JP2005261317A - Dimethylarginine dimethylaminohydrolase, its gene, recombinant dna and method for producing dimethylarginine dimethylaminohydrolase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain dimethylarginine dimethylaminohydrolase, its gene, etc., and to use dimethylarginine dimethylaminohydrolase as a diagnosing enzyme in an assaying kit. <P>SOLUTION: The protein comprises a specific amino acid sequence derived from Sinorhizobium and has dimethylarginine dimethylaminohydrolase activity. The protein comprises an amino acid sequence in which one or several amino acids are deleted, substituted or added in the protein amino acid sequence and has dimethylarginine dimethylaminohydrolase activity. The protein comprises an amino acid sequence having ≥80% homology to the amino acid sequence of the protein and has dimethylarginine dimethylaminohydrolase activity. The gene encodes the enzyme. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to dimethylarginine dimethylaminohydrolase, its gene, recombinant DNA, and a method for producing dimethylarginine dimethylaminohydrolase.

  Asymmetric dimethylarginine (hereinafter abbreviated as ADMA) is known as an endogenous inhibitor of NO synthase in the human body, and may be one of the controls for the amount of NO produced in the body. Has been suggested (see Non-Patent Document 1). As the role of NO, vasoconstriction / relaxation control, biological defense by NO produced from macrophages, utilization as a neurotransmitter, etc. are currently known.

  In recent years, research on diseases related to NO has progressed, and the relationship with ADMA has been known in arteriosclerosis, hypertension, pregnancy toxemia and the like (see Non-Patent Document 2 and Non-Patent Document 3). That is, it has been suggested that the above-mentioned diseases such as arteriosclerosis can be diagnosed by measuring the amount of ADMA.

  Currently, ADMA is measured by a method using HPLC (see Non-Patent Document 4, Non-Patent Document 5, Non-Patent Document 6, Non-Patent Document 7, Non-Patent Document 8, and Non-Patent Document 9) or ELISA (see Patent Document 1). Has been. Furthermore, an enzymatic measurement method using dimethylarginine dimethylaminohydrolase (hereinafter abbreviated as DDAH) is also disclosed (see Patent Document 2).

  This measurement method is a method in which ADMA in a living body is converted to citrulline by DDAH and the amount of citrulline is measured. DDAH has been isolated from rats for the first time by Ogawa et al. (See Non-Patent Document 10), human-derived genes (see Non-Patent Documents 11 and 12), rat-derived genes (see Non-Patent Document 13), etc. Are known. However, it is difficult to extract and purify DDAH from living tissues in large quantities. Recombinant sDDAH known so far cannot be expressed in E. coli without adding a histidine tag, GST tag, etc. Slightly reported.

  As for microorganism-derived genes, recombinant DDAH has been reported (see Non-Patent Document 14 and Non-Patent Document 15).

  However, no example of purifying a natural enzyme from a microbial culture has been reported. Recombinant DDAH has been reported to be expressed in E. coli with a histidine tag added. However, detailed enzymatic properties are not clear. In addition, Pseudomonas aeruginosa-derived enzyme, which seems to be the strongest among the microorganism-derived enzymes that have reported specific activity against ADMA, has a small amount of arginine deiminase activity, so it is sufficient when used in a highly sensitive ADMA measurement kit. That's not true.

  Furthermore, in order to purify DDAH to which a tag is added, it is useful to use a dedicated resin for trapping the tag, and it is often necessary to treat the tag with a dedicated protease after purification.

  As described above, DDAH known in the art is not suitable for mass purification because it has a low expression level or uses a dedicated resin or protease, and has a very difficult point in preparing a measurement kit. .

International Publication No. 98/49199 Pamphlet US Pat. No. 6,358,699 PNAS 99, 13527-13532, 2002 Lancet 339, 572-575, 1992 Circulation 98, 1842-1847, 1998 J. Chromatogr. B 692, 257-262, 1997 Biochem.Biophys.Res.Commun. 219, 598-603, 1996 J. Chromatogr. B 742, 199-203, 2000 J. Chromatogr. B 659, 185-207, 1994 Anal.Biochem.303, 131-137, 2002 Anal. Biochem. 318, 13-17, 2003 J. Biol. Chem. 264, 10205-10209, 1989 Eur. J. Biochem. 258, 863-868, 1998 Biochem. J. 343, 209-214, 1999 Biochim. Biophys. Acta 1337, 6-10, 1997 Biochem. J. 343, 209-214, 1999 Molecular Microbiology 33, 1278-1279, 1999

  The problem to be solved by the present invention is to provide dimethylarginine dimethylaminohydrolase, its gene, recombinant DNA and a method for producing dimethylarginine dimethylaminohydrolase.

