U.S. patent application number 09/794359 was filed with the patent office on 2001-10-18 for process for producing optically active amino compounds.
This patent application is currently assigned to Kaneka Corporation. Invention is credited to Hasegawa, Junzo, Ikenaka, Yasuhiro, Iwasaki, Akira, Kizaki, Noriyuki, Ogura, Masahiro, Yamada, Yukio.
Application Number | 20010031487 09/794359 |
Document ID | / |
Family ID | 17867103 |
Filed Date | 2001-10-18 |
United States Patent
Application |
20010031487 |
Kind Code |
A1 |
Yamada, Yukio ; et
al. |
October 18, 2001 |
Process for producing optically active amino compounds
Abstract
The method for preparing an optically active (R)-amino compound
characterized by the method comprising stereoselectively carrying
out amino group transfer by action of an (R)-form-specific
transaminase in the co-presence of a ketone compound (amino
acceptor), and an amino compound (amino donor) of a racemic form or
an (R)-form, to give an optically active (R)-amino compound.
According to the present invention, it is made possible to easily
prepare at a high yield the optically active (R)-amino compounds
and the like having an aryl group and the like at their 1-position,
which have been conventionally difficult to prepare.
Inventors: |
Yamada, Yukio;
(Kakogawa-shi, JP) ; Iwasaki, Akira; (Akasahi-shi,
JP) ; Kizaki, Noriyuki; (Takasago-shi, JP) ;
Ikenaka, Yasuhiro; (Akashi-shi, JP) ; Ogura,
Masahiro; (Ono-shi, JP) ; Hasegawa, Junzo;
(Akashi-shi, JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
Kaneka Corporation
|
Family ID: |
17867103 |
Appl. No.: |
09/794359 |
Filed: |
February 28, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09794359 |
Feb 28, 2001 |
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09064750 |
Apr 23, 1998 |
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6221638 |
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09064750 |
Apr 23, 1998 |
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PCT/JP96/03054 |
Oct 21, 1996 |
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Current U.S.
Class: |
435/128 ;
435/193 |
Current CPC
Class: |
Y10S 435/83 20130101;
C12N 9/1096 20130101; C12P 41/006 20130101; C12P 13/001 20130101;
C12P 13/005 20130101; C12P 13/04 20130101; C12P 13/008
20130101 |
Class at
Publication: |
435/128 ;
435/193 |
International
Class: |
C12P 013/00; C12N
009/10 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 23, 1995 |
JP |
7-299013 |
Claims
1. A method for preparing an optically active (R)-amino compound
characterized by the method comprising stereoselectively carrying
out amino group transfer by action of an (R)-form-specific
transaminase in the co-presence of a ketone compound (amino
acceptor) represented by the following general formula (I):
6wherein n is 0 to 5; m is 0 or 1; and X represents an
unsubstituted aryl group having 6 to 14 carbon atoms, an aryl group
having 6 to 14 carbon atoms and having one or more substituents
selected from the group consisting of an alkyl group having 4 to 15
carbon atoms, a hydroxyl group, a fluorine atom, a chlorine atom, a
bromine atom, an iodine atom, a nitro group, a carboxyl group, and
a trifluoromethyl group, or a methoxyl group, and an amino compound
(amino donor) of an achiral form, a racemic form, or an (R)-form
represented by the general formula (II): 7wherein X.sub.1 and
X.sub.2 independently represent a hydrogen atom; a straight-chain
alkyl group having 1 to 10 carbon atoms; a branched alkyl group
having 5 to 12 carbon atoms; an unsubstituted aryl group having 6
to 14 carbon atoms; an aryl group having 6 to 14 carbon atoms and
having one or more substituents selected from the group consisting
of an alkyl group having 1 to 4 carbon atoms, a hydroxyl group, a
fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a
nitro group, and a carboxyl group; an aralkyl group having 7 to 16
carbon atoms and having one or more substituents selected from the
group consisting of an alkyl group having 1 to 4 carbon atoms, a
hydroxyl group, a fluorine atom, a chlorine atom, a bromine atom,
an iodine atom, a nitro group, and a carboxyl group; or a
hydroxymethyl group or a hydroxyethyl group, to give an optically
active (R)-amino compound represented by the general formula (IV):
8wherein n, m, and X have the same definitions as those of n, m,
and X in the general formula (I), respectively.
2. The method for preparing an optically active (R)-amino compound
according to claim 1, characterized in that in the general formula
(II), X.sub.1 is an alkyl group having 2 to 10 carbon atoms, a
phenyl group, or a naphthyl group, and X.sub.2 is an alkyl group
having 1 or 2 carbon atoms.
3. The method for preparing an optically active (R)-amino compound
according to claim 1, characterized in that the amino donor
represented by the general formula (II) is an alkyl ester of
D-alanine, the alkyl group having 1 to 8 carbon atoms.
4. The method for preparing an optically active (R)-amino compound
according to claim 1, wherein the amino donor represented by the
general formula (II) is (R)-1-phenylethylamine,
(R)-1-naphthylethylamine, (R)-1-methylpropylamine,
(R)-2-aminopentane, (R)-2-amino-1-propanol, (R)-1-methylbutylamine,
(R)-1-phenylmethylamine, (R)-1-amino-1-phenyletha- nol,
(R)-2-amino-2-phenylethanol, (R)-3-aminoheptane,
(R)-1-amino-3-phenylpropane, (R)-2-amino-4-phenylbutane,
(R)-2-amino-3-phenylpropanol, (R)-3,4-dimethoxyaminopropane,
(R)-1-methylheptylamine, benzylamine, (S)-2-phenylglycinol,
3-aminophenylbutane, L-phenylalaninol,
(R)-2-amino-1-methoxypropane, D-alanine methyl ester, D-alanine
ethyl ester, or a racemic compound thereof.
5. The method for preparing an optically active (R)-amino compound
according to any one of claims 1 to 4, characterized in that in the
general formula (I) and the general formula (IV), n and m are n=1
and m=0.
6. The method for preparing an optically active (R)-amino compound
according to any one of claims 1 to 5, characterized in that in the
general formula (I) and the general formula (IV), X is phenyl,
2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl,
2,4-dimethoxyphenyl, 3,4-dimethoxyphenyl, 3-trifluoromethylphenyl,
or methoxyl.
7. The method for preparing an optically active (R)-amino compound
according to any one of claims 1 to 6, characterized in that the
amino acceptor represented by the general formula (I) and the amino
donor represented by the general formula (II) are brought into
contact with a culture of microorganisms, separated bacterial
cells, treated bacterial cells, or immobilized bacterial cells
which produce (R)-form specific transaminases.
8. The method for preparing an optically active (R)-amino compound
according to any one of claims 1 to 6, characterized in that the
amino acceptor represented by the general formula (I) and the amino
donor represented by the general formula (II) are brought into
contact with cell-free extracts of microorganisms, crudely purified
enzymes, purified enzymes, or immobilized enzymes which produce
(R)-form specific transaminases.
9. The method for preparing an optically active (R)-amino compound
according to any one of claims 1 to 8, wherein the microorganism
for producing the transaminase is a microorganism belonging to the
genus Arthrobacter.
10. The method for preparing an optically active (R)-amino compound
according to claim 9, wherein the microorganism belonging to the
genus Arthrobacter is Arthrobacter species (Arthrobacter sp.)
KNK168 (FERM BP-5228).
11. The method for preparing an optically active (R)-amino compound
according to any one of claims 1 to 10, characterized by adding to
a medium, when culturing the microorganism for producing the
transaminase, one or more members selected from the group
consisting of (RS)-1-methylpropylamine, (RS)-1-phenylethylamine,
(RS)-1-methylbutylamine, (RS)-3-amino-2,2-dimethylbutane,
(RS)-2-amino-1-butanol, and (R)- or
(RS)-1-(3,4-dimethoxyphenyl)aminoprop- ane as an inducer for the
enzyme.
