U.S. patent application number 13/093610 was filed with the patent office on 2013-06-13 for novel carbonyl reductase, gene therefor and use thereof.
This patent application is currently assigned to KANEKA CORPORATION. The applicant listed for this patent is Noriyuki KIZAKI, Daisuke MORIYAMA, Naoaki TAOKA, Makoto UEDA, Yoshihiko YASOHARA. Invention is credited to Noriyuki KIZAKI, Daisuke MORIYAMA, Naoaki TAOKA, Makoto UEDA, Yoshihiko YASOHARA.
Application Number | 20130149769 13/093610 |
Document ID | / |
Family ID | 36227695 |
Filed Date | 2013-06-13 |
United States Patent
Application |
20130149769 |
Kind Code |
A1 |
KIZAKI; Noriyuki ; et
al. |
June 13, 2013 |
NOVEL CARBONYL REDUCTASE, GENE THEREFOR AND USE THEREOF
Abstract
The present invention is to provide a process for efficiently
producing an optically active alcohol including
(R)-3-hydroxy-3-phenylpropanenitrile. One of the features of the
present invention is a polypeptide having an activity of
asymmetrically reducing 3-oxo-3-phenylpropanenitrile isolated from
a microorganism belonging to the genus Candida to product
(R)-3-hydroxy-3-phenylpropanenitrile, DNA encoding the polypeptide
and a transformant of producing the polypeptide. Another feature of
the present invention is a process for producing an optically
active alcohol such as (R)-3-hydroxy-3-phenylpropanenitrile by
reducing a carbonyl compound such as 3-oxo-3-phenylpropanenitrile
by use of the polypeptide or the transformant.
Inventors: |
KIZAKI; Noriyuki; (Hyogo,
JP) ; UEDA; Makoto; (Hyogo, JP) ; MORIYAMA;
Daisuke; (Hyogo, JP) ; TAOKA; Naoaki; (Hyogo,
JP) ; YASOHARA; Yoshihiko; (Hyogo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KIZAKI; Noriyuki
UEDA; Makoto
MORIYAMA; Daisuke
TAOKA; Naoaki
YASOHARA; Yoshihiko |
Hyogo
Hyogo
Hyogo
Hyogo
Hyogo |
|
JP
JP
JP
JP
JP |
|
|
Assignee: |
KANEKA CORPORATION
Osaka
JP
|
Family ID: |
36227695 |
Appl. No.: |
13/093610 |
Filed: |
April 25, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11665065 |
Apr 11, 2007 |
8008461 |
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PCT/JP2005/019269 |
Oct 20, 2005 |
|
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13093610 |
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Current U.S.
Class: |
435/190 |
Current CPC
Class: |
C12P 7/04 20130101; C12P
13/002 20130101; C12P 41/002 20130101; C12N 9/0006 20130101; C12P
7/62 20130101 |
Class at
Publication: |
435/190 |
International
Class: |
C12N 9/00 20060101
C12N009/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 27, 2004 |
JP |
2004-312365 |
Claims
1. An isolated polypeptide selected from the group consisting of:
(a) an isolated polypeptide having an amino acid sequence of SEQ ID
NO: 2, and (b) an isolated polypeptide with at least 90% amino acid
sequence identity to SEQ ID NO: 2, and wherein said polypeptide has
an activity of asymmetrically reducing 3-oxo-3-phenylpropanenitrile
represented by the formula (1) below; ##STR00011## to produce
(R)-3-hydroxy-3-phenylpropanenitrile represented by the formula (2)
below; ##STR00012##
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional application of U.S. patent application
Ser. No. 11/665,065, filed Apr. 11, 2007 (allowed), which is a 371
National Stage Entry of International Application No.
PCT/JP05/19269, filed Oct. 20, 2005, which claims benefit from
Japanese Patent Application No. 2004-312365, filed Oct. 27, 2004,
the contents of each of which are incorporated herein by reference
in their entirety.
TECHNICAL FIELD
[0002] The present invention relates to a polypeptide (carbonyl
reductase) having an activity of asymmetrically reducing
3-oxo-3-phenylpropanenitrile represented by the formula (1)
below:
##STR00001##
[0003] to produce (R)-3-hydroxy-3-phenylpropanenitrile represented
by the formula (2) below:
##STR00002##
[0004] and isolated from a microorganism having said activity; DNA
encoding the polypeptide; a vector containing the DNA; and a
transformant transformed with the vector.
[0005] The present invention also relates to a process for
producing an optically active alcohol, in particular,
(R)-3-hydroxy-3-phenylpropanenitrile represented by the formula (2)
above using the polypeptide or the transformant.
[0006] (R)-3-hydroxy-3-phenylpropanenitrile is a useful compound as
an intermediate of a pharmaceutical product such as a
.beta.-adrenaline receptor blocker.
[0007] The entire disclosure including the specification, claims,
drawings and abstract of Japanese Patent No. 2004-312365 (filed
Oct. 27, 2004) is incorporated herein by references in its
entirety.
BACKGROUND ART
[0008] As a process for producing optically active
3-hydroxy-3-phenylpropanenitrile, there are known 1) a process for
deriving it from an optically active precursor substance (Patent
Documents 1 and 2); 2) a process for optically resolving racemic
3-hydroxy-3-phenylpropanenitrile with a hydrolyzing enzyme such as
lipase (Patent Document 3, Non-Patent Document 2); 3) a process for
condensing benzaldehyde and acetonitrile in the presence of an
asymmetric ligand and a metallic catalyst (Non-patent document 3);
4) a process for reducing an carbonyl group of
3-oxo-3-phenylpropanenitrile in the presence of an asymmetric
ligand or an asymmetric catalyst (Patent Document 4 and Non-Patent
Document 4); and 5) a process for reducing an carbonyl group of
3-oxo-3-phenylpropanenitrile by use of a microorganism (Non-Patent
Documents 5 and 6). [0009] Non-Patent Document 1: Bulletin of the
Korean Chemical Society, 23, 1693 (2002) [0010] Non-Patent Document
2: Advanced Synthesis & Catalysis, 344, 947 (2002) [0011]
Non-Patent Document 3: Organic Letters, 5, 3147 (2003) [0012]
Non-Patent Document 4: Tetrahedron: Asymmetry, 3, 677 (1992) [0013]
Non-Patent Document 5: Tetrahedron: Asymmetry, 11, 3693 (2000)
[0014] Non-Patent Document 6: Organic Letters, 1, 1879 (1999)
[0015] Patent Document 1: US. Patent Application Laid-Open No.
2004/110985 [0016] Patent Document 2: Japanese Patent Application
Laid-Open No. 5-92946 [0017] Patent Document 3: National
Publication of International Patent Application No. 2004-520039
[0018] Patent Document 4: Japanese Patent Application Laid-Open No.
2003-201269
BRIEF SUMMARY OF THE INVENTION
[0019] The processes disclosed in the aforementioned documents
individually have drawbacks. In the process 1), an expensive
precursor substance is required. In the process 2), since a
theoretical yield is 50%, a half of a raw material is wasted. In
the processes 3) and 4), an expensive asymmetric ligand or
asymmetric catalyst is required. In the process 5), a productivity
per reaction volume is low. Any one of these processes is far from
an economic process.
[0020] In view of the aforementioned circumstances, an object of
the present invention is to provide a useful polypeptide in
producing (R)-3-hydroxy-3-phenylpropanenitrile, DNA encoding the
polypeptide, a vector containing the DNA, and a transformant
transformed with the vector.
[0021] Another object of the present invention is to provide
processes for effectively producing various types of optically
active alcohols including (R)-3-hydroxy-3-phenylpropanenitrile, by
use of the polypeptide and the transformant.
