JP4274767B2 - (R) -Amidase gene that selectively hydrolyzes amide bond and its use - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an amidase protein that stereoselectively hydrolyzes an amide bond, a DNA encoding the protein, a recombinant DNA molecule and a vector containing the DNA, the vector or a recombinant host molecule having the recombinant DNA molecule or vector, The present invention also relates to a method for producing chiral carboxylic acids and / or chiral carboxylic acid amides useful as intermediate materials for medicines and agricultural chemicals by allowing the protein or the transformed host cell or a processed product thereof to act on the carboxylic acid amides.
[0002]
[Prior art]
Conventionally, a method for producing (R) -carboxylic acids and / or (S) -carboxamides useful as biomedical / agrochemical intermediates by hydrolyzing racemic nitrogen-containing heterocycle-containing carboxamides. Pseudomonas azotoformans IAM1603 strain or treated product thereof (see Patent Document 1), Pseudomonas sp. MCI3433 strain or Pseudomonas sp. MCI3434 strain or treated product thereof (see Patent Document 2) Acting on racemic N-tertbutylpiperazine-2-carboxamide to obtain (R) -piperazine-2-carboxylic acid and / or (S) -N-tertbutylpiperazine-2-carboxylic acid amide with high optical purity The method is known.
[0003]
However, for example, in the technique described in Patent Document 1, cells or treated products thereof are used as a hydrolysis catalyst. However, the enzyme content for catalyzing the target reaction is insufficient, and the enzyme itself is unstable. In order to catalyze the reaction, it is necessary to prepare a large amount of microbial cells, and further, a great load is imposed on the purification of the reaction product, which is not an economically advantageous method. In addition, for example, in the technique described in Patent Document 2, the stereoselectivity of the microbial cell or its processed product is insufficient, so that the optical purity of the (R) -isomer carboxylic acid produced is insufficient, The (S) -form carboxamide provides a product with high optical purity, but it is not an economically advantageous method because of its low yield.
[0004]
[Patent Document 1]
JP-A-10-276794
[Patent Document 2]
JP-A-10-327890
[0005]
[Problems to be solved by the invention]
An object of the present invention is to obtain a novel amidase having the ability to stereoselectively hydrolyze the (R) -form of a nitrogen-containing heterocyclic ring-containing carboxylic acid amide, and a DNA encoding the same, and to use the amidase Another object of the present invention is to provide a method for producing (R) -nitrogen-containing heterocycle-containing carboxylic acids and / or (S) -nitrogen-containing heterocarboxylates having high optical purity.
[0006]
[Means for Solving the Problems]
As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that Pseudomonas sp. MCI3434 strain, which is known to selectively hydrolyze (R) -N-alkylpiperazine-2-carboxamide, although insufficient. From which an enzyme that selectively hydrolyzes (R) -N-alkylpiperazine-2-carboxamide was isolated, and a DNA encoding the enzyme was obtained. We found that a stereoselective amidase-rich recombinant organism or a high-concentration amidase solution produced using this DNA proceeds a higher stereoselective carboxylic acid amide hydrolysis reaction with higher productivity. . Furthermore, a protein having a high degree of coincidence (65%) with the amino acid sequence of the amidase is isolated from Pseudomonas aeruginosa strain PAO1, and a transformed host cell having the DNA is prepared. It has been found that the protein to be produced acts on a racemic nitrogen-containing complex-containing carboxamide to hydrolyze the amide bond specifically in the (R) -form, thereby completing the present invention.
[0007]
  That is, according to the present invention, a stereoselective amidase having the following physicochemical properties is provided.
(A) The apparent molecular weight measured by SDS-PAGE is about 29,000-31,000;
(B) The optimum pH of the amidase reaction when (RS) -piperazine-2-carboxamide or (RS) -N-tertbutylpiperazine-2-carboxamide is used as a substrate is around pH 8;
(C) The optimal temperature of the amidase reaction when using (RS) -piperazine-2-carboxamide or (RS) -N-tertbutylpiperazine-2-carboxamide as a substrate is 45-50 ° C;
(D) stable at a temperature of 45 ° C. or lower near pH 8.0;
(E) p-chloromercurybenzoic acid, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Pb²⁺Completely inhibits activity;
(F) When (RS) -piperazine-2-carboxamide is used as a substrateThe amide bond(R) -bodyHas the activity of selectively hydrolyzing to produce (R) -piperazine-2-carboxylic acid; and
(G) Subunit structure is monomerIt is.
[0008]
  According to another aspect of the present invention, a protein having any of the following amino acid sequences is provided.
(A) the amino acid sequence set forth in SEQ ID NO: 1;
(BDistributionColumn index1An amino acid sequence in which one to a plurality of amino acids are deleted, added, or substituted in the described amino acid sequence,(RS)-Piperazine-2-carboxamideAs a substrateThe amide bond is hydrolyzed selectively in the (R) -formTo produce (R) -piperazine-2-carboxylic acidAmino acid sequenceColumn.
[0009]
  According to still another aspect of the present invention, a DNA having any one of the following base sequences is provided.
(A) a base sequence encoding the amino acid sequence set forth in SEQ ID NO: 1;
(BDistributionColumn index1An amino acid sequence in which one to a plurality of amino acids are deleted, added, or substituted in the described amino acid sequence,(RS)-Acts on piperazine-2-carboxamide and hydrolyzes its amide bond selectively in (R) -formTo produce (R) -piperazine-2-carboxylic acidA nucleotide sequence encoding an amino acid sequence having the activity of:
(CDistributionThe nucleotide sequence set forth in Column No. 2;
(D) DistributionColumn index2A nucleotide sequence in which one to a plurality of bases are deleted, added, or substituted in the described nucleotide sequence,(RS)-Piperazine-2-carboxamideAs a substrateThe amide bond is hydrolyzed selectively in the (R) -formTo produce (R) -piperazine-2-carboxylic acidBase encoding a protein having the activityColumn.
[0010]
According to still another aspect of the present invention, a recombinant DNA molecule or vector comprising the above-described DNA of the present invention is provided.
According to still another aspect of the present invention, there is provided a transformed host cell having the above-described recombinant DNA molecule or vector of the present invention.
According to still another aspect of the present invention, the following general formula (I)
[0011]
[Chemical 7]
[0012]
(In the formula, A represents an optionally substituted hydrocarbon chain having 2 to 5 main chain atoms, the hydrocarbon chain may contain a double bond, and may be a nitrogen atom or a nitrogen atom. And R and R ′ each independently represents a hydrogen atom, an optionally substituted alkyl group or an optionally substituted aryl group, where n is 0 or 1))(RS)-The amide bond of (R) -form is selectively hydrolyzed by allowing the above-described amidase or protein of the present invention, or a transformed host cell or a processed product thereof to act on carboxylic acid amides.do it,The following general formula (II)
[0013]
[Chemical 8]
[0014]
(In the formula, A, R ′ and n are as defined above.)
Represented byA method for producing optically active (R) -carboxylic acids, wherein (R) -carboxylic acids are produced and then isolated;And / orRemainThe following general formula (III)
[0015]
[Chemical 9]
[0016]
(In the formula, A, R, R ′ and n are as defined above.)
(S)-Optically active, characterized by isolating rubonamides(S)-CarvoneAcidProvided is a method for producing amides.
[0017]
The present invention is described in detail below.
[0018]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments and implementation methods of the present invention will be described in detail.
(1) Amidase and protein of the present invention
The present invention relates to a stereoselective amidase having the following physicochemical properties.
