JP2000210083A - PRODUCTION OF SULFATE-REDUCING BACTERIUM CYTOCHROME c3 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To inexpensively and readily obtain the subject compound useful for studying an electron transfer mechanism of a protein by culturing a recombinant aerobic bacterium having a plasmid containing a cytochrome c3 gene of a sulfate-reducing bacterium and manifesting the gene. SOLUTION: The objective sulfate-reducing bacterium cytochrome c3 is useful as a material for a rectifying device and a molecular element or for a research material of an electron transferring mechanism of a protein, and further enabling to recover a useful material by bacterial leaching and to propagate environmental cleaning by a heavy metal treatment, thus greatly contribute to development of the industrial and research fields and is inexpensively and readily obtained by culturing a recombinant aerobic bacterium [e.g. Shewanella putrefaciens (FERM accession number FERM P-16752] containing a cytochrome c3 gene such as a sulfate-reducing bacterium [e.g. Desulfovibrio vulgaris Miyazaki F. (IAM accession number IAM2604)] in a bacterium body and expressing the gene.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for producing large amounts and inexpensively cytochrome c ₃ of sulfate-reducing bacteria, as well as to novel recombinant aerobic and recombinant plasmids used in this method.

[0002]

BACKGROUND OF THE INVENTION sulfate reducing bacteria cytochrome c ₃ has four c-type heme in the molecule, show very less noble oxidation-reduction potential (-300 mV vs. SHE) compared to other type c heme Heme protein. This protein is the only heme protein that can directly exchange electrons with a metal electrode without using a mediator or the like. In addition, the sulfate-reducing bacterium cytochrome c ₃ does not show conductivity in an oxidized form (in a state where no electrons are present), but shows a reduced form (in a state where electrons are present).
Shows conductivity equivalent to that of a metal complex. For these reasons, attention has been paid research material for the sulfate reducing bacteria cytochrome c ₃ molecular wires and organic semiconductor, so-called bio-elements such rectifying element material, also to achieve this, as imitation object.

[0003] Moreover, sulfate reducing bacteria, including cytochrome c ₃ and this has the ability to reduce the uranium and zinc alone. Utilizing this ability, for uranium, it is possible to perform bacterial leaching for recovering U (IV) from a dilute uranium-containing solution U (VI). In addition, heavy metals such as zinc can be rendered harmless by reducing them, or can be treated with heavy metals such that they can be easily removed as precipitates. Therefore, sulfate-reducing bacteria and cytochrome c ₃ are attracting attention as microorganisms and proteins that enable leaching of useful substances and environmental purification by heavy metal treatment.

Conventionally, sulfate-reducing bacterium cytochrome c ₃ is obtained by culturing sulfate-reducing bacteria in a large amount and ultrasonically destroying the collected cells.
It was obtained by performing purification by cation exchange and gel filtration. When a jar fermenter capable of mass culture is used, 100 g of wet cells of sulfate-reducing bacteria can be obtained by culturing for 2 days, and by producing this, 20 mg of cytochrome c ₃ can be obtained.

[0005] With the above prior art, it is possible to obtain a sulphate reducing bacteria cytochrome c _3. However, sulfate-reducing bacteria are a kind of obligate anaerobic bacterium and cannot grow in the presence of oxygen. In addition, harmful hydrogen sulfide is generated during the growth process. Therefore, to mass-cultivate sulfate-reducing bacteria, an anaerobic culture tank in which nitrogen or argon gas is bubbled and the culture device is kept at a positive pressure so as not to be exposed to oxygen, that is, a so-called anaerobic jar fermenter is used. Need. Further, it is necessary to provide a device for adsorbing the discharged gas on activated carbon or the like without leaking the gas to the other and safely treating the gas. As described above, in order to win the cytochrome c ₃ in large quantities there are many difficulties in terms of equipment and expenses.