[0022] The present inventors isolated a polypeptide having an
activity of asymmetrically reducing 3-oxo-3-phenylpropanenitrile to
produce (R)-3-hydroxy-3-phenylpropanenitrile from a microorganism
having the activity. Furthermore, they isolated DNA encoding the
polypeptide and succeeded in creating a transformant capable of
producing the polypeptide by using the DNA in high yield. Moreover,
they found that various types of useful optically active alcohols
including (R)-3-hydroxy-3-phenylpropanenitrile can be efficiently
produced by use of the polypeptide or the transformant. Based on
these, the present invention was accomplished.
[0023] More specifically, one of the features of the present
invention is a polypeptide having an activity of asymmetrically
reducing 3-oxo-3-phenylpropanenitrile to produce
(R)-3-hydroxy-3-phenylpropanenitrile. Another feature of the
present invention is DNA encoding the polypeptide. Still another
feature of the present invention is a vector containing the DNA. A
further feature of the present invention is a transformant
containing the vector. Still a further feature of the present
invention is a process for producing an optically active alcohol
including (R)-3-hydroxy-3-phenylpropanenitrile, by use of the
polypeptide or the transformant.
[0024] The present invention can provide a practical process for
producing a useful and optically active alcohol including
(R)-3-hydroxy-3-phenylpropanenitrile.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 shows the method and structure of a recombinant
vector pNTCMG1.
DETAILED DESCRIPTION OF THE INVENTION
[0026] The present invention will be more specifically explained
below.
[0027] Gene manipulation such as isolation of DNA, preparation of a
vector, and formation of a transformant can be performed in
accordance with the method described in publications such as
Molecular Cloning 2nd Edition (Cold Spring Harbor Laboratory Press,
1989), unless otherwise specified. Furthermore, the reference
symbol "%" used in the description of the specification refers to %
(w/v), unless otherwise specified.
[0028] 1. Polypeptide and its Source
[0029] The polypeptide of the present invention is one having an
activity of asymmetrically reducing 3-oxo-3-phenylpropanenitrile to
produce (R)-3-hydroxy-3-phenylpropanenitrile. Such a polypeptide
can be isolated from a microorganism having the activity. The
microorganism from which the polypeptide of the present invention
is derived is not particularly limited. For example, yeast
belonging to the genus Candida may be mentioned. Particularly
preferably, Candida magnoliae strain NBRC0661 may be mentioned. The
microorganism is available from an independent organization, the
National Institute of Technology and Evaluation, Biological
Resource Center (NBRC, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba,
292-0818, JAPAN).
[0030] As a medium for culturing a microorganism from which the
polypeptide of the present invention is derived, any liquid
nutrition medium generally used and containing a carbon source,
nitrogen source, inorganic salts, and organic nutrients, etc., can
be used as long as the microorganism can be proliferated in the
medium.
[0031] 2. Isolation of Polypeptide
[0032] Isolation of the polypeptide of the present invention from a
microorganism from which the polypeptide is derived can be
generally performed by using known protein purification methods in
appropriate combination, for example, as described below. First,
the microorganism is cultured in an appropriate medium and
microorganism's cells are collected from the culture solution by
centrifugation or filtration. The obtained cells are disrupted by
an ultrasonic homogenizer or a physical means using glass beads,
and thereafter, cell residues are centrifugally removed to obtain a
cell-free extract. Subsequently, the cell-free extract is subjected
to a process such as salting-out (e.g., precipitation with ammonium
sulfate and precipitation with sodium phosphate, etc.),
precipitation with a solvent (precipitation of a protein fraction
by acetone, ethanol or the like), dialysis, gel filtration
chromatography, ion exchange chromatography, reversed-phase
chromatography, and ultrafiltration, singly or in combination. In
this manner, the polypeptide of the present invention is isolated
from the cell-free extract.
[0033] 3. Reduction Reaction
[0034] The activity of reducing 3-oxo-3-phenylpropanenitrile can be
determined, for example, by adding a 0.5 mM
3-oxo-3-phenylpropanenitrile as a substrate, 0.25 mM coenzyme NADPH
and a crude enzyme to a 100 mM phosphate buffer (pH 6.5) containing
0.33% (v/v) dimethylsulfoxide, allowing the mixture to react at
30.degree. C. for one minute, measuring absorbance of the resultant
reaction solution at a wavelength of 340 nm, and calculating the
activity from the reduction rate of the absorbance.
[0035] The optical purity of 3-hydroxy-3-phenylpropanenitrile
produced in the aforementioned reaction can be measured by
capillary gas chromatography (column: CHIRALDEX G-TA manufactured
by Tokyo Chemical Industry Co., Ltd., .phi. 0.25 mm.times.20 m,
column temperature: 130.degree. C., carrier gas: helium (150 kPa),
detection: FID).
[0036] 4. DNA
[0037] The DNA of the present invention is one encoding the
polypeptide of the present invention mentioned above. Any DNA may
be used as long as it can express the polypeptide in a host cell to
which the DNA is introduced in accordance with a method as
described later. The DNA may optionally contain a non-translation
region. As long as the polypeptide can be obtained, one skilled in
the art can obtain such DNA from a microorganism from which the
polypeptide is derived, in accordance with a known method, for
example, the method set forth below.
[0038] First, the isolated polypeptide of the present invention is
digested with an appropriate endopeptidase. The resultant peptide
fragments are separated by reverse-phase HPLC. Subsequently, the
whole or part of amino acid sequences of these peptide fragments
are determined, for example, by an API492-type protein sequencer
(manufactured by Applied Biosystems).
[0039] Based on the amino acid sequence data thus obtained, a PCR
(Polymerase Chain Reaction) primer is synthesized for amplifying a
part of DNA encoding the polypeptide. Subsequently, the chromosomal
DNA of a microorganism from which the polypeptide is derived is
prepared by a general DNA isolation method, for example, a method
of Vissers et al. (Appl. Microbiol. Biotechnol., 53, 415 (2000)).
PCR is performed using the chromosomal DNA as a template and the
PCR primer described above to amplify a part of the DNA encoding
the polypeptide and the base sequence thereof is determined. The
determination of the base sequence can be performed for example, by
an A31373-type DNA Sequencer (manufactured by Applied
Biosystems).
[0040] When a part of DNA encoding the polypeptide is clarified,
the whole sequence of the DNA can be determined by use of, for
example, i-PCR method (Nucl. Acids Res., 16, 8186 (1988)).
[0041] As an example of DNA of the present invention thus obtained,
mention may be made of DNA containing a base sequence represented
by SEQ ID NO: 1 of the sequence listing. Furthermore, DNA that is
hybridized, under stringent conditions, with the DNA having a base
sequence represented by SEQ ID NO: 1 of the sequence listing and a
complementary base sequence thereof and that is encoding a
polypeptide having an activity of asymmetrically reducing
3-oxo-3-phenylpropanenitrile to produce
(R)-3-hydroxy-3-phenylpropanenitrile may be also included in the
DNA of the present invention.
[0042] DNA that is hybridized, under stringent conditions, with the
DNA having a base sequence represented by
[0043] SEQ ID NO: 1 of the sequence listing and a complementary
base sequence thereof refers to one that specifically forms a
hybrid with the DNA having a base sequence represented by SEQ ID
NO: 1 of the sequence listing and a complementary base sequence
thereof when a colony hybridization method, plaque hybridization
method, Southern hybridization method or the like is performed.
[0044] The stringent conditions herein refer to those in which
hybridization is performed at 65.degree. C. in an aqueous solution
having a composition of 75 mM trisodium citrate, 750 mM sodium
chloride, 0.5% sodium dodecyl sulfate, 0.1% bovine serum albumin,
0.1% polyvinylpyrrolidone and 0.1% Ficoll 400 (manufactured by
Amersham Bioscience), followed by washing with an aqueous solution
having a composition of 15 mM trisodium citrate, 150 mM sodium
chloride, and 0.1% sodium dodecyl sulfate at 60.degree. C.
