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WO2013054447A1 - Procédé de production de γ-glu-x-gly ou d'un sel de celui-ci, et glutathion synthase mutante - Google Patents

Procédé de production de γ-glu-x-gly ou d'un sel de celui-ci, et glutathion synthase mutante Download PDF

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WO2013054447A1
WO2013054447A1 PCT/JP2011/073715 JP2011073715W WO2013054447A1 WO 2013054447 A1 WO2013054447 A1 WO 2013054447A1 JP 2011073715 W JP2011073715 W JP 2011073715W WO 2013054447 A1 WO2013054447 A1 WO 2013054447A1
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amino acid
acid sequence
glu
seq
gly
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Japanese (ja)
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崇敬 伊藤
西内　博章
五十嵐　俊介
一雄 山岸
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味の素株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
    • C12Y603/02—Acid—amino-acid ligases (peptide synthases)(6.3.2)
    • C12Y603/02003—Glutathione synthase (6.3.2.3)

Definitions

  • the present invention relates to a method for producing a peptide represented by ⁇ -Glu-X-Gly (X represents an amino acid selected from the group consisting of Val, nVal, Leu, Abu, and Ile; the same shall apply hereinafter) or a salt thereof, And a mutant glutathione synthetase.
  • CaSR Calcium sensing receptor
  • Patent Document 1 Several ⁇ -glutamyl peptides such as ⁇ -Glu-Met, ⁇ -Glu-Thr, and ⁇ -Glu-Val-Gly have been reported to have a kokumi imparting effect (Patent Document 1). .
  • Patent Document 2 an S- or O-carboxyalkylated ⁇ -glutamyl or ⁇ -aspartyl peptide ester group has been reported as a body taste substance (Patent Document 2).
  • these peptides and esters impart a rich taste to foods like GSH, they do not have a reduced thiol group (—SH group) unlike GSH.
  • Patent Document 2 a substance having a reduced thiol group such as GSH is unstable, and its titer decreases with the formation of a disulfide bond. Therefore, these rich taste imparting peptides that do not have a reduced thiol group are considered useful in terms of stability and the like.
  • Non-patent Document 4 As a knowledge about foods containing ⁇ -glutamyldipeptide, there is a report that various ⁇ -glutamyldipeptides were detected from Gouda cheese aged for about 44 weeks (Non-patent Document 4). In this document, various ⁇ -glutamyldipeptides such as ⁇ -Glu-Ala, ⁇ -Glu-Glu, and ⁇ -Glu-Gln were detected, and the content of ⁇ -glutamyldipeptide was 3590 ⁇ mol / kg dry weight at the maximum. It has been reported.
  • Non-patent Document 5 an example of fermentation broth analysis of Micrococcus glutamicus.
  • fermentation broth was applied to various columns to separate peptides and ⁇ -Glu-Glu, ⁇ -Glu-Val, and ⁇ -Glu-Leu were isolated. It is unknown how much was contained in the broth.
  • GSH is produced in vivo by ⁇ -Glu-Cys being produced from Glu and Cys by ⁇ -glutamylcysteine synthase, and Gly being further bound by glutathione synthase.
  • ⁇ -glutamylcysteine synthetase is encoded by GSH1 gene or gshA gene
  • glutathione synthetase is encoded by GSH2 gene or gshB gene
  • Non-patent Document 3 a method for enzymatically producing theanine ( ⁇ -glutamylanilide) using the side reaction of ⁇ -glutamylcysteine synthetase of Escherichia coli has been reported (Patent Document 3, non-patent document). Reference 6).
  • Patent Document 7 an example in which the substrate specificity of ⁇ -glutamylcysteine synthetase of Proteus mirabilis to amino acids other than Cys has been reported has been reported (Non-patent Document 7).
  • ⁇ -glutamylcysteine synthetase is known to have a wide substrate specificity.
  • Non-Patent Document 8 Non-Patent Document 9
  • ⁇ -Glu-Abu ⁇ -glutamyl- ⁇ -aminobutyric acid
  • Gly for glutathione synthase derived from Escherichia coli Has reported a method for producing ⁇ -Glu-Abu-Gly (Patent Document 4), but for X other than Abu, it has been shown whether ⁇ -Glu-X can be a substrate for glutathione synthase. Absent.
  • ⁇ -Glu-Val and ⁇ -Glu-nVal were used for glutathione synthase derived from rat liver.
  • Gly have been studied (Non-patent Document 10), but ⁇ -Glu-Val is not a substrate, and ⁇ -Glu-nVal is a substrate, but the reactivity is very low. It was considered difficult to apply this enzyme to the production of ⁇ -glutamyl tripeptide using these ⁇ -glutamyl compounds as substrates.
  • Patent Document 5 discloses a yeast that produces ⁇ -Glu-Cys having a mutant of glutathione synthetase, and T47I, P54L, and G387D are disclosed as mutation points. The effect on the production of ⁇ -Glu-X-Gly is not known.
  • ⁇ -Glu-X-Gly is produced using glutathione synthetase derived from Saccharomyces cerevisiae or a mutant glutathione synthetase having a specific mutation.
  • Non-Patent Documents 11 and 12 As a method for producing ⁇ -Glu-X-Gly, an example in which ⁇ -Glu-Lys-Gly is generated using ⁇ -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.
  • the present invention provides a method for producing a peptide represented by ⁇ -Glu-X-Gly (X represents an amino acid selected from the group consisting of Val, nVal, Leu, Abu, and Ile) or a salt thereof. This is the issue.
  • ⁇ -Glu-X-Gly is produced by the action of Saccharomyces cerevisiae-derived glutathione synthetase or mutant glutathione synthetase using ⁇ -Glu-X and Gly as raw materials.
  • the present inventors acted on ⁇ -Glu-X by using ⁇ -glutamylcysteine synthetase, and Saccharomyces cerevisiae-derived glutathione synthetase or mutant glutathione synthetase using Glu, X, and Gly as raw materials.
  • -Gly was found to form. Based on these findings, the present invention has been completed.
  • the present invention is as follows.
  • ⁇ -Glu-X-Gly (1) (In the formula, X represents an amino acid selected from the group consisting of Val, nVal, Leu, Abu, and Ile.)
  • ⁇ -Glu-X-Gly (1) (In the formula, X represents an amino acid selected from the group consisting of Val, nVal, Leu, Abu, and Ile.)
  • ⁇ -Glu-X-Gly (1) (In the formula, X represents an amino acid selected from the group consisting of Val, nVal, Leu, Abu, and Ile.)
  • a method for producing a peptide represented by the formula: A step of producing ⁇ -Glu-X-Gly by allowing glutathione synthetase and ⁇ -glutamylcysteine synthetase to act on Glu, X, and Gly, The method wherein the glutathione synthetase is a protein selected from the following (A) to (D): (A) The following (a) to (c): (A) the amino acid sequence shown in SEQ ID NO: 2, (B) an amino acid sequence comprising one or several amino acid substitutions, deletions, insertions or additions in the amino acid sequence shown in SEQ ID NO: 2; (C) an amino acid sequence having 90% or more homology to the amino acid sequence shown in SEQ ID NO: 2; In any one of the amino acid
  • the mutation is a mutation corresponding to one or more mutations selected from the following: S28T, M29V, F34Y, S44T, F100L, L104V, I126L, F127Y, L132 (I, M, V), T149V, V150 (A, C, F, G, H, I, M, N, S, T, Y ), S153 (A, T), F154C, S158T, S191T, L195V, L199V, I223V, V224I, Q225 (D, E, N), R226Q, N227D, E228D, N230Q, V236I, L237I, L252V, T253S, F254Y, Y284F, T286S, Y288F, T289S, T290S, T291S, D292E, A313S, L320V, S321T, S323T, E386 (D, N), G389A
  • the mutation is a mutation corresponding to a mutation selected from the following: S28T, F34Y, S44T, L132M, V150A, V150C, V150F, V150G, V150H, V150I, V150M, V150N, V150S, V150T, V150Y, S191T, L195V, Q225D, V236I, L237I, E386D, I445V, Y446F, (S28T + S ), (S28T + L132M), (S28T + L195V), (S28T + E386D), (S28T + Y446F), (S44T + L132M), (S44T + L195V), (S44T + E386D), (S44T + Y446F), (L132M + L195V), (S44T + E386D), (S44T + Y446F), (L132M + L195V), (L132M + E386D), (S44
  • the method wherein the glutathione synthetase and / or ⁇ -glutamylcysteine synthetase is a culture of microorganisms having the activity of the enzyme or a processed product of the culture. [7] The method, wherein the glutathione synthetase and / or ⁇ -glutamylcysteine synthetase is a purified enzyme. [8] The method, wherein the glutathione synthetase and / or ⁇ -glutamylcysteine synthetase is an immobilized enzyme.
