JP2783660B2 - Iron oxidase gene isolated from Thiobacillus ferrooxidans. - Google Patents

Description

The present invention relates to an iron oxidase gene derived from Thiobacillus ferrooxidans which is an absolute autotrophic bacterium,
More specifically, by transforming Thiobacillus ferrooxidans using this enzyme gene, which is important in obtaining the energy required for the growth and growth of this bacterium, the above bacterial cells widely used in bacterial leaching and other mining industries are transformed. In addition to creating cells with increased iron oxidizing ability, it can be produced using other microorganisms such as Escherichia coli,
The resulting enzyme can be used for sensors and the like.

[Background Art] Bacteria of the genus Thiobacillus are commonly found near sulfide ore deposits, and bacterial leaching of ores (Bacterial leachin) is considered.
g). Although there are few cases of operation in Japan, it is carried out in the United States, Canada, Australia, Chile, South Africa, etc., and it is said that 18-25% of the total copper production in the United States is due to this bacterial leaching . It is also a property of this bacterium. Treatment of mine wastewater using the ability to oxidize ferrous iron (Fe ²⁺ ) to ferric iron (Fe ³⁺ ), removal of iron in hydrometallurgy, treatment of reducing gas such as H ₂ S gas, etc. Plants are operating in various parts of Japan.

Wastewater treatment involves the treatment of ferrous iron contained in mine wastewater. When ferrous iron is treated, it must be neutralized with slaked lime, which is a strong alkali. In addition, the volume of sediment generated is large, and handling properties such as sedimentation are poor. In the case of using iron, inexpensive calcium carbonate fine powder, which is an inexpensive weak alkali, can be used, and other handling characteristics are significantly improved, so that a significant cost reduction can be produced.

Further, when used in the deironing process of hydrometallurgy, heating is not required, and a large cost reduction has been achieved, such as easy separation from the recovered material in the next step. Furthermore, when used for H ₂ S gas treatment, it can be safely treated at normal temperature and normal pressure, and has great advantages such as lower running cost as compared with other methods. [Applicatio
n of Iron−Oxidizing Bacteria to Extractive Mettal
lurgy; Metallurgical Reviel of MMIJ, vol.3, No.1, Apri
l (1986)].

As described above, it can be said that the genus Thiobacillus is an industrially very useful cell.

By the way, among the bacteria belonging to the genus Thiobacillus, particularly useful in the above-mentioned applications is Thiobacillus ferrooxidans, which is an autotrophic bacterium (Autotrophic bacterium).
Bacteria), and its growth rate is extremely slow as compared with aerobic heterotrophic bacteria (Heterotrophic Bacteria).

By the way, the doubling time of E. coli is about 20 minutes,
It takes about 6 to 10 hours for Thiobacillus ferrooxidans. In addition, the cell yield of each medium was about 6 g,
Due to the fact that it is about 0.1 g, the size of the reaction vessel for industrial use of the genus Thiobacillus greatly affects the production cost of facilities and equipment, and also the processing cost. There have been some restrictions.
To solve this problem, attempts have been made to obtain bacteria having a high growth rate by random screening, as in other microbial industries, but there is a limit in the nature of the microorganism itself.

For this reason, the present inventors have been developing a host-vector system and the like with the aim of improving bacteria by genetic manipulation (Japanese Patent Application No. 1-218425). The present invention relates to a novel vector using a mercury resistance gene derived from Thiobacillus as a selection marker, and its ultimate purpose is to carry a gene that enhances another ability and transform the vector. One method of this capacity enhancement is to clone and utilize genes of enzymes and coenzymes derived from Thiobacillus.

To the best of our knowledge to date, the gene cloned from Thiobacillus ferrooxidans with the aim of enhancing the growth ability and the oxidation rate is the RuBPCase gene (Japanese Patent Application No. 2-139300) by the present applicant. Only iron oxidase.

