JPH07143882A - Decomposition of ethylene chloride and recombinant plasmid and transformant for decomposition - Google Patents

Abstract

PURPOSE:To decompose ethylene chloride at high decomposition rate in high efficiency by using a transformant transformed with a recombination plasmid having high decomposition capacity of ethylene chloride such as trichloroethylene and tetrachloroethylene. CONSTITUTION:The recombination plasmid to be used in this process contains (A) a hybrid gene containing, as an active site, a gene fragment todC1bphA2A3A 4 produced by linking a partial gene todC1 of a structural gene coding a toluene- decomposition enzyme originated from Pseudomonas putida F1 to a partial gene bphA2A3A4 of a structural gene coding a biphenyl decomposition enzyme originated from Pseudomonas pseudoalcaligenes, (B) a drug-resistant gene and (C) a gene replicable in a Gramnegative bacteria. The plasmid is introduced into a host bacterium cell and the obtained transformant is used for the decomposition of ethylene chloride.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a recombinant plasmid for degrading ethylene chloride capable of imparting a host cell with a capability of degrading ethylene chloride such as trichlorethylene and tetrachloroethylene, and a transformant for degrading ethylene chloride carrying this recombinant plasmid. And a method for decomposing ethylene chloride using this transformant.

[0002]

2. Description of the Related Art Ethylene chloride such as trichloroethylene and tetrachloroethylene is widely used as a solvent and the like, but it is poisonous and is not easily decomposed by natural microorganisms, so that it causes soil and groundwater pollution in various places.

As a method for treating ethylene chloride, there is a method of removing it by adsorbing it onto activated carbon, but this method merely recovers ethylene chloride and cannot detoxify it, which is an essential solution. Not not. In addition, trichloroethylene and tetrachloroethylene can be decomposed by anaerobic treatment, but there is a problem in that highly toxic intermediate products such as dichloroethylene and vinyl chloride are produced.

The use of an enzyme or a microorganism that decomposes ethylene chloride can be an effective measure for solving such problems. Conventionally, as an enzyme that decomposes trichlorethylene, methane monooxygenase possessed by methane-utilizing bacteria,
Toluene monooxygenase possessed by toluene-assimilating bacteria,
Toluene dioxygenase and ammonia monooxygenase possessed by nitrifying bacteria are known. However, there is a problem in that the trichlorethylene degrading ability of a natural bacterium having the above-described trichlorethylene degrading ability is low, and trichlorethylene cannot be efficiently degraded.

On the other hand, in Japanese Patent Publication No. 2-503866, Pseudomonas mendocina KR-1 ( Pseudomo
It has been described that a toluene monooxygenase gene (toluene oxygenase decomposes trichloroethylene) derived from Nas mendocina KR-1) is introduced into a host bacterium, and this transformant is used to decompose trichlorethylene. However, this method uses toluene monooxygenase alone, and thus has a low trichlorethylene decomposing ability.

[0006] Also, Applied and Envir
operational Microbiology, Vo
l. 15, No. 12, 1989, p. 3162-31
66 (hereinafter referred to as Reference 1), the Pseudomonas putida F1-derived toluene dioxygenase gene ( t
odC1C2BA ) is introduced into Escherichia coli, and the transformant is used to decompose trichlorethylene. However, this transformant also has a low trichlorethylene degrading ability and cannot efficiently decompose trichlorethylene.

Jourlan of Bacter
iology, Vol. 175, No. 16,199
3, p. 5224-5232 (hereinafter referred to as Document 2)
Is a toluenedio derived from Pseudomonas putida F1.
Part of the gene for xygenase (todC1) And shoe
From Domonas Pseudo-Alcaligenes KF707
Some of the genes with biphenyl degradability (bphA2A3
A4BC) And a hybrid gene (includingtodC1
(F1)bphA2A3A4BC(KF707))
The plasmid pJHF10 having
Lid genetodC1bphA2A3A4BCIs Tor
It is disclosed to have en resolution. But this
In Reference 2, the plasmid pJHF10 is chlorinated by the host bacterium.
It does not describe imparting ethylene resolution.

