JP2003289864A - Histamine dehydrogenase gene, new recombinant dna and method for producing histamine dehydrogenase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a histamine dehydrogenase gene suitably available for histamine assay, a new recombinant DNA and a method for producing the histamine dehydrogenase. <P>SOLUTION: Disclosed is a histamine dehydrogenase gene encoding a protein of following (a) or (b): (a) a protein consisting of an amino acid sequence represented by SEQ ID NO.1 (refer to the specification) ; (b) a protein consisting of an amino acid sequence in which one or a plurality of amino acids are deleted, substituted or added from/for/to the amino acid sequence (a) and having a histamine dehydrogenase activity. Disclosed are also a new recombinant DNA featured by inserting the histamine dehydrogenase gene into a vector DNA, a transformant or transductant including the recombinant DNA, and a method for producing the histamine dehydrogenase featured by culturing the transformant or the transductant in a medium and by obtaining the histamine dehydrogenase from the culture preparation. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a histamine dehydrogenase gene and a novel recombinant which can be preferably used for histamine quantification performed in a wide range of fields such as food industry, fishery industry, food hygiene, medical treatment and analytical instrument industry. Body DNA
And a method for producing histamine dehydrogenase.

[0002]

BACKGROUND OF THE INVENTION Histamine is used not only for bacteria but also for animals.
It is present in almost all tissues of plants. It has been revealed that histamine is involved in a wide variety of biological functions such as cell proliferation, nucleic acid and protein synthesis, and regulation of enzyme activity. In addition, histamine has been shown to have various physiological actions in the human body such as hypotension, smooth muscle contraction, and glandular secretion promotion. In particular, histamine plays a role as a transmitter in allergic reactions. Is important because it can be a clue for diagnosing allergic reactions and analyzing pathological conditions.
Histamine is an amine having a molecular formula of C ₅ H ₉ N ₃ and a molecular weight of 111 and having a chemical structural formula described in the chemical reaction formula shown in “Chemical Formula 1” below. Histamine, which is rarely found in fresh seafood and meat, appears in the spoilage process and is contaminated with histidine-decarboxylase-active microorganisms, resulting in the free amino acid histidine in protein tissues. It is generated by the decarboxylation action (chemical reaction formula) shown in "Chemical formula 1" described below.

[0003]

[Chemical 1]

Furthermore, histamine sometimes causes allergic food poisoning. In the process of spoiling foods, various food components such as proteins, nucleic acids, polysaccharides and fats are decomposed by the action of microorganisms, and various kinds of spoilage products such as ammonia, hydrogen sulfide, amines and organic acids are produced. Especially in fish and shellfish that contain a large amount of free amino acids, amines that use amino acids as precursors are generated very early, so there is a risk of eating those that contain a large amount of these amines even when the freshness decline is not significant. . Of the amines, what must be noted in food hygiene is histamine described above, and when ingesting food containing this, a feeling of heat on the face,
Headache, urticaria, etc. appear, sometimes accompanied by symptoms such as diarrhea or vomiting, and allergy-like food poisoning is said to occur. Therefore, the determination of histamine is not only important for food hygiene but also provides a clue as to the extent of food spoilage.

From the above, the food industry, the fishery industry,
Obtaining histamine dehydrogenase, which can be suitably used for quantifying histamine performed in a wide range of fields such as food hygiene, medical treatment and analytical instrument industries, has an extremely important industrial significance. The production of histamine dehydrogenase has hitherto been carried out, for example, by culturing in a medium, a strain having a histamine dehydrogenase-producing ability, which belongs to the genus Rhizobium and is characterized by acting specifically on histamine and having the following physicochemical properties. , The histamine dehydrogenase was collected from the culture (see JP 2001-157579 A).
. However, in the above method for producing histamine dehydrogenase, not only is the production amount insufficient, but a surfactant must be added in the step of extracting the enzyme from the strain, the purification step becomes complicated, and the recovery rate becomes low, etc. There was a problem.

[0006]

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems of the prior art, and its object is to obtain a histamine dehydrogenase gene, a novel recombinant DNA and a histamine dehydrogenase. To provide a manufacturing method.

[0007]

The inventors of the present invention have made various studies in view of the above-mentioned problems, and as a result, Rhizobium (Rhiz)
We succeeded in isolating and structurally determining the histamine dehydrogenase gene derived from S. obium sp. 4-7 and obtained a recombinant DNA in which the gene encoding histamine dehydrogenase was inserted into vector DNA. This recombinant DNA was transformed into Escherichia (Escheri).
The present invention was completed based on these findings and found that when a strain having a histamine dehydrogenase-producing ability contained in a strain belonging to the genus Chia) is produced and cultured in a medium, histamine dehydrogenase is efficiently produced. . That is, the first invention is the following (a) or (b)
Is a histamine dehydrogenase gene encoding the protein of. (A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (b) a protein consisting of the amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence (a), and having histamine dehydrogenase activity The second invention is a histamine dehydrogenase gene consisting of the following DNA (a) or (b). (A) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2 (b) Hybridizing with DNA consisting of the nucleotide sequence complementary to the DNA consisting of the nucleotide sequence of (a) under stringent conditions, and histamine dehydrogenase activity A DNA encoding a protein having: A third invention is a novel recombinant DNA characterized in that the above-mentioned histamine dehydrogenase gene is inserted into a vector DNA. A fourth invention is a transformant or transductant containing the above recombinant DNA.
A fifth invention is a method for producing histamine dehydrogenase, which comprises culturing the above transformant or transductant in a medium and collecting histamine dehydrogenase from the culture.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.
The histamine dehydrogenase gene of the present invention can be isolated, for example, as follows. First, Rhizobium sp. 4-7 (FERM BP-6861) is cultured by, for example, the method described in JP 2001-157579 A, and chromosomal DNA is extracted from the obtained strain. The chromosomal DNA is prepared from the bacterial cells by, for example, Cur
rentProtocols in Molecular
Biology (WILEYInterscience
e, 1989) and the like. Then, the above-mentioned microorganism is treated, for example, in Japanese Patent Laid-Open No. 2001-1.
The histamine dehydrogenase can be obtained by culturing by the method described in Japanese Patent No. 57579 or the like and purifying the histamine dehydrogenase. The resulting histamine dehydrogenase is treated with lysyl endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.) under denaturing conditions and fragmented.
The above fragment is, for example, Capcell Pak C18
It was fractionated by reverse-phase high performance liquid chromatography using SG300 (manufactured by Shiseido Co., Ltd.) and the like, and the amino acid sequences of several kinds of fragment peptides were determined, eg, Procise 492
It is determined using a protein sequencer (manufactured by Applied Biosystems) or the like.

