KR20090123174A - Composition for removing parathion comprising ophb gene or protein - Google Patents

Abstract

PURPOSE: A composition for removing pesticide containing ophB gene or its expressed protein is provided to use in bioremediation of soil polluted with parathion. CONSTITUTION: A composition for removing pesticide contains ophB gene having sequence which encodes amino acid of the sequence number 5 or its expressed protein. The ophB gene is isolated from Burkholderia sp. JBA3 having organic phosphate group pesticide dissolving ability. The organic phosphate group pesticide is parathion, EPN (o-ethyl O-p-nitrophenyl phenylphosphonothioate), fenitrothion, or methyl parathion.

Description

{Composition for removing parathion comprising ophB gene or protein}

The present invention relates to a pesticide removal composition comprising an ophB gene or an expression protein thereof, and more particularly to an ophB gene having a base sequence encoding an amino acid sequence of SEQ ID NO: 5 or a pesticide removal composition comprising the expression protein thereof. It is about.

Currently, the most widely used pesticides belong to the organophosphorus group [17]. Organophosphate insecticides inhibit the general activity of acetylcholine esterases, causing accumulation of acetylcholine at synapses and eventually convulsions, paralysis and death in insects and mammals. [14]. Among the organophosphate compounds, parathion (O, O-diethyl-O-4-nitrophenyl phosphorothioate; parathion) is one of the strongest toxic substances registered with the Environmental Protection Agency [5]. In Korea, a huge amount of parathion is produced and used for rice farming and horticulture [11]. Parathion residues that have spread to the environment through agriculture and nonpoint-source discharge can be harmful to wildlife. Thus, detoxification of parathions is important for land and aquatic environments. Since hydrolysis of parathion phosphoester bonds significantly reduces parathion toxicity, bacterial organophosphorus hydrolase (OPH) genes are attracting attention for bioremediation [2, 4].

One of the isolates identified as Burkholderia species was observed to have no sequence similarity to the parathion hydrolysis gene of most bacterial strains that hydrolyze previously reported organophosphates. In the present invention, we cloned the parathion-degrading soil bacterium and cloned its parathionase gene and successfully expressed it in E. coil strain.

Accordingly, the present inventors have diligently studied to overcome the problems of the prior art, by separating and cloning the parathion hydrolysis gene from soil bacteria using parathion as a nutrient source, and producing a transformant using the cloned gene. Confirmed that the parathion can be hydrolyzed, the present invention was completed.

Therefore, a main object of the present invention is to provide a pesticide removal composition comprising an ophB gene or an expression protein thereof that can be used for biopurification of soil contaminated with organophosphate pesticides.

Another object of the present invention is to provide a transformed cell comprising a recombinant vector inserting the gene which can be used for biopurification of soil contaminated with organophosphate pesticides.

Still another object of the present invention is to provide a method for biological purification of soil comprising treating the transformed cells or proteins having the nucleotide sequence of SEQ ID NO: 5 to soil containing organophosphate pesticides.

According to one aspect of the invention, the present invention provides a composition for removing a pesticide comprising an ophB gene having an nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 5 or an expression protein thereof.

In the present invention, the ophB gene is Burkholderia sp. Which is a microorganism having the resolution of organophosphate pesticides first isolated and identified by the present inventors. The gene is isolated from the JBA3 strain, and refers to a gene encoding the amino acid sequence of ORF2 found in the essential region of the activity among the genes (ORF1, ORF2, ORF3) having the hydrolytic activity of the organophosphate compound.

Genes of enzymes that hydrolyze organophosphate compounds have been isolated from several bacteria. Of these, the cloned opd genes [12, 15] in the Philippines and the United States showed 88% similarity to opdA genes [5] isolated in Australia. In the present invention, the isolated ophB gene (cloning the parathion hydrolysis gene and calling it ophB . See Example 3) did not show similarity with the hydrolysis gene of bacteria previously reported to hydrolyze the parathion [6, 7, 12, 15].