(A) The apparent molecular weight measured by SDS-PAGE is about 29,000-31,000;
(B) The optimum pH of the amidase reaction when (RS) -piperazine-2-carboxamide or (RS) -N-tertbutylpiperazine-2-carboxamide is used as a substrate is around pH 8;
(C) The optimal temperature of the amidase reaction when using (RS) -piperazine-2-carboxamide or (RS) -N-tertbutylpiperazine-2-carboxamide as a substrate is 45-50 ° C;
(D) stable at a temperature of 45 ° C. or lower near pH 8.0;
(E) p-chloromercurybenzoic acid, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Pb²⁺Completely inhibits activity; and
(F) The stereoselectivity of the amidase reaction when (RS) -piperazine-2-carboxamide is used as a substrate is (R) -isomer selective, and the E value is 30-60.
[0019]
Examples of the amidase of the present invention include, for example, proteins represented by the amino acid sequence of SEQ ID NO: 1 or 3, homologs thereof, and an enzyme that hydrolyzes piperazine-2-carboxamide selectively (R) -form A protein having activity, or a protein containing in its molecule the amino acid sequence set forth in SEQ ID NO: 1 or 3 or the amino acid sequence of a homologue thereof.
[0020]
Here, the homologue of the protein of the present invention is a protein having a homology of 60% or more, preferably 70% or more, more preferably 90% or more with the amino acid sequence shown in SEQ ID NO: 1 or 3, which is piperazine- A protein having an activity of acting on 2-carboxamide and hydrolyzing its amide bond selectively in the (R) -form. These homologs include proteins represented by amino acid sequences in which one to a plurality of amino acids are deleted, added or substituted in the amino acid sequence shown in SEQ ID NO: 1 or 3, and include piperazine-2-carboxamide. Also included are proteins that act and act to hydrolyze the amide bond selectively in the (R) -form.
[0021]
Incidentally, the homology search of the protein can be performed using, for example, a FASTA program or a BLAST program. As a result of homology search of amino acid sequence described in SEQ ID NO: 1 using BLAST program for amino acid sequence database such as Protein Data Bank (PDB), it showed the highest amino acid identity among known proteins Was conserved hypothetical protein PA3598 (65%) (Stover, CK, et al. [Nature, (2000), 406 (6799): 959-964]) of Pseudomonas aeruginosa PAO1 strain described in SEQ ID NO: 2. . Next, as a highly homologous protein, conserved hypothetical protein (NCBI accession number: CAC47075) of Sinolizobium meriroti 1012 strain was searched, and its amino acid identity was 36% (Galibert, F. et al. [Science, (2001), 293 (5530): 668-672]), and it could not be said that there was significant homology.
[0022]
In addition, “a protein containing in its molecule the amino acid sequence of SEQ ID NO: 1 or 3 or its homologous amino acid sequence” means a protein interaction, a ligand binding function or another special function at the N-terminus or C-terminus of the protein. It means a fusion protein to which the protein possessed is bound.
Examples of the fusion protein include Gilbert H., et al. Using ordinary molecular genetic techniques. J. et al. [Mol. Microbiol. 4, 759-767, (1990)] and Shoseyov O., et al. (US Pat. No. 5,496,934) reported by adding an amino acid sequence having a cellulose binding function to the N-terminus or C-terminus of the protein of the present invention, and binding a histidine hexamer having a nickel-binding function. , Those that gave specific binding ability to the protein, and KimG. J. et al. [Biotechnol. Bioeng. 68, 211-217, (2000)], and the like. The protein of the present invention is fused with a protein having other functions.
[0023]
Since the amino acid sequence of the protein of the present invention has been clarified, as described later, a DNA probe prepared based on a base sequence encoding part or all of the amino acid sequence of SEQ ID NO: 1 or 3 is used. By isolating DNA encoding a protein having (R) -selective-piperazine-2-carboxamide hydrolysis activity from any microorganism having (R) -piperazine-2-carboxamide hydrolysis activity it can. In addition, DNA encoding a protein having (R) -isomer selective piperazine-2-carboxamide hydrolysis activity can also be synthesized using a DNA synthesizer. Furthermore, a large amount of the protein can be produced by molecular genetic techniques using the obtained DNA, and the protein can be isolated therefrom by a conventional method. It can also be purified from a microorganism having the above DNA, for example, cultured cells of Pseudomonas sp. MCI3434 strain.
[0024]
The Pseudomonas sp. MCI3434 strain and Pseudomonas aeruginosa PAO1 strain are both known microorganisms, the former being deposited and registered as FERM P-16170 at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary, and the latter being American type culture. In the collection (ATCC), Pseudomonas aeruginosa ATCC 47085 and its genomic DNA are registered as ATCC 47085D and are readily available.
[0025]
As a method for isolating the protein of the present invention from cultured cells of microorganisms, general protein purification methods can be applied. For example, the above microorganisms are grown by culturing in a general microorganism nutrient medium, preferably a medium containing nutritive broth as a nitrogen source and malic acid as a carbon source, and then collected by centrifugation and subjected to ultrasonic disruption. A cell-free extract is obtained by treatment or French press treatment. From the cell-free extract, the target protein can be isolated by utilizing the difference in the physicochemical properties of the protein by operations such as centrifugation, column chromatography, and electrophoresis. As a more recommended technique, a cell-free extract is prepared from the above microorganism, and the protein of the present invention can be purified using ion exchange chromatography, hydrophobic chromatography, adsorption chromatography, size exclusion chromatography, or the like. Alternatively, as will be described later, a recombinant organism, preferably Escherichia coli (hereinafter referred to as “this”), which incorporates a DNA containing the DNA represented by SEQ ID NO: 2 or 4 downstream of an appropriate expression promoter and a ribosome binding sequence. A cell-free extract is prepared from the cultured cells of E. coli) by ultrasonic disruption and centrifugation, and the protein of the present application is prepared using ammonium sulfate fractionation and ion exchange chromatography. It can also be isolated.
[0026]
The physical and enzymatic properties of a protein having the amino acid sequence of SEQ ID NO: 1 isolated from Pseudomonas sp. MCI3434 strain are shown below.
1. Molecular weight: 30128 (estimated from amino acid sequence)
29000 (measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (hereinafter sometimes referred to as “SDS-PAGE”))
36000 (measured by gel filtration method)
2. Subunit structure: Monomer
3. Optimum pH of amidase reaction: pH 8.0
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 this protein: p-chloromercurybenzoic acid, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Pb²⁺. 7. Substrate specificity: High amidase activity for β-alanine amide, D-glutamine amide, and (R) nitrogen-containing heterocyclic carboxylic acid amides. More preferred substrates include (R) -piperazine-2-carboxamide, piperidine-3-carboxamide, (R) -N-tert-butylpiperazine-2-carboxamide, and the like.
8). Stereoselectivity: E value is 59 when reacted with racemic piperazine-2-carboxamide.
[0027]
The physical and enzymatic properties of the protein having the amino acid sequence of SEQ ID NO: 3 isolated from Pseudomonas aeruginosa PAO1 are shown below.
1. Molecular weight: 29590 (estimated from amino acid sequence)
32000 (SDS-PAGE)
87000 (gel filtration)
2. Subunit structure: Homodimer
3. Optimum pH of amidase reaction: pH 8.0
4). Amidase reaction optimum temperature: 50 ° C
5). Heat resistance: stable at 45 ° C. or lower.