12. The method for preparing an optically active (R)-amino compound
according to claim 1, characterized by carrying out the reaction at
a pH of not less than 5 and not more than 12 in the amino group
transfer reaction.
13. The method for preparing an optically active (R)-amino compound
according to claim 1, characterized by adding a surfactant or a
fatty acid as a reaction accelerator upon reaction in the amino
group transfer reaction.
14. A method for preparing an optically active (S)-amino compound,
characterized by the method comprising stereoselectively carrying
out amino group transfer reaction by action of an (R)-form-specific
transaminase to an amino compound of a racemic form represented by
the general formula (V) in the presence of a ketone compound (amino
acceptor) represented by the general formula (III), to give an
optically active (S)-amino compound represented by the general
formula (VI): 9 10wherein X.sub.1 and X.sub.2 independently
represent a hydrogen atom; a straight-chain alkyl group having 1 to
10 carbon atoms; a branched alkyl group having 5 to 12 carbon
atoms; an unsubstituted aryl group having 6 to 14 carbon atoms; an
aryl group having 6 to 14 carbon atoms and having one or more
substituents selected from the group consisting of an alkyl group
having 1 to 4 carbon atoms, a hydroxyl group, a fluorine atom, a
chlorine atom, a bromine atom, an iodine atom, a nitro group, and a
carboxyl group; an aralkyl group having 7 to 16 carbon atoms and
having one or more substituents selected from the group consisting
of an alkyl group having 1 to 4 carbon atoms, a hydroxyl group, a
fluorine atom, a chlorine atom, a bromine atom, an iodine atom, a
nitro group, and a carboxyl group; or a hydroxymethyl group or a
hydroxyethyl group; and wherein n is 0 to 5; m is 0 or 1; and X
represents an unsubstituted aryl group having 6 to 14 carbon atoms,
an aryl group having 6 to 14 carbon atoms and having one or more
substituents selected from the group consisting of an alkyl group
having 4 to 15 carbon atoms, a hydroxyl group, a fluorine atom, a
chlorine atom, a bromine atom, an iodine atom, a nitro group, a
carboxyl group, and a trifluoromethyl group, or a methoxyl
group.
15. An (R)-form-specific transaminase obtainable from a culture of
a microorganism belonging to the genus Arthrobacter.
16. The (R)-form-specific transaminase according to claim 15,
wherein the transaminase contains an amino acid sequence of:
Glu-Ile-Val-Tyr-Thr-His-- Asp-Thr-Gly-Leu-Asp-Tyr in the
neighborhood of an amino terminal of the enzyme protein.
Description
[0001] This application is a continuation-in-part application of
PCT/JP96/03054, the entire contents of which are incorporated
herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to optically active amino
compounds having an aryl group and the like at the 1-position,
which can be used as intermediates for pharmaceuticals and
agricultural chemicals. 1-(3,4-Dimethoxyphenyl)-2-aminopropane,
which is one of the desired compounds of the present invention, is
an important compound as intermediates for CL316, 243 (J. D. Bloom
et al., J. Med. Chem. 35, 3081-3084 (1992)) and analogous compounds
thereof, which are promising as antidiabetics and agents for
antiobesity under development.
[0003] In addition, (S)-2-amino-1-methoxypropane, which is another
desired compound is a useful compound which can be used as
intermediates for herbicides.
BACKGROUND ART
[0004] Processes for preparing optically active amino compounds
having an aryl group and the like at the 1-position by using an
enzyme include a report in Nakamichi et al. (Appl. Microbiol.
Biotechnol., 33, 634-640 (1990)) and Japanese Patent Examined
Publication No. Hei 4-11194. It is disclosed in these publications
that an (S)-form can be efficiently prepared by transferring an
amino group to 1-(substituted phenyl)-2-propanones by using an
enzyme. Further, Japanese Patent Examined Publication No. Hei
4-11194 also discloses a preparation of an (R)-form; however, the
present inventors have conducted additional experiment to examine
microorganisms and substrate disclosed in Japanese Patent Examined
Publication No. Hei 4-11194 and found that the reproducibility by
means of this method is very poor, and thereby making it difficult
to use this method for practical purposes. Further, Stirling et al.
disclose a method in which only the (S)-form is decomposed by
actions of an .omega.-amino acid transaminase to a racemic amino
compound produced by an organic reaction, to thereby obtain the
remaining (R)-form (Japanese Patent Laid-Open No. Hei 3-103192).
However, in this method, since the (S)-form is undesirably
decomposed to obtain the (R)-form, the yield against the substrate
is lowered to 50% or less. Accordingly, this method cannot be
considered to be advantageous from the aspect of costs. In
addition, Stirling et al., the authors as above, also disclose a
method in which only (S)-amino compounds are prepared from a
ketone-form by using an .omega.-amino acid transaminase in the
presence of an amino donor. However, optically active (R)-amino
compounds cannot be produced by this method.
DISCLOSURE OF INVENTION
[0005] Accordingly, an object of the present invention is to
provide a method for preparing optically active (R)-amino compounds
by actions of microbial enzymes efficiently and inexpensively.
[0006] The present inventors have found a microorganism from soil
which can prepare optical active (R)-amino compounds with good
yield by carrying out an amino group transfer to a ketone compound
having an aryl group, and the like at 1-position (R)-form
specifically and efficiently. Further studies have been made on the
reaction using this microorganism, and the present invention has
been completed.
[0007] Specifically, the present invention, in essence, pertains
to:
[0008] [1] A method for preparing an optically active (R)-amino
compound characterized by the method comprising stereoselectively
carrying out amino group transfer by action of an (R)-form-specific
transaminase in the co-presence of a ketone compound (amino
acceptor) represented by the following general formula (I): 1
[0009] wherein n is 0 to 5; m is 0 or 1; and X represents an
unsubstituted aryl group having 6 to 14 carbon atoms, an aryl group
having 6 to 14 carbon atoms and having one or more substituents
selected from the group consisting of an alkyl group having 4 to 15
carbon atoms, a hydroxyl group, a fluorine atom, a chlorine atom, a
bromine atom, an iodine atom, a nitro group, a carboxyl group, and
a trifluoromethyl group, or a methoxyl group, and an amino compound
(amino donor) of an achiral form, a racemic form, or an (R)-form
represented by the general formula (II): 2
[0010] wherein X.sub.1 and X.sub.2 independently represent a
hydrogen atom; a straight-chain alkyl group having 1 to 10 carbon
atoms; a branched alkyl group having 5 to 12 carbon atoms; an
unsubstituted aryl group having 6 to 14 carbon atoms; an aryl group
having 6 to 14 carbon atoms and having one or more substituents
selected from the group consisting of an alkyl group having 1 to 4
carbon atoms, a hydroxyl group, a fluorine atom, a chlorine atom, a
bromine atom, an iodine atom, a nitro group, and a carboxyl group;
an aralkyl group having 7 to 16 carbon atoms and having one or more
substituents selected from the group consisting of an alkyl group
having 1 to 4 carbon atoms, a hydroxyl group, a fluorine atom, a
chlorine atom, a bromine atom, an iodine atom, a nitro group, and a
carboxyl group; or a hydroxymethyl group or a hydroxyethyl group,
to give an optically active (R)-amino compound represented by the
general formula (IV): 3
[0011] wherein n, m, and X have the same definitions as those of n,
m, and X in the general formula (I), respectively;
[0012] [2] The method for preparing an optically active (R)-amino
compound described in item [1] above, characterized in that in the
general formula (II), X.sub.1 is an alkyl group having 2 to 10
carbon atoms, a phenyl group, or a naphthyl group, and X.sub.2 is
an alkyl group having 1 or 2 carbon atoms;
[0013] [3] The method for preparing an optically active (R)-amino
compound described in item [1] above, characterized in that the
