JP2003250558A - New amidase and gene encoding the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing optically active carboxylic acids and amides which are industrially useful compounds as intermediate raw materials for medicines, agrochemicals, or the like. <P>SOLUTION: A new stereoselective amide hydrolase derived from Pseudomonas azotoformans IAM1603, and a DNA encoding the enzyme are provided. The chiral carboxylic acid or amide (including an N-alkylamide), especially the chiral nitrogen-containing heterocyclic carboxylic acid or the amide thereof is produced by stereoselectively hydrolyzing an amide by using the amide hydrolase. The amide hydrolase has excellent activities and stereoselectivity. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a polypeptide having a novel stereoselective amidase activity, a DNA encoding the peptide, a recombinant vector incorporating the DNA, and a recombinant organism having the DNA or the recombinant vector. Regarding Furthermore, the present invention relates to a method for producing chiral carboxylic acids and / or chiral carboxylic acid amides, which are useful as intermediate raw materials for medicines and agricultural chemicals, by allowing the polypeptide to act on carboxylic acid amides.

[0002]

2. Description of the Related Art The following methods are available for producing chiral carboxylic acids and / or carboxamides useful as intermediates for medical and agricultural chemicals by hydrolyzing racemic nitrogen-containing heterocycle-containing carboxamides by biotechnology. Are known.

As a method for selectively hydrolyzing (R) -nitrogen-containing heterocycle-containing carboxylic acid amides, there is disclosed in Japanese Patent Laid-Open No.
-276794 discloses Pseudomonas azotoformans IAM1603 strain or a treated product thereof, and JP-A-10-327890 discloses a Pseudomonas sp. MCI3433 strain or Pseudomonas sp. MCI3434 strain or a treated product thereof as a racemic N-tert. (R) -piperazine-2- having high optical purity by acting on butylpiperazine-2-carboxamide
A method for obtaining a carboxylic acid and / or (S) -N-piperazine-2-carboxylic acid amide is known.

On the other hand, as a method for selectively hydrolyzing (S) -nitrogen-containing heterocycle-containing carboxylic acid amides, Japanese Patent Application Laid-Open No. 8-56652 discloses Pseudomonas putida, Pseudomonas fluorescens, Klebsiella terrigena. Species or Klebsiella pneumoniae microorganisms or treated products thereof are allowed to act on racemic proline amide, piperazine-2-carboxamide or pipecolic acid amide, and (S) -carboxylic acids and / or (R ) -Carboxylic acid amides are described in JP-A-10-12728.
No. 0 discloses a mycoplana bacterium or a treated product thereof as racemic piperidine-2-carboxamide or N.
-Alkylpiperidine-2-carboxamide, a method for obtaining high optical purity (S) -piperidine-2-carboxylic acid and / or (R) -N-alkylpiperazine-2-carboxamide is described. Japanese Unexamined Patent Publication (Kokai) No. 10-276794 discloses that N-tert-butylpiperazine-2- is a bacterial cell of Leucobacterium, Rhizobium, or Mycobacterium, or a treated product thereof.
A high optical purity of (S) -piperazine-2-carboxylic acid, and / or (R) -N-t by acting on carboxamide.
A method for obtaining ert butylpiperazine-2-carboxamide is described.

However, in these methods, the cells or treated products thereof are used as a hydrolysis catalyst, but the content of the enzyme that catalyzes the desired reaction is small or the enzyme itself is unstable, and the reaction is sufficiently catalyzed. In order to do so, it was not an economically advantageous method because a large amount of bacterial cells had to be prepared.

[0006]

The object of the present invention is to obtain a novel amidase having the ability to stereoselectively hydrolyze nitrogen-containing heterocycle-containing carboxylic acid amides, and a DNA encoding the same, and Another object of the present invention is to provide a method for producing nitrogen-containing heterocycle-containing carboxylic acids and / or amides thereof having high optical purity using amidase.

[0007]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have found that a stereospecific amidase that selectively hydrolyzes (R) -N-alkylpiperazine-2-carboxamide. Pseudomonas azotoformans IAM1 known to have the activity of
An enzyme that selectively hydrolyzes (S) -N-alkylpiperazine-2-carboxamide was isolated from strain 603, and the DNA encoding it was obtained. It has been found that a recombinant organism rich in stereoselective amidase produced using this DNA or a high-concentration amidase solution promotes higher productivity and higher stereoselective hydrolysis reaction of carboxamides, The present invention has been completed.

That is, according to the present invention, a stereoselective amidase having the following physicochemical properties is provided. (1) Molecular weight: 34,000 when measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 32000 when measured by gel filtration; (2) Subunit constitution: monomer (3) L-proline amide as substrate Optimum pH of amidase reaction when used: around pH 9 (4) Optimum temperature of amidase reaction when reacted at pH 8 using L-prolinamide as a substrate: around 45 ° C. (5) Heat resistance at pH 8: 40 (6) Compounds that completely inhibit the amidase activity of the present polypeptide, which are stable at ℃ or below: Phenylhydrazine, Zn ²⁺ , Ag ⁺ , Cd
²⁺ , Hg ²⁺ (7) Effect of EDTA: Addition of 1 mM EDTA to the reaction solution promotes enzyme activity.

According to another aspect of the present invention, any one of the following polypeptides is provided. (1) a polypeptide having the amino acid sequence represented by SEQ ID NO: 1; (2) an amino acid sequence in which one to a plurality of amino acids are deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 1 A polypeptide having a stereoselective amidase activity; or (3) a polypeptide having an amino acid sequence having 70% or more homology with the amino acid sequence represented by SEQ ID NO: 1 and having a stereoselective amidase activity. ;

[0010] According to still another aspect of the present invention, there is provided a DNA encoding the above-mentioned polypeptide of the present invention. According to still another aspect of the present invention, any one of the following DNAs is provided. (1) a DNA having the base sequence represented by SEQ ID NO: 2; (2) a base sequence having 1 to a plurality of bases deleted, substituted and / or added in the base sequence represented by SEQ ID NO: 2 And has a stereoselective amidase activity by hybridizing under stringent conditions with a DNA encoding a polypeptide having a stereoselective amidase activity; or (3) a DNA having the base sequence represented by SEQ ID NO: 2. DNA encoding the polypeptide:

According to still another aspect of the present invention, there is provided a recombinant vector having the above-mentioned DNA of the present invention. According to still another aspect of the present invention, there is provided a transformant having the above-described DNA or recombinant vector of the present invention.

According to still another aspect of the present invention, the following general formula (I)

[Chemical 4] (In the formula, A is an alkyl group which may be substituted with a substituent selected from a hydroxyl group, an alkoxy group, a thiol group, an alkylthio group, an aryl group and a heteroaryl group, or a carbon atom to which A is bonded and the carbon atom A group capable of forming a nitrogen-containing heterocyclic ring together with an amino group bonded to an atom, R is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted aryl group. The amide bond of the (S) -form is selectively hydrolyzed by allowing the amidase, polypeptide, or transformant of the present invention to act on the carboxylic acid amides represented by General formula (II)

[Chemical 5] (In the formula, A has the same meaning as above.) (S)-
Carboxylic acids or / and the following general formula (III)

[Chemical 6] (In the formula, A and R have the same meanings as described above.) A method for producing an optically active carboxylic acid and / or amide, which comprises isolating the (R) -form carboxylic acid amide represented by Will be provided.

Preferably, A is a group capable of forming a nitrogen-containing heterocycle together with the carbon atom to which A is bonded and the amino group bonded to the carbon atom. Preferably,
The reaction is performed in the coexistence of ethylenediaminetetraacetic acid.

[0014] According to still another aspect of the present invention, the above-mentioned DNA of the present invention and one or more kinds of DNA showing 50% or more homology with the DNA are randomly recombined with each other. Provided is a method for producing a novel polypeptide having selective amidase activity.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described in detail below. (I) Amidase and Polypeptide of the Present Invention The present invention relates to a stereoselective amidase having the following physicochemical properties. (1) Molecular weight: 34,000 when measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 32000 when measured by gel filtration; (2) Subunit constitution: monomer (3) L-proline amide as substrate Optimum pH of amidase reaction when used: around pH 9 (4) Optimum temperature of amidase reaction when reacted at pH 8 using L-prolinamide as a substrate: around 45 ° C. (5) Heat resistance at pH 8: 40 (6) Compounds that completely inhibit the amidase activity of the present polypeptide, which are stable at ℃ or below: Phenylhydrazine, Zn ²⁺ , Ag ⁺ , Cd
Effect of ²⁺ , Hg ²⁺ (7) EDTA: Addition of 1 mM EDTA to the reaction solution promotes enzyme activity, preferably 1.
It is accelerated by a factor of 1 or more, more preferably by a factor of 1.5 or more.

