JPH0698778A - Recombinant dna containing putrescine oxidase gene, transformant containing the dna and production of putrescine oxidase using the dna - Google Patents

Abstract

PURPOSE:To provide a recombinant DNA containing a putrescine oxidase(PUO) gene, a transformant containing the DNA and a process for producing PUO by using the transformant. CONSTITUTION:A DNA coding PUO is separated and purified and its base sequence is determined. A transformant containing integrated gene is prepared and cultured to produce PUO.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a recombinant DN containing a putrescine oxidase (hereinafter referred to as PUO) gene.
A, a transformant containing the same, and a method for producing PUO using the same. The PUO obtained in the present invention is an enzyme that catalyzes the oxidation reaction of putrescine, cadaverine, which is one of the diamines in the body, or spermidine, which is the polyamine in the body, and is used for the determination of diamines and polyamines in the clinical test field.

[0002]

2. Description of the Related Art PUO has been found in Micrococcus rubens [Agricultural Biological Chemistry, Vol. 30, p. 1202 (1966)] by Adachi etc. No. 18984), genus Nocardia (JP-A-57-164786), Arthrobacter
Production from the genus (Japanese Patent Laid-Open No. 60-41481) and the like has been reported, and further, genus Aspergillus, Rhizopu
s), Mucor, Penicillium
genus, Diaporthe, Aureobasidium, Gibberell
a) Genus, Fusarium genus, Cylindrocarpon genus, Cladospo
rium, Byssochlamys, Paecilomyces, Scopulariopsis
ulariopsis), Gliocladium
Genus, genus Monascus, Arthroderma
genus roderma, genus Phytophtora, genus Gloeophyllum, Geotricam
chum), Oidiodendron genus,
Verticillium genus, Trichoderma genus, Talaromyces genus,
Absidia genus (above, JP-A-62-275681
No.), Pseudomonas genus (Japanese Patent Laid-Open No. 62-3
The one derived from No. 35878) is known.

However, at present, no analysis has been made on these PUO genes. Therefore, the present situation is that no research has been conducted on the production of recombinant PUO.

[0004]

The conventional PUO production has many points to be improved in terms of the supply amount, etc., and the inventors of the present invention code PUO as a result of earnest research to solve the above problems. We have succeeded in purifying and isolating DNA and determining its nucleotide sequence.

Furthermore, a method for producing PUO was completed by obtaining a transformant incorporating the gene and culturing the transformant.

[0006] Based on the above results, the way to efficient mass production of PUO is opened, and further, PU by protein engineering is used.
It also opened the way for modification of the O specificity.

[0007]

In the present invention, the above-mentioned PUO-producing bacterium can be used as a source of DNA containing the PUO gene. For example, Micrococcus roseus (Micr
ococcus roseus) IFO3768.

Cloning with Escherichia coli is carried out for the purpose of isolating the PUO gene from the chromosomal DNA of Micrococcus roseus IFO 3768.

As a probe for selecting the PUO gene, a synthetic DNA having a nucleotide sequence deduced from a partial amino acid sequence of the PUO protein is used as a label, and a fragment population of chromosomal DNA containing the PUO gene is analyzed by Southern hybridization. After limiting, cloning is performed using Escherichia coli as a host, and a DNA fragment containing the PUO gene is obtained by the colony hybridization method.

For determining the partial amino acid sequence of the enzyme, for example, known literature [Eur. J. Biochem., Vol. 1, pp. 80-91 (196
An automated amino acid sequencer to which the method described in [7)] is applied can be used.

In order to deduce the DNA sequence of a gene, when there are a plurality of genetic codes corresponding to amino acids, the most likely one should be selected in consideration of the frequency of use of the genus Micrococcus, or the known literature [Nucleic Acids Res ., 13: p. 8927 (1985)] by using inosine for the third character.

Synthetic DNA can be produced, for example, by using an automatic DNA synthesizer. Labeling of synthetic DNA is carried out, for example, by publicly known literature [Proc. Natl. Acad. Sci. USA, 74.
Vol., 560-564], and phosphorylating the 5 ′ end with γ- ³² P-ATP using T4 polynucleotide kinase.