amino donor represented by the general formula (II) is an alkyl
ester of D-alanine, the alkyl group having 1 to 8 carbon atoms;
[0014] [4] The method for preparing an optically active (R)-amino
compound described in item [1] above, wherein the amino donor
represented by the general formula (II) is (R)-1-phenylethylamine,
(R)-1-naphthylethylamine, (R)-1-methylpropylamine,
(R)-2-aminopentane, (R)-2-amino-1-propanol, (R)-1-methylbutylamine,
(R)-1-phenylmethylamine, (R)-1-amino-1-phenyletha- nol,
(R)-2-amino-2-phenylethanol, (R)-3-aminoheptane,
(R)-1-amino-3-phenylpropane, (R)-2-amino-4-phenylbutane,
(R)-2-amino-3-phenylpropanol, (R)-3,4-dimethoxyaminopropane,
(R)-1-methylheptylamine, benzylamine, (S)-2-phenylglycinol,
3-aminophenylbutane, L-phenylalaninol,
(R)-2-amino-1-methoxypropane, D-alanine methyl ester, D-alanine
ethyl ester, or a racemic compound thereof;
[0015] [5] The method for preparing an optically active (R)-amino
compound described in any one of items [1] to [4] above,
characterized in that in the general formula (I) and the general
formula (IV), n and m are n=1 and m=0;
[0016] [6] The method for preparing an optically active (R)-amino
compound described in any one of items [1] to [5] above,
characterized in that in the general formula (I) and the general
formula (IV), X is phenyl, 2-methoxyphenyl, 3-methoxyphenyl,
4-methoxyphenyl, 2,4-dimethoxyphenyl, 3,4-dimethoxyphenyl,
3-trifluoromethylphenyl, or methoxyl;
[0017] [7] The method for preparing an optically active (R)-amino
compound described in any one of items [1] to [6] above,
characterized in that the amino acceptor represented by the general
formula (I) and the amino donor represented by the general formula
(II) are brought into contact with a culture of microorganisms,
separated bacterial cells, treated bacterial cells, or immobilized
bacterial cells which produce (R)-form-specific transaminases;
[0018] [8] The method for preparing an optically active (R)-amino
compound described in any one of items [1] to [6] above,
characterized in that the amino acceptor represented by the general
formula (I) and the amino donor represented by the general formula
(II) are brought into contact with cell-free extracts of
microorganisms, crudely purified enzymes, purified enzymes, or
immobilized enzymes which produce (R)-form-specific
transaminases;
[0019] [9] The method for preparing an optically active (R)-amino
compound described in any one of items [1] to [8] above, wherein
the microorganism for producing the transaminase is a microorganism
belonging to the genus Arthrobacter;
[0020] [10] The method for preparing an optically active (R)-amino
compound described in item [9] above, wherein the microorganism
belonging to the genus Arthrobacter is Arthrobacter species
(Arthrobacter sp.) KNK168 (FERM BP-5228);
[0021] [11] The method for preparing an optically active (R)-amino
compound described in any one of items [1] to [10] above,
characterized by adding to a medium, when culturing the
microorganism for producing the transaminase, one or more members
selected from the group consisting of (RS)-1-methylpropylamine,
(RS)-1-phenylethylamine, (RS)-1-methylbutylamine,
(RS)-3-amino-2,2-dimethylbutane, (RS)-2-amino-1-butanol, and (R)-
or (RS)-1-(3,4-dimethoxyphenyl)aminoprop- ane as an inducer for the
enzyme;
[0022] [12] The method for preparing an optically active (R)-amino
compound described in item [1] above, characterized by carrying out
the reaction at a pH of not less than 5 and not more than 12 in the
amino group transfer reaction;
[0023] [13] The method for preparing an optically active (R)-amino
compound described in item [1] above, characterized by adding a
surfactant or a fatty acid as a reaction accelerator upon reaction
in the amino group transfer reaction;
[0024]
[0025] [14] A method for preparing an optically active (S)-amino
compound, characterized by the method comprising stereoselectively
carrying out amino group transfer reaction by action of an
(R)-form-specific transaminase to an amino compound of a racemic
form represented by the general formula (V) in the presence of a
ketone compound (amino acceptor) represented by the general formula
(III), to give an optically active (S)-amino compound represented
by the general formula (VI). 4
[0026] [15] An (R)-form-specific transaminase obtainable from a
culture of a microorganism belonging to the genus Arthrobacter;
and
[0027] [16] The (R)-form-specific transaminase described in item
[15] above, wherein the transaminase contains an amino acid
sequence of:
[0028] Glu-Ile-Val-Tyr-Thr-His-Asp-Thr-Gly-Leu-Asp-Tyr in the
neighborhood of an amino terminal of the enzyme protein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 is a graph showing the optimal pH for the enzyme of
the present invention.
[0030] FIG. 2 is a graph showing the stable pH for the enzyme of
the present invention.
BEST MODE FOR CARRYING OUT THE INVENTION
[0031] The present invention will be described in detail below.
First, the reaction scheme in the present invention is shown as
follows. 5
[0032] The present inventors have carried out repeated screening to
separate bacteria from domestic soil which have an ability of
producing (R)-amnino compounds in (R)-form selectivity using
ketones as substrates, and consequently have found that bacteria
belonging to the genus Arthrobacter have strong activity for
catalyzing this reaction. Among them, the bacteriological natures
of Arthrobacter sp. KNK168 (FERM BP-5228), a typical example
thereof, are shown as follows.
1 Cell Morphology Rod (Coryne type) Gram Staining Positive Spore
Formation None Motility None Colony Morphology Round, regular,
entire, yellow, smooth, glossy, semi-translucent, convex, 2 m in
diameter (Bennett's agar medium) Growth (30.degree. C.) +
(37.degree. C.) - Catalase + Oxidase - OF Test (glucose) -
(oxidative) Cell Wall No mycolic acid; diamino acid is lysine.
Fatty Acid Analysis Almost all the acids present are three-branched
iso and anteiso acid.
[0033] From the aspects that an (R)-form amine, such as
(R)-1-phenylethylamine, is used as an amino donor and that the
resulting product is an optically active (R)-amino compound as
described in detail below, the transaminase produced by this
bacterium is obviously different from an enzyme of Brevibacterium
linens IFO12141 used in Nakamichi et al. (Appl. Microbiol.
Biotechnol., 33, 634-640 (1990)) and an .omega.-amino acid
transaminase used in Stirling et al. (Japanese Patent Laid-Open No.
Hei 3-103192), in which an (S)-amine is used as an amino donor and
the resulting product is an optically active (S)-amino
compound.
[0034] Further, the transaminase derived from Arthrobacter which
can be used in the present invention is different from the
above-mentioned enzyme derived from Brevibacterium in many other
aspects. For example, there are differences from the aspect that
inorganic ammonium salts such as ammonium chloride, and 1-amino
acids (being (S)-amines) such as glutamic acid and aspartic acid
cannot be used as an amino donor (Table 1).
2 TABLE 1 Transaminase of Enzyme Used in Amino Group Present
Nakamichi Donor Invention et al. Inorganic - + Ammonium Salt
L-Amino Acid - +
[0035] In addition, when the transaminase derived from Arthrobacter
which can be used in the present invention is compared with the
.omega.-amino acid transaminase used in Stirling et al. (Japanese
Patent Laid-Open No. Hei 3-103192), in addition to the obvious
difference regarding the above-mentioned substrate specificity,
there are differences from the aspect that the transaminase derived
from Arthrobacter does not act to .omega.-amino acids such as
.beta.-alanine and 4-aminobutyric acid, and .omega.-amines such as
n-butylamine and putrescine, and DL-3-aminobutyric acid, and the
like, and that the transaminase derived from Arthrobacter is
affected only a little by reaction inhibitors such as hydroxylamine
and phenylhydrazine.
[0036] In addition, as other enzymes similar to the enzyme used by
the present inventors, benzylamine transaminase is disclosed in
Okada et al. (Japanese Patent Laid-Open No. Hei 6-178685), wherein
the benzylamine transaminase strongly acts to benzylamine in the
presence of pyruvic acid to form 1-alanine and benzaldehyde.
