JP2007295803A - Gene cluster involving oxidative decomposition passage of polychlorinated biphenyls/polycyclic aromatic hydrocarbons and method for utilizing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a gene cluster and a group of enzymes involving oxidative decomposition passage of polychlorinated biphenyls (PCB)/polycyclic aromatic hydrocarbons and to provide a utilization method in environmental purification field thereof. <P>SOLUTION: A method for decomposing PCB comprises acquiring a gene encoding α and β subunits of a new aromatic ring dioxygenase and having a specific amino acid sequence, a gene encoding a dihydrodiol dehydrogenase and further a gene encoding a dioxygenase cleaving an aromatic ring from a bacterium of the genus Paenibacillus and using an enzymatic protein derived from these genes. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a group of genes involved in the oxidative degradation pathway of polychlorinated biphenyls / polycyclic aromatic hydrocarbons, and a method for using the same, and more particularly, polychlorinated biphenyls / polycyclic aromatic hydrocarbons decomposition activity. Aromatic ring dioxygenase gene, aromatic ring dihydrodiol / dehydrogenase gene, genes comprising an aromatic ring cleaved dioxygenase gene, enzymes encoded by these genes, and environmental purification thereof It relates to usage in the field.

  With recent industrial development, various harmful chemical substances are released and leaked into the environment, and the destruction of the global environment has been progressing, causing serious social problems. Among them, aromatics such as polychlorinated biphenyls (hereinafter sometimes referred to as “PCB”) and polycyclic aromatic hydrocarbons (hereinafter sometimes referred to as “PAH”) classified as organochlorine compounds. Family-based hazardous chemicals are compounds that are chemically very stable in living organisms and the environment, and are also lipophilic compounds. For this reason, when it spreads from the soil to the groundwater system and the ocean, it accumulates in the adipose tissue in the living organisms that inhabit, and is concentrated through the food chain. Bring. In recent studies, toxicity as an endocrine disrupting substance has been reported, and there is a concern that even a very small amount of pollution may adversely affect the environment. For this reason, it was indispensable to quickly establish a purification technique for decomposing and detoxifying harmful chemical substances such as PCB and PAH.

  Heat treatment technology, chemical treatment technology, biological treatment technology, etc. have been developed as technologies for decomposing and purifying harmful chemical substances such as PCB and PAH. Among them, the biological treatment technology is a technology for decomposing and detoxifying harmful chemical substances by utilizing the substance conversion ability of bacteria. And since it can reduce the concentration of harmful chemical substances such as PCB and PAH without producing harmful decomposition products at the time of decomposition, and can be processed under advantageous conditions in terms of cost and treatment equipment, Research is underway for its practical application. In particular, PCB, which is an organochlorine compound, is difficult to be decomposed by microorganisms in the natural world. Therefore, it is necessary to actively use useful microorganisms that can exhibit high decomposing ability for PCBs and enzymes produced by the useful microorganisms.

Conventionally, as biological treatment techniques for decomposing harmful chemical substances, chimeric enzymes constructed from Pseudomonas pseudoalcaligenes and dioxygenase derived from Burkholderiacepacia (see, for example, Patent Document 1), benzene ring hydroxylase isolated from the genus Nocardioides (for example, , See Patent Document 2), and angular dioxygenase isolated from Terrabacter genus (see, for example, Patent Document 3) has been reported. Furthermore, a gene group encoding a protein group involved in oxidative degradation of dibenzofuran has been isolated from Paenibacillus genus YK5 (see, for example, Non-Patent Document 3).

  In these compounds, the compounds to be decomposed are limited to monocyclic aromatic compounds or polycyclic aromatic compounds. Generally, environmental pollution sites are complexly contaminated with PCBs, PAHs, dioxins, monocyclic aromatic compounds, etc., so these enzymes with low substrate specificity are not sufficient for efficient purification. It was.