Thiobacillus ferrooxidans is ferrous (Fe ²⁺ )
The energy required for growth is obtained by oxidizing iron into ferric iron (Fe ³⁺ ). The main mechanism of oxidation of ferrous iron is currently described as follows.

First, the electrons extracted from ferrous iron are passed to the iron oxidase, then to the cytochrome c-552, and finally to the enzyme via the cytochrome oxidase to generate water, and the energy generated at this time is Converts to chemical energy such as ATP and NADH.

In the oxidation of 1 mol of ferrous iron, the generated energy is 8.5 Kcal / mo
le, the free energy efficiency is 3%, but the complete oxidation of glucose is 688 Kcal / mole, the alcohol fermentation of glucose is 57 Kcal / mole, and the free energy efficiency of respiration and fermentation is about 40% and 30%, respectively. It is understood from these comparisons that the energy required for the growth of Thiobacillus ferrooxidans is low and the growth rate is low.

[Problems to be Solved by the Invention] As described above, it is difficult to improve the operation only by simply using the conventional Thiobacillus ferrooxidans, and the growth ability and the oxidizing ability of Thiobacillus ferrooxidans themselves by some means. However, the present inventors have previously developed a novel vector using a mercury resistance gene derived from Thiobacillus ferrooxidans as a selection marker, and then a DNA encoding a RuBPCase gene that is important in carbonic acid assimilation. A fragment was obtained.

Subsequent research suggests that by improving the electron transport system of Thiobacillus ferrooxidans, the oxidizing ability of the bacterium could be enhanced. As a gene that enhances the oxidizing ability of a genus bacterium, a DNA fragment encoding an iron oxidase has been desired.

[Means for Solving the Problems] The present inventors have conducted intensive studies in order to solve such problems, and as a result, using the Thiobacillus ferrooxidans Fel strain (Microtechnical Research Bacteria No. 11479), The present invention has been able to provide the present invention by extracting and purifying DNA and cloning it into Escherichia coli.

That is, the present invention provides a DNA fragment encoding a thiobacillus iron oxidase comprising a base sequence and / or an amino acid sequence as shown in FIG. 4, and a DNA fragment encoding a thiobacillus iron oxidase.
Provision of a novel plasmid characterized by integration into a plasmid derived from E. coli, and genomic DN from Thiobacillus
A first step of extracting and purifying A; a second step of cutting this genomic DNA and a plasmid derived from E. coli with the same restriction enzyme, and then ligating each; the plasmid obtained in the second step Into E.coli,
Third step of screening a plasmid containing an iron oxidase gene from a plate culture colony by a colony hybridization method; after digesting the plasmid obtained in the third step with a restriction enzyme, a DNA fragment containing an iron oxidase gene is recovered. , E.coli
A method for constructing a novel plasmid and a method for producing an iron oxidase, comprising: a fourth step of ligating the plasmid to an expression vector plasmid.

[Action] In the method of the present invention, Thiobacillus ferrooxidans obtains energy required for growth by oxidizing ferrous iron (Fe ²⁺ ) to ferric iron (Fe ³⁺ ).
The main mechanism of iron oxidation is currently described as follows.

First, electrons extracted from ferrous iron are passed to iron oxidase, then to cytochrome c-552, and finally to oxygen via cytochrome oxidase to produce water, and the energy generated at this time is converted to ATP and ATP. Convert to chemical energy such as NADH.

For this reason, by cloning the gene for iron oxidase, the first enzyme in this electron transfer system, ligating it to the vector of iron oxidizing bacteria and returning it to the same bacterial body, the production capacity of the enzyme increases. It is thought that together with this, the overall iron oxidizing ability can be increased.
Cloning and expression were performed in the following process.