A polymerase chain reaction (hereinafter abbreviated as PCR) primer is prepared based on the above amino acid sequence. At this time, in consideration of the degeneracy of codons, a mixed base is used to prepare a primer for a portion where a plurality of bases are degenerate. Using the prepared primers, PCR is performed using the previously obtained chromosomal DNA of Rhizobium sp. 4-7 as a template. The amplified DNA fragment is used as a vector DNA, for example, pT7Blue T Vector.
(Manufactured by Takara Shuzo) or the like to obtain a recombinant plasmid. The nucleotide sequence of the inserted DNA in the plasmid can be determined by, for example, the multicapillary DNA analysis system CEQ20.
00 (manufactured by Beckman Coulter, Inc.) and the like, and those having at both ends a nucleotide sequence that correctly encodes the amino acid sequence of the peptide used for the PCR primer design are selected. The amplified DNA fragment obtained as described above is a part (partial gene) of the histamine dehydrogenase gene of the present invention. Then, a new PCR primer for inverse PCR is synthesized based on the base sequence of the obtained DNA.

Then, the chromosomal DNA is completely digested with various restriction enzymes and then subjected to a usual agarose gel electrophoresis method, and Southern blot analysis using the above-mentioned partial gene as a probe is carried out. As a result, the chain length of the DNA fragment containing the histamine dehydrogenase gene can be determined. Then, the chromosomal DNA is enzymatically treated with the restriction enzyme used for Southern blot analysis, and then self-ligated. Inverse PCR is performed using this as a template and the above-mentioned primers. When a plurality of DNA fragments are amplified, the amplified DNA fragment having the size estimated from the chain length obtained by the Southern blot analysis is the DNA fragment containing the histamine dehydrogenase gene. These D
By determining the base sequence of the NA fragment and ligating it,
The entire base sequence of the histamine dehydrogenase gene can be determined. Then, the amino acid sequence of the polypeptide translated by the gene having the base sequence is determined. This amino acid sequence is as shown in SEQ ID NO: 1.
The gene encoding the amino acid sequence thus determined is the histamine dehydrogenase gene of the present invention. In the amino acid sequence shown in SEQ ID NO: 1, one or more, preferably several amino acids have been deleted, substituted or added, and the histamine dehydrogenase gene encoding an amino acid sequence that brings about histamine dehydrogenase activity is , All are included in the present invention. Then, in order to obtain a histamine dehydrogenase gene encoding an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 and which brings about histamine dehydrogenase activity, any method can be used. Often, for example, a site-directed mutagenesis method, which is a well-known technique for producing a point mutation or a deletion mutation in a gene; a gene is selectively cleaved, and then a selected nucleotide is removed or added, and the gene is ligated. The method of doing so includes an oligonucleotide mutation induction method and the like.

It is highly probable that these DNAs encode a polypeptide which exerts histamine dehydrogenase activity, and it is possible to prepare transformants and select those having activity. In order to obtain a gene substantially identical to the histamine dehydrogenase gene of the present invention, hybridization is carried out under stringent conditions with a probe having the DNA having the nucleotide sequence of SEQ ID NO: 2 or its complementary strand, or a part thereof. However, those encoding a polypeptide having histamine dehydrogenase activity can be selected. The term "stringent conditions" as used herein refers to conditions under which only specific hybrids are selectively formed and a signal is detected, but nonspecific hybrids are not formed. Such conditions are slightly different depending on the individual species, but can be easily determined by only examining a few salt concentrations or temperatures during hybridization and washing by a conventional method. Such conditions include, for example, item (3) in the embodiment described below.
Since the specific signal can be observed in (5) and (5), hybridization was performed using DIG Easy H.
It is performed at 37 to 42 ° C. overnight using yb reagent (manufactured by Roche Diagnostics). Washing is 0.5 x SSC,
Perform twice for 15 minutes with 0.1% SDS. The washing temperature is 45 ° C or higher, preferably 52 ° C or higher, more preferably 57 ° C or higher. DNAs that hybridize under such conditions have a high probability of encoding a peptide having histamine dehydrogenase activity, but also include those having a mutation that loses histamine dehydrogenase activity. But for those,
After transformation, it can be easily removed by measuring the histamine dehydrogenase-producing ability of the transformant.

Since the recombinant DNA containing the histamine dehydrogenase gene obtained as described above does not have a promoter for high expression in E. coli, the transformant having the recombinant DNA has low histamine dehydrogenase productivity. . Therefore, a high histamine dehydrogenase-producing strain is obtained by the following procedure. First, based on the nucleotide sequence obtained by the above operation, an oligonucleotide containing 5'terminal or 3'terminal of histamine dehydrogenase gene and having about 12 bases each before and after it (oligonucleotide of 30 bases in total, 3'terminal side is (Complementary strand) is synthesized and used as a primer. An NdeI site is incorporated in this synthetic primer, and the product amplified by PCR is digested with NdeI to allow the coding region to be obtained. That is, PCR is performed using chromosomal DNA as a template and synthetic primers, and the resulting product is digested with NdeI to obtain a DNA in the region encoding histamine dehydrogenase.

