JPH11253162A - Dna encoding monoamine oxidase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a DNA fragment containing a monoamine oxidase gene and useful for application to a biosensor and a material-producing process, a gene aiagnosis and treatment, or the like by partially degrading a chromosome DNA of Micrococcus luteus under a specific condition. SOLUTION: This DNA fragment is obtained by partially degrading a chromosome DNA of Micrococcus luteus such as IAM 1099 strain by a restriction enzyme Sau3AI, has a restriction map shown by the formula I, and contains DNA fragment having 2.8 kb largeness separated to largenesses of 2.6 kb and 0.2 kb, 1.7 kb and 1.1 kb, and 1.8 kb and 1.0 kb by BamH I, SaIII and Kpn I sites respectively. Preferably, a recombinant monoamine oxidase is produced by culturing a transformer transformed by a recombinant vector including the DNA encoding monoamine oxidase included in the DNA fragment, or the DNA encoding the monoamine oxidase comprising the amino acid sequence of formula II.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a DNA encoding monoamine oxidase (hereinafter sometimes abbreviated as monoamine oxidase protein) (hereinafter sometimes abbreviated as monoamine oxidase gene), and a DNA fragment containing the gene. (Hereinafter sometimes abbreviated as a monoamine oxidase fragment), a recombinant vector containing the gene, a transformant carrying the gene, and a method for producing a recombinant monoamine oxidase using the transformant.

[0002]

2. Description of the Related Art Monoamine oxidase [EC 1.4.
3.9] is widely found in animals, plants and microorganisms,
Monoamines that have strong bioactivity in cells, ie, tyramine, dopamine
e) It is known as an enzyme that regulates the level of bioactive amines such as adrenaline and noradrenaline.

[0003] Monoamine oxidase (MAO) is classified into a copper-containing MAO and a FAD-containing MAO by a coenzyme. Animal-derived FAD-containing MAOs are further classified into type A and type B by substrates and inhibitors.

It has been reported that mutation of glutamine to a stop codon in the structural gene of MAO containing AAD FAD in animals is associated with diseases such as mental retardation.
As described above, several MAOs of animal or microbial origin have been studied in relation to the metabolism of catecholamines, which are neurotransmitters, but their structures and catalytic mechanisms have not been elucidated.

[0005] Regarding the monoamine oxidase gene, when it is derived from a microorganism, a gene derived from Escherichia coli [J. Ferment. Bioeng., 77, p315-319, 199] is used.
4], a gene derived from mold (Aspergillus niger) [Mol. G
en. Genet., 247, p430-438, 1995] has just been isolated and sequenced.

[0006] MAO from Micrococcus luteus
Is quite different from animal-derived enzymes [Me
th. Enzymol., 17B, p722-726, 1971], and there is no report on gene isolation so far.

[0007]

In order to elucidate the structure and catalytic mechanism of MAO and to develop its use, it is desired to isolate monoamine oxidase genes having various properties and different origins.

[0008] The present invention has been made with the primary object of providing a monoamine oxidase gene and a DNA fragment containing the gene.

[0009]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, the monoamine oxidase gene can be isolated from Micrococcus luteus by using gene recombination techniques. Thus, the present invention has been completed.

That is, the present invention provides a micrococcus
Luteus chromosomal DNA is obtained by partial digestion with the restriction enzyme Sau3AI, has the restriction map shown in FIG. 1, and is 2.6 kb, 0.2 kb, 1.7 kb, 1.7 kb and 1. kb by the BamHI, SalII, and KpnI sites, respectively. 1 kb,
Provided is a DNA fragment containing at least a DNA fragment of about 2.8 kb, which is divided into 1.8 kb and 1.0 kb.

[0011] Micrococcus luteus is preferably IAM1099 strain. Further, the present invention provides the DNA
D encoding monoamine oxidase contained in the fragment
Provide NA.

[0012] The above-mentioned DNA is specifically composed of the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2 in which one or several amino acids have been deleted, substituted or added. DNA encoding monoamine oxidase, and SEQ ID NO: 1
And DNA encoding a protein having a monoamine oxidase activity that hybridizes under stringent conditions to a DNA consisting of the nucleotide sequence described in SEQ ID NO: 1 or a DNA consisting of the nucleotide sequence described in SEQ ID NO: 1.

Further, the present invention provides a recombinant vector obtained by ligating the above DNA to a vector, a transformant transformed by introducing the above DNA, and culturing the transformant. Provided is a method for producing a recombinant monoamine oxidase, which comprises collecting a monoamine oxidase from a culture.