  Therefore, as a result of intensive studies for solving the above-mentioned problems, the inventors of the present invention have unknown functions, and the gene sequence derived from Sinorhizobium meliloti encodes a protein having a novel dimethylarginine dimethylaminohydrolase activity. Further, the present inventors have found that the dimethylarginine dimethylaminohydrolase gene can be expressed very efficiently without adding a tag, thereby completing the present invention.

That is, the present invention provides the following inventions.
(1) Dimethylarginine dimethylaminohydrolase having the following physicochemical properties:
(A) Action: Acts on asymmetric dimethylarginine to catalyze a reaction that produces dimethylamine and citrulline.
(B) Substrate specificity: Does not act on arginine.
(2) Dimethylarginine dimethylaminohydrolase having the following physicochemical properties:
(A) Action: Acts on asymmetric dimethylarginine to catalyze a reaction that produces dimethylamine and citrulline.
(B) Substrate specificity: Does not act on arginine.
(C) Optimum pH: 7.0 to 10.0
(3) Dimethylarginine dimethylaminohydrolase having the following physicochemical properties:
(A) Action: Acts on asymmetric dimethylarginine to catalyze a reaction that produces citrulline and dimethylamine.
(B) Substrate specificity: Does not act on arginine.
(C) Optimum pH: 7.0 to 10.0
(D) Stable pH range: pH 4.0-10.0
(E) Temperature range suitable for action: 30 to 40 ° C
(F) Thermal stability: 70% or more activity remains after heat treatment at 40 ° C. for 10 minutes.
(G) Molecular weight: about 31,000 (SDS-PAGE)
(4) The following dimethylarginine dimethylaminohydrolase (a), (b) or (c).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) consisting of an amino acid sequence in which one to several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1, and A protein having dimethylarginine dimethylaminohydrolase activity (c) a protein comprising an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 1 and having dimethylarginine dimethylaminohydrolase activity (5) A dimethylarginine dimethylaminohydrolase gene encoding the dimethylarginine dimethylaminohydrolase of (a), (b) or (c).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) consisting of an amino acid sequence in which one to several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1, and A protein having dimethylarginine dimethylaminohydrolase activity (c) a protein having an amino acid sequence of 80% or more homology with the amino acid sequence represented by SEQ ID NO: 1 and having dimethylarginine dimethylaminohydrolase activity (6) Item ( 5) Recombinant DNA characterized by inserting the gene described in vector DNA.
(7) A transformant or transductant containing the recombinant DNA according to item (6).
(8) A method for producing dimethylarginine dimethylaminohydrolase, comprising culturing the transformant or transductant according to item (7) in a medium and collecting dimethylarginine dimethylaminohydrolase from the culture.

  According to the present invention, a method for producing dimethylarginine dimethylaminohydrolase, its gene, recombinant DNA, and dimethylarginine dimethylaminohydrolase is provided, which can be easily used as a diagnostic enzyme in a measurement kit. The aminohydrolase gene is extremely useful industrially because it is supplied at low cost and in large quantities without adding a tag.

  Hereinafter, the present invention will be described in detail.

  The DDAH of the present invention is an enzyme that acts on asymmetric dimethylarginine to catalyze a reaction that produces citrulline and dimethylamine, and does not act on arginine. Any enzyme having this action is included in the DDAH of the present invention.

Dimethylarginine + H ₂ O → Dimethylamine + citrulline

Further, as one form of DDAH, for example, DDAH that does not act on arginine that has the action of catalyzing the above reaction, DDAH that has the action of catalyzing the above reaction and acts at pH 7.0 to 10.0, and the above reaction And DDAH, which has an action of catalyzing NO, and acts at pH 7.0 to 10.0 and does not act on arginine.