Southern hybridization is carried out according to the method of Southern et al. Described in a known document [J. Mol. Biol., 98, 503-517 (1975)], and the above-mentioned chromosome D is used.
By cleaving NA with a restriction enzyme and performing separation according to the fragment length by agarose gel electrophoresis, DNA is adsorbed to a nitrocellulose filter and hybridized with a labeled synthetic DNA probe, and an autoradiogram is taken. It can be carried out. The restriction enzymes used are
Examples include BamHI and EcoRI.

Then, an agarose gel described in, for example, a known document [Anal. Biochem., 101, 339-341 (1980)] was used to collect a DNA fragment assembly containing a chromosomal DNA fragment to which the probe hybridizes. It can be recovered according to the DNA extraction method from.

Next, in order to perform colony hybridization, the above-mentioned recovered DNA is inserted and ligated into vector DNA to prepare a recombinant DNA. Incorporation of chromosomal DNA into vector DNA is well known in the art [J. Mol. Biol., 9
6, pp. 171-184 (1975)], the chromosomal DNA and the vector DNA are cleaved with a restriction enzyme and then ligated with a ligase.

Examples of vector DNA include plasmid DNA, and pBR322 and pUC19 are particularly preferable. Examples of the ligase include T4 DNA ligase.

Introduction of recombinant DNA into Escherichia coli can be carried out, for example, by publicly known literature [J. Mol. Biol., 53, 159-162 (197).
0)] and the calcium chloride treatment method. Escherichia coli to be used is Escherichia coli (Escher
ichia coli) JM109 strain is preferred.

The selection of the strain containing the recombinant DNA containing the PUO gene can be carried out by, for example, the known literature [Nucleic Acids Re
S., Vol. 7, pages 2115 to 2136 (1979)] by the colony hybridization method using the synthetic DNA as a probe.

That is, Escherichia coli introduced with recombinant DNA was spread on L-broth agar medium containing ampicillin, and after overnight culture, the resulting ampicillin-resistant transformant was replicated on a nitrocellulose filter and further for 2 to 3 hours. The cells are cultured on L-broth agar medium containing ampicillin, lysed and the DNA is fixed to detect positive colonies to which the synthetic DNA hybridizes.

Next, from the positive strains, for example, known literature [Pr
oc. Natl. Acad. Sci. USA, 57, 1514-1521 (1
967)], the plasmid is extracted and purified and digested with various restriction enzymes to prepare a restriction enzyme map, and again it is confirmed by Southern hybridization that a plurality of synthetic DNA probes hybridize.

First, the base sequence of the fragment to which the synthetic DNA probe hybridizes is determined, and then the base sequences before and after the sequence are also determined to confirm the existence of an open reading frame encoding the known amino acid sequence of PUO. To determine the nucleotide sequence of DNA, DNA fragments cloned in M13 phage M13mp18 and M13mp19 were prepared by publicly known literature [Proc. Natl. Acad. Sci. U.
SA, 74, 5463-5467 (1977)].

The DNA base sequence determined by the above operation and the deduced amino acid sequence are shown in SEQ ID NO: 1.

The upper part of SEQ ID NO: 1 shows the DNA base sequence containing the PUO structural gene and the lower part shows the deduced amino acid sequence.

The nucleotide sequence of the mutant gene which has undergone changes such as nucleotide substitution, deletion, insertion and transfer can also be confirmed by the method described above.

A transformant having high productivity is obtained by connecting an Escherichia coli promoter upstream of the translation initiation site of the obtained PUO gene, ligating it with an E. coli vector such as plasmid vector pUC19, and introducing it into E. coli. Can be obtained.

The transformant can be confirmed by extracting the plasmid and examining the cleavage pattern by various restriction enzymes. The amount of PUO produced by the transformant strain is such that the transformant strain is shake-cultured in an L-broth medium containing ampicillin to induce gene expression with IPTG, and after further culturing, the cells are collected and washed, By carrying out SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on the crude enzyme preparation obtained by disrupting it by a method such as ultrasonication, the excessively produced PUO protein can be confirmed.

The PUO activity can also be confirmed using the PUO activity measuring method described later. However, when cultured at 37 ° C., which is the optimum growth temperature for E. coli, most of the PUO enzyme proteins produced form an inactive inclusion body, and the detected PUO activity value is low.

