactants for purposes such as maintaining enzyme stability and improving reaction rate. Examples of surfactants include nonionic surfactants (e.g., Triton® X-100) and ionic surfactants. Examples of ionic surfactants include cationic surfactants, anionic surfactants, and amphoteric surfactants. The aqueous solution may also contain polyethylene glycol for purposes such as improving reaction rate.
[0060] The temperature of the aqueous solution is not particularly limited as long as the L-amino acid ligase is active, and examples include 2 to 50°C, 16 to 50°C, and 25 to 50°C. The pH of the aqueous solution may be, for example, 5 to 9.5, and preferably 7.5 to 9.0. The time for which the L-amino acid ligase is allowed to act is not particularly limited, and examples include 1 to 72 hours.
[0061] The present invention will be described more specifically below based on examples, but the present invention is not limited to these examples.
[0062] <Experiment 1: Design of L-amino acid ligase> Using a mutant (BaYwfE_I112V / H378R) of the L-amino acid ligase Bacillus subtilis ATTC21555 strain YwfE (Sequence ID 7, hereinafter also referred to as "BaYwfE") in which the isoleucine residue at position 112 is replaced with a valine residue and the histidine residue at position 378 is replaced with arginine as a template, 3035 similar sequences were obtained from Blastp and used as a sequence library. Here, the analysis conditions for Blastp were set to Max target sequences of 5000 and the expected threshold value to 1.0E-6. Regarding the similar sequences obtained above, please refer to Reference 1 (Miyata, A., Chisuga, T., Kambe, A., Miyata, R., Kawamura, Y., Takeda, H., Ito, S., and Nakano, S. (2024). Design of Ancestral Sortase E that is Applicable in Protein Biomaterial Synthesis. ACS Catalysis, 14(5), 3514-3523) and Reference 2 (Nakano, S., Niwa, M., Asano, Y., and Ito, S. (2019). Following the evolutionary track of a highly specific l-arginine oxidase by reconstruction and biochemical analysis of ancestral and native enzymes. Applied and Environmental Microbiology, 85(11)). By applying the method used in e00345-19.), we identified six amino acid residues (positions 90, 135, 181, 272, 312, and 349) that are important for the classification of L-amino acid ligases.The eight main combinations of these residues are: (1) Ala at position 90, Ala at position 135, Trp at position 181, Thr at position 272, Ile at position 312, Met at position 349; (2) Asp at position 90, Ala at position 135, Trp at position 181, Thr at position 272, Ile at position 312, Met at position 349; (3) Asp at position 90, Ala at position 135, Trp at position 181, Val at position 272, Leu at position 312, Met at position 349; (4) Asp at position 90, Ala at position 135, Tyr at position 181, Thr at position 272, Val at position 312, Ala at position 349; (5) Glu at position 90, Ala at position 135, 181 Sequences belonging to the following groups were obtained from a sequence library: (6) Trp at position 272, Thr at position 272, Ile at position 312, Leu at position 349, (7) Glu at position 90, Ala at position 135, Trp at position 181, Thr at position 272, Ile at position 312, Met at position 349, (8) Glu at position 90, Ala at position 135, Trp at position 181, Val at position 272, Leu at position 312, Met at position 349, (8) Glu at position 90, Ala at position 135, Tyr at position 181, Thr at position 272, Ile at position 312, Leu at position 349. A total of 16 artificial sequences were obtained for each group using the consensus design method and the ancestor sequence reconstruction method. Two sequences (SEQ ID NO: 2 and SEQ ID NO: 3) that showed high identity with the template sequence (73% and 69%) were named FcYwfE and AncYwfE-3, respectively, and used in the following experiments. Hereinafter, FcYwfE and AncYwfE-3 may be referred to as "modified YwfE". When the protein with the amino acid sequence showing the closest identity to modified YwfE was investigated, it was found to be the ATP-grasp domain-containing protein from Bacillus pumilus (WP_099727991, SEQ ID NO: 1, also referred to as "BpYwfE" below). Table 1 above shows the mutation sites of FcYwfE and AncYwfE-3 compared to BpYwfE. FcYwfE and AncYwfE-3 share eight common mutations with BpYwfE: Q78H, G90E, M104I, I112V, K285E, E361D, M374I, and H378R.