1) Determination of part of the amino acid sequence of iron oxidase extracted from Thiobacillus ferrooxidans Fe1 strain 2) Preparation of two types of synthetic DNA probes based on the sequence obtained in 1) 3) Thiobacillus ferrooxidans Fe1 strain Genome D
Extraction and purification of NA 4) Cloning of DNA fragment obtained in 3) into E. coli plasmid 5) Selection of E. coli transformed with E. coli plasmid cloned in 4) using probe synthesized in 2) 6) 5) Extraction of plasmid DNA from Escherichia coli selected in step 2 and cutting out of the iron oxidase gene portion of this plasmid 7) Determination of base sequence of DNA fragment obtained in 5) and determination of amino acid sequence deduced therefrom 8) 5) Construction of Escherichia coli Plasmid for Expression of Thiobacillus ferrooxidans Iron Oxidase Gene by Ligation of the Obtained Thiobacillus ferrooxidans Iron Oxidase Gene Portion and Escherichia coli Expression Vector 9) This is the production of iron oxidase derived from Thiobacillus ferrooxidans.

In this case, the Thiobacillus ferrooxidans Fe1 strain 1) was isolated by Shioda et al. From the former Matsuo mine (J. Gen. Appl. Microbiol vol. 28. p331-342 “Bacteri”).
al pyrite oxidation I ", The effect of pure and mixe
d cultures of Thiobacillus ferrooxidans and Thioba
cillus thiooxidans on release of iron), the present inventors extracted iron oxidase from this Thiobacillus ferrooxidans Fel strain (FEMS Microbiology Letters 50).
(1988) p169-172 "Fe (II) -oxidizing enzyme puri
fied from Thiobacillus ferrooxidans), the amino acid sequence on the N-terminal side was determined, and two types of synthetic probes were prepared based on the knowledge.

Next, after extracting and purifying the genomic DNA of Thiobacillus ferrooxidans Fe1 strain (Example 1), the restriction enzyme E
The fragment was cut with coRI, and the fragment was ligated to the EcoRI site of a linker cloning site of Escherichia coli plasmid pUC18 to prepare a hybrid DNA.

Next, Escherichia coli was transformed using the obtained hybrid DNA, and the Escherichia coli was cultured on a plate medium. When colony hybridization was performed on about 5,000 obtained colonies using two types of synthetic probes, a plurality of positive colonies having the same size plasmid were selected, and Escherichia coli DH5α which formed these colonies was selected.
When the plasmid was examined, it was classified into two types in which the EcoR I site of the genomic DNA contained in the iron oxidase of Thiobacillus ferrooxidans Fe1 strain was inserted in the opposite direction to the EcoRI site of pUC18, These new plasmids were named pTIO11, pTIO12. The physical map of the EcoRI fragment containing the Thiobacillus ferrooxidans iron oxidase gene cloned into these plasmids is shown in FIG.

Then, use the restriction enzyme sites in pTIO 11, pTIO 12,
Deletion plasmids were prepared, and DNA sequencing was performed by the dideoxy method using these plasmids as templates. Based on the results, the genomic DNA base sequence of the region encoding the iron oxidase of Thiobacillus ferrooxidans was determined. The base sequence and amino acid sequence of these genomic DNAs are shown in FIG.

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

[Example] This example is an exemplification, and does not limit the present invention, and the following operations may be modified or modified without departing from the present invention as apparent to those skilled in the art. Changes are possible.

The methods for restriction enzymes, medium, agarose electrophoresis, hybridization, and transformation used in this example are as follows.

Enzyme source: Restriction enzymes Kpn I, Nae I, Ava I, Cla I, EcoR I, Ec
oR V, Pst I, Sal I, Eco47 III, BamH I, Ssp I, Hpa
I, Sph I, Xho I, Stu I, Sma I (Takara Shuzo) and T4 DNA
The ligase used was commercially available according to the attached instructions.

Medium: 9K medium of A. Silverman et al. (1) Inorganic salt medium (NH ₄ ) ₂ SO ₄ 3 g K ₂ HPO ₄ 0.5 g MgSO ₄ .7H ₂ O 0.5 g KCl 0.1 g Ca (NO ₃ ) ₂ ) 0.01 g 700 ml Adjust the pH to 5.5 with sulfuric acid.