The amount of active PUO protein can be increased by preventing the formation of inclusion bodies by culturing at a lower temperature, for example 30 ° C. or 26.5 ° C.

Further, as a method for efficient gene expression, for example, known literature [Methods in Enzymology, 100 volumes,
60-96 (1983)], the amount of the gene product can be increased by deleting the DNA between the promoter and the translation initiation point by using exonuclease III and S1 nuclease in combination.

Examples of the above transformants include Escherichia coli JM109 containing a plasmid in which the PUO gene derived from the chromosome of Micrococcus roseus IFO 3768 is introduced into pUC19 by the method described above.

As the medium used for the production of PUO by the microorganism of the present invention, any synthetic medium or natural medium containing a carbon source, a nitrogen source and an inorganic substance, which is generally used in the culture of microorganisms, can be used. You can Substances preferably added in a trace amount include antibiotics for maintaining stable plasmids. For example, in the case of a recombinant DNA derived from the plasmid vector pUC19 having an ampicillin resistance gene, ampicillin is contained in an amount of 30 to 100 μg /
It can also be added at a concentration of ml.

Cultivation of the microorganism of the present invention can be carried out in the same manner as in the culture of general microorganisms. Since PUO is accumulated in the microbial cells, it can be recovered by a normal microbial cell disruption method.

The activity of PUO was measured as follows. 4-aminoantipyrine 10 mg, phenol 0.2 m
l, peroxidase 5mg 0.2M phosphate buffer (pH 7.0)
Prepare the coloring reagent by dissolving in 100 ml. This coloring reagent 1.5
0.5 ml of 10 mM putrescine and 0.5 ml of sample are added to ml and the temperature is 35 ℃.
Then, the change in absorbance at 505 nm per minute is measured. One unit is the amount of enzyme that produces 1 μmol of hydrogen peroxide per minute.

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

【Example】

Example 1 Cloning of PUO gene by Escherichia coli (a) Micrococcus roseus IFO which is a PUO-producing bacterium
Preparation of chromosomal DNA from 3768 strain Micrococcus roseus IFO 3768 strain polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.) 0.5 g, yeast extract (manufactured by Difco) 0.5
pH containing 0.1g of glucose and glucose (manufactured by Kokusan Kagaku) in 1L
From cells obtained by culturing in 7.0 medium at 30 ° C for 30 hours with shaking [Agric. Biol. Chem., 44, 367-381 (1980)]
Chromosomal DNA was prepared according to the method described in 1.
7 ml of a mg / ml DNA solution was obtained.

(B) Preparation of a synthetic DNA probe that is considered to have a partial sequence of PUO gene Purified product of PUO or a product thereof [protein, nucleic acid, enzyme,
31, 629-p. (1986)], and about 0.1 μg of two kinds of peptides obtained by decomposing with lysyl endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.) according to the method described in [Automatic Amino Acid Sequencer (Applied Biosystems). Manufactured) was used to examine the amino acid sequence at the N-terminal and from the middle at three positions.

Based on the obtained amino acid sequence, a synthetic DNA of 26mer was prepared by changing all the third letters of the codon to inosine.
Sequence (SEQ ID NO: 2 and SEQ ID NO: 3) 26me synthesized as a mixture containing two kinds in total and possible major base sequences
One kind of r synthetic DNA (SEQ ID NO: 4) was synthesized using an automatic DNA synthesizer (manufactured by Beckman).

In SEQ ID NO: 2 to SEQ ID NO: 4, the upper part shows the determined partial amino acid sequence of PUO protein, and the lower part shows the base sequence of the synthetic DNA prepared corresponding thereto. In addition,-*-indicates an unknown part.

(C) Southern Hybridization 10 μl of the above chromosomal DNA solution prepared from Micrococcus roseus IFO 3768 strain, 2 μl of 10 times concentration of BamHI buffer, 10 mM Tris-hydrochloride buffer containing 1 mM EDTA (p
H7.6) (hereinafter referred to as TE) 6 μl and BamHI (Takara Shuzo) 2 μl were mixed and reacted at 37 ° C. for 3 hours to completely decompose.