[0014]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The monoamine oxidase fragment of the present invention is generally used for preparing Micrococcus luteus (Microc luteus).
occus luteus) (for example, IAM1099 strain etc.) by partial digestion with a restriction enzyme such as Sau3AI.

Specifically, it can be obtained by the following steps (1) and (2). (1) The above Micrococcus luteus IAM1099 was cultured according to a conventional method, cells were collected from the culture, the cells were crushed, and then ammonium sulfate fractionation, DEAE-
A purified MAO product is obtained by the treatment of cellulose column chromatography, hydroxyapatite column chromatography, and gel filtration [Meth. Enzymol., 17B, p72.
2-726, 1971].

(2) The monoamine oxidase fragment of the present invention is obtained by determining the N-terminal amino acid sequence of the purified protein and screening the Micrococcus luteus chromosomal DNA bank with the corresponding oligo DNA probe.

The above screening can be performed as follows. First, Micrococcus Luteus I
Chromosomal DNA is extracted from the culture of AM1099. This chromosomal DNA is converted to an appropriate restriction enzyme, for example, Sau3AI.
Is partially decomposed using.

Various DNA fragments obtained are inserted into a plasmid vector, for example, pUC118 (Takara Shuzo) to prepare a library. Using this plasmid bank, Escherichia coli, for example, JM109 (Takara Shuzo)
To form a colony. Next, plasmid DNA was extracted from the colonies, electrophoresed on an agarose gel, transferred to a nitrocellulose membrane, and synthesized oligonucleotides corresponding to the N-terminal sequence of monoamine oxidase (SEQ ID NO: 3) (for example, SEQ ID NO: 4). Southern hybridization using a probe having the nucleotide sequence shown in (1) as a probe. Thus, the DN containing the inserted MAO gene from the chromosome of Micrococcus luteus IAM1099 was inserted.
The A fragment can be confirmed.

In the present invention, the monoamine oxidase gene of the present invention was determined based on one of its base sequences as shown in SEQ ID NO: 1. Based on this, "oligo 1000M" DNA synthesizer (manufactured by Beckman) and the like were used. Can be synthesized using a commercially available DNA synthesizer. In addition, Micrococcus luteus (Micrococcu
luteus) can be obtained from a monoamine oxidase fragment obtained from a chromosomal DNA bank such as IAM1099 by the method described above by the method described below.

First, the monoamine oxidase fragment obtained above is cut out using an appropriate restriction enzyme such as Sau3AI, and the obtained DNA fragment is subcloned into an appropriate cloning vector, for example, pUC118 (manufactured by Takara Shuzo). E. coli JM109 strain (Takara Shuzo) is transformed. This transformant is cultured under appropriate antibiotic selection, and cells are recovered from the culture. A plasmid was extracted from the cells by a known method such as the alkali-SDS method, and the DNA introduced into the plasmid was extracted.
By determining the nucleotide sequence of the fragment, the nucleotide sequence of the DNA fragment containing the monoamine oxidase gene of the present invention is determined. As a method for determining the base sequence of this DNA fragment, for example, a dideoxynucleotide enzyme method [Dideoxy ch
ain termination method; Sanger F., et al., Proc. Natil.
Acad. Sci. USA, Vol. 74, p. 5463 (1977)]. The monoamine oxidase gene can be identified from the open reading frame present in the nucleotide sequence of this DNA fragment.

The monoamine oxidase protein encoded by the monoamine oxidase gene of the present invention comprises, for example, the amino acid sequence represented by SEQ ID NO: 2, and the DNA encoding the same comprises, for example, the amino acid sequence shown in SEQ ID NO: 1 It has a nucleotide sequence up to 1329th. The amino acid sequence of the monoamine oxidase protein is expected to include differences between strains of Micrococcus luteus and differences that can occur between individuals due to spontaneous mutations, etc., and thus encode such homologous mutant proteins. DNA is also included in the present invention.

In addition, the natural monoamine oxidase protein encoded by the monoamine oxidase gene obtained as described above may have one or several deletions, substitutions, or deletions as long as its function is not substantially impaired. A mutation that introduces an addition can be introduced into the monoamine oxidase gene by a method such as a mutagenesis technique using PCR or a mutagen such as NTG or hydroxylamine. The activity of the resulting modified monoamine oxidase protein was confirmed by transforming an Escherichia coli monoamine oxidase mutant (ST222) using the mutant monoamine oxidase gene, and allowing this transformant to grow under appropriate antibiotic selection. You can do it by seeing if. DNAs encoding proteins obtained by these techniques are also included in the monoamine oxidase gene of the present invention.