On the other hand, Rhodococcus globerulus P6-derived biphenyl dioxygenase (see, for example, Non-Patent Document 1) has been reported as an enzyme that degrades PCB. However, no knowledge about the decomposition of PCBs with 3 or more chlorines has been reported, and only PCBs with a limited structure could be decomposed. In general, a problem in PCB contamination purification is a highly-degradable PCB with 5 or more chlorines, which is difficult to decompose, and therefore biphenyldioxygenase derived from Rhodococcusgloberulus P6, which has not been reported to be degradable to 3 or more PCBs. The practical value in the field of PCB contamination purification was limited.

Furthermore, it has been reported that a Bacillus genus JF8 strain having the ability to decompose PCB and naphthalene has been isolated (see, for example, Patent Document 4 and Non-Patent Document 2). However, the Bacillus genus JF8 strain only showed a resolution as low as 20% with respect to the 5-chlorine substitute of PCB. Further, from the Bacillus genus JF8 strain, biphenyl and dioxygenase involved in PCB degradation have not been identified.

Therefore, both the above PCB-decomposing bacteria and the enzymes produced by these bacteria have a low ability to decompose high-chlorine-substituted PCBs with a large number of chlorine substitutions (for example, 5 chlorine-substituted or more), and PCBs with a limited structure. Could only be decomposed. Therefore, from the viewpoint of the resolution of the high chlorine-substituted PCB, it is still insufficient for application to the environmental purification field, and there has been a demand for the appearance of PCB-degrading bacteria that can efficiently decompose the high-chlorine-substituted PCB. . In addition, most of the conventionally known PCB-degrading bacteria and PCB-degrading enzymes are of the genus Rhodococcus and Pseudomonas (see FIG. 2), and the degrading enzymes produced by these bacteria are considered to have low substrate specificity. It was.

In order to solve the above problems, a bacterium having a characteristic capable of degrading PCB and PAH, Paenibacillus genus KBC101 strain was isolated by the present inventors (see, for example, Patent Document 5). Such a bacterium is phylogenetically different from the genus Rhodococcus and Pseudomonas, and was reported as the first PCB-degrading bacterium isolated from the genus Paenibacillus . And since it has a decomposition rate of about 40% even for high chlorine substituted PCBs having 5-9 chlorine substitutions, which are difficult to decompose by the conventionally known biodegradation treatment, practical application in the field of environmental purification is expected. It had been.

  However, when such bacteria are actually used in the field of contamination purification, specific bacteria monitoring is unavoidable for the efficient execution of the pollution purification business. In addition, if it can be used as an enzyme agent that can be easily handled and controlled in its effects, efficient contamination purification can be performed.

JP 2000-69979 A JP 2002-65268 A Japanese Patent Laid-Open No. 2003-25056 Japanese Patent Laid-Open No. 11-10122 JP 2005-245280 A Heterologous Expression of Biphenyl Dioxygenase-Encoding Gene from a Gram-Positive Broad-Spectrum Polychlorinated Biphenyl Degrader and Characterization of Chlorobiphenyl Oxidation by the gene Products. Characterization of chlorinated biphenyl oxidation by the gene product) J. Bacteriol., March 1997, pages 1924-1930 Isolation and characterization of thermophilic Bacillus sp. JF8 capable of degrading polychlorinated biphenyls and naphthalene., FEMS Microbiology Letters 1999 9 1st, Volume 178, Issue 1, Pages 87-93 Isolation and characterization of a gene cluster for dibenzofuran degradation in a new dibenzofuran-utilizing bacterium Paenibacillus sp. Strain YK5. (A novel dibenzofuran-utilizing bacterium, isolation and characterization of a gene cluster for dibenzofuran degradation in Paenibacillus genus YK5), Arch Microbiol., January 2006, Vol.184, No.5, pp.305-315

  Therefore, in view of the above circumstances, the present invention has a wide range of substrate specificities, and a degradation enzyme having characteristics capable of degrading PCB and PAH, and also a high chlorine-substituted PCB having a large number of chlorine substitutions, and the enzyme An object of the present invention is to provide a method for purifying contamination using the above. Furthermore, in order to improve the efficiency of pollution purification, aromatic compound-degrading bacteria genes that have a wide range of substrate specificities and are capable of degrading PCBs and PAHs, as well as high-chlorine-substituted PCBs with a high number of chlorine substitutions. The objective is to provide a method for decontamination using specific monitoring at the level.