The obtained DNA is a DNA sequence containing the expression region such as promoter, operator and ribosome binding site derived from Escherichia coli lactose operon (The
Operon, p. 227, Cold Spring
(Harbor Laboratory, 1980). The vector DNA used may be plasmid DNA or bacteriophage DNA. For example, the vector pUTE500K 'shown in item (7) of the Example (Japanese Patent Laid-Open No. 08-2
No. 05861) can be used. Using the obtained recombinant DNA, for example, E. coli K-1
2, preferably E. coli JM109 (manufactured by Toyobo), DH
Each strain is obtained by transforming 5α (Takara Shuzo Co., Ltd.) or the like or transducing them. Production of histamine dehydrogenase using the transformant or transductant having the histamine dehydrogenase-producing ability obtained as described above, for example, a strain belonging to the genus Escherichia, can be performed as follows. In order to cultivate the above-mentioned microorganism, it may be cultivated by a usual solid culturing method, but it is preferable to employ a liquid culturing method as much as possible.

As the medium for culturing the above-mentioned microorganisms, for example, yeast extract, peptone, meat extract, corn steep liquor or soybean or wheat malt exudate, etc., one or more nitrogen sources, potassium dihydrogen phosphate, Used is one to which one or more inorganic salts such as dipotassium hydrogen phosphate, magnesium sulfate, ferric chloride, ferric sulfate or manganese sulfate are added, and if necessary, sugar raw materials, vitamins and the like are appropriately added. . The initial pH of the medium is
It is suitable to adjust to 7-9. In addition, the culture is 30 ~
It is preferably carried out at 42 ° C., preferably around 37 ° C. for 6 to 24 hours by aeration and agitation deep culture, shaking culture, static culture and the like. After the culture is completed, a usual enzyme collecting means can be used to collect histamine dehydrogenase from the culture. The cells are separated from the culture by an operation such as filtration or centrifugation and washed. It is preferable to collect histamine dehydrogenase from the cells. In this case, the bacterial cells can be used as they are,
Ultrasonic crusher, French press, a method of destroying bacterial cells using various disrupting means such as Dynamill, a method of lysing bacterial cell wall by using a cell wall lysing enzyme such as lysozyme,
It is preferable to collect histamine dehydrogenase from the cells by a method of extracting the enzyme from the cells using a surfactant such as Triton X-100.

In order to isolate histamine dehydrogenase from the crude enzyme solution thus obtained, a method commonly used for enzyme purification can be used. For example, it is preferable to perform an appropriate combination of ammonium sulfate salting-out method, organic solvent precipitation method, ion exchange chromatography method, gel filtration chromatography method, adsorption chromatography method, electrophoresis method and the like.

The physicochemical properties of the obtained histamine dehydrogenase are as follows. (1) Action: 1 mol of histamine is produced by oxidative deamination reaction in the presence of an electron acceptor to produce 1 mol of 4-imidazolylacetaldehyde and 1 mol of ammonia. (2) Substrate specificity: It acts specifically on histamine. That is, it acts specifically on histamine but has no or weak action on other amines. (3) Optimum pH and stable pH range: The optimum pH is 9.0.
~ 11.5. The stable pH range of this enzyme is 7.0-1.
It is 1.5. (4) Suitable temperature range for action: 50 mM phosphate buffer (pH
8.0), the suitable temperature range for working is 65 to 75 ° C. (5) Stable up to around 60 ° C in 15-minute treatment (pH 8.0). Above that, it will be deactivated rapidly. (6) Molecular weight: about 150,000 (gel filtration method). The subunit is about 71,000 (SDS-polyacrylamide gel electrophoresis method). (7) Method for measuring titer: The enzyme titer is measured by the following method, and the amount of the enzyme that produces 1 μmol of 4-imidazolylacetaldehyde in 1 minute is 1 unit (1 U). 5
2.4 ml of 0 mM phosphate buffer (pH 8.0), 0.3
mM 1-Methoxy PMS aqueous solution 0.1 ml,
0.3 ml of 1 mM WST-8 aqueous solution, 0.1 ml of 10 mM histamine solution and 0.1 ml of this enzyme solution are added, and the reaction is performed at 30 ° C. for 30 to 60 minutes. The activity of this enzyme is the reduced form of 1-Met produced in this enzyme reaction.
The color development of WST-8 produced by the reaction with hoxy PMS is measured by the absorbance at 460 nm.

The present enzyme is extremely useful for quantifying histamine in fish meat to be measured or for quantifying a trace amount of histamine contained in body fluids such as human serum or urine when measuring freshness of fish meat. is there. And by using this enzyme, among various amines contained in fish meat or body fluid to be measured, it does not act on cadaverine or putrescine, etc., which do not need to be detected, and acts well only on histamine of interest, It becomes possible to quantify this accurately. Also, cadaverine or putrescine is
It is an amine that is produced at the same time as histamine when fish meat rots, and it was necessary to separate and remove these by some method in the conventional enzymatic assay of histamine, whereas in the assay of histamine using this enzyme, However, because of its excellent characteristics, there is an advantage that a troublesome and time-consuming separation operation is completely unnecessary.