Furthermore, DDAH etc. which have the effect | action which catalyzes the said reaction, and also have the physicochemical property as described in the following (a)-(e) or combined those suitably are also mentioned as this invention DDAH.
(A) Optimal pH: pH 7.0 to 10.0
For example, 200 mM acetate buffer (pH 4.0 to 6.0), 200 mM potassium phosphate buffer (pH 7.0 to 8.5), and 200 mM glycine buffer (pH 8.0 to 11.0) are used as buffers. At each pH, an enzyme reaction is performed at a temperature of 37 ° C. to obtain an optimum pH. For example, the DDAH of the present invention having physicochemical properties of pH 7.0 to 10.0, preferably pH 9.0 to 10.0 as the optimum pH.
(B) Suitable temperature range: 30-40 ° C
For example, using a reaction solution having the same composition as the reaction solution in the activity measurement method described later, the activity of the enzyme is measured at various temperatures to determine the optimum temperature range of action. For example, the DDAH of the present invention having a physicochemical property of 20 to 50 ° C., preferably 30 to 40 ° C., as the temperature range of action.
(C) Thermal stability: 70% or more activity remains after heat treatment at 40 ° C. for 10 minutes.
For example, after treatment with 40 mM potassium phosphate buffer (pH 8.0) at 40 ° C. for 10 minutes, the residual activity of the enzyme is measured. For example, the DDAH of the present invention in which the activity remains at 50% or more, preferably 70% or more under the above treatment conditions.
(D) Stable pH range: pH 4.0-10.0
For example, 200 mM acetate buffer (pH 3.0 to 6.0), 200 mM potassium phosphate buffer (pH 6.5 to 8.5), and 200 mM glycine buffer (pH 8.0 to 12.0) are used as buffers. Used, after treatment at 30 ° C. for 10 minutes at each pH, the residual activity of the DDAH of the present invention is measured. Examples of the stable pH range include the present invention DDAH having physicochemical properties of pH 4.0 to 10.0, preferably pH 8.0 to 10.0.
(E) Molecular weight: about 31,000 (SDS-PAGE)
For example, the molecular weight is determined by the SDS-PAGE method using Multigel 4/20 (Daiichi Chemicals Co., Ltd.). For example, the DDAH of the present invention having a molecular weight of 25,000 to 60,000, preferably 30,000 to 40,000 (SDS-PAGE).

  Examples of the method for measuring the enzyme activity of the DDAH of the present invention include a method using tritium-labeled ADMA or a method for measuring citrulline produced by the enzyme reaction. Hereinafter, as an example, a method for measuring citrulline will be described. Hereinafter, ADMA is used as a substrate for the measurement of the activity of the DDAH of the present invention unless otherwise specified. The enzyme titer is defined as 1 U for the amount of enzyme that produces 1 μmol of citrulline per minute when measured using ADMA as a substrate.

A. Preparation of reagents (1) Reagent 1: Color developing solution A
500 mg of antipyrine, 278 mg of iron sulfate are dissolved in 50% sulfuric acid and made up to a volume of 100 ml.
(2) Reagent 2: Color developing solution B
Dissolve 800 mg of diacetyl monooxime in 5% acetic acid and make up to 100 ml.
(3) Reagent 3: Substrate solution (10 mM; final concentration 1 mM)
Dissolve 25 mg of asymmetric dimethylarginine in ion-exchanged water to a constant volume of 9.1 ml. When symmetric dimethylarginine or L-arginine is used as a substrate, 25 mg and 16 mg are dissolved in ion-exchanged water and a solution having a constant volume of 9.1 ml is used.
(4) Reagent 4: 200 mM potassium phosphate buffer (pH 8.0)
(5) Reagent 5: Mixed color developer Reagents 1 and 2 are mixed in equal amounts immediately before use.

B. Measurement method Mix 0.25 ml of reagent 4 and 0.2 ml of enzyme solution, and preheat at 37 ° C. for 5 minutes. Thereafter, 0.05 ml of Reagent 3 is added and mixed well, and then incubated at 37 ° C. for 20 minutes. Add 0.25 ml of reagent 5 and stir well, then react at 85 ° C. for 40 minutes. After cooling, the absorbance at 466 nm is measured with a spectrophotometer. The control solution was the same as described above except that 100 μl of ion exchange water was added instead of 100 μl of reagent 3. Using this pre-prepared citrulline instead of the reagent 3 and ion-exchanged water instead of the enzyme solution, a graph is prepared in which the relationship with the amount of dye produced is examined. Using this graph, the micromolar amount of citrulline produced per minute at 37 ° C. is calculated, and this value is used as the activity unit in the enzyme solution. When measuring whether or not it acts on arginine, it can be measured in the same manner as described above by using reagent 3 (substrate solution) using L-arginine as a substrate instead of asymmetric dimethylarginine.