(2) adjusting the pH to 1.4 with ferrous solution FeSO ₄ · 7H ₂ O 44.22g total 300ml sulfuric acid.

(1) and (2) were separately separated in an autoclave at 120 ° C for 1 hour.
Sterilize for 0 minutes, mix after cooling and adjust.

B.LB medium (pH 7.5) Tryptone 1% Yeast extract 0.5% NaCl 1% C.M9 medium Na ₂ HPO ₄ · H ₂ O 6g KH ₂ PO ₄ 3g NaCl 0.5g NH ₄ Cl 1g Total amount 1 (dw) Adjust the pH to 7.4 and autoclave 1M MgSO ₄ 2ml Autoclave 20% glucose 10ml Filter sterilized 1M CaCl ₂ 0.1ml Autoclave Mix each separately Agarose Gel electrophoresis: TAE buffer (40mM Tris, 5mM sodium acetate, 1mM ED
A gel prepared by dissolving 0.7% agarose powder in TA; pH 7.8) was used. After electrophoresis, the gel was immersed in 0.5 μg / ml ethidium bromide solution to stain DNA, and an ultraviolet lamp (3
02 nm) and photographed the fluorescence. Polaroid for shooting
An MP-4 land camera was used.

Hybridization: Southern hybridization is performed using a nylon membrane filter [Amersham Hybond N
-N)]. In the Southern method, DNA was subjected to alkaline blotting on a filter. Hybridization using synthetic probes can be performed using the Current Protocol in Mole
cular Biology [Edited by Frederick M. Ausubel et al
(1987), Published by Greene Publishing Associates
and Wiley Interscienec].

Colony hybridization was performed using the method of Granstein and DSHogness, Pr.
oc.Natl.Acad.Sci.USA 72: 3961-3965, 1975),
Nylon membrane filter (Amersham Chemical, BN
This was performed using NG82 or Paul, Biodyne A).

Transformation method of Escherichia coli Escherichia coli DH5α, used as a host for transformation of CSR603, was prepared by the method of Hanahan (D. Hanahan, J. Mol. Biol. 166: 557-
580,1983) and made it a competent cell. The transformation efficiency of this competent cell is usually 1 × 10 ⁷ μg cells / μg p
UC18 DNA.

Example 1 Determination of Amino Acid Sequence of Iron Oxidase Extracted from Thiobacillus ferrooxidans Using Thiobacillus ferrooxidans Fe1 strain,
The extraction means developed by the present inventors, [FEMS Microbiology
Letters 50 (1988) p169-172.Fe (II) -oxidizing en
The N-terminal amino acid sequence was determined by subjecting the iron oxidase extracted by zyme purified from Thiobacillus ferrooxidans to an amino acid gas phase sequencer (Applied Biosystems, Inc., 470A), and the results are shown in FIG.

Example 2 Based on the amino acid sequence shown in FIG. 1, a DNA synthesizer (Milligen / Bio Research CYCLONE ^™ DNA
SYNTHESIZER) to prepare two types of synthetic probes. The results are shown in FIG.

Example 3 Genomic DAN from Thiobacillus ferrooxidans
Collection of Thiobacillus ferrooxidans Fe1 strain into 250 ml of 9K medium of Silverman et al., Culture at 30 ° C. for 2 days, centrifugation, collection of low pH washing solution (0.16M MgSO ₄ .7)
2 times with 9K inorganic salt medium containing H ₂ O; adjusted to pH 1.9 with sulfuric acid)
The plate was washed once with a high pH washing solution (25 mM phosphate buffer containing 0.3 M sucrose and 10 mM EDTA; pH 8.0), and then once with a 50 mM phosphate buffer (pH 7.4).

Next, 4 ml of a solution consisting of 2.5 mg / ml lysozyme, 0.05 M glucose, 25 mM Tris-HCl (pH 8.0) and 10 mM EDTA was added, and the mixture was allowed to stand at 0 ° C. for 10 minutes.