[0008]

SUMMARY OF THE INVENTION An object of the present invention is to provide a recombinant plasmid for degrading ethylene chloride, which has a high ethylene chloride decomposing ability. Another object of the present invention is to provide a transformant for degrading ethylene chloride, which is obtained by introducing the above recombinant plasmid into a host bacterium. Another object of the present invention is to propose an efficient method for degrading ethylene chloride using the above transformant.

[0009]

The present invention provides the following recombinant plasmid for degrading ethylene chloride, a transformant, and a method for degrading ethylene chloride using the same. (1) Pseudomonas Putida F1 ( Pseudom
onas putida F1) -derived structural gene partial gene todC1 encoding a toluene-degrading enzyme,
Pseudomonas shoe de alkali monocytogenes (Pseud
gene fragment to which a partial gene bphA2A3A4 of a structural gene encoding a biphenyl-degrading enzyme derived from Omonaspuseudoalcaligenes ) is ligated to
A recombinant plasmid for degrading ethylene chloride, which contains a hybrid gene containing dC1bphA2A3A4 as an active site, a drug resistance gene, and a gene replicable in Gram-negative bacteria, and is replicable in Gram-negative bacteria. (2) A transformant for degrading ethylene chloride, which is obtained by introducing the recombinant plasmid according to (1) above into a host bacterium. (3) A method for decomposing ethylene chloride, which comprises decomposing ethylene chloride using the transformant described in (2) above.

The ethylene chloride to be decomposed in the present invention includes mono-, di-, tri- and / or tetrachloroethylene, but trichloroethylene and / or tetrachloroethylene is particularly suitable as a decomposition target.

The hybrid gene used in the present invention is
At least including gene gene fragment TodC1bphA2A3A4, other todC1bphA2A3A4, bphB or C is bonded thereto todC1bp
hA2A3A4B or todC1bphA2A3A4
Those indicated by BC are also included. todC1 portion structural gene (hereinafter, toluene referred degradation genes) encoding toluene degradation enzyme derived from Pseudomonas putida F1 is todC1 part of TodC1C2BA. Also bphA2A3A4 , bphA2A3A4B or b
The phA2A3A4BC portion is a structural gene encoding a biphenyl-degrading enzyme derived from Pseudomonas pseudoalcaligenes (hereinafter referred to as a biphenyl-degrading gene) bp
It is a part or the whole of hA1A2A3A4BC .

The source of the toluene-degrading gene todC1C2BA is Pseudomonas isolated from nature by David T. Gibson et al., University of Iowa, USA.
You can raise the Puchida F1. In addition to this, a strain in which the toluene-degrading gene derived from Pseudomonas putida F1 is incorporated can be used as a source of todC1C2BA .

Examples of such strains include, for example, FERM P-13966 in the Institute of Biotechnology, Institute of Biotechnology, Industrial Technology Institute.
Deposited as Ethericia coli KWI-10
( Escherichia coli KWI-10) and the like. This strain is a vector plasmid pU
C119 (ATCC No. 37461, ATCCCa
talogue of Recombinant DN
A Materials 2nd edition, 1
Since it has the plasmid pJHF3051 in which the todC1C2BA gene is integrated in (991), this plasmid pJHF3051 can also be used as a source.
To obtain the gene todC1C2BA from these strains, it can be excised with a restriction enzyme.

The gene todC1C2BA is recognized as a gene encoding toluene dioxygenase which oxidizes toluene to cis-toluene dihydrodiol. Protein and gene group todC1C involved in the decomposition process of toluene into cis-toluenedihydrodiol
The relationship with 2BA is shown in the following reaction formula [1].