In the present invention, a gene that hybridizes under stringent conditions generally refers to a nucleotide sequence having 70%, preferably 80% homology with the nucleotide sequence of SEQ ID NO: 1. With respect to these nucleotide sequences, those whose monoamine oxidase activity was confirmed in the protein encoded by the nucleotide sequence by the above-mentioned method are included in the monoamine oxidase gene of the present invention.

The monoamine oxidase gene of the present invention or the monoamine oxidase fragment containing the same is ligated to an appropriate vector to prepare a recombinant vector.
Expression can be achieved by transforming an appropriate host microorganism with this recombinant vector. Accordingly, the present invention also provides a recombinant vector obtained by ligating a monoamine oxidase gene to a vector, and a transformant transformed by introducing the gene.

In preparing a recombinant vector, usually,
A monoamine oxidase gene, together with a promoter suitable for the host microorganism, and the 5'-end of the coding region of the gene is linked downstream of the promoter,
Insert into vector. Alternatively, a monoamine oxidase gene may be inserted into an expression vector containing a promoter.

In addition to the expression method using such an expression vector, a homologous recombination technique for directly introducing a DNA fragment containing a monoamine oxidase gene linked to a promoter into the chromosome of a host microorganism [AAVertes, et al., Bi
osci.Biotechnol.Biochem., Vol. 57, p. 2036 (1993),
Etc.), or a technique of introducing using a transposon or insertion sequence [AAVertes et al., Molecular Microbio
l, Vol. 11, p. 739 (1994), etc.].

The expression vector is not particularly limited as long as it can be replicated and propagated in a host microorganism, and any of a plasmid vector and a phage vector can be used. Specific plasmid vectors include pBR322, pUC18, pHSG298, pU
C118, pSTV28, pTWV228, pHY30
0PLK (the above plasmid vectors can be purchased from Takara Shuzo Co., Ltd.), pKK223-3, pTrc
99A (these can be purchased from, for example, Pharmacia), pCRY31, pCRY3KE, pCRY3KX
(U.S. Pat. No. 5,185,262) or derivatives thereof.

As the phage vector, (λF
ixII vector (available from Stratagene) and the like. As the promoter for expressing the monoamine oxidase gene of the present invention, a promoter possessed by a host microorganism can be generally used, but it is not limited thereto, and a prokaryotic base for initiating transcription of the monoamine oxidase gene is used. Any promoter may be used as long as it is a sequence.
When a microorganism capable of functioning as a host, the promoter specific to the obtained monoamine oxidase gene is used, the promoter may be used as it is.

The host into which the monoamine oxidase gene is introduced is not particularly limited, but bacteria of the genus Escherichia and Bacillus are used.
Bacteria, Serratia bacteria, Pseudomonas bacteria, Corynebacterium (Coryneb)
acterium bacterium, Brevibacacteri
um) bacteria, Rhodococcus bacteria, Lactobacillus bacteria, Streptomyces bacteria, Thermus
A genus bacterium, a Streptococcus genus bacterium and the like can be suitably used. Known methods can be used for introducing the gene into the host microorganism.

The method for producing a recombinant monoamine oxidase of the present invention is characterized in that the transformant obtained as described above is cultured, and the monoamine oxidase is collected from the culture. Here, the culture refers to the cells of the transformant or / and the medium after culturing (the culture supernatant when a liquid medium is used for culturing).

The above transformant can be cultured in a nutrient medium containing a carbon source, a nitrogen source, an inorganic salt, various vitamins and the like, which is usually used for culturing Micrococcus luteus. Other culture conditions may be those commonly used for culturing Micrococcus luteus.

The monoamine oxidase can be collected from the culture according to the method described in Meth. Enzymol., 17B, p722-726, (1971).

[0033]

The present invention will be described more specifically with reference to the following Examples, which do not limit the scope of the present invention.

(A) Purification of monoamine oxidase protein from Micrococcus luteus and determination of N-terminal sequence Micrococcus luteus IAM1099 was cultured according to the method described in Meth. Enzymol., 17B, p722-726, (1971). After the cells were collected and crushed, the MAO purified product was obtained from the cell extract by ammonium sulfate fractionation, DEAE-cellulose column chromatography, hydroxyapatite column chromatography, and gel filtration. .

