JPH1175842A - Gene encoding nitrous oxide reductase and the nitrous oxide reductase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new gene capable of producing a microorganism and a plant having enhanced absorption and decomposition of nitrogen oxides, by transferring the cell of the microorganism and the plant to the gene, comprising the gene for encoding a nitrous oxide reductase having a specific amino acid sequence. SOLUTION: This new gene encodes a nitrous oxide reductase comprising an amino acid sequence of the formula and is useful for improving the function of an enzyme for decomposing nitrous oxide into nitrogen or for designing the molecule of an artificial reducing catalyst and for creating a microorganism and a plant having enhanced absorption and decomposition of nitrogen oxides by transferring the gene into the cell of the microorganism and the plant. The gene is obtained by culturing Achromobacter cycloclastes, destroying the microbial cell with a surfactant, extracting a gene DNA, preparing a gene library by a conventional method, carrying out gene amplification (RCR) by using the gene of the partial sequence of the enzyme and performing a screening by the use of the prepared gene as a probe.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a gene encoding a nitrous oxide reductase capable of reducing and decomposing nitrous oxide (N ₂ O), which is one of the global warming gases and also a nitrogen oxide, to nitrogen. And its nitrous oxide reductase.
More specifically, it relates to the gene for nitrous oxide reductase of denitrifying bacteria and its nitrous oxide reductase, to improve the function of the enzyme for decomposing nitrous oxide to nitrogen, and to molecularly design an artificial reduction catalyst. It is useful as a molecular design guideline, and is also useful for creating microorganisms and plants with enhanced absorption and decomposition of nitrogen oxides.

[0002]

2. Description of the Related Art Nitrous oxide reductase is an enzyme that utilizes cytochrome or the like as an electron donor and catalyzes a reaction for reducing nitrous oxide to nitrogen. This enzyme has been found, for example, in denitrifying bacteria and catalyzes the last step of the denitrification reaction that reduces nitric acid to nitrogen (reducing nitrous oxide to nitrogen). The structure is a monomer or dimer, and it is known that copper ions exist in the active center (energy metabolism of microorganisms, written by Takeo Yamanaka).

[0003]

SUMMARY OF THE INVENTION Nitrous oxide reductase is widely distributed in denitrifying bacteria, for example, Pseudomonas.
(Eur. J. Biochem. 192, 591, 19)
90), Paracoccus (Eur. J. Bioc)
hem. , 218, 49, 1993), Alcalig.
ens (Eur. J. Biochem., 208, 3).
1, 1992), rhizobia (J. Bacteriol, 1).
78, 1505, 1996). However, these enzymes are metal enzymes having copper as an active center, and their activities are easily deactivated by oxygen. Furthermore, the decomposition mechanism of nitrous oxide by these enzymes and the three-dimensional structure at the molecular level are being studied.

At present, Achromo, one of the denitrifying bacteria,
Few studies at the genetic level have been reported on nitrous oxide reductase originating from Bacter cycles. The present invention relates to Achromobacter cycloclass, one of the denitrifying bacteria.
a gene encoding a nitrous oxide reductase having a high ability to decompose nitrous oxide to nitrogen, and a gene having a high ability to decompose nitrous oxide to nitrogen; It is intended to provide a nitrogen reductase.

[0005]

SUMMARY OF THE INVENTION The present invention relates to Achromobacter cycloclus, one of denitrifying bacteria.
tes focuses on the production of nitrous oxide reductase, and Achromobacter cyclocycles
A tes gene library was prepared, a gene encoding nitrous oxide reductase was cloned therefrom, and the nucleotide sequence was determined to complete the gene.

The gene encoding nitrous oxide reductase of the present invention has the amino acid sequence represented by SEQ ID NO: 1. The gene encoding the nitrous oxide reductase of the present invention has the nucleotide sequence shown in SEQ ID NO: 2. The nitrous oxide reductase of the present invention has the amino acid sequence represented by SEQ ID NO: 1.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION

Embodiment 1 FIG. Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to the following Examples.