This reaction solution was electrophoresed on a 1.0% agarose gel [J. Mol. Biol., 98, 503-517 (1975)].
Nitrocellulose filter (S &
DNA was adsorbed on S). Next, synthetic DNA 10p
mol, [γ- ³² P] ATP 30 pmol, 10 times concentration of kaination buffer 3 μl, T4 polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.) 3 μl and TE to make a total of 40 μl, and after reacting at 37 ° C. for 1 hour, Sephadex G -50 (Pharmacia) column was used to purify the synthetic DNA probe, and 6 times concentrated pH 7.5 NET buffer (0.15M NaCl 1mM
EDTA 15mM Tris), 0.5% NK40 (manufactured by Sigma),
Hybridization was performed overnight at 65 ° C.

After washing for 1 hour at 65 ° C. using a 4-fold concentrated SSC solution, it was confirmed by autoradiogram that all three kinds of probes hybridized at a position of about 4.5 Kb.

(D) Insertion of BamHI degradation product of chromosomal DNA of approximately 4.5 Kb into a vector A large amount of BamHI degradation product of the chromosomal DNA obtained in (c) above was prepared and subjected to 1.0% agarose gel electrophoresis. Anal. B
iochem., 101, 339-341 (1980)], a fragment of about 4 Kb was recovered. The recovered DNA solution was ethanol-precipitated to a 20 μl solution containing about 0.4 μg of DNA.

On the other hand, the plasmid vector pUC19 (about 400
μg / ml) 10 μl, 10 times concentration of BamHI buffer 5 μl, TE
After mixing 33 μl and 2 μl of BamHI and reacting at 37 ° C. for 2 hours, ethanol precipitation was performed and the precipitate was dissolved in 40 μl of TE and E. coli alkaline phosphatase (Takara Shuzo) 3
μl was added and mixed, and the dephosphorylation reaction was carried out at 65 ° C. for 1 hour.

Further, after phenol treatment, ethanol precipitation was carried out, and the precipitate was dissolved in 20 μl of TE and subjected to a ligase reaction. That is, the ligase reaction is based on the previously prepared chromosome DN.
20 μl of A fragment solution, pU treated with BAP after digestion with BamHI
C19 solution 13μl, 10 times concentration of ligase buffer 5μl, 100m
M dithiothreitol (DTT) solution 5 μl, 10 mM AT
5 μl of P and 2 μl of T4 DNA ligase (Takara Shuzo) were mixed and reacted at 4 ° C. for 24 hours, and the whole amount was used for transformation.

(E) Transformation of Escherichia coli Transformation was carried out by using the Escherichia coli JM109 strain as a host bacterium by the calcium chloride method described above. That is, the host is L-
After growing in 10 ml of broth to the mid-logarithmic growth phase (OD 650 nm≈0.4), the cells were collected by centrifugation, suspended in 5 ml of ice-cold 50 mM calcium chloride solution, stored in ice water for 30 minutes, and then centrifuged again. The cells were collected by separation, suspended in 500 μl of the same calcium chloride solution, and stored in ice water for 2 hours, then (d).
After adding the DNA solution obtained in step 1 and holding it for 1 hour,
Heat treatment was performed at 42 ° C. for 2 minutes.

2 ml of L-broth was added to this treatment solution,
After shaking culture for 1 hour, ampicillin 50 μg / ml, IPT
The cells were seeded on an L-broth agar medium containing G 2 mM and X-gal 2.5 × 10 ⁻³ % and cultured at 37 ° C. overnight.

(F) Colony Hybridization: Among the transformants obtained in (e), only white colonies, that is, those having a recombinant plasmid, were placed on a new L-broth agar medium and another L-broth agar medium. Inoculate the above nitrocellulose filter, [Nucleic Acids Res., Volume 7,
2115 to 2136 (1979)], colony hybridization was carried out using the same probe as in Southern hybridization, and one positive strain was obtained.

(G) Confirmation of Cloning of PUO Gene [Proc. Natl. Acad. Sci. USA, 57]
Vol., Pp. 1514-1521 (1967)], prepare a plasmid, cleave it with various restriction enzymes, analyze by 1% agarose gel electrophoresis, and
Upon hybridization, a 4.4 Kb insert fragment was included, and it was found that the synthetic DNA hybridizes to the AccI-SmaI 400 bp fragment.

M13 phage M13mp18 for each fragment
When the nucleotide sequence was determined by the dideoxy sequencing method using M13mp19 and M13mp19, an open reading frame encoding a known amino acid sequence was found. So PUO
The entire base sequence of the gene was determined in the same manner. This plasmid is designated as pPUO1.