The amino acid sequence at the N-terminus was determined using 1000 pmol of the purified product. Based on the determined N-terminal sequence, the corresponding oligonucleotide was
It was synthesized using a 394 DNA / RNA synthesizer manufactured by Applied Biosystems. The determined amino acid sequence (SEQ ID NO: 3) and the synthesized sequence (SEQ ID NO: 4) are as follows. Asn Pro His Val Val Ile Val Gly Ala Gly Phe Ala Gly AA (T / C) CCI CA (T / C) GTI GTI ATI GTI GGI GCI GGI TT (T / C) GC

(B) Micrococcus luteus (Micr
Extraction of total DNA of Lactococcus luteus) Micrococcus luteus IAM1099 was added to LB medium [composition: 10 g of tryptone, 5 g of yeast extract, and 5 g of sodium chloride in distilled water to make 1 liter]. After culturing, the cells were collected.

The obtained cells were lysozyme at 10 mg / ml.
[Composition: 10 mM NaCl, 2
0 mM Tris buffer (pH 8.0), 1 mM EDT
A.2Na] in 15 ml. Proteinase K was added to the suspension at a final concentration of 100 μg / ml and
Incubated for 1 hour at ° C. Next, sodium dodecyl sulfate was added to a final concentration of 0.5%,
The cells were lysed by incubating at 0 ° C. for 6 hours. An equal amount of a phenol / chloroform solution was added to the obtained lysate, and the mixture was gently shaken at room temperature for 10 minutes.
The mixture was centrifuged at 5,000 xg for 20 minutes at -12 ° C, and the supernatant fraction was collected. Sodium acetate was added to the supernatant fraction to a concentration of 0.3 M, and then twice the amount of ethanol was gently added. Capture the DNA between the aqueous and ethanol layers with a glass rod,
This was washed with 70% ethanol and air-dried. The obtained DNA was added to a solution [composition: 10 mM Tris buffer (p
H7.5) [1 mM EDTA · 2Na] (5 ml) was added, and the mixture was allowed to stand at 4 ° C. overnight, and then subjected to an experiment.

(C) Obtaining Sau3AI Partially Degraded Fragment of Chromosomal DNA Containing Monoamine Oxidase Gene 1 μg of the chromosome obtained in the above (B) was subjected to restriction enzyme Sau3.
Partial decomposition was performed using AI.

Each final concentration was 50 mM Tris-
Hydrochloric acid buffer (pH 7.9), 10 mM MgCl ₂ , 2
0 mM dithiothreitol, 1 mM ATP, T4D
NA ligase 1 unit / 50 μl, pUC118 vector BamHI digestion dephosphorylated product (Takara Shuzo)
Each component was added so as to be 50 ng / 50 μl and the chromosome Sau3AI partial degradation product 250 ng / 50 μl,
The reaction was performed at 16 ° C. for 3 hours to bind the chromosomal Sau3AI partial degradation product.

Then, a conventional method [J. Mol. Biol., 53, 159 (1
970)] and using the resulting solution in E. coli JM.
109 strains (Takara Shuzo) were transformed. The resulting transformant was spread on a selection medium [composition: 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, 15 g of agar, and 50 mg of ampicillin in distilled water to make 1 liter] and cultured at 42 ° C. for 16 hours. .

The strain grown on this medium is subjected to a conventional method [Molecular cloning, Cold Spring Ha
rbor Laboratory Press (1989)]
Plasmid DNA was extracted from the culture solution, the plasmid was cut with a restriction enzyme SalI, and electrophoresis was performed using a 0.7% agarose gel. The DNA was transferred from this agarose gel onto a nylon membrane, and an oligonucleotide probe (SEQ ID NO: 4) synthesized based on the N-terminal sequence of MAO determined in (A) above was labeled, and then used as a probe. Southern hybridization was performed.

The terminal label is obtained by using the above-mentioned synthesized oligonucleotide probe with a T4 polynucleotide kinase (manufactured by Takara Shuzo) by adding a 5 ′ terminal phosphate group to [γ-
^[ P] was performed by radioisotope labeling with [ ³² P] ATP.
l. Biochem., 158, 307-315 (1986)]. Southern hybridization can be carried out by a conventional method [Molecular Cloning, Cold Spring Harbor Laborat.
ory Press (1989)].

As a result, it was found that a plasmid containing a DNA fragment having a size of about 2.8 kb of the restriction enzyme Sau3AI partially hybridized with the probe, and that the MAO gene was contained in the DNA fragment. Became clear.