The present invention is a nitrous oxide reductase consisting essentially of the amino acid sequence shown in SEQ ID NO: 1. However, since this enzyme is expressed at a specific site in bacterial cell membranes, it has a special sequence called a signal peptide at the head of its amino acid sequence. This sequence is essential for enzyme transport but not for the activity of the mature enzyme. Therefore, an enzyme having a sequence lacking this signal peptide sequence and still maintaining nitrous oxide reductase activity is included.

The gene encoding the nitrous oxide reductase of the present invention typically has the nucleotide sequence shown in SEQ ID NO: 2.

[0010] In the present invention, Achromobac
The tercycles were cultured, the cells were disrupted with a surfactant, and the gene DNA was extracted. To construct a gene library, the extracted DNA and plasmid pUC18 are cut with the same restriction enzyme, ligated, and introduced into Escherichia coli by a conventional method (for example, Molecular Cloning).
ning, 2nd Ed. ).

Next, the purified Achromobate
The partial amino acid sequence of the nitric oxide reductase of r cycloclusteres was determined, and a gene having a short sequence synthesized based on the partial sequence of this amino acid was used as a primer to perform gene amplification (PCR). By hybridizing with the gene of the library using the paired gene as a probe, a gene encoding nitrous oxide reductase was obtained from this library.

The nitrous oxide reductase gene of the present embodiment is used to elucidate the mechanism for decomposing nitrous oxide of the enzyme and to improve its function, to provide a molecular model for molecular design of an artificial reduction catalyst, It is considered useful for the creation of microorganisms and plants with enhanced absorption and decomposition.

The gene thus obtained was analyzed by a fluorescent DNA sequencer to obtain the amino acid sequence shown in SEQ ID NO: 1 and the gene encoding nitrous oxide reductase shown in SEQ ID NO: 2. Hereinafter, a method for obtaining a gene encoding nitrous oxide reductase will be described.

(1) Achromobacter cy
Extraction of DNA from A. Chromlastes Cycloblastes
s in a culture solution (Yeast extract 10
(G), Bact peptone 10 (g), KN
O.sub.32 (g / L)) for one week at 30 (.degree. C.), followed by collection by a centrifuge (8,000.times.g, 30 (mi).
n)). 0.4 (g) of the cells were added to 2 (ml) of Tris-HCl buffer (Tris-HCl 50 (mM), pH 7.4,
EDTA 5 (mM)).
2 (ml) of 10 (%) SDS (sodium lauryl sulfate) was added, and the mixture was shaken at room temperature for 1 hour to lyse the cells. When the solution becomes completely clear, about 2 (ml)
Was added and the mixture was stirred at room temperature for 1 hour.

Next, the solution was centrifuged (8,000).
× g, 30 (min)). The upper layer was carefully removed, and 10 (ml) of ethanol cooled to -20 (° C) was gently added to the upper layer. The inoculation stick was carefully placed in the upper layer of ethanol, and the vicinity of the boundary with the lower layer was stirred to extract DNA from the lower layer aqueous solution. The extracted DNA is dried once and then 1 (m
1) TE buffer (10 (mM) Tris-HC, 1
mM EDTA), ethanol precipitation, extraction and drying were repeated twice, and finally 0.5 (ml) of TE buffer (10 (mM) Tris-HC, 1 mM
EDTA).

(2) Achromobacter cy
Determination of Partial Amino Acid Sequence of Cycloblastes Nitrous Oxide Reductase
s from the method of Averill et al. (Biochem. B
iophys. Res. Commun. 166,72
9, 1990). About 120 (μg) of the purified enzyme was added to 20 (mM) Tris
-HCl buffer was exchanged, and the volume was changed to 200 (μ
l). After heating at 90 (° C.) for 10 minutes to denature the protein, 2 (mg / ml) of trypsin was added to 2 (μm).
l) In addition, the mixture was reacted at 37 (° C) for 2 hours. Thereafter, the solution was heated at 100 (° C.) for 15 minutes to inactivate trypsin, and the peptide fragments were fractionated by reverse phase chromatography.