[0063] <Experiment 2: Construction of YwfE and modified YwfE expression strains> A fragment of the ywfE gene derived from Bacillus subtilis ATCC21555 strain, which was codon-optimized for E. coli, with an 8×His tag sequence added to the C-terminus, was inserted into the BamHI and XhoI sites of the E. coli expression vector pET-28a(+) to construct a BaYwfE expression plasmid. The obtained plasmid was introduced into the E. coli BL21(DE3) strain, and the resulting strain was designated as the BaYwfE strain. Using the same method, a strain was obtained in which a plasmid containing a BaYwfE mutant (hereinafter referred to as "BaYwfE_I112V / H378R") containing two amino acid substitutions (I112V and H378R) was introduced, and this strain was designated as the BaYwfE_I112V / H378R strain. Furthermore, a strain was obtained by introducing a plasmid containing a YwfE mutant (i.e., a BpYwfE mutant) of Bacillus pumilus NRRL B-12025 strain having two amino acid substitutions (I112V and H378R), and this strain was designated as the BpYwfE_I112V / H378R strain. In addition, strains were obtained by introducing plasmids containing FcYwfE and AncYwfE-3, respectively, and these strains were designated as the FcYwfE strain and the AncYwfE-3 strain.
[0064] <Experiment 3: Comparison of Carnosine Synthesis Activity Using Cell Extracts> The BaYwfE strain, BaYwfE_I112V / H378R strain, and BpYwfE_I112V / H378R strain prepared in Experiment 2 were each inoculated into 2 mL of kanamycin-containing LB medium and cultured overnight in test tubes at 37°C and 120 rpm. Subsequently, 50 μL of the pre-culture solution was inoculated into 5 mL of kanamycin-containing LB medium and cultured in test tubes at 37°C and 120 rpm for 2 hours. Isopropyl-β-thiogalactopyranoside (IPTG) was then added to a final concentration of 0.4 mM, and the culture was further cultured overnight at 30°C and 120 rpm. 1.5 mL of the obtained culture solution was placed in a microtube, centrifuged (5000 × g, 10 minutes) to collect the cells, and then washed once with 50 mM Tris-HCl (pH 9.0). The obtained bacterial cells were suspended in 500 μL of 50 mM Tris-HCl (pH 9.0) and sonicated. The resulting lysate was centrifuged (14400 × g, 4°C, 15 minutes) to remove residue and obtain a cell extract.
[0065] Using the obtained cell extract as the crude enzyme solution, 200 μL of a reaction solution with the following composition was prepared and reacted at 37°C for 1 hour. The reaction was then stopped by adding 40 μL of 0.5 M EDTA (pH 8.0). 20% (v / v) crude enzyme solution, 60 mM ATP, 30 mM MgSO4, 30 mM β-alanine, 30 mM L-histidine, 50 mM Tris-HCl buffer (pH 9.0) (adjusted to pH 9.0 with 8N NaOH).
[0066] After the reaction, the amount of carnosine produced was quantified by high-performance liquid chromatography (HPLC) under the following conditions. The results are shown in Figure 1. Compared to BaYwfE, BaYwfE_I112V / H378R and BpYwfE_I112V / H378R, which have the I112V and H378R mutations, showed higher carnosine synthesis activity, and it was found that BaYwfE_I112V / H378R had the highest carnosine synthesis activity. In other words, it was found that the eight mutations common to FcYwfE and AncYwfE-3—Q78H, G90E, M104I, I112V, K285E, E361D, M374I, and H378R—significantly improve the activity of YwfE. (HPLC conditions) Column: Inertsil ODS-4 150 mm × 4.6 mm I.D., 5 μm Mobile phase: 200 mM ammonium dihydrogen phosphate, 5.0 mM sodium 1-octanesulfonate 4% (v / v), acetonitrile pH 2.0 (adjusted with phosphoric acid) Temperature: 40°C Detector: UV: 220 nm Flow rate: 0.8 mL / min Injection volume: 10 μL Analysis time: 30 min
[0067] <Experiment 4: In vitro specific activity evaluation of modified YwfE> The BaYwfE_I112V / H378R strain, FcYwfE strain, and AncYwfE-3 strain prepared in Experiment 2 were inoculated into 2 mL of Overnight Expression® Instant TB Medium and cultured overnight in test tubes at 37°C and 120 rpm. 2 mL of the resulting culture solution was placed in a microcentrifuge tube and centrifuged (10000 × g, 4°C for 3 minutes) to collect the cells. Subsequently, enzyme solutions were obtained using the Xtractor® Buffer Kit (Clontech Laboratories, Inc.) and Capturem His-tagged purification mini prep kit (Clontech Laboratories, Inc.) according to the user manuals provided with the kits.