(D) Preparation of Map of Sau3AI 2.8 kb Fragment of Chromosomal DNA Containing Monoamine Oxidase Gene The plasmid DNA obtained in Example (C) was digested with restriction enzymes BamHI, KpnI and SalI, respectively. These were subjected to electrophoresis on a 0.8% agarose gel to determine the size of the inserted DNA fragment containing the monoamine oxidase fragment.

The size of the cleaved DNA fragment was determined by converting the E. coli lambda phage (λphase) DNA into the restriction enzyme Hind.
Calculated based on the standard line drawn by the migration distance of the DNA fragment of known molecular weight obtained by cleavage with III on the same agarose gel.

2.6 kb and 0.2 kb by the restriction enzyme BamHI, 1.8 kb and 1.0 kb by the KpnI,
SalI generated fragments of 1.7 kb and 1.1 kb in size. Based on this, a map was prepared as shown in FIG.

(E) Determination of the entire nucleotide sequence of the monoamine oxidase gene The partially degraded Sau3AI fragment having a size of about 2.8 kb was treated with the restriction enzyme Sau3AI at 37 ° C. and partially degraded again. The pUC118 vector (Takara Shuzo) was completely digested with the restriction enzyme BamHI. The obtained vector DNA fragment and the partially degraded DNA fragment were mixed, and the final concentrations of the mixed solutions were 50 mM Tris-HCl buffer (pH 7.9), 10 mM MgCl ₂ , and 20 mM.
Dithiothreitol, 1 mM ATP, T4 DNA ligase Each component was added so as to be 1 unit / 50 μl, and the vector DNA fragment and the partially degraded DNA fragment were combined.

Similarly, Sau3A having a size of about 2.8 kb
The partially degraded fragment of I was treated with a restriction enzyme TaqI at 37 ° C. to partially degrade. The pUC118 vector (Takara Shuzo) was completely digested with the restriction enzyme AccI. The obtained vector DNA fragment and the partially degraded DNA fragment were mixed, and the final concentration of each mixture was 50 mM Tris-HCl buffer (pH 7.9), 10 mM MgCl 2.
₂ , 20 mM dithiothreitol, 1 mM ATP,
T4 DNA ligase 1 unit / 50 μl of each component was added, and the vector DNA fragment and partially digested DN were added.
A fragment was ligated.

Then, a conventional method [J. Mol. Biol., 53, 159 (1
970)], Escherichia coli JM109 (Takara Shuzo) was transformed using each of the obtained solutions. The resulting transformant was spread on a selection medium [composition: 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, 15 g of agar, and 50 mg of ampicillin in distilled water to make 1 liter] and cultured at 42 ° C. for 16 hours. .

The strain grown on the selective medium was inoculated into an LB culture solution containing 10 μg / ml of ampicillin (final concentration: 10 g of tryptone, 5 g of yeast extract, and 5 g of NaCl in distilled water to make 1 liter). And this is 3
The cells were cultured at 7 ° C for 7 hours. The culture was incubated at 4 ° C for 10 minutes
The cells were collected by centrifugation at 000 × g. From the collected cells, an alkali-SDS method [T. Maniatis, EF F
ritsch, J. Sambrook, Molecular Cloning, p. 90-91 (198
2)] to extract the plasmid.

Using the extracted plasmid DNA, the nucleotide sequence of the DNA fragment inserted into the vector pUC118 was determined. The base sequence of the DNA fragment inserted into pUC118 was determined using a die primer cycle sequence kit manufactured by Perkin-Elmer. Then, the ligation of these individual sequences is performed by using the sequence analysis software Auto Assembler (Autoas
sembler). As a result, the above 2.8 kb
DNA fragment partially digested with Sau3AI of Aspergillus niger was 27.3% homologous to a monoamine oxidase protein of a known mold (Aspergillus niger), and partially homologous to a monoamine oxidase protein of Escherichia coli.
It was found to contain a gene encoding a protein having 7% homology (open reading frame described in SEQ ID NO: 1) (shown as MAO in FIG. 1). In addition, the open reading
The N-terminus of the frame completely matched the amino acid sequence determined in (A) above, and was confirmed to contain a monoamine oxidase gene derived from Micrococcus luteus.

[0052]

EFFECT OF THE INVENTION Using the monoamine oxidase gene provided by the present invention, a DNA fragment containing the gene, and the recombinant monoamine oxidase protein, the various properties of the enzyme are analyzed, etc. Application, genetic diagnosis by elucidating the etiology,
It is thought that treatment and the like will be possible.