[0015]

[Chemical 1] [In the reaction formula, ISP is iron-sulfur protein (iron-
sulfur protein), FDX represents ferredoxin, Rtase represents a reductase, red represents a reduced form, ox represents an oxidant, and TOL represents toluene. ]

As shown in the above reaction formula [1],
Electron transport system from the NADH reductase, ferredoxin and iron - is composed of three proteins of sulfur protein (component constituting the toluene dioxygenase), each of which genes TodC1C2, heritage <br/> gene todB and gene Coded by todA .

[0017] Pseudomonas putida F1-derived to
The restriction enzyme map of the dC1C2BA gene is shown below.

[Chemical 2] Regarding todC1C2BA , reference 1 and Th described above.
e Journalof Biological Ch
emissary, Vol. 264, No. 264. 25, 198
9, p. 14940-14946 and the like.

In order to obtain the partial gene todC1 which constitutes the todC1C2BA gene, it is possible to excise it from the above-mentioned source with a restriction enzyme, and to use the plasmid pJH.
It can also be chemically synthesized by polymer chain reaction (PCR) using F3051 as a template DNA.

Biphenyl degradation gene bphA1A2A3
As a source of A4BC , the inventor Furukawa separated from the natural world, and the FERM P-82
Pseudomonas pseudo-Alcaligenes KF707 ( Pseudomonas
Pseudoalcaligenes KF707) can be illustrated.

The mycological properties of this strain are as follows. [Mycological properties] Gram; Negative bacillus; 0.7 x 1.5 to 2.0 µm polar flagella; Single growth at 41 ° C; Positive Optimal growth temperature; 35 ° C Assimilability; Glucose, succinic acid, lactic acid , Pyruvate Based on the above bacteriological properties, a search based on Virgiase and Manual of Cystima Take of Bacteriology, 9th edition revealed that Pseudomonas pseudoalcalenes ( Pseudomonas pseudoalcohol
alignes ). The details of this strain and the gene bphA1A2A3A4BC are described in JP-A-61-282085.

The same strain as Pseudomonas pseudoalcaligenes KF707 has been deposited at the Institute of Biotechnology, and has been deposited as FERM P-13965. For the sake of convenience, this strain is referred to as Pseudomonas pseudoalcaligenes KWI-11 ( Pseudomon
as pseudoalcaligenes KWI-
11).

Gene bphA1A2A3 from this strain
For excision of A4BC, the strain was grown overnight in, for example, L medium (10 g of bactryptone , 5 g of yeast extract, 5 g of salt, 1 liter of distilled water), and lysozyme-S was added.
It can be performed by preparing lysed chromosomal DNA by the DS method and then cleaving the chromosomal DNA (1 μg) with a restriction enzyme Xho I.

The restriction enzyme map of the biphenyl-degrading gene cut out by such a method is shown below. In the restriction map shown below, the gene bphA1A2A3A4B
C is contained.

[Chemical 3]

The gene bphA1A2A3A4 is recognized as a gene encoding a biphenyl dioxygenase which oxidizes biphenyl to dihydrodiol. Proteins and genes involved in the degradation process of biphenyl bph
The relation with A1A2A3A4BC is shown in the following reaction formula [4].

[0025]

[Chemical 4] [In the reaction formula, ISP is iron-sulfur protein (iron-
Sulfur protein), FDX is ferredoxin (ferredoxin), FRase is ferredoxin reductase (ferredoxin re).
duct), red is a reductant, ox is an oxidant, B
PH represents biphenyl. ]

As shown in the above reaction formula [4],
The electron transfer system from NADH in the first stage reaction of biphenyl decomposition is ferredoxin reductase, ferredoxin, and three kinds of proteins of iron-sulfur protein (constituent components constituting biphenyl dioxygenase).
The genes are bphA4 and bp , respectively.
It is encoded by hA3 and the gene bphA1A2 . The enzyme that catalyzes the second-stage reaction is the gene bp
hB , the enzyme that catalyzes the third step reaction is the gene bphC
Coded by. These genes form the operon.