In using cytochrome c ₃ as a research material for biodevices, it is also necessary to clarify the relationship between its oxidation-reduction potential and electron transfer rate and the structure of the protein. This relationship can be clarified by artificially substituting a specific amino acid constituting a protein with another amino acid, and examining the effect of the substitution on the redox potential and the electron transfer rate. This method requires the development of a system for artificially replacing an amino acid with another amino acid, a so-called gene recombination system. In this regard, conventionally, it has been tried production by E. coli sulfate reducing bacteria cytochrome c _3. However, since there are four hemes in one molecule of the protein, production of holoproteins retaining heme has not been reported to date. on the other hand,
Although expression has been reported in a system using a photosynthetic bacterium as a host, the expression level of cytochrome c ₃ is 10
It is extremely small, 1/0, and cannot be called a mass production system.

[0007]

[0008] The present invention has been made in view of the above circumstances, and an object thereof is to provide a method for inexpensively and easily fed sulfate reducing bacteria cytochrome c _3.

[0008]

To achieve the above object SUMMARY OF THE INVENTION The present invention, culturing a recombinant aerobic bacterium harboring a plasmid containing the sulfate-reducing bacteria cytochrome c ₃ genes in bacteria, the gene The present invention provides a method for producing cytochrome c ₃ , comprising a step of producing cytochrome c ₃ by causing expression.

In this method, the sulfate-reducing bacteria cytochrome c ₃
By culturing in a special recombinant aerobic bacteria introduced with the gene, the transgene was expressed in aerobic bacteria, cytochrome c ₃ to produce. Then, the sulfate reducing bacteria cytochrome c ₃ thus produced is isolated and purified. Aerobic pongus can be easily cultured because it does not require special culture equipment such as obligate anaerobic bacteria and does not release environmentally regulated substances. As a result, it is possible to obtain a sulphate reducing bacteria cytochrome c ₃ inexpensively and in large quantities.

[0010] The present invention also to obtain a recombinant aerobic bacteria used for the above method, provides a plasmid containing the sulfate-reducing bacteria cytochrome c ₃ gene.

Further, the present invention provides a recombinant aerobic bacterium transformed with the above plasmid.

[0012]

DETAILED DESCRIPTION OF THE INVENTION The present inventors have, as a material of the bio-element, taken sulfate reducing bacteria cytochrome c ₃ is the only protein which performs direct electron transfer to the electrode, aerobic sulfate reducing bacteria cytochrome c ₃ As a result of conducting various studies on bacterial expression, the present invention has been achieved.

Hereinafter, the present invention will be described in detail.

In the present invention, incorporated into a suitable vector sulfate reducing bacteria cytochrome c ₃ gene, by the aerobic bacteria transformed with this, to create a recombinant aerobic bacteria. The sulfate reducing bacteria cytochrome c ₃ gene source, is not particularly limited as long as sulfate-reducing strain having this gene can be used, for example, the following strains.

Desulfovibrio vulgaris Miyazaki F; this bacterium was deposited at the Institute of Applied Microbiology of the University of Tokyo Microorganisms and Microalgae under the deposit number IAM2604. And can be sold if desired.

Desulfovibrio vulgaris Hilldenborough; this strain has been deposited with the American Type Culture Collection under ATCC Deposit No. ATCC 29579 and can be ordered if desired. .

[0017] as long to obtain these sulfate reduction strains by methods well known to those skilled in the art, it can be taken out cytochrome c ₃ gene from the strain. For example, sulfate reducing bacteria cytochrome c ₃ gene taken from desulfohydrase Vibrio Buno Regal Miyazaki F has a nucleotide sequence set forth in SEQ ID NO: 1.

The gene thus obtained is inserted into an appropriate vector that can be expressed in aerobic bacteria. One example of such a vector is the broad host plasmid vector pRK4
15 (Gene (1998) 70: 191-197). In addition,
In the examples described below, this plasmid vector pR
To K415, by incorporating desulfohydrase Vibrio Buno Regal Miyazaki F from sulfate reducing bacteria cytochrome c ₃ gene, was created recombinant broad host plasmid PRKM1. In this recombinant plasmid pRKM1, the sulfate-reducing bacterium cytochrome c ₃ gene is connected downstream of the promoter in the sulfate-reducing bacterium.