However, the benzylamine transaminase has a similar optical
specificity to that of the above-mentioned enzyme derived from
Brevibacterium or to that of the .omega.-amino acid transaminase
from the aspect that the optical activity of the product by the
transaminase action of this enzyme is that of L-alanine of an
(S)-form, and it can be said that the benzylamine transaminase is
completely different from the enzyme derived from Arthrobacter
which can be used in the present invention.
[0037] Further, when these enzymes are compared, it seems that
there are some differences in influence caused by reaction
inhibitors such as phenylhydrazine, D-penicillamine, and
p-chloromercuribenzoic acid.
[0038] The differences with the .omega.-amino acid transaminase
derived from Pseudomonas sp. F-1 (Agric. Biol. Chem., 42, 2363-2367
(1978), Agric. Biol. Chem., 43, 1043-1048 (1979), J. Biol. Chem.,
258, 2260-2265 (1983)) which have been well studied among the
.omega.-amino acid transaminases, and with the benzylamine
transaminase of Okada et al. (Japanese Patent Laid-Open No. Hei
6-178685) are summarized and shown in Table 2. In a case of the
transaminase action in the present invention, the data of the
purified enzyme are used, and in cases of those by other two
enzymes, the data of the purified enzymes which are disclosed in
each of the literature publication and patent publication are used.
Pyruvic acid is used as an amino acceptor in all cases.
3 TABLE 2 Transaminase Transaminase of Present .omega.-Amino Acid
of Okada Invention Transaminase et al. Substrate (Relative
(Relative (Relative Specificity Activity: %) Activity: %) Activity:
%) .beta.-Alanine 0 100 0 4-Aminobutyric 0 40 acid DL-3- 0 96
Aminobutyric acid n-Butylamine 0 60 0 Benzylamine 0.8 45 100
Putrescine 0 55 0 .beta.-Phenethylamine 0 54 9 Amino Group Donor -
+ + L-Alanine Inhibitor (Relative (Relative (Relative (1 mM each)
Activity: %) Activity: %) Activity: %) (without 100 100 100
addition) Hydroxylamine 17 0 30 (0.1 mM) Phenylhydrazine 82 6 27
D-Penicillamine 93 65 0 p-Chloromercuri- 53 100 9 benzoic acid (0.1
mM) CuSO.sub.4 44 5 (0.5 mM) Gabaculine 24 0
[0039] The activity of the transaminase (intracellular enzyme)
which can be used in the present invention against the typical
substrates of the .omega.-aminotransferases is shown in Table 3. In
the case of using an .omega.-amino acid or an .omega.-amine as an
amino donor, the .omega.-aminotransferase activity is extremely
low, and the transferase used in the present invention does not act
to .beta.-alanine which is a typical substrate of .omega.-amino
acid-pyruvic acid aminotransferase. The transferase used in the
present invention shows an especially high activity to
(R)-1-phenylethylamine. Therefore, it is quite different from the
(o-amino to transaminase in the substrate specificity.
4 TABLE 3 Relative Amino Donor Amino Acceptor Activity (%)
.beta.-Alanine Pyruvic acid 0 4-Aminobutyric 2-Ketoglutaric 0 acid
acid 2,5-Diaminovalerate 2-Ketoglutaric 2 (.alpha.,.omega.-amino
acid) acid DL-Ornithine 2-Ketoglutaric 0 acid DL-Lysine
2-Ketoglutaric 0 acid Putrescine 2-Ketoglutaric 0
(.alpha.,.omega.-Diamine) acid .alpha.-2,4-Diamino- Pyruvic acid 0
butyric acid Taurine 2-Ketoglutaric 0 acid DL-Asparagine
2-Ketoglutaric 0 acid DL-Glutamine 2-Ketoglutaric 0 acid
(R)-1-Phenylethyl- Pyruvic acid 100 amine (Control)
[0040] The (R)-form-specific transaminase produced by Arthrobacter
species (Arthrobacter sp.) KNK168 (FERM BP-5228) used in the
present invention has an amino acid sequence of SEQ ID NO:1 in
Sequence Listing:
[0041] Glu-Ile-Val-Tyr-Thr-His-Asp-Thr-Gly-Leu-Asp-Tyr in the
neighborhood of an amino terminal of the enzyme protein.
[0042] In a case where the amino group transfer action which can be
used in the present invention is carried out, the enzymatic
activity is increased by addition of an inducer upon culturing
microorganisms. The above inducers include one or more members of
amines, such as (RS)-1-methylpropylamine, (RS)-1-phenylethylamine,
(RS)-1-methylbutylamine, (RS)-3-amino-2,2-dimethylbutane,
(RS)-2-amino-1-butanol, and (R)- or
(RS)-1-(3,4-dimethoxyphenyl)aminoprop- ane.
[0043] The transaminase derived from Arthrobacter which can be used
in the present invention can be used in various forms. In other
words, not only cultured microorganisms, separated bacterial cells
and treated bacterial cells, but also cell-free extracts, crudely
purified enzymes, purified enzymes, and the like can be used.
Further, immobilized products of these cells, immobilized products
of the treated bacterial cells, immobilized products of enzyme
proteins to immobilizing carriers (for example, anionic exchange
resins) and the like can be also used. Here, the immobilization can
be carried out by conventional methods (for example, Japanese
Patent Laid-Open No. Sho 63-185382).
[0044] Supporting materials which can be used in the immobilization
include various kinds of anionic exchange resins, such as various
amines, ammonium salts and diethanolamine type resins having
functional groups. Suitable examples thereof include
phenol-formaldehyde anionic exchange resins such as Duolite A568
and DS17186 (registered trademark of Rohm and Haas Company);
polystyrene resins, such as Amberlite IRA935, IRA945, and IRA901
(registered trademark of Rohm and Haas Company), Lewatit OC1037
(registered trademark of Bayer A. G.), and Diaion EX-05 (registered
trademark of MITSUBISHI KASEI CORPORATION), and the like. In
addition, supporting materials, such as DEAE-cellulose, can be
used. Further, cross-linking agents are usually used in order to
make the adsorption of enzyme more firm and stable. A preferable
example of the cross-linking agents includes glutaraldehyde. The
enzymes used include purified enzymes, as well as enzymes at
various levels of the purification, such as partially purified
enzymes, solutions of disrupted cells, and cell-free extracts.
[0045] As a preparation method of the immobilized enzyme,
conventional preparation methods may be employed, including, for
example, a method wherein the cross-linking treatment is carried
out after adsorption of an enzyme to a supporting material.
[0046] In other words, in the present invention, it is preferable
that the amino acceptor represented by the general formula (I) and
the amino donor represented by the general formula (II) are brought
into contact to a culture of microorganisms, separated bacterial
cells, treated bacterial cells, or immobilized bacterial cells
which produce (R)-form-specific transaminase, or into contact to
cell-free extracts of microorganisms, crudely purified enzymes,
purified enzymes, or immobilized enzymes which produce
(R)-form-specific transaminases.
[0047] The amino acceptors which can be used in the present
invention include ketones represented by the general formula (I).
In the formula, n is 0 to 5; m is 0 or 1; and X represents an
unsubstituted aryl group having 6 to 14 carbon atoms, an aryl group
having 6 to 14 carbon atoms and having one or more substituents
selected from the group consisting of an alkyl group having 4 to 15
carbon atoms, a hydroxyl group, a fluorine atom, a chlorine atom, a
bromine atom, an iodine atom, a nitro group, a carboxyl group, and
a trifluoromethyl group, or a methoxy group.
[0048] Among them, a compound with n=1 and m=0, for example,
1-aryl-2-propanone or 1-methoxy-2-propanone, is preferable. In
addition, the compound in which the aryl group is a phenyl group, a
2-methoxyphenyl group, a 3-methoxyphenyl group, a 4-methoxyphenyl
group, a 2,4-dimethoxyphenyl group, a 3,4-dimethoxyphenyl group, or
a 3-trifluoromethylphenyl group is also preferable.