Example 2 PUO by E. coli transformant
The inclusion site of E. coli Ribosome recognition site, so-called SD sequence, is found upstream of the translation initiation site of the PUO gene cloned from Micrococcus roseus IFO 3768. PUO
Was produced.

At this time, in order to eliminate the possibility of intervening a sequence that inhibits transcription between the promoter and the translation initiation site, the translation initiation point was deleted as follows.

30 μl of a solution containing about 0.2 μg of pPUO1, 4 μl of 10 times concentration BamHI buffer, RNase A (Boehringer)
2 mg / ml solution 4 μl, BamHI (Takara Shuzo) 1 μl, StuI
(Takara Shuzo) 1 μl was mixed and reacted at 37 ° C for 2 hours, then 1
% Agarose gel electrophoresis was performed, and the 3.3 Kb fragment containing the gene was extracted from the agarose gel as described above and dissolved in 10 μl of TE.

On the other hand, about 1 μg of the plasmid vector pUC18
Containing 16 μl, 10 × concentration BamHI buffer 2 μl, BamHI
1 μl and HincII (manufactured by Takara Shuzo) were mixed, reacted at 37 ° C. for 2 hours, precipitated with ethanol, and dissolved in 10 μl of TE.

The both were combined and further mixed with 2.5 μl of a 10-fold concentration ligase buffer, 1.5 μl of 10 mM ATP and 1 μl of T4 DNA ligase, reacted at 4 ° C. overnight, and the whole amount was used for transformation. The Escherichia coli JM109 strain was used as a host, and the plasmid was isolated from the transformant by the alkaline method described in [Nucleic Acids Res., 7: 1513-1523 (1979)], and after digestion with various restriction enzymes, 1 % PU gel with 3.3Kb gene fragment inserted into pUC18 by agarose gel electrophoresis
I confirmed O11.

Next, 30 μl of a solution containing about 0.2 μg of pPUO11, 1
0x KpnI buffer 4μl, RNaseA 2mg / ml solution 4μ
l and 1 μl of KpnI (Takara Shuzo) were mixed, reacted at 37 ° C. for 2 hours, precipitated with ethanol and dissolved in 34 μl of TE to give 10
Double-concentration BamHI buffer 4 μl and BamHI 1 μl were added, and 37 ℃
The reaction was carried out for 2 hours and ethanol precipitation was carried out again.

The precipitate was dissolved in 60 μl of TE, 7 μl of a 10-fold concentrated exonuclease III buffer and 3 μl of exonuclease III (Takara Shuzo) were added, and the mixture was reacted at 23 ° C., and 23 μl after 10, 20 and 30 minutes. To another tube, 2.6 μl of 10 times concentrated S1 nuclease buffer and S1
10 U of nuclease (Takara Shuzo) was added, reacted at 23 ° C for 15 minutes, ethanol-precipitated, dissolved in 30 µl of TE, 10 times concentration of Klenow enzyme buffer 4 µl, 1 mM dNTP.
4 μl of a mixed solution (Takara Shuzo) and 1 μl of Klenow enzyme were added, and the mixture was reacted at 23 ° C. for 30 minutes, followed by ethanol precipitation.

After dissolving the precipitate in 20 μl of TE, 2.5 μl of 10 times concentration ligase buffer, 1.5 μl of 10 mM ATP, T4DN
1 μl of A ligase was mixed, reacted overnight at 4 ° C., and the whole amount was used for transformation. E. coli JM109 strain was used as a host.

Plasmids were isolated from 50 kinds of transformants by the above-mentioned method and confirmed by restriction enzymes. As a result, the upstream region of PUO gene was successfully deleted to various extents. A plasmid in which DNA has been deleted from the inside up to 12 bases upstream from the translation initiation site.
pPUO4 was selected. This plasmid retains up to 5 bases out of 6 bases considered to be the SD sequence.

This Escherichia coli JM109 (pAB2-del28) was cultured in 10 ml of L-broth containing 50 μg / ml of ampicillin at 37 ° C. overnight, and 100 μl of this was inoculated into 10 ml of L-broth containing 50 μg / ml of fresh ampicillin. Then, the cells were cultivated at 37 ° C. with shaking until OD 650 ≈0.3, IPTG was added to 2 mM to induce gene transcription, and the cells were further cultured for 6 hours.