Next, by transforming the recombinant plasmid in a suitable aerobic bacterial host to obtain a recombinant aerobic bacteria containing sulfate reducing bacteria cytochrome c ₃ gene. As the aerobic bacterial host, for example, Shewanella putrefaciens can be used. This fungus has been deposited with the National Collection of Industrial Marine Bacteria in the United Kingdom under the deposit number NCIMB400. Further, by screening from this Schwanner aptrifaciens, it is possible to obtain a rifampicin-resistant strain of Schwanner aptrifaciens DSP.
-10 (Shewanella putrefaciens DSP-10) was isolated, and this strain can be particularly preferably used.
In addition, this Schwanner ptrifaciens DSP-10
Was filed on April 3, 1998 for the purposes of this patent application,
Deposited under the deposit number FERM P-16750 at the Patent Microorganisms Depositary Center, National Institute of Biotechnology and Industrial Technology.

General methods for transforming the above-mentioned recombinant plasmid into aerobic bacteria include the calcium chloride method, the Tfb method using Cohen, etc., and electroporation. However, it should be noted that when the above-mentioned Schwanner ptrifaciens is used as a host, it cannot be transformed by the calcium chloride method and the Tfb method. In this case, as described in Examples below, E. coli (e.g., E.
coli S17-1) is preferably used. That is, the recombinant plasmid is once transformed into Escherichia coli, and Schwanneruptrifaciens is transformed by using the conjugation between Escherichia coli and Schwaneruptrifaciliens. Also, transformation by electroporation seems to be possible.

[0021] The above-mentioned recombinant Swannerup trifaciens DSP-10 (FERM P-16750) is obtained by transfecting the above-mentioned recombinant wide-range host plasmid pRKM1 into the above-mentioned DSP-10 (FERM P-16750). pRKM1) was deposited on April 3, 1998 with the Patent Microorganisms Depositary of the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology for deposit of the Patent Application No. FERM.
Deposited as P-16675.

In the present invention, sulfate-reducing bacterium cytochrome c ₃ can be produced by culturing the recombinant aerobic bacteria obtained as described above in an aerobic atmosphere. For example, if the above-mentioned recombinant Schwanella is cultured, the sulfate-reducing bacterium cytochrome c ₃ can be produced in the cells of Schwanella.
Is synthesized, and it may be separated and purified.
Because Schwanella, an aerobic pongus, is an aerobic bacterium,
Since no special culturing device is required for culturing and no environmentally regulated substances are released, culturing can be performed easily. for that reason,
It is possible to obtain a sulphate reducing bacteria cytochrome c ₃ inexpensively and in large quantities.

[0023]

EXAMPLES be described in detail in Example was expressed cytochrome c ₃ of sulfate-reducing bacteria in the aerobic bacteria below.

[0024] Example 1: set with full coding region of sulfate reducing bacteria Miyazaki F strain cytochrome c ₃ gene Schwarz Ne interrupt Reference tumefaciens in expression plasmids pRKM1 intended for building sulfate reducing bacteria Miyazaki F strain cytochrome c ₃ protein Recombinant plasmid pKM300
All structural gene including the promoter region of the cytochrome c ₃ protein isolated from a: (.. Protein Seq Data Anal (1993) 5 193-196 can be prepared by the method described in), digested with restriction enzymes Aat II and Sph I After that, the Aat II-Sp
The hI gene fragment (900 base pairs) was blunt-ended using T4 DNA polymerase, which was then pUC11
Eight vectors (manufactured by Takara Shuzo Co., Ltd.) were inserted into the Sma I site in the multicloning site (the nucleotide sequence of the Aat II-Sph I gene fragment is as shown in SEQ ID NO: 1).