The column for reverse phase chromatography is TOS
OH TSK80Tm, conditions are flow rate 0.6 (ml / mi
n), separation solution is solution A: 0.1% TFA (Trifluoroacet)
ic acid) Solution B: Fragments were eluted with 80% acetonitrile, 0.1% TFA with a linear gradient such that Solution B became 100% after 75 minutes. The separated fragment was an amino acid sequencer (Perkin Elmer A).
For analysis by BI 473A), after removing acetonitrile and the like with a freeze dryer, 0.1 (%) TF
A, solubilized in 200 (μl). PVDF (poly
In order to immobilize this peptide on a vinylidene difluoride (membrane) membrane, first, a PVDF membrane was applied to 10 (μm
l) The sample was wetted with methanol, about 100 (μl) of the sample was brought into contact with the sample, and the sample was transferred to an absorption filter pressed against the back side of the membrane. After drying, 5 (μl)
Fixative solution (Biobrene 20 (μl), 0.1
(%) TFA 10 (μl), MeOH 70 (μ
l)), and after air drying, 15 (μl) of 0.1
(%) TFA was contacted for 15 seconds, excess TFA was wiped off with a Kimwipe, and finally 4 (μl) of methanol was dropped and air-dried. The membrane was set on an amino acid sequencer and subjected to automatic analysis. As a result, the amino acid sequence shown in SEQ ID NO: 3 and the amino acid sequence shown in SEQ ID NO: 4 were obtained.

(3) Preparation of Probe for Cloning Nitrous Oxide Reductase Gene In order to amplify the DNA of the probe by gene amplification (PCR), the DNA was replaced with SEQ ID NO: 5 based on the amino acid sequence shown in SEQ ID NO: 3. The sense strand having the nucleotide sequence shown in FIG.
An antisense strand having the nucleotide sequence shown in Table 1 was chemically synthesized.

Gene amplification was performed as follows. A
chromobacter cycles
1 (μg) of DNA, 5 (pmol) of the sense strand shown in SEQ ID NO: 5, 5 (pmol) of the antisense strand shown in SEQ ID NO: 6, and 0 (μl) of liquid 100 containing Premix (Pharmacia) .5 (ml) tube and overlaid with mineral oil, astec PC-700
94 (° C) 1 (min), 50 (° C) with gene amplifier
The cycle of 1.5 (min), 72 (° C.) and 2.5 (min) was repeated 40 times to amplify the DNA.

The obtained DNA was confirmed by electrophoresis, purified by ethanol precipitation, and finally TE buffer (10 mM
Tris-HCl, 1 mM EDTA). The amplified gene was labeled for detection by chemiluminescence. As a labeling agent, for example, a kit that binds HRP (horseradish peroxidase) to a gene with glutaraldehyde was used (Amersham).
Company).

(4) Preparation of Gene Library Achromobacter cyclocluster
s DNA (about 100 (μg)) was added to Pst I and Hin.
3 hours with about 10 units of dIII restriction enzyme
Decomposed at 7 ° C. After that, ethanol precipitation was performed and TE
Buffer (10 (mM) Tris-HC, 1 mM ED
TA) and dissolved in saccharose by density gradient centrifugation (0
(%) → 40 (%), horizontal rotor, 18 hours). A DNA fragment having about 4,000 to 10,000 base pairs was precipitated with ethanol and ligated to a vector (pUC18) cut with each restriction enzyme. The ligation reaction was performed with about 1 (μg) of Achromobacter c.
cyclastes DNA, 0.2 (μg) p
UC18, ligation mix (Takara Shuzo Ligation Kit, Solut) was added to a total volume of about 10 (μl).
Ion I) was added in an equal amount, and a ligation reaction was performed at 16 (° C) for 1 hour. This reaction solution was used for E. coli JM-109 (Takara Shuzo competent cell, 200 (μl)).
And allowed to stand on ice for 1 hour.

Thereafter, a culture solution (Nippon Gene, Hi-Competent Broth) was added to the solution, and the mixture was cultured at 37 (° C.) for 1 hour. Finally, a Petri dish containing an agar LB medium (Polypepton 10 (g), Ye)
ast Extract (5 g), NaCl 10
(G), Bact Agar 15 (g), Ampci
llin 250 (μg / L)) by 50 (μl),
Dispensed into 20 petri dishes. On the next day, a petri dish in which 200 to 500 JM-109 colonies grew was selected.
The colonies were transferred by contacting the colonies with a nylon membrane (Hybond N +, manufactured by Amersham).
The transfer time is about 1 minute. After the transfer, the nylon membrane was washed with 10% SDS, 0.5N NaOH, 0.5%
M Tris-HCl (pH 8), 2 × SSC (0.
3M NaCl, 0.03M Na-citrate)
The DNA was adsorbed by contacting for 2 to 5 minutes in this order, and the DNA was immobilized by UV irradiation for 2 minutes.