In the present invention, ORF1 in the gene having the hydrolytic activity of the organophosphate compound showed a 79% similarity with the transposae (GenBank Accession No. EAM31871) of Burkholderia vietnamiensis G4 IS4. This indicates that the ophB gene may be part of a transposon like other organophosphorus compound hydrolysis genes [8, 16]. JBA3 was observed to have seven plasmids. In the curing experiment using the sodium dodecyl sulfate method [3], the fifth largest plasmid was lost and the strain showed rapid growth in glucose and succinate like wild type, but wild type. In contrast, parathion was not hydrolyzed to paranitrophenol. When each plasmid was extracted from the gel and subjected to PCR using a primer specific for the ophB gene, a PCR product of the desired size was obtained from the fifth largest plasmid. Thus, the ophB gene is located in the plasmid DNA like bacteria that hydrolyze other organophosphate compounds [15, 18]. Several other parathional degradation pathways are known but include all pathway hydrolysis steps [17]. Hydrolysis of organophosphorus compounds reduces the toxicity to mammals by several orders of magnitude, which is very useful for the detoxification of parathions. JBA3 rapidly hydrolyzes parathion to p -nitrophenol and makes it completely mineralized and detoxified. The hydrolysis gene also acts on other organophosphorus insecticides, which can be an additional advantage in bioremediation.

In the composition of the present invention, the ophB gene is preferably inserted into an expression vector. As used herein, the term "expression vector" refers to a plasmid, virus or other medium into which the ophB gene cloned in the present invention can be inserted or introduced. The ophB gene sequence cloned according to the present invention may be operably linked to an expression control sequence, wherein said operably linked gene sequence and expression control sequence comprise a single marker that includes a selection marker and a replication origin. May be included in an expression vector. Said “poerably linked” may be genes and expression control sequences linked in such a way as to enable gene expression when the appropriate molecule is bound to the expression control sequences. The "expression control sequence" refers to a DNA sequence that controls the expression of a polynucleotide sequence operably linked in a particular host cell. Such regulatory sequences include promoters for performing transcription, any operator sequence for controlling transcription, sequences encoding suitable mRNA liposome binding sites, and sequences that control the termination of display and translation. Examples of the plasmids include E. coli-derived plasmids (pBR322, pBR325, pUC118 and pUC119, pET-22b (+)), Bacillus subtilis-derived plasmids (pUB110 and pTP5) and yeast-derived plasmids (YEp13, YEp24 and YCp50). The virus may be an animal virus such as a retrovirus, adenovirus or vaccinia virus, or an insect virus such as baculovirus. A vector suitable for introducing the ophB gene according to the present invention into a host cell may be used, and preferably, a vector designed to facilitate ophB expression induction may be used.

Recombinant vectors comprising the ophB gene of the present invention can be introduced into a host cell using methods known in the art. As a method for introducing the recombinant vector according to the present invention into a host cell, a technique known in the art may be used. For example, calcium chloride (CaCl ₂ ) and a heat shock method, a particle total impact method (particle gun bombardment, silicon carbide whiskers, sonication, electroporation, and precipitation by PEG (polyethylenglycol) may be used.

In a specific embodiment of the present invention, the opUC-transduced pUC19-ORF2 recombinant vector was inserted into p UC19 using EcoR I and calf intestinal alkaline phosphatase (see Example 3).

In the composition of the present invention, the pesticide is preferably an organic phosphate insecticide, more preferably parathion (parathion), o-ethyl Op-nitrophenyl phenylphosphonothioate (EPN), fenitrothion or methyl parathion (methyl parathion) It is characterized by the).

According to another aspect of the present invention, the present invention provides a cell transformed with an expression vector inserted with the ophB gene having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 5.

The cell used for transformation may be any cell that can be transformed with an expression vector into which the ophB gene is inserted, but E. coli is preferable. Here, E. coli may be a known strain, for example, E. coli DH10B, E. coli JM109, E. coli DH5α, E. coli SF130, etc., but the expression conditions of the gene and the amount of protein expressed, etc. Considering this, it is more preferable to use E. coli DH10B.

According to another aspect of the present invention, the present invention provides a bioremediation of soil comprising treating a transformed cell of claim 6 or a protein having an amino acid sequence of SEQ ID NO: 5 to a soil containing an organophosphate pesticide. Provide a method.