[0049] The amino donors used in the present invention include amino
compounds represented by the general formula (II) of an achiral
form, a racemic form, or an (R)-form. In the general formula (II),
X.sub.1 and X.sub.2 independently represent a hydrogen atom; a
straight-chain alkyl group having 1 to 10 carbon atoms; a branched
alkyl group having 5 to 12 carbon atoms; an unsubstituted aryl
group having 6 to 14 carbon atoms; an aryl group having 6 to 14
carbon atoms having one or more substituents selected from the
group consisting of an alkyl group having 1 to 4 carbon atoms, a
hydroxyl group, a fluorine atom, a chlorine atom, a bromine atom,
an iodine atom, a nitro group, and a carboxyl group; an aralkyl
group having 7 to 16 carbon atoms and having one or more
substituents selected from the group consisting of an alkyl group
having 1 to 4 carbon atoms, a hydroxyl group, a fluorine atom, a
chlorine atom, a bromine atom, an iodine atom, a nitro group, and a
carboxyl group; or a hydroxymethyl group or a hydroxyethyl
group.
[0050] Among the above-mentioned amines, amines in which X.sub.1 is
an alkyl group having 2 to 10 carbon atoms, a phenyl group or a
naphthyl group, and X.sub.2 is an alkyl group having 1 or 2 carbon
atoms, or alkyl esters of D-alanine, the alkyl group having 1 to 8
carbon atoms, are particularly preferable. Concrete examples of the
amines include, for instance, (R)-1-phenylethylamine,
(R)-1-naphthylethylamine, (R)-1-methylpropylamine,
(R)-2-aminopentane, (R)-2-amino-1-propanol, (R)-1-methylbutylamine,
(R)-1-phenylmethylamine, (R)-1-amino-1-phenyletha- nol,
(R)-2-amino-2-phenylethanol, (R)-3-aminoheptane,
(R)-1-amino-3-phenylpropane, (R)-2-amino-4-phenylbutane,
(R)-2-amino-3-phenylpropanol, (R)-3,4-dimethoxyaminopropane,
(R)-1-methylheptylamine, benzylamine, (S)-2-phenylglycinol,
3-aminophenylbutane, L-phenylalaninol,
(R)-2-amino-1-methoxypropane, D-alanine methyl ester, D-alanine
ethyl ester, and the like.
[0051] The concentrations of the substrates used in the reaction
are as follows. It is preferred that the concentration of the amino
acceptor is from 0.1 to 10%, preferably from 3 to 5%. Regarding the
concentration of the amino donor, it is preferred that the
concentration of an (R)-form, in the case of chiral amine, is about
80 to 150 mol % to the amino acceptor. In addition, racemic amino
compounds can be also used as amino donors. In such cases, however,
twice the concentration as that of the (R)-form is needed.
[0052] The pH during the reaction is usually a pH of from 5.0 to
12.0, preferably a pH of from 7.0 to 10.0. The temperature during
the reaction is usually from 25.degree. to 40.degree. C.,
preferably from 30.degree. to 35.degree. C.
[0053] Further, the yield of reaction may be increased by adding a
surfactant, including sodium dodecyl sulfate (SDS), Triton X-100,
cetyl trimethylammonium bromide (CTAB), or the like, or a fatty
acid, including linoleic acid, oleic acid, or the like, in an
amount of from 0.1 to 10%.
[0054] The optically active amino compounds which are prepared by
the method of the present invention are the (R)-amino compounds
represented by the general formula (IV). In the formula, n, m, and
X, respectively, has the same definitions as those in the general
formula (I).
[0055] Concrete examples of the compounds include, for instance,
(R)-1-phenyl-2-aminopropane, (R)-1-phenyl-3-aminobutane,
(R)-1-(3,4-dimethoxyphenyl)-2-aminopropane,
(R)-3-(trifluoromethylphenyl)- -2-aminopropane,
(R)-3-(p-methoxyphenyl)-2-aminopropane,
(R)-4-(p-methoxyphenyl)-2-aminobutane,
(R)-4-(3',4'-methylenedioxyphenyl)- -2-aminobutane,
(R)-4-(p-hydroxyphenyl)-2-aminobutane,
(R)-1-(3-trifluoromethylphenyl)-2-aminopropane,
(R)-2-amino-1-methoxyprop- ane, and the like.
[0056] By the preparation method of the present invention, for
example, in a case where 1-(3,4-dimethoxyphenyl)-2-propanone used
as an amino acceptor and (R)-1-phenylethylamine used as an amino
donor, each having a concentration of 3%, are reacted by using
Arthrobacter species KNK168 (FERM BP-5228) for about 20 hours, 75%
or more of 1-(3,4-dimethoxyphenyl)- -2-propanone can be converted
to (R)-1-(3,4-dimethoxyphenyl)-2-aminopropan- e with an optical
purity of 99%ee or more by the amino group transfer action.
[0057] The quantitative analysis of the optically active amino
compound obtained as the reaction product can be carried out by
means of high-performance liquid chromatography. For example, the
quantitative analysis can be carried out by separating the reaction
mixture by using a reversed phase column (Cosmosil 5C.sub.18-AR,
NACALAI TESQUE, INC., and the like) and using 25% acetonitrile as a
moving phase, and the detected absorptions at 210 nm were compared
with the control. In addition, there are several methods for
measuring the optical purity. For example, the analysis of the
optical purity can be carried out by binding the resulting
optically active amino compound to N-carboxy-1-leucine anhydride,
and the like, in order to form a diastereomer, and thereafter
separating and quantitatively analyzing this diastereomer by the
above-mentioned high-performance liquid chromatography method.
[0058] The method for separating the optically active amino
compound, the desired compound, after the reaction can be carried
out by the conventional method in which extraction by an organic
solvent and distillation are combinably used. For example, ethyl
acetate, toluene, and the like can be used as the extraction
solvent. First, the reaction solution is made acidic and the
ketones are removed therefrom by extraction. Thereafter, the
reaction mixture is made alkaline, and separate the amines
including the desired compound by extraction. Further, by further
means of distillation of the extracted fraction, the desired
optically active amino compound can be isolated.
[0059] In the transaminase which can be used in the present
invention, when acted the racemic amino compound represented by the
general formula (V) in the presence of the ketone compound (amino
acceptor) represented by the general formula (III), only its
(R)-form is selectively converted to ketone compounds, because the
transaminase only acts to the (R)-form, so that the (S)-form
remains unchanged, and thus, collected as the amino compound.
Therefore, the (S)-amino compounds represented by the general
formula (VI) can be easily prepared from racemic amino compounds by
using the transaminase of the present invention.
[0060] For example, in a case where racemic forms of
1-(3,4-dimethoxyphenyl)-2-aminopropane or 2-amino-1-methoxypropane
are reacted with pyruvic acid used as an amino acceptor using
Arthrobacter species KNK168 (FERM BP-5228), the (R)-form amino
compound is converted to the ketone form by the amino group
transfer, so that the (S)-form amino compound can be obtained at
about 50% yield in a concentrated state up to a high optical purity
level.
[0061] The present invention will be described in further detail by
means of the working examples, but the scope of the present
invention is by no means limited to these examples.
EXAMPLE 1
[0062] Each 2 g of soil samples collected at various places 20 in
this country was suspended in 5 ml of physiological saline. 0.2 ml
of the supernatant thereof was added to 4 ml of an S medium (2 g/L
KH.sub.2PO.sub.4, 2 g/L K.sub.2HPO.sub.4, 0.3 g/L
MgSO.sub.4.7H.sub.2O, 5 g/L glycerol, 3 g/L NaCl, 1 g/L yeast
extract powder, 0.004 g/L FeSO.sub.4.7H.sub.2O, 0.0005 g/L
ZnSO.sub.4.7H.sub.2O, 0.0005 g/L MnCl.sub.2. 4H.sub.2O (pH 7.5);
and 2-oxoglutaric acid or pyruvic acid, filtrated by a
microorganisms exclusion filter after a treatment of autoclaving,
and (R)-1-(3,4-dimethoxyphenyl)-2-aminopropane being added so as to
give final concentrations of 1.5 g/L and 1.0 g/L, respectively).