Preferably, after hybridization is performed in the aforementioned
conditions, washing is performed by use of an aqueous solution
having a composition of 15 mM trisodium citrate, 150 mM sodium
chloride, and 0.1% sodium dodecyl sulfate, at 65.degree. C., and
more preferably, washing is performed with an aqueous solution
containing 1.5 mM trisodium citrate, 15 mM sodium chloride, and
0.1% sodium dodecyl sulfate, at 65.degree. C.
[0045] 5. Polypeptide
[0046] As an example of the polypeptide of the present invention,
mention can be made of a polypeptide consisting of the amino acid
sequence represented by SEQ ID NO: 2 of the sequence listing and
encoded by the base sequence represented by SEQ ID NO: 1 of the
sequence listing.
[0047] Furthermore, the polypeptide having a homology of not less
than a certain value with the polypeptide consisting of the amino
acid sequence represented by SEQ ID NO: 2 of the sequence listing,
and having an activity of asymmetrically reducing
3-oxo-3-phenylpropanenitrile to produce
(R)-3-hydroxy-3-phenylpropanenitrile is functionally equivalent to
the polypeptide and included in the present invention.
[0048] The homology of the sequence herein is expressed, for
example, by an identity value relative to the entire sequence when
two amino acid sequences are analyzed and compared by a homology,
search program FASTA (W.R. Pearson. & D.J. Lipman P.N.A.S.
(1988) 85:2444-2448). As a polypeptide equivalent to that
consisting of the amino acid sequence represented by SEQ ID NO: 2
of the sequence listing, mention may be made of a polypeptide
having a homology of 70% or more to the polypeptide, preferably 80%
or more, and more preferably 90% or more.
[0049] Such a polypeptide can be obtained by, for example, ligating
DNA, which hybridizes, under stringent conditions, with the DNA
having a base sequence represented by SEQ ID NO: 1 of the sequence
listing and a complementary base sequence thereof as previously
described, to an appropriate vector, introducing into an
appropriate host cell, and expressing the vector. Alternatively,
such a polypeptide can be also obtained by processing the
polypeptide consisting of amino acid sequence represented by SEQ ID
NO: 2 of the sequence listing to substitute, insert, delete or add
an amino acid(s) in accordance with known methods described, for
example, in Current Protocols in Molecular Biology (John Wiley and
Sons, Inc., 1989). The number of amino acids to be replaced,
inserted, deleted or added in the aforementioned polypeptide is not
particularly limited as long as the activity of asymmetrically
reducing 3-oxo-3-phenylpropanenitrile to produce
(R)-3-hydroxy-3-phenylpropanenitrile is not significantly
decreased; however, the number of amino acids is 20 amino acids or
less, preferably 5 or less, and further preferably 2 or 1.
[0050] 6. Vector and Transformant
[0051] The vector for use in introducing the DNA of the present
invention into a microbial host and expressing the DNA introduced
in the microbial host is not particularly limited as long as the
gene encoded by the DNA can be expressed in an appropriate
microbial host. Examples of such a vector include a plasmid vector,
phage vector and cosmid vector. Besides these, a shuttle vector
capable of exchanging a gene with another host strain may be
used.
[0052] Such a vector generally contains regulators such as lac UV5
promoter, trp promoter, trc promoter, tac promoter, lpp promoter,
tufB promoter, recA promoter, and pL promoter and can be suitably
used as an expression vector containing an expression unit operably
linked with the DNA of the present invention. For example, pUCNT
(U.S. Pat. No. 6,083,752) can be suitably used.
[0053] The term "regulatory element" used in the specification
refers to a base sequence having a functional promoter and any of
transcription elements (such as an enhancer, CCAAT box, TATA box,
and SPI site).
[0054] The term "operably linked" used in the specification means
that various regulatory elements such as a promoter and an enhancer
for regulating the expression of a gene are ligated to the gene in
a functional state in a host cell. It is known to one skilled in
the art that the type and kind of regulatory element may vary
depending upon the host.
[0055] Examples of the expression vector of the present invention
include pNTCM (described later) having the DNA represented by SEQ
ID NO: 1 introduced in pUCNT.
[0056] As a host cell to which a vector containing the DNA of the
present invention is introduced, mention may be made of bacterial
cells, yeasts, fungi, vegetable cells and animal cells. In view of
introduction and expression efficiencies, bacterial cells, in
particular, Escherichia coli, is preferable. The vector containing
the DNA of the present invention can be introduced into a host cell
by a known method. When E. coli is used as a host cell, the vector
can be introduced into the host cell by using, for example, a
commercially available E. coli HB101 competent cell (manufactured
by Takara Bio Inc.).
[0057] As an example of a transformant according to the present
invention, mention may be made of E. coli HB101(pNTCM) FERM
BP-10418 (described later). The transformant has been deposited as
of Sep. 26, 2005 under the aforementioned accession number with an
independent organization, the International Patent Organism
Depositary of the National Institute of Advanced Industrial Science
and Technology (located at Tsukuba Central 6, 1-1-1 Higashi,
Tsukuba, Ibaraki, 305-8566, Japan). Note that the original national
deposition date of Oct. 23, 2003 for the strain has been
transferred to international deposition based on the Budapest
Treaty.
[0058] 7. Polypeptide Having a Coenzyme Regeneratidn Activity
[0059] When the polypeptide of the present invention is reacted in
contact with a compound having a carbonyl group and a coenzyme such
as NADPH, it asymmetrically reduces the compound having a carbonyl
group to produce an optically active alcohol. At this time, as the
reaction proceeds, a coenzyme such as NADPH is converted into an
oxidation type. However, when the reaction of the polypeptide of
the present invention is performed in the presence of a polypeptide
having an ability (hereinafter referred to as "coenzyme
regeneration ability") to convert the oxidation-type coenzyme into
a reduction type and the compound serves as a substrate for the
polypeptide, the amount of an expensive coenzyme can be greatly
reduced. Examples of the polypeptide having a coenzyme regeneration
ability include hydrogenase, formic acid dehydrogenase, alcohol
dehydrogenase, aldehyde dehydrogenase, glucose-6-phosphate
dehydrogenase and glucose dehydrogenase. Preferably, glucose
dehydrogenase may be used.
[0060] 8. Use of Transformant
[0061] When a transformant containing DNA encoding the polypeptide
is used in place of the polypeptide of the present invention, an
optically active alcohol can be also produced. Furthermore, even if
a transformant containing both DNA encoding the polypeptide of the
present invention and DNA encoding the polypeptide having a
coenzyme regeneration ability is used, an optically active alcohol
can be also produced. In particular, when a transformant containing
both DNA encoding the polypeptide of the present invention and DNA
encoding the polypeptide having a coenzyme regeneration ability is
used, an optically active alcohol can be produced more effectively
because it is not necessary to separately prepare/add an enzyme for
regenerating the coenzyme.
[0062] Note that, with respect to a transformant containing DNA
encoding the polypeptide of the present invention or a transformant
containing both DNA encoding the polypeptide of the present
invention and DNA encoding the polypeptide having a coenzyme
regeneration ability, not only cultured bacterial cells of the
transformant but also a processed product of the transformant can
be used to produce an optically active alcohol. The "processed
product of the transformant" refers to cells treated with a
surfactant and an organic solvent, dried cells, disrupted cells,
crude cell-extract or a mixture thereof. Besides these, it refers
to immobilized cells fixed by means of a known means such as a
crosslinking method, physical adsorption method and entrapment
method.