[0018]

The present invention will be described in more detail with reference to the following examples. (Example) Cloning of histamine dehydrogenase gene (1) Preparation of Rhizobium sp 4-7 chromosomal DNA Rhizobium sp 4-7 (FERM BP-6861) was cultured by the method described in JP 2001-157579 A, etc., The cells were collected by centrifugation. GNOME from this fungus body
Using the DNA Isolation Kit (Funakoshi), 0.75 mg of chromosomal DNA was obtained.

(2) Acquisition of partial gene Next, histamine dehydrogenase was purified from the above microorganism by the method described in JP 2001-157579 A, and the obtained histamine dehydrogenase was added to 0.1%.
100 mM Tris-HCl buffer (pH 9.
In 0), it was treated with lysyl endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.) at 37 ° C. overnight for fragmentation. The above fragments were fractionated by reverse phase high performance liquid chromatography using Capcell Pak C18 SG300 (manufactured by Shiseido Co., Ltd.). The amino acid sequences of lysyl endopeptidase untreated protein and several fragment peptides were
The analysis was carried out by the Edman method using a locise 492 protein sequencer (manufactured by Applied Biosystems). Based on the N-terminal and internal amino acid sequences obtained by the above method, a plurality of PCR primers having mixed bases at degenerate codons were prepared. Using the chromosomal DNA prepared in Example item (1) as a template, Ex
PCR was performed using Taq DNA polymerase (Takara Shuzo). The PCR reaction was carried out with a PCR master cycler gradient (manufactured by Eppendorf) at thermal denaturation 94 ° C. for 2 minutes, annealing 50 ° C. for 30 seconds, extension reaction 72.
30 cycles were carried out under conditions of 1 ° C for 1 minute and 30 seconds. As a result, the sense primer is 5'-ATGMGNGAYAA
YAARTAYGAYATHYTNTTYGARCC-
3 '(35 mer, A is adenine, C is cytosine, G
Is guanine, T is thymine, H is adenine or cytosine or thymine, M is adenine or cytosine, N is adenine or cytosine or guanine or thymine, R is adenine or guanine, and Y is thymine or cytosine. N-terminal amino acid sequence of the corresponding protein: Met Arg Asp
Asn Lys Tyr Asp Ile Leu Phe
Glu Pro), antisense primer is 5'-Y
TTNGTRTAARAARTTNGGNCCRTTTDA
T-3 ′ (27mer, amino acid sequence of corresponding protein: Ile Asn Gly Pro Asn Ph
e Tyr Thr Lys), 0.3 kbp
A gene fragment corresponding to the degree was amplified. Amplified DN
The A fragment was recovered from the gel after 1% agarose gel electrophoresis and incorporated into pT7Blue T Vector (Takara Shuzo) to obtain a recombinant plasmid. The base sequence of the inserted DNA in the plasmid was calculated using the multicapillary DN.
A analysis system CEQ2000 (manufactured by Beckman Coulter, Inc.) was used. As a result, the DNA fragment (3
51 bp) had, at both ends, a nucleotide sequence that correctly encodes the amino acid sequence of the peptide used in the PCR primer design. In addition, the internal amino acid sequence determined by the protein sequencer was present in the amino acid sequence deduced from the determined nucleotide sequence. Therefore, it was revealed that the obtained amplified DNA fragment was a part (partial gene) of the histamine dehydrogenase gene of the present invention.

(3) Southern blot analysis of Rhizobium sp. 4-7 chromosomal DNA 1 Next, 2 μ of the chromosomal DNA prepared in Example item (1)
g is the restriction enzyme BamHI, ClaI, EcoRI, E
coRV, HindIII, NdeI, PstI, Sa
It was digested with 1I and SmaI at 37 ° C for 5 hours. The obtained restriction enzyme digested DNA was subjected to 0.7% agarose gel electrophoresis. After the migration, the DNA was transferred to a nylon membrane (Hybond-N +, Amersham Pharmacia Biotech) by Southern blotting. As a hybridization probe, PCR amplification was performed in the presence of PCR Dig labeling mix (Roche Diagnostics) using the plasmid gene obtained in Example item (2) as a template and M13 primer. I used one. The reaction conditions and the like of PCR were the same as those in the above-mentioned item (2) of the example. 2x the above nylon membrane
After washing with SSC (0.3M NaCl, 0.03M sodium citrate; pH 7.0), hybridization was performed using a DIG system according to a user guide (Roche Diagnostics). Washing the nylon membrane after hybridization is
2 × SSC containing 0.1% SDS (15 mM NaCl,
1.5 mM sodium citrate; pH 7.0) at room temperature for 5 minutes, twice, and then 0.1 × SDS-containing 0.1 ×
Performed twice in SSC at 68 ° C. for 15 minutes. as a result,
BamHI, ClaI, EcoRI, EcoRV, Hi
ndIII, NdeI, PstI, SalI, SmaI
Of each digestion product of about 8.0 kbp, about 2.5 kbp,
About 11 kbp, about 5.6 kbp, about 18 kbp, about 22
kbp, about 11 kbp, about 6.3 kbp, about 12 kbp
A signal derived from the probe hybridized at the position was observed.