  Thus, the DDAH of the present invention is a DDAH that acts on asymmetric dimethylarginine to produce dimethylamine and citrulline and catalyzes the reaction shown in the previous formula. The DDAH of the present invention, which has a combination of specific properties or appropriate combinations, can also be mentioned. Since these DDAHs of the present invention efficiently act on asymmetric dimethylarginine in biomaterials or samples, they are effectively used as an enzyme for measuring asymmetric dimethylarginine. Furthermore, the enzyme of the present invention that can be sufficiently produced as a recombinant without adding a tag and does not act on arginine is particularly preferred as the present invention DDAH when used as a clinical diagnostic enzyme.

  The DDAH of the present invention can be obtained from nature by searching for enzymes originating from microorganisms, animals, plants and the like. For example, when searching for a microorganism having the ability to produce the DDAH of the present invention, the microorganism is cultured in a medium to which an enzyme production inducer such as asymmetric dimethylarginine is added, the resulting microbial cell is disrupted, and the asymmetric dimethylarginine is obtained. The microorganism having the ability to produce the DDAH of the present invention can be obtained by examining the DDAH activity as a substrate. The microorganism used here may be newly separated from the soil, and further, a microorganism obtained from a microorganism storage organization or the like may be used. Furthermore, homology search may be performed using DDAH gene sequences known so far, and information on gene sequences with unknown functions may be obtained and cloned.

  Further, the DDAH of the present invention also includes the DDAH of the present invention obtained by modifying the natural DDAH of the present invention by genetic engineering techniques or methods such as mutation treatment.

  Examples of the modification method include irradiating the organism producing DDAH with ultraviolet rays, X-rays, radiation, etc., or ethyl methanesulfonate, N-methyl-N′-nitro-N-nitrosoguanidine, Examples include a method of obtaining a modified microorganism of the present invention DDAH by contacting a mutagen such as nitric acid, and obtaining the present invention DDAH from the obtained microorganism. In general, the DDAH of the present invention can be obtained by modifying a gene encoding DDAH or the like having different properties by using genetic engineering techniques.

Furthermore, as this invention DDAH, this invention DDAH etc., such as (a), (b) or (c), are mentioned, for example.
(A) DDAH comprising the amino acid sequence represented by SEQ ID NO: 1.
(B) DDAH containing an amino acid sequence in which one to several amino acids are deleted, substituted and / or added in the amino acid sequence represented by SEQ ID NO: 1.
(C) DDAH containing an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 1.

  Here, “from 1 to several amino acids are deleted, substituted and / or added” means, for example, an arbitrary number of 1 to 20, preferably 1 to 10, more preferably 1 to 5. Is deleted, substituted and / or added.

  “Showing 80% or more homology” is not particularly limited as long as the homology with the amino acid sequence represented by SEQ ID NO: 1 or 3 is 80% or more, for example, 80% or more, preferably 90%. % Or more, most preferably 95% or more.

  Examples of the DDAH of the present invention include DDAH obtained by cloning a natural DDAH gene derived from chromosomal DNA of Sinorhizobium meliloti ATCC 51124, introducing it into an appropriate vector host system, and expressing it. . Further, it can be obtained from various sources of DDAH obtained from the natural world.

Next, a method for obtaining the gene of the present invention will be described.
In order to obtain the gene of the present invention, a commonly used gene cloning method is used. For example, a homology search is performed using a publicly available database of DDAH gene sequences known so far, and homologs with high homology are picked up. Primers are designed so that structural genes can be amplified from the obtained homologous gene information. Chromosomal DNA or mRNA is extracted from the microbial cells having the DDAH homolog of the present invention or various cells by a conventional method, for example, the method described in Current Protocols in Molecular Biology (WILEY Interscience, 1989). Using the primer capable of amplifying the above structural gene, the gene DNA of the present invention is amplified by polymerase chain reaction (PCR method) or reverse transcription polymerase chain reaction (RT-PCR) using chromosomal DNA or mRNA as a template. Can be obtained. For example, the gene of the present invention of Sinorhizobium meliloti ATCC 51124 can be obtained as follows.

