Amidase, gene for the same, vector, transformant, and method for production of optically active carboxylic acid amide and optically active carboxylic acid by using any one of those items
View Patent ↗The present invention has its object to provide a novel polypeptide having amidase activity to selectively hydrolyze S-enantiomer in racemic nipecotamide, a DNA encoding the polypeptide, a vector containing the DNA, a transformant transformed with the vector, and a method for producing an optically active carboxylic acid amide and an optically active carboxylic acid in which a racemic carboxylic acid amide is hydrolyzed with the polypeptide or the transformant.
1. An isolated DNA which encodes a polypeptide which is any one of the following polypeptides (b), and (c):
(b) a polypeptide comprising an amino acid sequence derived from the amino acid sequence set forth in SEQ ID NO:1 by substitution, insertion, deletion, and/or addition of 90 or less amino acids, and having activity to selectively hydrolyze S-enantiomer in racemic nipecotamide and wherein the polypeptide is not SEQ ID NO: 1; and
(c) a polypeptide having sequence identity of 80% or higher to the amino acid sequence set forth in SEQ ID NO:1, and having activity to selectively hydrolyze S-enantiomer in racemic nipecotamide and wherein the polypeptide is not SEQ ID NO: 1.
2. An isolated DNA which is any one of the following DNAs (a), (b), and (c):
(b) a DNA hybridizable under a stringent condition with a DNA comprising a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO:2, and encoding a polypeptide having activity to selectively hydrolyze S-enantiomer in racemic nipecotamide, and wherein the DNA does not comprise SEQ ID NO: 2,
wherein the stringent condition is a condition where hybridization is carried out in the presence of 0.7 to 1.0 M NaCl, at 65° C., followed by washing the filter with 0.2-fold concentration of SSC solution at 65° C.; and
(c) a DNA having sequence identity of 80% or higher to the nucleotide sequence set forth in SEQ ID NO:2, and encoding a polypeptide having activity to selectively hydrolyze S-enantiomer in racemic nipecotamide, and wherein the DNA does not comprise SEQ ID NO: 2.
3. The DNA according to claim 2 , which has sequence identity of 85% or higher to the nucleotide sequence set forth in SEQ ID NO:2, and encodes the polypeptide having activity to selectively hydrolyze S-enantiomer in racemic nipecotamide.
4. A vector comprising an isolated DNA which encodes a polypeptide which is any one of the following polypeptides (a), (b), and (c):
(a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:1;
(b) a polypeptide comprising an amino acid sequence derived from the amino acid sequence set forth in SEQ ID NO:1 by substitution, insertion, deletion, and/or addition of 90 or less amino acids, and having activity to selectively hydrolyze S-enantiomer in racemic nipecotamide; and
(c) a polypeptide having sequence identity of 80% or higher to the amino acid sequence set forth in SEQ ID NO:1, and having activity to selectively hydrolyze S-enantiomer in racemic nipecotamide.
5. A transformant which is producible by transformation of a host microorganism with the vector according to claim 4 , wherein said host microorganism is not Cupriavidus sp.
6. The transformant according to claim 5 , wherein the host microorganism is Escherichia coli.
7. The isolated DNA according to claim 1 which encodes the polypeptide which has sequence identity of 85% or higher to the amino acid sequence set forth in SEQ ID NO:1, and has activity to selectively hydrolyze S-enantiomer in racemic nipecotamide.
8. The DNA according to claim 1 , which is isolated from a microorganism belonging to the genus Cupriavidus.
9. The DNA according to claim 8 , wherein the microorganism belonging to the genus Cupriavidus is Cupriavidus sp. KNK-J915 strain (FERM BP-10739).
10. A vector comprising an isolated DNA which is any one of the following DNAs (a), (b), and (c):
(a) a DNA comprising the nucleotide sequence set forth in SEQ ID NO:2;
(b) a DNA hybridizable under a stringent condition with a DNA comprising a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO:2, and encoding a polypeptide having activity to selectively hydrolyze S-enantiomer in racemic nipecotamide,
wherein the stringent condition is a condition where hybridization is carried out in the presence of 0.7 to 1.0 M NaCl, at 65° C., followed by washing the filter with 0.2-fold concentration of SSC solution at 65° C.; and
(c) a DNA having sequence identity of 80% or higher to the nucleotide sequence set forth in SEQ ID No:2, and encoding a polypeptide having activity to selectively hydrolyze S-enantiomer in racemic nipecotamide.