In order to solve the above-mentioned problems, the present inventors have obtained a subunit α gene of an aromatic ring dioxygenase from a bacterium belonging to the genus Paenibacillus and having a resolution of PCB and PAH, particularly a high-chlorine-substituted PCB having a large number of chlorine substitutions. In addition, the present inventors have succeeded in cloning the subunit β gene, the aromatic ring dihydrodiol / dehydrogenase gene, and the aromatic ring-cleaving dioxygenase gene, thereby completing the present invention. Furthermore, as a result of earnest research, the present invention was completed by constructing an efficient decontamination method using the gene and the enzyme encoded by the gene.

  In order to achieve the above object, the present invention is shown in the following [1] to [22].

[1] An aromatic compound oxidative degradation operon containing the following DNA (I) or (II):
(I) DNA comprising the base sequence shown in sequence recognition number 1 in the sequence listing
(II) comprising DNA that hybridizes with DNA consisting of the base sequence shown in sequence recognition number 1 in the sequence listing under stringent conditions and encodes a protein having oxidative degradation activity against aromatic compounds DNA forming an aromatic compound oxidative degradation operon
[2] The aromatic compound oxidative degradation operon according to [1] above, comprising DNA encoding a protein having oxidative degradation activity for at least one of polychlorinated biphenyls and polycyclic aromatic hydrocarbons.
[3] A gene encoding the protein shown in (A) or (B) below.
(A) a protein having the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing (B) substitution, deletion, insertion of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing, or A protein comprising an amino acid sequence including an addition and having a function as an α subunit of an aromatic ring dioxygenase [4] A gene including the following DNA (a) or (b):
(A) DNA comprising the base sequence shown in sequence recognition number 2 in the sequence listing
(B) a DNA that hybridizes with a DNA comprising the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing under stringent conditions and encodes a protein having a function as an α subunit of an aromatic ring dioxygenase
[5] The above [3] or [4], which encodes a protein having a function as an α subunit of an aromatic ring dioxygenase having a decomposition activity on at least one of polychlorinated biphenyls and polycyclic aromatic hydrocarbons Genes.
[6] A gene encoding a protein shown in (C) or (D) below.
(C) a protein having the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing (D) substitution, deletion, insertion of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing, or A protein comprising an amino acid sequence including an addition and having a function as a β subunit of an aromatic ring dioxygenase [7] A gene including the following DNA (c) or (d):
(C) DNA consisting of the base sequence represented by sequence recognition number 4 in the sequence listing
(D) a DNA that hybridizes with a DNA comprising the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing under stringent conditions and encodes a protein having a function as a β subunit of an aromatic ring dioxygenase
[8] The above [6] or [7], which encodes a protein having a function as a β subunit of an aromatic ring dioxygenase having a decomposition activity on at least one of polychlorinated biphenyls and polycyclic aromatic hydrocarbons Genes.
[9] A gene encoding a protein shown in (E) or (F) below.
(E) a protein having the amino acid sequence shown in SEQ ID NO: 9 in the sequence listing (F) substitution, deletion, insertion of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 9 in the sequence listing, or A protein comprising an amino acid sequence containing an addition and having a function as an aromatic ring dihydrodiol dehydrogenase [10] A gene comprising DNA of the following (e) or (f):
(E) DNA comprising the base sequence shown in SEQ ID NO: 8 in the sequence listing
(F) a DNA that hybridizes with a DNA comprising the base sequence shown in SEQ ID NO: 8 in the sequence listing under stringent conditions and encodes a protein having a function as an aromatic ring dihydrodiol dehydrogenase
[11] The gene according to [9] or [10], which encodes a protein having a function as an aromatic ring dihydrodiol / dehydrogenase having a decomposition activity on at least one of polychlorinated biphenyls and polycyclic aromatic hydrocarbons .
[12] A gene encoding a protein shown in (G) or (H) below.