After harvesting the cells, 10 mM phosphate buffer (KH ₂ PO ₄ -NaO
H) After washing with pH 7.0, 50 mM NaCl, suspending in 2 ml of the same buffer solution, disrupting the cells by ultrasonic waves, centrifuging at 10,000 rpm for 20 minutes, and measuring the PUO activity of the supernatant. An activity of about 0.1 U was obtained.

SDS-PAGE of the centrifugal residue showed that a large amount of a protein having a molecular weight of 52 kDa was expressed. This indicates that the majority of PUO is produced as inclusion bodies in this system.

On the other hand, when the culturing temperature was lowered to 30 ° C. and 26.5 ° C. and the same culturing was carried out, at 30 ° C., about 1.22 per 1 ml of the culturing was carried out.
U activity was obtained, which was about 0.86 U at 26.5 ° C. This indicates that the formation of inclusion bodies can be prevented by lowering the culture temperature.

[0062]

INDUSTRIAL APPLICABILITY According to the present invention, DN containing a PUO gene
The A fragment is separated, a plasmid having the DNA fragment is obtained, E. coli is transformed with the plasmid, and the transformant is cultured to produce a large amount of PUO, which is 5 to 10 times that of the conventional method. Can be collected.

[0063]

[Sequence list]

 SEQ ID NO: the length of one sequence: 2386 Type of sequence: nucleic acid sequence CTGCAGGGTG TGCGGGGAGT CGGCGACGGC TGGACGGCCA TGATCGGGAT CATGGCCGTG 60 TGCTGGGCGT AGCAGTCCCG GTAGGACCGC GCCCAGCTCT CGAGGGCCCG GTCCCACGGG 120 ACGGTGCCGA AGGCGGAGCA GTCGATGCCC TGGTTGATCG CGTCCTGGAC CCGGATCAGC 180 ACCTCGCGCT TCGACGGCGT GTGGTTGTAC AGGGCCGAGG TGGAGCTCAG CTCCCGGCGC 240 GACGTCGGTC ATCGTCACCG CAACCGAGCC CCGGGTGGCC ACGATCTTGA TCGCGCGCTT 300 CGTGATCCGC TCCTCGGAGA GCACCGGATG GCGCGGCGAC CGCGACGCCG AGGGGCGGGG 360 CCGGCGGGAG CCATGAGCAG ATCCTCCTGC GGTCGGGGCA TGTGGCGCTT CCTCCTGAGT 420 CTAGGTGCAA CGAGTTGGTG ACGGCTCTTG ACGAGCACGG GCCGGAGCCC ATTTAATGAA 480 CCTAATTCAT TTTAGTGTGA AGGAGAGCAC A GTG ACC GAT CAA CGC ACC CTG GGC 535 Met Thr Asp Gln Arg Thr Leu Gly AGC GAG ACC GCG ATC GAG CGC GAC GTC GTC GTC GTG GGG GCC GGC 580 Ser Glu Thr Ala Ile Glu Arg Asp Val Val Val Val Gly Ala Gly CCC GCC GGG CTC ATG GCC GCC CGC ACG CTC GTG GCC GCC GGG CGG 625 Pro Ala Gly Leu Met Ala Ala Arg Thr Leu Val Ala Ala Gly Arg ACC GTG GCG GTC CTC GAG GCC CGC GAC CGC GTG GGC GGG CGC ACC 670 Thr Val Ala Val Leu Glu Ala Arg Asp Arg Val Gly Gly Arg Thr TGG TCG AAG ACC GTG GAC GGC GCG TTC CTG GAG ATC GGC GGG CAG 715 Trp Ser Lys Thr Val Asp Gly Ala Phe Leu Glu Ile Gly Gly Gln TGG ATC TCC CCC GAC CAG ACC GAG CTG CTG GCG TTG GTC GAC GAG 760 Trp Ile Ser Pro Asp Gln Thr Glu Leu Leu Ala Leu Val Asp Glu CTC GGC CTG GAG ACC TAC CAG CGC TAC CGC GAG GGC GAG TCC GTC 805 Leu Gly Leu Glu Thr Tyr Gln Arg Tyr Arg Glu Gly Glu Ser Val TAC CTG GCG CCG GAC GGG ACC CGC CAC ACC TAC ACC GGC TCG ATG 850 Tyr Leu Ala Pro Asp Gly Thr Arg His Thr Tyr Thr Gly Ser Met TTC CCG GCG GGG GAG TCC ACG ATC GTC GAG ATG GAG AAG CTC GTC 895 Phe Pro Ala Gly Glu Ser Thr Ile Val Glu Met Glu Lys Leu Val GCG CTC CTG GAC GGG CTC GTG GCC GAG ATC GGC GCC ACC GAG CCG 940 Ala Leu Leu Asp Gly Leu Val Ala Glu Ile Gly Ala Thr Glu Pro TGG GCG CAC CCG GCG GCC CGG GAG CTG GAC ACC ATC TCC TTC CAC 985 Trp Ala His Pro Ala