Specific examples of the amidase of the present invention include the polypeptide represented by the amino acid sequence of SEQ ID NO: 1,
A polypeptide which is a homologue thereof and has stereoselective amidase activity, and a polypeptide which contains the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of a homologue thereof in the molecule.

The homologue of the polypeptide of the present invention means 1 to a plurality (preferably 1 to 20, more preferably 1 to 15) of the amino acid sequence represented by SEQ ID NO: 1.
More preferably 1 to 10, more preferably 1 to 7
Individual, particularly preferably about 1 to 5) amino acids are deleted,
A polypeptide having a substituted and / or added amino acid sequence and having stereoselective amidase activity; or 70% or more, preferably 80% or more, more preferably 90% or more with the amino acid sequence represented by SEQ ID NO: 1. , A polypeptide having an amino acid sequence having homology of 95% or more, particularly preferably 97% or more, and having stereoselective amidase activity;

Further, "a polypeptide containing the amino acid sequence of SEQ ID NO: 1 in the molecule" means a polypeptide having a protein interaction, a ligand binding function or another special function at the N-terminal or C-terminal of the polypeptide. It means a fusion polypeptide in which

By the way, the homology search of the above-mentioned polypeptide can be carried out by using, for example, the FASTA program or the BLAST program. Protein
Information Resource (PI
BL) for amino acid sequence databases such as R)
As a result of performing a homology search for the amino acid sequence shown in SEQ ID NO: 1 using the AST program, it was found that Sinorhizobium meritioti (Sinorhizobibi) showed a high amino acid identity among known proteins.
um meliliti) 's putative pro
line iminopeptidase prote
in (66%), and meso-Rhizobium roti (Mes
orhizobium loti) proline iminopeptidase (58%).

Examples of fusion polypeptides include, for example:
Using the usual molecular genetic techniques, Mol. Micro
biol. 4, 759-767, (1990) Gilbert H. et al. J. Reported in US Pat. No. 5,496,934 and Shoseyov.
O. The amino acid sequence having a cellulose-binding function as reported by et al. Is added to the N-terminal or C-terminal of the polypeptide of the present application, and the hexamer of histidine having a nickel-binding function is bonded to the polypeptide to specifically bind to the polypeptide. Given by Biotechnol. Bioe
ng. , 68, 211-217, (2000) in Kim G. et al. J. And the like, in which the polypeptide of the present invention is fused with a polypeptide having another function by a method as reported by et al.

The polypeptide of the present invention has a stereoselective amidase activity, and the stereoselective amidase activity means the optically active carboxylic acid obtained by stereoselectively hydrolyzing the amide bond in the compound. And / or the activity of producing a carboxylic acid amide.

Since the amino acid sequence of the polypeptide has been clarified by the present invention, a DNA probe prepared on the basis of a base sequence encoding a part or all of the amino acid sequence of SEQ ID NO: 1 will be described later. By using it, a DNA encoding a polypeptide having a stereoselective amidase activity is isolated from any microorganism having a stereoselective amidase activity, or a polypeptide having a stereoselective amidase activity is isolated using a DNA synthesizer. By synthesizing the encoding DNA, it can be used to produce a large amount of the polypeptide by a molecular genetic method, and the polypeptide can be isolated by a usual method.
It can also be purified from microorganisms having the above DNA, for example, cultured bacterial cells of Pseudomonas azotoformans IAM1603 strain.

The Pseudomonas azotoformans IAM1603 strain is a known microorganism and is available from IAM Culture Collection, Institute for Molecular and Cellular Biology, University of Tokyo.

As a method for isolating the polypeptide of the present invention from cultured bacterial cells of a microorganism, a general protein purification method can be applied. For example, the above-mentioned microorganisms are cultured in a general microbial nutrient medium, preferably a medium containing nutrient broth as a nitrogen source and malic acid as a carbon source, and then sufficiently grown and then recovered by centrifugation and ultrasonically disrupted. A cell-free extract is obtained by treatment or French press treatment. From this cell-free extract, the desired polypeptide can be isolated by operations such as centrifugation, column chromatography, and electrophoresis, utilizing the difference in the physicochemical properties of proteins. A more recommended procedure is to prepare a cell-free extract, ion exchange chromatography,
The subject polypeptides can be purified using hydrophobic chromatography, affinity chromatography, size exclusion chromatography, gel excision from electrophoresis, and the like. Alternatively, as described below, a recombinant organism, preferably an Escherichia coli (Escherichia coli), in which a vector having the DNA of SEQ ID NO: 2 downstream of an appropriate expression promoter and a ribosome binding sequence is incorporated.
chia coli; hereinafter, E. The cell-free extract is prepared from the cultured bacterial cells of E. coli) by ultrasonication and centrifugation, and the polypeptide of the present invention is isolated using ammonium sulfate fractionation, ion exchange chromatography, and hydrophobic chromatography.

The physical and enzymatic properties of the polypeptide having the amino acid sequence shown in SEQ ID NO: 1 isolated from Pseudomonas azotoformans IAM1603 strain are shown below. 1. Molecular weight: 34000 (sodium dodecyl sulfate-
Polyacrylamide gel electrophoresis, hereinafter SDS-PAG
Abbreviated as E. ) 32000 (gel filtration) 2. Subunit composition: Monomer 3. Optimum pH of amidase reaction: pH9 4. Optimum temperature for amidase reaction: 45 ° C 5. Heat resistance: Stable at 45 ° C or lower. 6. Compounds that completely inhibit the amidase activity of the present polypeptide: phenylhydrazine, Zn ²⁺ , Ag ⁺ , C
d ²⁺ , Hg ²⁺ . 7. Effect of EDTA: Addition of 1 mM EDTA to the reaction solution promotes enzyme activity about 1.8 times.

Further, the polypeptide of the present invention is obtained by the Fmoc method.
(Fluorenylmethyloxycarbonyl method), tBoc method (t-
It can also be produced by a chemical synthesis method such as a butyloxycarbonyl method). In addition, Kuwawa Trading (US Advanced
Chem Tech), Perkin lmajavan (Perki USA
n Elmer), Amersham Pharmacia Biotech (Ame
rsham Pharmacia Biotech), Aloca (Protein T
echnology Instrument Co., Kurabo (US Synthecell
-Vega), Japan Perceptive Limited (US Per
It can also be chemically synthesized using a peptide synthesizer such as manufactured by Septive Co., Ltd., Shimadzu Corporation.

(II) DNA of the present invention The DNA of the present invention means DN encoding the above-mentioned polypeptide.
A or a homolog thereof. Examples of the DNA encoding the above-mentioned polypeptide include DNA encoding the amino acid sequence set forth in SEQ ID NO: 1, and specific examples thereof include those containing the base sequence represented by SEQ ID NO: 2.

DNA encoding the polypeptide of the present invention
Is a homologue of SEQ ID NO: 2, which has a base sequence in which one to a plurality of bases have been deleted, substituted and / or added in the base sequence represented by SEQ ID NO: 2, and encodes a polypeptide having stereoselective amidase activity. DNA; or DNA that hybridizes with the DNA having the nucleotide sequence represented by SEQ ID NO: 2 under stringent conditions and encodes a polypeptide having a stereoselective amidase activity.

The above-mentioned DNA homologue is a method for introducing site-directed mutagenesis into the DNA of SEQ ID NO: 2 (Nucleic Ac
id Res. 10, pp. 6487 (1982), M
methods of Enzymol. 100, pp. Four
48 (1983), Molecular Clonin
g 2nd Edt. , Cold Spring Ha
rbor Laboratory Press (198
9), PCR A Practical Approa
ch IRL Press pp. 200 (199
It can be obtained by those skilled in the art by appropriately introducing substitutions, deletions, insertions and / or addition mutations using 1)) and the like.

In the present specification, "DNA which hybridizes under stringent conditions" means that DNA is used as a probe and colony hybridization method, plaque hybridization method, Southern blot hybridization method or the like is used. The term "base sequence" of the obtained DNA means, for example, 0.7 to 1.0 M using a filter on which DNA derived from a colony or plaque or a fragment of the DNA is immobilized.
After performing hybridization at 65 ° C. in the presence of NaCl, the filter is washed at 65 ° C. using 0.1 to 2 × SSC solution (1 × SSC composition is 150 mM sodium chloride, 15 mM sodium citrate). DNAs and the like that can be identified by

Hybridization is performed by Moleccu.
lar Cloning: A Laboratory
Manual, 3rd Ed. (Ed. Sambro
okJ. and Russsel D. W. ), Co
ld Spring Harbor Laborato
ry Press, Cold Spring Harb
Or, New York, 2001, hereinafter abbreviated as "Molecular cloning 3rd edition") and the like.