In detail, the transformed cells are cultured in a nutrient medium, and then cultured in a soil contaminated with an organic phosphate pesticide, such as parathion, by spraying and mixing the microorganisms to induce a hydrolysis reaction of microorganisms, or sequence. Proteins having an amino acid sequence of No. 5 can be separated and purified in large quantities by methods known in the art and directly applied to contaminated soil.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1. JBA3 Parathion-Degrading Strains Burkholderia  sp. Isolation of JBA3

Completely mixed parathione dissolved in dichloromethane (Cheil Chemicals, Korea) is a final concentration of 100 μg / g soil (100 μg per 1 g soil) of soil samples obtained from rice cultivation all over the country. Treated as possible. The treated soil was incubated with periodic mixing at room temperature. After 5 weeks of parathion treatment, 1 g of soil was treated with a minimum medium containing parathion (100 μg / ml) in grams per liter: KH ₂ PO ₄ (1 · 36), Na ₂ HPO ₄ (1 · 41), ( _{NH 4) 2 SO 4 (0} · 3), MgSO 4 .7H 2 O (0 · 05), CaCl 2 .H 2 O (0 · 0058), trace metal solution (5 ml per liter). The trace metal solution contained (in grams per liter) FeSO 4 .7H 2 O (0 · 55), ZnSO 4 .7H 2 O (0 · 23), MnSO 4 .7H 2 O (0 · 34), Co (NO 3) 2. _{6H 2 O (0 · 075)} , CuSO 4 .5H 2 O (0 · 047), and (NH 4) 6 Mo 7 O 24 .4H 2 O (0 · 025)) [13] tube containing 3 ml Transferred to and incubated with shaking at 28 ° C. After several parathion-degradation cultures in fresh medium, the appropriate dilution was added to PTYG agar medium (peptone-tryptone-yeast extract-glucose; (in grams per liter) peptone (0 · 25), tryptone (0 · 25), yeast extract ( 0 · 5), glucose (0 · 5), magnesium sulfate (0 · 03), calcium chloride (0 · 003)) [1, 13], and each colony having a distinctive form before pure strain isolation. Was tested for parathion digestion in fresh medium.

Among the isolates, JBA3 was able to quickly use parathion as the sole carbon and energy source, and further showed sequence similarity when analyzed by various PCR primer pairs targeting the previously reported parathionase enzyme gene. [6, 12, 15]. 16S rDNA (GenBank Accession No. EF495209) of the isolate was analyzed using the previously reported method [9, 10]. JBA3 was closely related to Burkholderia glathei LMG 14190 (U96935) among type strains with 98% similarity. JBA3 is a gram-negative bacterium of bacillus and catalase / oxidase positive. JBA3 was able to absorb glucose, D-mannose, D-mannitol, N-acetyl-glucosamine, gluconate and malate, and was positive for Voges-Proskauer (VP) test and nitrate reduction. JBA3 produced acids from D-mannitol, inositol, D-sorbitol, L-rhamnose and D-sucrose. Based on 16S rDNA sequence identity, the strains were harvested from Burkholderia sp. Named JBA3.

Example 2. Parathion Degradation

After incubation in PTYG medium, cells were harvested, washed and suspended in minimal medium. Aliquots of suspended cells were inoculated at a final concentration of OD ₆₀₀ = 0.005 in a pair of flasks containing 250 ml of minimal medium containing 433 μM of parathion (Riedel-de Ha? N, Seelze, Germany). . All cultures were incubated with shaking at 28 ℃. It was used to determine the concentration of the nitrophenol (p -nitrophenol) - 1 ml of the culture medium was removed at regular cell growth and parathion and p. Cell growth was measured at an optical density of 600 nm.

For quantification of parathion and p -nitrophenol, 1 ml of acetonitrile was added to 1 ml of the culture solution, mixed thoroughly, and centrifuged. A portion of the supernatant was used to measure at optical densities 270 nm (parathion measurement) and 410 nm ( p -nitrophenol measurement) using a spectrophotometer and reversed phase HPLC (Waters, Milford, USA). The concentrations of parathion and p -nitrophenol were calculated using a standard curve prepared from known concentrations of parathion and p -nitrophenol in the same medium. JBA3 completely hydrolyzed parathion to p -nitrophenol (FIG. 1). Once most of the parathion residues were hydrolyzed, the accumulated p -nitrophenols began to be used as an accessory to cell growth by JBA3. At 67 hours, p -nitrophenol was fully used and cell density began to decrease. These results indicate that JBA 3 first hydrolyzes most of the parathion before using the hydrolysis product of parathion, ie p -nitrophenol, as the carbon and energy source.