The resulting culture was subjected to an enrichment culture at
30.degree. C. for 3 to 7 days. Each 0.2 ml of the culture in which
the bacteria were grown was spread on an S-medium plate containing
1.5% of agar, and the colonies were grown by culturing at
30.degree. C. The grown colonies were subjected to shaking culture
in the S-medium. After harvesting the cells, they were suspended in
0.25 ml of a reaction mixture containing a 0.1 M carbonic acid
buffer (pH 8.5), 50 mM pyruvic acid and 30 mM
(R)-1-(3,4-dimethoxyphenyl)-2-aminopropane, and the components were
reacted with stirring at 30.degree. C. for 24 hours.
[0063] The resulting reaction mixture was separated by thin-layer
chromatography (Kieselgel 60F254 (Merck); developing solvent being
diethyl ether:methanol:aqueous ammonia solution (27%)=50:50:2). The
decrease of the substrates was detected by ninhydrin, and the
formation of the resulting product,
1-(3,4-dimethoxyphenyl)-2-propanone, was detected by 0.4% of
2,4-dihydrophenyl hydrazine. With regard to the strains in which
the formation of the products was confirmed, the presence or
absence of the (R)-form-specific amino group transfer action
activity was examined by carrying out its reverse reaction.
Specifically, the cultured cells were added to a reaction mixture
containing 0.6% of 1-(3,4-dimethoxyphenyl)-2-propanone and 0.6% of
(R)-1-phenylethylamine, and the components were reacted at
30.degree. C. for 2 days. As a result, the Arthrobacter species
KNK168 strain was found to have the (R)-form-specific amino group
transfer activity.
EXAMPLE 2
[0064] When culturing the Arthrobacter species KNK168 strain by the
method described in Example 1, (RS)-1-methylpropylamine,
(RS)-1-methylbutylamine- , (RS)-3-amino-2,2-dimethylbutane,
(RS)-2-amino-1-butanol, or (RS)-1-phenylethylamine was added to the
medium in place of (R)-1-(3,4-dimethoxyphenyl)-2-aminopropane
((R)-DMA). The cells were separated from the medium, and the
reaction was carried out at 30.degree. C. for 20 hours using the
above cells in a rel .tion mixture containing 1% of
1-(3,4-dimethoxyphenyl)-2-propanone and 1% of
(RS)-1-methylpropylamine. As a result, an increase of the
reactivity was observed as shown in Table 4.
5 TABLE 4 Inducer (R)-DMA Formed (%) (RS)-1-Methylpropylamine 22.3
(RS)-1-Methylbutylamine 13.0 (RS)-1-Phenylethylamine 8.0
(RS)-3-Amino-2,2-dimethylbutane 18.6 (RS)-2-Amino-1-butanol 12.9
(R)-DMA 0.9 No Addition 0.3
EXAMPLE 3
[0065] The Arthrobacter species KNK168 strain was inoculated to an
R medium (5 g/L KH.sub.2PO.sub.4, 5 g/L K.sub.2HPO.sub.4, 3 g/L
NaCl, 1 g/L MgSO.sub.4.7H.sub.2O, 0.005 g/L FeSO.sub.4.7H.sub.2O,
0.001 g/L ZnSO.sub.4.7H.sub.2O, 0.001 g/L MnCl.sub.2.4H.sub.2O (pH
7.5), 15 g/L glycerol, 2 g/L yeast extract powder, 8 g/L PRO-EX
(BANSYU CHOMIRYO CO., LTD.); pyruvic acid, filtrated by a
microorganisms exclusion filter after a treatment of autoclaving,
and (RS)-1-methylpropylamine being added so as to give final
concentrations of 1.6 g/L and 2.0 g/L, respectively, and being
adjusted to pH 7.2), and then subjected to shaking culture at
30.degree. C. for 24 hours. The cultured cells were harvested by
centrifugation, and the harvested cells were suspended in a
reaction mixture (pH 8.5) containing 1% of
1-(3,4-dimethoxyphenyl)-2-propanone, 0.1% of Triton X-100, and 1%
of an amino donor shown in Table 2. The components were reacted at
35.degree. C. for 20 hours. As shown in Table 5, it was found that
(RS)-1-phenylethylamine, (RS)-1-methylbutylamine,
(RS)-1-methylpropylamine, (RS)-2-aminopentane, and the like,
functioned as amino donors, among which (RS)-1-phenylethylamine was
most highly preferable.
6 TABLE 5 Amino Group Donor (R)-DMA Formed (%)
(RS)-1-Phenylethylamine 61.3 (RS)-1-Methylbutylamine 54.7
(RS)-1-Methylpropylamine 28.2 (RS)-2-Aminopentane 56.9
(RS)-2-Amino-1-propanol 9.6 (RS)-2-Amino-2-phenylethanol 48.4
(RS)-2-Amino-3-phenylpropanol 22.7 (RS)-2-Amino-4-phenylbutane 27.6
(RS)-1-Amino-2-phenylpropane 1.8 (RS)-1-Phenylmethylamine 3.1
(RS)-3-Aminoheptane 7.4 (RS)-l-Naphthylethylamine 34.2 D-Alanine
methyl ester 3.5 D-Alanine ethyl ester 4.2 D-Alanine 3.0
EXAMPLE 4
[0066] The cells obtained by culturing the Arthrobacter species
KNK168 strain in the R medium were suspended in a reaction mixture
containing 2% of 1-(3,4-dimethoxyphenyl)-2-propanone and 4% of
(RS)-1-phenylethylamine. The surfactants and fatty acids shown in
Table 6 were added thereto, and the mixture was reacted at
30.degree. C. at a pH of 8.5 for 20 hours. As a result, it was
found that the rate of reaction was increased by addition of sodium
dodecyl sulfate, linoleic acid, oleic acid, or the like to the
reaction mixture.
7 TABLE 6 Additives (%) (R)-DMA Formed (%) Triton X-100 0.1 31.6
0.5 34.0 Sodium dodecyl sulfate 0.1 45.3 0.5 42.2 1.0 46.3 2.0 50.1
3.0 51.9 4.0 53.1 Cetyl trimethyl ammonium 0.1 29.1 bromide (CTAB)
Linoleic Acid 2.9 45.1 Oleic Acid 2.9 44.9 (No Addition) 28.1
EXAMPLE 5
[0067] Using the cells obtained by culturing the Arthrobacter
species KNK168 strain in the R medium, the components were reacted
at 35.degree. C. for 20 hours, while making the pH of a reaction
mixture variable from 7.5 to 9.5, the reaction mixture containing
2% of 1-(3,4-dimethoxyphenyl)- -2-propanone, 1.4% of
(RS)-1-phenylethylamine, and 0.1% of sodium dodecyl sulfate. As a
result, the rate of reaction at a pH of 8.5 to 9.0 was favorable as
shown in Table 7.
8 TABLE 7 Reaction pH (R)-DMA Formed (%) 7.5 16.8 8.0 30.8 8.5 40.0
9.0 41.6 9.5 36.1
EXAMPLE 6
[0068] 30 ml of overnight preculture of the Arthrobacter species
KNK168 strain was inoculated to 1.5 liter of a J medium (5 g/L
KH.sub.2PO.sub.4, 5 g/L K.sub.2HPO.sub.4, 1 g/L NaCl, 1 g/L
MgSO.sub.4.7H.sub.2O, 0.005 g/L FeSO.sub.4.7H.sub.2O, 0.001 g/L
ZnSO.sub.4.7H.sub.2O, 0.001 g/L MnCl.sub.2.4H.sub.2O, 0.0005 g/L
CuSO.sub.4.5H.sub.2O (pH 7.5), 40 g/L glycerol, 3 g/L yeast extract
powder, 20 g/L PRO-EX (BANSYU CHOMIRYO CO., LTD.), a pH being
adjusted to 7.5) in a 2 liter-mini jar, and the bacterium was
cultured at 30.degree. C. at 0.5 vvm at 450 rpm for 43 hours while
keeping and adjusting the culture to a pH of 7.5. Incidentally,
from the start of culturing, (RS)-1-methylpropylamine filtered by a
microorganisms exclusion filter was added so as to have a final
concentration of 4 g/L after 14 hours. The final cell concentration
(OD.sub.610) was about 29 at the end of the culture, and the
transaminase activity was 0.3 unit per the culture at that time.