[0063] The transformant containing both DNA encoding the
polypeptide of the present invention and DNA encoding the
polypeptide having a coenzyme regeneration ability can be obtained
by introducing both DNA encoding the polypeptide of the present
invention and DNA encoding the polypeptide having a coenzyme
regeneration ability into a single vector and inserting the vector
to a host cell. Or it may be also obtained by introducing these two
types of DNA fragments into two different types of vectors of a
heterothallism group, respectively, and inserting these two types
of vectors to a single host cell.
[0064] As an example of a vector into which both DNA encoding the
polypeptide of the present invention and DNA encoding a polypeptide
having a coenzyme regeneration ability are introduced, mention may
be made of pNTCMG1 (described later), which is the aforementioned
expression vector pNTCM having a glucose dehydrogenase gene derived
from Bacillus megaterium introduced therein. Furthermore, as an
example of a transformant containing both DNA encoding the
polypeptide of the present invention and DNA encoding a polypeptide
having a coenzyme regeneration ability, mention may be made of E.
coli HB101(pNTCMG1) (described later) obtained by transforming E.
coli HB101 with the vector.
[0065] Culturing a transformant containing DNA encoding the
polypeptide of the present invention and a transformant containing
both DNA encoding the polypeptide of the present invention and DNA
encoding the polypeptide having a coenzyme regeneration ability can
be performed in a liquid nutrition medium generally used and
containing a carbon source, nitrogen source, inorganic salts and
organic nutrients, etc., as long as they are proliferated.
[0066] The activity of the polypeptide having a coenzyme
regeneration activity in a transformant can be measured by a
conventional method. For example, the activity of glucose
dehydrogenase can be obtained by adding 100 mM glucose, 2 mM
coenzyme NADP or NAD and an enzyme to a 1M tris-hydrochloric acid
buffer (pH 8.0) and allowing the reaction mixture to react at
25.degree. C. for one minute, measuring absorbance at a wavelength
of 340 nm, and calculating an increasing rate of the
absorbance.
[0067] 9. Production of Optically Active Alcohol
[0068] Production of an optically active alcohol using either the
polypeptide of the present invention or a transformant containing
DNA encoding the polypeptide can be performed by adding a compound
containing a carbonyl group serving as a substrate, a coenzyme such
as NADPH and the polypeptide of the present invention or the
transformant containing DNA encoding the polypeptide to an
appropriate solvent, and stirring the mixture while adjusting the
pH.
[0069] On the other hand, when a reaction is performed using the
polypeptide of the present invention in combination with a
polypeptide having a coenzyme regeneration ability, the polypeptide
having a coenzyme regeneration ability (e.g., glucose
dehydrogenase) and a compound serving as a substrate thereof (e.g.,
glucose) are further added to the reaction composition mentioned
above. Note that, also in the latter case, when the transformant
containing both DNA encoding the polypeptide of the present
invention and DNA encoding the polypeptide (e.g., glucose
dehydrogenase) having a coenzyme regeneration ability or a
processed product thereof is used, it is not necessary to
separately add the polypeptide (e.g., glucose dehydrogenase) having
a coenzyme regeneration ability.
[0070] The reaction may be performed in an aqueous solvent or in a
mixture of an aqueous solvent and an organic solvent. Examples of
the organic solvent include toluene, ethyl acetate, n-butyl
acetate, hexane, isopropanol, diisopropyl ether, methanol, acetone,
and dimethylsulfoxide. The reaction may be performed at a
temperature of 10.degree. C. to 70.degree. C. and the pH of the
reaction solution is maintained at 4 to 10. The reaction is
performed in a batch system or a continuous system. In the case of
the batch system, a reaction substrate is added in an initial
concentration of 0.1% to 70% (w/v).
[0071] 10. Substrate and Product
[0072] Examples of the compound having a carbonyl group serving as
a substrate, include 3-oxo-3-phenylpropanenitrile represented by,
for example, the formula (1) below:
##STR00003##
[0073] ethyl 4-chloro-3-oxobutyrate represented by the formula (3)
below:
##STR00004##
[0074] 2-chloro-1-(3'-chlorophenyl)ethanone represented by the
formula (5) below:
##STR00005##
, and
[0075] tert-butyl (S)-6-benzoyloxy-5-hydroxy-3-oxohexanoate
represented by the formula (7) below:
##STR00006##
However, the compound containing a carbonyl group is not
particularly limited as long as it can be reduced in the
aforementioned reaction conditions and converted into an optically
active alcohol.
[0076] In the aforementioned reaction conditions, when
3-oxo-3-phenylpropanenitrile represented by the formula (1) above
is used as a substrate, (R)-3-hydroxy-3-phenylpropenenitrile
represented by the formula (2) below:
##STR00007##
can be obtained.
[0077] When ethyl 4-chloro-3-oxobutyrate represented by the formula
(3) above is used as a substrate, ethyl
(S)-4-chloro-3-hydroxybutyrate represented by the formula (4)
below:
##STR00008##
can be obtained.
[0078] When 2-chloro-1-(3'-chlorophenyl)ethanone represented by the
formula (5) above is used as a substrate,
(R)-2-chloro-1-(3'-chlorophenyl)ethanol represented by the formula
(6) below:
##STR00009##
can be obtained.
[0079] Furthermore, when tert-butyl
(S)-6-benzoyloxy-5-hydroxy-3-oxohexanoate represented by the
formula (7) above is used as a substrate, tert-butyl
(3R,5S)-6-benzoyloxy-3,5-dihydroxyhexanoate represented by the
formula (8) below:
##STR00010##
can be obtained.
[0080] 11. Purification and Analysis
[0081] An optically active alcohol produced by the reaction can be
purified by a conventional method, for example, by extracting a
reaction solution containing an optically active alcohol produced
by the reaction with an organic solvent such as ethyl acetate and
toluene, removing the organic solvent by distillation under reduced
pressure, and subjecting the resultant mixture to distillation,
recrystallization or chromatographic process.
[0082] The amounts of 3-oxo-3-phenylpropanenitrile and
(R)-3-hydroxy-3-phenylpropanenitrile and the optical purity of
(R)-3-hydroxy-3-phenylpropanenitrile can be obtained by capillary
gas chromatography (column: CHIRALDEX G-TA (IDO, 25 mm.times.20 m:
manufactured by Tokyo Chemical Industry Co., Ltd.), column
temperature: 130.degree. C., carrier gas: helium (150 kPa).
detection: FID).
[0083] The amounts of ethyl 4-chloro-3-oxobutyrate and ethyl
(S)-4-chloro-3-hydroxybutyrate can be obtained by gas
chromatography (column: PEG-20M, Chromosorb WAW DMCS 10% 80/100
mesh (ID 3 mm.times.1 m; manufactured by G-L Sciences), column
temperature: 150.degree. C., carrier gas: nitrogen, detection:
FID). Furthermore, the optical purity of ethyl
(S)-4-chloro-3-hydroxybutyrate is measured by high performance
liquid chromatography (column: Chiralcel OB (ID 4.6 mmx 250 mm;
manufactured by Daicel Chemical Industries Ltd.), eluent:
n-hexane/isopropanol=9/1, flow rate: 0.8 ml/min, detection: 215 nm,
column temperature: room temperature).
[0084] The amounts of 2-chloro-1-(3'-chlorophenyl)ethanone and
(R)-2-chloro-1-(3'-chlorophenyl)ethanol can be obtained by high
performance liquid chromatography (column: YMC-Pack ODS A-303 (ID
4.6 mm.times.250 mm), manufactured by YMC, eluent:
water/acetonitrile=1/1, flow rate: 1 ml/min, detection: 210 nm,
column temperature: room temperature). Furthermore, the optical
purity of (R)-2-chloro-1-(3'-chlorophenyl)ethanol can be measured
by high performance liquid chromatography (column: Chiralcel OJ (ID
4.6 mm.times.250 mm; manufactured by Daicel Chemical Industries
Ltd.), eluent: n-hexane/isopropanol=39/1, flow rate: 1 ml/min,
detection: 254 nm, column temperature: room temperature).