Example 3. Cloning of Parathion Hydrolysis Genes

Total DNA of strain JBA3 was extracted using Wizard Genomic DNA Purification Kit (Promega, Madison, USA) and incompletely digested with Sau3A I. Using a QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany), only 2-4 kb DNA fragments were extracted and treated with p UC19 vector (ATCC 37254) treated with BamH I and calf intestinal alkaline phosphatase (Promega, Madison, USA). Inserted. After transforming it into E. coli DH10B, it was smeared into LB medium containing 100 μg / ml of ampicillin (gram per lieter, trytone (10), yeast extract (5), NaCl (5)), and its surface was coated. Covered with parathionized 1PS filter (Whatman, Maidstone, UK). Parathion treatment submerged the 1PS filter in 9 ml acetone with 10 μl of parathion and allowed the acetone to dry completely. After incubation at 37 ° C. for one day, p -nitrophenol yellow colonies were separated and tested for parathion hydrolysis in the minimum parathion medium in the same manner as described above (in which Parathion was added to the minimum medium in Example 1). Two strains showing parathion hydrolysis were selected and PCR was performed using primers M13-F (SEQ ID NO: 2) and M13-R (SEQ ID NO: 3) (Bioneer, Daejon, Korea), and PCR products were completely cleaved using Sau3A I. It was. When electrophoresed after cleavage, the two PCR products showed the same band pattern, and thus, only one strain was selected and the nucleotide sequence of the DNA fragment inserted into the vector was analyzed using primer extension. The sequence was analyzed using ABI3730 sequencer (Applied Biosystems) of the Center for Agricultural Sciences.

The total length of the DNA fragment inserted into the vector was 3,668 bp (Genbank Accession No. EF495210, SEQ ID NO: 1) and contained one complete ORF (ORF2) and two truncated ORFs (ORF1 and ORF3) (see Figure 2). ). ORF2 (1.578 bp, SEQ ID NO: 4) contained the genetic information of a 526 amino acid protein product, and ORF3 (1,288 bp) contained the genetic information of an incomplete protein product of 429 amino acids. Therefore, in the present invention it was named by the ORF2 ophB (organophosphorus hydrolase from Burkholderia sp .; organophosphorous compound hydrolase of Burkholderia).

Example 4. Parathion Hydrolysis Using ORF2 of Parathion Hydrolysis Gene

Only ORF2 was PCR amplified from the 3.7 kb DNA fragment to see if ORF2 alone could hydrolyze the parathion with p -nitrophenol. In this case, BP / f (5'-CTGGAAATCAAGGAAATCCG, SEQ ID NO: 6) and BP / r-4 (5'-TTACTGTTGCAGAGCAGATG, SEQ ID NO: 7) were used. PCR products were cloned using pGEM-T easy vector (Promega, MA, USA), and the extracted plasmid was used as a template, and PCR was performed using primers M13-F and M13-R. The PCR product was inserted into the gel after electrophoresis, extraction and p UC19 after cutting with EcoR I EcoR I and treated with calf intestinal alkaline phosphatase.

In all cases, the transcriptional direction of ORF2 was opposite to the lacZ promoter of p UC19. Burkholderia sp. When JBA3, E. coli DH10B / pUC19-3.7-kb DNA fragment, E. coli DH10B / pUC19-ORF2 were inoculated with a minimum medium containing 75 μM of parathion at OD ₆₀₀ = 0.036 ~ 0.045 and shaken at 28 ° C E. coli DH10B / pUC19-ORF2 completely hydrolyzed parathion to p -nitrophenol (FIG. 3).

Burkholderia sp. JBA3 took about 5 hours to decompose parathion into p -nitrophenol, whereas E. coli DH10B / p UC19-3.7-kb DNA fragment and E. coli DH10B / p UC19-ORF2 took about 16 hours. . The reason for this may be that the expression level of the gene cloned into E. coli DH10B is relatively low or that a complete ORF3 or other unknown enzyme is required.

Example 5 Degradation of Other Insecticides

E. coli DH10B / p UC19-ORF2 was tested for hydrolysis of other organophosphorus insecticides. Organophosphorus insecticides included in the test were EPN ( o -ethyl O -p-nitrophenyl phenylphosphonothioate), fenitrothion ( O , O -dimethyl O -p-nitro-m-tolyl phosphorothioate), methyl parathion (methyl parathion; O , O- dimethyl O- p-nitrophenyl phosphorothioate) was used. Hydrolysis was judged by the appearance of yellow (their hydrolysis products) by the formation of p -nitrophenol or 3-methyl-4-nitrophenol. E. coli DH10B / p UC19-ORF2 hydrolyzed phenythrothione and methylparathion and EPN hydrolyzed at a relatively slow rate.