Ala Arg Glu Leu Asp Thr Ile Ser Phe His CAC TGG CTG CGG CAG CAC TCC GAC GAC GAG GCC GCC TGC AGC AAC 1030 His Trp Leu Arg Gln His Ser Asp Asp Glu Ala Ala Cys Ser Asn ATC GGG ATC TTC GTG GCC GGC GGG ATG CTG ACC AAG CCC GCG CAC 1075 Ile Gly Ile Phe Val Ala Gly Gly Met Leu Thr Lys Pro Ala His GCG TTC TCC GTG CTG CAG GCC GTG CTC ATG GCC GCC TCC GCC GGG 1120 Ala Phe Ser Val Leu Gln Ala Val Leu Met Ala Ala Ser Ala Gly TCC TTC TCG AAC CTC GTG GAC GAG GAC TTC ATC CTC GAC CGG CGC 1165 Ser Phe Ser Asn Leu Val Asp Glu Asp Phe Ile Leu Asp Arg Arg GTG GTC GGC GGC ATG CAG TCC GTC TCG GAG ACC ATG GCG GCG GAG 1210 Val Val Gly Gly Met Gln Ser Val Ser Glu Thr Met Ala Ala Glu CTG GGC GAG GAC GTC GTC TTC CTG GAC ACC CCG GTG CGC ACG ATC 1255 Leu Gly Glu Asp Val Val Phe Leu Asp Thr Pro Val Arg Thr Ile CGC TGG GCC GGT GAC GGC GGC ACC TAC GCC GAG CAC GTC CCG GGC 1300 Arg Trp Ala Gly Asp Gly Gly Thr Tyr Ala Glu His Val Pro Gly ACC CCG GTG ACC GTG TGG TCC GAC CGG CTC ACG GTG CGG GCG AAG 1345 Thr Pro Val Thr Val Trp Ser Asp Arg Leu Thr Val Arg Ala Lys GAC GTG GTC GTG GC C GTC CCG CCC AAC CTC TAC TCC CGG ATC TCC 1390 Asp Val Val Val Ala Val Pro Pro Asn Leu Tyr Ser Arg Ile Ser TTC GAG CCG CCC CTG CCG CGG CTG CAG CAC CAG ATG CAC CAG CAC 1435 Phe Glu Pro Pro Leu Pro Arg Leu Gln His Gln Met His Gln His CAG TCG CTG GGC CTC GTG ATC AAG GTG CAC GCC GTC TAC GAG ACG 1480 Gln Ser Leu Gly Leu Val Ile Lys Val His Ala Val Tyr Glu Thr CCC TTC TGG CGG GAC AAG GGC CTC TCC GGC ACC GGC TTC GGC GCC 1525 Pro Phe Trp Arg Asp Lys Gly Leu Ser Gly Thr Gly Phe Gly Ala CAC GAG CTC TCC CAG GAG GTC TAC GAC AAC ACC AAC CAC GGG GAC 1570 His Glu Leu Ser Gln Glu Val Tyr Asp Asn Thr Asn His Gly Asp CCG CGC GGC ACC CTG GTC GGC TTC GTC TCG GAC GAG CGG GCC GAC 1615 Pro Arg Gly Thr Leu Val Gly Phe Val Ser Asp Glu Arg Ala Asp GAG CTC TTC GGC CTG CCC GCC GAG GAG CGC CGC CGG CTG ATC CTC 1660 Glu Leu Phe Gly Leu Pro Ala Glu Glu Arg Arg Arg Leu Ile Leu GAG TCC CTC TCC CAC TAC CTG GGG GAG GAG GCC CTG CAC CCG GTG 1705 Glu Ser Leu Ser His Tyr Leu Gly Glu Glu Ala Leu His Pro Val GTC TAC TAC GAG TC C GAC TTC GGC TCC GAG GAG TGG ACC CGC GGC 1750 Val Tyr Tyr Glu Ser Asp Phe Gly Ser Glu Glu Trp Thr Arg Gly GCC TAC GCC GCC AGC TAC GAC CTC GGC GGC CTG CAC CGC TAC GGC 1795 Ala Tyr Ala Ala Ser Tyr Asp Leu Gly Gly Leu His Arg Tyr Gly GCC CAC CAG CGC ACC CCG GTG GGC CCG ATC CGC TGG GCC TGC TCC 1840 Ala His Gln Arg Thr Pro Val Gly Pro Ile Arg Trp Ala Cys Ser GAC CTC GCG GCC GAG GGC TAC CAG CAC GTC GAC GGC GCC CTG CGG 1885 Asp Leu Ala Ala Glu Gly Tyr Gln His Val Asp Gly Ala Leu Arg CAG GGC CGG CTC GCC GCG GCC GAG GTC CTC GGC GCC GGC TCG CTG 1930 Gln Gly Arg Leu Ala Ala Ala Glu Val Leu Gly Ala Gly Ser Leu ACG GGC GCG GAG CGA TGA CC ACCGCCGGGG TGCCGCGCTA CGTGGTCGGC 1980 Thr Gly Ala Glu Arg *** TACAGCGGCG ACCGGCGCAG CCGGGACGCC GTGCGGCTGG GCAGTCGCGC TCGCGCGCGA 2040 CGTGGGCGCC GAGCTCGAGA TCGTCCTCGT GCTGCGCGCG GACGACCCGT ACCAGCAGGT 2100 CTACCCGCCG GTGGGGGACA TCTCGCCCGT GCTCCGCCGG CAGGCCCACC AGTGGCTGCG 2160 CGAGGGCCCG CCGCCCTGGT GCCCGAGGGG ATCACGGCCC GCACCCACGT GCGGGAGGCG 2220 CGGTCCGTGC CCACCGGCC T GGTCGAGGCC GCCACCGAGC TGGGGCCGCG ATGATCGTGG 2280 TCGGCGCCGG GACGGGCGGC GGCCGCTTCA CCCTGGGTTC CGTGGTGAAC GCCTGCTGCA 2340 CAGCTCGCCG GTCCCCGTGG CGCTCGCCCC GCGCCGGTAT CCCGGG 2386