(4) Acquisition of partial fragment of histamine dehydrogenase gene by inverse PCR method 1 Based on the above results, about 8.0 kbp BamHI fragment, about 2.5 kbp ClaI fragment, about 5.6 kbp EcoRV fragment, about Inverse PCR was performed using the 6.3 kbp SalI fragment. BamHI, Cla
Chromosomal DNA 10 digested with I, EcoRV and SalI
μg was separated by 1.0% agarose gel electrophoresis, and the agarose gel at the position corresponding to each size was cut out. The DNA fragment was extracted and purified from the gel by GENE CLEAN II (manufactured by Funakoshi Co., Ltd.)
4 Self-ligation was performed using DNA Ligase (manufactured by Roche). Inverse PCR was performed according to a conventional method using the above ligation product as a template. A plurality of PCR primers were prepared based on the partial gene sequence determined in Example item (2). KOD Das
h heat denaturation 94 using h polymerase (manufactured by Toyobo Co., Ltd.)
℃, 30 seconds, annealing 67-72 ℃, 2 seconds, extension reaction 7
30 cycles were performed under conditions of 2 ° C. and 4 minutes. As a result, the sense primer is 5'-TCATCGAACTGCG
CCTGTGGGAAGAC-3 ′ (21 of SEQ ID NO: 2
5 to 240 bp, 26 mer), antisense primer is 5'-TTCTTCCGCAATGTGTGGAC
CTATC-3 ′ (65 to 42 bp of SEQ ID NO: 2, 24
Amplified DNA from ClaI digestion product
(About 2.5 kbp) and amplified D from EcoRV digests
It was possible to obtain NA (5.6 kbp). Each amplified DNA fragment was electrophoresed on an agarose gel to which Crystal Violet was added, and S. N.
A. P. Gel Purification Kit
(Manufactured by Invitrogen). This is the TOPOTA Cloning Kit (Invitr
(manufactured by Ogen) and incorporated into the pCR-XL-TOPO vector. The base sequence of the inserted DNA in the plasmid was determined by the multicapillary DNA analysis system CEQ2.
000 (manufactured by Beckman Coulter, Inc.). As a result of analysis of a DNA fragment of 2.5 kbp derived from a ClaI digest, 971 bp from the 5 ′ end to the ClaI cleavage site of the gene encoding histamine dehydrogenase and the nucleotide sequence information of 557 bp upstream from the 5 ′ end were obtained. The internal amino acid sequence determined by the protein sequencer was present in the amino acid sequence deduced from the determined nucleotide sequence. Therefore, it was revealed that the obtained amplified DNA fragment was a part (partial gene) of the histamine dehydrogenase gene of the present invention. For the amplified DNA derived from the EcoRV digest,
Since the cleavage site of coRV was present at a position 433 bp from the 5'end of the histamine dehydrogenase gene and had gene information 5'upstream from it,
From this, it was not possible to obtain further downstream gene information encoding histamine dehydrogenase.

(5) Southern blot analysis of Rhizobium sp. 4-7 chromosomal DNA 2 Cl is a gene encoding histamine dehydrogenase
In order to obtain information downstream of the aI cleavage site, Southern blot analysis was performed again in the same manner as in Example item (3). That is, the chromosome DN adjusted in Example item (1)
A2 μg was added to the restriction enzymes BamHI, BanII, Eco
RV, EcoT14I, HincII, MluI, Sp
It was digested with hI and XhoI at 37 ° C. for 5 hours. The obtained restriction enzyme digested DNA was subjected to 0.7% agarose gel electrophoresis. After the migration, the DNA was transferred to a nylon membrane (Hybond-N +, Amersham Pharmacia Biotech) by Southern blotting. As a hybridization probe, the plasmid into which the amplified DNA derived from the ClaI digestion product obtained in Example (4) was inserted was used as a template, and the sense primer 5'-TTAA was used.
GGAAGCGGTTTGGCGACACGAC-3 '
(635 to 659 bp of SEQ ID NO: 2, 25 mer),
Antisense primer is 5'-TGAAGTCCGAG
AACACCCTGGGCCAC-3 ′ (949 to 925 bp of SEQ ID NO: 2, 25 mer) was prepared and PCR was performed.
PCR amplification was performed in the presence of Dig labeling mix (manufactured by Roche Diagnostics). Regarding the PCR reaction conditions and the like, the annealing temperature was 62 ° C. under the conditions of the above-mentioned item (2) of the example. The above nylon membrane was applied to 2 × SSC (0.3M NaCl, 0.0
After washing with 3M sodium citrate; pH 7.0), DI
Hybridization was performed using a G system according to a user guide (manufactured by Roche Diagnostics). After the hybridization, the nylon membrane was washed with 2 × SSC (15%) containing 0.1% SDS.
mM NaCl, 1.5 mM sodium citrate; pH
7.0) at room temperature for 5 minutes, twice, and then 0.1% S
It was performed twice at 0.1 ° SSC containing DS at 68 ° C for 15 minutes. As a result, BamHI, BanII, EcoR
V, EcoT14I, HincII, MluI, Sph
I, XhoI digestion products of about 3.9 kbp, about 1.6 kbp, about 1.3 kbp, about 0.7 kbp, about 0.9 kbp, about 8.1 kbp, about 2.5 kbp, about 1.1 kbp, respectively. A signal derived from the probe hybridized to the position was observed. Based on the molecular weight of histamine dehydrogenase, the number of coding genes is about 2 kbp.
It is presumed that Sph from Example item (4)
It was expected that the fragment of SphI contained the full length gene encoding histamine dehydrogenase, as it was known that the I cleavage site was located approximately 130 bp upstream of the 5'end of the gene.