As described above, according to the present invention Burkholderia sp. JBA3 was found to have no sequence similarity with previously known parathion hydrolysis genes. In addition, we cloned the parathion-degrading soil bacteria and cloned its parathionase gene and successfully expressed them in E. coil strains. Therefore, the composition containing the parathion hydrolysis gene of the present invention or the expression protein thereof, or cells transformed with the recombinant vector into which the gene of the present invention is inserted can be used for bioremediation of parathion-contaminated soil.

[references]

1 shows the use of parathion with the growth of JBA3.

Symbols: ●, cell density; ○, parsione; ▽, p-nitrophenol. Error bars indicate standard deviation.

2 is Burkholderia sp. Restriction map of the 3.7 kb DNA fragment cloned from JBA3 is shown. DNA length was expressed in bp. White arrow is ORF. Black arrows indicate the position of the primers used to PCR ORF2.

3 is Burkholderia sp. Parathion hydrolytic activity of JBA3 (▽), E. coli DH10B / pUC19-3.7-kb DNA fragment (○), E. coli DH10B / pUC19-ORF2 (▼), E. coli DH10B / pUC19 (●). Error bars represent standard deviations.

<110> Seoul National University Industry Foundation <120> Composition for removing parathion comprising ophB gene or          protein <160> 7 <170> KopatentIn 1.71 <210> 1 <211> 3668 <212> DNA <213> Burkholderia sp. JBA3 <400> 1 gatcaggcat gggccagcca caaaagcttt gtgcggaagt ctgacgatga atccgacagc 60 gccgatggtg gtggcaactt caaaggcgag aagcgcagca acgagacgca tgaatccaag 120 acggataccg atgcgaggct ctaccgcaag ggcaacactg ccagcgagtt gcgcttcatg 180 ggtcatacgc tgagcgacaa ccgtcacggc ttgattgcca gcgccgtcgt gaccaccgcc 240 gacggctacg ccgaacgtga agcggccaag gtcatgatca acgatgcccg gcaagccctc 300 ggggatccgg agcgcgagat cacgctgggc gctgacaagg gctacgacgc gcaggaattc 360 atcgacacct gcattgaact gggcgtcacg ccgcatgtcg cgcagaacaa gtcgggccgc 420 aagtcggcgg tgcccgacgc cattgctcag agcgagggct atgcgctgtc gcagcgcaag 480 cgcaagttga tcgagcaagg cttcggctgg gccaagactg tcggtggcat tcggcaggtg 540 atggtgcgtg gactcaagcg cgtcgaccag atgttcgtgc tgaacatggc cgccttcaac 600 ttggttcgca tgcgtacctt gggacaggtc cgtccgctgg gtgcgcaatg aagggaatcg 660 gcggcaaagg gaccgcgaaa aagctggaaa tcaaggaaat ccgacctcga catcccgcat 720 gacgaaaaga tcgatgccaa ctcgtcggac gggctgaacg aggctgcgat tcggaccagt 780 atttcagcag cctgttagcc gcaaggtgtc aggcgaatcc tttcctaaat tcaaaggaaa 840 caacaatgaa gacaagcgtt catctcagtt tgccgctcct tgcggtagtc agtacagtag 900 cgttggttgc gtgcgggggg gacgggggca acccagtcgt agcgccggca cccgagccgt 960 tgaccgttgc caaggcaagt tgcgccactg gaaaccgccc ggaaacgggc ctgcagggcc 1020 aagtggacgc gtccctccgg gcgaacggct tcaatggatt caactgcaac ttagacctcg 1080 tgggtagcta caaaggtgac ggcgccggtg tttcctttgc gtccttcaag gtttatgcgg 1140 gacgcacgtg tacctaccat tcgacctcca cgggtcaccc ggttggcttg cagaacccgg 1200 gcgtgaccgt gctggatatc acggacccgg ccacgcccat ccgaacggca tcacttacct 1260 cgattgcgat gctcgatccg ttggagtcga tgcggctaaa