The DNA homologue has a homology of 60% or more, preferably 70% or more, more preferably 80% or more, particularly preferably 90% or more with the DNA having the nucleotide sequence of SEQ ID NO: 2. Is mentioned.

DNA encoding the polypeptide of the present invention
Can be obtained, for example, by the following method. First, using a chromosomal DNA prepared from a microorganism having stereoselective amidase activity, a plasmid library or a phage library is prepared by a conventional method. Next, using the information of the partial amino acid sequence of the polypeptide, a partial fragment of DNA encoding the polypeptide is obtained, and a DNA probe is prepared by a conventional method.
This DNA probe was used to identify the DNA encoding the polypeptide of the present invention from the chromosomal DNA library described above,
By subcloning an appropriate DNA, a DNA encoding the polypeptide can be obtained.

More specifically, the DNA according to the present invention can be obtained by the following method. 1. Pseudomonas Azotoformance IAM16
The amidase of the present application was purified from
Chromosomal DN of Pseudomonas azotoformans IAM1603 based on the information of terminal and internal amino acid sequences
Degenerate PCR (abbreviation of polymerase chain reaction) is performed using A as a template to obtain a partial fragment of DNA encoding a polypeptide having the desired stereoselective amidase activity. This is labeled with a nucleic acid containing a radioisotope to prepare a DNA probe. 2. Pseudomonas Azotoformance IAM16
Chromosomal DNA is extracted from No. 03 and partially digested with an appropriate restriction enzyme (for example, FbaI). Southern hybridization is carried out using the DNA fragment obtained in 3.2 and the DNA probe prepared in 1. The DNA specified in 4.3 is extracted and purified, ligated to an appropriate plasmid vector, and Escherichia coli is transformed. Colony hybridization is performed using the DNA probe prepared in 5.1 to obtain the plasmid of the clone containing the target DNA sequence. The plasmid obtained in 6.5 was used to integrate Pseudomonas azotoformans IAM1 into a vector.
The nucleotide sequence of the DNA derived from 603 is determined. 7. An open reading frame for the amino acid sequence of the stereoselective amidase is found in the determined DNA base sequence, and it is confirmed that the entire coding region for amidase is present in the DNA fragment obtained in the above step.

In the above process, DNA and E. coli as a recombinant host were used. General operations necessary for handling E. coli are performed by those skilled in the art, and can be easily carried out, for example, according to Molecular Cloning 3rd Edition.
Commercially available products can be used for all of the enzymes and reagents used, and unless otherwise specified, these objects can be completely achieved according to the use conditions specified for the product.
In particular, the above 1. Extraction of total DNA from the bacterium is performed, for example, by Saito et al. [Biochim. Biophy
s. Acta, 72, 619-629 (1963)].
It can be performed according to the method of. Further, for labeling of DNA, any conventionally used radioisotope or a non-radioactive compound such as digoxigenin, biotin or fluorescein can be used.
^TM II random prime labeling
system (manufactured by Amersham Pharmacia Biotech Co., Ltd.) and AlkPhos ^™ Direct La
belling and Detection Sys
tem (manufactured by Amersham Pharmacia Biotech Co., Ltd.) can be easily used. In addition, the above 4. Examples of vectors include pBluescript
tSK (−) (manufactured by Stratagene) or the like can be used. Further, the above 6. For the determination of the DNA base sequence in, the known method described in Molecular Cloning, Third Edition, etc. can be used. For example, Li
-Cor DNA Sequencer model
It can be easily carried out by using a device such as 4000L according to the attached manual instruction.

(III) Recombinant Vector of the Present Invention and Transformant A stereoselective amidase expression vector is obtained by inserting the DNA encoding the polypeptide of the present invention obtained in (II) above into a known expression vector. Will be provided.
In addition, a stereoselective amidase can be obtained by culturing a recombinant organism transformed with this expression vector.

The host cell to be transformed for expressing the stereoselective amidase of the present invention is not particularly limited as long as the host itself does not adversely affect this reaction, and a host vector system is established. Bacteria, actinomycetes, yeasts, molds and other such microorganisms, insect cells, plant cells and animal cells can be preferably used. Specifically, the following microorganisms and higher organism cells can be mentioned.

Bacillus, genus Escherichia
us), Pseudomonas
Genus, Brevibacterium
m) genus, Corynebacte
Rium genus, Rhodococcus
s) genus, Streptomyces
s) genus, Saccharomyces
s), Shizosaccharomyces
aromyces, Pichia, Candida, Aspergillus (A)
spergillus), silkworm, rapeseed, soybean, etc.

The above-mentioned host cells are preferably microorganisms of the genera Escherichia, Bacillus, Pseudomonas, Brevibacterium, Corynebacterium, and Rhodococcus, and particularly preferably Escherichia, Pseudomonas, Brevibacterium. Genus and Corynebacterium.

In order to produce the polypeptide of the present invention in vivo, the DNA of the present invention is introduced into a vector which is stably present in the host organism, or it is directly introduced into the host genome by a technique such as homologous recombination. D of the present invention
It is necessary to introduce a NA to prepare a recombinant DNA molecule and to transcribe / translate its genetic information.

At this time, a promoter and a ribosome binding site (RBS) were provided upstream of the 5'side of the DNA chain of the present invention,
More preferably, it is preferable to incorporate a transcription terminator at the 3'side downstream. As the promoter, RBS and transcription termination factor, RBS, which is known to function in cells used as a host, RBS
And a transcription termination factor are not particularly limited, and vectors, promoters, RBs usable in these various organisms
S and the transcription termination factor are described in detail, for example, in Molecular Cloning 3rd Edition and "Basic Microbiology Course 8 Genetic Engineering / Kyoritsu Shuppan".

Specifically, for example, the genus Escherichia, particularly E. For E. coli, pBR and pU are used as vectors.
C-type plasmids include lac (β-galactosidase), trp (tryptophan operon), tac, t
Examples include promoters derived from rc, lpp, λ phage PL, PR and the like. In addition, as transcription termination factors, trpA-derived, phage-derived, ribosomal RN
Examples include those derived from A and lpp. Thus, E. When E. coli is used as a polypeptide synthesis system, the target polypeptide is a recombinant E. coli vector containing the DNA of the present invention. constitutively with the growth of E. coli, or isopropylthio-β-D-galactoside (hereinafter I
It is inductively produced by the addition of a polypeptide synthesis inducer such as PTG).

The stereoselective amidase can be produced in vivo by the method described above. in vitro using the cell-free protein synthesis system of E. coli or wheat germ
The amidase can also be produced with o. For example,
E. In E. coli, Kigawa et al. [FEBS Lett
ers, 442, 15-19 (1999)], for the synthetic system using wheat germ, Madin et al. [Proc.
Natl. Acad. Sci. USA, 97, 559-
564 (2000)].

The transformant having the DNA of the present invention can be cultured, and the amidase of the present invention can be isolated and purified from the culture by a known method. The method of culturing the transformant having the DNA of the present invention can be carried out according to a usual method used for culturing a host. When the transformant of the present invention is a prokaryote such as Escherichia coli, or a eukaryote such as yeast,
The medium for culturing these microorganisms may be a natural medium or a synthetic medium as long as it contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the microorganisms and can efficiently culture the transformants. . Culturing is preferably carried out under aerobic conditions such as shaking culture or deep aeration stirring culture, the culture temperature is usually 15 to 40 ° C., and the culture time is usually 5 hours to 7 hours.
It is a day. The pH is maintained at 3.0 to 9.0 during the culture. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like. If necessary, antibiotics such as ampicillin and tetracycline may be added to the medium during the culturing.

As a medium for culturing a transformant obtained by using animal cells as host cells, RP which is generally used
M11640 medium [The Journal of the American Medical As
sociation, 199, 519 (1967)], Eagle's MEM medium [Scienc
e, 122, 501 (1952)], DMEM medium (Virology, 8, 396 (19
59)], 199 medium (Proceeding of the Society for the B
iological Medicine, 73, 1 (1950)] or a medium obtained by adding fetal bovine serum or the like to these mediums. Culturing is usually carried out for 1 to 7 days under conditions such as pH 6 to 8 at 30 to 40 ° C. and in the presence of 5% CO ₂ . If necessary, antibiotics such as kanamycin and penicillin may be added to the medium during the culture.

