JP5978579B2 - Method for producing γ-Glu-XY - Google Patents

Description

  The present invention relates to a method for producing γ-Glu-XY (X represents an amino acid or amino acid derivative excluding Cys and its derivatives, Y represents an amino acid or amino acid derivative, and the same applies hereinafter).

  Calcium sensing receptor (CaSR), a G protein classified as class C, has been reported to respond to glutathione (γ-Glu-Cys-Gly) (GSH), one of γ-glutamyl compounds ( Non-patent document 1), but its physiological meaning has not been clarified. In addition, this CaSR was also present in the tongue cells and was thought to exhibit some taste response (Non-Patent Document 2), but recently, this CaSR is involved in human kokumi recognition. (Non-Patent Document 3). In the same document, it is reported that not only GSH, which has been recognized as a rich body, but also several γ-glutamyl compounds respond to CaSR as well.

  Peptides represented by the general formula γ-Glu-X or γ-Glu-X-Gly (X represents an amino acid or amino acid derivative excluding Cys and its derivatives), such as γ-Glu-Met, γ-Glu-Thr, It has been reported that γ-Glu-Val-Gly has a savoring effect (Patent Document 1). In addition, an S- or O-carboxyalkylated γ-glutamyl or β-aspartyl peptide ester group has been reported as a body taste substance (Patent Document 2). Although these peptides and esters impart a rich taste to foods like GSH, they do not have a reduced thiol group (—SH group) unlike GSH. In general, it is known that a substance having a reduced thiol group such as GSH is unstable, and its titer decreases with the formation of a disulfide bond (Patent Document 2). Therefore, a body taste imparting peptide such as γ-Glu-X or γ-Glu-X-Gly that does not have a reduced thiol group is considered to be useful in terms of stability and the like.

  As knowledge about the food containing γ-glutamyldipeptide, there is a report that various γ-glutamyldipeptides were detected from Gouda cheese that was aged for about 44 weeks (Non-patent Document 4). In this document, various γ-glutamyldipeptides such as γ-Glu-Ala, γ-Glu-Glu, and γ-Glu-Gln were detected, and the content of γ-glutamyldipeptide was 3590 μmol / kg dry weight at the maximum. It has been reported.

  Moreover, as another knowledge regarding γ-glutamyldipeptide, an example of fermentation broth analysis of Micrococcus glutamicus can be given (Non-patent Document 5). In the same document, it was reported that fermentation broth was applied to various columns to separate peptides and γ-Glu-Glu, γ-Glu-Val, and γ-Glu-Leu were isolated. It is unknown how much was contained in the broth.

  Thus, although there are some reports on foods and fermentation broths containing γ-Glu-X, no reports are found on those containing γ-Glu-X-Gly.

As research on biosynthesis of γ-glutamyl compounds, many studies on GSH or its precursor γ-Glu-Cys have been made. It is known that GSH is produced in vivo by γ-Glu-Cys being produced from Glu and Cys by γ-glutamylcysteine synthase, and Gly being further bound by glutathione synthase. It is known that γ-glutamylcysteine synthetase is encoded by GSH1 gene or gshA gene, and glutathione synthetase is encoded by GSH2 gene or gshB gene, and their properties have been investigated.

  For example, a method for enzymatically producing theanine (γ-glutamylanilide) using the side reaction of γ-glutamylcysteine synthetase of Escherichia coli has been reported (Patent Document 3, non-patent document). Reference 6). In addition, an example in which the substrate specificity of a γ-glutamylcysteine synthetase of Proteus mirabilis for amino acids other than Cys has been reported (Non-patent Document 7). As described above, γ-glutamylcysteine synthetase is known to have a wide substrate specificity.

  On the other hand, regarding the substrate specificity of glutathione synthase, there is a method for producing γ-Glu-Abu-Gly from γ-Glu-Abu and Gly using α-aminobutyric acid (Abu), which is a Cys derivative. It has been reported (Patent Document 4). In addition, with regard to the substrate specificity of glutathione synthase derived from Escherichia coli and three kinds of plants (soybean, wheat, and corn), examples of examining amino acids other than Gly as amino acids that bind to γ-Glu-Cys have been reported. (Non-patent document 8, Non-patent document 9). However, in all cases, in the biosynthesis of γ-Glu-XY, X is an examination example using γ-Glu-X as a substrate where X is Cys or its derivative, and X is an amino acid other than Cys and its derivative It is not shown whether γ-Glu-X can be a substrate for glutathione synthetase when it is an amino acid derivative. Only in the study of the substrate specificity of glutathione synthase derived from rat liver, γ-Glu-Val and γ-Glu-nVal (γ-glutamylnorvaline) have been tried (Non-patent Document 10). Γ-Glu-Val is not a substrate, and γ-Glu-nVal is a substrate, but its reactivity is very low, so this enzyme is a γ-glutamyl tripeptide using these γ-glutamyl compounds as substrates. It was considered difficult to apply to manufacturing.

  In addition, as a method for producing γ-Glu-XY, an example in which γ-Glu-Lys-Gly is generated using γ-GTP has been reported (Non-Patent Documents 11 and 12). When using, there is also a problem of how to prepare Lys-Gly used as a substrate.

WO2007 / 055393 WO2007 / 042288 WO2006 / 121055 Japanese Patent Laid-Open No. 62-163698

J. Biol. Chem., 281, 8864-8870 (2006) Biochem. Biophys. Res. Commun., 378, 414-418 (2009) J. Biol. Chem., 285, 1016-1022 (2010) J. Agric. Food. Chem., 57, 3738-3748 (2009) J. Biol. Chem., 240, 2508-2511 (1965) Biosci. Biotechnol. Biochem., 73, 2677-2683 (2009) Agric. Biol. Chem., 45, 2839-2845 (1981) Biochem. Biophys. Res. Commun., 352, 351-359 (2007) Biochem. J., 391, 567-574 (2005) J. Biol. Chem., 254, 5184-5190 (1979) Recent Highlights in Flavor Chemistry & Biology, [Proceedings of the Wartburg Symposium on Flavor Chemistry and Biology], 8th, Eisenach, Germany, Feb. 27-Mar. 2, 227-232 (2007) Annals of the New York Academy of Sciences, 613, 647-651 (1990)

  An object of the present invention is to provide a method for producing γ-Glu-XY (X represents an amino acid or an amino acid derivative excluding Cys and its derivatives, and Y represents an amino acid or an amino acid derivative).

  The present inventors act on glutathione synthase using γ-Glu-X (X represents an amino acid or an amino acid derivative excluding Cys and its derivatives) and Y (Y represents an amino acid or an amino acid derivative) as raw materials. As a result, it was found that γ-Glu-XY was produced. Further, the present inventors have found that γ-Glu-XY is produced by using γ-glutamylcysteine synthetase and glutathione synthetase with Glu, X, and Y as raw materials. Based on these findings, the present invention has been completed.

  That is, the present invention is as follows.

(1) Synthesis of glutathione synthetase and γ-glutamylcysteine in a raw material containing Glu, X (X represents an amino acid or amino acid derivative excluding Cys and its derivatives), and Y (Y represents an amino acid or amino acid derivative) A method for producing γ-Glu-XY, comprising a step of producing γ-Glu-XY by acting an enzyme.
(2) γ-Glu-X (X represents an amino acid or amino acid derivative excluding Cys and its derivatives) and Y (Y is an amino acid or amino acid derivative) and G- A method for producing γ-Glu-XY, comprising a step of producing Glu-XY.
(3) The method for producing γ-Glu-XY, wherein the glutathione synthetase and / or γ-glutamylcysteine synthetase is a culture of a microorganism having the activity of the enzyme or a processed product of the culture.
(4) The method for producing γ-Glu-XY, wherein the glutathione synthetase and / or γ-glutamylcysteine synthetase is a purified enzyme.
(5) The method for producing γ-Glu-XY, wherein the glutathione synthetase and / or γ-glutamylcysteine synthetase is an immobilized enzyme.
(6) The glutathione synthetase and / or γ-glutamylcysteine synthetase is selected from the group consisting of Saccharomyces cerevisiae, Candida utilis, and Escherichia coli. The method for producing γ-Glu-XY, wherein
(7) Glu, X (X represents an amino acid or an amino acid derivative excluding Cys and its derivatives), and Y (wherein the expression of glutathione synthetase gene and / or γ-glutamylcysteine synthetase gene is enhanced. Y represents an amino acid or an amino acid derivative), and is cultured in a medium supplemented with at least one amino acid and / or amino acid derivative selected from the group consisting of producing and accumulating γ-Glu-XY in the medium, A method for producing γ-Glu-XY, which comprises collecting γ-Glu-XY from a culture.
(8) At least one amino acid and / or amino acid selected from the group consisting of Glu, X (X represents an amino acid or an amino acid derivative excluding Cys and its derivatives), and Y (Y represents an amino acid or an amino acid derivative) Culturing a microorganism having the ability to produce a derivative and having enhanced expression of a glutathione synthetase gene and / or a γ-glutamylcysteine synthetase gene to produce γ-Glu-XY in the medium; A method for producing γ-Glu-XY, characterized by accumulating and collecting γ-Glu-XY from the culture.
(9) The method for producing γ-Glu-XY, wherein the microorganism is a microorganism belonging to the genus Escherichia or Corynebacterium.
(10) The method for producing γ-Glu-XY, wherein the microorganism belonging to the genus Escherichia is Escherichia coli and the microorganism belonging to the genus Corynebacterium is Corynebacterium glutamicum.
(11) The γ-Glu-XY, wherein the glutathione synthetase gene and / or the γ-glutamylcysteine synthetase gene is derived from a microorganism selected from the group consisting of Saccharomyces cerevisiae, Candida utilis, and Escherichia coli. Manufacturing method. (12) The method for producing γ-Glu-X-Gly, wherein Y is Gly.
(13) The method for producing γ-Glu-Val-Gly, wherein X is Val.
(14) The method for producing γ-Glu-nVal-Gly, wherein X is nVal.

  According to the present invention, γ-Glu-XY can be produced.

The figure which shows construction of plasmid pET-GSH2. The figure which shows construction of plasmid pET-gshB. The figure which shows construction of plasmid pET-GSH1. The figure which shows construction of plasmid pQE-gshA. The figure which shows construction of plasmid pET-ScGSH1.

(1) γ-Glu-XY produced by the present invention
According to the present invention, γ-Glu-XY, which is a γ-glutamyl tripeptide, can be produced. Glu in γ-Glu-XY represents glutamic acid. “-” Represents a peptide bond. “γ” in γ-Glu means that X is bonded via a carboxyl group at the γ-position of glutamic acid.

  “X” represents any of 19 natural amino acids excluding Cys among 20 natural amino acids, or an amino acid derivative excluding Cys derivatives. “Y” represents any of the 20 natural amino acids or amino acid derivatives. In the present invention, X and Y are independently selected.

  The 20 natural amino acids include glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), serine (Ser), threonine (Thr), cysteine (Cys), methionine. (Met), neutral amino acids such as asparagine (Asn), glutamine (Gln), proline (Pro), acidic amino acids such as aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg), histidine Basic amino acids such as (His) and aromatic amino acids such as phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp) are included.

  Examples of amino acid derivatives include norvaline (nVal), norleucine (nLeu), tert-leucine (tLeu), hydroxyproline (Hyp), α-aminobutyric acid, and β-aminobutyric acid. Among the amino acid derivatives, examples of the Cys derivative include α-aminobutyric acid and β-aminobutyric acid.

  In the present invention, Y is preferably Gly. That is, γ-Glu-X-Gly is preferable as γ-Glu-XY.

  When Y is Gly, X is preferably a hydrophobic amino acid or a hydrophobic amino acid derivative. Examples of the hydrophobic amino acid include Val, Ala, Leu, and Phe, and examples of the hydrophobic amino acid derivative include nVal. When X is a hydrophobic amino acid or a hydrophobic amino acid derivative, the effect of imparting the rich taste of γ-Glu-X-Gly is high.

  As γ-Glu-XY, γ-Glu-Val-Gly or γ-Glu-nVal-Gly is particularly preferable.

  In the present invention, one kind of γ-Glu-XY may be produced, or two or more kinds of γ-Glu-XY may be produced.

  The amino acids and amino acid derivatives are both L-forms. In the present invention, γ-Glu-XY and Glu, X, Y, and γ-Glu-X used as raw materials may all be free forms or salts thereof, or a mixture thereof. . Examples of the salt include sulfate, hydrochloride, carbonate, ammonium salt, sodium salt, and potassium salt.