6). Compounds that completely inhibit the amidase activity of this protein: N-ethylmaleimide, p-chloromercurybenzoic acid, Cu²⁺, Zn²⁺, Ag⁺, Cd²⁺, Hg²⁺, Tl⁺, Pb²⁺.
7. Stereoselectivity: E value is 30 when reacted with racemic piperazine-2-carboxamide.
[0028]
(2) DNA of the present invention
The DNA of the present invention is a DNA encoding the above-mentioned (1) amidase and protein of the present invention. This DNA includes DNA encoding the homologue of the above protein (hereinafter sometimes referred to as “DNA homologue”).
As DNA which codes the said protein, what contains the base sequence represented by sequence number 2 or 4 is mentioned, for example.
The DNA homolog encoding the protein of the present invention is a DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added to the amino acid sequence set forth in SEQ ID NO: 1 or 3. including.
[0029]
Those skilled in the art will be able to introduce site-directed mutagenesis (Nucleic Acids Res. 10, pp. 6487 (1982), Methods Enzymol. 100, pp. 448 (1983), Molecular Cloning 2nd Edt) into the DNA described in SEQ ID NO: 2 or 4. , Cold Spring Harbor Laboratory Press (1989) PCR A Practical Approach IRL Press pp.200 (1991)) can be used to obtain DNA homologues as appropriate by introducing substitution, deletion, insertion and / or addition mutations. It is.
[0030]
The DNA homologue also includes DNA that hybridizes with the base sequence represented by SEQ ID NO: 2 or 4 or a complementary sequence thereof under stringent conditions and encodes a protein having the above activity.
In this specification, “DNA that hybridizes under stringent conditions” means DNA obtained by using a DNA as a probe and using a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like. After performing hybridization at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using, for example, a colony or plaque-derived DNA or a filter on which the DNA fragment is immobilized, Examples include DNA that can be identified by washing the filter under a condition of 65 ° C. using a 0.1 to 2 × SSC solution (the composition of 1 × SSC is 150 mM sodium chloride, 15 mM sodium citrate).
[0031]
Hybridization was performed using Molecular Cloning: A Laboratory Manual, 3rd Ed. (Ed. Sambrook J. and Russell D.W.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001, and hereinafter may be abbreviated as “Molecular Cloning 3rd Edition”. ) And the like.
[0032]
The DNA encoding the protein of the present invention can be obtained, for example, by the following method. First, SEQ ID NO: 2 by amino acid sequence homology search using a BLAST program for a microorganism having amide bond hydrolysis activity of (R) -piperazine-2-carboxamide or an amino acid sequence database such as Protein Data Bank (PDB). 4 or a chromosomal DNA prepared from a microorganism found to have a homologue thereof, a plasmid library or a phage library is prepared by a conventional method. Next, using the information on the partial amino acid sequence of the protein, a partial fragment of DNA encoding the protein is obtained, and a DNA probe is prepared by a conventional method. By using this DNA probe, DNA encoding the protein of the present invention is identified from the chromosomal DNA library described above, and DNA encoding the protein can be obtained by subcloning an appropriate DNA.
[0033]
More specifically, the DNA according to the present invention can be obtained by the following method.
1. Based on the N-terminal of purified (R) -selective amidase obtained from Pseudomonas sp. MCI3434 and information on the internal amino acid sequence, degenerate PCR (abbreviation of polymerase chain reaction) using the chromosomal DNA of Pseudomonas sp. MCI3434 as a template. (Knoth, K., et al., Nucleic Acids Res., (1988), 16: 10932, and Lee, CC, et al., Science, (1988), 239: 1288-1291), and obtaining (R) -form A partial fragment of DNA encoding a protein having selective amidase activity is obtained. This is labeled with a nucleic acid containing a radioisotope to prepare a DNA probe.
2. Chromosomal DNA is extracted from Pseudomonas sp. MCI3434 strain and partially degraded with an appropriate restriction enzyme (eg, FbaI).
3. Southern hybridization is performed using the DNA fragment obtained in 2 above and the DNA probe prepared in 1.
4). The DNA specified in 3 above is extracted and purified, bound to an appropriate plasmid vector, and transformed into E. coli.
5). Colony hybridization is performed using the DNA probe prepared in 1 above, and a clone plasmid containing the target DNA sequence is obtained.
6). Using the plasmid obtained in 5 above, the base sequence of the DNA derived from Pseudomonas sp. MCI3434 incorporated into the vector is determined.
7. The reading frame of 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 of the amidase is present in the DNA fragment obtained in the above step.
[0034]
DNA in the above process and E. coli as a recombinant host. The general operations required for handling E. coli are those performed by those skilled in the art, and can be easily performed according to, for example, the 3rd edition of Molecular Cloning. Commercially available products can also be used for the enzymes and reagents used, and unless otherwise specified, the purpose can be completely achieved if the use conditions specified for the products are followed. In particular, the above 1. Extraction of chromosomal DNA from strains in, for example, Saito et al. [Biochim. Biophys. Acta, 72, 619-629 (1963)]. For labeling of DNA, any of the conventionally used radioisotopes or non-radioactive compounds such as digoxigenin, biotin, fluorescein, etc. can be used.^TMII random prime labeling system (Amersham Pharmacia Biotech) and AlkPhos^TM  It can be easily carried out using Direct Labeling and Detection System (Amersham Pharmacia Biotech Co., Ltd.).
[0035]
4. above. As a vector in p, for example, pBluescriptSK (-) (Stratagene) can be used.
Above 6. The DNA base sequence in can also be determined by a known method described in Molecular Cloning 3rd edition and the like. For example, it can be easily carried out by using a device such as Li-cor DNA Sequencer model 4000L according to the attached manual instructions.
[0036]
Furthermore, by recombining the DNA having the base sequence represented by SEQ ID NO: 2 or 4 or a DNA homolog thereof, and the DNA having a base sequence highly homologous with any of them in vivo or in vitro DNA having a new base sequence having amidase activity can be obtained. In this case, “highly homologous” refers to a nucleotide sequence having a nucleic acid level identity of 50% or more. For random recombination of DNA in vivo, see Cherry J. et al. R. [Nat. Biotechnol. 17, 33-334 (1999)], and random recombination of DNA in vitro, see Stemmer W. et al. P. C. [Proc. Natl. Acad. Sci. USA, 91, 10747-10651 (1994)], or Zhao H. et al. [Nat. Biotechnol. 16, 258-261 (1998)]. In particular, CrameriA. [Nature, 391, 288-291 (1998)], recombination in vitro using several kinds of DNA encoding a hydrolase having high homology with each other to achieve specific activity and stereoselectivity. It is possible to obtain DNA encoding a stereospecific amidase with improved
[0037]
(3) Vector and transformant of the present invention
A stereoselective amidase expression vector is provided by inserting the DNA encoding the protein of the present invention obtained in (2) above into a known expression vector. Moreover, a transformant (recombinant) can be obtained by transforming a biological cell with this expression vector, and a stereoselective amidase can be obtained by culturing the obtained transformant.
[0038]
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 the enzyme reaction, and a host vector system has been established. Bacteria, actinomycetes, yeasts, molds, and other microorganisms, insect cells, plant cells, and animal cells can be suitably used. Specific examples include microorganisms and higher biological cells as shown below.
[0039]
Escherichia genus, Bacillus genus, Pseudomonas genus, Brevibacterium genus, Corynebacterium genus, Rhodococcus genus, Streptomyces genus Streptomyces , Shizosaccharomyces genus, Pichia genus, Candida genus, Aspergillus genus, persimmon, rapeseed, soybean, and the like.