(6) Acquisition of partial fragment of histamine dehydrogenase gene by inverse PCR method 2 Based on the above results, inverse PCR was carried out using a SphI fragment of about 2.5 kbp. 10 μg of the SphI-digested chromosomal DNA was separated by 1.0% agarose gel electrophoresis, and the agarose gel at the position corresponding to each size was cut out. Gel from GENE CLEAN II
The DNA fragment was extracted and purified by (Funakoshi Co., Ltd.)
The NA fragment was self-ligated using T4 DNA Ligase (manufactured by Roche). Inverse PC using the above ligation product as a template according to a conventional method
R was done. A plurality of PCR primers were prepared based on the partial gene sequence determined in Example item (4).
KOD Dash polymerase for PCR (manufactured by Toyobo Co., Ltd.)
Using heat denaturation 94 ° C., 30 seconds, annealing 63-70
C., 2 seconds, extension reaction 72.degree. C., 4 minutes, 30 cycles were performed. As a result, 5'- as a sense primer
GTGCCGCCAGGGGTGTCTCGACTTCA
-3 '(925 to 949 bp of SEQ ID NO: 2, 25 me
r), 5'-TGCAT as an antisense primer
TGGATTTGTTGAGAGCCGGG-3 '
When (25-49 bp upstream from 5'end, 25 mer) was used, amplified DNA (about 1.5
kbp) could be obtained. Cry the amplified DNA fragment
Electrophoresis was carried out on an agarose gel containing stal violet, and the gel was subjected to S. N. A. P. Gel Pu
It was recovered using a Ricification Kit (manufactured by Invitrogen). This is pT7Blue T Ve
It was incorporated into ctor (Takara Shuzo). The base sequence of the inserted DNA in the plasmid was changed to multicapillary DNA.
It was determined using an analysis system CEQ2000 (manufactured by Beckman Coulter, Inc.). DNA from SphI digest
As a result of analyzing the fragment of 1.5 kbp, the remaining gene encoding histamine dehydrogenase and the nucleotide sequence information of 229 bp downstream from the 3'end were obtained. The internal amino acid sequence determined by the protein sequencer was present in the amino acid sequence deduced from the determined nucleotide sequence. Therefore, the obtained amplified DNA fragment is a part (partial gene) of the histamine dehydrogenase gene of the present invention.
It turned out to be

(7) Cloning of full-length histamine dehydrogenase gene and acquisition of histamine dehydrogenase producing strain First, based on the nucleotide sequence obtained by the above-mentioned operation, an oligonucleotide containing a 5'end or a 3'end of the histamine dehydrogenase gene (total An oligonucleotide of 30 bases and a complementary strand on the 3'-terminal side were designed. The NdeI site was incorporated into this primer, and the product amplified by PCR was digested with NdeI to allow the coding region to be obtained. That is, 5'-AAGCAGT as a sense primer
GAGGAACCATATGCGCGATAACA-
3 '(30 mer), 5'-TCGATCAATCCCTCATATGTCAC as an antisense primer
GCTGGGCTC-3 ′ (30mer) was synthesized. The SphI-digested chromosomal DNA was separated by 1.0% agarose gel electrophoresis, the agarose gel at the position corresponding to the size of 2.5 kbp was cut out, and the S.I. N.
A. P. Gel Purification Kit (I
The gene fragment was recovered using nvitrogen). Using this as a template, synthetic primers and KOD Pl
heat denaturation 94 using us polymerase (manufactured by Toyobo Co., Ltd.)
℃, 15 seconds, annealing 56 ℃, 30 seconds, extension reaction 68
30 cycles were carried out under the conditions of 3 ° C for 30 minutes. As a result, amplified DNA (about 2.1 kb) derived from the SphI digest was obtained.
p) was obtained, which could be digested with NdeI to obtain the DNA for the region encoding histamine dehydrogenase.

The obtained DNA is used as a DNA sequence containing the expression regions such as promoter, operator and ribosome binding site derived from Escherichia coli lactose operon (The
Operon, p. 227, Cold Spring
The recombinant plasmid pHMDH1 DNA was obtained by inserting the vector carrying the Harbor Laboratory, 1980) into the NdeI site of pUTE500K '(described in JP-A-08-205861).
D. M. Morrison's Method (Methods i
n Enzymology, 68, p. 326-33
1, 1979), recombinant plasmid DNAp
E. coli JM109 using HMDH1
(Toyobo Co., Ltd.) was transformed to obtain a transformant, E. coli JM109 (pHMDH5001). The nucleotide sequence of the inserted DNA in the plasmid was determined using a multicapillary DNA analysis system CEQ2000 (manufactured by Beckman Coulter, Inc.). The nucleotide sequence was compared with that of a clone previously obtained by the inverse PCR method. For a portion where PCR error was considered to be contained in either side, PCR was carried out for multiple lots using the fragment obtained by treating the chromosomal DNA as described above with SphI as a template for the portion including the vicinity thereof. It was determined by comparing the nucleotide sequences. As a result, pHMD
No PCR error was included in H5001. The determined nucleotide sequence of the histamine dehydrogenase gene is shown in SEQ ID NO: 2, and the amino acid sequence of the polypeptide translated from the DNA sequence is shown in SEQ ID NO: 1. The ORF of the histamine dehydrogenase gene is 2
It was found to be composed of 079 bp and 693 amino acids. In addition, pHMDH5001 is FERM BP-
Deposited as 7981. The resulting E. coli (E.
coli) JM109 (pHMDH5001) 1m
TY medium containing M isopropyl-β-D-thiogalactopyranoside (1% Bacto tryptone, 0.5% Bacto yeast extract, 0.5% NaCl, pH)
After culturing with shaking at 37 ° C. for 10 hours at 7.0), the histamine dehydrogenase activity was measured.
It was 07 U / ml.

[0026]

INDUSTRIAL APPLICABILITY According to the present invention, histamine dehydrogenase can be efficiently produced, so that the present invention is industrially very useful.