[0085] The amounts of tert-butyl
(S)-6-benzoyloxy-5-hydroxy-3-oxohexanoate and tert-butyl
(3R,5S)-6-benzoyloxy-3,5-dihydroxyhexanoate and furthermore, the
diastereomer excess ratio of tert-butyl
(3R,5S)-6-benzoyloxy-3,5-dihydroxyhexanoate can be obtained by high
performance liquid chromatography (column: YMC-Pack ODS A-303 (ID
4.6 mm.times.250 mm), manufactured by YMC, eluent:
water/acetonitrile=1/1, flow rate: 1 ml/min, detection: 210 nm,
column temperature: room temperature).
[0086] As described in the foregoing, according to the present
invention, the polypeptide of the present invention can be
efficiently produced and a process for producing a useful and
optically active alcohol represented by
(R)-3-hydroxy-3-phenylpropanenitrile can be provided by use of the
polypeptide.
EXAMPLES
[0087] The present invention will be more specifically described by
way of Examples below, which will not be construed as limiting the
invention. Note that specific manipulation methods regarding
recombinant DNA techniques used in the following Examples are
described in the following publications: [0088] Molecular Cloning
2nd Edition (Cold Spring Harbor Laboratory Press, 1989), [0089]
Current Protocols in Molecular Biology (Greene Publishing
Associates and Wiley-Interscience).
Example 1
Purification of Polypeptide
[0090] In accordance with the following method, a polypeptide from
Candida magnoliae strain NBRC0661, having an activity of
asymmetrically reducing 3-oxo-3-phenylpropanenitrile to produce
(R)-3-hydroxy-3-phenylpropanenitrile was separated and purified as
a single product. Purification operation was performed at 4.degree.
C. unless otherwise specified.
[0091] The reduction activity to 3-oxo-3-phenylpropanenitrile was
obtained by dissolving a substrate, 3-oxo-3-phenylpropanenitrile so
as to have a final concentration of 0.5 mM and a coenzyme NADPH in
a final concentration of 0.25 mM in a 100 mM phosphate buffer (pH
6.5) containing 0.33% (v/v) of dimethylsulfoxide, further adding a
crude enzyme solution, reacting the mixture at 30.degree. C. for
one minute, measuring absorbance of the reaction solution at a
wavelength of 340 nm, and calculating a reduction rate of the
absorbance. In the reaction conditions, the activity of oxidizing 1
.mu.mol of NADPH to NADP per minute was defined as 1 unit.
[0092] (Culturing of Microorganism)
[0093] In a 30 L jar fermentor (manufactured by B. E. Marubishi Co.
Ltd.), 18 L of liquid medium was prepared containing 4% of glucose,
0.3% of yeast extract, 0.7% of potassium dihydrogenphosphate, 1.3%
of diammonium hydrogen phosphate, 0.1% of sodium chloride, 0.08% of
magnesium sulfate 7-hydrate, 0.006% of zinc sulfate 7-hydrate,
0.009% of ferrous sulfate 7-hydrate, 0.0005% of copper sulfate
5-hydrate, 0.001% of mangan sulfate 4-6 hydrate, and 0.01% of
Adecanol LG-109 (manufactured by NFO corporation). The liquid
medium was sterilized by vapor at 120.degree. C. for 20
minutes.
[0094] To this medium, 180 ml of a culture solution of Candida
magnoliae strain NERC0661 previously cultured in the same type of
medium was inoculated. The culture medium was cultured with
stirring at a rotation rate of 250 rpm and supplying air at a rate
of 5.0 NL/min at 30.degree. C. and while adjusting the lower pH
value to 5.5 by adding a 30% (w/w) aqueous sodium hydroxide
solution dropwise. Culturing was performed for 30 hours, while
adding 655 g of a 55% (w/w) aqueous glucose solution each of 18
hours, 22 hours and 26 hours after initiation of culturing.
[0095] (Preparation of Cell-Free Extraction)
[0096] Bacterial cells were centrifugally collected from the
culture solution above and washed with a 0.8% aqueous sodium
chloride solution. The bacterial cells were suspended in a 100 mM
phosphate buffer (pH 7.0) containing 5 mM of .beta.-mercaptoethanol
and 0.1 mM phenyl methane sulfonyl fluoride and disrupted by a
Dyno-mill (manufactured by Willy A. Bachofen AG.). Thereafter, cell
residues were centrifugally removed from the disrupted material to
obtain 1900 ml of a cell-free extract.
[0097] (Removal of Nucleic Acid)
[0098] To the cell-free extract obtained above, 100 ml of a 5%
aqueous protamine sulfate solution was added. The mixture was
stirred overnight and centrifuged to remove a precipitate.
[0099] (Fractionation with Ammonium Sulfate)
[0100] After nucleic acids were removed in the above, the pH of the
crude enzyme solution was adjusted to 7.0 by ammonia water. While
maintaining the pH, ammonium sulfate was added and dissolved so as
to obtain 50% saturation. The precipitate generated was
centrifugally removed. To the supernatant, ammonium sulfate was
further added and dissolved so as to obtain 60% saturation while
maintaining the pH to 7.0 in the same manner as above. The
generated precipitate was centrifugally collected. The precipitate
was dissolved in a 10 mM phosphate buffer (pH 7.0) and dialyzed
against the same buffer overnight to obtain active fractions.
[0101] (DEAF TOYOPEARL Column Chromatography)
[0102] The active fractions obtained by fractionation with ammonium
sulfate were loaded to a DEAE-TOYOPEARL 650M column (400 ml,
manufactured by TOSOH Corporation), which was previously
equilibrated with a 10 mM phosphate buffer (pH 7.0) to adsorb the
active fractions. After the column was washed with the same buffer,
the active fractions were eluted by linear gradient with NaCl (0 M
to 0.5 M). The active fractions were collected and dialyzed against
a 10 mM phosphate buffer (pH 7.0) overnight.
[0103] (Blue Sepharose Column Chromatography)
[0104] The active fractions obtained by DEAE-TOYOPEARL column
chromatography were loaded to Blue Sepharose CL-6B column (50 ml,
manufactured by Amersham Biosciences), which was previously
equilibrated with a 10 mM phosphate buffer (pH 7.0) to adsorb the
active fractions. After the column was washed with the same buffer,
the active fractions were eluted by linear gradient with NaCl (0 M
to 1 M). The active fractions were collected and dialyzed against a
10 mM phosphate buffer (pH 7.0) overnight.
[0105] (2',5'-ADP Sepharose Column Chromatography)
[0106] The active fractions obtained by Blue sepharose column
chromatography were loaded to 2',5'-ADP sepharose column (20 ml,
manufactured by Amersham Biosciences), which was previously
equilibrated with a 10 mM phosphate buffer (pH 7.0) to adsorb the
active fractions. After the column was washed with the same buffer,
the active fractions were eluted by linear gradient with NaCl (0 M
to 1 M). The active fractions were collected and dialyzed against a
10 mM phosphate buffer (pH 7.0) overnight.
[0107] (HiTrap Phenyl HP Column Chromatography)
[0108] To the active fractions obtained by 2',5'-ADP sepharose
column chromatography, ammonium sulfate was added so as to obtain a
final concentration of 35%. The mixture was loaded to a HiTrap
Phenyl HP column (manufactured by Amersham Biosciences), which was
previously equilibrated with a 10 mM phosphate buffer (pH 7.0)
containing 35% ammonium sulfate, to adsorb the active fractions.