cgggcaacga cagattttgg 1320 tggcgagcaa cggctacgca ggcgtcggcg gtccggaggt ggacgtctac gatttgtcta 1380 gtgactgtcg cactccccag cttctggctt cagcgcccgt cgggaccgga gcggacggtg 1440 gcgtttacac acccaattcg ccattgtcca aggggcatga aggggccatt tcccctgacg 1500 gactgacgta ctacatggga gactttacga ataacgtcta ccgggcaatt gatattacgg 1560 atcctgccaa gccgaagcag atcgccgtat ttgacatgaa gggccttccc ggcaaagcgc 1620 atggtctgtc cgtgagcgca gacggcaacc gggtttacgc agctgcgtat ggactgccta 1680 cattggcgca ggttgccgac ccggccgcgt atcccatcaa cgggtttgtt gtgctcgaca 1740 cttcggaagt acaagcgcgc aagccgaacg cggctatcac gctcatatct acagccttcc 1800 acaaggacgg ttcagcgtca cagcacacct tgcctatgaa ggtcgcgggc aagtcgtatc 1860 tggtgcaggt cgacgagggc ggatcggccg gcgtcctaac tgacgccggg ttcaaggctg 1920 cgtgcgccgc caaactgcca ccgtttccga tggctcgaat ctacgacata caggacgaca 1980 gcgacccgaa gcttgtttct aagctcatgc tggaaacgca cgatcccgcc aactgcgcca 2040 aggtcgctcc ggacgtagtg ggactcggct tatatgctta tggcagtcac ttctgctcgg 2100 tggacaatcg cgagaacgcc acagcacttg cgtgtgccta ttttaattcc ggcattcgcg 2160 tctttgacat tcgggatcct gccaaaccga aggaactcgc gtacttcaat cccgctagcg 2220 gtccgttgtt gccgggatcg gctcacgcga tgttgcagca gtggcgcgag ggggggccgg 2280 acttgtgtgg cgcgcagctc catttcgact ttgatcgcaa acagctagtg tcagcgtgca 2340 tggacaacgg ggttcaagtt cttcagttcg ctccgaatac atggccgttt gcacagagtg 2400 tggcatctgc tctgcaacag taaagcgtca atcttcgatg agtgagcgtc gcgaacctac 2460 ctgtgcccga atggccgtgc tcggggttcg catacttctt ttgggcgccc tgagcgcgcc 2520 gctttcagcg tctgcgcact tcttcgcgca gccgtacaca atgccggtgc cgttccaggt 2580 atacgcacta gcggtggggc tggccttggg actctccttt cttctgatag gcgtctttgc 2640 cgcggttccg acgcttggac caagcgcccc tgaggcagga tgcccggttc ctgcgtcaag 2700 gcttgccaga tgggcttcgg gtgttggccg ggtacttagt gtggcgcttc tagtgctgtg 2760 tattgccaca ggggcagtgg gcacgaagaa tccgttcacc aatttcaata tgtccttctt 2820 ctggatcata tttgtgctgg tgattcctta cgcggtcgcg ctgctgggaa atttttattc 2880 caaagtcaat ccttggctgg cgatcgtcga taccctcgaa aaggtgctcg gagttccttg 2940 cggaaaactt tcttatccaa aagggttagg gcactatccg gcacttttac tctacataat 3000 ttttatctgg ctcgaattat ttggggcttt gacgccgcgt gggctgtcgc tatgccttgt 3060 tgcgtacacc gtcaccaatt tgtgcggtgc atggctggtg ggttctcgag actggttccg 3120 ctattgcgag ttctttggtg tcatgatgca tttttttgca tttctcgcgc ctttaagaag 3180 ggcgaggaat gcgtgtttcg ccaaagaggc ggcaaccgcc gtcggtttca gcctttctgt 3240 ttttgtgata ttcatgcttt cgtcaaccgc tttcgacgga gtgcactcga cgttgccgtt 3300 cgctcatctg tattgggtct gggtgcttcc gctcatgtcc cccttgctag aagcgctagg 3360 cgcaaacacg cgcgggctcg cgacctcgct ctactatgtt tggcagtggg caatgctatt 3420 ggttatgttg gtgttttacg tgcttgtcta taccgcgttc atctggatgg ccaggttcgt 3480 ggcgcgttcg cagcatccaa tccaaatgct catgaatagg ttcgcacttt cgcttattcc 3540 tgttgcattt gtctaccacg tcgcacatta cttcacgttg attttctcgc agggtgccca 3600 actggtgcgc attgtttccg acccgtttgg ctggggttgg aatttgtttg gaacggcgaa 3660 cgttgatc 3668 <210> 2 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> M13-F <400> 2 gtaaaacgac ggccagt 17 <210> 3 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> M13-R <400> 3 caggaaacag ctatgac 17 <210> 4 <211> 1578 <212> DNA <213> Burkholderia sp. JBA3 <400> 4 atgaagacaa gcgttcatct cagtttgccg ctccttgcgg tagtcagtac agtagcgttg 60 gttgcgtgcg ggggggacgg gggcaaccca gtcgtagcgc cggcacccga gccgttgacc 120 gttgccaagg caagttgcgc cactggaaac cgcccggaaa cgggcctgca gggccaagtg 180 gacgcgtccc tccgggcgaa cggcttcaat ggattcaact gcaacttaga cctcgtgggt 240 agctacaaag gtgacggcgc cggtgtttcc tttgcgtcct tcaaggttta tgcgggacgc 300 acgtgtacct accattcgac ctccacgggt cacccggttg gcttgcagaa cccgggcgtg 360 accgtgctgg atatcacgga cccggccacg cccatccgaa cggcatcact tacctcgatt 420 gcgatgctcg atccgttgga gtcgatgcgg ctaaacgggc aacgacagat tttggtggcg 480 agcaacggct acgcaggcgt cggcggtccg gaggtggacg tctacgattt gtctagtgac 540 tgtcgcactc cccagcttct ggcttcagcg cccgtcggga ccggagcgga cggtggcgtt 600 tacacaccca attcgccatt gtccaagggg catgaagggg ccatttcccc tgacggactg 660 acgtactaca tgggagactt tacgaataac gtctaccggg caattgatat tacggatcct 720 gccaagccga agcagatcgc cgtatttgac atgaagggcc ttcccggcaa agcgcatggt 780 ctgtccgtga gcgcagacgg caaccgggtt tacgcagctg cgtatggact gcctacattg 840 gcgcaggttg ccgacccggc cgcgtatccc atcaacgggt ttgttgtgct cgacacttcg 900 gaagtacaag cgcgcaagcc gaacgcggct atcacgctca tatctacagc cttccacaag 960 gacggttcag cgtcacagca caccttgcct atgaaggtcg cgggcaagtc gtatctggtg 1020 caggtcgacg agggcggatc ggccggcgtc ctaactgacg ccgggttcaa ggctgcgtgc 1080 gccgccaaac tgccaccgtt tccgatggct cgaatctacg acatacagga cgacagcgac 1140 ccgaagcttg tttctaagct catgctggaa acgcacgatc ccgccaactg cgccaaggtc 1200 gctccggacg tagtgggact cggcttatat gcttatggca gtcacttctg ctcggtggac 1260 aatcgcgaga acgccacagc acttgcgtgt gcctatttta attccggcat tcgcgtcttt 1320 gacattcggg atcctgccaa accgaaggaa ctcgcgtact tcaatcccgc tagcggtccg 1380 ttgttgccgg gatcggctca cgcgatgttg cagcagtggc gcgagggggg gccggacttg 1440 tgtggcgcgc agctccattt cgactttgat cgcaaacagc tagtgtcagc gtgcatggac 1500 aacggggttc aagttcttca gttcgctccg aatacatggc cgtttgcaca gagtgtggca 1560 tctgctctgc aacagtaa 1578 <210> 5 <211> 525 <212> PRT <213> Burkholderia sp. JBA3 <400> 5 Met Lys Thr Ser Val His Leu Ser Leu Pro Leu Leu Ala Val Val Ser   1 5 10 15 Thr Val Ala Leu Val Ala Cys Gly Gly Asp Gly Gly Asn Pro Val Val              20 25 30 Ala Pro Ala Pro Glu Pro Leu Thr Val Ala Lys Ala Ser Cys Ala Thr          35 40 45 Gly Asn Arg Pro Glu Thr Gly Leu Gln Gly Gln Val Asp Ala Ser Leu      50 55 60 Arg Ala Asn Gly Phe Asn Gly Phe Asn Cys Asn Leu Asp Leu Val Gly  65 70 75 80 Ser Tyr Lys Gly Asp Gly Ala Gly Val Ser Phe Ala Ser Phe Lys Val                  85 90 95 Tyr Ala Gly Arg Thr Cys Thr Tyr His Ser Thr Ser Thr Gly His Pro             100 105 110 Val Gly Leu Gln Asn Pro Gly Val Thr Val Leu Asp Ile Thr Asp Pro         115 120 125 Ala Thr Pro Ile Arg Thr Ala Ser Leu Thr Ser Ile Ala Met Leu Asp     130 135 140 Pro Leu Glu Ser Met Arg Leu Asn Gly Gln Arg Gln Ile Leu Val Ala 145 150 155 160 Ser Asn Gly Tyr Ala Gly Val Gly Gly Pro Glu Val Asp Val Tyr Asp                 165 170 175 