Further, 500 µ of 0.25 M EDTA was added, and the mixture was allowed to stand at 0 ° C for 10 minutes. Then, 500 µ of 10% SDS was added to lyse the cells.
50μ of 20mg / ml proteinase K
K) was added and incubated at 37 ° C. for 2 hours to degrade the protein. An equal volume of a phenol: chloroform (1: 1) mixture was added to the solution, and the protein was removed three times. The nucleic acid portion was precipitated with isopropanol, centrifuged, and dried. For further purification, density gradient centrifugation using cesium chloride (20 ° C., 55,000 rpm, 16 hours) and dialysis (one day and night) were performed to obtain purified genomic DNA.

[Example 4] Southern hybridization between a genomic DNA of Thiobacillus ferrooxidans and a synthetic probe synthesized from an amino acid sequence of an iron oxidase derived from Thiobacillus ferrooxidans Purification of Thiobacillus ferrooxidans Fe1 strain obtained in Example 3 Genomic DNA was converted to Kpn I, Nae I, Ava I, Cla
I, EcoR I, EcoR V, Pst I, Sal I, EOC47 III, BamH
Cleavage was performed with restriction enzymes consisting of I, SspI, HpaI, SphI, XhoI, StuI, and SmaI, and 2 μg thereof was subjected to agarose electrophoresis.

Procedures for subsequent Southern hybridization: DNA depurination reaction on gel, denaturation reaction, nylon membrane filter (Amersham
Blotting on High Bond-N) was performed according to the protocol of Amersham.

Next, prehybridization (6 × SSC, 5 × Den
hardts solution, 0.05% sodium pyrophosphate, 100μg
/ ml boiled salmon sperm DNA, 0.5% sodium dodecyl s
After that, hybridization (6 × SSC, 1 × Denhardts solution, 100 μg / ml boiled) was performed with two kinds of DNA probes labeled with ³² P prepared by the method described below.
salmon sperm DNA, 0.05% sodium pyrophosphate).

The synthetic probe was labeled with [ ³² P] radiation as follows by cination. 2.5-250 pmol synthetic probe, 10 × kinase buffer (0.5 Tris · Cl (pH7.6) 0.1MM
gCl ₂ 50 mM dithiothreitol 1 mM spermidine 1 mM EDTA
Incubated 50μCi [γ- ³² P] ATP.25-50U T4 polynucleotide kinase scan mixture of 37 ° C. for 30 minutes, unreacted Sephadex G-50 mini-column [.gamma. ³²
[P] ATP was removed.

The genomic DNA of the EcoRI fragment of the Thiobacillus ferrooxidans Fe1 strain and the probe strongly hybridize at around 4 kb, and it is considered that a fragment of this size encodes the iron oxidase gene.

[Example 5] Cloning of iron oxidase gene contained in genomic DNA of Thiobacillus ferrooxidans Fe1 strain Genomic DNA of 1 µg of Thiobacillus ferrooxidans Fe1 strain obtained in Example 3 was subjected to restriction enzyme EcoRI (Takara Shuzo).
(The excised fragment is referred to as a thiobacillus iron oxidase fragment), and the DNA of E. coli plasmid pUC18 (physical map is shown in FIG. 5) similarly digested with EcoRI.
2 μg was ligated with T4 DNA ligase.

Next, the resulting ligated DNA mixture was taken up into DH5α cells, and then mixed with ampicillin 50 μg / ml, 5-bromo-4-chloro-3-indolyl-β-D-galactoside (Xgal) 40 μg / ml, isopropyl Plate medium of LB medium containing 1 mM -β-D-thiogalactoside (IPTG) (1.5% agar)
The mixture was applied to the above nylon membrane filter and cultured at 37 ° C for one day.