(5) Screening and Gene Analysis of Nitrous Oxide Reductase Gene A nylon membrane on which DNA has been immobilized is put into a prehybridization solution (manufactured by Amersham) and shaken (1 hour, 42 ° C.) to remove HRP. The labeled probe DNA (0.1 (μg)) was added, and the mixture was further stirred at 42 (° C.) for 4 to 15 hours. The nylon membrane is washed with a washing solution (manufactured by Amersham) (20 minutes, 42 ° C.).
After further washing with 2 × SSC solution (20 minutes, room temperature),
It was placed in an ECL detection solution (manufactured by Amersham) for 1 minute and exposed on an X-ray film. Hind III and Pst
From the gene library prepared with the two restriction enzymes I and I, the petri dish colonies (positive colonies) located at each of the spots (total of about 100) exposed to the X-ray film were extracted with 2 (ml) liquid LB After placing in a medium and culturing all day long, the plasmid was isolated using an alkaline method kit (PerkinElmer ABI).

The obtained plasmid is fluorescently labeled by a cycle sequence method (Perkin Elmer ABI) using appropriate primers (M4 or Rv, both manufactured by Takara Shuzo), and the gene analyzer ABI is used as it is.
Prism 310 (Perkin Elmer AB
I company).

As a result, the base sequence of the plasmid containing one cloned gene obtained from each of the two gene libraries was identified as Achromoba.
fully identical with partial amino acid sequence of Cter Cycloclastes nitrous oxide reductase (N ₂ OR), the two clones nitrous oxide reductase (N ₂ O
R) was found to have DNA encoding the gene. Synthetic primers were prepared based on the determined nucleotide sequences, and the nucleotide sequences of all the genes were determined as shown in SEQ ID NO: 2. The above-mentioned probe was a DNA encoding the nucleotide sequences 182 to 1406 of SEQ ID NO: 2.

[0026]

According to the gene encoding the nitrous oxide reductase according to the present invention, it comprises the amino acid sequence shown in SEQ ID NO: 1. By incorporating this gene into microorganisms and plant cells, It is possible to create microorganisms and plants with enhanced absorption and decomposition of oxides.

According to the gene encoding the nitrous oxide reductase according to the present invention, which has the nucleotide sequence shown in SEQ ID NO: 2, the gene can be incorporated into microorganisms and plant cells to absorb nitrogen oxides. -It is possible to create microorganisms and plants with enhanced degradation.

The nitrous oxide reductase according to the present invention comprises the amino acid sequence represented by SEQ ID NO: 1,
This enzyme becomes a metal enzyme having copper as an active center, and is not inactivated by oxygen, and the function for decomposing nitrous oxide is improved. Further, it is possible to construct a device having a high ability of absorbing and decomposing nitrogen oxides by using the present enzyme.

[0029]

[Sequence list]

SEQ ID NO: 1 Sequence length: 643 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genonic DNA Origin Organism name: Acronobacter Cycloclusteres (Ac)
Strain name: IAM 1013 Characteristic of sequence Characteristic of characteristic: cellular Characteristic determination method: E Sequence: Met Glu Ser Lys Glu His Lys Gly Leu Ser Arg Arg Ala Leu Phe Ser 1 5 10 15 Ala Thr Ala Gly Ser Ala Ile Leu Ala Gly Thr Val Gly Pro Ala Ala 20 25 30 Leu Ser Leu Gly Ala Ala Gly Leu Ala Thr Pro Ala Arg Ala Ala Thr 35 40 45 Gly Ala Asp Gly Ser Val Ala Pro Gly Lys Leu Asp Asp Tyr Tyr Gly 50 55 60 Phe Trp Ser Ser Gly Gln Thr Gly Glu Met Arg Ile Leu Gly Ile Pro 65 70 75 80