[0027]

[Sequence list]                   SEQUENCE LISING <110> KIKKOMAN CORPORATION <120> A HISTAMINE DEHYDROGENASE GENE, A NOVEL RECOMBINANT DNA, AN D A PROCESS FOR PRODUCING A HISTAMINE DEHYDROGENASE <130> P2272 <160> 2 <210> 1 <211> 693 <212> PRT <213> Rhizobium sp. 4-7 <400> 1      Met Arg Asp Asn Lys Tyr Asp Ile Leu Phe Glu Pro Val Arg Ile Gly                 1 5 10 15                 Pro His Ile Ala Lys Asn Arg Phe Tyr Gln Val Pro His Cys Asn Gly                           20 25 30                Gly Gly Tyr Arg Asp Pro Ser Ala Ala Ala Ala Met Arg Gly Ile Lys                        35 40 45                Ser Glu Gly Gly Trp Gly Val Ile Phe Thr Glu Gln Thr Glu Met His                    50 55 60               His Thr Ser Glu Ile Thr Pro Phe Ile Glu Leu Arg Leu Trp Glu Asp                65 70 75 80                 Lys Asp Ile Pro Gly Leu Arg Arg Met Ser Asp Ala Met Lys Val His                                85 90 95                 Gly Ala Leu Ala Gly Ile Gln Leu Ala Tyr Ser Gly Ile Asn Gly Pro                           100 105 110               Asn Phe Tyr Thr Lys Glu Val Pro Leu Ala Pro Ser Ala Leu Pro Ile                        115 120 125                Arg Thr Phe Thr Asn Asp Pro Val Gln Ala Arg Ala Leu Asp Lys Gln                   130 135 140                 Asp Ile Lys Asn Leu Arg Arg Trp Phe Val Asn Ala Ala Lys Arg Ser               145 150 155 160               Lys Ile Ala Gly Phe Asp Leu Ile Cys Leu Tyr Gly Ala His Gly Phe                               165 170 175               Gly Ile Phe Gln His Phe Leu Ser Arg Ala Thr Asn Gln Arg Thr Asp                           180 185 190                 Glu Tyr Gly Gly Ser Leu Glu Asn Arg Ser Arg Phe Ala Arg Glu Val                       195 200 205                 Val Glu Asp Ile Lys Glu Ala Val Gly Asp Thr Thr Ala Ile Thr Met                   210 215 220                Arg Val Ser Leu Asp Glu Thr Ile Gly Glu Leu Gly Phe Ser Asn Ala               225 230 235 240                Glu Val Arg Glu Phe Val Glu Met Asn Ala Asn Leu Pro Asp Leu Trp                                245 250 255                  Asp Leu Ala Gln Gly Thr Trp Glu Asp Cys Ser Gly Pro Ser Arg Phe                            260 265 270                 Lys Glu Glu Gly Ala Gln Glu Ile Leu Val Lys Gly Ile Arg Glu Leu                       275 280 285               Ser Ser Lys Pro Val Val Gly Val Gly Arg Phe Thr Ser Pro Asp Val                   290 295 300               Met Ala Arg Met Val Arg Gln Gly Val Leu Asp Phe Ile Gly Cys Ala               305 310 315 320                Arg Pro Ser Ile Ala Asp Pro Phe Leu Pro Lys Lys Ile Glu Glu Gly                               325 330 335               Arg Ile Glu Asp Ile Arg Glu Cys Ile Gly Cys Asn Ile Cys Ile Thr                           340 345 350              Gly Asp Met Thr Met Ser Ile Ser Arg Cys Thr Gln Asn Pro Thr Phe                       355 360 365      Met Glu Glu Trp Arg Lys Gly Trp His Pro Glu Arg Met Asn Ala Lys                   370 375 380                Gly Asp Ser Asn Thr Val Leu Val Val Gly Ala Gly Pro Ala Gly Leu               385 390 395 400                Glu Ala Thr Arg Ala Leu Ser Leu Arg Gly Tyr Asp Val Thr Leu Ala                                405 410 415                 Glu Ala Thr Thr Thr Leu Gly Gly Arg Val Ala Arg Glu Arg Leu Leu                            420 425 430                 Pro Gly Leu Ser Ala Trp Gly Arg Val Val Asp Tyr Arg Gln Tyr Gln                       435 440 445               Ile Ser Gln Arg Thr Asn Val Glu Thr Tyr Phe Asp Ser Arg Leu Thr                   450 455 460                Ala Glu Asp Val Leu Gly Phe Gly Phe Glu His Val Ala Ile Ala Thr               465 470 475 480               Gly Ser His Trp Arg Arg Asp Gly Val Ala Arg Gln His Val Val Pro                               485 490 495                Met Pro Ile Asp Pro Ser Met Thr Val Trp Thr Pro Asp Asp Ile Met                           500 505 510                Ala Lys Val His Pro Glu Asn Leu Ser Gly Lys Thr Val Val Val Tyr                       515 520 525                Asp Asp Asp His Tyr Tyr Met Gly Gly Val Met Ala Glu Val Met Ala                   530 535 540                Lys Ala Gly Ala Lys Val Ile Leu Val Thr Ser Ser Ala Tyr Val Ser               545 550 555 560               Asp Trp Thr Arg Asn Thr Leu Glu Gln Gly Ala Ile His Val Arg Leu                                565 570 575                Asp Asp Leu Gly Val Asp Ile Arg Leu Asn Arg Gly Val Thr Ala Ile                            580 585 590                 Arg Ala Gly Glu Val Glu Thr Asn Cys Val Tyr Thr Gly Lys Arg Ser                       595 600 605                 Ala Ile Gly Cys Asp Ala Val Leu Met Val Ala Ser Arg Thr Ser Glu                   610 615 620               Asp Gln Leu Phe Asn Asp Leu Ile Ala Arg Gln Gly Asp Trp Pro Asp               625 630 635 640                Ala Gly Ile Lys Ser Val Lys Ile Ile Gly Asp Ala Ala Ala Pro Ala                               645 650 655             Pro Ile Ala Trp Ala Thr Tyr Ala Gly His Arg Tyr Ala Arg Glu Leu                          660 665 670               Asp Thr Pro Asp Ile Gly Asp Asp Leu Pro Phe Arg Arg Glu Val Thr                       675 680 685                Gln Leu Glu Pro Ala          690