(2) Enzyme used in the present invention Glutathione synthase used in the present invention recognizes γ-Glu-X as a substrate and binds to Y to catalyze a reaction that produces γ-Glu-XY. It does not specifically limit as long as it has activity. In the present invention, the activity that catalyzes the reaction is referred to as glutathione synthetase activity. The glutathione synthetase used in the present invention does not need to exhibit glutathione synthetase activity for all X and Y, but glutathione synthetase for the amino acid or amino acid derivative selected as X and the amino acid or amino acid derivative selected as Y. What is necessary is just to show activity. In addition, when multiple types of γ-Glu-XY are produced, it is necessary that a single glutathione synthase has an activity to catalyze a reaction that generates all of the multiple types of γ-Glu-XY. Alternatively, multiple types of glutathione synthetase may be used depending on X and / or Y. In the present invention, glutathione synthase has an activity of catalyzing the reaction of generating GSH or γ-Glu-Cys derivative-Gly from γ-Glu-Cys or γ-Glu-Cys derivative and Gly. You may or may not have.

The γ-glutamylcysteine synthetase used in the present invention is not particularly limited as long as it has an activity of recognizing X as a substrate and catalyzing a reaction for producing γ-Glu-X by binding to Glu. In the present invention, the activity catalyzing the reaction is referred to as γ-glutamylcysteine synthetase activity. The γ-glutamylcysteine synthetase used in the present invention does not have to exhibit γ-glutamylcysteine synthetase activity with respect to any X, but exhibits γ-glutamylcysteine synthetase activity with respect to the amino acid or amino acid derivative selected as X. That's fine. In addition, when multiple types of X are used as a substrate, it is not necessary for a single γ-glutamylcysteine synthetase to exhibit γ-glutamylcysteine synthetase activity for all of the multiple types of X. γ-glutamylcysteine synthetase may be used. In the present invention, γ-glutamylcysteine synthetase may have an activity of catalyzing a reaction of generating γ-Glu-Cys or γ-Glu-Cys derivative from Glu and Cys or Cys derivative. It does not have to be good.

  The glutathione synthetase used in the present invention and the γ-glutamylcysteine synthetase used in the present invention are collectively referred to as enzymes used in the present invention.

  Glutathione synthase is generally encoded by the GSH2 gene or the gshB gene. Hereinafter, glutathione synthetase encoded by the GSH2 gene may be referred to as “Gsh2 protein” or “Gsh2”. In addition, glutathione synthetase encoded by the gshB gene may be described as “GshB protein” or “GshB”.

  γ-glutamylcysteine synthetase is generally encoded by the GSH1 gene or the gshA gene. Hereinafter, γ-glutamylcysteine synthetase encoded by the GSH1 gene may be referred to as “Gsh1 protein” or “Gsh1”. In addition, γ-glutamylcysteine synthetase encoded by the gshA gene may be described as “GshA protein” or “GshA”.

  The origin of the enzyme used in the present invention is not particularly limited, and an enzyme derived from any organism such as a microorganism, a plant or an animal can be used, but an enzyme derived from a microorganism is preferred. Examples of the microorganism include eukaryotic microorganisms such as yeast, and bacteria such as enteric bacteria and coryneform bacteria. Hereinafter, the gene encoding the enzyme used in the present invention will be exemplified.

  The gshB gene of Escherichia coli K12 MG1655 strain corresponds to positions 3089900 to 3090850 in the genome sequence registered as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990) in the NCBI database. The gshA gene of the MG1655 strain corresponds to a complementary sequence of the 2812905 to 2814461 positions in the genomic sequence. The base sequence of the gshB gene of Escherichia coli K12 W3110 strain and the amino acid sequence encoded by the same gene are shown in SEQ ID NOs: 7 and 8, respectively. Further, the nucleotide sequence of the gshA gene of the W3110 strain and the amino acid sequence encoded by the same gene are shown in SEQ ID NOs: 17 and 18, respectively.

  The base sequences of the GSH1 gene and GSH2 gene of Saccharomyces cerevisiae are disclosed in Saccharomyces Genome Database (http://www.yeastgenome.org/), respectively. In addition, the base sequences of Candida utilis GSH1 and GSH2 genes are disclosed in US Pat. No. 7,553,638. The base sequence of the GSH2 gene of Saccharomyces cerevisiae S288C strain (ATCC No. 26108) and the amino acid sequence encoded by this gene are shown in SEQ ID NOs: 1 and 2, respectively. In addition, the base sequence of the GSH1 gene of Saccharomyces cerevisiae S288C strain (ATCC No. 26108) and the amino acid sequence encoded by the gene are shown in SEQ ID NOs: 21 and 22, respectively. Further, the base sequence of the GSH1 gene of Candida utilis ATCC22023 strain and the amino acid sequence encoded by the same gene are shown in SEQ ID NOs: 11 and 12, respectively.

Since the base sequence of the gene encoding glutathione synthetase or γ-glutamylcysteine synthetase may differ depending on the organism from which the enzyme is derived, the gene encoding them may be SEQ ID NO: 1, 7, 11, or It may be a variant of the base sequence shown in FIG. Variants of each gene can be searched by BLAST or the like with reference to the nucleotide sequence shown in SEQ ID NO: 1, 7, 11, or 17 (http://blast.genome.jp/). Moreover, the variant of each gene contains the homologue of each gene. As a homologue of each gene, for example, a base of SEQ ID NO: 1, 7, 11, or 17 using a chromosome of any organism, for example, a eukaryotic microorganism such as yeast, or a bacterium such as intestinal bacteria or coryneform bacteria, as a template. Examples include genes that can be amplified by PCR using synthetic oligonucleotides prepared on the basis of sequences.

  Moreover, as long as the gene encoding the enzyme used in the present invention encodes a protein having glutathione synthetase activity or γ-glutamylcysteine synthetase activity, the amino acid sequence of the protein encoded by the above gene, for example, the sequence A gene encoding a protein having a sequence including substitution, deletion, insertion or addition of one or several amino acids at one or several positions in the amino acid sequence of No. 2, 8, 12, or 18. May be. The “one or several” differs depending on the position of the protein in the three-dimensional structure of the amino acid residue and the type of the amino acid residue, but specifically preferably 1 to 20, more preferably 1 to 10, More preferably, it means 1-5. One or several amino acid substitutions, deletions, insertions or additions described above are conservative mutations in which the function of the protein is maintained normally. A typical conservative mutation is a conservative substitution. Conservative substitution is, for example, when the substitution site is an aromatic amino acid, between Phe, Trp, and Tyr, and when the substitution site is a hydrophobic amino acid, between Leu, Ile, and Val, a polar amino acid Is an amino acid having a hydroxyl group between Lys, Arg and His when it is a basic amino acid, and between Asp and Glu when it is an acidic amino acid. In some cases, it is a mutation that substitutes between Ser and Thr. Specifically, substitutions considered as conservative substitutions include substitution from Ala to Ser or Thr, substitution from Arg to Gln, His or Lys, substitution from Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln substitution, Cys to Ser or Ala substitution, Gln to Asn, Glu, Lys, His, Asp or Arg substitution, Glu to Gly, Asn, Gln, Lys or Asp Substitution, substitution from Gly to Pro, substitution from His to Asn, Lys, Gln, Arg or Tyr, substitution from Ile to Leu, Met, Val or Phe, substitution from Leu to Ile, Met, Val or Phe, Replacement of Lys with Asn, Glu, Gln, His, or Arg Met to Ile, Leu, Val or Phe substitution, Phe to Trp, Tyr, Met, Ile or Leu substitution, Ser to Thr or Ala substitution, Thr to Ser or Ala substitution, Trp to Phe or Substitution to Tyr, substitution from Tyr to His, Phe or Trp, and substitution from Val to Met, Ile or Leu. In addition, amino acid substitutions, deletions, insertions, additions, or inversions as described above include naturally occurring mutations (mutants or variants) such as cases based on individual differences or species differences of microorganisms from which genes are derived. Also included by

  Furthermore, the gene having a conservative mutation as described above is 80% of the entire amino acid sequence of the protein encoded by the gene as described above, for example, the entire amino acid sequence of SEQ ID NO: 2, 8, 12, or 18. Or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more, and has glutathione synthetase or γ-glutamylcysteine synthetase activity It may be a gene encoding a protein. In the present specification, “homology” may refer to “identity”.

In addition, the gene encoding the enzyme used in the present invention is hybridized under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a complementary sequence of the nucleotide sequence shown in SEQ ID NO: 1, 7, 11, or 17. It may be a DNA encoding a protein that is soybean and has glutathione synthetase activity or γ-glutamylcysteine synthetase activity. Here, “stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, highly homologous DNAs, for example, 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more DNAs having homology. Are hybridized and DNAs with lower homology do not hybridize with each other, or normal Southern hybridization washing conditions of 60 ° C., 1 × SSC, 0.1% SDS, preferably 60 ° C. Washing once, more preferably 2-3 times at a salt concentration and temperature corresponding to 0.1 × SSC, 0.1% SDS, more preferably 68 ° C., 0.1 × SSC, 0.1% SDS The conditions to do are mentioned.

  The probe may be part of the complementary sequence of the gene. Such a probe can be prepared by PCR using an oligonucleotide prepared on the basis of a known gene sequence as a primer and a DNA fragment containing these base sequences as a template. For example, when a DNA fragment having a length of about 300 bp is used as a probe, hybridization washing conditions include 50 ° C., 2 × SSC, and 0.1% SDS.

  Moreover, the gene encoding the enzyme used in the present invention can be used in its natural form, but may be modified to have an optimal codon according to the codon usage frequency of the host to be used.

(3) Preparation of enzyme used in the present invention The method for obtaining the enzyme used in the present invention is not particularly limited. The enzyme used in the present invention can be prepared from an organism having the activity of the enzyme, for example, a wild strain or a mutant strain of a microorganism having the activity of the enzyme. In addition, the enzyme used in the present invention can be prepared from an organism with enhanced activity of the enzyme, and as such an organism, expression of a gene encoding the enzyme used in the present invention by genetic engineering techniques can be used. Is an enhanced transformant. The phrase “enzyme activity is enhanced” is not limited to increasing the enzyme activity in a microorganism that inherently has the enzyme activity, and is a microorganism that does not inherently have the enzyme activity. And imparting the activity of the enzyme.

  The method for enhancing the expression of the gene exemplified below is not limited to enhancing the expression of the gene encoding the enzyme used in the present invention, and is involved in the biosynthesis of any gene, for example, L-amino acid described later. It can also be used to enhance the expression of a gene that encodes the enzyme.

  When constructing transformants by genetic engineering techniques and mass-producing proteins, the hosts to be transformed include bacterial cells, actinomycetes cells, yeast cells, fungal cells, plant cells, insect cells and animal cells. Can be used. As the host to be transformed, microbial cells are preferably used. Examples of microorganisms for which host-vector systems have been developed include Escherichia, Pseudomonas, Corynebacterium, Arthrobacter, and Bacillus. ) Genus bacteria, Aspergillus fungi and the like. Especially, Escherichia coli (E. coli) or Corynebacterium glutamicum (C. glutamicum) is used suitably.

Enhancement of gene expression can be achieved, for example, by replacing the promoter of the gene on the chromosome with a stronger one. Examples of such a promoter include T7 promoter, trp promoter, lac promoter, tac promoter, and PL promoter. Moreover, the substitution to a strong promoter can be used in combination with the improvement of the translation efficiency of the gene and the increase in the copy number of the gene, which will be described later.

  In addition, enhancement of gene expression can be achieved, for example, by improving gene translation efficiency. It is known that substitution of several nucleotides in the spacer region between the ribosome binding site (RBS) and the start codon, particularly in the sequence immediately upstream of the start codon, has a great influence on the translation efficiency of mRNA. The translation efficiency can be improved by modifying.

  Further, enhancement of gene expression can be achieved, for example, by increasing the copy number of the gene.

  An increase in gene copy number can be achieved by introducing the gene of interest onto the chromosome. Introduction of a gene onto a chromosome can be performed using, for example, homologous recombination. For example, multiple copies of a gene can be introduced into a chromosome by performing homologous recombination with a sequence having multiple copies in the chromosome as a target. Examples of sequences having multiple copies in the chromosome include, but are not limited to, repetitive DNA or an inverted repeat sequence at the end of a transposable element. It is also possible to incorporate a gene into a transposon and transfer it to introduce multiple copies of the gene into the chromosome, as disclosed in US Pat. No. 5,595,889.

  An increase in the copy number of a gene can also be achieved by introducing a vector containing the target gene into a host organism. As a vector to be used, a multicopy vector is preferable. In order to select a transformant, the vector preferably has a marker such as an ampicillin resistance gene. For example, examples of multi-copy vectors that can be used for Escherichia coli include pUC-based plasmids, pBR322-based plasmids, and plasmids having a ColE1 origin of replication such as derivatives thereof. Here, the “derivative” means a plasmid modified by base substitution, deletion, insertion, addition or inversion. The modification here includes mutagenesis using mutagen and UV irradiation, and modifications caused by natural mutation and random mutation. Suitable vectors that can be used for Escherichia coli include pTrc99A (Pharmacia), pUC (Takara Bio), pPROK (Clontech), pKK233-2, which are commercially available expression vectors having a strong promoter. (Clontech), pET (Novagen), pQE (Qiagen) and the like. Examples of other vectors include shuttle vectors for Escherichia coli-Bacillus subtilis such as pHY300PLK, pGK12, pLF14, and pLF22, and phage vectors such as 11059, 1BF101, M13mp9, and Mu phage (JP-A-2-109985). And transposons such as Mu, Tn10, Tn5 (Berg, DE and Berg, CM, Bio / Technol., 1, 417 (1983)) can be used. For example, when a transposon is used as a vector, as described above, the gene copy number increases when the target gene is transferred and introduced onto the chromosome.