First, after culturing the above-mentioned microorganisms, the obtained cells are frozen in liquid nitrogen, and then physically ground using a mortar or the like to obtain a fine powdery cell piece. Chromosomal DNA is extracted from the piece by a usual method using a commercially available DNA extraction kit or the like. Primers used in PCR capable of amplifying a structural gene from the gene sequence of the DDAH homolog of the present invention are synthesized. PCR is performed using these primers and the obtained chromosomal DNA as a template to obtain a DNA fragment encoding the DDAH of the present invention. Amplified DNA can be cloned according to a conventional method. The amplified DNA is inserted into an appropriate vector to obtain recombinant DNA. For cloning, for example, commercially available kits or vectors such as TA Cloning Kit (Invitrogen) include pUC119 (manufactured by Sakai Shuzo), pBR322 (manufactured by Sakai Shuzo), pMAL-C2 (manufactured by NEW England Labs), pBluescript II. Plasmid vector DNA such as SK ⁺ (Stratagene), pKK223-3 (Amersham Biotech), or bacteriophage vector DNA such as λENBL3 (Stratagene), λDASH II (Funakoshi) can be used.

  Using the recombinant DNA thus obtained, for example, E. coli K12, preferably E. coli JM109, DH5α (both manufactured by Toyobo Co., Ltd.), XL1-Blue [manufactured by Funakoshi Co., Ltd.], etc. are transformed or transduced. A transformant or transductant containing recombinant DNA can be obtained. Transformation can be performed, for example, by the method of D.M. Morrison (Methods in Enzymology, 68, 326-331, 1979). Transduction can be performed, for example, by the method of B. Hohn (Methods in Enzymology, 68, 299-309, 1979). As host cells, in addition to E. coli, other bacteria, yeasts, filamentous fungi, actinomycetes and other microorganisms or animal cells can be used as appropriate.

  The entire base sequence of the amplified DNA can be analyzed using, for example, a LI-COR MODEL 4200L sequencer (manufactured by LI-COR), a 370A DNA sequence system (manufactured by PerkinElmer), or the like. Analysis of the obtained gene of the present invention confirms the translated polypeptide, that is, the amino acid sequence of the present DDAH.

  An example of the gene of the present invention includes a gene containing DNA consisting of the base sequence represented by SEQ ID NO: 2. Plasmid pT7sDDAH containing DNA consisting of the base sequence represented by SEQ ID NO: 2 has been deposited as FERM P-19735 at the National Institute of Advanced Industrial Science and Technology (AIST).

  Further, various modified DDAHs of the present invention can be obtained by modifying the gene of the present invention.

  As a method for modifying the gene, any known method can be used, for example, a method in which the recombinant DNA is contacted with a chemical mutagen such as hydroxyamine or nitrous acid, or a random method using a PCR method. A point mutation method such as conversion to a site, a site-directed mutagenesis method that is a well-known technique for generating a site-specific substitution or deletion mutation using a commercially available kit, and selectively cleaving this recombinant DNA Then, a method of removing or adding selected oligonucleotides and linking them, that is, an oligonucleotide mutagenesis method and the like can be mentioned. Next, the recombinant DNA after the treatment can be purified using a desalting column, QIAGEN (Qiagen) or the like, and various recombinant DNAs can be obtained.

  By these modifications, for example, it hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the full length of DNA consisting of the base sequence represented by SEQ ID NO: 2 or a portion of 15 or more bases thereof, and A DNA encoding a protein having DDAH activity or a full-length DNA consisting of the base sequence represented by SEQ ID NO: 2 or a portion having 15 bases or more of which has a homology of 80% or more and encodes a protein having DDAH activity The gene of the present invention comprising the DNA to be obtained is obtained.

  Furthermore, by modifying the gene of the present invention, for example, the amino acid sequence represented by SEQ ID NO: 1 consists of an amino acid sequence in which one to several amino acids are deleted, substituted and / or added, and has DDAH activity. The DDAH of the present invention, such as a protein having a DDAH activity, comprising a protein having the amino acid sequence having 80% or more homology with the protein having the amino acid sequence represented by SEQ ID NO: 1, or the like.