[0040]
The host cells are preferably Escherichia, Bacillus, Pseudomonas, Brevibacterium, Corynebacterium, and Rhodococcus microorganisms, and particularly preferably Escherichia, Pseudomonas, Brevibacterium, and Coryne. It is a bacterium belonging to the genus Bacteria.
In order to produce the protein of the present invention in vivo, the DNA of the present invention is introduced into a vector that is stably present in the host organism, or directly into the host genome by a technique such as homologous recombination. To create a recombinant DNA molecule and to transcribe and translate the genetic information.
[0041]
At this time, it is necessary to incorporate a promoter and a ribosome binding site (RBS) on the 5 'upstream side of the DNA strand of the present invention, and more preferably a transcription terminator on the 3' downstream side. The promoter, RBS and transcription terminator are not particularly limited as long as they are known to function in cells used as hosts, RBS and transcription terminators, and vectors usable in these various organisms. The promoter, RBS, and transcription terminator are described in detail in, for example, Molecular Cloning 3rd Edition and “Microbiology Basic Course 8 Genetic Engineering / Kyoritsu Shuppan”.
[0042]
Specifically, for example, Escherichia, particularly E. coli. As for E. coli, vectors include pBR and pUC plasmids, and promoters derived from lac (β-galactosidase), trp (tryptophan operon), tac, trc, lpp, λ phage PL, PR, and the like. Examples of transcription termination factors include trpA-derived, phage-derived, ribosomal RNA-derived, and lpp-derived. In this way, E.I. When using E. coli as a protein synthesis system, the target protein is a recombinant E. coli vector containing the DNA of the present application. It is produced constitutively along with the growth of E. coli or inductively by adding a protein synthesis inducer such as isopropylthio-β-D-galactoside (hereinafter IPTG).
[0043]
In addition to producing a stereoselective amidase in vivo by the method described above, The amidase can also be produced in vitro using a cell-free protein synthesis system of E. coli or wheat germ. For example, E.I. In E. coli, Kagawa et al. [FEBS Letters, 442, 15-19 (1999)], and for the synthesis system using wheat germ, Madin et al. [Proc. Natl. Acad. Sci. USA, 97, 559-564 (2000)].
[0044]
(4) Production of optically active carboxylic acids and / or amides using the amidase of the present invention
In the present invention, the protein of the present invention or the transformant obtained above is represented by the following general formula (I):
[0045]
[Chemical Formula 10]
[0046]
(In the formula, A represents an optionally substituted hydrocarbon chain having 2 to 5 main chain atoms, the hydrocarbon chain may contain a double bond, and may be a nitrogen atom or a nitrogen atom. And R and R ′ each independently represents a hydrogen atom, an optionally substituted alkyl group or an optionally substituted aryl group, where n is 0 or 1), the amide bond of the (R) -form is selectively hydrolyzed by acting on a carboxylic acid amide represented by the following general formula (II):
[0047]
Embedded image
[0048]
(Wherein, A, R ′ and n are as defined above) (R) -form carboxylic acids represented by the formula: and / or the following general formula (III)
[0049]
Embedded image
[0050]
(Wherein A, R, R ′ and n are as defined above) (S) -form carboxylic acid amides are isolated and optically active carboxylic acids and / or Or it is the method of manufacturing amides.
In the above general formula (I), A represents an optionally substituted hydrocarbon chain having 2 to 5 main chain atoms, and the hydrocarbon chain may contain a double bond, or nitrogen. One hetero atom other than an atom or a nitrogen atom may be contained. That is, A is a group that forms a nitrogen-containing heterocyclic group together with the nitrogen atom and carbon atom to which it is bonded, and in the formed nitrogen-containing heterocyclic group, a hetero atom other than the nitrogen atom is present. It may be included. Here, examples of the hydrocarbon chain A substituent include a halogen atom, an alkyl group, an aryl group, a hydroxyl group, an amino group, a nitro group, a carboxyl group, and an alkoxy group.
[0051]
The nitrogen-containing heterocyclic group is preferably a 5- to 7-membered nitrogen-containing heterocyclic group, more preferably a nitrogen-containing heterocyclic group containing 1 or 2 nitrogen atoms and no other heteroatoms. . Preferable specific examples of the nitrogen-containing heterocyclic group include piperidyl group, piperazyl group, pyrazyl group and 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 the same atoms or groups as those of the substituents R ′ and A, That is, a halogen atom, an optionally substituted alkyl group, an optionally substituted aryl group, a hydroxyl group, an amino group, a nitro group, a carboxyl group, an alkoxy group, and the like can be given. Of the alkyl group.
[0052]
In the general formula (I), the substituents R and R ′ represent a hydrogen atom, an optionally substituted alkyl group, or an optionally substituted aryl group.
Examples of the alkyl group include straight-chain, branched-chain, and cyclic alkyl groups. Of these, those having 1 to 6 carbon atoms are preferable, and examples of the aryl group include a phenyl group and a naphthyl group.
[0053]
Examples of the substituent of the alkyl group or aryl group include the same groups as those described as the substituent of the hydrocarbon chain of A.
Specific examples of the alkyl group or aryl group which may be substituted include methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, t-butyl group, cyclohexyl group, benzyl group, trifluoro group. Examples include a methyl group, 1-hydroxycarbonylethyl group, phenyl group, tolyl group and the like.
[0054]
The substituent R is preferably a hydrogen atom or an alkyl group having 1 to 4 carbon atoms or a phenyl group having a substituent, more preferably a hydrogen atom or an alkyl group having 1 to 4 carbon atoms and a p-nitrophenyl group. It is.
In this production method, the recombinant organism accumulating the protein of the present invention, the processed product of the recombinant organism, the cell-free protein synthesis reaction solution in which the protein of the present invention is accumulated and / or the treated product react as an enzyme catalyst. Added to the system. Specifically, the cells obtained by culturing the above-mentioned recombinant organisms, or processed products treated by known techniques such as treatment with organic compounds such as toluene and surfactant, freeze-drying treatment, physical disruption treatment, etc. Then, the stereoselective amidase activity fraction of the present invention is taken out from the reaction solution of the cell-free protein synthesis system, and further from the processed product of the recombinant organism and the cell-free protein synthesis reaction solution as a crudely purified fraction or a purified product. It is also possible to use it. Furthermore, it is also possible to use a recombinant organism, a recombinant organism treatment product, an enzyme fraction, and the like obtained as described above immobilized on a carrier such as polyacrylamide gel and carrageenan gel. Hereinafter, the term “high-concentration enzyme preparation” is used as a term encompassing all the concepts of the above-mentioned recombinant organisms, cell-free protein synthesis reaction products, processed products thereof, enzyme fractions, and immobilized products.
[0055]
In this reaction, racemic carboxylic acid amides, which are reaction raw materials, may be subject to substrate inhibition if the substrate concentration is too high. Therefore, the concentration is usually 0.1 mM to 1 M, and more preferably 10 mM. Add to the reaction at a concentration of ~ 500 mM. These may be added all at once at the start of the reaction, but can also be added continuously or intermittently in order to improve the accumulated concentration of the product.
[0056]
This reaction is carried out in an aqueous medium or in a mixed liquid of the aqueous medium and an organic solvent. Examples of the aqueous medium include water or a buffer solution such as an aqueous potassium phosphate solution or an aqueous tris-hydrochloric acid solution. Examples of organic solvents include alcohols such as ethanol and propanol; ketones such as acetone; ethers such as tetrahydrofuran; esters such as ethyl acetate and butyl acetate; aromatic hydrocarbons such as toluene; Halogenated hydrocarbons; aliphatic hydrocarbons such as n-hexane; organic compounds such as dimethyl sulfoxide can be used.