  A transformant with enhanced gene expression can be obtained by transforming a host with an expression plasmid in which a DNA fragment obtained by sequentially linking a promoter and a gene to the above vector is inserted. Some vectors contain a promoter suitable for gene expression. When such a vector is used and a gene is linked downstream to a promoter contained in the vector, a separate promoter is used in the vector. There is no need to insert. Further, a terminator, which is a transcription termination sequence, may be linked downstream of the gene. Examples of the terminator include T7 terminator, fd phage terminator, T4 terminator, tetracycline resistance gene terminator, Escherichia coli trpA gene terminator, and the like.

  The method for transformation is not particularly limited, and a conventionally known method can be used. For example, to introduce a vector into Escherichia coli, D. M.M. Morrison's method (Methods in Enzymology 68, 326 (1979)) or a method in which recipient cells are treated with calcium chloride to increase DNA permeability (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 (1970)) and the like, which can be carried out by a method used for transformation of ordinary Escherichia bacteria.

  The culture conditions of the transformant in which the expression of the gene encoding the enzyme used in the present invention obtained as described above is enhanced, and the conditions for inducing production of the enzyme are the type of marker, the type of promoter, and the type used. What is necessary is just to select suitably according to the kind etc. of a host. For example, the medium used for culturing Escherichia coli transformants is preferably a medium used for culturing normal Escherichia coli such as 2 × YT medium, M9-casamino acid medium, LB medium, etc. it can.

  After culturing, the microbial cells are collected and crushed or dissolved to obtain an enzyme-containing solution. For crushing the cells, for example, ultrasonic crushing, French press crushing or glass bead crushing can be suitably used. When cells are lysed, for example, egg white lysozyme, peptidase treatment, or an appropriate combination thereof can be used. In addition, when a residue remains after crushing or melt | dissolving, a residue can be removed by centrifugation etc. as needed. For example, when the target enzyme is present in the culture supernatant, the culture supernatant can be used as an enzyme-containing solution.

  Recovery of the enzyme from the enzyme-containing solution can be performed by a known method used for enzyme purification. Such methods include ammonium sulfate precipitation, gel filtration chromatography, ion exchange chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography and the like. When the target enzyme is produced as a fusion protein with another protein or peptide, the target enzyme can be purified by affinity chromatography using the affinity for the fused protein or peptide.

  In the above description, the preparation of the enzyme using the transformant with enhanced expression of the gene encoding the enzyme used in the present invention has been described. However, in other cases, it can be appropriately modified and applied as necessary. . For example, even when a wild strain or a mutant strain of a microorganism having the activity of the enzyme used in the present invention is used as an enzyme acquisition source, the cell is grown to produce the target enzyme, and the enzyme is prepared. Can do.

  In the method of the present invention, an enzyme purified as described above may be used, or an unpurified enzyme may be used. Examples of the unpurified enzyme include a culture supernatant obtained by culturing a microorganism that produces the enzyme, and an enzyme-containing solution such as a crushed product of the microorganism. In the method of the present invention, the enzyme can be used in the state of an immobilized enzyme immobilized on an arbitrary carrier. Further, for example, a culture containing microbial cells producing an enzyme, a microbial cell separated and recovered from the culture, a microbial cell processed product obtained by immobilizing the microbial cell, acetone treatment, lyophilization treatment, etc. It can also be used as

(4) Method for Producing γ-Glu-XY The method for producing γ-Glu-XY of the present invention (hereinafter also referred to as the method of the present invention) is characterized by utilizing the activity of glutathione synthetase. To do.

(4-1) Production of γ-Glu-XY by enzymatic method γ-Glu-XY can be produced by an enzymatic method using an enzyme.

  In the first aspect of the method of the present invention (hereinafter also referred to as the first aspect), γ-Glu-XY is produced by allowing glutathione synthetase to act on a raw material containing γ-Glu-X and Y. A process for producing γ-Glu-XY. That is, in the first embodiment, Y is added to γ-Glu-X in the reaction raw material by the action of glutathione synthetase to produce γ-Glu-XY.

  In the first embodiment, γ-Glu-X used as a substrate may be produced by any method. For example, γ-Glu-X can be produced by an enzymatic reaction using γ-glutamylcysteine synthetase using a raw material containing Glu and X. Further, for example, γ-Glu-X can be obtained by recovering from the cells or culture supernatant of a microorganism having the ability to produce γ-Glu-X. The microorganism having the ability to produce γ-Glu-X may be a wild strain or a mutant strain of a microorganism, or a transformant produced using a genetic engineering technique. A transformant having the ability to produce γ-Glu-X can be obtained, for example, by introducing a gene encoding γ-glutamylcysteine synthetase into an appropriate strain by a known technique.

  The first aspect of the method of the present invention may further include a step of generating γ-Glu-X by the method as described above. That is, for example, in one aspect of the first aspect (hereinafter also referred to as the second aspect), γ-Glu-X is produced by allowing γ-glutamylcysteine synthetase to act on a raw material containing Glu and X. Γ-Glu-XY is produced by allowing glutathione synthase to act on the raw material containing γ-Glu-X and Y produced in the step (hereinafter also referred to as step (A)) and in step (A) This is a method for producing γ-Glu-XY, including a step (hereinafter also referred to as step (B)). That is, in the second embodiment, γ-Glu-X is produced from Glu and X by the action of γ-glutamylcysteine synthase, and Y is further added by the action of glutathione synthase to form γ-Glu-XY. Generate.

  In the second embodiment, step (A) and step (B) may proceed separately. For example, γ-Glu-X produced in step (A) may be recovered, and step (B) may be advanced using the recovered γ-Glu-X. Further, for example, after the production of γ-Glu-X in step (A) reaches equilibrium, step (B) may be advanced by adding glutathione synthase, Y, or both to the reaction system. .

  In the second embodiment, the step (A) and the step (B) may proceed simultaneously. Both steps may proceed simultaneously during the entire reaction process, or may proceed simultaneously during only a part of the reaction process. When both steps are allowed to proceed simultaneously, both enzymes and Glu, X, and Y may coexist in the reaction system. For example, by allowing all of them to coexist at the start of the reaction, both steps can be started simultaneously. In addition, after the start of the step (A), the step (B) may be advanced by adding glutathione synthase, Y, or a combination thereof to the reaction system at an arbitrary point in the reaction process.

  A further aspect of the method of the present invention (hereinafter, also referred to as the third aspect) is to allow γ-Glu-X to react with a raw material containing Glu, X, and Y by causing γ-glutamylcysteine synthetase and glutathione synthetase to act. This is a method for producing γ-Glu-XY, which comprises a step of generating -Y. That is, in the third embodiment, γ-Glu-X is produced from Glu and X by the action of γ-glutamylcysteine synthase, and Y is further added by the action of glutathione synthase to form γ-Glu-XY. It is thought to generate.

  In the third aspect, γ-Glu-XY can be produced by allowing γ-glutamylcysteine synthetase and glutathione synthetase, and Glu, X, and Y to coexist in the reaction system. Further, for example, γ-glutamylcysteine synthetase may be acted first, and then glutathione synthetase may be additionally added to the reaction system at any time point. In addition, when glutathione synthetase is made to act additionally in the third aspect, Glu, X, or both may not remain in the reaction system at the time when glutathione synthetase acts.

  Hereinafter, γ-Glu-X and Y in the first aspect, Glu, X, γ-Glu-X, and Y in the second aspect, and Glu, X, and Y in the third aspect are generically named. Also called “substrate”. Hereinafter, the glutathione synthetase in the first embodiment, and the γ-glutamylcysteine synthetase and the glutathione synthetase in the second and third embodiments are also collectively referred to as “enzymes”.

  In any embodiment, the enzyme reaction is preferably performed in water or an aqueous solvent such as a buffer. The enzyme reaction may be performed, for example, by containing each substrate and each enzyme in the reaction solution, or may be performed by supplying a reaction solution containing each substrate to the immobilized enzyme. The pH of the reaction solution is not particularly limited as long as the enzyme used functions, and can be appropriately set according to the properties of the enzyme used. The pH of the reaction solution is, for example, preferably 5 to 10, more preferably 7 to 9, and particularly preferably 7.5 to 8.5.

  The concentration of each substrate in the reaction solution is not particularly limited. For example, it is preferably 1 μM or more, more preferably 100 μM or more, and particularly preferably 1 mM or more. Moreover, although the upper limit of a density | concentration is not specifically limited, For example, it is 2M or less normally, and it is preferable that it is 1M or less. In addition, for example, from the viewpoint of yield, it may be preferable to adjust the concentration of each substrate in the reaction solution. That is, for example, in the first embodiment, it may be preferable that the ratio of the concentrations of γ-Glu-X and Y is close to 1: 1. In the second or third embodiment, Glu, X, and It may be preferable that the Y concentration ratio is close to 1: 1: 1. Depending on the nature of the enzyme used, it may be preferable that there is a difference in the concentration of each substrate.

  The concentration of each enzyme in the reaction solution or the concentration of each immobilized enzyme is not particularly limited, and various conditions such as enzyme properties, X type, Y type, raw material concentration, reaction temperature, and reaction time. What is necessary is just to set suitably according to. The concentration of each enzyme in the reaction solution is, for example, preferably 1 μg / ml to 100 mg / ml, and more preferably 10 μg / ml to 10 mg / ml. The concentration of each immobilized enzyme is, for example, preferably 2 to 200 mg / g carrier, and more preferably 10 to 100 mg / g carrier.

It is preferable that a factor essential for the enzyme reaction is present in the reaction solution. Factors essential for the enzymatic reaction include ATP in both the reaction with glutathione synthetase and the reaction with γ-glutamylcysteine synthetase. ATP is used as an energy source for peptide bond formation. In the method of the present invention, ATP may be supplied to the reaction system so that the enzyme used in the present invention can use ATP for peptide bond formation. For example, ATP can be supplied as a powder or as an aqueous solution to a reaction solution for generating and accumulating γ-Glu-XY. Further, practically, an ATP regeneration system may be used instead of adding expensive ATP directly. That is, by adding a purified enzyme having an ATP regeneration ability, a crude enzyme, a treated bacterial cell, or a catalyst such as a bacterial cell and a substrate used for ATP regeneration to the reaction system, -Generation of Glu-XY can be continued. As a method for using the ATP regeneration system, specifically, a method of adding a cultured microbial cell of C. ammoniagenes and a carbon source (Biosci. Biotechnol. Biochem., 65, 644). -650 (2001)) a method of adding polyphosphate kinase and polyphosphate (J. Biosci. Bioeng., 91, 557-563 (2001)), a method of adding creatine phosphorylase and creatine phosphate, and the like.

Depending on the enzyme used, it may be preferable that various cations such as Mg ²⁺ are present in the reaction solution. In such a case, it is preferable to use a reaction solution composition in which the cation is not chelated, but there is no limitation as long as the action of the cation is maintained. For example, as a buffer solution with a low possibility that a cation is chelated, a HEPES buffer, a MES buffer, a GTA wide area buffer, etc. are mentioned. Furthermore, any other component may be present in the reaction solution as long as the production of γ-Glu-XY can be achieved. For example, organic solvents such as alcohols, esters, ketones, and amides can be contained.

  The reaction temperature is not particularly limited as long as the enzyme used functions, and can be appropriately set according to the properties of the enzyme. The reaction temperature is, for example, usually 10 ° C to 80 ° C, preferably 20 ° C to 70 ° C. For example, when an enzyme derived from Escherichia coli is used, the reaction temperature is preferably 30 ° C to 40 ° C.

  The reaction time is not particularly limited, and may be set as appropriate according to various conditions such as enzyme properties and reaction temperature. The reaction time is usually preferably from 5 minutes to 200 hours, for example.

  In any embodiment, in the course of the reaction, each substrate, each enzyme, and other components may be added to the reaction system alone or in any combination. These components may be added once or a plurality of times, or may be added continuously. Further, uniform conditions from the start of the reaction to the end of the reaction may be used, and the conditions may be changed in the course of the reaction. Note that changing the conditions in the course of the reaction includes spatially changing conditions such as reaction temperature and enzyme concentration in the case of using an immobilized enzyme, for example. For example, in the case where the immobilized enzyme is used in the second or third aspect, γ-glutamylcysteine synthetase and glutathione synthetase may be mixed and arranged separately. Good.

