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US 9,428,783 B2Granted
DNA encoding dipeptide-synthesizing enzyme (variants), bacterium belonging to the genus Escherichia, and methods for producing dipeptides using thereof
AssigneeAjinomoto Co., Inc.
InventorsSergey Vasilievich Smirnov, Pavel Mikhailovich Sokolov, Takayuki Ito
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Abstract
The present invention describes novel bacterial L-amino acids α-ligases, which catalyzing reaction of dipeptide formation having an acidic L-amino acid such as L-Asp or L-Glu at the N-terminus. The method for producing dipeptides using said L-amino acids α-ligases and a bacterium of the family Enterobacteriaceae, particularly a bacterium belonging to the genus Escherichia, which has been modified to contain the DNA encoding said L-amino acids α-ligases, is described.
Claims15
A method for producing a dipeptide or a salt thereof, comprising:
- (a) reacting L-amino acids, L-amino acid derivatives, or salts thereof under appropriate conditions in the presence of a protein having dipeptide-synthesizing activity such that a dipeptide or a salt thereof is produced;
- (b) accumulating the dipeptide or a salt thereof in an appropriate solvent;
- and
- (c) collecting the dipeptide or a salt thereof from the appropriate solvent,
- wherein the protein is selected from the group consisting of:
- a protein having the amino acid sequence of SEQ ID NO:
- 2, 4, or 6;
- a variant of a protein having the amino acid sequence of SEQ ID NO:
- 2, 4, or 6 in which no more than fifteen amino acid residues of the amino acid sequence of SEQ ID NO:
- 2, 4, or 6 are changed by substitution, deletion, insertion, or addition, and wherein the variant protein has the dipeptide-synthesizing activity;
- and
- a protein having an amino acid sequence having not less than 95% sequence identity to the amino acid sequence of SEQ ID NO:
- 2, 4, or 6 and wherein the protein has the dipeptide-synthesizing activity,
- wherein the dipeptide comprises two L-amino acids, two L-amino acid derivatives, or an L-amino acid and an L-amino acid derivative, and
- wherein the L-amino acids or the L-amino acid derivatives are selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and a lower alkyl ester of L-phenylalanine, wherein the lower alkyl ester of L-phenylalanine is methyl, ethyl or propyl ester of the L-phenylalanine.
12 more elements
A method for producing a dipeptide or a salt thereof, comprising:
- (a) cultivating a bacterium in a culture medium comprising an L-amino acid or an L-amino acid derivative;
- (b) accumulating the dipeptide in the bacterium, the culture medium, or both;
- and
- (c) collecting the dipeptide from the bacterium, the culture medium, or both,
- wherein the bacterium is a dipeptide-producing bacterium belonging to the genus Escherichia transformed with a recombinant DNA comprising a DNA encoding a protein having dipeptide-synthesizing activity,
- wherein the DNA encoding the protein having a dipeptide-synthesizing activity is selected from the group consisting of:
- a DNA having the nucleotide sequence of SEQ ID NO:
- 1, 3, or 5;
- a DNA encoding a protein having the amino acid sequence of SEQ ID NO:
- 2, 4, or 6;
- a DNA encoding a variant of a protein having the amino acid sequence of SEQ ID NO:
- 2, 4, or 6 in which no more than fifteen amino acid residues of the amino acid sequence of SEQ ID NO:
- 2, 4, or 6 are changed by substitution, deletion, insertion, or addition, and wherein the variant protein has the dipeptide-synthesizing activity;
- and
- a DNA encoding a protein having an amino acid sequence having not less than 95% sequence identity to the amino acid sequence of SEQ ID NO:
- 2, 4, or 6 and wherein the protein has the dipeptide-synthesizing activity,
- wherein the dipeptide comprises two L-amino acids, two L-amino acid derivatives, or an L-amino acid and an L-amino acid derivative, and
- wherein the L-amino acids or the L-amino acid derivatives are selected from the group consisting of L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and a lower alkyl ester of L-phenylalanine, wherein the lower alkyl ester of L-phenylalanine is methyl, ethyl or propyl ester of the L-phenylalanine.
15 more elements
Dependencies are inferred from the claim text.