[0068] The protein concentration of the obtained enzyme solution was quantified using Quick Start® Bradford Protein Assay (Bio-Rad Laboratories, Inc.). Bovine serum albumin (BSA) of known concentration was used as the standard protein. 200 μL of the reaction solution with the following composition was prepared and reacted at 37°C for 2 hours. The reaction was stopped by adding 40 μL of 0.5 M EDTA (pH 8.0). 50 μg / mL Enzyme 12.5 mM ATP 12.5 mM MgSO4 12.5 mM β-Alanine 12.5 mM L-Histidine 100 mM Tris-HCl buffer (pH 9.0) (adjusted to pH 9.0 with 8N NaOH)
[0069] After the reaction, the amount of carnosine produced was quantified by high-performance liquid chromatography (HPLC), as in Experiment 3. The results are shown in Figure 2. Compared to BaYwfE_I112V / H378R, FcYwfE and AncYwfE-3 showed significantly higher carnosine synthesis activity.
[0070] <Experiment 5: Comparison of Alanylglutamine Synthesis Activity Using Cell Extracts> The BaYwfE_I112V / H378R, FcYwfE, and AncYwfE-3 strains prepared in Experiment 2 were inoculated into 5 mL of Overnight Expression® Instant TB Medium and cultured in test tubes at 37°C and 120 rpm for 24 hours. The culture solution was collected from the obtained culture medium so that the cell volume at OD562 nm was 10, placed in a microcentrifuge tube, and centrifuged (15000 × g, 4°C for 3 minutes) to collect the cells. Subsequently, an enzyme solution was obtained using the Xtractor® Buffer Kit (Clontech Laboratories, Inc.), with some modifications to the user manual included with the kit. Specifically, the cells were suspended in 1 mL of physiological saline and washed by centrifuging (15000 × g, 4°C for 3 minutes). The collected bacterial cells were suspended in 978 μL of xTractor™ Buffer, 2 μL of 5 units / mL DNase I solution, and 10 μL of 100× Lysozyme solution, and allowed to stand at room temperature for 15 minutes. The supernatant obtained by centrifugation was then used as the enzyme solution.
[0071] The protein concentration of the obtained enzyme solution was quantified using Quick Start® Bradford Protein Assay (Bio-Rad Laboratories, Inc.). Bovine serum albumin (BSA) of known concentration was used as the standard protein. 200 μL of the reaction solution with the following composition was prepared and reacted at 37°C for 2 hours. The reaction was stopped by adding 40 μL of 0.5 M EDTA (pH 8.0). 20 μg / mL enzyme 48 mM ATP 24 mM MgSO4 24 mM L-alanine 24 mM L-glutamine 40 mM Tris-HCl buffer (pH 9.0) (adjusted to pH 9.0 with 8N NaOH)
[0072] After the reaction, the product was quantified using high-performance liquid chromatography (HPLC) and a Hitachi High-Tech Science L-8900 high-performance amino acid analyzer under the following conditions. The results are shown in Figure 3. Both FcYwfE and AncYwfE-3 showed alanylglutamine synthesis activity. Column: SCX UG80 4.6 mm × 150 mm, 50 μm (Osaka soda) Mobile phase: 50 mM sodium dihydrogen phosphate pH 2.5 (adjusted with phosphoric acid) Temperature: 40°C Detector: UV: 210 nm Flow rate: 1.9 mL / min Injection volume: 10 μL Analysis time: 60 min
Claims
1. Proteins described in (A), (B), or (C) below. (A) Proteins containing an amino acid sequence in the amino acid sequence shown in Sequence ID No. 1, having the following substitutions: a substitution from the glutamine residue at position 78 to histidine, a substitution from the glycine residue at position 90 to glutamic acid, a substitution from the methionine residue at position 104 to isoleucine, a substitution from the isoleucine residue at position 112 to valine, a substitution from the lysine residue at position 285 to glutamic acid, a substitution from the glutamic acid residue at position 361 to aspartic acid, a substitution from the methionine residue at position 374 to isoleucine, and a substitution from the histidine residue at position 378 to arginine; (B) A protein having L-amino acid ligase activity, comprising an amino acid sequence in which the amino acid sequence shown in Sequence ID No. 1 has the substitution of a glutamine residue at position 