Ser Met Arg Glu Leu Met Arg Val Pro Val Phe Asn Arg Cys Ser Ala 85 90 95 Thr Gly Trp Gly Gln Thr Asn Glu Ser Ile Arg Ile His Gln Arg Thr 100 105 110 Met Thr Glu Lys Thr Lys Lys Gln Leu Ala Ala Asn Gly Lys Lys Ile 115 120 125 His Asp Asn Gly Asp Leu His His Val His Met Ser Phe Thr Asp Gly 130 135 140 Lys Tyr Asp Gly Arg Tyr Leu Phe Met Asn Asp Lys Ala Asn Thr Arg 145 150 155 160

Val Ala Arg Val Arg Cys Asp Val Met
Lys Thr Asp Ala Ile Leu Glu 165 170 175 Ile Pro Asn Ala Lys Gly Ile His Gly Met Arg Pro Gln Lys Trp Pro 180 185 190 Arg Ser Asn Tyr Val Phe Cys Asn Gly Glu Asp Glu Ala Pro Leu Val As 195 200 205 Asn Gly Ser Thr Met Thr Asp Val Ala Thr Tyr Val Asn Ile Phe 210 215 220 Thr Ala Val Asp Ala Asp Lys Trp Glu Val Ala Trp Gln Val Lys Val 225 230 235 240

Ser Gly Asn Leu Asp Asn Cys Asp Ala Asp Tyr Glu Gly Lys Trp Ala 245 250 255 Phe Ser Thr Ser Tyr Asn Ser Glu Met Gly Met Thr Leu Glu Glu Met 260 265 270 Thr Lys Ser Glu Met Asp His Val Val Val Phe Asn Ile Ala Glu Ile 275 280 285 Glu Lys Ala Ile Lys Ala Gly Gln Tyr Glu Glu Ile Asn Gly Val Lys 290 295 300 Val Val Asp Gly Arg Lys Glu Ala Lys Ser Leu Phe Thr Arg Tyr Ile 305 310 315 320

Pro Ile Ala Asn Asn Pro His Gly Cys Asn Met Ala Pro Asp Arg Lys 325 330 335 His Leu Cys Val Ala Gly Lys Leu Ser Pro Thr Val Thr Thr Val Leu Asp 340 345 350 350 Thr Thr Lys Phe Asp Ala Leu Phe Tyr Asp Asn Ala Glu Pro Arg Ser 355 360 365 Ala Val Val Ala Glu Pro Glu Leu Gly Leu Gly Pro Leu His Thr Ala 370 375 380 Phe Asp Gly Arg Gly Asn Ala Tyr Thr Ser Leu Phe Leu Asp Ser Gln 385 390 395 400

Val Val Lys Trp Asn Ile Asp Glu Ala Ile Arg Ala Tyr Ala Gly Glu 405 410 415 Lys Ile Asn Pro Ile Lys Asp Lys Leu Asp Val Gln Tyr Gln Pro Gly 420 425 430 His Leu Lys Thr Val Met Gly Glu Thr Leu Asp Ala Ala Asn Asp Trp 435 440 445 445 Leu Val Cys Leu Cys Lys Phe Ser Lys Asp Arg Phe Leu Asn Val Gly 450 455 460 Pro Leu Lys Pro Glu Asn Asp Gln Leu Ile Asp Ile Ser Gly Asp Lys 465 470 475 475 480

Met Val Leu Val His Asp Gly Pro Thr Phe Ala Glu Pro His Asp Ala 485 490 495 Ile Ala Val Ser Pro Ser Ile Leu Pro Asn Ile Arg Ser Val Trp Asp 500 505 510 Arg Lys Asp Pro Leu Trp Ala Glu Thr Arg Lys Gln Ala Glu Ala Asp 515 520 525 Glu Val Asp Ile Asp Glu Trp Thr Glu Ala Val Ile Arg Asp Gly Asn 530 535 540 Lys Val Arg Val Tyr Met Thr Ser Val Ala Pro Ser Phe Ser Gln Pro 545 550 555 555 560