  • microorganism is a microorganism belonging to the genus Escherichia or Corynebacterium.
  • microorganism is Escherichia coli or Corynebacterium glutamicum.
  • the mutant glutathione synthetase wherein the mutation is a mutation corresponding to a mutation selected from the following: S28T, F34Y, S44T, L132M, V150A, V150C, V150F, V150G, V150H, V150I, V150M, V150N, V150S, V150T, V150Y, S191T, L195V, Q225D, V236I, L237I, E386D, I445V, Y446F, (S28T + S ), (S28T + L132M), (S28T + L195V), (S28T + E386D), (S28T + Y446F), (S44T + L132M), (S44T + L195V), (S44T + E386D), (S44T + Y446F), (L132M + L195V), (S44T + E386D), (S44T + Y446F), (L132M + L195V
  • ⁇ -Glu-X-Gly (X represents an amino acid or an amino acid derivative selected from the group consisting of Val, nVal, Leu, Abu, and Ile) can be produced.
  • the present invention also provides a mutant glutathione synthetase that can be used for the production of ⁇ -Glu-X-Gly.
  • ⁇ -Glu-X-Gly produced by the present invention
  • the peptide produced by the present invention has the following formula (1): ⁇ -Glu-X-Gly (1)
  • X represents an amino acid selected from the group consisting of Val, nVal, Leu, Abu, and Ile.
  • Glu in the formula (1) represents glutamic acid.
  • “-” Represents a peptide bond.
  • “ ⁇ ” in ⁇ -Glu means that X is bound to glutamic acid via a carboxyl group at the ⁇ -position of glutamic acid.
  • X represents an amino acid selected from the group consisting of valine (Val), norvaline (nVal), leucine (Leu), ⁇ -aminobutyric acid (Abu), and isoleucine (Ile). That is, X is selected from aliphatic neutral amino acids. X is preferably Val or nVal.
  • one type of ⁇ -Glu-X-Gly may be produced, or two or more types of ⁇ -Glu-X-Gly may be produced.
  • ⁇ -Glu-X-Gly and Glu, X, Gly, and ⁇ -Glu-X used as raw materials may all be free forms or salts thereof, or a mixture thereof.
  • the salt include sulfate, hydrochloride, carbonate, ammonium salt, sodium salt, and potassium salt.
  • the glutathione synthase used in the present invention recognizes ⁇ -Glu-X as a substrate and binds to Gly to form a peptide that is a glutathione derivative, that is, ⁇ -Glu-X-Gly. Has the activity of catalyzing the reaction to produce 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 any X, and may exhibit glutathione synthetase activity for the amino acid or amino acid derivative selected as X.
  • glutathione synthase when two or more ⁇ -Glu-X-Gly are produced, a single glutathione synthase catalyzes a reaction that produces all of the two or more ⁇ -Glu-X-Gly. It is not necessary to have such activity, and two or more glutathione synthetases may be used depending on X.
  • glutathione synthase has an activity of catalyzing the reaction of generating GSH or ⁇ -Glu-Cys derivative-Gly from ⁇ -Glu-Cys or ⁇ -Glu-Cys derivative and Gly. You may or may not have.
  • Examples of the glutathione synthetase used in the present invention include a protein encoded by the GSH2 gene of Saccharomyces cerevisiae.
  • the base sequence of the GSH2 gene of Saccharomyces cerevisiae is disclosed in Saccharomyces Genome Database (http://www.yeastgenome.org/).
  • 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.
  • the glutathione synthetase used in the present invention may be a glutathione synthetase, for example, a protein variant having the amino acid sequence shown in SEQ ID NO: 2, as long as it has glutathione synthetase activity. Such a variant may be referred to as a “conservative variant”.
  • the conservative variant may be, for example, a homologue or artificially modified form of the glutathione synthetase.
  • the gene encoding glutathione synthetase is a protein having glutathione synthetase activity, that is, if it encodes the glutathione synthetase or a conservative variant thereof, It may be a variant.
  • the gene encoding the homolog of glutathione synthetase can be easily obtained from a public database by BLAST search or FASTA search using the GSH2 gene of Saccharomyces cerevisiae as a query sequence, and a chromosome such as yeast can be used as a template. Thus, it can be obtained by PCR using oligonucleotides prepared based on these known gene sequences as primers.
  • the glutathione synthetase gene can be substituted, deleted, inserted or added at one or several positions in the amino acid sequence as long as it encodes a protein having glutathione synthetase activity. It may be a gene encoding a protein having a sequence to be included.
  • the “one or several” differs depending on the position of the amino acid residue in the three-dimensional structure of the protein and the type of amino acid residue, but specifically preferably 1 to 20, more preferably 1 to 10, More preferably, it means 1 to 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.
  • 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
  • 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 mutations (mutants or variants) such as cases based on individual differences or species differences of microorganisms from which genes are derived. Also included by mutations (mutants or variants) such as cases based on individual differences or species differences of microorganisms from which genes are derived. Also included by
  • a gene having a conservative mutation as described above is 80% or more, preferably 90% or more, more preferably 95% or more, more preferably 97% or more, particularly preferably 99% or more with respect to the entire amino acid sequence. It may be a gene encoding a protein having a homology of at least% and having glutathione synthetase activity. In the present specification, “homology” may refer to “identity”.
  • the glutathione synthase gene encodes a protein having glutathione synthetase activity by hybridizing under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a complementary sequence to the whole or a part of the base sequence. It may also be DNA that does.
  • 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.
  • the probe used for the hybridization may be a part of a complementary sequence of a gene.
  • 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.
  • hybridization washing conditions include 50 ° C., 2 ⁇ SSC, and 0.1% SDS.
  • the glutathione synthase gene can be used in its natural form, but it may be modified to have an optimal codon according to the codon usage frequency of the host to be used.
  • examples of the glutathione synthetase used in the present invention include glutathione synthetase having a specific mutation.
  • the “specific mutation” will be described later.
  • the glutathione synthetase having the “specific mutation” is also referred to as “mutant glutathione synthetase”.
  • the glutathione synthetase having no “specific mutation” is also referred to as “wild-type glutathione synthetase”.
  • wild type as used herein is not limited to those obtained in nature unless it has the “specific mutation”.
  • Examples of the wild-type glutathione synthase include proteins encoded by the above-mentioned Saccharomyces cerevisiae GSH2 gene and conservative variants thereof that do not have the “specific mutation”.
  • mutant glutathione synthetase may be a protein encoded by the above-mentioned Saccharomyces cerevisiae GSH2 gene or a conservative variant thereof, except that it has the “specific mutation”.
  • the mutant glutathione synthase may be a protein having the amino acid sequence shown in SEQ ID NO: 2 except having the “specific mutation”.
  • the mutant glutathione synthase has one or several amino acid substitutions, deletions, insertions in the amino acid sequence shown in SEQ ID NO: 2 except that it has the “specific mutation”.
  • it may be a protein having an amino acid sequence including an addition.
  • the mutant glutathione synthase has 80% or more, preferably 90% or more, more preferably, with respect to the amino acid sequence shown in SEQ ID NO: 2 except that it has the “specific mutation”.