[0053]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1329 Number of chains: double-stranded Sequence type: nucleic acid Topology: linear Sequence type: Genomic DNA Origin: Organism name: Micrococcus lute
teus) Strain name: IAM1099 Sequence characteristics: Symbol indicating characteristics: CDS Location: 1..329 Method for determining characteristics: E sequence GTG AGC AAC CCG CAT GTC GTG ATC GTC GGA GCC GGC TTC GCC GGC CTG 48 Val Ser Asn Pro His Val Val Ile Val Gly Ala Gly Phe Ala Gly Leu 1 5 10 15 GTG GCC GCC CGT GAA CTG CAG ATG GCA GGC GTG GAC GTG GAG ATC GTG 96 Val Ala Ala Arg Glu Leu Gln MET Ala Gly Val Asp Val Glu Ile Val 20 25 30 GAG GCC CGC GAC CGC GTG GGC GGC CGC GCC TGG ACC GAG GAG CGC ATG 144 Glu Ala Arg Asp Arg Val Gly Gly Arg Ala Trp Thr Glu Glu Arg Met 35 40 45 GGC CGT CCC CTG GAA CTG GGC GCC ACG TGG GTG CAC TGG ATG CAG CCG 192 Gly Arg Pro Leu Glu Leu Gly Ala Thr Trp Val His Trp Met Gln Pro 50 55 60 CAC GTG TGG AGC GAG ATC ACC CGC TAC GAC CAG AGC ATC TAC CCC AGC 240 His Val Trp Ser Glu Ile Thr Arg Tyr Asp Gln Ser Ile Tyr Pro Ser 65 70 75 80 CCG TTC TGC GAC GAC GCC TAC TGG ATC ACC GGG GGC CGG GTG GAG CAC 288 Pro Phe Cys Asp Asp Ala Tyr Trp Ile Thr Gly Gly Arg Val Glu His 85 90 95 GGC ACC GAG GCG GAC CTG GAT GCC GCT CTG GCC CGC CCC ATG GCC AAG 336 Gly Thr Glu Ala Asp Leu Asp Ala Ala Leu Ala Arg Pro Met Ala Lys 100 105 110 ATC TTC GAG GAC TCG CGG GAG TTC TTC CCG TAC CCG TAC GAG CCC CTG 384 Ile Phe Glu Asp Ser Arg Glu Phe Phe Pro Tyr Pro Tyr Glu Pro Leu 115 120 125 CAC GTG CTG GAC GAG AGC AGC GGC AGC ACC CCC GAG CTG CGG GAG CGC 432 His Val Leu Asp Glu Ser Ser Gly Ser Thr Pro Glu Leu Arg Glu Arg 130 135 140 TTC CGC GCG GCG GAC CAG GGC AGT GTC CTG GAC TGC CTC AAG GGC GGC 480 Phe Arg Ala Ala Asp Gln Gly Ser Val Leu Asp Cys Leu Lys Gly Gly 145 150 155 160 160 GAC TTC ACC CAG GAG GAG CGG GAC CTG TGC GAC GCG TAC TGG TCC GCC 528 Asp Phe Thr Gln Glu Glu Arg Asp Leu Cys Asp Ala Tyr Trp Ser Ala 165 170 175 GCG TAC ATC GGG GAC CCG CAC CAG GGG TCA CCG CTC ATG GCC AAG CAG 576 Ala Tyr Ile Gly Asp Pro His Gln Gly Ser Pro Leu Met Ala Lys Gln 180 185 190 TGG GCG GCG CTG TCC GAC CAC CGG TTG AGC CTG GTG GAC GAG CAG ACC 624 Trp Ala Ala Lela Ser Asp His Arg Leu Ser Leu Val Asp Glu Gln Thr 195 200 205 CTG CGC TTC AAG CTC ACC CAC GGC ATG CGG GGA CTG TAC GAG AAC ATC 672 Leu Arg Phe Lys Leu Thr His Gly Met Arg Gly Leu Tyr Glu Asn Ile 210 215 220 GCC GCG GAC CTG CGC TGC CCC ATC CGC CTG AAC ACC CCG GTC ACG GCG 720 Ala Ala Asp Leu Arg Cys Pro Ile Arg Leu Asn Thr Pro Val Thr Ala 225 230 235 240 GTC GAC CAC CGC TCC GAC GGC GCC ACG GTC ACC CTG GGC ACC GGC GAG 768 Val Asp His Arg Ser Asp Gly Ala Thr Val Thr Leu Gly Thr Gly Glu 245 250 255 AAG ATC TCG TGC GAC AGC GTG ATC GTC ACG GTG CCG GTG GGG GCG CTG 816 Lys Ile Ser Cys Asp Ser Val Ile Val Thr Val Pro Val Gly Ala Leu 260 265 270 270 CCA ACC ATC GAG TTC ACC CCG GGC CTG CCC TCG GGG ATG CGC ACC GTG 864 Pro Thr Ile Glu Phe Thr Pro Gly Leu Pro Ser Gly Met Arg Thr Val 275 280 285 ATC GAC CAG CGC TGG AAC TCC ACG GGC TGC AAG ATC TGG GTC AAG GTC 912 Ile Asp Gln Arg Trp Asn Ser Thr Gly Cys Lys Ile Trp Val Lys Val 290 295 300 AAG GGC CAC CAC AGC ATC CTG GGC TAC GCC CCC ACC CCT CAC AAG GCC 960 Lys Gly His His Ser Ile Leu Gly Tyr Ala Pro Thr Pro His Lys Ala 305 310 315 320 GCC GTG TTC CGC AGC GAG TTC TTC ATG GAC GAC GAC ACC ACC ATC TGC 1008 Ala Val Phe Arg Ser Glu Phe Phe Met Asp Asp Asp Thr Thr Ile Cys 325 330 335 GTG GGC TTC GGC TCC CAC CAC GAC GCC GTG GAC CTC ACC GAC CCG CGG 1056 Val Gly Phe Gly Ser His His Asp Ala Val Asp Leu Thr Asp Pro Arg 340 345 350 GAC GCC CAG GCA ATC GTG GAC CAG TGG CGC CCC GAC CTT GAG GTC GTG 1104 Asp Ala Gln Ala Ile Val Asp Gln Trp Arg Pro Asp Leu Glu Val Val 355 360 365 GAC TGC ACG GGC CAC GAC TGG GTG GCG GAC AGG TGG AGC GGT CAG GCG 1152 Asp Cys Thr Gly His Asp Trp Val Ala Asp Arg Trp Ser Gly Gln Ala 370 375 380 TGG GCC ACG CTG CGC TCA GGG CAG TTC ACC AAC GGC TGG CAC CAC TTC 1200 Trp Ala Thr Leu Arg Ser Gly Gln Phe Thr Asn Gly Trp His His Phe 385 390 395 400 400 CGC TCC ACC GAC TCG CGG CTG CGC TTC GCC GGG GCG GAC TGG GCG CGC 1248 Arg Ser Thr Asp Ser Arg Leu Arg Phe Ala Gly Ala Asp Trp Ala Arg 405 410 415 GGC TGG CGC GGC GTG GTG GTG GAC GGC GCC ATC GAG ACG GGC CTG TCC 1296 Gly Trp Arg Gly Val Val V al Asp Gly Ala Ile Glu Thr Gly Leu Ser 420 425 430 ACC GCC CGG GAC GTC CTC CGG GAC ATC CGC GCC 1329 Thr Ala Arg Asp Val Leu Arg Asp Ile Arg Ala 435 440