Using this vector, E. coli strain JM109 was used.
Was transformed, from several clones, the orientation of the promoter of the cytochrome c ₃ gene a plasmid was extracted a direction opposite to the lac promoter on pUC118 vector to further purification (building pUCM1).

Next, this pUCM1 vector was
After digestion with XbaI and EcoRI, the XbaI-EcoRI
The gene fragment (900 base pairs) was transformed into XbaI-Ec of pRK415 (Gene (1998) 70: 191-197) which is a broad host range vector.
Incorporated at the oRI site (construction of pRKM1). FIG. 1 shows an outline of the above construction procedure. Example 2: Shewanella putrefaciens DS with expression plasmid pRKM1
Transformation of P-10 Using the expression plasmid pRKM1 obtained in Example 1, E. coli strain S17-1 was transformed. Next, the transformant E. coli S17-1 (pRKM1) was cultured with shaking at 37 ° C. overnight in 3 mL of LB medium containing 200 mg / L of streptomycin and 15 mg / L of tetracycline as antibiotics. 1 mL of this culture
Was subcultured in 100 mL of the same medium, and cultured for further 2 hours under the same conditions (Culture E). Since the transformant is resistant to tetracycline, only a transformant can be obtained by this culture.

On the other hand, the Schwanner rapture science DSP
Since the -10 strain (FERMP-16750) is a rifampicin resistant strain, shaking culture was performed overnight at 30 ° C. in 3 mL LB medium in the presence of 10 mg / L rifampicin. 2 mL of this is 100 mL
And cultured for 3 hours under the same conditions (culture S).

The 100 mL cultures of the cultures E and S were cultured for 2 hours and 3 hours, respectively, and then centrifuged (60 mL).
(00 rpm × 10 min), and the cells were collected and suspended in an LB medium containing no antibiotic. Thereafter, the cells were centrifuged again (6000 rpm × 10 min), and the cells were collected to wash the cells. Next, after suspending each of these cells in about 1 mL of LB medium, both were aseptically mixed at a ratio of about 2: 1. Next, 100 mL of this was taken, spread on an LB agar plate, and
The cells were cultured at 0 ° C. for 4 hours in a gas phase incubator.
As a result, heterologous conjugation with E. coli transformants
Transformation of Schwanner ptrifaciens DSP-10 strain is performed. Next, mixed bacteria that grew on the whole plate,
Use a platinum loop to scrape it, and remove it with 100 mL of L
After suspending in a B medium, the cells were spread on an LB agar plate containing 10 mg / L rifampicin and 15 mg / L tetracycline, and cultured at 30 ° C. overnight. Pick up some growing colonies and pick up 3 mg L containing 10 mg / 1 rifampicin and 15 mg / L tetracycline.
The cells were inoculated into a B medium and cultured at 30 ° C. overnight with shaking. The transformant Schwannerptorifaciens DSP-10 (pRKM1) is resistant to both rifampicin and tetracycline, whereas the transformed E. coli S17-1 (pR
KM1) is only resistant to tetracycline and has no rifampicin resistance. Therefore, in this selective culture, Schwaneruptrifaciens DSP-10 (pRKM1)
Only strains grow. These are centrifuged (6000 rpm × 1).
0 min), the cells were collected, suspended in 50% glycerol, quenched with liquid nitrogen, and stored at -80 ° C. The thus-obtained recombinant Schwanner ptrifaciens DSP-10 (p
RKM1) (FERMP-16752) was used as a seed for the following experiments.