After the column was washed with the same buffer, the active
fractions were eluted by linear gradient with NaCl (35% to 0%). The
active fractions were collected and dialyzed against a 10 mM
phosphate buffer (pH 7.0) overnight to obtain a purified
polypeptide sample.
Example 2
Gene Cloning
[0109] (Preparation of PCR Primer)
[0110] The purified polypeptide obtained in Example 1 was denatured
in the presence of BM urea, and thereafter digested with lysyl
end-peptidase (manufactured by Wako Pure Chemical Industries Ltd.)
derived from Achromobacter. The amino acid sequences of the
obtained peptide fragments were determined by an ABI492-type
protein sequencer (manufactured by PerkinElmer). Based on the
putative DNA sequence from the amino acid sequence, a primer
1:5'-cargarcaytaygtntggcc-3' (SEQ ID NO: 3 of the sequence listing)
and primer 2: 5'-atygcrtcnggrtadatcca-3' (SEQ ID NO: 4 of the
sequence listing) were synthesized for amplifying a part of the
gene encoding the polypeptide by PCR.
[0111] (PCR Amplification of Gene)
[0112] Chromosomal DNA was extracted from bacterial cells of
Candida magnoliae strain NBRCO661 cultured in the same manner as in
Example 1 in accordance with the method of Visser et al. (Appl.
Microbial. Biotechnol., 53, 415 (2000)). Subsequently, PCR was
performed using the DNA primers 1 and 2 prepared above, and the
chromosomal DNA thus obtained as a template. As a result, a DNA
fragment of about 0.5 kbp, which was conceivably a part of the
desired gene, was amplified. PCR was performed by using TaKaRa Ex
Taq (manufactured by Takara Bio Inc.) as DNA polymerase under
reaction conditions according to the instructions. The DNA fragment
was cloned to plasmid pT7Blue T-Vector (manufactured by Novagen)
and the base sequence thereof was analyzed by use of ABI PRISM Dye
Terminator Cycle Sequencing Ready Reaction Kit (manufactured by
PerkinElmer) and an ARI 373A DNA Sequencer (manufactured by
PerkinElmer). The resultant base sequence is shown in SEQ ID NO: 5
of the sequence listing.
[0113] (Determination of the Entire Sequence of Desired Gene by
i-PCR Method)
[0114] The chromosomal DNA of Candida magnoliae strain NBRCOGG1
prepared above was completely digested with restriction enzyme
PstI. A mixture of the obtained DNA fragments was treated with T4
ligase to form intramolecular cyclization. Using this as a
template, i-PCR method (Nucl. Acids Res., 16, 8186 (1988)) was
performed. In this manner, the entire base sequence of a gene
containing the base sequence represented by SEQ ID NO: 5 above. The
results are shown in SEQ ID NO: 1 of the sequence listing. The
aforementioned i-PCR was performed using TaKaRa LA Taq (Takara Bio.
Inc.) as DNA polymerase under the reaction conditions according to
the instructions. The amino acid sequence encoded by the base
sequence represented by SEQ ID NO: 1 is shown in SEQ ID NO: 2.
Example 3
Construction of Expression Vector
[0115] PCR was performed using primer 3:
5'-gtgcatatgtcttctcttcacgctcttg-3' (SEQ ID NO: 6 of the sequence
listing) and primer 4: 5'-ggcgaattcttattaaacggtagagccattgtcg-3'
(SEQ ID NO: 7 of the sequence listing), and chromosomal DNA of
Candida magnoliae strain NBRC0661 obtained in Example 2 as a
template. As a result, a double stranded DNA was obtained having an
NdeI recognition site added to the portion of the initiation codon
of a gene consisting of the base sequence represented by SEQ ID NO:
1 of the sequence listing and an EcoRI recognition site added
immediately after the termination codon. PCR was performed using
TaKaRa LA Taq (manufactured by Takara Bio Inc.) as DNA polymerase
under the reaction conditions according to the instructions. The
DNA was digested with NdeI and EcoRI and inserted between the NdeI
recognition site and the EcoRI recognition site downstream of the
lac promoter of plasmid pUCNT (U.S. Pat. No. 6,083,752). In this
manner, a recombination vector pNTCM was constructed.
Example 4
Construction of Expression Vector Further Containing Glucose
Dehydrogenase Gene
[0116] PCR was performed using primer 5:
5'-gccgaattctaaggaggttaacaatgtataaa-3' (SEQ ID NO: 8 of the
sequence listing) and primer 6: 3'-gcggtcgacttatccgcgtcctgcttgg-5'
(SEQ ID NO: 9 of the sequence listing), and plasmid pGDK1 (Eur. J.
Biochem., 186, 389 (1989)) as a template. As a result, a double
stranded DNA was obtained in which a ribosome connecting sequence
of E. coli was added to a position by 5 bases upstream of the
initiation codon of a glucose dehydrogenase gene (hereinafter
referred to as "GDH") derived from Bacillus megaterium strain
LAM1030, further an EcoRI cleaving site was added to the site
immediately therebefore, and an SalI cleaving site was added to
immediately after the termination codon. The DNA fragment thus
obtained was digested with EcoRI and SalI and inserted between the
EcoRI recognition site and the SalI recognition site downstream of
the lac promoter of plasmid pUCNT (U.S. Pat. No. 6,083,752). In
this manner, a recombination vector pNTG1 was constructed.
[0117] Subsequently, a double stranded DNA was prepared having an
NdeI recognition site added to the portion of the initiation codon
of a gene consisting of the base sequence represented by SEQ ID NO:
1 of the sequence listing and an EcoRI recognition site added to
the portion immediately after the termination codon, in the same
manner as in Example 3. The resultant DNA was inserted between the
NdeI recognition site and the EcoRI recognition site of the
recombinant vector pNTG1 mentioned above. In this manner, a
recombinant plasmid pNTCMG1 was constructed. The preparation method
and structure of pNTCMG1 are shown in FIG. 1.
Example 5
Preparation of Transformant
[0118] Using the recombinant vector pNTCM constructed in Example 3,
the E. coli HB101 competent cell (manufactured by Takara Bio Inc.)
was transformed to obtain E. coli HB101(pNTCM). This transformant
has been deposited as of Sep. 26, 2005 under the accession number
of FERM BP-10418 at an independent organization, the International
Patent Organism Depositary of the National Institute of Advanced
Industrial Science and Technology (located at Tsukuba Central 6,
1-1-1 Higashi, Tsukuba, Ibaraki, 305-8566, Japan). Note that the
original national deposition date of Oct. 23, 2003 for the strain
has been transferred to international deposition based on the
Budapest Treaty.
[0119] Similarly, the E. coli HB101 competent cell (manufactured by
Takara Bio Inc.) was transformed with the recombinant vector
pNTCMG1 constructed in Example 4 to obtain E. coli
HB101(pNTCMG1).
Example 6
Expression of Gene in Transformant
[0120] Two types of transformants obtained in Example 5, and E.
coli HB101(pUCNT), which is a transformant containing a vector
plasmid pUCNT, were separately inoculated in 50 ml of 2.times.YT
medium (1.6% of tryptone, 1.0% of yeast extract, 0.5% of NaCl, pH
7.0) containing 200 .mu.g/ml ampicillin and cultured while shaking
at 37.degree. C. for 24 hours. Bacterial cells were centrifugally
collected and suspended in 50 ml of 100 mM phosphate buffer (pH
6.5). The suspended bacterial cells were disrupted by an UH-50 type
ultrasonic homogenizer (manufactured by SMT) and thereafter
bacterial debris was centrifugally removed to obtain a cell-free
extract. The reduction activity to 3-oxo-3-phenylpropanenitrile and
GDH activity of the cell-free extract were measured and expressed
as relative activities in Table 1. In each of the two types of
transformants obtained in Example 5, the reduction activity to
3-oxo-3-phenylpropanenitrile was observed. Furthermore, the
expression of the GDH activity was observed in E. coli
HB101(pNTCMG1) containing a GDH gene. The reduction activity to
3-oxo-3-phenylpropanenitrile was measured by the method described
in Example 1. The GDH activity was obtained by adding 0.1M glucose,
2 mM coenzyme NADP and a crude enzyme solution to 1M tris
hydrochloride buffer (pH 8.0), performing a reaction at 25.degree.