Then ³² P adjusted as described in Example 4.
Colony hybridization was carried out using the synthetic probe labeled with, and a plurality of colonies having the same size plasmid were selected. In this case, the plasmid of E. coli DH5α that formed these colonies was p
EcoR of genomic DAN containing the iron oxidase gene of Thiobacillus ferrooxidans Fe1 strain against EcoRI site of UC18
It was found that the I fragment was two types of novel plasmids inserted in the reverse direction, and these were named pTIO11 and pTIO12 and shown in FIG.

Example 6 Determination of the base sequence of the DNA encoding the iron oxidase of Thiobacillus ferrooxidans Fe1 strain and the amino acid sequence of the iron oxidase The novel plasmids of pTIO11 and pTIO12 obtained in Example 5 were each fragmented Cleavage was performed at the restriction enzyme site inside to create a deletion plasmid.

Subsequently, these plasmids were alkali-denatured (Hattle and Sakaki, 1986) and sequenced by the dideoxynucleotide chain termination method (Sanger method) as a plasmid template. In this case, the sequences were 7-DEAZAS Sequencing kit (Takara Shuzo), Sequanase ^™ kit (Toyobo) and [α- ³² P] dCTP.
(NEN or Amersham drug: 400 Ci / mmol).

Primer M1 (15Mer, 5 ') which hybridizes with 16 bp upstream of Hind III and 8 bp upstream of EcoRI of pUC18 and pUC19.
-AGTCACGACGTTGTA-3 ') and primer RV (17mer, 5'
-CAGGAAACAGCTATGAC-3 ') was manufactured by Takara Shuzo.

Next, sequence analysis of the obtained plasmid was performed by the following means. That is, the nucleotide sequence and the amino acid sequence were obtained by the Genetic informatio of SDC-GENETYX.
When analyzed using the n Processing Program (Software development), as shown in Fig. 4, Thiobacillus
The entire nucleotide sequence of the DNA fragment encoding the iron oxidase of ferrooxidans and the entire amino acid sequence of the iron oxidase could be determined.

Example 7 Construction of Escherichia coli Plasmid for Expression of the Iron Oxidase Gene of Thiobacillus ferrooxidans From the 4 kb fragment of the novel plasmid of pTIO11 cloned in Example 5, about 300 as shown in FIG.
The bp Hind III-Nae I fragment was recovered and
A plasmid inserted into the Hind III-Sma I restriction enzyme site of 223-3 (Pharmacia) was prepared and named pTIO110 as shown in FIG.

Similarly, about 200 bp Sph I-Ec as shown in FIG.
The oRV fragment was recovered and this fragment was
-3, a synthetic DNA linker having an EcoR I site and a Sph I site on one side was ligated to this fragment (this fragment is an iron oxidase structural gene). It lacks a part of glycine, serine and methosine at the N-terminal side of this, and is therefore synthesized inside the synthetic DNA linker so that the base sequence encoding glycine, serine and methosine can be supplemented when ligated. The plasmid was inserted into the EcoR I-Sma I restriction enzyme site of pKK223-3 (manufactured by Pharmacia), and this was inserted into pTIO as shown in FIG.
Named 100.

[Example 8] Expression of Thiobacillus ferrooxidans iron oxidase in Escherichia coli The maxicell method and the method of Sancar et al. (A. Sancar et al., J. Bacteriol. 137: 692-693,197) were used as plasmid-producing proteins.
According to 9), the following analysis was conducted.

The new plasmids pTIO 11, pTIO 12 obtained in Example 5
And the new plasmids pTIO 110, pTIO 10 obtained in Example 7
0 and pKK223-3 were used to transform CSR603. Next, the transformed CSR603 was cultured aerobically at 37 ° C. (9 M medium containing 1% casamino acid and 0.1 μg / ml thiamine), and then irradiated with ultraviolet light (50 J / m ² ) for 45 seconds, and then the cells were cultured.
Treated with 150 μg / ml D-cycloserine.