Ser Phe Thr Val Lys Glu Gly Asp Glu Val Thr Val Ile Val Thr Asn 565 570 575 Leu Asp Glu Ile Asp Asp Leu Thr His Gly Phe Thr Met Gly Asn His 580 585 590 Gly Val Ala Met Glu Val Gly Pro Gln Gln Thr Ser Ser Val Thr Phe 595 600 605 Val Ala Ala Asn Pro Gly Val Tyr Trp Tyr Tyr Cys Gln Trp Phe Cys 610 615 620 His Ala Leu His Met Glu Met Arg Gly Arg Met Phe Val Glu Pro Lys 625 630 635 640 640 Gly Ala (Stop)

SEQ ID NO: 2 Sequence length: 1929 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genonic DNA Origin Organism name: Acronobacter Cycloclastes (Ac)
Strain name: IAM1013 Characteristic of sequence Characteristic of characteristic: Cellular Characteristic method for determining characteristic: E Sequence: ATG GAA TCA AAG GAA CAC AAG GGA CTA AGC CGG CGA GCA CTT TTC AGC 48 Met Glu Ser Lys Glu Lys Gly Leu Ser Arg Arg Ala Leu Phe Ser 1 5 10 15 GCG ACT GCA GGC AGC GCC ATT CTG GCG GGC ACT GTA GGG CCG GCC GCA 96 Ala Thr Ala Gly Ser Ala Ile Leu Ala Gly Thr Val Gly Pro Ala Ala 20 25 30 CTC AGC CTC GGC GCT GCA GGA TTG GCG ACA CCG GCC CGT GCG GCC ACG 144 Leu Ser Leu Gly Ala Ala Gly Leu Ala Thr Pro Ala Arg Ala Ala Thr 35 40 45 GGT GCC GAC GGC TCG GTC GCA CCG GGC AAG CTC GAC GAT TAC TAC TAC GGC 192 Gly Ala Asp Gly Ser Val Ala Pro Gly Lys Leu Asp Asp Tyr Tyr Gly 50 55 60 TTC TGG TCC TCC GGT CAG ACC GGC GAG ATG CGC ATT CTC GGC ATT CCC 240 Phe Trp Ser Ser Gly Gln Thr Gly Glu Met Arg Ile Leu Gly Ile Pro 65 70 75 80

TCG ATG CGC GAA CTG ATG CGC GTG CCG GTG TTC AAC CGT TGC TCG GCC 288 Ser Met Arg Glu Leu Met Arg Val Pro Val Phe Asn Arg Cys Ser Ala 85 90 95 ACC GGT TGG GGG CAG ACC AAT GAA TCG ATC CGG ATC CAT CAG CGC ACG 336 Thr Gly Trp Gly Gln Thr Asn Glu Ser Ile Arg Ile His Gln Arg Thr 100 105 110 A TG ACG GAA AAG ACG AAG AAG CAG CTT GCC GCC AAC GGC AAG AAA ATC 384M et Thr Glu Lys Thr Lys Lys Gln Leu Ala Ala Asn Gly Lys Lys Ile 115 120 125 CAC GAC AAC GGC GAC CTG CAT CAC GTC CAT ATG TCC TTC ACC GAC GGT 432 His Asp Asn Gly Asp Leu His His Val His Met Ser Phe Thr Asp Gly 130 135 140 AAA TAT GAC GGT CGC TAC CTG TTC ATG AAC GAC AAG GCC AAT ACG CGC 480 Lys Tyr Asp Gly Arg Tyr Leu Phe Met Asn Asp Lys Ala Asn Thr Arg 145 150 155 160

GTG GCG CGC GTG CGC TGC GAC GTG ATG AAG ACC GAC GCC ATC CTG GAG 528 Val Ala Arg Val Arg Cys Asp Val Met Lys Thr Asp Ala Ile Leu Glu 165 170 175 ATC CCC AAC GCC AAG GGC ATC CAC GGC ATG CGT CCG CAG AAA TGG CCG 576 Ile Pro Asn Ala Lys Gly Ile His Gly Met Arg Pro Gln Lys Trp Pro 180 185 190 CGT TCA AAC TAT GTG TTC TGC AAC GGC GAG GAC GAG GCC CCG CTC GTC 624 Arg Ser Asn Tyr Val Phe Cys Asn Gly Glu Asp Glu Ala Pro Leu Val 195 200 205 AAC GAC GGC TCG ACG ATG ACG GAC GTG GCG ACC TAC GTG AAC ATC TTC 672 Asn Asp Gly Ser Thr Met Thr Asp Val Ala Thr Tyr Val Asn Ile Phe 210 215 220 ACC GCC GTC GAT GCC GAC AAG TGG GAA GTG GCT TGG CAG GTG AAG GTC 720 Thr Ala Val Asp Ala Asp Lys Trp Glu Val Ala Trp Gln Val Lys Val 225 230 235 240