  In addition, for example, in the second or third embodiment, when the preferable conditions on which glutathione synthetase and γ-glutamylcysteine synthetase act are different from each other, it is suitable for γ-glutamylcysteine synthetase in the course of the reaction. It may be effective to change the conditions to conditions suitable for glutathione synthase.

  In the method of the present invention, the production of γ-Glu-XY can be confirmed by any technique used for detecting or identifying a compound. Such techniques include HPLC, LC / MS, GC / MS, and NMR.

  The γ-Glu-XY produced according to the present invention can be isolated using a commonly used peptide purification method. For example, γ-Glu-XY can be isolated from the reaction supernatant after removing solids by centrifugation or the like, by combining operations such as ion exchange resin, membrane treatment, and crystallization. it can.

(4-2) Production of γ-Glu-XY by fermentation method γ-Glu-XY is not limited to the enzyme method described above, and can also be produced by fermentation using microorganisms.

Specifically, for example, a microorganism having the activities of γ-glutamylcysteine synthetase and glutathione synthetase is cultured, γ-Glu-XY is produced from Glu, X, and Y, and the microbial body and / or culture is performed. Γ-Glu-XY can be accumulated in the liquid. Glu, X, and Y used as substrates may be added to the medium or biosynthesized by the microorganism used. The microorganism having the activities of γ-glutamylcysteine synthetase and glutathione synthetase is preferably a microorganism having enhanced activity of γ-glutamylcysteine synthetase and / or glutathione synthetase.

  In addition, for example, a microorganism having the activity of glutathione synthase is cultured to produce γ-Glu-XY from γ-Glu-X and Y, and γ-Glu-X- Y can be accumulated. Γ-Glu-X and Y used as substrates may be added to the medium or may be biosynthesized by the microorganism used. The microorganism having activity of glutathione synthetase is preferably a microorganism having enhanced activity of glutathione synthetase.

  Accumulated γ-Glu-XY can be recovered from the culture. In any of the above cases, γ-Glu-XY is preferably accumulated in the culture medium.

  Hereinafter, as in the enzyme method, Glu, X, and Y, or γ-Glu-X and Y used as substrates in each aspect of the fermentation method are collectively referred to as “substrate”. A microorganism that produces γ-Glu-XY is also simply referred to as “microorganism”.

  The microorganism is not particularly limited. For example, bacteria belonging to the family Enterobacteriaceae or coryneform bacteria can be appropriately modified as necessary. Examples of the modification herein include enhancement of the activity of γ-glutamylcysteine synthetase and / or glutathione synthetase, and addition or enhancement of the ability to produce one or more substrates described below.

  The bacteria belonging to the family Enterobacteriaceae include the genus Escherichia, the Enterobacter genus, the Pantoea genus, the Klebsiella genus, the Serratia genus, the Erwinia genus, and the Salmonella genus. And bacteria belonging to the genus Morganella. Specifically, those belonging to the family Enterobacteriaceae can be used according to the classification described in the NCBI (National Center for Biotechnology Information) database (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax .cgi? id = 91347). As bacteria belonging to the family Enterobacteriaceae, bacteria belonging to the genus Escherichia can be preferably used.

  Examples of Escherichia bacteria include those listed in the book by Neidhardt et al. (Neidhardt, FCet.al., Escherichia coli and Salmonella Typhimurium, American Society for Microbiology, Washington DC, 1208, table 1), for example, Escherichia coli it can. Examples of the wild strain of Escherichia coli include the K12 strain or a derivative thereof, Escherichia coli MG1655 strain (ATCC No. 47076), the W3110 strain (ATCC No. 27325), and the like.

  The coryneform bacterium is a group of microorganisms defined in Bergey's Manual of Determinative Bacteriology, 8th edition, page 599 (1974), aerobic, gram-positive, Non-acidic and microorganisms classified as gonococci having no sporulation ability can be used. As the coryneform bacteria, bacteria belonging to the genus Corynebacterium can be preferably used. Coryneform bacteria were previously classified as genus Brevibacterium but are now integrated as Corynebacterium (Int.J. Syst. Bacteriol., 41, 255 (1991)), and coryneform bacteria. Includes Brevibacterium and Microbatterium bacteria that are very closely related to the genus Bacteria. As a bacterium belonging to the genus Corynebacterium, for example, Corynebacterium glutamicum can be preferably used.

  These strains can be obtained, for example, from the American Type Culture Collection (address P.O. Box 1549, Manassas, VA 2010812301 Parklawn Drive, Rockville, Maryland 20852, United States of America). That is, a corresponding registration number is assigned to each strain, and distribution can be received using this registration number. The registration number corresponding to each strain is described in the catalog of American Type Culture Collection (see http://www.atcc.org/).

  The microorganism may have the ability to produce one or more substrates. When the microorganism has the ability to produce one or more substrates, the one or more substrates biosynthesized by the microorganism can be directly used for the production of γ-Glu-XY.

  The ability to produce a substrate may mean the ability to produce a substrate in the medium or in the cells and accumulate it to the extent that it can be recovered in the medium or from the cells when the microorganism is cultured in the medium. For example, a microorganism having the ability to produce a substrate preferably produces a substrate in an amount of 0.05 g / L or more, more preferably 0.1 g / L or more, particularly preferably 0.2 g / L or more. It can be a microorganism that can. In addition, since the substrate biosynthesized by the microorganism can be used as it is for the production of γ-Glu-XY, the ability to produce the substrate means that the substrate can be used in the medium or in the cells without adding the substrate to the medium. It may mean the ability to produce γ-Glu-XY and accumulate it to the extent that it can be recovered in the medium or from the cells.

  As a microorganism having the ability to produce a substrate, for example, a known L-amino acid producing bacterium can be suitably used. In addition, the microorganism having the ability to produce a substrate may originally have the ability to produce a substrate, or may be modified to have the ability to produce a substrate. In addition, the microorganism having the ability to produce a substrate may be a microorganism having an enhanced ability to produce a substrate compared to a wild strain or a parent strain. The ability to produce a substrate can be imparted to or enhanced by a known technique.

  In order to give microorganisms the ability to produce substrates, such as L-amino acids, the acquisition of auxotrophic mutants, analog resistant strains or metabolic control mutants, and the expression of L-amino acid biosynthetic enzymes were enhanced. Conventional methods such as the creation of recombinant strains that have been adopted for breeding coryneform bacteria or bacteria belonging to the genus Escherichia can be applied (Amino Acid Fermentation, Academic Publishing Center, Inc., May 30, 1986, first edition issued) Pp. 77-100). Here, in the breeding of L-amino acid-producing bacteria, the auxotrophy, analog resistance, metabolic control mutation and other properties imparted may be single or two or more. In addition, L-amino acid biosynthetic enzymes whose expression is enhanced may be used alone or in combination of two or more. Furthermore, imparting properties such as auxotrophy, analog resistance, and metabolic regulation mutation may be combined with enhancement of biosynthetic enzymes.

  To obtain an auxotrophic mutant having L-amino acid-producing ability, an analog-resistant strain of L-amino acid, or a metabolically controlled mutant, the parent strain or wild strain is subjected to normal mutation treatment, that is, irradiation with X-rays or ultraviolet rays, N-methyl-N'-nitro-N-nitrosoguanidine (NTG) or ethyl methanesulfonate (EMS), etc. It can be obtained by selecting those that show tolerance or metabolic control mutations and have the ability to produce L-amino acids.

Examples of amino acid-producing bacteria that serve as hosts for fermentation production of γ-Glu-XY include the following. As an L-glutamic acid-producing bacterium, a recombinant strain in which an mviN gene having a V197M mutation is introduced into an odhA-deficient strain obtained from Corynebacterium glutamicum (Brevibacterium lactofermentum) ATCC 13869 (JP 2010- 161970), Pantoea agglomerans strain AJ13355 (patent No. 4285582) into which gltA (citrate synthase) gene derived from Brevibacterium lactofermentum was introduced, belonging to the genus Escherichia, and the tyrosine residue at position 397 of glutamine synthetase And a strain having a mutant glutamine synthetase substituted with the amino acid residue (US Patent Application Publication No. 2003-0148474). Examples of L-valine-producing bacteria include Escherichia coli VL1970 strain (US Pat. No. 5,658,766), strains belonging to the genus Escherichia, which have a mutation that requires lipoic acid for growth and / or a mutation that lacks H ⁺ -ATPase. In addition to these properties, a bacterium belonging to the genus Escherichia in which a DNA fragment containing an ilvGMEDA operon that expresses at least each gene of ilvG, ilvM, ilvE, and ilvD and does not express threonine deaminase activity has been introduced into the cell ( WO96 / 06926) and the like can be exemplified.

  Further, L-amino acid-producing ability can be imparted or enhanced by enhancing the enzyme activity involved in L-amino acid biosynthesis. Enhancing the activity of an enzyme involved in L-amino acid biosynthesis can be achieved, for example, by enhancing the expression of a gene encoding the enzyme.

  In addition, the microorganism may be modified so that the ability to take up each substrate added to the medium is improved. In addition, the microorganism may be modified so that the ability to excrete γ-Glu-XY outside the microbial cell is improved.

  In addition, the modification of microorganisms such as enhancement of activity of glutathione synthetase and / or γ-glutamylcysteine synthetase, addition or enhancement of the ability to produce a substrate, and any other modification may be performed in any order. Good.

  The culture conditions for culturing the microorganism are not particularly limited as long as the microorganism can grow, and the conditions usually used for culturing the microorganism can be appropriately modified as necessary. For example, as the medium, a normal medium containing a carbon source, a nitrogen source, inorganic ions, and other components as required can be used. The culture can be performed, for example, at 25 ° C. to 37 ° C., at a pH of 5 to 8 and under aerobic conditions for 5 to 200 hours. In addition, an inorganic or organic acidic or alkaline substance, ammonia gas or the like can be used for pH adjustment.

  In culturing microorganisms, each substrate is added to the medium. Each substrate may be contained in the medium from the beginning of the culture, or may be added to the medium at any time during the culture. The timing of addition can be appropriately changed according to conditions such as culture time, but as an example, it is preferably 0 to 50 hours before, more preferably 0.1 to 24 hours, and particularly preferably 0. It can be added 5 to 6 hours before. Each substrate may be added one or more times, or may be added continuously.

  The kind of the substrate added to the medium can be appropriately changed according to the ability of the microorganism to produce the substrate. For example, if the microorganism does not have the ability to produce any substrate, all the substrate is added to the medium. “All substrates” refers to “Glu, X, and Y” or “γ-Glu-X and Y”, depending on the mode of the fermentation process. In addition, when the microorganism has an ability to produce one or more substrates, the one or more substrates may or may not be added to the medium. Further, when the microorganism has an ability to produce all the substrates, any substrate may not be added to the medium, and may be added. That is, in a fermentation method, a microorganism that does not have the ability to produce any substrate, or a microorganism that has the ability to produce one or more substrates, is at least all substrates other than the substrate produced by the microorganism. By culturing in the added medium, γ-Glu-XY can be produced. In the fermentation method, the ability to produce at least all substrates other than the substrate added to the medium in a medium to which no substrate is added, or a medium to which one or more substrates are added. Γ-Glu-XY can be produced by culturing microorganisms having the same.

  The concentration of each substrate is not particularly limited. For example, the initial concentration is preferably 1 μM or more, more preferably 100 μM or more, and particularly preferably 1 mM or more. Moreover, although the upper limit of a density | concentration is not specifically limited, For example, it is 2M or less normally, and it is preferable that it is 1M or less. In addition, for example, when each substrate is added to the medium during the culture, the concentration of each substrate may be appropriately changed according to the mode of addition or the like. In addition, for example, from the viewpoint of yield, it may be preferable to adjust the concentration of each substrate in the medium.

  Prior to culture in a medium containing each substrate, pre-culture may be performed. The medium used for pre-culture may or may not contain each substrate.

  The ATP used for producing the peptide in the fermentation method may be ATP produced by energy metabolism in a microorganism that produces γ-Glu-XY. It is also possible to improve the production efficiency of γ-Glu-XY by coexisting a microorganism with enhanced ATP regeneration system with a microorganism that produces γ-Glu-XY. ). Examples of the co-culture system include a method in which a microorganism having an enhanced ATP regeneration system by normal energy metabolism or a microorganism having an ability to regenerate ATP by the action of polyphosphate kinase coexist in a culture solution (Japanese Patent Publication No. 7-16431). No. 6-69386) can be used.

  Hereinafter, the present invention will be described more specifically with reference to examples. In addition, this invention is not limited at all by the following examples.

Example 1: Construction of Saccharomyces cerevisiae-derived glutathione synthetase gene (GSH2) expression plasmid pET-GSH2 The expression plasmid pET-GSH2 encoding the glutathione synthetase of Saccharomyces cerevisiae S288C strain (ATCC No. 26108) It was constructed according to the procedure and introduced into Escherichia coli. An outline of the construction procedure is shown in FIG.

