Protein capable of catalyzing transamination...

Details Protein capable of catalyzing transamination... Protein capable of catalyzing transamination...

2000-03-16

2002-07-02

Slobodyansky, Elizabeth (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

Reexamination Certificate

active

06413752

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a protein capable of catalyzing transamination stereoselectively, a gene encoding said protein and uses thereof.
2. Description of the Related Art
An optically active amino compound represented by Formula (3):
X
1
—C*H(NH
2
)—(CH
2
)
m
—R
1
  (3)
(wherein X
1
is an optionally substituted C
1
-C
9
alkyl group, an optionally substituted C
6
-C
14
aryl group, an optionally substituted C
7
-C
17
arylalkyl group, an optionally substituted C
4
-C
12
heteroaryl group, an optionally substituted C
5
-C
15
heteroarylalkyl group, an amino group, an aminocarbonyl group, a hydroxyl group, a thiol group, a guanidyl group, a cyano group, a halogen atom or a hydrogen atom, R
1
is a C
1
-C
6
alkyl group, a carboxyl group, C
2
-C
6
alkyloxycarbonyl group or a hydrogen atom, m is an integer of 0 to 6, and * is an asymmetric carbon atom, is a compound which is useful as an intermediate for synthesizing a compound utilizable in various applications.
For example, an optically active form of an amino group-containing compound represented by Formula (10):
X
5
—(CH
2
)
r
—CH(NH
2
)—R
9
  (10)
(wherein X
5
is an optionally substituted phenyl or an optionally substituted naphthyl group, R
9
is a C
1
-C
6
alkyl group, and r is an integer of 0 to 4), is a compound which is useful as an intermediate for synthesizing a pharmaceutical such as a diabetes-treating agent, an anti-obesity agent, a bronchodilator and the like, as well as a pesticide such as a fungicide, a herbicide and the like, while an optically active amino compound represented by Formula (8):
(wherein X
4
is an optionally substituted C
6
-C
14
aryl group, an optionally substituted C
4
-C
12
heteroaryl group, an optionally substituted C
1
-C
3
alkyl group, an amino group, an aminocarbonyl group, a hydroxyl group, a thiol group, a guanidyl group or a hydrogen atom, R
7
and R
8
may be the same or different and each is a hydrogen atom or a C
1
-C
3
alkyl group, p is an integer of 0 to 3 and q is an integer of 0 to 2) is a compound which is useful as an intermediate for synthesizing a pharmaceutical such as a growth-disorder treating agent, anti-coagulant, carcinostatic and the like.
A known biocatalyst used in a production of an optically active amino compound described above is, for example, a transaminase derived from a microorganism belonging to the genus Arthrobacter which has an ability to produce an optically active amine compound from a certain ketone compound as a starting material (WO97/15682, WO98/48030). A D-amino acid transaminase derived from a microorganism belonging to the genus of Bacillus which has an ability to produce an optically active D-amino acid from a certain keto acid compound as a starting material (JP-B-5-5472) is also known.
SUMMARY OF THE INVENTION
The objective of the present invention is to provide a novel protein which has an excellent ability to produce an optically active amino compound efficiently, a gene encoding said protein and uses thereof.
Considering such circumstances, the present inventors have intensively investigated biocatalysts for producing an optically active amino compound. As a result, they have found a novel protein capable of catalyzing transamination stereoselectively and a gene encoding said protein and uses thereof, and have accomplished the present invention.
That is, the present invention provides:
1. A protein having any one of the following amino acid sequences (hereinafter referred to as “the protein of the present invention”):
(a) an amino acid sequence represented by Sequence ID No. 1;
(b) an amino acid sequence comprising an amino acid substitution occurring at a part corresponding to a part of an amino acid sequence represented by Sequence ID No. 1;
(c) an amino acid sequence of a protein capable of converting acetophenone to an optically active 1-phenylethylamine in the presence of a racemic mixture of sec-butylamine, said amino acid sequence showing an amino acid identity with the amino acid sequence represented by Sequence ID No. 1 of 60% or higher;
(d) an amino acid sequence of a protein capable of converting acetophenone to an optically active 1-phenylethylamine in the presence of a racemic mixture of sec-butylamine and having a molecular weight of about 37 kDa as a monomer, said amino acid sequence showing an amino acid identity with the amino acid sequence represented by Sequence ID No. 1 of 80% or higher; and,
(e) an amino acid sequence of a protein capable of converting acetophenone to an optically active 1-phenylethylamine in the presence of a racemic mixture of sec-butylamine and derived from a microorganism belonging to the genus Mycobacterium, said amino acid sequence showing an amino acid identity with the amino acid sequence represented by Sequence ID No. 1 of 60% or higher.
2. A protein having an amino acid sequence represented by Sequence ID No. 1.
3. A protein having an amino acid sequence comprising an amino acid substitution from threonine to alanine occurring at a position corresponding to amino acid's position No.2 of an amino acid sequence represented by Sequence ID No. 1;
4. A protein having a molecular weight of about 37 kDa as a monomer which is obtainable from
Mycobacterium aurum
SC-S423 and which is capable of converting acetophenone to an optically active 1-phenylethylamine in the presence of a racemic mixture of sec-butylamine.
5. A gene encoding a protein having any one of the following amino acid sequences (hereinafter referred to as “the gene of the present invention”):
(a) an amino acid sequence represented by Sequence ID No. 1;
(b) an amino acid sequence comprising an amino acid substitution occurring at a part corresponding to a part of an amino acid sequence represented by Sequence ID No. 1;
(c) an amino acid sequence encoded by the nucleotide sequence of nucleotide's positions No. 1 to No. 1017 in the nucleotide sequence represented by Sequence ID No. 2;
(d) an amino acid sequence of a protein capable of converting acetophenone to an optically active 1-phenylethylamine in the presence of a racemic mixture of sec-butylamine, said amino acid sequence showing an amino acid identity with the amino acid sequence represented by Sequence ID No. 1 of 60% or higher;
(e) an amino acid sequence of a protein capable of converting acetophenone to an optically active 1-phenylethylamine in the presence of a racemic mixture of sec-butylamine and having a molecular weight of about 37 kDa as a monomer, said amino acid sequence showing an amino acid identity with the amino acid sequence represented by Sequence ID No. 1 of 80% or higher; and,
(f) an amino acid sequence of a protein capable of converting acetophenone to an optically active 1-phenylethylamine in the presence of a racemic mixture of sec-butylamine and obtainable from a microorganism belonging to the genus Mycobacterium, said amino acid sequence showing an amino acid identity with the amino acid sequence represented by Sequence ID No. 1 of 60% or higher.
6. A gene having any of the following nucleotide sequences (hereinafter also referred to as “the gene of the present invention”):
(a) a nucleotide sequence of nucleotide's positions No. 1 to No. 1017 in the nucleotide sequence represented by Sequence ID No. 2; and,
(b) a nucleotide sequence comprising a nucleotide substitution from adenine to guanine occurring at a position corresponding to nucleotide's position No.4 of a nucleotide sequence of nucleotide's positions No. 1 to 1017 in the nucleotide sequence represented by Sequence ID No. 2;
(c) a nucleotide sequence of about 1020 bp which is amplified by PCR using as primers an oligonucleotide having the nucleotide sequence of nucleotide's positions No. 1 to 28 in the nucleotide sequence represented by Sequence ID No. 2 or an oligonucleotide having the nucleotide sequence represented by Sequence ID No. 11, and an oligonucleotide having a complementary nucleotide sequence to the nucleotide sequence of nucleotide's positions No. 999 to 1020

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