SEQ ID NO: 2 Sequence length: 443 Sequence type: amino acid Topology: linear Sequence type: protein Sequence Val Ser Asn Pro His Val Val Ile Val Gly Ala Gly Phe Ala Gly Leu 1 5 10 15 Val Ala Ala Arg Glu Leu Gln MET Ala Gly Val Asp Val Glu Ile Val 20 25 30 Glu Ala Arg Asp Arg Val Gly Gly Arg Ala Trp Thr Glu Glu Arg Met 35 40 45 Gly Arg Pro Leu Glu Leu Gly Ala Thr Trp Val His Trp Met Gln Pro 50 55 60 His Val Trp Ser Glu Ile Thr Arg Tyr Asp Gln Ser Ile Tyr Pro Ser 65 70 75 80 Pro Phe Cys Asp Asp Ala Tyr Trp Ile Thr Gly Gly Arg Val Glu His 85 90 95 Gly Thr Glu Ala Asp Leu Asp Ala Ala Leu Ala Arg Pro Met Ala Lys 100 105 110 Ile Phe Glu Asp Ser Arg Glu Phe Phe Pro Tyr Pro Tyr Glu Pro Leu 115 120 125 His Val Leu Asp Glu Ser Ser Gly Ser Thr Pro Glu Leu Arg Glu Arg 130 135 140 Phe Arg Ala Ala Asp Gln Gly Ser Val Leu Asp Cys Leu Lys Gly Gly 145 150 155 160 Asp Phe Thr Gln Glu Glu Arg Asp Leu Cys Asp Ala Tyr Trp Ser Ala 165 170 175 Ala Tyr Ile Gly Asp Pro His Gln Gly Ser Pro Leu Met Ala Lys Gln 180 185 190 Trp Ala Ala Leu Ser Asp His Arg Leu Ser Leu Val Asp Glu Gln Thr 195 200 205 Leu Arg Phe Lys Leu Thr His Gly Met Arg Gly Leu Tyr Glu Asn Ile 210 215 220 Ala Ala Asp Leu Arg Cys Pro Ile Arg Leu Asn Thr Pro Val Thr Ala 225 230 235 240 Val Asp His Arg Ser Asp Gly Ala Thr Val Thr Leu Gly Thr Gly Glu 245 250 255 Lys Ile Ser Cys Asp Ser Val Ile Val Thr Val Pro Val Gly Ala Leu 260 265 270 Pro Thr Ile Glu Phe Thr Pro Gly Leu Pro Ser Gly Met Arg Thr Val 275 280 285 Ile Asp Gln Arg Trp Asn Ser Thr Gly Cys Lys Ile Trp Val Lys Val 290 295 300 Lys Gly His His Ser Ile Leu Gly Tyr Ala Pro Thr Pro His Lys Ala 305 310 315 320 Ala Val Phe Arg Ser Glu Phe Phe Met Asp Asp Asp Thr Thr Ile Cys 325 330 335 Val Gly Phe Gly Ser His His Asp Ala Val Asp Leu Thr Asp Pro Arg 340 345 350 Asp Ala Gln Ala Ile Val Asp Gln Trp Arg Pro Asp Leu Glu Val Val 355 360 365 Asp Cys Thr Gly His Asp Trp Val Ala Asp Arg Trp Ser Gly Gln Ala 370 375 380 380 Trp Ala Thr Leu ArgSer Gly Gln Phe Thr Asn Gly Trp His His Phe 385 390 395 400 Arg Ser Thr Asp Ser Arg Leu Arg Phe Ala Gly Ala Asp Trp Ala Arg 405 410 415 Gly Trp Arg Gly Val Val Val Asp Gly Ala Ile Glu Thr Gly Leu Ser 420 425 430 Thr Ala Arg Asp Val Leu Arg Asp Ile Arg Ala 435 440