78 to histidine, a glycine residue at position 90 to glutamic acid, a methionine residue at position 104 to isoleucine, an isoleucine residue at position 112 to valine, a lysine residue at position 285 to glutamic acid, a glutamic acid residue at position 361 to aspartic acid, a methionine residue at position 374 to isoleucine, and a histidine residue at position 378 to arginine, wherein one or more amino acid residues other than those substituted are substituted, deleted, inserted, or added; (C) A protein having 90% or more identity with an amino acid sequence having the substitutions described above, and having L-amino acid ligase activity, in the amino acid sequence shown in Sequence ID No. 1, the substitution of the glutamine residue at position 78 to histidine, the glycine residue at position 90 to glutamic acid, the methionine residue at position 104 to isoleucine, the isoleucine residue at position 112 to valine, the lysine residue at position 285 to glutamic acid, the glutamic acid residue at position 361 to aspartic acid, the methionine residue at position 374 to isoleucine, and the histidine residue at position 378 to arginine.
2. The amino acid sequence is subject to the following substitutions: substitution of alanine at position 26 to glutamic acid, substitution of serine at position 67 to aspartic acid, substitution of proline at position 82 to aspartic acid, substitution of leucine at position 92 to isoleucine, substitution of arginine at position 94 to lysine, substitution of alanine at position 101 to valine, substitution of valine at position 153 to isoleucine, substitution of glutamine at position 156 to lysine, substitution of serine at position 162 to glutamic acid, substitution of aspartic acid at position 198 to glutamic acid, substitution of asparagine at position 215 to lysine, substitution of alanine at position 232 to glutamic acid, The protein according to claim 1, further comprising a substitution of alanine at position 243 to serine, a substitution of glutamine at position 287 to lysine, a substitution of arginine at position 295 to lysine, a substitution of glutamine at position 365 to glutamic acid, a substitution of tyrosine at position 372 to histidine, a substitution of valine at position 402 to isoleucine, a substitution of lysine at position 418 to phenylalanine, a substitution of valine at position 420 to alanine, a substitution of serine at position 422 to alanine, a substitution of threonine at position 463 to lysine, a substitution of isoleucine at position 464 to leucine, and a deletion of glycine at position 473.
3. The protein according to claim 1, wherein the amino acid sequence further comprises a substitution of a glutamic acid residue at position 239 with aspartic acid, an insertion of threonine at position 474, an insertion of leucine at position 475, and an insertion of glutamic acid at position 476.
4. Proteins described in (A'), (B'), or (C') below: (A') Proteins containing the amino acid sequence shown in SEQ ID NO: 2 or 3; (B') Proteins containing an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted, or added in the amino acid sequence shown in SEQ ID NO: 2 or 3, and which have L-amino acid ligase activity; (C') Proteins having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 2 or 3, and which have L-amino acid ligase activity.
5. The protein according to claim 4, which is a protein comprising the amino acid sequence shown in SEQ ID NO: 2 or 3.
6. A polynucleotide encoding a protein according to any one of claims 1 to 5.
7. A recombinant vector comprising the polynucleotide described in claim 6.
8. A transformant into which the recombinant vector described in claim 7 has been introduced.
9. A method for producing a dipeptide, comprising reacting a protein described in any one of claims 1 to 5 with one or more amino acids.
10. The manufacturing method according to claim 9, wherein the dipeptide is an imidazole peptide.
11. The manufacturing method according to claim 9, wherein the dipeptide is carnosine or alanylglutamine.