(1-1) Construction of Yeast Expression Plasmid pAUR-GSH2 First, the yeast expression plasmid pAUR-GSH2 was commissioned to Takara Bio and constructed according to the following procedure.

  Primer A (SEQ ID NO: 3) and primer B (SEQ ID NO: 4) prepared based on the base sequence (SEQ ID NO: 1) of GSH2 gene of Saccharomyces cerevisiae S288C strain, and chromosomal DNA of Saccharomyces cerevisiae S288C strain as a template The sequence containing the GSH2 gene was amplified by PCR. Primer A is obtained by adding a KpnI recognition sequence and a partial sequence of yeast expression plasmid pAUR123 to the 5 'end of the region containing the start codon of the GSH2 gene of the chromosomal DNA of Saccharomyces cerevisiae S288C. Primer B includes a base sequence complementary to the C-terminal base sequence of the GSH2 gene, a base sequence complementary to a sequence encoding a His tag, a base sequence complementary to a stop codon (TAA), an XbaI recognition sequence, and yeast A partial sequence of the expression plasmid pAUR123 is added. PCR was performed according to the manual using PrimeSTAR Max DNA polymerase (manufactured by Takara Bio Inc.). The amplified fragment was introduced into the KpnI-XbaI site of the yeast expression plasmid pAUR123 (Takara Bio) using In-Fusion Advantage PCR Kit (Takara Bio) to obtain the yeast expression plasmid pAUR-GSH2. .

(1-2) Construction of Escherichia coli expression plasmid pET-GSH2 Subsequently, an Escherichia coli expression plasmid pET-GSH2 was constructed by the following procedure.

  Primer C (SEQ ID NO: 5) and Primer D (SEQ ID NO: 6) prepared from the base sequence of the GSH2 gene of Saccharomyces cerevisiae S288C strain (SEQ ID NO: 1) were purchased from Nippon Bioservice. Primer C is obtained by adding a base sequence containing an NdeI recognition sequence to the 5 'end of the region containing the start codon of the GSH2 gene in the chromosomal DNA of Saccharomyces cerevisiae S288C strain. Primer D is obtained by adding a base sequence containing an XhoI recognition sequence to the 5 'end of a base sequence complementary to the base sequence outside the stop codon of the GSH2 gene of pAUR-GSH2.

  A sequence containing the GSH2 gene was amplified by PCR using Primer C and Primer D and the above-described pAUR-GSH2 as a template. PCR was performed using plasmid DNA, 0.2 μmol / L of each primer, 1.25 units of PrimeSTAR HS DNA polymerase (Takara Bio), 10 μL of 5 × PrimeSTAR buffer (Takara Bio), 2.5 mmol / L of dNTP. 50 μl of a reaction solution containing (dATP, dGTP, dCTP, and dTTP) is prepared, heated at 98 ° C. for 10 seconds, and then subjected to 30 steps of 98 ° C. for 10 seconds, 56 ° C. for 5 seconds, and 72 ° C. for 2 minutes. Repeatedly, it was further heated at 72 ° C. for 1 minute.

3 μl of the reaction solution after PCR was subjected to agarose gel electrophoresis, and after confirming that an approximately 1.5 kb DNA fragment corresponding to the GSH2 gene fragment had been amplified, Ethhachinate (manufactured by Nippon Gene) was used from the remaining reaction solution. The DNA fragment was purified using and dissolved in 25 μl of dH ₂ O. Next, using the total amount of the obtained DNA solution, the DNA fragment was cleaved with restriction enzymes NdeI and XhoI, then purified using MinElute Reaction Cleanup Kit (Qiagen), and 15 μl of Buffer EB (Qiagen) Dissolved in.

  1 μg of the expression plasmid pET-21a (+) (manufactured by Novagen) was cleaved with restriction enzymes NdeI and XhoI, purified using MinElute Reaction Cleanup Kit, and dissolved in 15 μl of Buffer EB. Next, using the entire amount of the obtained DNA solution, the DNA fragment was dephosphorylated with Alkaline Phosphatase (Cal intestine) (CIAP), purified using MinElute Reaction Cleanup Kit, and dissolved in 10 μl of Buffer EB.

  The about 1.5 kb DNA fragment containing the GSH2 gene obtained above and the about 5.4 kb DNA fragment of the expression plasmid pET-21a (+) obtained above were prepared using TaKaRa Ligation Kit Ver. 2.1 (manufactured by Takara Bio Inc.) was reacted at 16 ° C. for 30 minutes for ligation. Escherichia coli DH5α strain competent cells (manufactured by Takara Bio Inc.) were transformed by the heat shock method using the reaction solution, and the transformant was LB [10 g / L bactotryptone containing 50 μg / ml ampicillin. (Difco), 5 g / L yeast extract (Difco), 5 g / L sodium chloride (Wako)]] After application on an agar medium, the cells were cultured overnight at 37 ° C.

A plasmid was extracted from the grown transformant colony by a known method, and its nucleotide sequence was determined by a known method. The obtained plasmid is a plasmid in which a GSH2 gene derived from Saccharomyces cerevisiae S288C strain added with a sequence encoding a His tag at the 3 ′ end is ligated downstream of the T7 promoter.
It was named GSH2. The base sequence of the GSH2 gene derived from Saccharomyces cerevisiae S288C strain and the amino acid sequence encoded thereby are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

  Subsequently, Escherichia coli BL21 (DE3) strain competent cell (manufactured by Novagen) was transformed with pET-GSH2 by the heat shock method, and the transformant was LB agar medium containing 50 μg / ml ampicillin. Then, the cells were cultured overnight at 37 ° C. A plasmid was extracted from the grown colonies of the transformant according to a known method, and the structure was analyzed using a restriction enzyme to confirm that pET-GSH2 was retained. The Escherichia coli BL21 (DE3) strain carrying this pET-GSH2 was named Escherichia coli BL21 (DE3) / pET-GSH2.

Example 2: Purification of C-terminal His-tagged recombinant Gsh2 Test tube containing 3 mL of LB medium containing 100 μg / ml ampicillin of Escherichia coli BL21 (DE3) / pET-GSH2 obtained in Example 1 And inoculated with shaking at 37 ° C. for 16 hours. Of the obtained culture solution, 2 ml was inoculated into a test tube containing 100 ml of LB medium. After shaking culture at 37 ° C. for 2 hours, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mmol / L, and further cultured at 30 ° C. for 4 hours. The culture broth was centrifuged to obtain wet cells.

Purification of the His-tagged protein from the supernatant obtained by suspending the wet cells in 10 ml of a 100 mmol / L Tris-HCl buffer solution containing 300 mM sodium chloride, pH 8.0 and sonicating, followed by centrifugation Kit Ni Sepharose 6
The His-tagged recombinant Gsh2 was purified using Fast Flow (manufactured by GE Healthcare) according to the manual, and then desalted using a PD-10 column (manufactured by GE Healthcare) according to the manual. This purified and desalted Gsh2 was used as purified Gsh2 in the subsequent experiments.

Example 3: Production of γ-glutamyltripeptide using purified Gsh2 Using purified Gsh2 derived from Saccharomyces cerevisiae S288C strain obtained in Example 2, γ-glutamylvaline (γ-Glu-Val) or γ-glutamylnor The production of γ-glutamyl tripeptide using valine (γ-Glu-nVal) as a substrate was examined. 200 μl of a reaction solution (pH 8.0) having the following composition was prepared and reacted at 37 ° C. for 16 hours.
[Reaction solution composition]
Purified Gsh2 12.4 μg / 200 μl
Tris-HCl (pH 8.0) 100 mmol / L
γ-Glu-Val or γ-Glu-nVal 12.5mmol / L
Glycine 12.5mmol / L
Adenosine triphosphate (ATP) 12.5mmol / L
Magnesium sulfate 12.5mmol / L
Dithiothreitol (DTT) 2 mmol / L

After completion of the reaction, the reaction product was analyzed by HPLC. The analysis conditions are as follows. (1) HPLC: HITACHI L-2000 series (2) Separation column: Synergi 4 μ Hydro-RP 80A, inner diameter 4.6 mm, length 250 mm, particle size 4 μm (manufactured by Phenomenex)
(3) Column temperature: 40 ° C
(4) Mobile phase A: 50 mM phosphate buffer (pH 2.5)
(5) Mobile phase B: Acetonitrile (6) Flow rate: 1.0 ml / min
(7) Elution conditions: Elution was performed using a mixture of mobile phase A and mobile phase B. The ratio of the mobile phase B to the mixed liquid is as follows. 0 minutes (0%), 0 minutes to 5 minutes (0% to 2.5%), 5 minutes to 15 minutes (2.5%), 15 minutes to 30 minutes (2.5% to 40%), 30 Min to 30.1 min (40% to 0%), 30.1 min to 50 min (0%).
(8) Detection: UV210nm

  As a result of the above measurement, the peaks of the respective reaction products are the peaks of the samples of γ-glutamylvalylglycine (γ-Glu-Val-Gly) and γ-glutamylnorvalylglycine (γ-Glu-nVal-Gly). Retention times were in agreement and determined to be γ-Glu-Val-Gly and γ-Glu-nVal-Gly. As a result of the quantification, the γ-Glu-Val-Gly concentration was 0.13 mmol / L, and the γ-Glu-nVal-Gly concentration was 6.0 mmol / L.

  Further, a fraction in which the retention time coincided with the peak of the standard γ-Glu-nVal-Gly was collected by HPLC, and confirmed to be γ-Glu-nVal-Gly using a mass spectrometer. Specifically, the fractionated peptide was fluorescently derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and detected by LC-MS / MS. The procedure is as follows.

  To 2.5 μl of a preparative fraction solution diluted to an appropriate concentration or 2.5 μl of a standard solution containing 1 μM γ-Glu-nVal-Gly, 2.5 μl of MilliQ water, 5 μM internal standard solution (L-alanine- 3,3,3-d3, Sigma (Ala-D3), labeled with a stable isotope) 5 μl, and borate buffer (supplied with AccQ-Flor (registered trademark) reagent kit manufactured by Nihon Waters) 30 μl Was added. To this mixture, 10 μl of AQC reagent solution (prepared by dissolving the reagent powder of the above reagent kit in 1 ml of acetonitrile) was added. The obtained mixture was heated at 55 ° C. for 10 minutes, and 100 μl of a 0.1% formic acid aqueous solution was added to prepare an analytical sample.

The analytical sample was then separated by reverse phase liquid chromatography. The separation conditions are as follows.
(1) HPLC: Agilent 1200 series (2) Separation column: Unison UK-Phenyl, inner diameter 2.0 mm, length 100 mm, particle diameter 3 μm (manufactured by Imtakt)
(3) Column temperature: 40 ° C
(4) Mobile phase A: Aqueous solution of 25 mM formic acid aqueous solution adjusted to pH 6.0 with aqueous ammonia (5) Mobile phase B: Methanol (6) Flow rate: 0.25 ml / min
(7) Elution conditions: Elution was performed using a mixture of mobile phase A and mobile phase B. The ratio of the mobile phase B to the mixed liquid is as follows. 0 minutes (5%), 0 minutes to 17 minutes (5% to 40%), 17 minutes to 17.1 minutes (40% to 80%), 17.1 minutes to 19 minutes (80%), 19 minutes to 19.1 min (80% -5%), 19.1 min-27 min (5%).

Thereafter, a derivatized product of γ-Glu-nVal-Gly eluted under the above separation conditions was introduced into a mass spectrometer, and a mass chromatogram was obtained. The analysis conditions are as follows. (1) Mass spectrometer: AB Sciex API3200 QTRAP
(2) Detection mode: Selected Ion Monitoring (positive ion mode)
(3) Selected ions: Table 1

  The obtained results were analyzed using analysis software Analyst ver 1.4.2 (manufactured by AB Sciex). A peak whose retention time coincided with the peak of the standard γ-Glu-nVal-Gly was γ-Glu- It was confirmed that the peak was nVal-Gly.

Example 4: Construction of Escherichia coli-derived glutathione synthase gene (gshB) expression plasmid pET-gshB An expression plasmid pET-gshB encoding the glutathione synthetase of Escherichia coli K-12 W3110 strain was constructed by the following procedure. . An outline of the construction procedure is shown in FIG.

  Primer E (SEQ ID NO: 9) and Primer F (SEQ ID NO: 10) prepared from the base sequence of the gshB gene of Escherichia coli K-12 W3110 strain (SEQ ID NO: 7) were purchased from Nippon Bioservice. Primer E is obtained by adding a base sequence containing an NdeI recognition sequence to the 5 'end of the region containing the start codon of the gshB gene of chromosomal DNA of Escherichia coli K-12 W3110 strain. Primer F is obtained by adding a base sequence containing an XhoI recognition sequence to the 5 'end of a base sequence complementary to the C-terminal base sequence of the gshB gene.