Incidentally, the unit of enzymatic activity means the intensity of
the enzymatic activity which converts a 1 .mu.M substrate of
1-(3,4-dimethoxyphenyl)-2-propanone to
(R)-1-(3,4-dimethoxyphenyl)-2-aminopropane at 30.degree. C. in 1
minute.
[0069] The cells harvested from 1.5 liter of the culture were
suspended in a reaction mixture (pH 8.5) containing 45 g of
1-(3,4-dimethoxyphenyl)-2-- propanone, 28.3 g of
(R)-1-phenylethylamine, and 65.0 g of oleic acid. The components
were reacted at 30.degree. C. for 39 hours. As a result, 81.6% of
the substrate was converted to
(R)-1-(3,4-dimethoxyphenyl)-2-aminoprop- ane. After the pH of the
reaction mixture to 2.0 with hydrochloric acid was adjusted, the
reaction mixture was extracted with toluene to separate ketones by
migrating the ketones to the organic layer. After the pH of the
aqueous layer was adjusted to 12 with sodium hydroxide, the
reaction mixture was extracted with toluene again, and 32.9 g of
(R)-1-(3,4-dimethoxyphenyl)-2-aminopropane was contained x$ in the
organic layer. The separation of the desired compound from the
extract was carried out by distillation. As a result, 30.3 g of
(R)-1-(3,4-dimethoxyphenyl)-2-aminopropane was contained in the
main distillated fraction. The total yield obtained from the
sequential procedures was about 68.7%, and the optical purity was
about 99.6%ee ((R)-form).
EXAMPLE 7
[0070] The Arthrobacter species KNK168 strain was subjected to
shaking culture in a Sakaguchi flask containing 400 ml of the R
medium at 30.degree. C. for 30 hours. After harvesting the cells,
the cells were suspended in 40 ml of a 20 mM potassium phosphate
buffer (pH 6.8) containing 0.1% of 2-mercaptoethanol. After the
cells were disrupted by ultrasonic sound, the precipitates were
removed by centrifugation to obtain 34 ml of a cell-free extract
obtained as a supernatant.
[0071] Using 10 ml of the cell-free extract, the components were
reacted in 100 ml of a reaction mixture comprising a 0.1 M
Tris-hydrochloric acid buffer (pH 8.5) containing 0.5 g of
1-(3,4-dimethoxyphenyl)-2-propanone and 0.34 g of
(R)-1-phenylethylamine at 30.degree. C. for 24 hours. As a result,
0.36 g of (R)-(3,4-dimethoxyphenyl)-2-aminopropane was formed.
EXAMPLE 8
[0072] The Arthrobacter species KNK168 strain was cultured in the
same manner as in Example 7, and the cells were separated from 1 ml
of the culture. The cells were suspended in 1 ml of a reaction
mixture containing a 0.1 M Tris-hydrochloric acid buffer (pH 8.5),
40 mg of racemic 1-(3,4-dimethoxyphenyl)-2-aminopropane, 20 mg of
pyruvic acid, and 2% of sodium dodecyl sulfate. The components were
reacted at 30.degree. C. for 48 hours with stirring. As a result of
the analysis of the mixture after the reaction, 20.5 mg of
1-(3,4-dimethoxyphenyl)-2-amin- opropane remained, and its optical
purity was 96%ee ((S)-form).
EXAMPLE 9
[0073] The Arthrobacter species KNK168 strain was cultured in 3.5
liter of the J medium at 30.degree. C. for 43 hours using a 5 liter
mini-jar in the same manner as in Example 6. The cells were
harvested by centrifugation, and the harvested cells were suspended
in 1 liter of 20 mM potassium phosphate buffer (pH 6.8) containing
0.01% of 2-mercaptoethanol. The cells were disrupted by Dynomill
(Trade Mark, Switzerland), and 810 ml of the supernatant was
separated by centrifugation. Protamine sulfate was added to this
supernatant so as to give a concentration of 50 mg/ml, and nucleic
acids were removed. Ammonium sulfate was added thereto so as to
have a concentration of 30% to saturation, and the precipitated
protein was removed. Thereafter, ammonium sulfate was added again
so as to have a concentration of 60% to saturation, and the
precipitated protein was separated. This protein was dissolved in
the above-mentioned buffer, and it was dialyzed against the same
buffer. After the composition of the buffer was adjusted to 20%
(v/v) of glycerol, 0.3 M of NaCl, and 20 .mu.M of pyridoxal
phosphate, a DEAE-Sepharose, Fast-Flow (Pharmacia) column (.phi.
4.4 cm.times.20 cm) was charged with the buffer, which was eluted
with an NaCl linear concentration gradient of from 0.3 to 0.5 M.
After this active fraction was collected and dialyzed, ammonium
sulfate was added thereto so as to give a concentration of 0.2 M. A
Phenylsepharose (Pharmacia) column (.phi. 2.2 cm.times.17 cm) was
charged with the active fraction, which was eluted with an ammonium
sulfate linear concentration gradient of from 0.2 to 0 M. After the
concentration of the ammonium sulfate in the active fraction was
adjusted to 0.6 M, Butylsepharose (Pharmacia) column (.cent. 2.2
cm.times.17 cm) was charged with the active fraction, which was
eluted with an ammonium sulfate linear concentration gradient of
from 0.6 to 0.2 M. The active fraction was collected and
concentrated by ultrafiltration. Thereafter, the concentrated
fraction was subjected to electrophoresis with SDS-polyacrylamide
gel, and as a result, substantially a single band was formed at the
position corresponding to a molecular weight of about 37,000.
EXAMPLE 10
[0074] The amino acid sequence in the neighborhood of the amino
terminal of the purified enzyme protein obtained in Example 9 was
analyzed by a Gas-Phase Protein Sequencer (470A, Applied
Biosystems, Inc.). As a result, it was found that the above enzyme
had an amino acid sequence of SEQ ID NO:1 in Sequence Listing:
[0075] Glu-Ile-Val-Tyr-Thr-His-Asp-Thr-Gly-Leu-Asp-Tyr in the
neighborhood of the amino terminal end.
EXAMPLE 11
[0076] Using the purified enzyme obtained in Example 9,
reactivities to various amino compounds when using pyruvic acid as
an amino acceptor were evaluated. 150 .mu.l of a solution of the
purified enzyme diluted ten-folds was added to 150 .mu.l of a
substrate solution (0.1 M potassium phosphate buffer (pH 8.3), 40
mM pyruvic acid, 0.1 mM pyridoxal phosphate, and 40 mM various
amino compounds). The components were reacted at 30.degree. C. for
1 hour. The reaction tube was transferred into boiling water to
stop the reaction, and, thereafter, a part of the reaction mixture
was diluted five-folds. Ten .mu.l of 0.1 M boric acid buffer (pH
8.0) and 20 .mu.l of an ethanol solution of 80 mM of
4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) were added to 10
.mu.l of the above-mentioned diluted reaction mixture. The
components were reacted at 60.degree. C. for 1 minute, and,
thereafter, the reaction mixture was ice-cooled, and 460 .mu.l of 5
mM HCl was added thereto. This reaction mixture was analyzed by
high-performance liquid chromatography using fine pack C18-5 column
and using a 0.1 M potassium phosphate buffer (pH 6.5) and
CH.sub.3CN (90:10) as an eluent solution to detect formation of
alanine-NBD using excitation wavelength of 470 nm and using
detection wave of 530 mm. The results thereof are shown in Table 8
in terms of relative activity in the case of using
R-1-phenylethylalanine as a substrate.