<210> 2 <211> 2079 <212> DNA <213> Rhizobium sp. 4-7 <400> 2 atg cgc gat aac aag tac gac att ctc ttc gaa ccc gtc cgg ata ggt 48 cca cac att gcg aag aac cgc ttc tac cag gtc ccc cat tgc aac ggc 96 ggc ggc tat cgc gcgcc gcg gcc atg cgc gga atc aag 144 tcg gaa ggc ggg tgg ggt gtc atc ttc acc gag cag acc gag atg cac 192 cac acc tcg gag atc aca ccc ttc atc gaa ctg cgc ctg tgg gaa gac 240 cct ggt atc ag atg tcc gac gcc atg aag gtc cat 288 ggt gcg ctt gcc ggc atc cag ctc gcc tac tcc ggc atc aat ggt ccg 336 aac ttc tac acc aag gag gtt ccg ctg gca ctt ccg atacc 384c gtg cag gcg cgt gcc ttg gac aag cag 432 gat atc aaa aat ctt aga cgc tgg ttc gtc aac gcc gcc aag cgg tcg 480 aag atc gcc ggt ttc gat ctg atc tgt ctc tac ggc gca cac ggc tt ctg tca cgc gcg acc aac cag cgc acc gac 576 gag tac ggc gga agt ctc gag aac cgt tcg cgc ttt gcc cgc gaa gtc 624 gtc gag gac att aag gaa gcg gtt ggc gac gc cg acg acc gcg atg gag acg a tc ggc gag ctc ggt ttc tcc aat gcc 720 gag gtc cgg gag ttc gtc gag atg aac gcg aac ctg ccg gat ctg tgg 768 gat ctt gcg cag gga acc tgg gaag actgt tcc ggg cct tga cagag gag atc ctg gtc aag ggg atc cgc gag ctg 864 tcc tcg aag ccg gtt gtc ggc gtc ggc cgc ttc acg tcc ccc gat gtg 912 atg gca cgc atg gtc ctc atc gct cct cat cg cg tg gat ccg ttc ttg ccg aag aag atc gag gaa ggg 1008 cgc atc gaa gac atc cgc gaa tgc atc gga tgc aac atc tgc atc acc 1056 ggc gac atg acg atg tcg atc tgc acgg acacc gag acg cag cag cag cag cag cag cgc aag ggt tgg cac ccc gag cgg atg aac gcc aag 1152 ggt gac agc aac acc gtg ctc gtt gtg ggc gcc ggt ccg gcc ggc ctc 1200 gaa gcg aca cgc gcc acg48 gc gcc gg gg acg48 acc acg ctt ggc ggg cgc gtt gcg cgt gag cgc ttg ttg 1296 ccc ggt ctc tca gcc tgg ggc cgc gtc gtc gac tat cgt cag tat cag 1344 atc agc cag cgc acc aat gtc gag gct acgt tt gca gag gat gtg ctc ggc ttc ggg ttc gaa cat gtc gcg atc gca acc 1440 ggt tct cac tgg cgc cgt gac ggc gtt gca cga cag cac gtc gtg ccg 1488 atg ccg atc gat cct tcc atg acg atg acg atg acg 1536 gcc aag gtg cat ccc gaa aac ctg tcc ggc aag aca gtc gtc gtc tat 1584 gac gac gac cac tat tac atg ggc ggc gtc atg gcc gag gtc atg gcc 1632 aag gcc ggc gcc ag gtcgtg acc gtcgtg acg gtc tcc 1680 gac tgg acg cga aac acg ctc gag cag ggt gca atc cat gtg cgc ctt 1728 gac gat ctc ggg gtc gat atc cgc ctc aac cgc ggc gta acg gcc att 1776 cgc gcc gga gaa gtc tgcgag acc cga agc 1824 gcg atc ggc tgc gac gcg gtt ctc atg gtt gcg tca cgg acg tcc gag 1872 gat cag ctc ttc aac gac ctg att gcc cgg cag ggt gat tgg ccc gat 1920 gcc ggc atc aag aag atg gca ccc gcc 1968 ccg atc gca tgg gcg acc tat gcc ggt cat cgc tat gcc cgt gaa ctc 2016 gac aca ccc gat atc ggc gat gac ctc ccg ttc cgc cgc gaa gtc acg 2064 cag ctc gag acg a gcg
2079

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 9/06 C12N 5/00 AF term (reference) 4B024 AA03 AA11 BA08 CA03 DA06 EA04 GA11 HA01 4B050 CC04 DD02 EE10 LL03 4B065 AA01Y AA26X AB01 AC14 BA02 CA28 CA46

Claims (5)

[Claims] 1. A histamine dehydrogenase gene encoding the following protein (a) or (b). (A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (b) a protein consisting of the amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence (a), and having histamine dehydrogenase activity 2. A histamine dehydrogenase gene consisting of the following DNA (a) or (b): (A) DNA consisting of the nucleotide sequence shown in SEQ ID NO: 2 (b) Hybridizing with DNA consisting of the nucleotide sequence complementary to the DNA consisting of the nucleotide sequence of (a) under stringent conditions, and histamine dehydrogenase activity Encoding a protein having 3. A novel recombinant DNA, wherein the histamine dehydrogenase gene according to claim 1 or 2 is inserted into vector DNA. 4. A transformant or transductant containing the recombinant DNA according to claim 3. 5. A method for producing histamine dehydrogenase, which comprises culturing the transformant or transductant according to claim 4 in a medium, and collecting histamine dehydrogenase from the culture.