  • the mutant glutathione synthetase has the “specific mutation” in the amino acid sequence of the protein encoded by the GSH2 gene of Saccharomyces cerevisiae as long as it has glutathione synthetase activity. It may be a variant containing a mutation at a place other than “mutation of”. For such variants, the above-mentioned descriptions concerning variants of glutathione synthetase can be applied mutatis mutandis.
  • the mutant glutathione synthase has the “specific mutation” in the amino acid sequence shown in SEQ ID NO: 2, and further, one or several other than the “specific mutation” It may be a protein having an amino acid sequence including amino acid substitution, deletion, insertion, or addition, and having glutathione synthetase activity.
  • the mutant glutathione synthetase has a mutation in an amino acid residue corresponding to one or more amino acid residues selected from the following.
  • This mutation is a “specific mutation” in the present invention.
  • numbers indicate positions in the amino acid sequence shown in SEQ ID NO: 2, and letters on the left side of the numbers indicate amino acid residues before mutation. That is, for example, “S28” indicates the serine residue at position 28 in the amino acid sequence shown in SEQ ID NO: 2.
  • amino acid residue corresponding to the amino acid residue at position X in the amino acid sequence shown in SEQ ID NO: 2 refers to the alignment between the amino acid sequence of SEQ ID NO: 2 and the target amino acid sequence.
  • amino acid residue corresponding to S28 means an amino acid residue corresponding to the serine residue at position 28 in SEQ ID NO: 2, and one amino acid residue on the N-terminal side from position 28 is deleted.
  • the 27th amino acid residue counted from the N-terminus (the methionion residue encoded by the start codon is the first) is the “amino acid residue corresponding to S28”.
  • the 29th amino acid residue counted from the N-terminal is “an amino acid residue corresponding to S28”.
  • the mutant glutathione synthetase when the wild-type glutathione synthetase has the amino acid sequence shown in SEQ ID NO: 2, the mutant glutathione synthetase only needs to have a mutation in the amino acid residue in SEQ ID NO: 2.
  • the mutant glutathione synthetase when the wild-type glutathione synthetase is a variant of a protein having the amino acid sequence shown in SEQ ID NO: 2, the mutant glutathione synthetase is an amino acid residue corresponding to the amino acid residue in SEQ ID NO: 2. It suffices to have a mutation.
  • the mutant glutathione synthetase is an amino acid sequence represented by SEQ ID NO: 2, an amino acid sequence comprising one or several amino acid substitutions in the amino acid sequence represented by SEQ ID NO: 2, or a sequence number
  • the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 2 may have a mutation.
  • Alignment can be performed using, for example, known gene analysis software.
  • Specific software includes DNA Solutions from Hitachi Solutions and GENETYX from Genetics (Elizabeth C. Tyler et al., Computers and Biomedical Research, 24 (1), 72-96, 1991; Barton GJ et) al., Journal of molecular biology, 198 (2), 327-37. 1987).
  • mutant glutathione synthetase preferably has a mutation in an amino acid residue corresponding to one or more amino acid residues selected from the following among the above amino acid residues.
  • the numbers and the meanings of the characters to the left of the numbers are the same as described above.
  • the amino acid residue after the mutation may be any amino acid residue other than the original amino acid residue as long as the mutant glutathione synthetase has glutathione synthetase activity.
  • the substrate specificity for “X” of ⁇ -Glu-X may be the same between wild-type glutathione synthetase and mutant glutathione synthetase. Well, it can be different. For example, when the wild-type glutathione synthetase can recognize ⁇ -Glu-Cys as a substrate, the mutant glutathione synthetase may or may not recognize ⁇ -Glu-Cys as a substrate.
  • Specific mutations at the positions include mutations corresponding to mutations selected from the following.
  • the numbers and the meanings of the characters to the left of the numbers are the same as described above.
  • the character on the right side of the number or the character in parentheses on the right side of the number indicates the amino acid residue after mutation. That is, for example, “S28T” indicates a mutation in which the Ser residue at position 28 in the amino acid sequence shown in SEQ ID NO: 2 is replaced with a Thr residue.
  • “L132 (I, M, V)” is a substitution of the Lys residue at position 132 in the amino acid sequence shown in SEQ ID NO: 2 with any amino acid residue selected from Ile, Met, and Val. The mutation is shown.
  • mutantation corresponding to the mutation of the amino acid residue at position X in the amino acid sequence shown in SEQ ID NO: 2 refers to the alignment between the amino acid sequence of SEQ ID NO: 2 and the target amino acid sequence.
  • mutation corresponding to S28T refers to a mutation in which an amino acid residue corresponding to the Ser residue at position 28 in the amino acid sequence shown in SEQ ID NO: 2 is substituted with a Thr residue.
  • mutations in which the synthetic activity of ⁇ -Glu-Val-Gly or ⁇ -Glu-Nva-Gly is confirmed are preferable.
  • Specific examples of such a mutation include a mutation corresponding to a mutation selected from the following. In the following notation, the meanings of the numbers, the characters on the left side of the numbers, and the characters on the right side of the numbers or the characters in the parentheses on the right side of the numbers are the same as described above.
  • mutations corresponding to mutations selected from the following are more preferable.
  • the meanings of the numbers, the characters on the left side of the numbers, and the characters on the right side of the numbers are the same as described above.
  • mutations corresponding to the mutations shown below include mutations corresponding to the mutations shown below.
  • the meanings of the numbers, the characters on the left side of the numbers, and the characters on the right side of the numbers are the same as described above.
  • the combination of two or more mutations represented by “+” indicates double mutation or multiple mutations. That is, for example, “S28T + S44T” indicates a double mutation of S28T and S44T.
  • multiple mutations may be described as “(S28T + S44T)” with parentheses.
  • Mutant glutathione synthetase can be obtained by modifying the gene encoding wild-type glutathione synthetase so that the encoded glutathione synthetase has the “specific mutation” and expressing the resulting modified gene .
  • Introduction of such mutations is achieved by modifying the gene encoding wild-type glutathione synthase so that the amino acid residue at a specific position is replaced with another amino acid, for example, by site-directed mutagenesis. it can.
  • ⁇ -glutamylcysteine synthetase used in the present invention is selected from the amino acids represented by X, ie, valine, norvaline, leucine, ⁇ -aminobutyric acid, and isoleucine. It is not particularly limited as long as it has an activity of catalyzing a reaction of recognizing one or more amino acids as a substrate and binding to Glu to produce a peptide represented by ⁇ -Glu-X. In the present invention, the activity catalyzing the reaction is referred to as ⁇ -glutamylcysteine synthetase activity.
  • the ⁇ -glutamylcysteine synthetase used in the present invention does not need to exhibit ⁇ -glutamylcysteine synthetase activity for any X, and may exhibit ⁇ -glutamylcysteine synthetase activity for the amino acid selected as X.
  • ⁇ -glutamylcysteine synthetases may be used.
  • ⁇ -glutamylcysteine synthetase may have an activity of catalyzing a reaction of generating ⁇ -Glu-Cys or ⁇ -Glu-Cys derivative from Glu and Cys or Cys derivative. It does not have to be good.
  • the origin of the ⁇ -glutamylcysteine synthetase 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 one derived from a microorganism is preferred.
  • the microorganism include eukaryotic microorganisms such as yeast, and bacteria such as enteric bacteria and coryneform bacteria.
  • Examples of the ⁇ -glutamylcysteine synthetase used in the present invention include a protein encoded by the GSH1 gene of Saccharomyces cerevisiae.
  • the base sequence of the GSH1 gene of Saccharomyces cerevisiae is disclosed in Saccharomyces Genome Database (http://www.yeastgenome.org/).
  • the nucleotide sequence of the GSH1 gene of Saccharomyces cerevisiae S288C strain (ATCC No. 26108) and the amino acid sequence encoded by this gene are shown in SEQ ID NOs: 239 and 240, respectively.
  • examples of the ⁇ -glutamylcysteine synthetase used in the present invention include a protein encoded by the CSH1 gene of Candida utilis.
  • the base sequence of Candida utilis GSH1 gene is disclosed in US Pat. No. 7,553,638.