In order to isolate and purify the amidase of the present invention from the culture of the transformant, the usual protein isolation and purification methods may be used as described above. For example, when the amidase of the present invention is expressed in a dissolved state in cells,
After the completion of the culture, the cells are collected by centrifugation and suspended in an aqueous buffer solution, and then the cells are disrupted by an ultrasonic disruptor, a French press, a Manton Gaulin homogenizer, a Dynomill or the like to obtain a cell-free extract. From the supernatant obtained by centrifuging the cell-free extract, an ordinary protein isolation and purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Diethylaminoethyl (D
EAE) Sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical Co., Ltd.), etc. Anion exchange chromatography using resin, S-Se
Cation exchange chromatography using a resin such as pharose FF (Pharmacia), butyl sepharose,
Hydrophobic chromatography method using resin such as phenyl sepharose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric focusing, etc., alone or in combination. , A purified standard can be obtained.

When the amidase of the present invention is expressed by forming an insoluble substance in cells, cells are similarly collected, disrupted, and centrifuged to obtain a precipitate fraction.
After recovering the amidase by a usual method, the insoluble matter of the amidase is solubilized with a protein denaturing agent. The solubilized solution is diluted or dialyzed with a solution that does not contain a protein denaturant or a protein denaturant is diluted to such an extent that the amidase does not denature, and the amidase is formed into a normal three-dimensional structure. A purified preparation can be obtained by the same isolation and purification method.

(IV) Optical activity using the amidase of the present invention
Production of Organic Carboxylic Acids and / or Amides In addition, the present invention provides the following general formula (I)

[Chemical 7] (In the formula, A is an alkyl group which may be substituted with a substituent selected from a hydroxyl group, an alkoxy group, a thiol group, an alkylthio group, an aryl group and a heteroaryl group,
Alternatively, A is a group capable of forming a nitrogen-containing heterocycle by being integrated with the carbon atom to which A is bonded and the amino group bonded to the carbon atom, and R is a hydrogen atom or, even if substituted, A good alkyl group or an optionally substituted aryl group is shown. ) The amide bond of the (S) -form is selectively hydrolyzed by allowing the amidase, polypeptide or transformant of the present invention to act on the carboxylic acid amide represented by II)

[Chemical 8] (In the formula, A has the same meaning as above.) (S)-
Carboxylic acids or / and the following general formula (III)

[Chemical 9] (In the formula, A and R have the same meanings as described above.) The (R) -form carboxylic acid amides are isolated to produce optically active carboxylic acids and / or amides. It also relates to how to do it.

In the above general formula (I), the substituent A is an alkyl group which may be substituted with a substituent selected from a hydroxyl group, an alkoxy group, a thiol group, an alkylthio group, an aryl group and a heteroaryl group, or A nitrogen-containing heterocyclic group is a group that forms a nitrogen-containing heterocyclic ring together with the carbon atom to which A is bonded and the amino group bonded to the carbon atom. Heteroatoms may be included.

Specific examples of the above alkyl group include methyl group, ethyl group, propyl group, 1-methylpropyl group, butyl group, isobutyl group, pentyl group, isopentyl group,
Hydroxymethyl group, 1-hydroxyethyl group, 2-hydroxyethyl group, thiomethoxymethyl group, 2- (4-
Examples thereof include an alkyl group having 1 to 6 carbon atoms which may be substituted by the above-mentioned substituents such as a hydroxyphenyl) ethyl group and a 2- (3-indolyl) ethyl group.

The above-mentioned nitrogen-containing heterocyclic group is preferably a 5- to 7-membered nitrogen-containing heterocycle, more preferably a nitrogen-containing heterocycle containing 1 or 2 nitrogen atoms and containing no other heteroatoms. A ring group is mentioned. Specific preferred examples of the nitrogen-containing heterocyclic group include a piperidyl group, a piperazyl group, a pyrazyl group and a pyrrolidyl group.

The nitrogen-containing heterocyclic group may have a substituent as long as it does not inhibit the reaction. Specific examples of the substituent include a halogen atom, an alkyl group, an aryl group, a hydroxyl group and an amino group. , Nitro group, carboxylic acid group,
Examples thereof include an alkoxy group, and among these, an alkyl group having 1 to 4 carbon atoms is preferable.

As the substituent R in the above general formula (I),
A hydrogen atom, an optionally substituted alkyl group or an optionally substituted aryl group is shown.

The alkyl group may be a linear, branched or cyclic alkyl group, and preferably has 1 to 6 carbon atoms, and the aryl group may be a phenyl group or a naphthyl group.

Examples of the substituent of the above alkyl group or aryl group include the same groups as those described above as the substituent of the nitrogen-containing heterocycle of A.

Specific preferred examples of the above-mentioned optionally substituted alkyl group or aryl group include a methyl group,
Examples thereof include ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, t-butyl group, cyclohexyl group, benzyl group, trifluoromethyl group, 1-hydroxycarbonylethyl group, phenyl group, tolyl group and nitrophenyl group. To be

The above R is preferably a hydrogen atom or an optionally substituted alkyl group having 1 to 4 carbon atoms or a phenyl group having a substituent, and more preferably a hydrogen atom or 1 to 4 carbon atoms. Is an alkyl group and p-nitrophenyl group.

In this production method, a recombinant organism accumulating the polypeptide of the present invention, a treated product of the recombinant organism, a cell-free protein synthesis reaction solution in which the polypeptide of the present invention is accumulated, and / or a treated product thereof. Is added to the reaction system as an enzyme catalyst. Specifically, cells obtained by culturing the above recombinant organisms as they are, or treatment with an organic compound such as toluene or a surfactant, lyophilization treatment,
A stereoselective amidase active fraction of the present application from a treated product treated by a known method such as physical crushing treatment, the reaction solution of the cell-free protein synthesis system, the recombinant biological treatment product, and the cell-free protein synthesis reaction solution. It is also possible to take out the fraction as a crudely purified fraction or a purified product and use it. Moreover,
It is also possible to use the thus-obtained recombinant organisms, processed products of recombinant organisms, enzyme fractions and the like immobilized on a carrier such as polyacrylamide gel and carrageenan gel. Hereinafter, the term "high-concentration enzyme preparation" refers to the above recombinant organism, cell-free protein synthesis reaction product,
It is used as a term that includes all the concepts of those processed products, enzyme fractions, and immobilized products.

In this reaction, the racemic carboxylic acid amide, which is a reaction raw material, is usually 0.1 mM because the enzyme may be inhibited by the substrate if the substrate concentration is too high.
It is added to the reaction solution at a concentration of -1 M, more preferably at a concentration of 10-100 mM. These may be added all at once at the start of the reaction, or may be added continuously or intermittently in order to improve the accumulated concentration of the product.

This reaction is carried out in an aqueous medium or in a mixed solution of the aqueous medium and an organic solvent. As the above-mentioned aqueous medium, water, potassium phosphate aqueous solution, tris-
Examples of the organic solvent include alcohols such as ethanol and propanol; ketones such as acetone; ethers such as tetrahydrofuran; esters such as ethyl acetate and butyl acetate; and toluene. Aromatic hydrocarbons; halogenated hydrocarbons such as chloroform; aliphatic hydrocarbons such as n-hexane; organic compounds such as dimethyl sulfoxide can be used.

The reaction solution preferably proceeds in an alkaline state, but it is preferably pH 6.0-9.5, and more preferably pH 7.0-9.5. To satisfy this condition, a dilute acid or alkali solution may be added during the reaction as needed to maintain the pH in the desired range.

The reaction proceeds at a reaction temperature of 0-50 ° C., but it is more preferably maintained at 25-45 ° C.

In addition, ethylenediaminetetraacetic acid (EDT
Enzyme activity can be promoted by adding A) to the reaction solution. In this case, EDTA is 0.001-1
It is added at a concentration of 00 mM, more preferably 0.5-10 mM. The reaction solution is stirred and mixed if necessary.

The (S) -form carboxylic acid and / or the remaining (R) -form amide compound produced by the above reaction can be isolated by a conventional separation / purification method such as solvent extraction and chromatography. .

(V) By random combination
Production of Novel Polypeptide Further, the present invention provides a DNA having a nucleotide sequence represented by SEQ ID NO: 2 or a DNA homolog thereof and a DNA having 50% or more homology with the DNA in vivo or in v.
The present invention relates to a method for obtaining a DNA having a new nucleotide sequence having amidase activity by randomly recombining in vitro.

As a method for randomly recombining DNA in vivo, Nat. Biotechn
ol. , 17, 333-334 (1999)]. R. The method reported by et al.

As a method for randomly recombining DNA in vitro, Proc. Nat
l. Acad. Sci. USA, 91, 10747-1.
0751 (1994) in Stemmer W.
P. C. Et al., Nat. Biotechn
ol. , 16, 258-261 (1998),
Zhao H. Et al. And Nature,
391, 288-291 (1998).
mer A. The method reported by C. et al. These methods are particularly preferable.