(G) a protein having the amino acid sequence shown in SEQ ID NO: 11 in the sequence listing (H) substitution, deletion, insertion of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 11 in the sequence listing, or A protein comprising an amino acid sequence containing an addition and having a function as an aromatic ring-cleaving dioxygenase [13] A gene comprising the following DNA of (g) or (h).
(G) DNA comprising the base sequence shown in SEQ ID NO: 10 in the sequence listing
(H) a DNA that hybridizes with a DNA comprising the nucleotide sequence shown in SEQ ID NO: 10 in the sequence listing under a stringent condition and encodes a protein having a function as an aromatic ring-cleaving dioxygenase
[14] The gene according to [12] or [13], which encodes a protein having a function as an aromatic ring-cleaving dioxygenase having a decomposition activity for at least one of polychlorinated biphenyls and polycyclic aromatic hydrocarbons.
[15] An aromatic ring dioxygenase comprising a protein complex comprising the following protein (A) or (B) and (C) or (D).
(A) a protein having the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing (B) substitution, deletion, insertion of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing, or Protein (D) sequence recognition number of protein (D) sequence list which has amino acid sequence shown in sequence recognition number 5 of protein (C) sequence list which consists of amino acid sequence including addition and has a function as α-subunit of aromatic ring dioxygenase 5. A protein comprising an amino acid sequence containing substitution, deletion, insertion, or addition of one or several amino acid residues in the amino acid sequence shown in 5 and having a function as a β subunit of an aromatic ring dioxygenase [16 The above-mentioned [15] having a resolution for at least one of polychlorinated biphenyls and polycyclic aromatic hydrocarbons Aromatic ring dioxygenase.
[17] An aromatic ring dihydrodiol / dehydrogenase comprising the following protein (E) or (F):
(E) a protein having the amino acid sequence shown in SEQ ID NO: 9 in the sequence listing (F) substitution, deletion, insertion of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 9 in the sequence listing, or A protein comprising an amino acid sequence including an addition and having a function as an aromatic ring dihydrodiol dehydrogenase [18] The above [17] having a resolution for at least one of polychlorinated biphenyls and polycyclic aromatic hydrocarbons Aromatic ring dihydrodiol dehydrogenase.
[19] An aromatic ring-cleaving dioxygenase comprising the following protein (G) or (H):
(G) a protein having the amino acid sequence shown in SEQ ID NO: 11 in the sequence listing (H) substitution, deletion, insertion of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 11 in the sequence listing, or A protein comprising an amino acid sequence including an addition and having a function as an aromatic ring-cleaving dioxygenase [20] The resolution according to [19], which has a resolution for at least one of polychlorinated biphenyls and polycyclic aromatic hydrocarbons Aromatic ring cleavage dioxygenase.

According to the configuration of the above [1] to [20], an aromatic ring dioxygenase, an aromatic ring cleavage dioxygenase, and an aromatic ring dihydrodiol / dehydrogenase having high decomposition activity against PCB and PAH are provided. In particular, the present invention provides an enzyme having high practical value in the purification of PCB-contaminated systems, PAH-contaminated systems, and combined contaminated systems using PCB and PAH. In particular, the enzyme provided by the present invention is a novel enzyme obtained from a bacterium having a high resolution with respect to a high chlorine-substituted PCB having a chlorine substitution number of 5 or more, which has been difficult to decompose by a conventionally known biodegradation treatment technique. It is expected to have a wide range of substrate specificities including high chlorine substituted PCBs.
Furthermore, it has been found that the aromatic ring dioxygenase, aromatic ring cleavage dioxygenase, and aromatic ring dihydrodiol dehydrogenase of the present invention are encoded by a gene group that forms one operon derived from biphenyl. Yes.
In addition, since the amino acid sequences and base sequences of the aromatic ring dioxygenase, aromatic ring cleavage dioxygenase, and aromatic ring dihydrodiol / dehydrogenase of the present invention have been clarified, they can be produced at low cost and industrially using genetic engineering techniques. Mass production became possible.