C. for one minute, measuring absorbance at a wave length of 340 nm,
calculating an increasing rate of the absorbance. The enzymatic
activity for reducing 1 .mu.mol of NADP to NADPH per minute in the
reaction conditions was defined as 1 unit. The protein
concentration of the cell-free extract was measured by a protein
assay kit (manufactured by BIO-RAD).
TABLE-US-00001 TABLE 1 Relative RRG Relative GDH Name of bacterial
strain activity (U/mg) activity (U/mg) E. coli HB101(pUCNT)
<0.01 <0.01 E. coli HB101(pNTCM) 0.22 <0.01 E. coli
HB101(pNTCMG1) 0.13 269
Example 7
Production of (R)-3-hydroxy-3-phenylpropanenitrile Using
Transformant
[0121] To 50 ml of the cell-free extract of E. coli HB101(pNTCM)
prepared in the same manner as in Example 6, 2000 U of glucose
dehydrogenase (manufactured by Amano Enzyme Inc.), 3 g of glucose,
6 mg of NADP and 2 g of 3-oxo-3-phenylpropanenitrile were added.
The mixture was stirred at 30.degree. C. for 22 hours while
adjusting the pH of the mixture to 6.5 by adding 5 M sodium
hydroxide dropwise. After completion of the reaction, extraction
was performed with ethyl acetate. The solvent was removed and then
an extracted material was analyzed. As a result,
(R)-3-hydroxy-3-phenylpropanenitrile having an optical purity of
97.3% e.e. was obtained in a yield of 86.3%.
[0122] The amounts of 3-oxo-3-phenylpropanenitrile and
(R)-3-hydroxy-3-phenylpropanenitrile and the optical purity of
(R)-3-hydroxy-3-phenylpropanenitrile were obtained by capillary gas
chromatography (column: CHIRALDEX G-TA manufactured by Tokyo
Chemical Industry Co., Ltd., .phi. 0.25 mm.times.20 m, column
temperature: 130.degree. C., carrier gas: helium (150 kPa),
detection: FID).
Example 8
Production of (R)-3-hydroxy-3-phenylpropanenitrile Using
Transformant
[0123] To 50 ml of the cell-free extract of E. coli. HB101(pNTCMG1)
prepared in the same manner as in Example 6, 3 g of glucose, 6 mg
of NADP and 2 g of 3-oxo-3-phenylpropanenitrile were added. The
mixture was stirred at 30.degree. C. for 22 hours while adjusting
the pH of the mixture to 6.5 by adding 5 M sodium hydroxide
dropwise. After completion of the reaction, extraction was
performed with ethyl acetate. The solvent was removed and then an
extracted material was analyzed. As a result,
(R)-3-hydroxy-3-phenylpropanenitrile having an optical purity of
97.4% e.e. was obtained in a yield of 92.1%.
[0124] The amounts of 3-oxo-3-phenylpropanenitrile and
(R)-3-hydroxy-3-phenylpropanenitrile and the optical purity of
(R)-3-hydroxy-3-phenylpropanenitrile were obtained in the same
manner as in Example 7.
Production of (R)-2-chloro-1-(3'-chlorophenyl)ethanol Using
Transformant
[0125] To 50 ml of a culture solution of E. coli HB101(pNTCMG1)
prepared in the same manner as in Example 6, 5 g of glucose, 6 mg
of NADP, 4 g of 2-chloro-1-(3'-chlorophenyl)ethanone and 4 g of
toluene were added. The mixture was stirred at 30.degree. C. for 24
hours while adjusting the pH of the mixture to 6.5 by adding 5 M
sodium hydroxide dropwise. After completion of the reaction, the
reaction solution was extracted with toluene. The solvent was
removed and then an extracted material was analyzed. As a result,
(R)-2-chloro-1-(3'-chlorophenyl)ethanol having an optical purity of
82.0% e.e. was obtained in a yield of 90.5%.
[0126] The amounts of 2-chloro-1-(3'-chlorophenyl)ethanone and
(R)-2-chloro-1-(3'-chlorophenyl)ethanol were obtained by high
performance liquid chromatography (column: YMC-Pack ODS A-303 (ID
4.6 mm.times.250 mm), manufactured by YMC, eluent:
water/acetonitrile=1/1, flow rate: 1 ml/min, detection: 210 nm,
column temperature: room temperature). Furthermore, the optical
purity of (R)-2-chloro-1-(3'-chlorophenyl)ethanol was measured by
high performance liquid chromatography (column: Chiralcel OJ (ID
4.6 mm.times.250 mm; manufactured by Daicel Chemical Industries
Ltd., eluent: n-hexane/isopropanol=39/1, flow rate: 1 ml/min,
detection: 254 nm, column temperature: room temperature).
Example 10
Production of ethyl (S)-4-chloro-3-hydroxybutyrate Using
Transformant
[0127] To 50 ml of a culture solution of E. coli HB101(pNTCMG1)
prepared in the same manner as in Example 6, 6 g of glucose, 4 mg
of NADP were added. The reaction solution was stirred at 30.degree.
C. While the pH of the reaction solution was adjusted to pH 6.5 by
adding 5 M sodium hydroxide dropwise, 5 g in total of ethyl
4-chloro-3-oxobutyrate was added at a rate of 0.5 g/hour. Stirring
was continued for a further 5 hours. After completion of the
reaction, the reaction solution was extracted with ethyl acetate.
The solvent was removed and then an extracted material was
analyzed. As a result, ethyl (S)-4-chloro-3-hydroxybutyrate having
an optical purity of 80.5% e.e. was obtained in a yield of
93.1%.
[0128] The amounts of ethyl 4-chloro-3-oxobutyrate and ethyl
(S)-4-chloro-3-hydroxybutyrate were obtained by gas chromatography
(column: PEG-20M, Chromosorb WAW DMCS 10% 80/100 mesh (ID 3
mm.times.1 m; manufactured by G-L Sciences), column temperature:
150.degree. C., carrier gas: nitrogen, detection: FID).
Furthermore, the optical purity of ethyl
(S)-4-chloro-3-hydroxybutyrate is obtained by high performance
liquid chromatography (column: Chiralcel OB (ID 4.6 mm.times.250
mm; manufactured by Daicel Chemical Industries Ltd.), eluent:
n-hexane/isopropanol=9/1, flow rate: 0.8 ml/min, detection: 215 nm,
column temperature: room temperature).
Example 11
Production of tert-butyl 6-benzoyloxy-3,5-dihydroxyhexanoate Using
Transformant
[0129] To 40 ml of a culture solution of E. coli HB101(pNTCMG1)
prepared in the same manner as in Example 6, 3 g of glucose, 6 mg
of NADP, 4 g of tert-butyl
(S)-6-benzoyloxy-5-hydroxy-3-oxohexanoate and 1.6 g of toluene were
added. The reaction solution was stirred at 30.degree. C. for 24
hours while adjusting the pH of the reaction solution to pH 6.5 by
adding 5 M sodium hydroxide dropwise. After completion of the
reaction, the reaction solution was extracted with toluene. The
solvent was removed and then an extracted material was analyzed. As
a result, tert-butyl (3R,5R)-6-benzyloxy-3,5-dihydroxyhexanoate
having an excess rate of a diastereomer of 99.7% d.e. was obtained
in a yield of 92.3%.