Next, a method for producing the DDAH etc. of the present invention will be described.
A transformant or transductant containing the DDAH gene of the present invention is cultured in a medium, and the present DDAH or the like is recovered from the culture by using various commonly used protein separation methods. it can. As an example, the following method using a transformant or transductant containing the DDAH gene of the present invention can be adopted as a specific production method. First, transformants or transductants (hereinafter collectively referred to as “recombinant bacteria”) that produce the DDAH of the present invention are cultured. In this case, the culture may be performed by a solid culture method, but it is more preferable to employ a liquid culture method for culture. Examples of the culture medium for cultivating the microorganism include one or more nitrogen sources such as yeast extract, peptone, meat extract, corn steep liquor, soybean or wheat straw leachate, potassium dihydrogen phosphate, dihydrogen phosphate. One or more inorganic salts such as potassium, magnesium sulfate, ferric chloride, ferric sulfate or manganese sulfate are added, and further, saccharide raw materials, vitamins and the like are appropriately added as necessary. As an enzyme production induction substrate, it is also effective to improve the production amount to appropriately add an induction substrate suitable for a promoter, for example, IPTG.

  The initial pH of the medium is suitably adjusted to 7.0 to 9.0, for example. Cultivation is preferably performed, for example, at 25 to 42 ° C., preferably around 30 ° C. for 1 to 5 days, by aeration / agitation deep culture, shaking culture, stationary culture, or the like. In order to collect the DDAH of the present invention from the culture after completion of the culture, a normal enzyme collecting means can be used.

  Next, the cells are separated from the culture solution by, for example, operations such as filtration and centrifugation, and washed. The DDAH of the present invention is preferably collected from the cells. In this case, the bacterial cells can be used as they are, but the cell wall using a cell wall lytic enzyme such as lysozyme, a method of destroying the bacterial cells using various disrupting means such as an ultrasonic crusher, French press, dynomill, etc. It is preferable to collect the DDAH of the present invention from the microbial cells by a method for dissolving the microbial cells, a method for extracting an enzyme from the microbial cells using a surfactant such as Triton X-100, and the like.

  In order to purify and isolate the DDAH of the present invention from the crude enzyme solution thus obtained, a conventional method for enzyme purification can be used. For example, an ammonium sulfate salting-out method, an organic solvent precipitation method, an ion exchange chromatography method, a gel filtration chromatography method, a hydrophobic chromatography method, an adsorption chromatography method, an electrophoresis method and the like are preferably combined appropriately. In this way, the DDAH of the present invention can be isolated by showing an almost single band on SDS-PAGE. In addition, enzyme preparations with different degrees of purification can be prepared by appropriately combining the above purification methods.

  The DDAH of the present invention thus obtained is effectively used for measuring dimethylarginine described in US Pat. No. 6,358,699, for example. First, DDAH of the present invention is allowed to act on a sample containing asymmetric dimethylarginine to liberate citrulline. The amount of citrulline liberated is measured according to the above activity measurement method.

  Hereinafter, the present invention will be described more specifically with reference to examples.

(Database search)
BLAST database search was performed using rat-derived DDAH amino acid sequences. As a result, the existence of insect-derived or microbial-derived homologs in addition to rats and humans was revealed.

  Sinorhizobium meliloti ATCC51124 has been genome-analyzed, and the entire genome DNA information has been clarified. This gene has only 28-38% homology with known mammalian or microbial amino acid sequences, and the function or property of the enzyme encoded by the gene has been investigated so far. There wasn't.

[Cloning of the gene of the present invention (Sinorhizobium meliloti) and expression by transformants]
(1) Primer synthesis and DNA preparation A DNA primer capable of amplifying a structural gene based on a gene sequence derived from Sinorhizobium meliloti, which was found from amino acid sequence homology search, was prepared by contract synthesis of Sigma Genesis. Sinorhizobium meliloti was purchased from ATCC, and after reconstitution with the method specified by ATCC, 0.2 L of TY medium was inoculated and cultured at 30 ° C. and 120 rpm for 1 day. From this culture, the cells were collected by centrifugation at 12,000 rpm for 10 minutes. The obtained cells were suspended in 5 ml of TE buffer, added with an equal amount of TE saturated phenol, and then lysed by gentle shaking. After centrifugation at 12000 rpm for 2 minutes, the supernatant was taken out and further subjected to protein removal treatment with TE saturated phenol. Finally, DNA was recovered by ethanol precipitation to obtain 1.20 mg of DNA.