[21] The aromatic ring dioxygenase of [15] or [16] above, the aromatic ring dihydrodiol dehydrogenase of [17] or [18] above, and the aromatic ring cleavage dioxygenase of [19] or [20] above A method for decomposing an aromatic compound, wherein the aromatic compound is brought into contact with at least one of the above.

  According to the configuration of [21] above, it is possible to provide a method for decomposing an aromatic compound using an aromatic ring dioxygenase, an aromatic ring dihydrodiol / dehydrogenase, or an aromatic ring-cleaving dioxygenase having a high decomposing activity for PCB and PAH. It is possible to provide a decomposition method having a high practical value in the purification of contaminated systems, PAH contaminated systems, and combined contaminated systems using PCB and PAH. In addition, it is possible to provide a decomposition method capable of efficiently decomposing a high chlorine substituted PCB, which has been difficult to decompose by a conventionally known biodegradation processing technique.

[22] Any of the base sequence shown in sequence recognition number 2 in the sequence listing, the base sequence shown in sequence recognition number 4 in the sequence listing, the base sequence shown in sequence recognition number 8 or the base sequence shown in sequence recognition number 10 in the sequence listing Detection of Paenibacillus genus KBC101 strain deposited under the accession number FERM BP-08636 using an oligonucleotide having a part or all of the base sequence in at least one base sequence, or an oligonucleotide complementary to the oligonucleotide To detect bacteria.

According to the configuration of [22] above, it is possible to provide a bacterial detection method for detecting Paenibacillus genus KBC101 strain deposited under the accession number FERM BP-08636 having high resolution for PCB and PAH. In particular, it is possible to specifically detect Paenibacillus genus KBC101 strain having high resolution with respect to a high-chlorine-substituted PCB having a chlorine substitution number of 5 or more, which has been difficult to decompose by known biodegradation treatment techniques. With this bacteria detection method, it is possible to monitor the presence and dynamics of Paenibacillus genus KBC101 strain, and based on the detection results, it is possible to construct a highly efficient pollution purification system and shorten the purification treatment time. Become.

  Embodiments of the present invention will be described in detail below. However, the scope of the present invention is not limitedly interpreted by these embodiments.

[1] Oxidative Decomposition Mechanism of Aromatic Compound First, the oxidative decomposition mechanism of an aromatic compound will be described by taking biphenyl belonging to the aromatic compound as an example. As shown in FIG. 1, biphenyl is biphenyl dioxygenase (hereinafter sometimes referred to as “BphA”), dihydrodiol dehydrogenase (hereinafter sometimes referred to as “BphB”), 2,3-dihydroxybiphenyl. 1,2-dioxygenase (hereinafter sometimes referred to as “BphC”), 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (hereinafter sometimes referred to as “BphD”) .)). Specifically, BphA introduces two hydroxyl groups adjacent to biphenyl to produce cis-biphenyl dihydrodiol. Then, BphB removes two hydrogen molecules from the cis-biphenyl dihydrodiol to produce 2,3-dihydroxybiphenyl, and then meta-cleavages with BphC to produce 2-hydroxy-6-oxo-6, which is a yellow compound. -Phenylhexa-2,4-dienoic acid (hereinafter, “HOPDA” may be abbreviated) is produced. And BOPD decomposes HOPDA into benzoic acid and 2-hydroxypenta-2,4-dienoate. Then, it is decomposed to acetyl CoA by a group of enzymes called BphE, BphF, and BphG.