TCG GGC AAC CTC GAC AAT TGC GAT GCC GAC TAC GAG GGC AAG TGG GCG 768 Ser Gly Asn Leu Asp Asn Cys Asp Ala Asp Tyr Glu Gly Lys Trp Ala 245 250 255 TTC TCC ACC AGC TAC AAC TCC GAA ATG GGC ATG ACG CTG GAG GAG ATG 816 Phe Ser Thr Ser Tyr Asn Ser Glu Met Gly Met Thr Leu Glu Glu Met 260 265 270 ACC AAG TCC GAG ATG GAT CAT GTC GTC GTC TTC AAC ATC GCC GAA ATC 864 Thr Lys Ser Glu Met Asp His Val Val Val Phe Asn Ile Ala Glu Ile 275 280 285 GAG AAG GCC ATT AAG GCC GGC CAA TAT GAG GAG ATC AAC GGC GTC AAG 912 Glu Lys Ala Ile Lys Ala Gly Gln Tyr Glu Glu Ile Asn Gly Val Lys 290 295 300 GTG GTG GAC GGG CGC AAG GAG GCA AAG TCG CTC TTC ACG CGC TAC ATC 960 Val Val Asp Gly Arg Lys Glu Ala Lys Ser Leu Phe Thr Arg Tyr Ile 305 310 315 320

CCG ATC GCC AAC AAC CCC CAC GGC TGC
AAC ATG GCG CCG GAC AGG AAG 1008 Pro Ile Ala Asn Asn Pro His Gly Cys Asn Met Ala Pro Asp Arg Lys 325 330 335 CAT CTG TGC GTT GCC GGC AAG CTT TCG CCA ACC GTC ACC GTG CTG GAC 1056 His Leu Cys A Lys Leu Ser Pro Thr Val Thr Val Leu Asp 340 345 350 GTG ACG AAG TTC GAT GCC CTG TTC TAC GAC AAT GCC GAG CCG CGC AGC 1104 Val Thr Lys Phe Asp Ala Leu Phe Tyr Asp Asn Ala Glu Pro Arg Ser 355 360 365 GCA GTG GTT GCG GAA CCG GAA CTG GGC CTT GGC CCA TTG CAC ACC GCC 1152 Ala Val Val Ala Glu Pro Glu Leu Gly Leu Gly Pro Leu His Thr Ala 370 375 380 380 TTC GAC GGG CGC GGC AAC GCC TAT ACC TCG CTG TTC CTC GAC AGC CAG 1200 Phe Asp Gly Arg Gly Asn Ala Tyr Thr Ser Leu Phe Leu Asp Ser Gln 385 390 395 400

GTG GTA AAG TGG AAC ATC GAT GAG GCC ATC CGC GCC TAC GCC GGT GAG 1248 Val Val Lys Trp Asn Ile Asp Glu Ala Ile Arg Ala Tyr Ala Gly Glu 405 410 415 AAG ATC AAC CCG ATC AAG GAC AAG CTC GAC GTT CAG TAT CAA CCC GGC 1296 Lys Ile Asn Pro Ile Lys Asp Lys Leu Asp Val Gln Tyr Gln Pro Gly 420 425 430 CAC TTG AAG ACG GTG ATG GGC GAA ACG CTC GAT GCC GCC AAC GAC TGG 1344 His Leu Lys Thr Val Met Gly Glu Thr Leu Asp Ala Ala Asn Asp Trp 435 440 445 CTC GTT TGC CTG TGC AAA TTC TCC AAG GAC CGG TTC CTG AAT GTC GGC 1392 Leu Val Cys Leu Cys Lys Phe Ser Lys Asp Arg Phe Leu Asn Val Gly 450 455 460 CCG CTG AAG CCG GAA AAC GAT CAG TTG ATC GAC ATT TCC GGT GAC AAG 1440 Pro Leu Lys Pro Glu Asn Asp Gln Leu Ile Asp Ile Ser Gly Asp Lys 465 470 475 475 480