[0077] It is apparent from the Table 8 that (S)-2-phenylglycinol,
3-aminophenylbutane, and the like, showed favorable
reactivities.
9 TABLE 8 Relative Compound Activity (%) (R)-1-Phenylethylamine 100
(R)-3,4-Dimethoxyaminopropane 30 1-Methylheptylamine 47
2-Heptylamine 37 1-Methylpropylamine 1.2 Benzylamine 0.8
(S)-2-Phenylglycinol 180 3-Aminophenylbutane 84 L-Phenylalaninol
0.8
EXAMPLE 12
[0078] Using the purified enzyme obtained in Example 9,
reactivities to various carbonyl compounds using
(R)-1-phenylethylamine as an amino donor were evaluated. 50 .mu.l
of a solution of the purified enzyme was added to 200 .mu.l of a
substrate solution (0.1 M Tris-HCl buffer (pH 8.5), 25 mM
(R)-1-phenylethylamine, 0.1 mM pyridoxal phosphate, and 25 mM
various carbonyl compounds), and the components were reacted at
30.degree. C. for 1 to 3 hours. The reaction tube was transferred
into boiling water to stop the reaction, and, thereafter, the
reaction mixture was diluted five-folds with methanol. This diluted
solution was analyzed by high-performance liquid chromatography
using fine pack C18-5 column and using methanol/water (20:80) as an
eluent solution to quantitatively analyze acetophenone formed by
the reaction. The results thereof are shown in Table 9 in terms of
relative activity in the case of using pyruvic acid as a substrate.
It is apparent from the Table 9 that oxalacetic acid,
phenoxy-2-propanone, and the like also showed favorable
reactivities.
10 TABLE 9 Relative Compound Activity (%) Pyruvic acid 100
Oxalacetic acid 87 Glyoxylic acid 4 2-Ketobutyric acid 7 Ethyl
pyruvate 27 Ethyl acetoacetate 4 2-Decane 3 4-Methoxyphenyl acetone
14 1-(3,4-Dimethoxyphenyl)-2-propanone 7 Benzyl acetone 3
4-(4-Methoxyphenyl)-2-butanone 7 1-Phenyl-2-butanone 1 Phenyl
acetaldehyde 13 Ethyl benzoyl acetate 2 Phenoxy-2-propanone 83
Diacetyl 10 1-Methoxy-2-propanone 16 1-Tetralone 1 2-Acetylpyridine
19 3-Acetylpyridine 8 4-Acetylpyridine 27 3-Acetoxypyridine 1
2-Acetylpyrazine 17 2-Acetylfuran 2 2-Acetylthiophene 1
2-Acetylthiazole 10
EXAMPLE 13
[0079] The Arthrobacter species KNK168 strain was cultured in the
same manner as in Example 7, and the cells were separated from 1 ml
of the culture. The cells were suspended in 1 ml of a reaction
mixture containing a 0.1 M Tris-hydrochloric acid buffer (pH 8.5),
100 mM (R)-1-phenylethylamine, 100 mM methoxy-2-propanone, and 0.1%
of sodium dodecyl sulfate. The components were reacted at
30.degree. C. for 20 hours with stirring. As a result of the
analysis of the reaction mixture, methoxy-2-propanone, the
substrate, almost disappeared and converted to
2-amino-1-methoxypropane, and its optical purity was 99.4%ee
((R)-form).
EXAMPLE 14
[0080] The Arthrobacter species KNK168 strain was cultured in the
same manner as in Example 13, and the cells were separated from 1
ml of the culture. The cells were suspended in 1 ml of a reaction
mixture containing a 0.1 M Tris-hydrochloric acid buffer (pH 8.5),
50 mg of racemic 2-amino-1-methoxypropane, 37.5 mg of pyruvic acid,
and 0.1% of sodium dodecyl sulfate. The components were reacted at
30.degree. C. for 24 hours with stirring. As a result of the
analysis after the reaction, 23.4 mg of 2-amino-1-methoxypropane
remained, and its optical purity was 95%ee ((S)-form).
EXAMPLE 15
[0081] By carrying out the same reaction as in Example 14 using 200
mM racemic 2-amino-1-methoxypropane, and 150 mM n-butylaldehyde or
propionaldehyde, the components were reacted at 30.degree. C. for
20 hours. As a result of the analysis thereof, the (S)-form of
2-amino-1-methoxypropane was concentrated, and the ratio of the
(S)-form was 76% in a case of using n-butylaldehyde, and 58% in a
case of using propionaldehyde.
EXAMPLE 16
[0082] The influence of pH on the activity was evaluated by using
the purified enzyme obtained in Example 9. 0.1 ml of an enzyme
solution which was diluted suitably beforehand was added to 0.9 ml
of a solution containing 20 .mu.mol of
1-(3,4-dimethoxyphenyl)-2-propanone, 20 .mu.mol of
(R)-1-phenylethylamine, and 1 .mu.mol of pyridoxal phosphate, of
which the pH was adjusted using the buffer described as follows.
The components were reacted at 30.degree. C. for 1 hour. The used
buffers were an acetic acid buffer (CH.sub.3COONa being used as the
abbreviation; pH 4 to 6), a potassium phosphate buffer (KPB; pH 5
to 8), and a Tris-hydrochloric acid buffer (Tris-HCl; pH 7 to 10),
all of these buffers having final concentrations of 0.1 M. The
formed (R)-DMA in the reaction mixture after the reaction was
quantitatively analyzed by high-performance liquid chromatography.
The relative activity at each pH, relative to the activity at pH
8.5 as being 100%, is shown in FIG. 1. It was found that the
optimum pH of this enzyme is in the neighborhood of pH 8.5 (pH 7.5
to 9.0).
EXAMPLE 17
[0083] The influence of the pH on the stability of the enzyme was
evaluated by using the purified enzyme obtained in Example 9. After
the pH of the enzyme solution was adjusted by HCl or NaOH, the
reaction mixture was incubated at 20.degree. C. for 23 hours.
Thereafter, in the same manner as in Example 16, the components
were reacted at pH 8.5, and the formed (R)-DMA was quantitatively
analyzed. The results in which the relative activities of the
treated samples at each pH, relative to the activity of the
untreated enzyme solution as being 100%, are shown in FIG. 2. It
was found that this enzyme was most stable at a pH in the
neighborhood of 7.
EXAMPLE 18
[0084] The Arthrobacter species KNK168 strain was subjected to
shaking culture in the same manner as in Example 6 using
(RS)-1-methylpropylamine as an enzyme inducer. The cells harvested
from one liter of the culture were suspended in one liter of a 0.1
M Tris-hydrochloric acid buffer (pH 8.5), and 30 g of
1-(3-trifluoromethylphenyl)-2-propanone, 18 g of
(R)-1-phenylethylamine, and 44 g of oleic acid were added thereto.
The components were reacted at 30.degree. C. for 40 hours with
stirring. After the reaction, the resulting mixture was subjected
to extraction and distillation in the same manner as in Example 6,
to give 19.5 g of (R)-1-(3-trifluoromethylphenyl)-2-aminopropane.
The optical purity was 100%ee, and the yield was 65%.
[0085] Industrial Applicability
[0086] According to the present invention, it is made possible to
easily prepare at a high yield the optically active (R)-amino
compounds and the like having an aryl group and the like at their
1-position, which have been conventionally difficult to
prepare.
[0087] Identification Of Deposited Microorganism
[0088] (1) Name and Address of Depository Organization The Ministry
of International Trade and Industry, National Institute of
Bioscience and Human-Technology, Agency of Industrial Science and
Technology
[0089] 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan (zip
code 305)
[0090] (2) Date of Deposit
[0091] Sep. 8, 1995 (date of original deposit)
[0092] (3) Accession Number
[0093] FERM BP-5228
* * * * *