(2) PCR
A reaction solution was prepared with the following composition, and PCR was performed under the conditions of denaturation at 94 ° C. for 30 seconds, annealing at 62 ° C. for 30 seconds, extension reaction at 72 ° C. for 2 minutes and 30 cycles.
(Reaction solution composition)
0.6 μl of 10 μM primer (SEQ ID NO: 3)
0.6 μl of 10 μM primer (SEQ ID NO: 4)
2x 10x PCR buffer
dNTP 2 μl
1 mM magnesium chloride 0.8 μl
Taq polymerase 0.4U
Add to a final volume of 20 μl of H ₂ O.
After completion of PCR, the reaction solution was subjected to agarose gel electrophoresis. As a result, a band considered to be the target fragment was confirmed at about 0.8 kb, and the band was purified and extracted with RecoChip (Takara Shuzo).

(3) Analysis of purified DNA fragment When the purified DNA fragment was subjected to nucleotide sequence determination and analysis using a 370A DNA sequencing system (manufactured by PerkinElmer), the determined nucleotide sequence was determined from the DNA sequence in the database. It was the same. On the other hand, plasmid pT7-Blue (manufactured by Novagen) was digested with restriction enzyme EcoRV, and then ligated with the purified and extracted DNA fragment to transform E. coli JM109. The obtained plasmid pT7sDDAH has been deposited as FERM P-19735 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.

(4) Confirmation of activity Escherichia coli JM109 (pT7sDDAH) cells were treated with TY medium (1% bactotryptone, 0.5% bacto yeast extract, 0.5% NaCl, pH 7.0) containing 50 μg / ml ampicillin. ) After shaking culture at 10 ml at 28 ° C. to Klett 100, IPTG was added to a final concentration of 1 mM and further cultured for 3 hours. This culture solution was treated 4 times for 20 seconds under cooling on ice using an ultrasonic crusher (Ultrasonicgenerator, manufactured by Nissei). Place this in an Eppendorf tube, centrifuge at 12,000 rpm for 10 minutes using a microcentrifuge, separate the supernatant fraction and the precipitate fraction, and transfer the supernatant to another Eppendorf tube. When the DDAH activity was measured by the enzyme activity measurement method described above, JM109 (pT7sDDAH) had a DDAH activity of 0.23 U / ml.

(5) Manufacture of DDAH of the present invention derived from Sinorhizobium meliloti JM109 (pT7sDDAH) containing 50 μg / ml ampicillin in TY medium (1% bactotryptone, 0.5% bacto yeast extract, 0.5% NaCl, pH 7. 0) 10 ml was inoculated and cultured with shaking at 30 ° C. for 1 day. As a seed culture, inoculate 1 ml each of 0.1 L of the above medium in a 0.5 L Erlenmeyer flask, shake culture at 28 ° C. to Klett 100, add IPTG to a final concentration of 1 mM, Incubated for 3 hours. From this culture solution, the cells were collected by centrifugation (12000 rpm) for 10 minutes. The obtained microbial cells were stored frozen at -80 ° C.

  Frozen cells (300 ml) are suspended in 15 ml of buffer A (50 mM phosphate buffer, 5% glycerol, pH 8.0), and cooled on ice, using an ultrasonic crusher (Ultrasonicgenerator, manufactured by Nissei) for 20 seconds. Processed 4 times. The disrupted solution was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was applied to a Q Sepharose FF (Amersham Biotech) column (2.5 cm × 15 cm) previously equilibrated with buffer A. After washing with 150 ml of buffer A, elution was performed with a linear gradient of buffer A to buffer B (0.7 M sodium chloride, 50 mM phosphate buffer, 5% glycerol, pH 8.0). The active fraction was eluted with about 0.3M sodium chloride.

  The eluted active fraction was concentrated and dialyzed with Centriprep 10 (Amicon), and again subjected to Q Sepharose FF. Elution was performed with a linear gradient of buffer A to buffer B. The active fraction was eluted with about 0.3M sodium chloride.

  When the obtained active fraction was analyzed by SDS-PAGE, an almost single band was obtained (molecular weight about 31,000). The obtained active fraction was used to determine the following physicochemical properties.

(Physicochemical properties of the present invention DDAH derived from Sinorhizobium meliloti)
The physicochemical properties of the DDAH of the present invention obtained in Example 2 were as follows.
(A) Action and substrate specificity The activity of the sDDAH of the present invention was measured by using the enzyme activity measurement method described above, using asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and L-arginine as substrates. The DDAH of the present invention did not act on SDMA, L-arginine.