  Here, the aromatic ring dioxygenase that is the subject of the present invention is a group of enzymes that catalyze the reaction of incorporating molecular oxygen into the aromatic ring, and catalyzes the initial oxidation of the aromatic compound. Specifically, molecular oxygen of 2 atoms is introduced onto the aromatic ring, and as a result, the reaction of introducing two adjacent hydroxyl groups onto the aromatic ring is catalyzed. The dioxygenase that catalyzes the initial oxidative degradation in the degradation of biphenyl is referred to as biphenyl dioxygenase. Biphenyl dioxygenase is sometimes referred to as α subunit (hereinafter sometimes referred to as “BphA1”), β subunit (hereinafter sometimes referred to as “BphA2”), ferredoxin (hereinafter referred to as “BphA3”). A multi-component enzyme composed of ferredoxin reductase (hereinafter sometimes referred to as “BphA4”). Then, as shown in FIG. 1, the α subunit and the β subunit form a complex, catalyze the binding and oxygenation reaction with the substrate, the ferredoxin functions as an electron carrier, and the ferredoxin reductase is NAD or NADPH. And catalyzes the redox reaction of ferredoxin.

  In addition, the aromatic ring dihydrodiol dehydrogenase that is the subject of the present invention catalyzes a dehydrogenation reaction that removes hydrogen molecules from an aromatic dihydrodiol compound to produce an aromatic diol compound.

  The aromatic ring-cleaving dioxygenase that is the subject of the present invention introduces molecular oxygen of 2 atoms onto the aromatic diol compound, and as a result, introduces two adjacent hydroxyl groups onto the aromatic diol compound. Catalyses the reaction of meta-cleaving aromatic diol compounds.

[2] Cloning of the aromatic ring dioxygenase gene of the present invention The present inventors have identified a gene encoding an aromatic ring dioxygenase having characteristics capable of degrading PCB and PAH, as well as a high chlorine substituted PCB having a large number of chlorine substitutions. to isolate, Paenibacillus sp KBC101 strain (accession number: FERM BP-08636, hereinafter abbreviated as "KBC101 share") was cloned genomic library. Cloning is performed using a degenerate primer set designed based on the sequence conserved in the α-subunit gene of the aromatic-degrading bacterial dioxygenase, so that a part of the α-subunit gene of the aromatic ring dioxygenase can be obtained. Obtained an array. Positive clones were obtained by colony hybridization of the genomic library of KBC101 strain using such a partial sequence as a probe. Subsequently, after nucleotide sequencing of the cloned DNA fragment, homology search is presumed to encode aromatic ring dioxygenase α and β subunits, aromatic ring dihydrodiol dehydrogenase, and aromatic ring cleaved dioxygenase The gene was identified. The amino acid sequence encoded by such a gene showed homology with known aromatic ring dioxygenases, aromatic ring dihydrodiol dehydrogenases, and aromatic ring cleavage dioxygenases at an amino acid level of about 70 to 80% at maximum. . However, it was suggested that the enzyme group identified in the present invention is a novel enzyme group that is systematically different from the conventionally known oxidative degradation enzymes derived from PCB-degrading bacteria. Subsequently, an expression plasmid containing the gene identified in the present invention is constructed and found to have biphenyl conversion activity, which encodes a protein group having a function involved in the oxidative degradation pathway of aromatic compounds containing biphenyl. It was confirmed that it is a gene group.