[0057]
Although the reaction proceeds suitably in an alkaline state, the pH of the reaction solution is preferably pH 6.0 to 9.5, more preferably pH 7.0 to 9.5. In order to satisfy this condition, a dilute acid or alkali solution can be added as needed during the reaction to maintain the pH within a desired range.
The reaction proceeds at a reaction temperature of 0 to 50 ° C., but is more preferably maintained at 25 to 45 ° C. The reaction solution is stirred and mixed as necessary.
[0058]
Since the protein of the present invention has the effect of stereoselectively hydrolyzing the carboxylic acid amides represented by the above formula (I), (R) -form carboxylic acid is generated in the reaction solution, and (S) -Remaining amides remain.
The (R) -form carboxylic acid and / or the remaining (S) -form amide compound (amides) produced by the above reaction are isolated by a conventional separation / purification method such as solvent extraction or chromatography. be able to.
[0059]
【Example】
EXAMPLES The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples, and usual modifications in the technical field of the present invention can be made without departing from the gist thereof. .
Example 1: Purification of Pseudomonas sp. MCI3434 Stereoselective Amidase
Pseudomonas sp. MCI3434 strain was inoculated into 25.0 liters of the medium shown in Table 1, cultured at 25 ° C. for 8 hours, and the cells were obtained by centrifugation.
[0060]
[Table 1]
[0061]
The obtained cells were sonicated and a cell-free extract was prepared by centrifugation. This is adsorbed on DEAE-Toyopearl resin equilibrated with 10 mM potassium phosphate buffer (pH 7.0) and subjected to ion exchange chromatography, and a fraction having a stereoamidase activity of the eluted fraction containing 100 mM sodium chloride is obtained. This protein was isolated by sequential column chromatography using a Butyl-Toyopearl column using ammonium sulfate, DEAE-Toyopearl column, Gigapite column, and FPLC Superdex 200 again. In order to determine the molecular weight of the protein, it was 29000 (SDS-PAGE) and 36000 (size exclusion chromatography), respectively, as measured by size exclusion chromatography with SDS-PAGE and TSKgel G3000SW (Tosoh Corp.). The subunit composition was assumed to be monomeric.
[0062]
Example 2: Determination of N-terminal amino acid sequence of a stereoselective amidase derived from Pseudomonas sp. MCI3434
The N-terminal amino acid sequence of the purified stereospecific amidase obtained in Example 1 was analyzed using an automated Edman degradation amino acid sequencer Prosequencer 6625 (Millipore). The N-terminal amino acid sequence determined as described above is shown in SEQ ID NO: 5.
[0063]
Example 3: Acquisition of a partial fragment of DNA encoding a stereoselective amidase derived from Pseudomonas sp. MCI3434
Pseudomonas sp. MCI3434 strain was inoculated into 100 mL of TGY medium (Table 2) and cultured at 30 ° C. for 12 hours.
[0064]
[Table 2]
[0065]
Microbial cells were obtained by centrifugation, and chromosomal DNA of the microorganism was obtained using the phenol chloroform method. When a protein containing an amino acid sequence having a high sequence homology with the amino acid sequence of SEQ ID NO: 5 found in Example 2 was searched using the BLAST program, the amino acid sequences of several hydrolases derived from bacteria had the highest homology (30 About 60%). Therefore, Pseudomonas aeruginosa PAO1 strain unknown protein PA3598, Streptomyces coelicolor A3 (2) strain unknown hydrolase SCL6.12c (Redenbach, M et al. [Mol. Microbiol., ( 1996), 21 (1): 77-96)] and Hypothetical protein (Schneider, U. et al. [Appl. Environ. Microbiol., (1995), 61 (11): 3856-] of the PQQF 5 ′ region of Pseudomonas florescence. 3864]) was aligned, and two amino acid sequences that exist in common were selected (SEQ ID NO: 6: YRKTHL, SEQ ID NO: 7: DIEFPE). Based on these sequences, PCR primers shown in SEQ ID NOs: 8 and 9 (3434HA5 and 3434HA7) are designed, these primers are added in the presence of heat-resistant DNA polymerase, and PCR reaction is carried out using the MCI3434 strain chromosomal DNA as a template. went. When the product of this PCR reaction was subjected to agarose gel electrophoresis, 120 bp of DNA was specifically amplified, and this was extracted and purified from the agarose electrophoresis gel. A part of the DNA was cloned into pT7Blue T-Vector (Novagen), and the base sequence was determined using Li-cor DNA Sequencer model 4000L (the operation method was in accordance with the attached manual instructions) (SEQ ID NO: 10). . As a result of analyzing the obtained nucleotide sequence, it was revealed that the fragment amplified by PCR was a partial fragment of a DNA encoding a stereoselective amidase derived from Pseudomonas sp. MCI3434. The remainder of the 120 bp purified DNA amplified by the above Degenerate PCR was used as a rediprime.^TMIt was labeled with II random prime labeling system (Amersham Pharmacia Biotech Co., Ltd.) to obtain a DNA probe.
[0066]
Example 4: Isolation of a stereoselective amidase gene from Pseudomonas sp. MCI3434 strain and determination of its nucleotide sequence
Chromosomal DNA was extracted from Pseudomonas sp. MCI3434 strain by the method described in Example 3. This DNA was digested with PstI, EcoRV, or FbaI, and then Southern hybridization was performed using the DNA probe prepared in Example 3. The procedure was in accordance with the third edition of Molecular Cloning, and hybridization was performed at 42 ° C. in a buffer containing 40% formamide, 2 × SSC, and 0.1% SDS. As a result, DNA bands of about 2.1 kbp, about 5.5 kbp, and about 5.3 kbp were detected, respectively. These DNAs were extracted and purified from an agarose gel, inserted into the PstI, HincII or BamHI sites of pBluescriptSK (−) (Stratagene), respectively, and transformed into E. coli by a transformation method using calcium chloride. E. coli strain JM109.
[0067]
The obtained E.I. E. coli JM109 transformed strain was applied to LB agar medium containing 80 μg / mL ampicillin (1 L aqueous solution containing Bacto Trypton 10.0 g, Bacto Yeast Extract 5.0 g, sodium chloride 10.0 g, agar 15.0 g) And cultured overnight at 37 ° C. The resulting colonies were nitrocellulose membrane Hybond^TM  ECL^TM(Amersham Pharmacia Biotech Co., Ltd.) was used for hybridization at 42 ° C. in a buffer containing 40% formamide, 2 × SSC, and 0.1% SDS using the DNA probe obtained in Example 3. . As a result of the hybridization, plasmids (pRTB1-PstI, pRTB1-EcoRV, pRTB1-FbaI) were obtained from each positive clone, and the nucleotide sequence was determined using Li-cor DNA Sequencer model 4000L. The sequence of FbaI-PstI DNA fragment 6668 bp connected based on overlapping portions of each sequence is shown in SEQ ID NO: 11. From the homology of this 6668 bp DNA sequence with SEQ ID NO: 10, and the homology of the N-terminal amino acid sequence of the purified stereospecific amidase described in SEQ ID NO: 5 with the sequence when the 6668 bp DNA is translated into amino acids It was confirmed that the DNA sequence of No. 2 was DNA encoding this stereospecific amidase. SEQ ID NO: 1 describes the amino acid sequence of a stereoselective amidase derived from Pseudomonas sp. MCI3434 predicted from the DNA sequence.