ATG GTG CTG GTC CAT GAC GGC CCG ACC TTC GCC GAG CCG CAT GAC GCC 1488 Met Val Leu Val His Asp Gly Pro Thr Phe Ala Glu Pro His Asp Ala 485 490 495 ATC GCC GTC TCC CCC TCG ATC CTG CCG AAC ATC CGC TCG GTC TGG GAT 1536 Ile Ala Val Ser Pro Ser Ile Leu Pro Asn Ile Arg Ser Val Trp Asp 500 505 510 CGC AAG GAT CCG CTG TGG GCC GAA ACC CGC AAG CAG GCC GAG GCC GAT 1584 Arg Lys Asp Pro Leu Trp Ala Glu Thr Arg Lys Gln Ala Glu Ala Asp 515 520 525 GAG GTC GAC ATC GAC GAA TGG ACC GAG GCC GTG ATC CGC GAC GGC AAC 1632 Glu Val Asp Ile Asp Glu Trp Thr Glu Ala Val Ile Arg Asp Gly Asn 530 535 540 540 AAG GTT CGC GTC TAC ATG ACC TCG GTC GCA CCC AGC TTC AGC CAG CCG 1680 Lys Val Arg Val Tyr Met Thr Ser Val Ala Pro Ser Phe Ser Gln Pro 545 550 555 560

AGC TTT ACG GTG AAG GAG GGC GAC GAA GTC ACG GTC ATC GTC ACC AAT 1728 Ser Phe Thr Val Lys Glu Gly Asp Glu Val Thr Val Ile Val Thr Asn 565 570 575 CTC GAT GAA ATC GAT GAC CTT ACC CAT GGC TTC ACC ATG GGC AAT CAC 1776 Leu Asp Glu Ile Asp Asp Leu Thr His Gly Phe Thr Met Gly Asn His 580 585 590 GGC GTG GCG ATG GAG GTC GGC CCG CAA CAG ACG AGC TCC GTT ACC TTC 1824 Gly Val Ala Met Glu Val Gly Pro Gln Gln Thr Ser Ser Val Thr Phe 595 600 605 GTT GCG GCC AAT CCT GGC GTC TAC TGG TAC TAT TGC CAA TGG TTC TGC 1872 Val Ala Ala Ala Asn Pro Gly Val Tyr Trp Tyr Tyr Cys Gln Trp Phe Cys 610 615 620 620 CAT GCC CTG CAC ATG GAA ATG CGC GGC CGC ATG TTC GTG GAA CCG AAG 1920 His Ala Leu His Met Glu Met Arg Gly Arg Met Phe Val Glu Pro Lys 625 630 635 640 GGC GCC TGA Gly Ala Sto

SEQ ID NO: 3 Sequence length: 15 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence: Val Ala Pro Gly Lys Leu Asp Asp Tyr Tyr Gly Phe Trp Asp Gly 1 5 10 15

SEQ ID NO: 4 Sequence length: 20 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence: Phe Leu Asn Val Gly Pro Leu Lys Pro Glu Asn Asp Gln Leu Ile 1 5 10 15 Asp Ile Ser Gly Asp 20

SEQ ID NO: 5 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics Characteristic symbol: exon antisense : No Sequence: ACTACTACGGCTTCTGGTCC 20

SEQ ID NO: 6 Sequence length: 18 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA Sequence characteristics Characteristic symbol: exon antisense : Yes Sequence: TCTCGGNKCAGCGGGCCA 18

────────────────────────────────────────────────── ─── front page continued (51) Int.Cl. ⁶ identifications FI C12R 1:19)

Claims (3)

[Claims] 1. A gene encoding nitrous oxide reductase having the amino acid sequence shown in SEQ ID NO: 1. 2. A gene encoding nitrous oxide reductase having the nucleotide sequence shown in SEQ ID NO: 2. 3. A nitrous oxide reductase having the amino acid sequence shown in SEQ ID NO: 1.