Thereafter, the protein produced by the gene encoded by the plasmid was labeled with [ ³² S] methionine (NEN 1,000 Ci / mmol). These electrophoresis were then carried out according to the method of Laemmli [U.
K. Laemmli, Nature (London) 227: 680-685, 1970], and sodium salicylate was used for detection of ³⁵ S labeling peptide. In this case, pUC1 as a control
Escherichia coli CSR 603 containing 8 was similarly treated.

As described above, the plasmid pTIO 1 was obtained using the maximal method.
The proteins produced by the genes encoded by 1, pTIO 12, pTIO 110 and pTIO 100 were detected.

[Effects of the Invention] As described above, the present invention encodes an iron oxidase, which is the first enzyme in the electron transfer system that oxidizes ferrous iron (Fe ²⁺ ) of Thiobacillus ferrooxidans.

By using this gene or a vector incorporating the improved version of this gene, thiobacillus ferrooxidans can be transformed to enhance the oxidizing ability of the bacterium, and the bacterium can be produced using genus Thiobacillus or Escherichia coli. Iron oxidase can be applied to enzyme sensors and the like.

[Brief description of the drawings]

FIG. 1 is an amino acid sequence diagram of iron oxidase analyzed by an amino acid sequencer. FIG. 2 is a nucleotide sequence diagram of a probe synthesized based on FIG. FIG. 3 is a physical map of a structural gene. FIG. 4 is a sequence diagram showing the nucleotide sequence containing the iron oxidase gene and the amino acid sequence of the iron oxidase. FIG. 5 is a physical map of Escherichia coli plasmid pUC18. FIGS. 6 (a) and 6 (b) show the new plasmids pTIO11 and PTIO1.
2 is a physical map. FIG. 7 is a physical map of the new plasmid pTIO110, and FIG. 8 is a physical map of the new plasmid pTIO100.

Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12N 9/02 C12R 1:19) (72) Inventor Takeo Yamanaka 1-3-3 Echunakajima, Koto-ku, Tokyo 16-710 Echunakajima Housing (72) Inventor Yoshihiro Fukumori 3-24 Fujigaoka, Fujisawa-shi, Kanagawa RA-33 (72) Inventor Yoshihiro Yano 2-35-4, Kamiikedai, Ota-ku, Tokyo (72) Inventor Juichi Shiratori 162-1-1, Kawaguchicho, Hachioji-shi, Tokyo Ko Senuma Po 203 (72) Inventor Chihiro Inoue 12-2-17, Iijima Midorigaoka-cho, Akita-shi, Akita (72) Inventor Toshiyuki Takeshima 155-1 Tobukicho, Hachioji-shi, Tokyo 2nd ear Aoryo (56) Reference FEMS Microbiology Letters 50 (1988) P.M. 169 -172 (58) Fields investigated (Int. Cl. ⁶ , DB name) C12N 15/52-15/62 C12N 9/02-9/12 WPI (DIALOG) BIOSIS (DIALOG) GenBank / EMBL / DDBJ (G (ENETYX)

Claims (3)

(57) [Claims] 1. A DNA fragment encoding an iron oxidase of Thiobacillus ferrooxidans comprising the following base sequence and / or amino acid sequence: 2. The DAN fragment according to claim 1, which encodes an iron oxidase of Thiobacillus ferrooxidans.
A novel plasmid, which is incorporated into a plasmid of origin. 3. A first step of extracting and purifying genomic DNA from the genus Thiobacillus; a second step of cutting this genomic DNA and a plasmid derived from E. coli with the same restriction enzyme, and then ligating them. A third step of introducing the plasmid obtained in the second step into E. coli and screening a plasmid containing an iron oxidase gene from a plate culture colony by a colony hybridization method; 4. A method for constructing a novel plasmid, comprising: recovering the DNA fragment of claim 1 containing an iron oxidase gene after digestion with a restriction enzyme, and ligating the DNA fragment to an E. coli expression vector plasmid. .