Polypeptide having amidase activity and gene thereof
View Patent ↗It is an object of the present invention to provide a novel amidase that is useful for production of an optically active amino acid, and in particular, a D-amino acid, and a production method thereof. The present invention relates to a novel D-amidase isolated and purified from the Arthrobacter sp. KNK1101J, a gene encoding the above amidase, a recombinant plasmid comprising the above gene, and a transformant into which the above amidase gene has been introduced. In addition, the present invention also relates to a method for producing the amidase, comprising culturing the Arthrobacter sp. KNK1101J or the above transformant, and collecting the above amidase.
1. An isolated polypeptide consisting of:
(a) the amino acid sequence of SEQ ID NO: 1; or
(b) an amino acid sequence resulting from substitution, insertion, deletion, and/or addition of one amino acid in the amino acid sequence of SEQ ID NO: 1, wherein the polypeptide has amidase activity.
2. The isolated polypeptide according to claim 1 , wherein the polypeptide is derived from a microorganism belonging to the genus Arthrobacter.
3. The isolated polypeptide according to claim 2 , wherein the microorganism is Arthrobacter sp. KNK1101J (FERM BP-10192).
4. An isolated DNA encoding the polypeptide of claim 1 .
5. An isolated DNA consisting of:
(a) the nucleotide sequence of SEQ ID NO: 3;
(b) a nucleotide sequence that is capable of hybridizing, under stringent conditions, to the DNA having the nucleotide sequence that is complementary to the full-length nucleotide sequence of SEQ ID NO: 3, the stringent conditions being washing with an aqueous solution consisting of 1.5 mM trisodium citrate. 15 mM sodium chloride and 0.1% sodium dodecyl sulfate at 65° C. or
(c) a nucleotide sequence resulting from substitution, insertion, deletion, and/or addition of one nucleotide in the nucleotide sequence of SEQ ID NO: 3,
wherein the isolated DNA encodes a polypeptide having amidase activity.
6. A recombinant plasmid comprising the isolated DNA of claim 5 and a vector.
7. The recombinant plasmid according to claim 6 , wherein the vector is pUC18, pUC19, pBR322, pACYC184, pSC101, pT7Blue, or pUCNT.
8. The recombinant plasmid according to claim 6 , wherein the plasmid is pHA002.
9. A transformant, wherein the transformant is obtained by transformation of a host microorganism with the recombinant plasmid according to claim 6 .
10. The transformant according to claim 9 , wherein the host microorganism is Escherichia coli.
11. The transformant according to claim 9 , wherein the transformant is Escherichia coli HB101 (pHA002) (FERM BP-10193).
12. An isolated microorganism, wherein the isolated microorganism produces the polypeptide according to claim 1 and belongs to genus Arthrobacter.
13. The isolated microorganism according to claim 12 , the isolated microorganism being Arthrobacter sp. KNK1101J (FERM BP-10192) or a mutant thereof.
14. A method for producing an amidase, comprising
culturing a microorganism that is able to produce the polypeptide according to claim 1 ,
accumulating said polypeptide in the culture, and
collecting said polypeptide.
15. The method according to claim 14 , wherein the microorganism is a transformant obtained by transformation of a host microorganism with a recombinant plasmid, the recombinant plasmid comprising a DNA and a vector, wherein the DNA of the recombinant plasmid consists of
(a) the nucleotide sequence of SEQ ID NO: 3;
(b) a nucleotide sequence that is capable of hybridizing, under stringent conditions, to the DNA having the nucleotide sequence that is complementary to the full-length nucleotide sequence of SEQ ID NO: 3, the stringent conditions being washing with an aqueous solution consisting of 1.5 mM trisodium citrate, 15 mM sodium chloride and 0.1% sodium dodecyl sulfate at 65° C., or
(c) the nucleotide sequence resulting from substitution, insertion, deletion, and/or addition of one nucleotide in a nucleotide sequence of SEQ ID NO: 3,
wherein the DNA of the recombinant plasmid encodes a polypeptide having amidase activity.
16. The production method according to claim 14 , wherein the microorganism belongs to the genus Arthrobacter.
17. A recombinant plasmid comprising the isolated DNA of claim 4 and a vector.