[0029] Example 3: mass culture of recombinant Schwarz Ne interrupt Reference tumefaciens DSP-10 (pRKM1), the inoculum obtained in Expression Example 2 of sulfate reducing bacteria Miyazaki F strain cytochrome c ₃ proteins, of 10 mg / L 3 mL LB containing rifampicin and 15 mg / L tetracycline
The whole medium was inoculated into the medium and cultured at 30 ° C. overnight with shaking. Next, 100 mL of 10 mg / L rifampicin and 1
Subculture was carried out at 1% (v / v) with respect to an LB medium containing 5 mg / L tetracycline.
Shake at 30 ° C. overnight. In addition, 50m
L was transferred to 5 L of LB medium (containing 10 mg / L rifampicin and 30 mg / L tetracycline), and 71 jar fermenters (manufactured by Iwashiya Bioscience)
The aerobic culture was carried out at 30 ° C. for 24 hours under the air flow rate of 8 L / min. Under these conditions, the transformant, recombinant Schwanner ptrifaciens DSP-10,
The yield of (pRKM1) (FERM P-16752) was 25 g.

Recombinant Schwanner ptrifaciens DSP
25 g of wet cells of -10 (pRKM1) (FERM P-16752)
Was suspended in about twice the volume of 30 mM phosphate buffer (pH 7.0). This solution was subjected to ultrasonic crushing (SONIFIER250, BRANSON) at 72 W for 60 minutes in an ice bath. At this time, in order to suppress the increase in the solution viscosity due to the disruption of the cells and to carry out the hydrolysis treatment of the DNA, 1 / 10,000 of deoxyribonuclease I was added. After crushing, the solution was centrifuged (RP-42 HITACHI CP-; 29,000 rpm, 60 minutes), and the supernatant was collected. Streptomycin sulfate was added to the obtained supernatant, followed by stirring at 4 ° C for 1 hour. The amount of streptomycin sulfate added (Xg) was calculated by the following equation by measuring the absorbance at 960 nm based on the amount of DNA.

X = Abs _{260 nm} × C × V / 5000 C: dilution ratio V: supernatant volume After stirring, centrifugation was carried out at 15,000 rpm for 30 minutes, and the supernatant was placed in a dialysis tube (molecular weight cut-off 6,000).
8000) and 10 mM phosphate buffer (pH 7.
0) Dialysis (1 hour or more x 3 times) was performed with 10 L. The dialyzed solution was centrifuged again at 15000 rpm for 30 minutes, and the supernatant was subjected to cation exchange chromatography (SP-Sepharose, manufactured by Pharmacia). After protein is adsorbed on the resin, FPLC
(Fast Protein Liquid Chromatography) using a gradient elution method (N from 0M to 0.5M).
The fraction containing cytochrome c ₃ was collected by a gradient of aCl. Further, this fraction was concentrated by ultrafiltration and subjected to gel filtration chromatography (HiLoad 16/60 Superdex G-75p
g, Pharmacia Mori), and fractions considered to be cytochrome c ₃ were collected by absorption spectrum measurement. The resulting oxidized and reduced ultraviolet absorption spectra of the obtained cytochrome c ₃ were measured, and the cytochrome c calculated from the following equation was obtained.
Purity of ₃ I. (Purity Index). For pure cytochrome c ₃ , the ratio of the oxidized absorption (280 nm) to the reduced absorption (552 nm) is 3.0. Here, P. I. Of approximately 3.0 was assigned to cytochrome c ₃
It was a standard.

P. I. = Abs _552nm (reduced) / Abs
280 nm (oxidized form) Example 4: Recombinant Schwanner ptrifaciens DSP
Characterization of Cytochrome c ₃ standard purified from -10 (pRKM1) (FERM P-16752) Mass culture using 8 L / min air flow conditions using a 7 L jar fermenter (manufactured by Iwashiya Bioscience) Recombinant Schwanner ptrifaciens DSP-10 (pRK
M1) The yield of the cytochrome c ₃ sample derived from sulfate-reducing bacteria purified from (FERM P-16752) was 1.9 mg (0.29 mg / g of cells) per liter of the medium, and the yield of wild-type sulfate-reducing bacteria was 0.3mg / L culture (0.15m
g / g of cells) was found to be about 6 times more per liter of medium.