  A sequence containing the gshB gene was amplified by PCR using primer E and primer F and chromosomal DNA of Escherichia coli K-12 W3110 as a template. PCR includes chromosomal DNA, 0.2 μmol / L of each primer, 1.25 units of PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.), 10 μL of 5 × PrimeSTAR buffer (manufactured by Takara Bio Inc.), 2.5 mmol / L of dNTP each 50 μl of a reaction solution containing (dATP, dGTP, dCTP, and dTTP) was prepared, heated at 98 ° C. for 10 seconds, then treated at 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 1.6 minutes. This was repeated 30 times and further heated at 72 ° C. for 1 minute.

5 μl of the reaction solution after PCR was subjected to agarose gel electrophoresis, and after confirming that a DNA fragment of about 1.0 kb corresponding to the gshB gene fragment had been amplified, Ethhainate (manufactured by Nippon Gene) was used from the remaining reaction solution. The DNA fragment was purified using and dissolved in 30 μl of dH ₂ O. Next, using the entire amount of the obtained DNA solution, the DNA fragment was cleaved with restriction enzymes NdeI and XhoI, purified using MinElute Reaction Cleanup Kit, and dissolved in 15 μl of Buffer EB.

  An approximately 1.0 kb DNA fragment containing the gshB gene obtained above and an approximately 5.4 kb DNA fragment of the expression plasmid pET-21a (+) prepared in the same manner as in Example 1 (1-2) were used. TaKaRa Ligation Kit Ver. 2.1 (manufactured by Takara Bio Inc.) was reacted at 16 ° C. for 30 minutes for ligation. Escherichia coli BL21 (DE3) strain competent cells (manufactured by Novagen) were transformed with the reaction solution by a heat shock method, and the transformant was LB [10 g / L Bacterium containing 50 μg / ml ampicillin. Tryptone (manufactured by Difco), 5 g / L yeast extract (manufactured by Difco), 5 g / L sodium chloride (manufactured by Wako)] was applied to an agar medium and cultured at 37 ° C. overnight.

  A plasmid was extracted from the grown transformant colony by a known method, and its nucleotide sequence was determined by a known method. The obtained plasmid is a plasmid in which a gshB gene derived from Escherichia coli K-12 W3110 strain to which a sequence encoding a His tag is added at the 3 ′ end is ligated downstream of the T7 promoter, and the plasmid is expressed as pET-gshB. Named. In addition, Escherichia coli BL21 (DE3) strain carrying this pET-gshB was named Escherichia coli BL21 (DE3) / pET-gshB. The base sequence of the gshB gene derived from Escherichia coli K-12 W3110 strain and the amino acid sequence encoded thereby are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

Example 5: Purification of C-terminal His-tagged recombinant GshB Test tube containing 5 mL of LB medium containing 100 μg / ml ampicillin of Escherichia coli BL21 (DE3) / pET-gshB obtained in Example 4 And inoculated with shaking at 37 ° C. for 16 hours. 1 ml of the obtained culture solution was inoculated into a test tube containing 50 ml of LB medium. After shaking culture at 37 ° C. for 2 hours, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mmol / L, and the mixture was further cultured at 37 ° C. for 3 hours. The culture broth was centrifuged to obtain wet cells.

  The wet cells are suspended in 2 ml of BugBuster (manufactured by Novagen), gently shaken at room temperature for 20 minutes, and centrifuged to obtain a His-tagged protein purification kit, Ni Sepharose 6 Fast Flow ( GE Healthcare) was used to purify the His-tagged recombinant enzyme according to the manual, followed by desalting according to the manual using a PD-10 column (GE Healthcare). This purified and desalted GshB was used in subsequent experiments as purified GshB.

Example 6: Production of γ-glutamyltripeptide using purified GshB Using purified GshB derived from Escherichia coli K-12 W3110 obtained in Example 5, γ-glutamylvaline (γ-Glu-Val) or γ The production of γ-glutamyl tripeptide using γ-glutamyl norvaline (γ-Glu-nVal) as a substrate was examined. 200 μl of a reaction solution (pH 8.0) having the following composition was prepared and reacted at 37 ° C. for 16 hours.
[Reaction solution composition]
Purified GshB 43.6 μg / 200 μl
Tris-HCl (pH 8.0) 100 mmol / L
γ-Glu-Val or γ-Glu-nVal 12.5mmol / L
Glycine 12.5mmol / L
Adenosine triphosphate (ATP) 12.5mmol / L
Magnesium sulfate 12.5mmol / L
Dithiothreitol (DTT) 2 mmol / L

  After completion of the reaction, the reaction product was identified and quantified by HPLC under the same conditions as in Example 3. As a result, 2.6 mmol / L of γ-glutamylvalylglycine (γ-Glu-Val-Gly), 10.9 mmol / Production of L γ-glutamylnorvalylglycine (γ-Glu-nVal-Gly) was confirmed.

In addition, the fraction in which the retention time coincided with the peak of the standard γ-Glu-Val-Gly was collected by HPLC and confirmed to be γ-Glu-Val-Gly using a mass spectrometer. The specific analysis procedure is the same as the procedure when γ-Glu-nVal-Gly was analyzed in Example 3. The selected ions in the first and second mass analyzers in the γ-Glu-Val-Gly measurement conform to the selected ions of γ-Glu-nVal-Gly shown in Table 1.

Example 7: Construction of Candida utilis-derived γ-glutamylcysteine synthase gene (GSH1) expression plasmid pET-GSH1 The expression plasmid pET-GSH1 encoding γ-glutamylcysteine synthetase of Candida utilis ATCC22023 strain is shown below. It was constructed according to the procedure and introduced into Escherichia coli. An outline of the construction procedure is shown in FIG.

(7-1) Construction of Yeast Expression Plasmid pAUR-GSH1 First, the yeast expression plasmid pAUR-GSH1 was commissioned to Takara Bio and constructed according to the following procedure.

  Primer G (SEQ ID NO: 13) and primer H (SEQ ID NO: 14) prepared on the basis of the base sequence (SEQ ID NO: 11) of GSH1 gene of Candida utilis ATCC22023, and chromosomal DNA of Candida utilis ATCC 22023 as template The sequence containing the GSH1 gene was amplified by PCR. Primer G is obtained by adding a KpnI recognition sequence and a partial sequence of yeast expression plasmid pAUR123 to the 5 'end of the region containing the start codon of the GSH1 gene of chromosomal DNA of Candida utilis ATCC22023. Primer H includes a base sequence complementary to the C-terminal base sequence of the GSH1 gene, a base sequence complementary to a sequence encoding a His tag, a base sequence complementary to a stop codon (TAA), an XbaI recognition sequence, and yeast A partial sequence of the expression plasmid pAUR123 is added. PCR was performed according to the manual using PrimeSTAR Max DNA polymerase (manufactured by Takara Bio Inc.). The amplified fragment was introduced into the KpnI-XbaI site of the yeast expression plasmid pAUR123 (Takara Bio) using In-Fusion Advantage PCR Kit (Takara Bio) to obtain the yeast expression plasmid pAUR-GSH1. .

(7-2) Construction of Escherichia coli expression plasmid pET-GSH1 Subsequently, an Escherichia coli expression plasmid pET-GSH1 was constructed by the following procedure.

  Primer I (SEQ ID NO: 15) and Primer J (SEQ ID NO: 16) prepared from the base sequence of the GSH1 gene of Candida utilis ATCC22023 strain (SEQ ID NO: 11) were purchased from Nippon Bioservice. Primer I is obtained by adding a base sequence containing an NheI recognition sequence to the 5 'end of the region containing the start codon of the GSH1 gene of chromosomal DNA of Candida utilis ATCC22023. Primer J is obtained by adding a base sequence containing an XhoI recognition sequence to the 5 'end of a base sequence complementary to the base sequence outside the stop codon of the GSH1 gene of pAUR-GSH1.

  A sequence containing the GSH1 gene was amplified by PCR using Primer I and Primer J and the above-described pAUR-GSH1 as a template. PCR was performed using plasmid DNA, 0.2 μmol / L of each primer, 1.25 units of PrimeSTAR HS DNA polymerase (Takara Bio), 10 μL of 5 × PrimeSTAR buffer (Takara Bio), 2.5 mmol / L of dNTP. 50 μl of a reaction solution containing (dATP, dGTP, dCTP, and dTTP) is prepared, heated at 98 ° C. for 10 seconds, and then subjected to 30 steps of 98 ° C. for 10 seconds, 56 ° C. for 5 seconds, and 72 ° C. for 2 minutes. Repeatedly, it was further heated at 72 ° C. for 1 minute.

3 μl of the reaction solution after PCR was subjected to agarose gel electrophoresis, and after confirming that a DNA fragment of about 2.0 kb corresponding to the GSH1 gene fragment had been amplified, Ethhachinate (manufactured by Nippon Gene) was used from the remaining reaction solution. The DNA fragment was purified using and dissolved in 25 μl of dH ₂ O. Next, using the entire amount of the obtained DNA solution, the DNA fragment was cleaved with restriction enzymes NheI and XhoI, purified using MinElute Reaction Cleanup Kit, and dissolved in 15 μl of Buffer EB.

  The about 2.0 kb DNA fragment containing the GSH1 gene obtained above and the about 5.4 kb DNA fragment of the expression plasmid pET-21a (+) prepared in the same manner as in Example 1 (1-2) were used. Kit Ver. 2.1 (manufactured by Takara Bio Inc.) was reacted at 16 ° C. for 30 minutes for ligation. Escherichia coli DH5α competent cell (manufactured by Takara Bio Inc.) was transformed by the heat shock method using the reaction solution, and the transformant was LB [10 g / L bactotryptone containing 100 μg / ml ampicillin. (Difco), 5 g / L yeast extract (Difco), 5 g / L sodium chloride (Wako)]] After application on an agar medium, the cells were cultured overnight at 37 ° C.

  A plasmid was extracted from the grown transformant colony by a known method, and its nucleotide sequence was determined by a known method. The resulting plasmid is a plasmid in which the GSH1 gene derived from Candida utilis ATCC22023 strain, to which a sequence encoding a His tag is added at the 3 ′ end, is ligated downstream of the T7 promoter, and this plasmid was named pET-GSH1. . The base sequence of the GSH1 gene derived from Candida utilis ATCC22023 and the amino acid sequence encoded thereby are shown in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

  Subsequently, Escherichia coli Rosetta2 (DE3) pLysS strain competent cell (manufactured by Novagen) was transformed with the heat shock method using pET-GSH1, and the transformant was treated with 100 μg / ml ampicillin and 30 μg / ml. Was applied to an LB agar medium containing chloramphenicol, and cultured at 37 ° C. overnight. A plasmid was extracted from the grown colonies of the transformant according to a known method, and the structure was analyzed using a restriction enzyme to confirm that pET-GSH1 was retained. The Escherichia coli Rosetta2 (DE3) pLysS strain carrying this pET-GSH1 was named Escherichia coli Rosetta2 (DE3) pLysS / pET-GSH1.

Example 8: Purification of C-terminal His-tagged recombinant Gsh1 Escherichia coli Rosetta2 (DE3) pLysS / pET-GSH1 obtained in Example 7 was treated with 100 μg / ml ampicillin and 30 μg / ml chloramphenicol. A test tube containing 3 mL of LB medium was inoculated and cultured at 37 ° C. for 16 hours with shaking. Of the obtained culture solution, 2 ml was inoculated into a Sakaguchi flask containing 100 ml of LB medium. After shaking culture at 37 ° C. for 2 hours, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mmol / L, and further cultured at 30 ° C. for 4 hours. The culture broth was centrifuged to obtain wet cells.

  Purification of His-tagged protein from the supernatant obtained by suspending the wet cells in 10 ml of 100 mmol / L Tris-HCl buffer solution containing 300 mM sodium chloride, sonicating, and then centrifuging. Using a kit, Ni Sepharose 6 Fast Flow (GE Healthcare), purify His-tagged recombinant Gsh1 according to the manual, and then, using PD-10 column (GE Healthcare), remove according to the manual. Salt went. This purified and desalted Gsh1 was used as purified Gsh1 in subsequent experiments.

Example 9: Production of γ-glutamyl dipeptide using purified Gsh1 Using the purified Gsh1 derived from Candida utilis ATCC22023 obtained in Example 8, production of γ-glutamyl dipeptide using norvaline as a substrate was examined. 200 μl of a reaction solution (pH 8.0) having the following composition was prepared and reacted at 37 ° C. for 16 hours.
[Reaction solution composition]
Purified Gsh1 24.6 μg / 200 μl
Tris-HCl (pH 8.0) 100 mmol / L
Norvaline 12.5mmol / L
Glutamic acid 12.5mmol / L
Adenosine triphosphate (ATP) 12.5mmol / L
Magnesium sulfate 12.5mmol / L
Dithiothreitol (DTT) 2 mmol / L

  After completion of the reaction, the reaction product was identified and quantified by HPLC under the same conditions as in Example 3. As a result, production of 3.8 mmol / L γ-glutamylnorvaline (γ-Glu-nVal) was confirmed.