The hybrid gene includes a partial gene todC1 of the toluene degradation gene, a gene fragment todC1bphA2A3A4 that combines the partial gene bphA2A3A4 of the biphenyl degradation gene, which contains as an active site at least ethylene dichloride resolution is expressed, Still another gene fragment bphB or bp
hC may be added. Examples of such a hybrid gene include todC1bphA2A3A4 ,
Examples include, but are not limited to, todC1bphA2A3A4BC .

The above hybrid gene can be obtained, for example, as follows. Plasmid pHSG396 having a restriction enzyme Xho I cleavage site (Takara Shuzo Co., Ltd.)
( Manufactured by K.K. ) was digested with Xho I, and the DNA fragment ( bphA1A2A3A4 ) represented by the above restriction map [3] was cut into
( Including BC ) is ligated with T ₄ ligase to construct the recombinant plasmid pKTF10. Then from pUC118
Plasmid pUC118Δ S from which the Sac I site has been removed
Get ac I. This pUC118Δ Sac I is replaced with Xba I
And cut with Hin dIII, Xba from the pKTF10
BphA1A2A3A4B cut out in the I and Hin dIII
The DNA fragment containing C was ligated to pJHF18
To build.

On the other hand, the above-mentioned plasmid pJHF3051
Template D (containing todC1C2BA )
NA and 5'- by polymer chain reaction
Sac I cleavage site on the terminal side and Bgl on the 3'-terminal side
A todC1 fragment having a II cleavage site is synthesized.

[0030] The pJHF18 was removed bphA1 was cleaved with Sac I and Bgl II, which Sac I and Bgl
By ligation with the todC1 fragment cleaved with II, the hybrid gene todC1bphA2A3
The plasmid pJHF10 carrying A4BC is obtained.
Further, this plasmid was cleaved with Ppu MI to obtain bphB.
By removing C , the hybrid gene todC
The plasmid having the 1bphA2A3A4 pJHF10
1 is obtained.

The restriction enzyme map of the hybrid gene todC1bphA2A3A4 thus obtained is shown below.

[Chemical 5] [In the map, orf3 is the open reading frame 3 (open reading frame 3),
Ap ^r represents an ampicillin resistance gene. ]

The recombinant plasmid for degrading ethylene chloride of the present invention is todC which is a gene fragment ligated as described above.
It has a hybrid gene containing 1bphA2A3A4 as the active site. todC1bphA2A3A4
The protein formed from the biphenyldioxidase gene bphA1 has an iron-sulfur protein subunit (ISP subunit) formed from the gene tod.
It is an enzyme substituted with an ISP subunit formed from C1 . Since this protein (enzyme) has a high ethylene chloride decomposing ability, particularly a high decomposing ability for trichloroethylene or tetrachloroethylene, the recombinant plasmid of the present invention was introduced into a host bacterium, and the resulting transformant was used to produce ethylene chloride, Particularly, trichloroethylene or tetrachloroethylene can be efficiently decomposed.

The recombinant plasmid of the present invention can be constructed by ligating the following DNA fragments with ligase. 1) Hybrid gene containing the gene fragment todC1bphA2A3A4 as an active site 2) Drug resistance gene (marker) 3) Gene replicable in Gram-negative bacteria

As the drug resistance gene, ampicillin resistance gene (Am ^r ), chloramphenicol resistance gene, kanamycin resistance gene, tetracycline resistance gene and the like can be adopted. These drug resistance genes are
KT240 (ATCC No. 37258), pUC1
18 (ATCC No. 37462, all of which are commercially available from ATCC under these catalog numbers), pAC
It can be isolated from plasmids such as YC184, pKK223-3 and pTrc99A.