According to the above-mentioned method, several kinds of DNAs having high homology with each other and coding for a hydrolase are used for in
It is possible for those skilled in the art to carry out recombination in vitro and obtain a DNA encoding a polypeptide having a novel hydrolase activity. Also, the obtained DN
A novel polypeptide having amidase activity can be produced by introducing A into a suitable expression system according to the method described above to express the encoded polypeptide. . Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto, and ordinary modifications in the technical field of the present invention can be made without departing from the gist thereof.

[0069]

EXAMPLES Example 1 Isolation of Stereoselective Amidase Pseudomonas azotoformans IAM1603 strain was inoculated into 25.0 liters of BM medium having the following composition, cultured at 30 ° C. for 12 hours, and centrifuged. Bacteria were obtained. 125 g of the cells were ultrasonically disrupted and centrifuged to prepare a cell-free extract.

[0070]

[Table 1]

10 mM Tris-hydrochloric acid buffer solution (pH
8.0) Adsorbed on DEAE-Toyopearl resin (manufactured by Toso-Co., Ltd.) equilibrated with ion-exchange chromatography, and a fraction having a steric amidase activity of the elution fraction containing 50 mM sodium chloride. Butyl-Toyopearl using ammonium sulfate sequentially
l column (manufactured by Tosoh Corporation), Gigapite column (manufactured by Seikagaku Corporation), FPLC Superdex2
00 (manufactured by Amersham Pharmacia Biotech), Mo
NoQ (manufactured by Amersham Pharmacia Biotech) and again Superdex 200 (manufactured by Amersham Pharmacia Biotech) were subjected to column chromatography, and finally the active fraction obtained therefrom was gradient gel NPG-520L (Ato (strain)). The product of the present invention was isolated by carrying out Native-electrophoresis using the product and excising the active portion from the gel. To determine the molecular weight of the polypeptide, S
DS-PAGE and TSKgel G3000SW
When determined by size exclusion chromatography (manufactured by Tosoh Corporation), 34000 (SD
S-PAGE), and 32000 (size exclusion chromatography), the subunit composition was assumed to be monomeric.

Example 2: Analysis of partial amino acid sequence The purified stereospecific amidase obtained in Example 1 was digested with peptidase, and two kinds of partial peptides were separated by high performance liquid chromatography condition A (HPLC). HPLC condition A; Column: TSKgel ODS-120T (manufactured by Tosoh Corporation) Eluent: Gradient elution flow rate with water / trifluoroacetic acid / acetonitrile: 1.0 mL / min Detection: UV 210 nm Absorbance Obtained peptide fragment and Example The N-terminal amino acid sequence of the polypeptide purified by 1 was used as an automated Edman degradation amino acid sequencer HP G1005A pr.
The analysis was performed using an Otein Sequencing System (manufactured by Hewlett-Packard Co.). The respective amino acid sequences are shown in SEQ ID NOs: 3, 4, and 5.

Example 3: Acquisition of partial fragment of DNA of the present invention Pseudomonas azotoformans IAM1603 strain was inoculated into 5.0 mL of the above BM medium and cultured at 30 ° C for 12 hours. The microbial cells were obtained by centrifugation, and the chromosomal DNA of the microorganism was obtained by the phenol-chloroform method.
Got From the amino acid sequence found in Example 2, SEQ ID NOs: 6-10 (AF1, BF1, BR1, CF1, C
R1) PCR primer was designed and any two of these 5 kinds of primers were designed in the presence of heat-resistant DNA polymerase.
PCR was performed with the addition of seeds. When the product of this PCR was subjected to agarose gel electrophoresis, primers AF1 and CR
Since a 0.2 kbp DNA was specifically amplified in the reaction solution using 1, this was extracted and purified from an agarose electrophoresis gel. A part of the DNA is pT7BlueT-
Li-cor DNA Sequencer mode by cloning into Vector (manufactured by Novagen).
The nucleotide sequence was determined using 4000 L (the operating method was according to the attached manual instruction) (SEQ ID NO: 11). The rest of the 0.2 kbp purified DNA amplified by the above-mentioned Degenerate PCR was used as Redi.
prime ^TM II random prime lab
It was labeled with an elling system (Amersham Pharmacia Biotech Co., Ltd.).

Example 4: Southern hybridization of Pseudomonas azotoformans IAM1603 chromosomal DNA library Chromosomal DNA of Pseudomonas azotoformans IAM1603 was obtained by the method described in Example 3.
After digesting this DNA with FbaI, Southern hybridization was carried out using the 0.2 kbp labeled DNA prepared in Example 3 as a probe. The procedure was according to Molecular Cloning 3rd Edition, and hybridization was carried out at 42 ° C. in a buffer containing 40% formamide, 2 × SSC, 0.1% SDS. As a result, about 2 kbp
DNA band was detected, this DNA was extracted and purified from agarose gel, and pBluescript SK (-)
(Stratagene) was inserted into the BamHI site and transformed into E. coli by the transformation method using calcium chloride. c
It was introduced into the oli JM109 strain.

Example 5: Colony Hybridization and Subcloning The E. coli obtained in Example 4 was used. The transformed strain of E. coli JM109 was transformed with LB agar medium containing 80 μg / mL ampicillin (in 1 L of an aqueous solution, Bacto Trypton was added.
10.0g, Bacto Yeast Extrac
t 5.0 g, sodium chloride 10.0 g, agar 1
(Including 5.0 g) and cultured overnight at 37 ° C. The resulting colonies were nitrocellulose membrane Hybond ^™ E.
40% formamide, 2 × SSC, 0.1 was used for lifting with CL ^™ (manufactured by Amersham Pharmacia Biotech Co., Ltd.) and using the DNA probe obtained in Example 3.
Hybridization was performed at 42 ° C. in a buffer containing% SDS. As a result of hybridization, a plasmid (pSTB10) was obtained from a positive clone, and L
i-cor DNA Sequencer model
The nucleotide sequence was determined using 4000 L (SEQ ID NO: 12).

Example 6: Construction of plasmid for expression Using pSTB10 obtained in Example 5 above as a template, primers STB-F and STB-R (SEQ ID NOS: 13 and 1)
PCR was performed using 4). The obtained PCR reaction product was purified, digested with restriction enzymes HindIII and XbaI, and digested with the same enzymes to obtain pUC19 (Takara Shuzo Co., Ltd.).
(Manufactured by the same company) to prepare pSTB20, and then E. coli
JM109 was transformed by the calcium chloride method. 80
The transformant was applied to LB agar medium containing μg / mL ampicillin, and grown clones were obtained. col
i JM109 (pSTB20) was obtained.

Example 7: E. coli JM109 (p
Purification of Polypeptides with Stereoselective Amidase Activity from STB 20) E. 80 μg of E. coli JM109 (pSTB20)
After culturing in 2.5 L of LB medium containing / mL ampicillin,
About 10.0 g of bacterial cells was obtained by centrifugation. This is 10
The cell-free extract was obtained by suspending in 0 mM Tris-hydrochloric acid buffer (pH 8.0), sonicating and centrifuging. Ammonium sulfate was added thereto to obtain a 50-70% saturated ammonium sulfate fraction. Furthermore, this was dissolved / dialyzed in 10 mM Tris-hydrochloric acid buffer (pH 8.0), and then adsorbed on a DEAE-Toyopearl column equilibrated with the same buffer to give 10 mM containing 0-200 mM sodium chloride.
Elution was performed with Tris-hydrochloric acid buffer solution (pH 8.0) (linear concentration gradient). Ammonium sulfate was added to the fraction containing the desired activity so as to be 30% saturated, and the Butyl-Toyope was added.
hydrophobic column chromatography using arl,
Elution was performed with a linear gradient method of 30-0% saturated ammonium sulfate. After elution, a fraction containing activity was selected, desalted and concentrated to obtain a purified sample.

Example 8: Enzymatic properties of purified stereoselective amidase Using L-proline amide as a substrate, optimum pH and optimum temperature of amide hydrolysis reaction of the purified enzyme obtained in Example 7, In addition, the temperature tolerance of this enzyme and the influence of 1 mM inhibitor were analyzed by HPLC using L-proline generated by hydrolysis reaction.
It quantified by. The optimum temperature, temperature tolerance, and influence of the inhibitor were measured in 100 mM Tris-HCl buffer (pH 8). P is the optimum pH measurement
It was carried out in various buffer solutions having different H. Reaction is 5 at 30 ℃
Minutes, the reaction was stopped by the addition of ethanol, and HPL
The sample was C. The heat treatment for measuring temperature resistance was carried out by incubating for 5 minutes at each temperature, adjusting the temperature to 30 ° C., and measuring the activity. Regarding the inhibitor, after incubating the enzyme in the presence of 1 mM inhibitor at 30 ° C for 3 minutes, L-prolinamide was added and incubated at 30 ° C for 5 minutes, the reaction was stopped by the addition of ethanol, and the following HPLC was performed. The amount of L-proline produced using condition B was quantified.