SEQ ID NO: 3 Sequence length: 13 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Asn Pro His Val Val Ile Val Gly Ala Gly Phe Ala Gly 1 5 10

SEQ ID NO: 4 Sequence length: 25 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic nucleotide) Sequence characteristics Location: 6, 12 , 15, 18, 21, 24, 27, 30 Other information: N = I array AAYCCNCAYG TNGTNATNGT NGGNGCNGGN TTYGC 25

[Brief description of the drawings]

FIG. 1 DNA containing monoamine oxidase gene
It is a restriction map of a fragment.

──────────────────────────────────────────────────続 き Continued on front page (51) Int.Cl. ⁶ Identification symbol FI (C12N 1/21 C12R 1:19) (C12N 9/06 C12R 1:19)

Claims (8)

[Claims] 1. Chromosome D of Micrococcus luteus
NA obtained by partially digesting NA with a restriction enzyme Sau3AI, having a restriction map shown in FIG.
2.6 kb and 0.1 kb respectively according to the alII and KpnI sites.
2 kb, 1.7 kb and 1.1 kb, 1.8 kb and 1.0
D of about 2.8 kb, divided into kb sizes
A DNA fragment containing at least an NA fragment. 2. Micrococcus luteus is IAM1
The DNA fragment according to claim 1, which is strain 099. 3. A DNA encoding monoamine oxidase contained in the DNA fragment according to claim 1 or 2. 4. A DNA encoding a monoamine oxidase comprising the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2. 5. A DNA comprising the nucleotide sequence of SEQ ID NO: 1 or a DNA which hybridizes with a DNA comprising the nucleotide sequence of SEQ ID NO: 1 under stringent conditions and encodes a protein having a monoamine oxidase activity.
NA. 6. The DNA according to any one of claims 3 to 5,
And a recombinant vector obtained by ligating the vector to a vector. 7. The DNA according to any one of claims 3 to 5,
A transformant transformed by the introduction of A. 8. culturing the transformant according to claim 7,
A method for producing a recombinant monoamine oxidase, comprising collecting monoamine oxidase from the obtained culture.