  • the ⁇ -glutamylcysteine synthetase used in the present invention includes a protein encoded by the gshA gene of Escherichia coli.
  • the gshA gene of Escherichia coli K12 MG1655 strain corresponds to the complementary sequence of the 2812905 to 2814461 positions in the genome sequence registered as GenBank accession NC_000913 (VERSION NC_000913.2 GI: 49175990) in the NCBI database.
  • the ⁇ -glutamylcysteine synthetase used in the present invention may be a variant of a protein having the amino acid sequence as long as it has ⁇ -glutamylcysteine synthetase activity.
  • the above-mentioned descriptions concerning the glutathione synthase variants can be applied mutatis mutandis.
  • the present invention provides a yeast (also referred to as “the yeast of the present invention”) having a DNA encoding a mutant glutathione synthase.
  • the yeast of the present invention can be obtained by introducing DNA encoding a mutant glutathione synthetase into an appropriate strain of yeast, for example, a strain described later.
  • Introduction of a DNA encoding a mutant glutathione synthetase can be achieved, for example, by introducing a vector containing a DNA encoding a mutant glutathione synthetase into yeast and retaining the vector.
  • Introducing DNA encoding a mutant glutathione synthetase can be achieved, for example, by inserting a DNA encoding a mutant glutathione synthetase into a yeast chromosome by homologous recombination.
  • a technique for introducing such a gene into a host for example, a known technique can be used. Specifically, for example, the technique described in “(4) Preparation of enzyme used in the present invention” can be used.
  • the yeast of the present invention may be a budding yeast or a fission yeast.
  • Saccharomyces cerevisiae Saccharomyces cerevisiae
  • Candida utilis Candida utilis
  • genus Pichia pastel Piichia ⁇ ⁇ pastoris
  • Pichia genus Hansenula polymorpha (Hansenula morph)
  • yeast belonging to the genus Hansenula examples include yeast belonging to the genus Hansenula.
  • the fission yeast include yeast belonging to the genus Schizosaccharomyces pombe, such as Schizosaccharomyces pombe.
  • yeast belonging to the genus Saccharomyces is preferable, and Saccharomyces cerevisiae is more preferable.
  • the yeast of the present invention may be haploid or may have diploidity or higher ploidy.
  • Saccharomyces cerevisiae AJ14956 can be used as the Saccharomyces cerevisiae. Saccharomyces cerevisiae AJ14956 was deposited and commissioned on August 18, 2010 at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (address: postal number 305-8666, 1-chome, 1-chome, 1-chome, Tsukuba, Ibaraki) The number FERM BP-11299 is assigned.
  • Saccharomyces cerevisiae specifically, BY4743 strain (ATCC201390) and S288C strain (ATCC26108) can be used.
  • Candida utilis specifically, Candida utilis ATCC22023 strain can be used. These stocks can be sold, for example, from the American Type Culture Collection (address 12301 Parklawn Drive, Rockville, Maryland 20852 P.O. Box 1549, Manassas, VA 20108, United States of America). That is, a registration number corresponding to each strain is given, and it is possible to receive a sale using this registration number (see http://www.atcc.org/). The registration number corresponding to each strain is described in the catalog of American Type Culture Collection.
  • 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 enzyme activity, for example, a wild strain or a mutant strain of a microorganism having the enzyme activity.
  • the organism having the activity of the enzyme is preferably an organism having an enhanced activity of the enzyme. Examples of such an organism include a transformant in which expression of a gene encoding the enzyme used in the present invention is enhanced by genetic engineering techniques.
  • 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.
  • transformed hosts include bacterial cells, actinomycetes cells, yeast cells, fungal cells, plant cells, insect cells, and animals. Cells or the like can be used. As the host to be transformed, microbial cells are preferably used.
  • the microorganisms for which host-vector systems have been developed include Escherichia bacteria, Pseudomonas bacteria, Corynebacterium bacteria, Arthrobacter bacteria, Bacillus. ) Genus bacteria, Aspergillus fungi and the like. Among these, Escherichia coli (E. coli) or Corynebacterium glutamicum (C. glutamicum) is preferably used.
  • Enhancement of gene expression can be achieved, for example, by replacing the promoter of the gene on the chromosome with a stronger one.
  • a promoter examples include T7 promoter, trp promoter, lac promoter, tac promoter, and PL promoter.
  • 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.
  • enhancement of gene expression can be achieved, for example, by improving gene translation efficiency. It is known that the 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, greatly affects the translation efficiency of mRNA. The translation efficiency can be improved by modifying.
  • RBS ribosome binding site
  • enhancement of gene expression can be achieved, for example, by increasing the copy number of the gene.
  • Increase in gene copy number can be achieved by introducing the target gene onto the chromosome.
  • Introduction of a gene onto a chromosome can be performed using, for example, homologous recombination.
  • 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.
  • 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.
  • examples of sequences having many copies in the chromosome include autonomously replicating sequences (ARS) consisting of unique short repetitive sequences and rDNA sequences having about 150 copies.
  • ARS autonomously replicating sequences
  • the increase in the copy number of a gene can also be achieved by introducing a vector containing the target gene into a host organism.
  • a vector to be used a multicopy vector is preferable.
  • the vector preferably has a marker such as an ampicillin resistance gene.
  • 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.
  • 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.
  • vectors examples 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 (Japanese Patent Laid-Open No. 2-109855).
  • transposons such as Mu, Tn10, and Tn5 (Berg, DE and Berg, CM, Bio / Technol., 1, 417 (1983)) can be used.
  • a transposon 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.
  • vectors that can be used in yeast include plasmids having a CEN4 replication origin and multicopy plasmids having a 2 ⁇ m DNA replication origin.
  • 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 a vector as described above 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.
  • 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 of transformation is not particularly limited, and a conventionally known method can be used.
  • a conventionally known method can be used.
  • D. M.M. Morrison's method Methods in Enzymology 68, ⁇ ⁇ ⁇ ⁇ 3261979 (1979)
  • 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)
  • a method used for transformation of ordinary Escherichia bacteria can be carried out by a method used for transformation of ordinary Escherichia bacteria.
  • yeast transformation methods include protoplast method, KU method (H. Ito et al., J. Bateriol., 153-163 (1983)), KUR method (fermentation and industrial vol.43, p.630- 637 (1985)), electroporation method (Luis et al., FEMS micro microbiology Letters 165 (1998) 335-340), method using carrier DNA (Gietz RD and Schiestl RH, Methods Methods Mol. Cell. Biol. 5: 255-269 (1995)) and the like, a method usually used for yeast transformation can be employed.
  • 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.
  • 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.
  • the medium used for culturing the yeast transformant is not particularly limited as long as the yeast can grow, and a medium usually used industrially can be used.
  • an SD medium or a YPD medium can be suitably used.
  • the bacterial cells are collected and crushed or dissolved to obtain an enzyme-containing solution.
  • crushing the cells for example, ultrasonic crushing, French press crushing or glass bead crushing can be suitably used.
  • cells are lysed, for example, egg white lysozyme, peptidase treatment, or an appropriate combination thereof can be used.
  • a residue can be removed by centrifugation etc. as needed.
  • the target enzyme is present in the culture supernatant
  • the culture supernatant can be used as an enzyme-containing solution.
  • the 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.
  • the target enzyme 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.
  • 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.
  • an enzyme purified as described above may be used, or an unpurified enzyme may be used.
  • 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.
  • the enzyme can be used in the state of an immobilized enzyme immobilized on an arbitrary carrier.
  • a culture containing microbial cells producing an enzyme or a treated product of the culture may be used as the enzyme.
  • the treated product of the culture include cells separated and collected from the culture, and treated cells obtained by immobilizing, treating with acetone, freeze drying, and the like.
  • Method for producing ⁇ -Glu-X-Gly is characterized by utilizing the activity of glutathione synthetase. To do.
  • the first aspect of the method of the present invention includes a step of generating ⁇ -Glu-X-Gly by causing glutathione synthetase to act on ⁇ -Glu-X and Gly. , ⁇ -Glu-X-Gly. That is, in the first embodiment, Gly is added to ⁇ -Glu-X in the reaction raw material by the action of glutathione synthetase to produce ⁇ -Glu-X-Gly.