On the other hand, 2 L of LB medium (10 mg / mL of rifampicin and 3
0 mg / mL of tetracycline) and 30
℃ for 24 hours, the yield of the resulting cytochrome c ₃ preparations from cells 8g subjected to shaking culture, 1mg / culture (0.
5 mg / g of cells).

The molecular weight was confirmed by 15% SDS-polyacrylamide electrophoresis, and heme staining was performed. The results are shown in FIG. From this figure, it can be seen that the recombinant Schwanner ptrifaciens DSP-10 (pRKM1) (FERM P-16752)
Is Shewanella putrefaciens DSP-10 (pRKM1)
(FERM P-16750) and heme protein is expressed which is not present in, it was consistent with the molecular weight of the wild strain sulfate reducing bacteria cytochrome c _3.

Using this sample, the N-terminal 15 residue amino acid
No acid sequence was converted to Shimadzu PPSQ-10 protein sequencer
Analysis revealed that there was only one alanine residue at the end.
Except for the extra one,
Tochrome c_Three And matched. Also, freeze-dry this sample
After that, three times replacement with heavy water is performed,¹H-NMR
A measurement was made. The result is shown in FIG. Obvious from the figure
Like, cytochrome c _Three The standard spectrum of the wild strain
Sulfate reducing bacteria cytochrome c_Three Exactly matches the spectrum of
Was.

On the basis of the above results, recombinant Schwanner ptrifaciens DSP-10 (pRKM1) (FERM P-16
752) The purified cytochrome c ₃ sample was derived from the wild-type sulfate-reducing bacterium cytochrome c ₃ gene, and was confirmed to be a holoprotein bound to c-type heme.

[0037]

As described in the foregoing, according to the present invention it is possible to mass-produced easily and efficiently sulfate reducing bacteria cytochrome c _3, using a cytochrome c ₃ as the material of the rectifying element and molecular element Very useful for: Also,
The cytochrome c ₃ of the present invention is widely used as a research material for the electron transfer mechanism of proteins. However, the present invention allows substitution of amino acids constituting cytochrome c ₃ and provides a new perspective for studying the mechanism. Furthermore, it greatly contributes to the development of the industrial and research fields, for example, by enabling the recovery of useful substances by bacterial leaching and the spread of environmental purification by treating heavy metals.

[0038]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 873 Sequence type: Nucleic acid (Nucleic acid) Number of chains: Double-stranded Topology: Linear Sequence type: Genomic DNA Origin organism name: Deslfovibrio vulgaris) Strain name: Miyazaki F IAM2604 Characteristic of sequence Symbol representing characteristic: -35 signal Location: 187 ... 193 Method for determining characteristic: S Symbol representing characteristic: -10 signal Location: 210 ... 215 Characteristic determination method: S Characteristic symbol: sig peptide Location: 264 ... 332 Characteristic determination method: E Characteristic symbol: CDS Location: 333 ... 652 Characteristic determination method: E Sequence: GACGTCCAAT GGCGCAGGAA TCCGCAGAAA TCCGTCCTGT GGCCAGTACC CCGCCACATT 60 TGCCGCCAAT GCGCATTCTA ATGGACATCC GCGCCACCTT GTGCGGCACG GCACACGCGG 120 CGGCTTCGGA ATGAGGCAAC GTTTACCCCT AACCCACCAG AGTTCGCCGC ATTTTCCCGG 180 CCCGTCTTGA CACCGCACTACCGCG TTGGGAAATC CTTAACATGC 240 CTTGTGAAGG AGGTATTTCT GCGATGAAAA AGATGTTCCT CACCGGTGTG CTCGCGCTGG 300 CCGTTGCCAT CGCCATGCCC GCCCTTGCCG CCGCCCCGAA GGCCCCTGCC GACGGTCTGA 360 AGATGGACAA GACCAAGCAG CCCGTGGTCT TCAACCACTC GACCCACAAG GCCGTGAAGT 420 GTGGCGACTG TCACCACCCG GTCAACGGCA AGGAAGACTA CCAGAAGTGC GCCACCGCCG 480 GTTGCCACGA CAACATGGAC AAGAAGGACA AGTCCGCCAA GGGCTACTAC CACGCCATGC 540 ATGACAAGGG CACAAAGTTC AAGAGCTGCG TGGGCTGCCA CCTTGAAACC GCGGGCGCCG 600 ACGCCGCCAA GAAAAAGGAA CTGACGGGCT GCAAGGGCTC CAAGTGCCAT AGCTAGGCAC 660 TGCCGTCATT TTCCCGGCTT TCTGGGCCGT CCCTTCGGGG CGCCTTTGCG TTTCCGGGCC 720 GAATCAGGCC GGACCGAACT GGACCGGATC GAACTGGACC GGACCGAGCG GGGCCGGGCC 780 GTACCGAGTC GGGCCGCGCC GGACATGC GG GG GG