[0068]
Example 5: Search for proteins having homology to the amino acid sequence of stereoselective amidase derived from Pseudomonas sp. MCI3434
Using the amino acid sequence described in SEQ ID NO: 1 (274 residues), the BLAST program (http://www.ncbi.nlm.nih.gov./BLAST/) of National Center for Biotechnology Information (hereinafter NCBI) is used. We searched for highly protein. The protein with the highest amino acid identity was Conserved hypothetical protein PA3598 of Pseudomonas aeruginosa (strain PAO1) (amino acid identity: 65%). Next, the protein having the highest amino acid identity was Conserved hypothetical protein (NCBI accession number: CAC47075) of Synorizobium meriroti (1021 strain), and its amino acid identity was 36%.
[0069]
Example 6: Pseudomonas sp. MCI3434 strain-derived stereoselective amidase E. coli expression in E. coli
PCR was performed using pRTB1-EcoR as a template and primers RTB-F and RTB-R (SEQ ID NOs: 12 and 13). The obtained PCR reaction product was purified, digested with restriction enzymes HindIII and XbaI, and ligated to pUC19 (manufactured by Takara Bio Inc.) digested with the same enzymes to prepare pRTB1EX. E. coli JM109 was transformed by the calcium chloride method. The transformed strain was applied to an LB agar medium containing 80 μg / mL ampicillin, and a grown clone was obtained. E. coli JM109 (pRTB1EX) was obtained.
[0070]
Example 7: E.I. Purification of a protein having stereoselective amidase activity from E. coli JM109 (pRTB1EX)
E. E. coli JM109 (pRTB1EX) was cultured in 2.5 L of LB medium containing 80 μg / mL ampicillin, and then about 10.0 g of cells were obtained by centrifugation. This was suspended in 100 mM Tris-HCl buffer (pH 8.0) containing 0.1 mM dithiothreitol, 0.1 mM EDTA, and 5.0 mM 2-mercaptoethanol, and cell-free extract was obtained by sonication and centrifugation. Got. Ammonium sulfate was added thereto to obtain a 0 to 40% ammonium sulfate saturated fraction. Further, this was dissolved in 10 mM Tris-HCl buffer solution (pH 8.0) (buffer A) containing 0.1 mM dithiothreitol, 0.1 mM EDTA, and 5.0 mM 2-mercaptoethanol. The column was adsorbed on a DEAE-Toyopearl column equilibrated with, and eluted with buffer A containing 100 mM sodium chloride. Fractions having the desired steric amidase activity were collected and dialyzed against buffer A. This was applied to a MonoQ HR10 / 10 column of FPLC (manufactured by Amersham Pharmacia Biotech) and eluted with a linear concentration gradient method of buffer A containing 0 to 500 mM sodium chloride. After elution, a fraction containing activity was selected, desalted and concentrated to obtain a purified sample.
[0071]
Example 8: Enzymatic properties of a stereoselective amidase from Pseudomonas sp. MCI3434 strain
Using N-tert-butylpiperazine-2-carboxamide as a substrate, the optimum pH and temperature of the amide hydrolysis reaction of the purified enzyme obtained in Example 7, the temperature tolerance of the enzyme and the 1 mM inhibitor The effect was performed by quantifying the piperazine-2-carboxylic acid produced by the hydrolysis reaction by HPLC. The reaction was carried out at 30 ° C. for 20 minutes, and the reaction was stopped by adding ethanol to prepare an HPLC sample. In addition, the heat processing for measuring temperature tolerance performed incubation for 5 minutes in each temperature, measured the activity after adjusting temperature to 30 degreeC. As for the inhibitor, the enzyme was incubated at 30 ° C. for 5 minutes in the presence of 1 mM inhibitor, then N-tert-butylpiperazine-2-carboxamide was added, incubated at 30 ° C. for 20 minutes, and then reacted by adding ethanol. The piperazine-2-carboxylic acid produced was quantified using the following HPLC condition A.
[0072]
HPLC condition A
Column: Sumichiral OA-5000
Column temperature: 30 ° C
Eluent: 2 mM CuSO_Four  Aqueous solution
Flow rate: 1.0 mL / min
Detection: UV254nm absorbance
The results are summarized in Tables 3 and 4 below.
[0073]
[Table 3]
[0074]
[Table 4]
[0075]
Next, the substrate selectivity of the stereoselective amidase derived from Pseudomonas sp. MCI3434 strain was examined. The substrate compound was added at a concentration of 20 mM, and an enzyme reaction was performed at 30 ° C. in 100 mM Tris-HCl buffer (pH 8.0). The analysis was performed under the following HPLC condition B.
The activity against piperazine-2-carboxamide is defined as 100, and the relative activity is shown in Table 5.
[0076]
[Table 5]
[0077]
Pseudomonas sp. MCI3434-derived stereoselective amidase activity shows particularly strong activity for piperazine-2-carboxamide, piperidine-3-carboxamide, and β-alanineamide, and weak activity for D-glutamineamide and piperidine-4-carboxamide. Indicated. In addition, it showed significant activity against N-alkylamide compounds, but other L- or D-amino acid amides, peptides, aliphatic amides, aromatic amides and nitriles were not hydrolyzed.
[0078]
Example 9: Examination of stereoselectivity of Pseudomonas sp. MCI3434-derived stereoselective amidase
E. E. coli JM109 (pRTB1EX) was cultured in LB medium containing 80 μg / mL ampicillin and 0.5 mM IPTG, and the cells were obtained by centrifugation. The wet cell weight of this was measured, added to 1.0 mL of 100 mM Tris-HCl buffer (pH 8.0) containing 200 mM (RS) -piperazine-2-carboxamide, and added at 30 ° C. After the reaction for 1.1 hours, the reaction was stopped with ethanol, and the optical purity of piperazine-2-carboxylic acid produced under HPLC condition A was measured. The optical purity of (R) -piperazine-2-carboxylic acid is 97.4% e.e. e. And the E value was 59.0.
[0079]
Example 10: E.I. hydrolysis reaction of (RS) -N-tert-butylpiperazine-2-carboxamide using E. coli JM109 (pRTB1EX)
E. E. coli JM109 (pRTB1EX) was cultured in LB medium containing 80 μg / mL ampicillin and 0.5 mM IPTG, and the cells were obtained by centrifugation. The wet cells were weighed and 0.4% each in 1.0 mL of 100 mM Tris-HCl buffer (pH 8.0) containing 200 mM (RS) -N-tert-butylpiperazine-2-carboxamide, and Added to 2.0% and incubated at 30 ° C. Sampling was performed at regular intervals, and after stopping the reaction with ethanol, the piperazine-2-carboxylic acid produced under the above HPLC condition A was quantified and the optical purity was measured (FIG. 1). As a result, the optical purity of the produced (R) -piperazine-2-carboxylic acid was 99.7% and 99.5% e.e., respectively. e. Met.