Example 10: Construction of Escherichia coli-derived γ-glutamylcysteine synthetase gene (gshA) expression plasmid pQE-gshA An expression plasmid pQE-gshA encoding the γ-glutamylcysteine synthetase of Escherichia coli K-12 W3110 strain It was constructed according to the following procedure. An outline of the construction procedure is shown in FIG.

Primer K (SEQ ID NO: 19) and primer L (SEQ ID NO: 20) prepared from the base sequence of the gshA gene of Escherichia coli K-12 W3110 strain (SEQ ID NO: 17) were purchased from Nippon Bioservice. Primer K is Escherichia coli K-12
A nucleotide sequence containing an NcoI recognition sequence is added to the 5 ′ end of the region containing the start codon of the gshA gene of chromosomal DNA of the W3110 strain. Primer L is obtained by adding a base sequence containing an XhoI recognition sequence to the 5 ′ end of a base sequence complementary to the C-terminal base sequence of the gshA gene.

  A sequence containing the gshA gene was amplified by PCR using primer K and primer L, and chromosomal DNA of Escherichia coli K-12 W3110 as a template. PCR includes chromosomal DNA, 0.2 μmol / L of each primer, 1.25 units of PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.), 10 μL of 5 × PrimeSTAR buffer (manufactured by Takara Bio Inc.), 2.5 mmol / L of dNTP each 50 μl of a reaction solution containing (dATP, dGTP, dCTP, and dTTP) was prepared, heated at 98 ° C. for 10 seconds, then treated at 98 ° C. for 10 seconds, 55 ° C. for 5 seconds, and 72 ° C. for 1.6 minutes. This was repeated 30 times and further heated at 72 ° C. for 1 minute.

5 μl of the reaction solution after PCR was subjected to agarose gel electrophoresis, and after confirming that a DNA fragment of about 1.6 kb corresponding to the gshA gene fragment had been amplified, Etachinate (manufactured by Nippon Gene) was used from the remaining reaction solution. The DNA fragment was purified using and dissolved in 30 μl of dH ₂ O. Next, using the entire amount of the obtained DNA solution, the DNA fragment was cleaved with restriction enzymes NcoI and XhoI, and then the DNA fragment was purified using MinElute Reaction Cleanup Kit and dissolved in 15 μl of Buffer EB.

1 μg of expression vector pQE-TriSystem (manufactured by QIAGEN) was cleaved with restriction enzymes NcoI and XhoI, and then MinElute Reaction Cleanup
It was purified using Kit and dissolved in 15 μl of Buffer EB (manufactured by QIAGEN).

  An about 1.6 kb DNA fragment containing the gshA gene obtained above and an about 5.8 kb DNA fragment of the expression plasmid pQE-TriSystem obtained above were prepared using TaKaRa Ligation Kit Ver. 2.1 (manufactured by Takara Bio Inc.) was reacted at 16 ° C. for 30 minutes for ligation. Escherichia coli BL21 (DE3) strain competent cell (manufactured by Novagen) was transformed with the reaction solution by a heat shock method, and the transformant was LB [10 g / L bacterin containing 100 μg / ml ampicillin. Tryptone (manufactured by Difco), 5 g / L yeast extract (manufactured by Difco), 5 g / L sodium chloride (manufactured by Wako)] was applied to an agar medium and cultured at 30 ° C. overnight.

  A plasmid was extracted from the grown transformant colony by a known method, and its nucleotide sequence was determined by a known method. The obtained plasmid is a plasmid in which a gshA gene derived from Escherichia coli K-12 W3110 strain to which a sequence encoding a His tag is added at the 3 ′ end is ligated downstream of the T7 promoter. Named. In addition, Escherichia coli BL21 (DE3) strain carrying this pQE-gshA was named Escherichia coli BL21 (DE3) / pQE-gshA. The base sequence of the gshA gene derived from Escherichia coli K-12 W3110 strain and the amino acid sequence encoded thereby are shown in SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

Example 11: Purification of C-terminal His-tagged recombinant GshA Test tube containing 5 mL of LB medium containing 100 μg / ml ampicillin of Escherichia coli BL21 (DE3) / pQE-gshA obtained in Example 10 And inoculated with shaking at 30 ° C. for 16 hours. Of the obtained culture solution, 1 ml was inoculated into a Sakaguchi flask containing 50 ml of LB medium. After shaking culture at 30 ° C. for 5 hours, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mmol / L, and further cultured at 30 ° C. for 16 hours. The culture broth was centrifuged to obtain wet cells.

  The wet cells are suspended in 2 ml of BugBuster (manufactured by Novagen), gently shaken at room temperature for 20 minutes, and centrifuged to obtain a His-tagged protein purification kit, Ni Sepharose 6 Fast Flow ( GE Healthcare) was used to purify the His-tagged recombinant enzyme according to the manual, followed by desalting according to the manual using a PD-10 column (GE Healthcare). This purified and desalted GshA was used in subsequent experiments as purified GshA.

Example 12: Production of γ-glutamyl dipeptide using purified GshA Using purified GshA derived from Escherichia coli K-12 W3110 obtained in Example 11, production of γ-glutamyl dipeptide using valine or norvaline as a substrate was performed. investigated. 200 μl of a reaction solution (pH 8.0) having the following composition was prepared and reacted at 37 ° C. for 16 hours.
[Reaction solution composition]
Purified GshA 38.6 μg / 200 μl
Tris-HCl (pH 8.0) 100 mmol / L
Valine or norvaline 12.5mmol / L
Glutamic acid 12.5mmol / L
Adenosine triphosphate (ATP) 12.5mmol / L
Magnesium sulfate 12.5mmol / L
Dithiothreitol (DTT) 2 mmol / L

  After completion of the reaction, the reaction product was identified and quantified by HPLC under the same conditions as in Example 3. As a result, 1.1 mmol / L γ-Glu-Val, 5.6 mmol / L γ-Glu-nVal was produced. Was confirmed.

Example 13: Generation of tripeptide from amino acid using purified GshA and purified GshB Escherichia coli K-12 obtained in Example 11 Purified GshA derived from W3110 strain and Escherichia coli K-12 obtained in Example 5 Using purified GshB derived from W3110 strain, production of γ-glutamyltripeptide was examined. 200 μl of a reaction solution (pH 8.0) having the following composition was prepared and reacted at 37 ° C. for 16 hours. As a result, 0.048 mmol / L γ-Glu-Val-Gly and 1.2 mmol / L γ-Glu -NVal-Gly was produced.
[Reaction solution composition]
Purified GshA 19.3 μg / 200 μl
Purified GshB 21.8 μg / 200 μl
Tris-HCl (pH 8.0) 100 mmol / L
Valine or norvaline 12.5mmol / L
Glutamic acid 12.5mmol / L
Glycine 12.5mmol / L
Adenosine triphosphate (ATP) 12.5mmol / L
Magnesium sulfate 12.5mmol / L
Dithiothreitol (DTT) 2 mmol / L

Example 14: Construction of Saccharomyces cerevisiae-derived γ-glutamylcysteine synthase gene (GSH1) expression plasmid pET-ScGSH1 An expression plasmid pET-ScGSH1 (γ-glutamylcysteine synthetase encoded by the same gene is also referred to as ScGsh1) was constructed by the following procedure and introduced into Escherichia coli. An outline of the construction procedure is shown in FIG.

(14-1) Construction of Yeast Expression Plasmid pAUR-ScGSH1 First, the yeast expression plasmid pAUR-ScGSH1 was commissioned to Takara Bio and constructed according to the following procedure.

  Primer M (SEQ ID NO: 23) and N (SEQ ID NO: 24) prepared based on the base sequence (SEQ ID NO: 21) of GSH1 gene of Saccharomyces cerevisiae S288C strain, and chromosomal DNA of Saccharomyces cerevisiae S288C strain as template The sequence containing the GSH1 gene was amplified by PCR. Primer M is obtained by adding a KpnI recognition sequence and a partial sequence of yeast expression plasmid pAUR123 to the 5 'end of the region containing the start codon of the GSH1 gene in the chromosomal DNA of Saccharomyces cerevisiae S288C strain. Primer N includes a base sequence complementary to a sequence encoding a His tag in a base sequence complementary to the C-terminal base sequence of the GSH1 gene, a base sequence complementary to a stop codon (TAA), an XbaI recognition sequence, and yeast expression A partial sequence of plasmid pAUR123 is added. PCR was performed using PrimeSTAR Max DNA polymerase according to the manual.

The amplified fragment was introduced into the KpnI-XbaI site of the yeast expression plasmid pAUR123 using the In-Fusion Advantage PCR Kit based on the manual to prepare the yeast expression plasmid pAUR-ScGSH1.

(14-2) Construction of Escherichia coli expression plasmid pET-ScGSH1 Subsequently, an Escherichia coli expression plasmid pET-ScGSH1 was constructed by the following procedure.

  Primer O (SEQ ID NO: 25) and Primer P (SEQ ID NO: 26) prepared from the base sequence of the GSH1 gene of Saccharomyces cerevisiae S288C strain (SEQ ID NO: 21) were purchased from Nippon Bioservice. Primer O is obtained by adding a base sequence containing a SpeI recognition sequence to the 5 'end of the region containing the next of the start codon of the GSH1 gene of the chromosomal DNA of Saccharomyces cerevisiae S288C strain. Primer P is obtained by adding a base sequence including a SalI recognition sequence to the 5 'end of a base sequence complementary to the base sequence outside the stop codon of the ScGSH1 gene of pAUR-ScGSH1.

  A sequence containing the ScGSH1 gene was amplified by PCR using the primer O and the primer P and the above-described pAUR-ScGSH1 as a template. PCR was performed using plasmid DNA, 0.2 μmol / L of each primer, 1.25 units of PrimeSTAR HS DNA polymerase (Takara Bio), 10 μL of 5 × PrimeSTAR buffer (Takara Bio), 2.5 mmol / L of dNTP. 50 μl of a reaction solution containing (dATP, dGTP, dCTP, and dTTP) is prepared, heated at 98 ° C. for 10 seconds, and then subjected to 30 steps of 98 ° C. for 10 seconds, 56 ° C. for 5 seconds, and 72 ° C. for 2 minutes. Repeatedly, it was further heated at 72 ° C. for 1 minute.

3 μl of the reaction solution after PCR was subjected to agarose gel electrophoresis, and after confirming that a DNA fragment of about 2.0 kb corresponding to the ScGSH1 gene fragment had been amplified, the DNA was obtained from the remaining reaction solution using Ethachinate. The fragment was purified and dissolved in 25 μl dH ₂ O. Next, using the total amount of the obtained DNA solution, the DNA was cleaved with restriction enzymes SpeI and SalI, and then the DNA fragment was purified using MinElute Reaction Cleanup Kit and dissolved in 15 μl of Buffer EB.

After cutting 1 μg of the expression plasmid pET-21a (+) with restriction enzymes NheI and SalI, the DNA fragment was purified using MinElute Reaction Cleanup Kit and dissolved in 15 μl of Buffer EB. Next, using the entire amount of the obtained DNA solution, the DNA fragment was dephosphorylated with Alkaline Phosphatase, Calf intestine (CIAP), and then MinElute Reaction
It was purified using Cleanup Kit and dissolved in 10 μl of Buffer EB.

  The about 2.0 kb DNA fragment containing the ScGSH1 gene obtained above and the about 5.4 kb DNA fragment of the expression vector pET-21a (+) obtained above were prepared using TaKaRa Ligation Kit Ver. Using 2.1, the reaction was carried out at 16 ° C. for 30 minutes for ligation. Escherichia coli DH5α strain competent cell (manufactured by Takara Bio Inc.) was transformed by the heat shock method using the reaction solution, and the transformant was applied to an LB agar medium containing 100 μg / ml ampicillin. Cultured overnight at 37 ° C.

A plasmid was extracted from the grown colonies of the transformant according to a known method, and its nucleotide sequence was determined by a known method. The obtained plasmid was a plasmid in which a GSH1 gene derived from Saccharomyces cerevisiae S288C strain added with a sequence encoding a His tag sequence at the 3 ′ end was linked downstream of the T7 promoter, and was named pET-ScGSH1. The base sequence of the GSH1 gene derived from Saccharomyces cerevisiae S288C strain and the amino acid sequence encoded thereby are shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively.

  Subsequently, Escherichia coli Rosetta2 (DE3) pLysS strain competent cells were transformed with the heat shock method using pET-ScGSH1, and the transformants were transformed with 100 μg / ml ampicillin and 30 μg / ml chloramphenicol. After being applied to an LB agar medium containing the solution, the cells were cultured overnight at 37 ° C. A plasmid was extracted from the grown colonies of the transformant according to a known method, and the structure was analyzed using a restriction enzyme to confirm that pET-ScGSH1 was retained. The Escherichia coli Rosetta2 (DE3) pLysS strain carrying this pET-ScGSH1 was named Escherichia coli Rosetta2 (DE3) pLysS / pET-ScGSH1.