(B) Optimal pH
200 mM acetate buffer (pH 4.0 to 6.0), 200 mM potassium phosphate buffer (pH 7.0 to 8.5), and 200 mM glycine buffer (pH 8.0 to 11.0) were used as buffers. The enzyme reaction was carried out at a temperature of 37 ° C. The results are shown in FIG.
The sDDAH of the present invention showed the highest activity at pH 9.0-10.0. In addition, since the activity value in the vicinity of pH 9.0 was 50% or more even at pH 7.0 to 10.0, the optimum pH of the DDAH of the present invention is pH 7.0 to 10.0, and the most preferable optimum. The pH was judged to be pH 9.0-10.0.

(C) Optimum temperature range The activity of the enzyme was measured at various temperatures using a reaction solution having the same composition as the reaction solution in the activity measurement method described above. The results are shown in FIG. The temperature range showing 60% or more of the activity in the vicinity of 37 ° C., which is the temperature showing the highest activity, was 30 to 40 ° C.
From the above, the optimum temperature range of the DDAH of the present invention was determined to be 30 to 40 ° C.

(D) Thermal stability The results of thermal stability when treated with 200 mM potassium phosphate buffer (pH 8.0) at each temperature for 10 minutes are as shown in FIG. It was stable up to around 40 ° C.

(E) Stable pH Range As a buffer solution, 200 mM acetate buffer (pH 3.0 to 6.0), 200 mM potassium phosphate buffer (pH 6.5 to 8.5), 200 mM glycine buffer (pH 8.0 to 12) 0.0), the residual activity of the DDAH of the present invention was measured after treatment at 30 ° C. for 10 minutes at each pH. The results are shown in FIG. The pH showing the highest residual activity. The pH range showing an activity of 50% or more with respect to the activity around pH 10.0 was pH 4.0 to 10.0.
As mentioned above, it was judged that the stable pH range of this invention DDAH is pH 4.0-10.0.

(F) Molecular weight As a result of obtaining the molecular weight by SDS-PAGE method using Multigel 4/20 (manufactured by Daiichi Chemicals Co., Ltd.), the molecular weight of the DDAH of the present invention was about 31,000.

The figure which shows optimal pH of this invention DDAH. The figure which shows the range of the optimal temperature of this invention DDAH. The figure which shows the thermal stability of this invention DDAH. The figure which shows the stable pH range of this invention DDAH.

Claims (8)

Dimethylarginine dimethylaminohydrolase having the following physicochemical properties:
(A) Action: Acts on asymmetric dimethylarginine to catalyze a reaction that produces dimethylamine and citrulline.
(B) Substrate specificity: Does not act on arginine. Dimethylarginine dimethylaminohydrolase having the following physicochemical properties:
(A) Action: Acts on asymmetric dimethylarginine to catalyze a reaction that produces dimethylamine and citrulline.
(B) Substrate specificity: Does not act on arginine.
(C) Optimum pH: 7.0 to 10.0 Dimethylarginine dimethylaminohydrolase having the following physicochemical properties:
(A) Action: Acts on asymmetric dimethylarginine to catalyze a reaction that produces citrulline and dimethylamine.
(B) Substrate specificity: Does not act on arginine.
(C) Optimum pH: 7.0 to 10.0
(D) Stable pH range: pH 4.0-10.0
(E) Temperature range suitable for action: 30 to 40 ° C
(F) Thermal stability: 70% or more activity remains after heat treatment at 40 ° C. for 10 minutes.
(G) Molecular weight: about 31,000 (SDS-PAGE) The following (a), (b) or (c) dimethylarginine dimethylaminohydrolase.
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) consisting of an amino acid sequence in which one to several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1, and A protein having dimethylarginine dimethylaminohydrolase activity (c) A protein having an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 1 and having dimethylarginine dimethylaminohydrolase activity A dimethylarginine dimethylaminohydrolase gene encoding the dimethylarginine dimethylaminohydrolase of the following (a), (b) or (c).
(A) a protein comprising the amino acid sequence represented by SEQ ID NO: 1 (b) consisting of an amino acid sequence in which one to several amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1, and A protein having dimethylarginine dimethylaminohydrolase activity (c) A protein having an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 1 and having dimethylarginine dimethylaminohydrolase activity  A recombinant DNA, wherein the gene according to claim 5 is inserted into a vector DNA.  A transformant or transductant comprising the recombinant DNA according to claim 6. A method for producing dimethylarginine dimethylaminohydrolase, comprising culturing the transformant or transductant according to claim 7 in a medium and collecting dimethylarginine dimethylaminohydrolase from the culture.

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