(S)-&agr;-phenethylamine: pyruvate transaminase

Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing alpha or beta amino acid or substituted amino acid...

Reexamination Certificate

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C435S041000, C435S183000, C435S243000

Reexamination Certificate

active

06346402

ABSTRACT:

This application is the national phase under 35 U.S.C. § 371 of PCT International Application No. PCT/JP99/05997 which has an International filing date of Oct. 29, 1999, which designated the United States of America.
TECHNICAL FIELD
The present invention relates to an enzyme capable of efficiently converting a ketone compound to an optically active amino compound by transamination, and a process for preparing an optically active amino compound using the enzyme. The resulting optically active amino compound can be utilized as an intermediate for pharmaceuticals and agricultural chemicals.
BACKGROUND ART
As a process for biochemically preparing optically active amino compounds, there have been known processes for preparing optically active &agr;-phenethylamine by a method of asymmetric degradation of racemic &agr;-phenethylamine by a microorganism (Japanese Patent Laid-Open Nos. Hei 1-174398 and Hei 6-253891); by a method for aminating acetophenone by a microorganism (Japanese Patent Laid-Open Nos. Hei 4-365490 and Hei 6-253875), and the like. However, in these processes, the properties of the enzyme participating in the reaction, for instance, dehydrogenase, oxidase, ammonia-lyase, and the like, are not shown. Further, since the productivity of the optically active amino compounds by the above processes is also low, it would be thought that its practical use on an industrial scale is difficult. Also, these processes do not describe on the preparation of the optically active amino compounds other than &agr;-phenethylamine.
On the other hand, there has been reported that optically active 1-(4-methoxyphenyl)-2-aminopropane can be synthesized by subjecting 1-(4-methoxyphenyl)-2-propanone to transamination using a microorganism belonging to the genus Brevibacterium (Japanese Patent Laid-Open No. Sho 63-273486). The above publication discloses that there is an effect of adding a reduced coenzyme, for instance, nicotinamide adenine dinucleotide, in the reaction. From the above, the enzyme utilized is thought to be dehydrogenase, but it is not actually clear.
In addition, Japanese Examined Patent Publication No. Hei 4-11194 discloses that a microorganism belonging to the genus Brevibacterium produces (R)-1-(4-methoxyphenyl)-2-aminopropane from 1-(4-methoxyphenyl)-2-propanone in the presence of ammonium chloride. The present inventors have conducted an additional study on the microorganism and the substrate disclosed in the above publication, and as a result, they were found to be extremely poor in the reproducibility.
Further, Japanese Patent Laid-Open No. Hei 3-103192 discloses that an &ohgr;-amino acid transaminase is allowed to act on a racemic amino compound, to degrade only an (S) modification, thereby obtaining the remaining (R) modification. In the above publication, since the reaction of the transaminase is inhibited by a transaminase inhibitor such as gabaculine or hydroxylamine, the enzyme used is considered to be an &ohgr;-amino acid transaminase. However, it would be insufficient to specify an enzyme simply from the action of the inhibitor, and moreover, the reactivity of the enzyme used for the &ohgr;-amino acid is not disclosed. Also, according to this process, the amino compound having an opposite optical activity to a desired compound is undesirably degraded in order to obtain an optically active amino compound, as in the case of a process of asymmetrically degrading a microorganism (Japanese Patent Laid-Open Nos. Hei 1-174398 and Hei 6-253891). Therefore, there is a defect that the yield for the substrate is lowered to 50% or less, making it disadvantageous in costs.
DISCLOSURE OF INVENTION
An object of the present invention is to provide an enzyme capable of efficiently converting a ketone compound to an optically active amino compound by transamination; a process for preparing an optically active amino compound using the enzyme; and a method for culturing a microorganism producing the above enzyme.
As a result of screening from various soils, the present inventors have been able to obtain a microorganism having (S)-&agr;-phenethylamine:pyruvate transaminase activity, the transaminase activity capable of converting (S)-&agr;-phenethylamine to acetophenone in the presence of pyruvic acid, and able to isolate and purify the enzyme having the activity from the microorganism. Further, as a result of a detailed study on the reaction characteristics of the (S)-&agr;-phenethylamine:pyruvate transaminase, the present inventors have found that the enzyme has excellent characteristics of allowing to act not only to an &agr;-keto acid but also to a ketone compound other than the &agr;-keto acid by using (S)-&agr;-phenethylamine or the like as an amino group donor, converting them to a corresponding optically active amino compound, and the present invention has been accomplished thereby.
The gist of the present invention relates to:
[1] an (S)-&agr;-phenethylamine:pyruvate transaminase having the following physicochemical properties:
(A) action:
acting on optically active (S)-&agr;-phenethylamine and pyruvic acid, thereby catalyzing transamination for forming acetophenone and alanine, respectively; and
(B) substrate specificity:
(a) amino group donor:exhibiting activity to (S)-&agr;-phenethylamine but not exhibiting activity to each of &bgr;-alanine, taurine, putrescine, DL-ornithine and DL-lysine; and
(b) amino group receptor: exhibiting activity to pyruvic acid and glyoxylic acid;
[2] a process for preparing an optically active amino compound, characterized in that the process comprises acting the (S)-&agr;-phenethylamine:pyruvate transaminase of item [1] above on a ketone compound represented by the following general formula (1):
wherein p is 0 or 1; q is an integer of 0 to 8; r is an integer of 0 to 4; R is a substituted or unsubstituted aryl group having 6 to 14 carbon atoms, a heterocyclic group having 4 to 12 carbon atoms, carboxyl group, an alkoxycarbonyl group having 2 to 6 carbon atoms, methyl group or hydrogen atom; and X is hydroxyl group, carboxyl group, an alkoxycarbonyl group having 2 to 6 carbon atoms, or hydrogen atom, in the presence of an amino group donor, to thereby give an optically active amino compound having the configuration represented by the general formula (2):
wherein each of p, q, r, R and X has the same definitions as those of p, q, r, R and X in the general formula (1);
[3] a process for preparing an optically active amino compound, characterized in that the process comprises acting the (S)-&agr;-phenethylamine:pyruvate transaminase of item [1] on a racemic modification of an amino compound represented by the general formula (4):
wherein p is 0 or 1; q is an integer of 0 to 8; r is an integer of 0 to 4; R is a substituted or unsubstituted aryl group having 6 to 14 carbon atoms, a heterocyclic group having 4 to 12 carbon atoms, carboxyl group, an alkoxycarbonyl group having 2 to 6 carbon atoms, methyl group or hydrogen atom; and X is hydroxyl group, carboxyl group, an alkoxycarbonyl group having 2 to 6 carbon atoms, or hydrogen atom, in the presence of an amino group receptor, to thereby give an optically active amino compound having the configuration represented by the general formula (5):
wherein each of p, q, r, R and X has the same groups as those of p, q, r, R and X in the general formula (4); and
[4] a method for culturing a microorganism for producing (S)-&agr;-phenethylamine:pyruvate transaminase, comprising adding to a medium one or more compounds selected from the group consisting of propylamine, 1-butylamine, 2-butylamine, 2-pentylamine, isopropylamine and isobutylamine as an inducer for the enzyme when the microorganism for producing (S)-&agr;-phenethylamine:pyruvate transaminase is cultured.


REFERENCES:
patent: 4950606 (1990-08-01), Stirling et al.
patent: A2227073 (1990-09-01), None
patent: A6178685 (1994-06-01), None
Partial English Translation of Japanese Patent Laid-Open No. Hei 1-174398 (Jul. 10, 1989) with corresponding portions of Japanese publication.
English Ab

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