As the gene capable of replicating in Gram-negative bacterium, a gene necessary for autonomous replication in Gram-negative bacterium and a broad host range replication region containing a replication initiation site can be used. The broad host range replication region is the gene region required for the recombinant plasmid to grow independently and be stably maintained in Gram-negative bacteria.
Alternatively, a plasmid RSF1010-derived one or the like can be used, but the present invention is not limited thereto. RSF101
0 contains a gene and a replication initiation site required for autonomous replication in Gram-negative bacteria and is concentrated in a DNA region of about 5.8 kb. This region can be separated from not only RSF1010 but also plasmids such as pKT240 and pMMB22 using a nuclease such as a restriction enzyme.

The recombinant plasmid of the present invention preferably expresses ethylene chloride decomposing ability by a promoter such as lac promoter. Introduction of the recombinant plasmid into a host bacterium can be carried out by electroporation, calcium and rubidium treatment, transfer using a helper plasmid, Hanahan's method (“Mo
second edition of "Lecular Cloning", Cold
Spring Harbar Lab. It can be carried out very easily by a conventional method such as publishing. Gram-negative bacteria, such as Escherichia coli, whose recombinant plasmid is stably maintained are used as the host bacteria.

The transformant for degrading ethylene chloride of the present invention is a transformant into which the above recombinant plasmid has been introduced, and it is possible to efficiently degrade ethylene chloride, particularly trichloroethylene or tetrachloroethylene, using this transformant. it can. The decomposition of ethylene chloride can be carried out by a method such as aerobically culturing the transformant in a medium containing ethylene chloride, and complete dehalogenation can be carried out.

The culture of the transformant for degrading ethylene chloride is carried out by
It is carried out under conditions suitable for the growth of host bacteria, but as a carbon source and a nitrogen source, peptone, tryptone, yeast extract, etc.
Using sodium chloride, potassium chloride, etc. as inorganic salts,
PH of medium 5 to 8.5, preferably 6 to 7, temperature 15 to
It is desirable to culture aerobically at 40 ° C, preferably around 35 ° C.

[0039]

EXAMPLES Next, examples of the present invention will be described. The plasmids and microorganisms used in the examples are as follows. 1) pUC118 It is replicable in Gram-negative bacteria and has ampicillin resistance gene, lac promoter and multiple cloning site. The size is 3.2 kb. ATCC
No. ATCC (Amer with catalog number 37462
ican Type Culture Collect
Ion).

2) pJHF3051 Pseudomonas putida F1-derived toluene-degrading gene todC1C2BA is pUC119 (ATCC N
o. A 6.5 kbp plasmid integrated into 37461). This plasmid is Escherichia coli KWI-10.
Has been introduced to.

3) pHSG396 This is a pUC type cloning vector plasmid having a chloramphenicol resistance gene. It has a cloning site for Xho I, which is an excision restriction enzyme of the biphenyl degrading gene. No. 1 from Takara Shuzo Co., Ltd. 33
96 Catalog Number (Bio Technology
commercially available under catalog 1993).

4) The plasmid pJHF3051 in which Escherichia coli KWI-10 toluene-degrading gene todC1C2BA was incorporated was transformed into Escherichia coli JM.
A recombinant bacterium introduced in 109, which has been deposited under the accession number of the microorganism.

5) Pseudomonas pseudoalcaligenes KWI-11 A strain carrying the biphenyl-degrading gene bphA1A2A3A4BC as chromosomal DNA, deposited under the accession number of the microorganism. 6) Ethericia coli JM109 ATCC No. Commercially available from ATCC under the catalog number 53323 (previous catalog).

Example 1 A recombinant plasmid pJHF101 for degrading ethylene chloride was constructed by the procedure shown in FIGS. First, as shown in FIG. 1, Pseudomonas pseudo-Alcaligenes KWI-
This DNA was prepared by digesting 11 chromosomes with the restriction enzyme Xho I.
The fragment was cut with Xho I, plasmid pHSG396.
And was introduced into Escherichia coli JM109 to obtain a transformant. This transformant was sprayed with 2,3-dihydroxybiphenyl, and the yellowed strain was cultured.
In this strain, the DNA fragment shown in the above restriction enzyme map [3] containing the gene bphA1A2A3A4BC is p
The plasmid pKTF10 integrated in HSG396 is retained.