[Brief description of the drawings]

FIG. 1 is an explanatory diagram showing the outline of construction of an expression vector pRKM1.

Figure 2 is a wild strain sulfate reducing bacteria cytochrome c ₃ recombinant Schwarz Ne interrupt Reference tumefaciens DSP-10 (pRKM
1) 15% SDS- showing expression in (FERM P-16752)
It is a figure which shows the heme staining result by polyacrylamide electrophoresis.

FIG. 3 shows that wild-type sulfate reducing bacteria cytochrome c ₃
Recombinant Schwanner ptrifaciens DSP-10 (pRK
M1) shows an oxidized ¹ H-NMR spectrum showing agreement with cytochrome c ₃ expressed in (FERM P-16752).

Continued on the front page (51) Int.Cl. ⁷ Identification FI Theme Court II (Reference) (C12N 1/21 C12R 1:01) (C12P 21/02 C12R 1:01) (72) Inventor Alexandr Cappin United States of America, E-Belview Drive Number 8 276, California 91101, Pasadena, 276 (72) Inventor Hideo Akutsu 1928-4 Kawashima-cho, Asahi-ku, Yokohama, Kanagawa Prefecture (72) Inventor Kiyoshi Ozawa 89, Higashi-Kyogaoka, Asahi-ku, Yokohama, Kanagawa Prefecture -14 F term (reference) 4B024 AA03 AA11 BA80 CA04 DA05 EA04 GA11 HA01 4B064 AG35 CA02 CA19 CC24 4B065 AA01X AA01Y AB01 BA02 CA24 CA44 CA46

Claims (6)

[Claims] 1. A method comprising producing a cytochrome c ₃ by culturing a recombinant aerobic bacterium holding a plasmid containing a cytochrome c ₃ gene of a sulfate-reducing bacterium in the cells, and expressing the gene. the method of producing cytochrome c _3, characterized in that. 2. The aerobic bacterium according to claim 1, wherein the cytochrome c ₃ gene is incorporated into Schwanner aptrifaciens (NCIMB accession number NCIMB 400). the method of production of cytochrome c _3. 3. The method according to claim 1, wherein the sulfate-reducing bacterium is desulfovibrio vulgaris Miyazaki F (IAM deposit number IAM260).
The method of producing cytochrome c ₃ according to claim 1, wherein from 4). 4. The method according to claim 1, wherein the sulfate-reducing bacterium is desulfovibrio vulgaris hildenboro (ATCC accession number ATCC2).
The method of producing cytochrome c ₃ according to claim 1, characterized in that the 9579). 5. A include cytochrome c ₃ gene sulfate reducing bacteria, aerobic bacteria an expression plasmid, characterized in that to express the cytochrome c ₃ gene in the aerobic bacteria. Is 6. aerobic bacteria retaining the cytochrome c ₃ gene sulfate reducing bacteria, recombinant Schwarz ne interrupt Reference tumefaciens (FERM Deposit No. FERM P-1675
2).