  • ⁇ -Glu-X used as a substrate may be produced by any method.
  • ⁇ -Glu-X can be produced by an enzymatic reaction using ⁇ -glutamylcysteine synthetase using a raw material containing Glu and X.
  • ⁇ -Glu-X can be obtained by recovering from the cells or culture supernatant of a microorganism having the ability to produce ⁇ -Glu-X.
  • the microorganism having the ability to produce ⁇ -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 ⁇ -Glu-X can be obtained, for example, by introducing a gene encoding ⁇ -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 ⁇ -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), a step of producing ⁇ -Glu-X by causing ⁇ -glutamylcysteine synthetase to act on Glu and X (hereinafter referred to as “the second aspect”). Step (A)), and a step of producing ⁇ -Glu-X-Gly by allowing glutathione synthase to act on ⁇ -Glu-X and Gly produced in step (A) (hereinafter referred to as step (B)) And ⁇ -Glu-X-Gly.
  • ⁇ -Glu-X is produced from Glu and X in the reaction raw material by the action of ⁇ -glutamylcysteine synthetase, and Gly is further added by the action of glutathione synthase to give ⁇ -Glu- X-Gly is generated.
  • step (A) and step (B) may proceed separately.
  • ⁇ -Glu-X produced in step (A) may be recovered, and step (B) may be advanced using the recovered ⁇ -Glu-X.
  • step (B) may be advanced by adding glutathione synthase, Gly, or both to the reaction system. .
  • 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 performed simultaneously, both enzymes and Glu, X, and Gly may be present 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.
  • the step (B) may be advanced by adding glutathione synthase, Gly, or both 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 produce ⁇ -Glu-X-Gly by allowing ⁇ -glutamylcysteine synthetase and glutathione synthetase to act on Glu, X, and Gly.
  • a process for producing ⁇ -Glu-X-Gly That is, in the third embodiment, ⁇ -Glu-X is produced from Glu and X in the reaction raw material by the action of ⁇ -glutamylcysteine synthase, and Gly is further added by the action of glutathione synthase. X-Gly is considered to be generated.
  • ⁇ -Glu-X-Gly can be produced by allowing ⁇ -glutamylcysteine synthetase and glutathione synthetase, and Glu, X, and Gly to coexist in the reaction system. Further, for example, ⁇ -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.
  • ⁇ -Glu-X and Gly in the first aspect Glu, X, ⁇ -Glu-X, and Gly in the second aspect, and Glu, X, and Gly in the third aspect are collectively referred to. Also called “substrate”.
  • glutathione synthetase in the first embodiment, and the ⁇ -glutamylcysteine synthetase and the glutathione synthetase in the second and third embodiments are also collectively referred to as “enzymes”.
  • the enzyme reaction is preferably performed in an aqueous solvent such as water or a buffer solution.
  • 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. That is, for example, the enzyme reaction may be performed in a batch system or a column system. In the case of the batch type, necessary substrates and enzymes may be mixed in the reaction solution in the reaction vessel. The enzyme reaction may be performed by standing or may be performed with stirring. In the case of a column type, a reaction solution containing a necessary substrate may be passed through a column packed with an immobilized enzyme.
  • the pH of the reaction solution is not particularly limited as long as the enzyme used functions, and can be set as appropriate according to the properties of the enzyme used.
  • the pH of the reaction solution is, for example, preferably 6 to 11, more preferably 8 to 10, and particularly preferably 8.5 to 9.5.
  • the concentration of each substrate in the reaction solution is not particularly limited. For example, it is preferably 1 ⁇ M or more, more preferably 100 ⁇ M or more, and particularly preferably 1 mM or more. Moreover, although the upper limit of a density
  • the concentration ratio of each substrate is not particularly limited, but it may be preferable to adjust the concentration of each substrate in the reaction solution from the viewpoint of the yield of ⁇ -Glu-X-Gly and purification. That is, for example, in the first embodiment, it may be preferable that the ratio of the concentrations of ⁇ -Glu-X and Gly is close to 1: 1.
  • 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, Gly 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 ⁇ g / ml to 100 mg / ml, and more preferably 10 ⁇ g / 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.
  • an essential factor for the enzyme reaction is present.
  • Factors essential for the enzymatic reaction include ATP in both the reaction with glutathione synthetase and the reaction with ⁇ -glutamylcysteine synthetase.
  • ATP is used as an energy source for peptide bond formation.
  • ATP may be supplied to the reaction system so that the enzyme used in the present invention can use ATP for peptide bond formation.
  • ATP can be supplied as a powder or as an aqueous solution to a reaction solution for generating and accumulating ⁇ -Glu-X-Gly.
  • an ATP regeneration system may be used instead of adding expensive ATP directly.
  • a catalyst such as a purified enzyme, crude enzyme, fungus body, or treated cell product having ATP regeneration ability and a substrate used for ATP regeneration to the reaction system
  • a method for using the ATP regeneration system specifically, a method of adding a cultured cell of Corynebacterium ammoniagenes (C. amoniagenes) and a carbon source (Biosci. Biotechnol. Biochem., Bio 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, etc. .
  • 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.
  • 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.
  • other optional components may be present in the reaction solution as long as the production of ⁇ -Glu-X-Gly can be achieved.
  • 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 usually 10 ° C. to 80 ° C., preferably 15 ° C. to 60 ° C., more preferably 20 ° C. to 50 ° C., and further preferably 30 ° C. to 45 ° 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. Further, for example, when the reaction is performed in a column type, the liquid passing speed may be set so as to achieve the preferable reaction time.
  • 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 is not limited to changing the conditions in terms of time. For example, in the case of performing a column reaction using an immobilized enzyme, the reaction temperature, enzyme concentration, etc. Including spatially changing the condition. For example, in the case where the immobilized enzyme is used in the second or third aspect, ⁇ -glutamylcysteine synthetase and glutathione synthetase may be mixed and arranged separately. Good.
  • the preferable conditions on which glutathione synthetase and ⁇ -glutamylcysteine synthetase act are different from each other, it is suitable for ⁇ -glutamylcysteine synthetase in the course of the reaction. It may be effective to change the conditions to conditions suitable for glutathione synthase.
  • the production of ⁇ -Glu-X-Gly can be confirmed by any method used for detection or identification of a compound.
  • Such techniques include HPLC, LC / MS, GC / MS, and NMR.
  • ⁇ -Glu-X-Gly produced according to the present invention can be isolated using a commonly used peptide purification method.
  • ⁇ -Glu-X-Gly can be isolated from a reaction supernatant obtained by removing solids by centrifugation, if necessary, by combining operations such as ion exchange resin, membrane treatment, and crystallization. it can.
  • ⁇ -Glu-X-Gly may be isolated after disrupting the cells and the like.
  • Example 1 Construction of Saccharomyces cerevisiae-derived wild-type glutathione synthase gene (GSH2) expression plasmid pET-GSH2 Expression plasmid pET-GSH2 encoding the wild-type glutathione synthetase of Saccharomyces cerevisiae S288C strain (ATCC26108) It was constructed according to the following procedure and introduced into Escherichia coli. An outline of the construction procedure is shown in FIG.
  • 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. .
  • Primer C (SEQ ID NO: 5) and Primer D (SEQ ID NO: 6) prepared from the base sequence (SEQ ID NO: 1) of the GSH2 gene of Saccharomyces cerevisiae S288C strain 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.
  • the sequence containing the GSH2 gene was amplified by PCR using Primer C and Primer D and the above-mentioned pAUR-GSH2 as a template. PCR was performed using plasmid DNA, 0.2 ⁇ mol / L of each primer, 1.25 unit PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.), 10 ⁇ L of 5 ⁇ PrimeSTAR buffer (manufactured by Takara Bio Inc.), 2.5 mmol / L dNTP each. 50 ⁇ l 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.