  The base sequence of the DNA fragment containing the α-subunit and β-subunit of the aromatic ring dioxygenase obtained as described above, the aromatic ring dihydrodiol dehydrogenase, and the aromatic ring-cleaving dioxygenase is represented by SEQ ID NO: 1 in the sequence listing. Show. In this sequence, there are five open reading frames (hereinafter abbreviated as “ORF”). From the homology search, it is presumed from the upstream that it is the coding sequence of α-subunit, β-subunit, unknown function, dihydrodiol dehydrogenase, 2,3-dihydroxybiphenyl 1,2-dioxygenase of dioxygenase. The base sequence of each gene is shown in sequence recognition numbers 2, 4, 6, 8 and 10 in the sequence listing, and the encoded amino acid sequences are shown in sequence recognition numbers 3, 5, 7, 9 and 11 in the sequence listing.

  Furthermore, as a result of examination, it has been found that the gene identified in the present invention is subject to transcriptional regulation as a clustered operon. It has also been found that these are transcriptionally regulated by aromatic compounds such as biphenyl. In general, functionally related genes are clustered on the same operon, so that the enzyme involved in the oxidative degradation pathway of aromatic compounds will be analyzed in the future analysis of the above unknown sites. The possibility of coding is suggested.

[3] Enzyme Group Involved in Oxidative Degradation Pathway of Aromatic Compound of the Present Invention As an enzyme group involved in the oxidative degradation path of aromatic compound of the present invention, aromatic ring dioxygenase, aromatic ring dihydrodiol / dehydrogenase, aromatic ring cleavage Dioxygenase was found. These are novel enzymes discovered from Paenibacillus genus KBC101 strain, which is phylogenetically different from conventionally known PCB-degrading bacteria.

  Aromatic ring dioxygenase is a protein complex composed of four components: α subunit, β subunit, ferredoxin, and ferredoxin reductase.

  And what has the amino acid sequence of sequence recognition number 3 of a sequence table is illustrated as (alpha) subunit of the aromatic-ring dioxygenase of this invention.

  Examples of the β subunit of the aromatic ring dioxygenase of the present invention include those having the amino acid sequence of SEQ ID NO: 5 in the sequence listing.

  Regarding each component of ferredoxin and ferredoxin reductase, those having the activity can be used without particular limitation.

  Examples of the aromatic ring dihydrodiol dehydrogenase of the present invention include those having the amino acid sequence of SEQ ID NO: 9 in the sequence listing.

  Examples of the aromatic ring-cleaving dioxygenase of the present invention include those having the amino acid sequence of SEQ ID NO: 11 in the sequence listing.

  In addition, the enzyme of the present invention includes those having an amino acid sequence having a modified site where a specific amino acid is modified in the amino acid sequence of each of the above subunits, and having each function or activity. Here, the modification means that a modification comprising at least one of deletion, substitution, insertion and addition of one or several amino acids in the amino acid sequence of the protein serving as the basis of the modification has occurred. "Modification consisting of at least one of deletion, substitution, insertion and addition of one or several amino acids" means a known DNA recombination technique for a gene encoding a protein that is the basis of the modification, point mutation introduction It means that as many amino acids as can be deleted, substituted, inserted or added by a method or the like are deleted, substituted, inserted or added, and combinations thereof are also included.

  Such a variant can be obtained by screening a protein having the activity from mutants generated by natural or artificial mutation.

  Alternatively, the method for modifying such a gene, which can be obtained by modifying using the gene of the present invention described below, is not particularly limited, and for producing a modified protein known to those skilled in the art. Mutagenesis techniques can be used. For example, a known mutagenesis technique such as a site-directed mutagenesis method, a PCR abrupt induction method that introduces a point mutation using a PCR method, or a transposon insertion mutagenesis method can be used. A commercially available mutagenesis kit (for example, QuikChange (registered trademark) Site-directed Mutagenesis Kit (manufactured by Stratagene), etc.) may be used, and a DNA synthesis method such as a conventional phosphoramidite method may be used. Alternatively, it can be prepared by constructing a gene having a desired modification.