The above recombinant plasmid pKTF10 was added to Xba
It was cut with I and Hin dIII, bphA1A2A3A4B
It was cut out fragment of Xba I~ Hin dIII, including C. Then, pUC118 was cleaved with Sac I, treated with T ₄ polymerase and blunt-ended, and self-ligated to obtain a plasmid pU having the Sac I site deleted.
C118Δ Sac I was created. This pUC118Δ S
The multi-cloning site of ac I was replaced with Xba I and Hin
cut with dIII, ligated a fragment of Xba I through Hin dIII containing the BphA1A2A3A4BC, generating plasmid PJHF18.

On the other hand, as shown in FIG. 2, pJHF3051 ( todC1C2BA gene is integrated in this plasmid) retained in Escherichia coli KWI-10 was used as a template DNA, and 5'-TCTCTC GAGC was used.
TC GAAAAGTGAGAAGACAATGA-3 '(underlined indicates Sac I cleavage site) and 3'-CTTCCGCTGTGCGACTTAG TCTAGA ACGAA-
Using 5 ′ (underlined Bgl II cleavage site) two primers, GeneAmp PCR Kit (manufactured by Takara Shuzo Co., Ltd., trade name) was used to attach Sac I,
Gene tod having a Bgl II cleavage site at the other end
C1 was synthesized by PCR (polymer chain reaction). The number of times of amplification of DNA at this time was 20 times, and the conditions were such that denaturation of DNA was 95 ° C. for 30 seconds, primer annealing was 55 ° C. for 30 seconds, and primer extension was 72 ° C. for 1 minute.

PC containing todC1 obtained as described above
The R product was cleaved with Sac I and Bgl II. On the other hand, p
The JHF18 was cut with Sac I and Bgl II bphA1
After removing the to prepare a plasmid pJHF10 by ligating the Sac I through Bgl II fragment. By this operation, bphA1 of pJHF18 is replaced with todC1 . This plasmid is bphC1bphA2
It contains a hybrid gene fragment of A3A4BC .

Next, as shown in FIG. 3, p obtained above is obtained.
The JHF10 was cut with Ppu MI, cut out bphB
The fragment containing C was removed, and p was obtained by self-ligation.
JHF101 was created. This plasmid is todC1
It contains a hybrid gene fragment of bphA1A2A3A4 .

Example 2 The plasmid pJHF10 or pJHF101 obtained in Example 1 was introduced into Escherichia coli JM109 by the method of Hanahan to obtain a transformant. This E. coli is called E. coli JM109 (pJHF10) or E. coli JM109 (pJHF101).

Using these transformants, a trichlorethylene decomposition test was conducted as follows. E. coli JM109 (pJHF10) or E. coli JM109 (pJHF101) were cultured in LB medium at 37 ° C. and the absorbance at 600 nm (hereinafter A ₆₀₀
When it reached 1.0 to 2.0, the cells were collected by centrifugation and the cells were suspended in the following inorganic medium so that the A ₆₀₀ was 2.0.

Inorganic medium: Na ₂ HPO ₄ + KH ₂ PO ₄ (1M, pH6.8) 40ml Hunter's vitamin-free mineral base * 1 20ml (NH ₄ ) ₂ SO ₄ 1g distilled water 840ml * 1 Hunter's vitamin-free mineral base : nitrilotriacetic acid _{10.0 g MgSO 4 14.45 g CaCl 2} · 2H 2 O 3.335g (NH 4) 6 Mo 7 O 24 · 4H 2 O 9.25 mg FeSO 4 · 7H 2 O 99 mg Metals "44" * 2 50 ml distilled Water total 1000 ml * 2 Metals “44” (Metals “44”): Ethylenediaminetetraacetic acid 250.0 mg ZnSO ₄ / 7H ₂ O 109 5.0 mg (250 mg Zn) FeSO ₄ 7H ₂ O 500.0 mg (100 mg Fe) MnSO _{4 · H 2 O 154.0mg (50mg Mn} ) CuSO 4 · 5H 2 O 39.2mg (10mg Cu) Co (NO 3) 2 · 6H 2 O 24.8mg (5mg Co) Na 2 B 4 O 7 · 10H 2 O 17.7mg (2mg B) Add a few drops of sulfuric acid to prevent precipitation 100ml distilled water