SEQ ID NO: 2 Sequence length: 20 Sequence type: Amino acid sequence Thr Asp Gln Arg Thr Leu Gly Ser Glu Thr Ala Ile Glu Arg Asp 15 5'-ACI CTI GGI TCI GAI ACI GCI ATI GA Val Val Val Val * Gly 20

SEQ ID NO: 3 Sequence length: 20 Sequence type: Amino acid sequence Asp Val Val Val Ala Val Pro Pro Asn Leu Tyr Ser Arg Ile Ser 15 CTI CAI CAI CAI CGI CAI GGI GGI TT-5 'Phe Glu Pro Pro Leu 20

SEQ ID NO: 4 Sequence length: 13 Sequence type: Amino acid sequence Val His Ala Val Tyr Glu Thr Pro Phe Trp Pro Asp Lys 13 ATG CTC TGN GGN AAG ACC GGN CTG TT-5 '

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication C12R 1: 265) (C12N 1/21 C12R 1:19)

Claims (3)

[Claims] 1. A recombinant DNA in which a DNA containing a gene encoding putrescine oxidase is incorporated into a plasmid vector for E. coli. 2. Escherichia coli into which the recombinant DNA according to claim 1 has been introduced. 3. The Escherichia coli according to claim 2 is cultivated in a nutrient medium, and after the culture is made to produce putrescine oxidase,
A method for producing putrescine oxidase, which comprises collecting putrescine oxidase from the culture.