[0130] The amounts of tert-butyl
(S)-6-benzoyloxy-5-hydroxy-3-oxohexanoate and tert-butyl
(3R,5R)-6-benzyloxy-3,5-dihydroxyhexanoate and further the
excessive rate of a diastereomer of tert-butyl
(3R,5R)-6-benzyloxy-3,5-dihydroxyhexanoate were obtained by high
performance liquid chromatography (column: YMC-Pack ODS A-303 (ID
4.6 mm.times.250 mm), manufactured by YMC, eluent:
water/acetonitrile=1/1, flow rate: 1 ml/min, detection: 210 nm,
column temperature: room temperature).
Example 12
Substrate Specificity of Polypeptide
[0131] To 100 mM phosphate buffer (pH 6.5) containing 0.33% (v/v)
dimethylsulfoxide, a substrate, a carbonyl compound serving as a
substrate was dissolved so as to obtain a final concentration of 1
mM and a coenzyme NADPH in a final concentration of 0.25 mM. To the
mixture, the purified polypeptide prepared in Example 1 was added
in an appropriate amount and a reaction was performed at 30.degree.
C. for one minute. Based on a reduction rate of absorbance of the
reaction solution at a wavelength of 340 nm, the reduction activity
to each carbonyl compound was calculated. This was expressed by a
relative value to the activity to 3-oxo-3-phenylpropanenitrile as
being 100 in Table 2. As is apparent from Table 2, the polypeptide
of the present invention exhibits reduction activity to a wide
variety of carbonyl compounds.
TABLE-US-00002 TABLE 2 Substrate specificity of polypeptide
Relative Reaction substrate activity (%)
3-oxo-3-phenylpropanenitrile 100 2-acetylpyridine 1926
3-acetylpyridine 2107 4-acetylpyridine 15710 acetophenone 1023
m-nitroacetophenone 5056 p-chloroacetophenone 2347
p-fluoroacetophenone 722 3,4-dimethoxyacetophenone 2889
p-methylacetophenone 542 2-hydroxyacetophenone 1806
2-chloro-1-(3'-chlorophenyl)ethanone 10473 1-phenyl-2-butanone 1144
propiophenone 3010 benzylacetone 241 ethyl benzoylacetate 7704
2-butanone 181 2-hexanone 602 2-heptanone 782 chloroacetone 2468
hydroxyacetone 361 4-hydroxy-2-butanone 301 4-methyl-2-pentanone 60
cyclopropyl methyl ketone 181 methyl pyruvate 9450 ethyl
acetoacetate 903 benzyl acetoacetate 4936 ethyl
4-chloroacetoacetate 8547 2-keto-n-butyric acid 1866 oxalacetic
acid 421
[SEQUENCE LISTING]
Sequence CWU 1
1
91723DNACandida magnoliae 1atgtcttctc ttcacgctct tgttacaggt
gctagccgcg gtattggcga ggcctcggcc 60attaagctcg cccaagaggg ctacagcgtc
acgctcgcgt cgcgcggtgt cgacaagctg 120aacgaggtga aggccaagct
tcctgttgtg aagcagggcc aggagcacta cgtctggccg 180cttgatctca
gcgatgtgca ggcggcgctc gagttcaagg gcgcgccgct gcccgcgagc
240aagtacgacc tgtttgtttg caacgccggc gtcgcctcga tgtcgccgac
tgccgaccac 300gatgacgcgg actggcagca cattctgacc gtgaaccttt
cgagcccgat tgcgctcacg 360aagacgctcg tgaaggcggt aggcgagcgg
cccaaggaca agccgttcca catcgtgtac 420atctcttcgg tggttagccg
ccgcggcttc gcgggcgctg cggtgtacag cgcgtcgaag 480gctggtctcg
acggctttgc gcgctcgatt gcccgcgagc tcggcccgaa gggaatccat
540gtgaactctg tgcagcctgg tctcacgaag accgagatga caaccagctt
cgatcctccg 600gccgatctgc ccatcagcgg atggatctac ccggatgcaa
ttgccgatgc cgtggtgttc 660ttcgccaagt cgacgaacgt cacaggtgcg
aacatcatcg tcgacaatgg ctctaccgtt 720taa 7232240PRTCandida magnoliae
2Met Ser Ser Leu His Ala Leu Val Thr Gly Ala Ser Arg Gly Ile Gly1 5
10 15Glu Ala Ser Ala Ile Lys Leu Ala Gln Glu Gly Tyr Ser Val Thr
Leu 20 25 30Ala Ser Arg Gly Val Asp Lys Leu Asn Glu Val Lys Ala Lys
Leu Pro 35 40 45Val Val Lys Gln Gly Gln Glu His Tyr Val Trp Pro Leu
Asp Leu Ser 50 55 60Asp Val Gln Ala Ala Leu Glu Phe Lys Gly Ala Pro
Leu Pro Ala Ser65 70 75 80Lys Tyr Asp Leu Phe Val Cys Asn Ala Gly
Val Ala Ser Met Ser Pro 85 90 95Thr Ala Asp His Asp Asp Ala Asp Trp
Gln His Ile Leu Thr Val Asn 100 105 110Leu Ser Ser Pro Ile Ala Leu
Thr Lys Thr Leu Val Lys Ala Val Gly 115 120 125Glu Arg Pro Lys Asp
Lys Pro Phe His Ile Val Tyr Ile Ser Ser Val 130 135 140Val Ser Arg
Arg Gly Phe Ala Gly Ala Ala Val Tyr Ser Ala Ser Lys145 150 155
160Ala Gly Leu Asp Gly Phe Ala Arg Ser Ile Ala Arg Glu Leu Gly Pro
165 170 175Lys Gly Ile His Val Asn Ser Val Gln Pro Gly Leu Thr Lys
Thr Glu 180 185 190Met Thr Thr Ser Phe Asp Pro Pro Ala Asp Leu Pro
Ile Ser Gly Trp 195 200 205Ile Tyr Pro Asp Ala Ile Ala Asp Ala Val
Val Phe Phe Ala Lys Ser 210 215 220Thr Asn Val Thr Gly Ala Asn Ile
Ile Val Asp Asn Gly Ser Thr Val225 230 235 240320DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
3cargarcayt aygtntggcc 20420DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 4atygcrtcng grtadatcca
205442DNACandida magnoliae 5gcttgatctc agcgatgtgc aggcggcgct
cgagttcaag ggcgcgccgc tgcccgcgag 60caagtacgac ctgtttgttt gcaacgccgg
cgtcgcctcg atgtcgccga ctgccgacca 120cgatgacgcg gactggcagc
acattctgac cgtgaacctt tcgagcccga ttgcgctcac 180gaagacgctc
gtgaaggcgg taggcgagcg gcccaaggac aagccgttcc acatcgtgta
240catctcttcg gtggttagcc gccgcggctt cgcgggcgct gcggtgtaca
gcgcgtcgaa 300ggctggtctc gacggctttg cgcgctcgat tgcccgcgag
ctcggcccga agggaatcca 360tgtgaactct gtgcagcctg gtctcacgaa
gaccgagatg acaaccagct tcgatcctcc 420ggccgatctg cccatcagcg ga
442628DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 6gtgcatatgt cttctcttca cgctcttg 28734DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
7ggcgaattct tattaaacgg tagagccatt gtcg 34832DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
8gccgaattct aaggaggtta acaatgtata aa 32928DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
9gcggtcgact tatccgcgtc ctgcttgg 28
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