HPLC condition B column: Sumichiral OA-5000
(Manufactured by Sumika Chemical Analysis Service Co., Ltd.) Column temperature: 30 ° C. Eluent: 2 mM CuSO ₄ aqueous solution Flow rate: 1.0 mL / min Detection: UV254 nm absorbance The results are summarized in Tables 2 and 3 below.

[0080]

[Table 2]

[0081]

[Table 3]

The Km and Vmax of this enzyme for L-proline-p-nitroanilide were also 100 mM.
As a result of measuring at 30 ° C. in a Tris-hydrochloric acid buffer solution (pH 8.0), the Km value and the Vmax value were each 0.583 m.
M, and 80.9 U / 1 mg polypeptide (1 U is the amount of enzyme required for the enzyme to hydrolyze 1 μmole of L-proline-p-nitroanilide in 1 minute).

Example 9: Examination of substrate selectivity Using the purified enzyme obtained in Example 7, substrate selectivity was examined. Substrate compound was added at a concentration of 20 mM to give 100
An enzyme reaction was performed in mM Tris-hydrochloric acid (pH 8.0) at 30 ° C., and analysis was performed under the following HPLC condition C. HPLC condition C; Column: Schichiral OA-5000 or OA-5000L (manufactured by Sumika Analytical Center Co., Ltd.) Column temperature: 30 ° C Eluent: 2 mM CuSO ₄ aqueous solution Flow rate: 1.0 mL / min Detection: UV 254 nm Absorbance L -Assuming that the activity against prolinamide is 100, its relative activity is shown in Table 4 below.

[0084]

[Table 4]

The amidase activity of the polypeptide of the present invention is particularly strong for L-proline amide or L-proline-p-nitroanilide, and other piperazine-
2-Carboxamide, L-alanine amide, etc. showed weak activity. It also showed significant activity against N-alkylamide compounds.

Example 10: Recombinant E. Stereospecific amide hydrolysis reaction using E. coli coli JM109 (pSTB20) 80 μg /
LB containing mL of ampicillin and 0.5 mM IPTG
3.0 mL of the medium was inoculated and cultured with stirring at 37 ° C. for 12 hours. The cells were obtained by centrifugation at 3000 xg for 10 minutes, suspended in 3.0 mL of 0.9% NaCl, and then again separated by the same centrifugation. 100m of this fungus body
Suspend in M Tris-hydrochloric acid buffer (pH 8.0) to obtain 3.6.
mL of cell suspension was obtained. Of this, 400 μL is 40
100 mM containing 0 μL of 40 mM racemic prolinamide or racemic piperazine-2-carboxamide
After mixing with Tris-HCl buffer (pH 8.0), the mixture was incubated at 30 ° C. After 2 hours, the reaction was stopped with ethanol, and under the above HPLC condition C, the optical purity of proline or piperazine-2-carboxylic acid produced by hydrolysis was measured. As a result, no formation of D-proline or (R) -piperazine-2-carboxylic acid was observed, and the optical purity of L-proline or (S) -piperazine-2-carboxamide was almost 100%.

Example 11: Recombinant E. (RS) -N-tert-butylpiperazine-2-using E. coli
Hydrolysis reaction of carboxamide E. coli JM109 (pSTB20) 80 μg /
LB containing mL of ampicillin and 0.5 mM IPTG
The cells were cultured in a medium and the cells were obtained by centrifugation. The wet cell weight of this was measured, and 200 mM of racemic-N-tert.
-Butylpiperazine-2-carboxamide containing 100
The mixture was added to 1.0 mL of mM Tris-hydrochloric acid buffer (pH 8.0) at 0.28%, 1.41%, and 2.83%, and incubated at 30 ° C. After sampling at regular intervals and stopping the reaction with ethanol, the piperazine-2-carboxylic acid produced under the above HPLC condition B was quantified and the optical purity was measured. The result is shown in Figure 1.
Shown in. As can be seen from the result of FIG. 1, the generated (S)
-The optical purity of piperazine-2-carboxylic acid is 95.5%.
e. e. Met.

[0088]

INDUSTRIAL APPLICABILITY The present invention provides a novel amidase having the ability to stereoselectively hydrolyze nitrogen-containing heterocycle-containing carboxylic acid amides, and a DNA encoding the same. By allowing the amidase of the present invention to act on carboxylic acid amides, it becomes possible to produce chiral carboxylic acids and / or chiral carboxylic acid amides useful as intermediate raw materials for medicines and agricultural chemicals.

[0089]

[Sequence list] SEQUENCE LISTING <110> Mitsubishi Chemical Corporation <120> A novel amidase and a gene encoding it <130> A21129A <160> 14

[0090] <210> 1 <211> 310 <212> PRT <213> Pseudomonas Azotoformans <400> 1 Met Glu Phe Ile Glu Lys Ile Arg Glu Gly Tyr Ala Ala Phe Gly Ala   1 5 10 15 Tyr Gln Thr Trp Tyr Arg Val Thr Gly Asp Leu Ser Ser Gly Arg Thr              20 25 30 Pro Leu Val Val Ile His Gly Gly Pro Gly Cys Thr His Asp Tyr Val          35 40 45 Asp Ala Phe Lys Asp Val Ala Ala Ser Gly His Ala Val Ile His Tyr      50 55 60 Asp Gln Leu Gly Asn Gly Arg Ser Thr His Leu Pro Asp Lys Asp Pro  65 70 75 80 Ser Phe Trp Thr Val Gly Leu Phe Leu Glu Glu Leu Asn Asn Leu Leu                  85 90 95 Asp His Leu Gln Ile Ser Asp Asn Tyr Ala Ile Leu Gly Gln Ser Trp             100 105 110 Gly Gly Met Leu Gly Ser Glu His Ala Ile Leu Gln Pro Lys Gly Leu         115 120 125 Arg Ala Phe Ile Pro Ala Asn Ser Pro Thr Cys Met Arg Thr Trp Val     130 135 140 Ser Glu Ala Asn Arg Leu Arg Lys Leu Leu Pro Glu Gly Val His Glu 145 150 155 160 Thr Leu Leu Lys His Glu Thr Ala Gly Thr Tyr Gln Asp Pro Glu Tyr                 165 170 175 Leu Ala Ala Ser Arg Val Phe Tyr Asp His His Val Cys Arg Val Ile             180 185 190 Pro Trp Pro Glu Glu Val Ala Arg Thr Phe Ala Ala Val Asp Ala Asp         195 200 205 Pro Thr Val Tyr His Ala Met Ser Gly Pro Thr Glu Phe His Val Ile     210 215 220 Gly Ser Leu Lys Asp Trp Lys Ser Thr Gly Arg Leu Ser Ala Ile Asn 225 230 235 240 Val Pro Thr Leu Val Ile Ser Gly Arg His Asp Glu Ala Thr Pro Leu                 245 250 255 Val Val Lys Pro Phe Leu Asp Glu Ile Ala Asp Val Arg Trp Ala Leu             260 265 270 Phe Glu Asp Ser Ser His Met Pro His Val Glu Glu Arg Gln Ala Cys         275 280 285 Met Gly Thr Val Val Lys Phe Leu Asp Glu Val Cys Ser Ala Lys Tyr     290 295 300 Lys Val Leu Lys Ala Ser 305 310