10 ml of this bacterial solution was placed in a 125 ml vial bottle manufactured by GL Sciences, 10 mg / l of trichloroethylene (concentration when all dissolved in the solution) was added, and a Teflon-coated butyl rubber stopper was added.
Sealed with an aluminum cap. 30 this vial
The culture was carried out with shaking at 200 ° C. at 200 ° C., and 100 μl of the gas phase was periodically sampled with a gas tight syringe to perform a trichlorethylene decomposition test. As a control, Escherichia coli KWI-10 (Ethericia coli JM10
A recombinant bacterium in which the plasmid pJHF3051 was introduced into 9) was used. The results are shown in Fig. 4.

From the results shown in FIG. 4, all of the transformants of the Examples decomposed trichlorethylene at a decomposition rate about 3 times that of the control, and the hybrid gene is very effective for decomposition of trichlorethylene. I understand.

[0054]

The recombinant plasmid for degrading ethylene chloride of the present invention is todC derived from Pseudomonas putida F1.
Gene fragment in which 1 gene and bphA2A3A4 gene derived from Pseudomonas pseudoalcaligenes are linked
Since it contains todC1bphA2A3A4 as an active site that expresses ethylene chloride decomposing ability , an enzyme having a high ethylene chloride decomposing ability can be biosynthesized.

Since the transformant for degrading ethylene chloride of the present invention is transformed with the above recombinant plasmid, it has high ethylene chloride decomposing ability.

Since the method for decomposing ethylene chloride of the present invention uses the above transformant, ethylene chloride can be easily and efficiently decomposed at a high decomposition rate.

[0057]

[Sequence list]

SEQ ID NO: 1 Sequence Length: 32 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence TCTCTCGAGC TCGAAAAGTG AGAAGACAAT GA 32

SEQ ID NO: 2 Sequence Length: 30 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence AAGCAAGATC TGATTCAGCG TGTCGCCTTC 30

[Brief description of drawings]

1 is a schematic diagram showing the steps of constructing a plasmid pJHF101 in Example 1. FIG.

FIG. 2 is a schematic diagram showing steps of constructing a plasmid pJHF101 in Example 1.

FIG. 3 is a schematic diagram showing the steps of constructing the plasmid pJHF101 in Example 1.

FIG. 4 is a graph showing the results of Example 2.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location // (C12N 15/09 ZNA C12R 1:40) (C12N 15/09 ZNA C12R 1:38) ( C12N 1/21 C12R 1:19) (C12P 1/00 C12R 1:19) C12R 1:40) (C12N 15/00 ZNA A C12R 1:38)

Claims (3)

[Claims] 1. A Pseudomonas putida F1 ( Pse
partial gene todC of the structural gene encoding the toluene-degrading enzyme derived from udomonasputida F1)
1 and Pseudomonas Pseudo Alcaligenes ( Ps
eudomonas pseudoalcaligen
es ) derived from a structural gene encoding biphenyl-degrading enzyme partial gene bphA2A3A4 linked gene fragment todC1bphA2A3A4 as a hybrid gene containing as an active site, drug resistance gene, and a gene capable of replication in Gram-negative bacteria, Gram-negative A recombinant plasmid for degrading ethylene chloride that can replicate in bacteria. 2. A transformant for degrading ethylene chloride, which comprises the recombinant plasmid according to claim 1 introduced into a host bacterium. 3. A method for decomposing ethylene chloride, which comprises decomposing ethylene chloride using the transformant according to claim 2.