  • the approximately 1.5 kb DNA fragment containing the GSH2 gene obtained above and the approximately 5.4 kb DNA fragment of the expression plasmid pET-21a (+) obtained above were added to TaKaRa Ligation Kit Ver. 2.1 (manufactured by Takara Bio Inc.) was reacted at 16 ° C. for 30 minutes for ligation.
  • Escherichia coli DH5 ⁇ 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 ⁇ g / ml ampicillin. (Difco), 5 g / L yeast extract (Difco), 5 g / L sodium chloride (Wako)]]
  • the cells were cultured overnight at 37 ° C.
  • a plasmid was extracted from the grown colonies of the transformant by a known method, and its nucleotide sequence was determined by a known method.
  • the resulting plasmid was a plasmid in which the GSH2 gene derived from Saccharomyces cerevisiae S288C strain added with a sequence encoding a His tag at the 3 ′ end was ligated downstream of the T7 promoter, and the plasmid was named pET-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.
  • Escherichia coli BL21 (DE3) strain competent cell manufactured by Novagen
  • the transformant was LB agar medium containing 50 ⁇ g / ml ampicillin.
  • 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 wild-type Gsh2 Escherichia coli BL21 (DE3) / pET-GSH2 obtained in Example 1 was inoculated into a tube containing 3 mL of LB medium containing 100 ⁇ g / ml ampicillin at 37 ° C. Cultured with shaking for 16 hours. 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- ⁇ -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.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • Example 3 Production of ⁇ -glutamyl tripeptide using wild type Gsh2 Using purified Gsh2 (wild type) derived from Saccharomyces cerevisiae S288C obtained in Example 2, ⁇ -glutamylvaline ( ⁇ -Glu-Val) Alternatively, the production of ⁇ -glutamyl tripeptide using ⁇ -glutamylnorvaline ( ⁇ -Glu-nVal) as a substrate was examined. 200 ⁇ l of a reaction solution (pH 8.0) having the following composition was prepared and reacted at 37 ° C. for 16 hours.
  • a reaction solution pH 8.0
  • the peaks of the respective reaction products are the peaks of the samples of ⁇ -glutamylvalylglycine ( ⁇ -Glu-Val-Gly) and ⁇ -glutamylnorvalylglycine ( ⁇ -Glu-nVal-Gly). Retention times were in agreement and determined to be ⁇ -Glu-Val-Gly and ⁇ -Glu-nVal-Gly.
  • the ⁇ -Glu-Val-Gly concentration was 0.13 mmol / L
  • the ⁇ -Glu-nVal-Gly concentration was 6.0 mmol / L.
  • fractionated peptide was fluorescently derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and detected by LC-MS / MS.
  • AQC 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate
  • AQC reagent solution prepared by dissolving the above AccQ-Flor reagent kit reagent powder in 1 ml of acetonitrile
  • the obtained mixture was heated at 55 ° C. for 10 minutes, and 100 ⁇ l of a 0.1% formic acid aqueous solution was added to prepare an analytical sample.
  • Example 4 Construction of mutant GSH2 gene
  • Phusion High-Fidelity DNA Polymerase (FINNZYMES) was used, and various mutant GSH2 genes were supported.
  • PCR was performed using primers (SEQ ID NOs: 7 to 238) and pET-GSH2 constructed in Example 1 as a template.
  • PCR was performed using plasmid DNA, 0.5 ⁇ mol / L of each primer, 0.5 unit of Phusion High-Fidelity DNA polymerase, 5 ⁇ L of 5 ⁇ Phusion HF buffer (manufactured by FINNZYMES), 2.5 mmol / L of dNTP (dATP, dGTP). , DCTP, and dTTP) are prepared, heated at 98 ° C. for 30 seconds, and then the process of 98 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 2 minutes and 30 seconds is repeated 30 times. Was done. Tables 2 to 6 show the relationship between each mutation and the primer.
  • Escherichia coli JM109 strain competent cell (manufactured by Takara Bio Inc.) was transformed by the heat shock method using the reaction solution.
  • the body was applied to an LB agar medium containing 100 ⁇ g / ml ampicillin, and then cultured at 37 ° C. overnight.
  • Plasmids were extracted from the grown colonies of the transformant according to a known method, the nucleotide sequence of the DNA was confirmed by a known method, and various plasmids having a GSH2 gene mutated at the target position were obtained.
  • the expression plasmid of the GSH2 gene into which multiple mutations were introduced was prepared by sequentially introducing mutations in the same manner as described above using the expression plasmids of each mutant type GSH2 gene prepared by the above method as a template.
  • the plasmid into which each mutation was introduced was named by adding the form of each mutation to pET-GSH2.
  • a plasmid having a mutant GSH2 gene encoding a mutant Gsh2 having an E386D mutation has a mutant GSH2 gene encoding pET-GSH2 (E386D) and a mutant Gsh2 having two mutations E386D and Y446F.
  • the plasmid is designated pET-GSH2 (E386D + Y446F).
  • the expression plasmids of the mutant GSH2 gene may be collectively referred to as pET-GSH2 (XXX).
  • Escherichia coli BL21 (DE3) strain competent cell manufactured by Invitrogen
  • pET-GSH2 XXX
  • the transformant contained 100 ⁇ g / mL ampicillin.
  • the cells were cultured overnight at 37 ° C., and the obtained transformant was used for further studies.
  • Example 5 Purification of mutant Gsh2 100 ⁇ g of Escherichia coli BL21 (DE3) [Escherichia coli BL21 (DE3) / pET-GSH2 (XXX)] carrying pET-GSH2 (XXX) obtained in Example 4
  • a test tube containing 3 mL of LB medium containing / ml ampicillin was inoculated and cultured with shaking at 37 ° C. for 16 hours. 400 ⁇ l of the obtained culture solution was inoculated into a test tube containing 20 ml of LB medium. After shaking culture at 37 ° C.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the wet cells are suspended in 1 mL of BugBuster Master Mix (manufactured by Novagen), protein is extracted, and then centrifuged from the supernatant obtained from Ni Sepharose 6 Fast Flow (His-tagged protein purification kit, GE Healthcare). His-tagged recombinant Gsh2 was purified according to the manual, and purified Gsh2 (mutant) containing 500 mmol / L imidazole was obtained.
  • Example 6 Evaluation of ability to synthesize ⁇ -glutamyl tripeptide by mutant Gsh2 Using each purified Gsh2 (mutant) obtained in Example 5, ⁇ -glutamylcysteine ( ⁇ -Glu-Cys), ⁇ -glutamylvaline ( ⁇ -Glu-Val) or ⁇ -glutamylnorvaline ( ⁇ -Glu-nVal) as a substrate was examined for the production of ⁇ -glutamyltripeptide. 200 ⁇ l of a reaction solution (pH 8.0) having the following composition was prepared, and the reaction using ⁇ -Glu-Cys substrate was performed at 37 ° C. for 2 minutes, the reaction using ⁇ -Glu-Val substrate was performed for 30 minutes, and ⁇ -Glu.
  • a reaction solution pH 8.0
  • Example 7 Kinetic analysis of Gsh2 (7-1) Large-scale purification of wild-type Gsh2 and mutant Gsh2 In order to analyze the kinetics of wild-type Gsh2 and mutant Gsh2, purified enzyme was purified by the following procedure. Prepared.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • Example 8 Construction of ⁇ -glutamylcysteine synthetase gene (GSH1) expression plasmid pET-GSH1 derived from Saccharomyces cerevisiae Expression plasmid pET-GSH1 encoding the ⁇ -glutamylcysteine synthetase of S288C strain of Saccharomyces cerevisiae It was constructed according to the following procedure and introduced into Escherichia coli. An outline of the construction procedure is shown in FIG.
  • Primer E (SEQ ID NO: 241) and primer F (SEQ ID NO: 242) prepared based on the base sequence (SEQ ID NO: 239) 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 E is obtained by adding a KpnI recognition sequence and a partial sequence of the yeast expression plasmid pAUR123 to the 5 'end of the region containing the start codon of the GSH1 gene of the chromosomal DNA of the Saccharomyces cerevisiae strain.
