Chlorohydrin and hydroxycarboxylic ester asymmetric hydrolase gene
View Patent ↗The present invention is to provide a gene having asymmetric hydrolase activity which is useful for synthesis of an optically active carboxylic acid, its antipode ester, and lactone, and a hydroxycarboxylic ester asymmetric hydrolase enzyme (EnHCH) derived from Enterobacter sp . DS-S-75 strain (FERM BP-5494) which is bacteria belonging to the genus Enterobacter , a EnHCH gene shown by base sequence of SEQ.ID.NO: 1, a gene encoding a protein having an amino acid sequence of SEQ.ID.NO: 2, and E. coli DH5α (pKK-EnHCH) deposited to International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology as a deposition No. FERM BP-08466.
1. An isolated gene comprising a nucleotide sequence selected from:
(a) a nucleotide sequence described in SEQ. ID. NO: 1,
(b) a nucleotide sequence which encodes a protein having a chlorohydrin and hydroxycarboxylic ester asymmetric hydrolase activity and hybridizes with the nucleotide sequence described in SEQ. ID. NO: 1 under stringent conditions,
wherein the stringent conditions comprise hybridization using a DNA derived from a colony or plaque, or a filter to which a fragment of the DNA is fixed, in the presence of 0.7 to 1.0 M NaCl at 65° C., and then washing the filter with 0.1 to 2-fold of a SSC solution at 65° C.,
(c) a nucleotide sequence which encodes an amino acid sequence described in SEQ. ID. NO: 2, and
(d) a nucleotide sequence which encodes an amino acid sequence having a chlorohydrin and hydroxycarboxylic ester asymmetric hydrolase activity where the amino acid sequence is an amino acid sequence having 95% or more homology with the amino acid sequence described in SEQ. ID. NO: 2.
2. An isolated DNA which consists of the nucleotide sequence described in SEQ. ID. NO: 8.
3. A vector containing the gene according to claim 1 .
4. A vector containing an isolated gene comprising a nucleotide sequence selected from:
(a) a nucleotide sequence described in SEQ. ID. NO: 1,
(b) a nucleotide sequence which encodes a protein having a chlorohydrin and hydroxycarboxylic ester asymmetric hydrolase activity and hybridizes with the nucleotide sequence described in SEQ. ID. NO: 1 under stringent conditions,
wherein the stringent conditions comprise hybridization using a DNA derived from a colony or plaque, or a filter to which a fragment of the DNA is fixed, in the presence of 0.7 to 1.0 M NaCl at 65° C., and then washing the filter with 0.1 to 2-fold of an SSC solution at 65° C.,
wherein said vector further contains the DNA as defined in claim 2 .
5. The vector according to claim 4 , wherein said vector is plasmid pKK-EnHCH1.
6. An isolated transformed cell containing the gene as defined in claim 1 .
7. An isolated transformed cell containing the vector as defined in claim 5 .
8. An isolated transformed cell containing the vector as defined in claim 4 .
9. The transformant according to claim 6 , wherein the host is E. coil.
10. The transformant according to claim 7 , wherein the host is E. coli.
11. The transformant according to claim 8 , wherein the host is E. coli.
12. The transformant according to claim 6 , wherein the host is E. coli JM109 strain or DH5α strain.
13. The transformant according to claim 7 , wherein the host is E. coli JM109 strain or DH5α strain.
14. The transformant according to claim 8 , wherein the host is E. coli JM109 strain or DH5α strain.
15. A process for the preparation of a protein comprising an amino acid sequence selected from:
(a) an amino acid sequence described in SEQ. ID. NO: 2, and
(b) an amino acid sequence having a chlorohydrin and hydroxycarboxylic ester asymmetric hydrolase activity where the amino acid sequence is an amino acid sequence having 95% or more of homology with the amino acid sequence described in SEQ. ID. NO: 2, comprising culturing the isolated transformed cell of claim 6 .