[0091] <210> 2 <211> 933 <212> DNA <213> Pseudomonas Azotoformans <400> 2 atg gaa ttc atc gaa aaa atc cgc gaa ggg tat gcg gcg ttt ggc gcc 48 Met Glu Phe Ile Glu Lys Ile Arg Glu Gly Tyr Ala Ala Phe Gly Ala   1 5 10 15 tac caa acc tgg tac cgc gtc acc ggt gac ctc tcc agc ggt cgc acg 96 Tyr Gln Thr Trp Tyr Arg Val Thr Gly Asp Leu Ser Ser Gly Arg Thr              20 25 30 ccc ctg gtg gtt atc cac ggc ggc ccc gga tgc acc cac gat tac gtt 144 Pro Leu Val Val Ile His Gly Gly Pro Gly Cys Thr His Asp Tyr Val          35 40 45 gac gcg ttc aag gat gtc gcc gcc agc ggc cac gcc gtg atc cac tac 192 Asp Ala Phe Lys Asp Val Ala Ala Ser Gly His Ala Val Ile His Tyr      50 55 60 gac caa ctg ggc aac ggc cgc tcc act cat ctg ccc gac aaa gac cca 240 Asp Gln Leu Gly Asn Gly Arg Ser Thr His Leu Pro Asp Lys Asp Pro  65 70 75 80 tcc ttc tgg acc gta ggc ctg ttt ctc gaa gaa ctg aac aac ctg ctg 288 Ser Phe Trp Thr Val Gly Leu Phe Leu Glu Glu Leu Asn Asn Leu Leu                  85 90 95 gac cat ctg cag atc agc gat aac tac gcg atc ctc ggc cag tcc tgg 336 Asp His Leu Gln Ile Ser Asp Asn Tyr Ala Ile Leu Gly Gln Ser Trp             100 105 110 ggc ggc atg ctc ggc agc gaa cac gcg atc ctg caa ccc aaa ggc ctg 384 Gly Gly Met Leu Gly Ser Glu His Ala Ile Leu Gln Pro Lys Gly Leu         115 120 125 cgt gcg ttc atc ccg gcc aat tcg cca aca tgc atg cgc act tgg gtc 432 Arg Ala Phe Ile Pro Ala Asn Ser Pro Thr Cys Met Arg Thr Trp Val     130 135 140 agc gaa gcc aac cgc ctg cgc aaa tta ttg cct gaa ggc gtg cat gaa 480 Ser Glu Ala Asn Arg Leu Arg Lys Leu Leu Pro Glu Gly Val His Glu 145 150 155 160 acc ctg ctc aag cac gaa acc gcc ggc acc tat cag gac ccg gaa tac 528 Thr Leu Leu Lys His Glu Thr Ala Gly Thr Tyr Gln Asp Pro Glu Tyr                 165 170 175 ctc gcc gca tcg cgg gtg ttc tat gac cat cac gtg tgc cgg gtc atc 576 Leu Ala Ala Ser Arg Val Phe Tyr Asp His His Val Cys Arg Val Ile             180 185 190 cca tgg ccc gag gaa gtg gcg cgc acc ttc gcc gcg gtc gac gcc gac 624 Pro Trp Pro Glu Glu Val Ala Arg Thr Phe Ala Ala Val Asp Ala Asp         195 200 205 ccg acg gtg tac cac gcc atg agc ggc ccg acc gag ttc cat gtg atc 672 Pro Thr Val Tyr His Ala Met Ser Gly Pro Thr Glu Phe His Val Ile     210 215 220 ggc agc ttg aaa gac tgg aaa tcc act ggc cgc cta tcg gcg atc aac 720 Gly Ser Leu Lys Asp Trp Lys Ser Thr Gly Arg Leu Ser Ala Ile Asn 225 230 235 240 gta ccg acc ctg gtg atc tca ggc cgg cac gac gag gcc acg cca ctg 768 Val Pro Thr Leu Val Ile Ser Gly Arg His Asp Glu Ala Thr Pro Leu                 245 250 255 gta gtc aag ccg ttc ctg gat gaa atc gcc gat gtg cgc tgg gcg ctg 816 Val Val Lys Pro Phe Leu Asp Glu Ile Ala Asp Val Arg Trp Ala Leu             260 265 270 ttt gaa gac tcc agc cac atg ccc cat gtg gaa gaa cgc cag gcg tgc 864 Phe Glu Asp Ser Ser His Met Pro His Val Glu Glu Arg Gln Ala Cys         275 280 285 atg ggg act gtg gtg aag ttt ctg gat gag gtg tgt tcg gca aaa tac 912 Met Gly Thr Val Val Lys Phe Leu Asp Glu Val Cys Ser Ala Lys Tyr     290 295 300 aaa gtg ctc aag gcc agc tga 933 Lys Val Leu Lys Ala Ser Stop 305 310

[0092] <210> 3 <211> 10 <212> PRT <213> Pseudomonas Azotoformans <400> 3 Met Glu Phe Ile Glu Lys Ile Arg Glu Gly 1 5 10

[0093] <210> 4 <211> 10 <212> PRT <213> Pseudomonas Azotoformans <400> 4 Ser Thr Gly Arg Leu Ser Ala Ile Asn Val   1 5 10

[0094] <210> 5 <211> 10 <212> PRT <213> Pseudomonas Azotoformans <400> 5 Asp Val Ala Ala Ser Gly His Ala Val Ile   1 5 10

[0095] <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 6 atggagttca tcgagaagat c 21

[0096] <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 7 wssacsggsm gsytswssgc 20

[0097] <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 8 acgttgatsg csswsarsck scc 23

[0098] <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 9 gacggtsgcs gcswssggsc acgc 24

[0099] <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 10 gatsacsgcg tgsccsswsg c 21

[0100] <210> 11 <211> 186 <212> DNA <213> Pseudomonas Azotoformans <400> 11 atg gag ttc atc gag aag atc cgc gaa ggg tat gcg gcg ttt ggc gcc 48 Met Glu Phe Ile Glu Lys Ile Arg Glu Gly Tyr Ala Ala Phe Gly Ala   1 5 10 15 tac caa acc tgg tac cgc gtc acc ggt gac ctc tcc agc ggt cgc acg 96 Tyr Gln Thr Trp Tyr Arg Val Thr Gly Asp Leu Ser Ser Gly Arg Thr              20 25 30 ccc ctg gtg gtt atc cac ggc ggc ccc gga tgc acc cac gat tac gtt 144 Pro Leu Val Val Ile His Gly Gly Pro Gly Cys Thr His Asp Tyr Val          35 40 45 gac gcg ttc aag gat gtc gcc gcc acc ggc cac gcc gtc atc 186 Asp Ala Phe Lys Asp Val Ala Ala Thr Gly His Ala Val Ile      50 55 60