[0080]
Example 11: Cloning of a gene encoding Pseudomonas aeruginosa PAO1 strain unknown function protein PA3598
In order to obtain a DNA encoding the unknown function protein PA3598 of Pseudomonas aeruginosa strain PAO1 which was revealed to have the amino acid sequence of SEQ ID NO: 1 and the highest amino acid identity (65%) in Example 5, Stover, CK, et al. [Nature, (2000), 406 (6799): 959-964]) Primers PAE1 and PAE2 (SEQ ID NOs: 14 and 15) were designed based on the genomic DNA sequence of Pseudomonas aeruginosa PAO1 strain. . Using these primers, PCR was performed using the genomic DNA of P. aeruginosa PAO1 strain purchased from ATCC as a template DNA. The product of this PCR was subjected to agarose gel electrophoresis, and 0.96 kbp DNA was extracted and purified from the agarose electrophoresis gel. PT7Blue part of the DNA
The plasmid pTPA was obtained by cloning into T-Vector (manufactured by Novagen), and the base sequence of the inserted fragment was determined using Li-cor DNA Sequencer model 4000L (the operation method was in accordance with the attached manual instructions). No. 16) and the obtained DNA fragment was confirmed to be DNA encoding PA3598 described in SEQ ID NO: 3.
[0081]
Example 12 Pseudomonas aeruginosa PAO1 strain-derived PA3598-derived gene encoding E. coli expression in E. coli
PCR was performed using primers pAEX1 and PAEX2 (SEQ ID NOs: 17 and 18) using the plasmid pTPA containing the PA3598 gene prepared in Example 11 as a template. The obtained PCR reaction product was purified, digested with restriction enzymes HindIII and XbaI, and ligated to pUC19 (Takara Shuzo Co., Ltd.) digested with the same enzymes to prepare plasmid pAEX. E. coli JM109 was transformed by the calcium chloride method. The transformed strain was applied to an LB agar medium containing 80 μg / mL ampicillin, and a grown clone was obtained. E. coli JM109 (pAEX) was obtained.
[0082]
Example 13: E.I. Purification of a protein having stereoselective amidase activity from E. coli JM109 (pPAEX)
E. After culturing E. coli JM109 (pPAEX) in 2.5 L of LB medium containing 80 μg / mL ampicillin, about 10.0 g of cells were obtained by centrifugation. This was suspended in 100 mM Tris-HCl buffer (pH 8.0) containing 0.1 mM dithiothreitol, 0.1 mM EDTA, and 5.0 mM 2-mercaptoethanol, and cell-free extract was obtained by sonication and centrifugation. Got. To this was added sodium chloride to a concentration of 4 M, and 10 mM Tris-HCl buffer (pH 8.0) containing 4 M NaCl, 0.1 mM dithiothreitol, 0.1 mM EDTA, and 5.0 mM 2-mercaptoethanol. And passed through a butyl Toyopearl column equilibrated with 1. This was dialyzed with 10 mM Tris-HCl buffer (pH 8.0) (buffer A) containing 0.1 mM dithiothreitol, 0.1 mM EDTA, and 5.0 mM 2-mercaptoethanol, and then equilibrated with the same buffer. The sample was adsorbed on the DEAE-Toyopearl column and eluted with buffer A containing 150 mM sodium chloride. Fractions having the desired steric amidase activity were collected and dialyzed against buffer A. This was applied to a Superdex 200 HR10 / 30 column of FPLC (Amersham Pharmacia Biotech) and eluted with buffer A containing 150 mM sodium chloride. After elution, a fraction containing activity was selected, desalted and concentrated to obtain a purified sample.
[0083]
Example 14 Pseudomonas aeruginosa Enzymological properties of stereoselective amidase derived from PAO1 strain
Using (RS) -piperazine-2-carboxamide as a substrate, the optimum pH and temperature of the amide hydrolysis reaction of the purified enzyme obtained in Example 13, the temperature tolerance of this enzyme and the influence of 1 mM inhibitor Was performed by quantifying the piperazine-2-carboxylic acid produced by the hydrolysis reaction by HPLC. The reaction was carried out at 30 ° C. for 20 minutes, and the reaction was stopped by adding ethanol to prepare an HPLC sample. In addition, the heat processing for measuring temperature tolerance performed incubation for 5 minutes in each temperature, measured the activity after adjusting temperature to 30 degreeC. As for the inhibitor, the enzyme was incubated at 30 ° C. for 5 minutes in the presence of 1 mM inhibitor, (RS) -piperazine-2-carboxamide was added and incubated at 30 ° C. for 20 minutes, and then the reaction was performed by adding ethanol. After stopping, the piperazine-2-carboxylic acid produced | generated using the following HPLC conditions A was quantified.
The results are summarized in Tables 6 and 7 below.
[0084]
[Table 6]
[0085]
[Table 7]
[0086]
Example 15: Examination of stereoselectivity of Pseudomonas aeruginosa PAO1 strain-derived stereoselective amidase
E. E. coli JM109 (pAEX) was cultured in LB medium containing 80 μg / mL ampicillin and 0.5 mM IPTG, and the cells were obtained by centrifugation. The wet cell weight of this was measured, and it was added to 1.0 mL of 100 mM Tris-HCl buffer (pH 8.0) containing 200 mM (RS) -piperazine-2-carboxamide at 0.52%. After the reaction for 1.1 hours, the reaction was stopped with ethanol, and the optical purity of piperazine-2-carboxylic acid produced under HPLC condition A was measured. The optical purity of (R) -piperazine-2-carboxylic acid is 94.4% e.e. e. And the E value was 30.0.
[0087]
[Sequence Listing]
[0088]
[0089]
[0090]
[0091]
[0092]
[0093]
[0094]
[0095]
[0096]
[0097]
[0098]
[0099]
[0100]
[0101]
[0102]
[0103]
[0104]
[0105]

[Brief description of the drawings]
FIG. It is a figure which shows the production | generation of (R) -piperazine-2-carboxylic acid by E. coli JM109 (pRTB1EX). In the figure, ○ is (S) -piperazine-2-carboxylic acid (0.4% wet cell), ● is (R) -piperazine-2-carboxylic acid (0.4% wet cell), and □ is ( S) -piperazine-2-carboxylic acid (2.0% wet cell) and ▪ indicate (R) -piperazine-2-carboxylic acid (2.0% wet cell). (○ is not displayed because it overlaps with □.)

Claims (9)

A stereoselective amidase derived from Pseudomonas having the following physicochemical properties:
(A) The apparent molecular weight measured by SDS-PAGE is about 29,000-31,000;
(B) The optimum pH of the amidase reaction when (RS) -piperazine-2-carboxamide or (RS) -N-tertbutylpiperazine-2-carboxamide is used as a substrate is around pH 8;
(C) The optimal temperature of the amidase reaction when using (RS) -piperazine-2-carboxamide or (RS) -N-tertbutylpiperazine-2-carboxamide as a substrate is 45-50 ° C;
(D) stable at a temperature of 45 ° C. or lower near pH 8.0;
(E) Activity is completely inhibited by p-chloromercurybenzoic acid, Cu ²⁺ , Zn ²⁺ , Cd ²⁺ , Hg ²⁺ , Pb ²⁺ ;
(F) When (RS) -piperazine-2-carboxamide is used as a substrate, the activity to selectively hydrolyze the amide bond to (R) -form to produce (R) -piperazine-2-carboxylic acid. And (G) the subunit structure is a monomer. A protein having any of the following amino acid sequences.