Example 15: Purification of C-terminal His-tagged recombinant ScGsh1 Escherichia coli Rosetta2 (DE3) pLysS / pET-ScGSH1 obtained in Example 14 was treated with 100 μg / ml ampicillin and 30 μg / ml chloramphenicol. A test tube containing 3 mL of LB medium was inoculated and cultured at 37 ° C. for 16 hours with shaking. Of the obtained culture solution, 2 ml was inoculated into a Sakaguchi flask containing 100 ml of LB medium. After shaking culture at 37 ° C. for 2 hours, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.5 mmol / L, and further cultured at 30 ° C. for 4 hours. The culture broth was centrifuged to obtain wet cells.

  The wet cells are suspended in 5 mL of BugBuster Master Mix (manufactured by Novagen), protein is extracted, and from the supernatant obtained by centrifugation, Ni Sepharose 6 Fast Flow is used according to the instructions. The modified ScGsh1 was purified. Subsequently, desalting was performed using a PD-10 column (manufactured by GE Healthcare) according to the instructions. This purified and desalted ScGsh1 was used in the subsequent experiments as purified ScGsh1.

Example 16: Production of γ-glutamyldipeptide using purified ScGsh1 Using purified ScGsh1 derived from Saccharomyces cerevisiae S288C obtained in Example 15, γ using valine, norvaline, or α-aminobutyric acid (Abu) as a substrate -The production of glutamyl dipeptide was investigated. 200 μl of a reaction solution (pH 8.5) having the following composition was prepared and reacted at 30 ° C. for 24 hours.
[Reaction solution composition]
Purified ScGsh1 40.5 μg / 200 μl
Tris-HCl (pH 8.5) 50 mmol / L
Valine, norvaline, or α-aminobutyric acid 10 mmol / L
Glutamic acid 10 mmol / L
Adenosine triphosphate (ATP) 10 mmol / L
Magnesium sulfate 10 mmol / L
Dithiothreitol (DTT) 2 mmol / L
Glycerol 20% (Vol./Vol.)

  After completion of the reaction, the reaction product was analyzed by HPLC under the conditions described in Example 3. As a result, the peaks of the respective reaction products were γ-Glu-Val, γ-Glu-nVal, and γ-Glu-Abu. The peak of the sample coincided with the retention time, and it was judged to be γ-Glu-Val, γ-Glu-nVal, and γ-Glu-Abu. As a result of quantification, the γ-Glu-Val concentration was 0.5 mmol / L, the γ-Glu-nVal concentration was 4.7 mmol / L, and the γ-Glu-Abu concentration was 8.2 mmol / L.

Example 17: Generation of tripeptide from amino acids using purified ScGsh1 and purified Gsh2 Purified ScGsh1 derived from Saccharomyces cerevisiae S288C strain obtained in Example 15 and purified Gsh2 derived from Saccharomyces cerevisiae S288C strain obtained in Example 2 Was used to examine the production of γ-glutamyl tripeptide. When 200 μl of a reaction solution (pH 9.0) having the following composition was prepared and reacted at 30 ° C. for 8 hours, 0.11 mmol / L γ-Glu-Val and 0.08 mmol / L γ-Glu-Val- Gly produced.
[Reaction solution composition]
Purified ScGsh1 127 μg / 200 μl
Purified Gsh2 7 μg / 200 μl
Tris-HCl (pH 9.0) 50 mmol / L
Valine 10 mmol / L
Glutamic acid 10 mmol / L
Glycine 10 mmol / L
Adenosine triphosphate (ATP) 10 mmol / L
Magnesium sulfate 10 mmol / L
Dithiothreitol (DTT) 2 mmol / L
Glycerol 20% (Vol./Vol.)

  According to the present invention, γ-Glu-XY can be produced.

Description of Sequence Listing SEQ ID NO: 1: Base sequence of GSH2 gene derived from Saccharomyces cerevisiae SEQ ID NO: 2: Amino acid sequence of Gsh2 protein derived from Saccharomyces cerevisiae SEQ ID NO: 3: Forward primer for amplifying GSH2 gene derived from Saccharomyces cerevisiae (Primer A)
SEQ ID NO: 4: Reverse primer (primer B) for amplifying the GSH2 gene derived from Saccharomyces cerevisiae
SEQ ID NO: 5: Forward primer (primer C) for amplifying the GSH2 gene derived from Saccharomyces cerevisiae
SEQ ID NO: 6: Reverse primer (primer D) for amplifying the GSH2 gene derived from Saccharomyces cerevisiae
SEQ ID NO: 7: base sequence of gshB gene derived from Escherichia coli SEQ ID NO: 8: amino acid sequence of GshB protein derived from Escherichia coli SEQ ID NO: 9: forward primer for amplifying gshB gene derived from Escherichia coli (primer E)
SEQ ID NO: 10: Reverse primer (primer F) for amplifying the gshB gene derived from Escherichia coli
SEQ ID NO: 11: nucleotide sequence of GSH1 gene derived from Candida utilis SEQ ID NO: 12 amino acid sequence of Gsh1 protein derived from Candida utilis SEQ ID NO: 13: forward primer for amplifying GSH1 gene derived from Candida utilis (primer G)
SEQ ID NO: 14: Reverse primer (primer H) for amplifying the GSH1 gene derived from Candida utilis
SEQ ID NO: 15: Forward primer (primer I) for amplifying the GSH1 gene derived from Candida utilis
SEQ ID NO: 16: Reverse primer (primer J) for amplifying the GSH1 gene derived from Candida utilis
SEQ ID NO: 17: base sequence of gshA gene derived from Escherichia coli SEQ ID NO: 18: amino acid sequence of GshA protein derived from Escherichia coli SEQ ID NO: 19: forward primer for amplifying gshA gene derived from Escherichia coli (primer K)
SEQ ID NO: 20: Reverse primer (primer L) for amplifying the gshA gene derived from Escherichia coli
SEQ ID NO: 21: base sequence of GSH1 gene derived from Saccharomyces cerevisiae SEQ ID NO: 22: amino acid sequence of Gsh1 protein derived from Saccharomyces cerevisiae SEQ ID NO: 23: forward primer for amplifying GSH1 gene derived from Saccharomyces cerevisiae (primer M)
SEQ ID NO: 24: Reverse primer (primer N) for amplifying the GSH1 gene derived from Saccharomyces cerevisiae
SEQ ID NO: 25: Forward primer (primer O) for amplifying GSH1 gene derived from Saccharomyces cerevisiae
SEQ ID NO: 26: Reverse primer (primer P) for amplifying the GSH1 gene derived from Saccharomyces cerevisiae

Claims (9)

Production of γ-Glu-XY, including the step of producing γ-Glu-XY by allowing glutathione synthetase and γ-glutamylcysteine synthetase to act on raw materials containing Glu, X, and Y A method,
The γ-glutamylcysteine synthetase is selected from the following A) to I):
A) γ-glutamylcysteine synthetase derived from Saccharomyces cerevisiae microorganism B) γ-glutamylcysteine synthetase derived from Candida utilis microorganism C) Escherichia coli microorganism derived from Escherichia coli Glutamylcysteine synthetase D) Protein consisting of the amino acid sequence of SEQ ID NO: 12 E) Protein consisting of the amino acid sequence of SEQ ID NO: 18 F) Protein consisting of the amino acid sequence of SEQ ID NO: 22) 90% identity with the amino acid sequence of SEQ ID NO: 12 A protein having the above amino acid sequence and having γ-glutamylcysteine synthetase activity H) No amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 18 A protein having γ-glutamylcysteine synthetase activity I) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 22 and having γ-glutamylcysteine synthetase activity Glutathione synthetase is selected from the following J) to O),
J) Glutathione synthase derived from a microorganism of Saccharomyces cerevisiae K) Escherichia coli Glutathione synthase derived from a microorganism L) From the amino acid sequence of SEQ ID NO: 8 N) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 2 and having glutathione synthetase activity O) an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 8 A protein having the glutathione synthetase activity, wherein Y is Gly, and X is Val or nVal. A method for producing γ-Glu-XY, comprising a step of producing γ-Glu-XY by allowing glutathione synthetase to act on a raw material containing γ-Glu-X and Y,
The glutathione synthetase is selected from the following J) to O):
J) Glutathione synthase derived from a microorganism of Saccharomyces cerevisiae K) Escherichia coli Glutathione synthase derived from a microorganism L) From the amino acid sequence of SEQ ID NO: 8 N) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 2 and having glutathione synthetase activity O) an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 8 A protein having the glutathione synthetase activity, wherein Y is Gly, and X is Val or nVal.   The γ-Glu-XY according to claim 1 or 2, wherein the glutathione synthetase and / or γ-glutamylcysteine synthetase is a culture of a microorganism having the activity of the enzyme or a processed product of the culture. Manufacturing method.   The method for producing γ-Glu-XY according to any one of claims 1 to 3, wherein the glutathione synthetase and / or γ-glutamylcysteine synthetase is a purified enzyme.   The method for producing γ-Glu-XY according to any one of claims 1 to 4, wherein the glutathione synthetase and / or γ-glutamylcysteine synthetase is an immobilized enzyme. Glutathione synthetase gene and gamma - a microorganism expression guru Tamil cysteine synthetase gene has been enhanced, Glu, X, and at least one amino acid and / or amino acid derivative selected from the group consisting of Y is added It is a medium , characterized in that it is cultured in a medium to which at least X is added , γ-Glu-XY is produced and accumulated in the medium, and γ-Glu-XY is collected from the culture. A method for producing γ-Glu-XY,
The γ-glutamylcysteine synthetase is selected from the following A) to I):
A) γ-glutamylcysteine synthetase derived from Saccharomyces cerevisiae microorganism B) γ-glutamylcysteine synthetase derived from Candida utilis microorganism C) Escherichia coli microorganism derived from Escherichia coli Glutamylcysteine synthetase D) Protein consisting of the amino acid sequence of SEQ ID NO: 12 E) Protein consisting of the amino acid sequence of SEQ ID NO: 18 F) Protein consisting of the amino acid sequence of SEQ ID NO: 22) 90% identity with the amino acid sequence of SEQ ID NO: 12 A protein having the above amino acid sequence and having γ-glutamylcysteine synthetase activity H) No amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 18 A protein having γ-glutamylcysteine synthetase activity I) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 22 and having γ-glutamylcysteine synthetase activity Glutathione synthetase is selected from the following J) to O),
J) Glutathione synthase derived from a microorganism of Saccharomyces cerevisiae K) Escherichia coli Glutathione synthase derived from a microorganism L) From the amino acid sequence of SEQ ID NO: 8 N) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 2 and having glutathione synthetase activity O) an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 8 A protein having the glutathione synthetase activity, wherein Y is Gly, and X is Val or nVal. Glu, X, and has an ability to produce at least one amino acid and / or amino acid derivative selected from the group consisting of Y, productivity of Val is imparted or enhanced, and, glutathione synthetase gene and γ Culturing microorganisms with enhanced expression of glutamylcysteine synthase gene, generating and accumulating γ-Glu-XY in the medium, and collecting γ-Glu-XY from the culture A method for producing γ-Glu-XY, characterized by:
The γ-glutamylcysteine synthetase is selected from the following A) to I):
A) γ-glutamylcysteine synthetase derived from Saccharomyces cerevisiae microorganism B) γ-glutamylcysteine synthetase derived from Candida utilis microorganism C) Escherichia coli microorganism derived from Escherichia coli Glutamylcysteine synthetase D) Protein consisting of the amino acid sequence of SEQ ID NO: 12 E) Protein consisting of the amino acid sequence of SEQ ID NO: 18 F) Protein consisting of the amino acid sequence of SEQ ID NO: 22) 90% identity with the amino acid sequence of SEQ ID NO: 12 A protein having the above amino acid sequence and having γ-glutamylcysteine synthetase activity H) No amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 18 A protein having γ-glutamylcysteine synthetase activity I) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 22 and having γ-glutamylcysteine synthetase activity Glutathione synthetase is selected from the following J) to O),
J) Glutathione synthase derived from a microorganism of Saccharomyces cerevisiae K) Escherichia coli Glutathione synthase derived from a microorganism L) From the amino acid sequence of SEQ ID NO: 8 N) a protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 2 and having glutathione synthetase activity O) an amino acid sequence having 90% or more identity with the amino acid sequence of SEQ ID NO: 8 a protein consisting of a said Y is Gly having glutathione synthetase activity, and said X is Va l, manufacturing method.   The method for producing γ-Glu-XY according to claim 6 or 7, wherein the microorganism is a microorganism belonging to the genus Escherichia or Corynebacterium.   The method for producing γ-Glu-XY according to claim 8, wherein the microorganism belonging to the genus Escherichia is Escherichia coli, and the microorganism belonging to the genus Corynebacterium is Corynebacterium glutamicum.

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