Aminotransferase, gene encoding the same, and methods of using them
View Patent ↗The invention relates to a novel aminotransferase, DNA encoding the enzyme, a recombinant vector into which the DNA has been introduced, and a transformant into which the vector has been introduced. Further, the invention also relates to a method for producing an optically active amino compound utilizing the enzyme or transformant. The aminotransferase of the invention has an ability of efficiently converting a ketone compound, particularly a cyclic ketone compound to an optically active amino compound. According to the invention, a method for efficiently producing an optically active amino compound, particularly an optically active cyclic amino compound is provided.
1. An isolated DNA
(A) having the base sequence of SEQ ID NO: 2 in the sequence list; or
(B) being hybridizable with DNA complementary with the base sequence of SEQ ID NO: 2 in the sequence list under stringent conditions, and having an amino group transfer activity, which produces acetophenone and 1-benzyl-3-aminopyrrolidine from 1-benzyl-3-pyrrolidinone and optically active (S)-α-phenethylamine,
wherein said DNA encodes an aminotransferase enzyme, and
wherein the stringent conditions are conditions in which hybridization is carried out at 65° C. in the presence of NaCl of 0.7 M to 1.0 M with a filter on which a polynucleotide derived from a colony or plaque is immobilized, and then the filter is washed at 65° C. with a 2×SSC solution.
2. A vector having DNA as set forth in claim 1 .
3. A transformant obtained by transformation of a host cell with a vector as set forth in claim 2 .
4. An isolated DNA encoding an aminotransferase having physical and chemical properties (1) to (6):
(1) effect: the aminotransferase catalyzes an amino group transfer reaction that produces acetophenone and 1-benzyl-3-aminopyrrolidine from 1-benzyl-3-pyrrolidinone and optically active (S)-α-phenethylamine;
(2) substrate specificities:
(a) amino donor: the aminotransferase is active with (S)-α-phenethylamine, but substantially inactive with β-alanine, taurine, putrescine, DL-ornithine, and DL-lysine; and
(b) amino acceptor: the aminotransferase is active with pyruvic acid and glyoxylic acid;
(3) molecular weight: approximately 120,000 when measured by gel filtration, and approximately 53,000 when measured by SDS-polyacrylamide gel electrophoresis;
(4) an optimum pH of 7 to 9;
(5) an optimum temperature for activity of 30° C. to 50° C.; and
(6) a thermal stability after being treated with a pH of 7.0 and a temperature of 30° C. to 40° C. for 30 minutes, and wherein the enzyme maintains 90% or more of its activity that the enzyme had before treatment.
5. A vector having DNA as set forth in claim 4 .
6. A transformant obtained by transformation of a host cell with a vector as set forth in claim 5 .
7. The isolated DNA according to claim 1 , wherein said DNA encodes an aminotransferase comprising SEQ ID NO: 1.
8. The vector having DNA according to claim 2 , wherein said DNA encodes an aminotransferase comprising SEQ ID NO: 1.
9. The transformant according to claim 3 , wherein said DNA encodes an aminotransferase comprising SEQ ID NO: 1.
10. The isolated DNA according to claim 1 , wherein said DNA comprises the polynucleotide sequence of SEQ ID NO: 2.
11. The vector having DNA according to claim 2 , wherein said DNA comprises the polynucleotide sequence of SEQ ID NO: 2.
12. The transformant according to claim 3 , wherein said DNA comprises the polynucleotide sequence of SEQ ID NO: 2.