Leu Ser Ser Asp Cys Arg Thr Pro Gln Leu Leu Ala Ser Ala Pro Val             180 185 190 Gly Thr Gly Ala Asp Gly Gly Val Tyr Thr Pro Asn Ser Pro Leu Ser         195 200 205 Lys Gly His Glu Gly Ala Ile Ser Pro Asp Gly Leu Thr Tyr Tyr Met     210 215 220 Gly Asp Phe Thr Asn Asn Val Tyr Arg Ala Ile Asp Ile Thr Asp Pro 225 230 235 240 Ala Lys Pro Lys Gln Ile Ala Val Phe Asp Met Lys Gly Leu Pro Gly                 245 250 255 Lys Ala His Gly Leu Ser Val Ser Ala Asp Gly Asn Arg Val Tyr Ala             260 265 270 Ala Ala Tyr Gly Leu Pro Thr Leu Ala Gln Val Ala Asp Pro Ala Ala         275 280 285 Tyr Pro Ile Asn Gly Phe Val Val Leu Asp Thr Ser Glu Val Gln Ala     290 295 300 Arg Lys Pro Asn Ala Ala Ile Thr Leu Ile Ser Thr Ala Phe His Lys 305 310 315 320 Asp Gly Ser Ala Ser Gln His Thr Leu Pro Met Lys Val Ala Gly Lys                 325 330 335 Ser Tyr Leu Val Gln Val Asp Glu Gly Gly Ser Ala Gly Val Leu Thr             340 345 350 Asp Ala Gly Phe Lys Ala Ala Cys Ala Ala Lys Leu Pro Pro Phe Pro         355 360 365 Met Ala Arg Ile Tyr Asp Ile Gln Asp Asp Ser Asp Pro Lys Leu Val     370 375 380 Ser Lys Leu Met Leu Glu Thr His Asp Pro Ala Asn Cys Ala Lys Val 385 390 395 400 Ala Pro Asp Val Val Gly Leu Gly Leu Tyr Ala Tyr Gly Ser His Phe                 405 410 415 Cys Ser Val Asp Asn Arg Glu Asn Ala Thr Ala Leu Ala Cys Ala Tyr             420 425 430 Phe Asn Ser Gly Ile Arg Val Phe Asp Ile Arg Asp Pro Ala Lys Pro         435 440 445 Lys Glu Leu Ala Tyr Phe Asn Pro Ala Ser Gly Pro Leu Leu Pro Gly     450 455 460 Ser Ala His Ala Met Leu Gln Gln Trp Arg Glu Gly Gly Pro Asp Leu 465 470 475 480 Cys Gly Ala Gln Leu His Phe Asp Phe Asp Arg Lys Gln Leu Val Ser                 485 490 495 Ala Cys Met Asp Asn Gly Val Gln Val Leu Gln Phe Ala Pro Asn Thr             500 505 510 Trp Pro Phe Ala Gln Ser Val Ala Ser Ala Leu Gln Gln         515 520 525 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BP / f <400> 6 ctggaaatca aggaaatccg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BP / r-4 <400> 7 ttactgttgc agagcagatg 20  

Claims (6)

A pesticide removal composition comprising an ophB gene having an nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 5 or an expression protein thereof. The composition of claim 1, wherein the ophB gene is inserted into an expression vector. The composition of claim 1, wherein the pesticide is an organophosphate insecticide. The composition of claim 3, wherein the organophosphate insecticide is parathion, o-ethyl O-p-nitrophenyl phenylphosphonothioate (EPN), fenitrothion or methyl parathion. A cell transformed with an expression vector into which an ophB gene having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 5 is inserted. A method for bioremediation of soil comprising treating a transformed cell of claim 5 or a protein having an amino acid sequence of SEQ ID NO: 5 to a soil containing an organophosphate pesticide.

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