[0101] <210> 12 <211> 2104 <212> DNA <213> Pseudomonas Azotoformans <400> 12 tgatcacccc ttcggtgctg caactgcgca acaccccgcc cgactggcat gccttgtggt 60 gcagcgaagg gttctaccag atcgacccgg tgcaacatct ggccctgagc cgggtgtcgc 120 cgttcgtgtg gtcctacgac gtcaaggccg acaccccgct gcaaaagatc atcgacccct 180 gccacgcgcc ggtttcatcc tacctacatg accagcaatt gacctgcggg gtcagcgtgc 240 cgatccacct gccccgcggt ggctttgcct cgctgaccgg cctgcgtacc ggcaatgccc 300 gcaacgtgct gcaggatgcg cagcagaccc tgtcggattt cggcctgatt tcccatgcgt 360 tacaggaggc cgcctacccg ctgttcagca aagagctgcg cacttacccg catatccacc 420 tgaccaaacg cgaacgtgag tgcctcaaat gggctgccga cggcctgact gcggccgaaa 480 ttgccacaca actgagccgt tccctggcgg tagtcaccct gcacctggcc tcggccatgc 540 acaagctggg ggccaagaac cgcgtgcaag cggtggtgcg cgccacccat taccgcctgc 600 tcgacgattg accgccgggc aaaacctagc tgttttgcta gttacttaaa tttctttcac 660 ccattatcgt gtccccgatc cttttttcag agcagggcac gaa atg gaa ttc atc 715                                                 Met Glu Phe Ile                                                   1 gaa aaa atc cgc gaa ggg tat gcg gcg ttt ggc gcc tac caa acc tgg 763 Glu Lys Ile Arg Glu Gly Tyr Ala Ala Phe Gly Ala Tyr Gln Thr Trp   5 10 15 20 tac cgc gtc acc ggt gac ctc tcc agc ggt cgc acg ccc ctg gtg gtt 811 Tyr Arg Val Thr Gly Asp Leu Ser Ser Gly Arg Thr Pro Leu Val Val                  25 30 35 atc cac ggc ggc ccc gga tgc acc cac gat tac gtt gac gcg ttc aag 859 Ile His Gly Gly Pro Gly Cys Thr His Asp Tyr Val Asp Ala Phe Lys              40 45 50 gat gtc gcc gcc agc ggc cac gcc gtg atc cac tac gac caa ctg ggc 907 Asp Val Ala Ala Ser Gly His Ala Val Ile His Tyr Asp Gln Leu Gly          55 60 65 aac ggc cgc tcc act cat ctg ccc gac aaa gac cca tcc ttc tgg acc 955 Asn Gly Arg Ser Thr His Leu Pro Asp Lys Asp Pro Ser Phe Trp Thr      70 75 80 gta ggc ctg ttt ctc gaa gaa ctg aac aac ctg ctg gac cat ctg cag 1003 Val Gly Leu Phe Leu Glu Glu Leu Asn Asn Leu Leu Asp His Leu Gln  85 90 95 100 atc agc gat aac tac gcg atc ctc ggc cag tcc tgg ggc ggc atg ctc 1051 Ile Ser Asp Asn Tyr Ala Ile Leu Gly Gln Ser Trp Gly Gly Met Leu                 105 110 115 ggc agc gaa cac gcg atc ctg caa ccc aaa ggc ctg cgt gcg ttc atc 1099 Gly Ser Glu His Ala Ile Leu Gln Pro Lys Gly Leu Arg Ala Phe Ile             120 125 130 ccg gcc aat tcg cca aca tgc atg cgc act tgg gtc agc gaa gcc aac 1147 Pro Ala Asn Ser Pro Thr Cys Met Arg Thr Trp Val Ser Glu Ala Asn         135 140 145 cgc ctg cgc aaa tta ttg cct gaa ggc gtg cat gaa acc ctg ctc aag 1195 Arg Leu Arg Lys Leu Leu Pro Glu Gly Val His Glu Thr Leu Leu Lys     150 155 160 cac gaa acc gcc ggc acc tat cag gac ccg gaa tac ctc gcc gca tcg 1243 His Glu Thr Ala Gly Thr Tyr Gln Asp Pro Glu Tyr Leu Ala Ala Ser 165 170 175 180 cgg gtg ttc tat gac cat cac gtg tgc cgg gtc atc cca tgg ccc gag 1291 Arg Val Phe Tyr Asp His His Val Cys Arg Val Ile Pro Trp Pro Glu                 185 190 195 gaa gtg gcg cgc acc ttc gcc gcg gtc gac gcc gac ccg acg gtg tac 1339 Glu Val Ala Arg Thr Phe Ala Ala Val Asp Ala Asp Pro Thr Val Tyr             200 205 210 cac gcc atg agc ggc ccg acc gag ttc cat gtg atc ggc agc ttg aaa 1387 His Ala Met Ser Gly Pro Thr Glu Phe His Val Ile Gly Ser Leu Lys         215 220 225 gac tgg aaa tcc act ggc cgc cta tcg gcg atc aac gta ccg acc ctg 1435 Asp Trp Lys Ser Thr Gly Arg Leu Ser Ala Ile Asn Val Pro Thr Leu     230 235 240 gtg atc tca ggc cgg cac gac gag gcc acg cca ctg gta gtc aag ccg 1483 Val Ile Ser Gly Arg His Asp Glu Ala Thr Pro Leu Val Val Lys Pro 245 250 255 260 ttc ctg gat gaa atc gcc gat gtg cgc tgg gcg ctg ttt gaa gac tcc 1531 Phe Leu Asp Glu Ile Ala Asp Val Arg Trp Ala Leu Phe Glu Asp Ser                 265 270 275 agc cac atg ccc cat gtg gaa gaa cgc cag gcg tgc atg ggg act gtg 1579 Ser His Met Pro His Val Glu Glu Arg Gln Ala Cys Met Gly Thr Val             280 285 290 gtg aag ttt ctg gat gag gtg tgt tcg gca aaa tac aaa gtg ctc aag 1627 Val Lys Phe Leu Asp Glu Val Cys Ser Ala Lys Tyr Lys Val Leu Lys         295 300 305 gcc agc tgaatgctca agctggatgc attccaattg tgggaggggg cttgcccccg at 1685 Ala Ser     310 gacgggggat cagctgagtg atgggttgct gacacaccgc tatcgggggc aagccccctc 1745 ccacattcag acctcagtgg tttgaggctt tgcggccttc ttcgacactt ccgccaccaa 1805 cgggatttcc ctgccctgcg catcaaacaa cttgccgccc ttgaagtaat ccccatcacg 1865 cagttccgac acatcgcgaa agcacaagct acgctcggtc cccgccacaa ataccgactg 1925 ctggtccgag ttaccggcgg tgaaatggtt gaacgccagg ttcagcagga tcgccatgat 1985 tgccgatgag ctgatccccg agtggaaaat agtcgcgaac caagtcggga agtgatcgta 2045 gaagctcggt gccgcaatcg ggatcatgcc aaaaccgatg gaagtggcga cgatgatca 2104

[0102] <210> 13 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 13 cgatccaagc tttaaggagg aataggaaat ggaattcatc gaaaaaatcc g 51

[0103] <210> 14 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic DNA <400> 14 tgcatccatc tagagcattc agc 23

[Brief description of drawings]

FIG. 1 shows that recombinant E. E. coli (R
The measurement result of the hydrolysis reaction of (S) -N-tert-butylpiperazine-2-carboxamide is shown.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 9/80 C12N 15/00 ZNAA C12P 41/00 5/00 A (72) Inventor Makoto Ueda Yokohama, Kanagawa 1000, Kamoshida-cho, Aoba-ku, Aichi-shi Mitsubishi Chemical Corporation F-term (reference) 4B024 AA03 BA11 CA04 DA06 EA04 GA11 HA12 4B050 CC03 DD02 LL05 4B064 AE03 AE35 CA21 CB05 CD27 4B065 AA26X AA41Y AB01 AC14 BA02 CA17 CA31

Claims (10)

[Claims] 1. A stereoselective amidase having the following physicochemical properties. (1) Molecular weight: 34,000 when measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 32000 when measured by gel filtration; (2) Subunit constitution: monomer (3) L-proline amide as substrate Optimum pH of amidase reaction when used: around pH 9 (4) Optimum temperature of amidase reaction when reacted at pH 8 using L-prolinamide as a substrate: around 45 ° C. (5) Heat resistance at pH 8: 40 (6) Compounds that completely inhibit the amidase activity of the present polypeptide, which are stable at ℃ or below: Phenylhydrazine, Zn ²⁺ , Ag ⁺ , Cd
²⁺ , Hg ²⁺ (7) Effect of EDTA: Addition of 1 mM EDTA to the reaction solution promotes enzyme activity. 2. The polypeptide according to any one of the following: (1) a polypeptide having the amino acid sequence represented by SEQ ID NO: 1; (2) an amino acid sequence in which one to a plurality of amino acids are deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 1 A polypeptide having a stereoselective amidase activity; or (3) a polypeptide having an amino acid sequence having 70% or more homology with the amino acid sequence represented by SEQ ID NO: 1 and having a stereoselective amidase activity. ; 3. A DNA encoding the polypeptide according to claim 2. 4. One of the following DNAs. (1) a DNA having the base sequence represented by SEQ ID NO: 2; (2) a base sequence having 1 to a plurality of bases deleted, substituted and / or added in the base sequence represented by SEQ ID NO: 2 And has a stereoselective amidase activity by hybridizing under stringent conditions with a DNA encoding a polypeptide having a stereoselective amidase activity; or (3) a DNA having the base sequence represented by SEQ ID NO: 2. DNA encoding the polypeptide: 5. A recombinant vector having the DNA according to claim 3 or 4. 6. A transformant having the DNA according to claim 3 or 4 or the recombinant vector according to claim 5. 7. The following general formula (I): (In the formula, A is an alkyl group which may be substituted with a substituent selected from a hydroxyl group, an alkoxy group, a thiol group, an alkylthio group, an aryl group and a heteroaryl group, or a carbon atom to which A is bonded and the carbon atom A group capable of forming a nitrogen-containing heterocyclic ring together with an amino group bonded to an atom, R is a hydrogen atom, an optionally substituted alkyl group or an optionally substituted aryl group. ), The amidase according to claim 1, the polypeptide according to claim 2, or the transformant according to claim 6 is allowed to act on the carboxylic acid amide represented by
After selectively hydrolyzing the amide bond of the (S) -form,
The following general formula (II): (In the formula, A has the same meaning as above.) (S)-
Carboxylic acids or / and the following general formula (III): (In the formula, A and R have the same meanings as described above.) A method for producing an optically active carboxylic acid and / or amide, which comprises isolating the (R) -form carboxylic acid amide represented by . 8. The group according to claim 7, wherein A is a group capable of forming a nitrogen-containing heterocycle together with the carbon atom to which A is bonded and the amino group bonded to the carbon atom. Production method. 9. The production method according to claim 7, wherein the reaction is carried out in the presence of ethylenediaminetetraacetic acid. 10. The DNA according to claim 3 or 4.
And one or more types of D having 50% or more homology with the DNA
A method for producing a polypeptide having stereoselective amidase activity or a DNA encoding the same, which comprises randomly recombining NA.