  • Primer F includes a base sequence complementary to the sequence encoding the 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 according to the manual using PrimeSTAR Max DNA polymerase.
  • 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-GSH1.
  • Primer G (SEQ ID NO: 243) and Primer H (SEQ ID NO: 244) prepared from the base sequence (SEQ ID NO: 239) of the GSH1 gene of Saccharomyces cerevisiae S288C strain were purchased from Nippon Bioservice.
  • the primer is obtained by adding a base sequence containing a SpeI recognition sequence to the 5 'end of a region containing the next of the start codon of the GSH1 gene of chromosomal DNA of Saccharomyces cerevisiae S288C strain.
  • the primer is obtained by adding a base sequence containing a SalI 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.
  • PCR is a reaction containing plasmid DNA, 0.2 ⁇ mol / L of each primer, 1.25 unit of PrimeSTAR HS DNA polymerase, 10 ⁇ L of 5 ⁇ PrimeSTAR buffer, and 2.5 mmol / L of dNTP (dATP, dGTP, dCTP and dTTP).
  • dNTP dATP, dGTP, dCTP and dTTP.
  • the DNA fragment was purified using MinElute Reaction Cleanup Kit and dissolved in 15 ⁇ l of Buffer EB. Next, using the total amount of the obtained DNA solution, the DNA fragment was dephosphorylated with Alkaline Phosphatase, Calf intestine (CIAP), purified using MinElute Reaction Cleanup Kit, and dissolved in 10 ⁇ l of Buffer EB.
  • the approximately 2.0 kb DNA fragment containing the GSH1 gene obtained above and the approximately 5.4 kb DNA fragment of the expression vector pET-21a (+) obtained above were added to TaKaRa Ligation Kit Ver. Using 2.1, the reaction was carried out at 16 ° C. for 30 minutes for ligation. Escherichia coli DH5 ⁇ 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 ⁇ g / 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 base 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 ligated downstream of the T7 promoter and was named pET-GSH1.
  • 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: 239 and SEQ ID NO: 240, respectively.
  • Escherichia coli Rosetta2 (DE3) pLysS strain competent cells were transformed with the heat shock method using pET-GSH1, and the transformants were transformed with 100 ⁇ g / ml ampicillin and 30 ⁇ g / 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-GSH1 was retained.
  • the Escherichia coli Rosetta2 (DE3) pLysS strain carrying pET-GSH1 was named Escherichia coli Rosetta2 (DE3) pLysS / pET-GSH1.
  • Example 9 Purification of Gsh1 Escherichia coli Rosetta2 (DE3) pLysS / pET-GSH1 obtained in Example 8 was added to 3 mL of LB medium containing 100 ⁇ g / ml ampicillin and 30 ⁇ g / ml chloramphenicol. The test tube was inoculated and cultured with shaking at 37 ° C. for 16 hours. 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.
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside
  • the wet cells are suspended in 5 mL of BugBuster Master Mix (manufactured by Novagen), extracted from the protein, and centrifuged from the supernatant obtained by using Ni Sepharose 6 Fast Flow according to the instructions.
  • the modified Gsh1 was purified. Subsequently, the purified enzyme was desalted using a PD-10 column (GE Healthcare) according to the instructions. This purified and desalted Gsh1 was used as purified Gsh1 in subsequent experiments.
  • Example 10 Production of ⁇ -glutamyl dipeptide using Gsh1 Using the purified Gsh1 obtained in Example 9, production of ⁇ -glutamyl dipeptide using valine, norvaline, or ⁇ -aminobutyric acid (Abu) as a substrate was examined. . 200 ⁇ l of a reaction solution (pH 8.5) having the following composition was prepared and reacted at 30 ° C. for 24 hours.
  • the reaction product was analyzed by HPLC under the conditions described in Example 6.
  • the peaks of the respective reaction products were ⁇ -Glu-Val, ⁇ -Glu-nVal, and ⁇ -Glu-Abu.
  • the peak of the sample coincided with the retention time, and judged to be ⁇ -Glu-Val, ⁇ -Glu-nVal, and ⁇ -Glu-Abu.
  • the ⁇ -Glu-Val concentration was 0.5 mmol / L
  • the ⁇ -Glu-nVal concentration was 4.7 mmol / L
  • the ⁇ -Glu-Abu concentration was 8.2 mmol / L.
  • Example 11 Generation of ⁇ -glutamyl tripeptide from amino acid using mutant Gsh2 Using purified Gsh1 obtained in Example 9 and purified wild-type GSH2 or purified mutant Gsh2 (S44T + L132M + L195V) obtained in Example 7 The production of ⁇ -glutamyl tripeptide from amino acids was investigated. 200 ⁇ l of a reaction solution (pH 9.0) having the following composition was prepared and reacted at 30 ° C. for 8 hours.
  • a reaction solution pH 9.0
  • the reaction product was analyzed by HPLC under the conditions described in Example 6.
  • 0.08 mmol / L of ⁇ -Glu-Val-Gly was produced, whereas mutant type Gsh2 (S44T / L132M / L195V) was produced.
  • 0.11 mmol / L of ⁇ -Glu-Val-Gly was produced, and an increase in the amount of ⁇ -Glu-Val-Gly produced due to the Gsh2 mutation was observed.
  • Example 12 Construction of YCp-type plasmid for GSH2 expression
  • YCp-type plasmid which is a one-copy type plasmid
  • Y446F an expression plasmid pCAU-PGK1p-GSH2
  • WT wild type GSH2 expression plasmid
  • Y446F mutant type GSH2
  • URA3 was cloned into the EcoRI-AatII site of pUC19
  • H4ARS was cloned into the KpnI-EcoRI site
  • CEN4 was cloned into the KpnI site
  • the PGK1 promoter was cloned into the SmaI-BamHI site
  • PGK1p was produced.
  • the wild-type GSH2 fragment was amplified using the S288C strain genome as a template, using SEQ ID NOs: 253 and 254, The obtained DNA fragment was cloned into the BamHI-SphI site of pCAU-PGK1p.
  • WT YCp-type plasmid pCAU-PGK1p-GSH2
  • mutant GSH2 (Y446F)
  • SEQ ID NOs: 253 and 254 mutation was performed using plasmid pET-GSH2 (Y446F) having mutant GSH2 (Y446F) constructed in Example 4 as a template.
  • the type GSH2 (Y446F) fragment was amplified and the resulting DNA fragment was cloned into the BamHI-SphI site of pCAU-PGK1p.
  • a YCp-type plasmid pCAU-PGK1p-GSH2 (Y446F) that expresses mutant GSH2 (Y446F) was constructed.
  • Example 13 Acquisition of uracil-requiring strain (ura3 mutant)
  • uracil-requiring strain ura3 mutant
  • URA3 the method of Sofyanovich et al.
  • the DNA near URA3 excluding the gene was introduced into a Saccharomyces cerevisiae wild-type haploid (Mat ⁇ type) strain and obtained by disrupting the URA3 gene.
  • a URA3 gene-deficient strain exhibits 5-fluoroorotic acid (5-FOA) resistance. The specific procedure is shown below.
  • 500 bp upstream of URA3 was amplified by PCR using the chromosomal DNA of the wild type strain as a template.
  • 500 bp downstream of URA3 was amplified using the primers shown in SEQ ID NOs: 257 and 258.
  • the PCR conditions were 25 ° for heat denaturation at 94 ° C. for 10 seconds, annealing at 55 ° C. for 10 seconds, and extension at 72 ° C. for 1 minute.
  • Example 14 Acquisition of uracil-requiring gsh2 ⁇ 0 strain
  • the GSH2 gene of AJ14956 was disrupted by the following procedure.
  • PCR was carried out using the GSH2 disruption strain (available from Homozygous Diploid collection, available from Open Bio Systems) in the Saccharomyces cerevisiae gene disruption strain collection as a template, and the primers shown in SEQ ID NOs: 261 and 262 were used to amplify the DNA fragment. did.