(A) the amino acid sequence set forth in SEQ ID NO: 1;
(B) an amino acid sequence in which one to a plurality of amino acids are deleted, added or substituted in the amino acid sequence shown in SEQ ID NO: 1, and when (RS) -piperazine-2-carboxamide is used as a substrate , An amino acid sequence having an activity of hydrolyzing the amide bond selectively (R) -form to produce (R) -piperazine-2-carboxylic acid . DNA having any of the following base sequences:
(A) a base sequence encoding the amino acid sequence set forth in SEQ ID NO: 1;
(B) an amino acid sequence in which one to a plurality of amino acids are deleted, added or substituted in the amino acid sequence shown in SEQ ID NO: 1, and when (RS) -piperazine-2-carboxamide is used as a substrate , A base sequence encoding an amino acid sequence having an activity to hydrolyze the amide bond (R) -selectively to produce (R) -piperazine-2-carboxylic acid ; (C) the base described in SEQ ID NO: 2 Sequence;
(D) A base sequence in which one to a plurality of bases are deleted, added or substituted in the base sequence shown in SEQ ID NO: 2, and (RS) -piperazine-2-carboxamide is used as a substrate
When used, a base sequence encoding a protein having activity to hydrolyze the amide bond selectively to (R) -form to produce (R) -piperazine-2-carboxylic acid ; or (E) SEQ ID NO: 2 A base sequence that hybridizes under stringent conditions with the base sequence described in 1. or (R) -form when (RS) -piperazine-2-carboxamide is used as a substrate. A base sequence encoding a protein having an activity of selectively hydrolyzing to produce (R) -piperazine-2-carboxylic acid .   A recombinant DNA molecule or vector comprising the DNA of claim 3.   A transformed host cell comprising the recombinant DNA molecule or vector of claim 4. The following general formula (I)
(In the formula, A represents an optionally substituted hydrocarbon chain having 2 to 5 main chain atoms, the hydrocarbon chain may contain a double bond, and may be a nitrogen atom or a nitrogen atom. And R and R ′ each independently represents a hydrogen atom, an optionally substituted alkyl group or an optionally substituted aryl group, where n is 0 or The amidase or protein according to claim 1 or 2 or the transformed host cell according to claim 5 or a processed product thereof is allowed to act on (RS) -carboxylic acid amides represented by 1). The amide bond of the (R) -form is selectively hydrolyzed by the following general formula (II)
(In the formula, A, R ′ and n are as defined above.)
A method for producing an optically active (R) -carboxylic acid, wherein the (R) -carboxylic acid represented by the formula (1) is produced and then isolated. The following general formula (I)
(In the formula, A represents an optionally substituted hydrocarbon chain having 2 to 5 main chain atoms, the hydrocarbon chain may contain a double bond, and may be a nitrogen atom or a nitrogen atom. And R and R ′ each independently represents a hydrogen atom, an optionally substituted alkyl group or an optionally substituted aryl group, where n is 0 or represented by showing a 1) (RS) -. carboxylic acid amides, a DNA having any one of the nucleotide sequences of the protein or the following (B) ~ (D) having the amino acid sequence of the following (a) by the action of transformed host cells or their treated product having a recombinant DNA molecule or vector comprising, (R) - selectively hydrolyze the amide bond of the body, the following general formula (II)
(In the formula, A, R ′ and n are as defined above.)
A method for producing an optically active (R) -carboxylic acid, wherein the (R) -carboxylic acid represented by the formula (1) is produced and then isolated.
(A) an amino acid sequence represented by SEQ ID NO: 3, or an amino acid sequence in which one to a plurality of amino acids are deleted, added or substituted in the amino acid sequence represented by SEQ ID NO: 3, and (RS) -piperazine- When 2-carboxamide is used as a substrate , an amino acid sequence having an activity to hydrolyze the amide bond (R) -selectively to produce (R) -piperazine-2-carboxylic acid ;
(B) an amino acid sequence represented by SEQ ID NO: 3, or an amino acid sequence in which one to a plurality of amino acids are deleted, added or substituted in the amino acid sequence represented by SEQ ID NO: 3, and (RS) -piperazine- When 2-carboxamide is used as a substrate, a base sequence encoding an amino acid sequence having an activity to hydrolyze (R) -selectively the amide bond to produce (R) -piperazine-2-carboxylic acid ;
(C) a base sequence represented by SEQ ID NO: 4, or a base sequence in which one to a plurality of bases are deleted, added or substituted in the base sequence represented by SEQ ID NO: 4, wherein (RS) -piperazine- When 2-carboxamide is used as a substrate, a base sequence encoding a protein having an activity to selectively hydrolyze the amide bond to form (R) -piperazine-2-carboxylic acid ; or (D) a nucleotide sequence that hybridizes with the nucleotide sequence of SEQ ID NO: 4 or a complementary sequence thereof under stringent conditions, and when (RS) -piperazine-2-carboxamide is used as a substrate , its amide bond A base sequence encoding a protein having an activity to hydrolyze (R) -form selectively to produce (R) -piperazine-2-carboxylic acid . The following general formula (I)
(In the formula, A represents an optionally substituted hydrocarbon chain having 2 to 5 main chain atoms, the hydrocarbon chain may contain a double bond, and may be a nitrogen atom or a nitrogen atom. And R and R ′ each independently represents a hydrogen atom, an optionally substituted alkyl group or an optionally substituted aryl group, where n is 0 or The amidase or protein according to claim 1 or 2 or the transformed host cell according to claim 5 or a processed product thereof is allowed to act on (RS) -carboxylic acid amides represented by 1). After selectively hydrolyzing the amide bond of the (R) -form,
Formula (III)
(In the formula, A, R, R ′ and n are as defined above.)
A method for producing an optically active (S) -carboxylic acid amide characterized by isolating (S) -carboxylic acid amide represented by the formula: The following general formula (I)
(In the formula, A represents an optionally substituted hydrocarbon chain having 2 to 5 main chain atoms, the hydrocarbon chain may contain a double bond, and may be a nitrogen atom or a nitrogen atom. And R and R ′ each independently represents a hydrogen atom, an optionally substituted alkyl group or an optionally substituted aryl group, where n is 0 or A protein having the following amino acid sequence (A) or DNA having any one of the following base sequences (B) to (D): The following general formula (III), which remains after selectively hydrolyzing the amide bond of (R) -form by acting a transformed host cell having a recombinant DNA molecule or vector or a processed product thereof:
(In the formula, A, R, R ′ and n are as defined above.)
A method for producing an optically active (S) -carboxylic acid amide characterized by isolating (S) -carboxylic acid amide represented by the formula:
(A) an amino acid sequence represented by SEQ ID NO: 3, or an amino acid sequence in which one to a plurality of amino acids are deleted, added or substituted in the amino acid sequence represented by SEQ ID NO: 3, and (RS) -piperazine- When 2-carboxamide is used as a substrate, an amino acid sequence having an activity to hydrolyze the amide bond (R) -selectively to produce (R) -piperazine-2-carboxylic acid;
(B) the amino acid sequence set forth in SEQ ID NO: 3 or the amino acid sequence set forth in SEQ ID NO: 3
Wherein one or more amino acids are deleted, added or substituted, and when (RS) -piperazine-2-carboxamide is used as a substrate, the amide bond is (R) -selective A base sequence encoding an amino acid sequence having an activity of hydrolyzing to form (R) -piperazine-2-carboxylic acid;
(C) a nucleotide sequence represented by SEQ ID NO: 4 or a nucleotide sequence in which one to a plurality of bases are deleted, added or substituted in the nucleotide sequence represented by SEQ ID NO: 4, When 2-carboxamide is used as a substrate, a base sequence encoding a protein having an activity to selectively hydrolyze the amide bond to form (R) -piperazine-2-carboxylic acid; or
(D) A base sequence that hybridizes with the base sequence described in SEQ ID NO: 4 or a complementary sequence thereof under stringent conditions, and its amide bond when (RS) -piperazine-2-carboxamide is used as a substrate A base sequence encoding a protein having an activity to hydrolyze (R) -form selectively to produce (R) -piperazine-2-carboxylic acid.