  • AJ14956 competent cells were prepared using the Frozen EZ Yeast Transformation II kit from Zymo Research, the amplified DNA fragment was introduced into the competent cells, and a YPD plate containing G418 at a concentration of 75 ⁇ g / ml Spread to. From the emerged resistant strains, a strain in which the GSH2 gene was disrupted was selected to obtain gsh2 ⁇ 0 strain, which is a uracil-requiring GSH2 disruption strain.
  • Example 15 Acquisition of wild-type GSH2 and mutant-type GSH2-expressing yeast Strains that express each GSH2 by transforming the gsh2 ⁇ 0 strain constructed in Example 14 with each GSH2 expression plasmid constructed in Example 12 was bred. Specifically, in the same manner as in Example 14, a competent cell of gsh2 ⁇ 0 strain was prepared using the Frozen EZ Yeast Transformation II kit of Zymo Research, and each GSH2 expression plasmid was introduced to thereby obtain wild type GSH2.
  • the WT strain (gsh2 ⁇ 0 / pCAU-PGK1p-GSH2 (WT)) and the Y446F strain (gsh2 ⁇ 0 / pCAU-PGK1p-GSH2 (Y446F)) expressing the mutant GSH2 (Y446F) were obtained.
  • Example 16 Verification of Effect of Y446F Mutation
  • the effect of the Y446F mutation of glutathione synthetase on the ⁇ -Glu-Val-Gly content in bacterial cells was verified.
  • the WT strain and the Y446F strain were each inoculated into SD medium (50 ml in a 500 ml Sakaguchi flask) for 1 ase and cultured with shaking at 30 ° C. and 120 rpm for 24 hours.
  • SD medium composition Glucose 2% Nitrogen Base 1-fold concentration (10-fold concentration Nitrogen Base was prepared by dissolving 1.7 g of Bacto Yeast Nitrogen Base w / o Amino Acids and Ammonium Sulfate (Difco) and 5 g of ammonium sulfate in 100 ml of sterilized water. pH adjusted to about 5.2 and filter sterilized)
  • the absorbance of the obtained culture broth was measured so that the initial OD660 was 0.01 (absorbance was measured using DU640 SPECTROPHOTOMETER of BECKMAN COULTER).
  • the final concentration was 100 ppm ⁇ -Glu-.
  • Inoculated into SD medium containing Val 50 ml in a 500 ml Sakaguchi flask
  • the filter-sterilized Gly solution was added to the culture solution to a final concentration of 750 ppm, and the culture was continued.
  • 10 ODunits (bacteria contained in 1 ml of culture solution with OD660 of 1 is defined as 1 ODunit) from the culture medium cultured for 0 hour, 1 hour, and 4 hours after the addition of the Gly solution is collected by centrifugation. did. The supernatant was removed as much as possible, and the remaining cells were suspended in 15 ml of milliQ water. The cells were collected again by centrifugation and resuspended in 15 ml of milliQ water. By repeating this operation three times in total, the medium was completely removed from the cells. The obtained washed cells were suspended in about 0.5 ml of milliQ water and heated at 70 ° C. for 10 minutes. In this step, the extract contained in the cells was extracted. Next, the extract and the cell residue were separated by centrifugation.
  • the content of ⁇ -Glu-Val-Gly contained per dry cell was calculated from the amount of ⁇ -Glu-Val-Gly and the dry cell weight contained in a certain amount of the culture medium thus measured.
  • Table 13 shows changes with time in the ⁇ -Glu-Val-Gly content of the cells.
  • AQC reagent solution prepared by dissolving the above AccQ-Fluor reagent kit reagent powder in 1 mL of acetonitrile
  • the obtained mixture was heated at 55 ° C. for 10 minutes, and then 100 ⁇ L of a 0.1% formic acid aqueous solution was added to prepare an analysis sample.
  • a peptide represented by ⁇ -Glu-X-Gly or a salt thereof can be produced.
  • 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 NOs: 7 to 238 PCR primers for GSH2 mutation introduction
  • SEQ ID NO: 239 Base sequence of GSH1 gene derived from Saccharomyces cere

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Abstract

L'invention concerne un procédé de production d'un peptide représenté par la formule : γ-Glu-X-Gly (où X représente un acide aminé choisi dans le groupe consistant en Val, nVal, Leu, Abu et Ile) ou un sel de celui-ci. Le peptide est représenté par la formule : γ-Glu-X-Gly ou un sel de celui-ci peut être produit en laissant une glutathion synthase, par exemple une glutathion synthase mutante qui a une mutation spécifique, agir sur γ-Glu-X et Gly.
PCT/JP2011/073715 2011-10-14 2011-10-14 Procédé de production de γ-glu-x-gly ou d'un sel de celui-ci, et glutathion synthase mutante WO2013054447A1 (fr)

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EP4112725A4 (fr) * 2020-03-25 2023-09-27 CJ Cheiljedang Corporation Variant de glutamate-cystéine ligase et procédé de production de glutathion l'utilisant
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JPWO2015115612A1 (ja) * 2014-01-31 2017-03-23 味の素株式会社 変異型グルタミン酸−システインリガーゼ、及び、γ−グルタミルバリルグリシンの製造法
WO2015115612A1 (fr) 2014-01-31 2015-08-06 味の素株式会社 Glutamate-cystéine ligase mutante et procédé de fabrication de γ-glutamyl-valyl-glycine
US10113161B2 (en) 2014-01-31 2018-10-30 Ajinomoto Co., Inc. Mutant glutamate-cysteine ligase and method for manufacturing gamma glutamyl-valyl-glycine
US10508295B2 (en) 2014-03-05 2019-12-17 Ajinomoto Co., Inc. Gamma glutamyl-valine synthase, and method for producing gamma glutamyl-valyl-glycine
WO2015133547A1 (fr) * 2014-03-05 2015-09-11 味の素株式会社 γ-GLUTAMYL-VALINE SYNTHASE, ET PROCÉDÉ DE PRODUCTION DE γ-GLUTAMYL-VALYL-GLYCINE
US20160340707A1 (en) * 2014-03-05 2016-11-24 Ajinomoto Co., Inc. Gamma-glutamyl-valine synthase, and method for producing gamma-glutamyl-valyl-glycine
JPWO2015133547A1 (ja) * 2014-03-05 2017-04-06 味の素株式会社 γ−グルタミルバリン合成酵素、及び、γ−グルタミルバリルグリシンの製造法
EP3115463A4 (fr) * 2014-03-05 2018-02-14 Ajinomoto Co., Inc. Gamma-glutamyl-valine synthase, et procédé de production de gamma-glutamyl-valyl-glycine
CN104328092B (zh) * 2014-09-28 2017-03-15 邦泰生物工程(深圳)有限公司 一种谷胱甘肽合成酶突变体、编码基因及应用
JP2016166156A (ja) * 2015-03-10 2016-09-15 味の素株式会社 γ−グルタミルバリルグリシン結晶及びその製造方法
US11788109B2 (en) 2015-09-04 2023-10-17 Ajinomoto Co., Inc. Microorganism and method for producing gamma-glutamyl-valyl-glycine
WO2018084165A1 (fr) * 2016-11-01 2018-05-11 株式会社カネカ Enzyme modifiée et utilisation correspondante
US11142755B2 (en) 2018-02-27 2021-10-12 Ajinomoto Co., Inc. Mutant glutathione synthetase and method for producing gamma-glutamyl-valyl-glycine
EP3530749A1 (fr) 2018-02-27 2019-08-28 Ajinomoto Co., Inc. Synthétase de glutathione mutant et procédé de production de gamma-glu-val-gly
WO2021100846A1 (fr) * 2019-11-22 2021-05-27 株式会社カネカ Procédé de fabrication de substance utile
CN114761541A (zh) * 2019-11-22 2022-07-15 株式会社钟化 有用物质的制造方法
EP4112725A4 (fr) * 2020-03-25 2023-09-27 CJ Cheiljedang Corporation Variant de glutamate-cystéine ligase et procédé de production de glutathion l'utilisant

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