  Whether or not the gene into which the modification is introduced encodes a protein having a desired function or activity depends on whether or not the aromatic ring dioxygenase activity, aromatic ring dihydrodiol dehydrogenase activity of the recombinant protein produced based on the modified gene The aromatic ring-cleaving dioxygenase can be evaluated by measuring each. For example, the aromatic ring dioxygenase activity is preferably evaluated by expressing it together with other subunit genes.

  A person skilled in the art can easily predict a modification that retains the enzyme activity of the present invention upon modification of the amino acid sequence. Specifically, in the case of amino acid substitution, for example, polarity, charge, hydrophilicity from the viewpoint of retaining the protein structure. The amino acid can be substituted with an amino acid having properties similar to those of the amino acid before substitution in terms of property, hydrophobicity, and the like. Such substitutions are well known to those skilled in the art as conservative substitutions. In addition, for convenience such as subsequent purification and immobilization on a solid phase, those in which His-tag, FLAG-tag, etc. are added to the N- or C-terminus of the amino acid sequence are also preferably exemplified.

The aromatic ring dioxygenase, aromatic ring dihydrodiol dehydrogenase and aromatic ring-cleaving dioxygenase of the present invention can be prepared as follows. For example, it can be produced by isolating and purifying from a bacterium belonging to the genus Paenibacillus having PCB resolution, in particular, from the culture of the above-described KBC101 strain. Since the amino acid sequence of the enzyme of the present invention has been clarified, it can be produced in large quantities based on such sequence information. For example, the α-subunit and β-subunit of aromatic ring dioxygenase can be produced by using hybridization as a DNA probe prepared based on a base sequence encoding part or all of the amino acid sequence of SEQ ID NO: 3 or 5. According to the method, a nucleic acid molecule encoding the aromatic ring dioxygenase of the present invention can be prepared from a bacterium belonging to the genus Paenibacillus having PCB resolution, particularly preferably from the genomic DNA of KBC101 strain. Similarly, a nucleic acid molecule encoding the aromatic ring dioxygenase of the present invention can also be prepared by PCR using a part of the base sequence encoding the amino acid sequence of SEQ ID NO: 3 or 5 as a primer. Furthermore, a nucleic acid molecule encoding the aromatic ring dioxygenase of the present invention can be chemically synthesized using a DNA synthesis method such as a conventional phosphoramidite method. The nucleic acid molecule encoding the aromatic ring dioxygenase obtained as described above can be used to produce the aromatic ring dioxygenase of the present invention by a gene recombination technique described in detail below. An aromatic ring dihydrodiol dehydrogenase activity and an aromatic ring-cleaving dioxygenase can be produced in the same manner.

  Here, in the production of the enzyme of the present invention, the primer used when the PCR method is used can be designed and prepared based on a conventional method. For example, the primers used in the examples described below can be suitably used. As the primer, a chemical synthesis method based on the phosphoramidite method or the like, or a restriction enzyme fragment or the like when a target nucleic acid has already been obtained can be used. When preparing a primer based on a chemical synthesis method, it is designed based on the sequence information of the target nucleic acid prior to synthesis. The primer can be designed using, for example, primer design support software so as to amplify a desired region. After synthesis, the primer is purified by means such as HPLC. Moreover, when performing chemical synthesis, it is also possible to use a commercially available automatic synthesizer. Here, complementary means that the primer and the target nucleic acid can specifically bind according to the base pairing rule to form a stable double-stranded structure. Here, not only perfect complementarity, but only a few nucleobases fit along the base-pairing rule as long as the primer and target nucleic acid are sufficient to form a stable duplex structure with each other Even partial complementarity is acceptable. The number of bases must be long enough to specifically recognize the target nucleic acid, but if it is too long, a non-specific reaction is induced, which is not preferable. Accordingly, an appropriate length is determined depending on many factors such as target nucleic acid sequence information such as GC content, and hybridization reaction conditions such as reaction temperature and salt concentration in the reaction solution. It is 10-50 bases long.

  Furthermore, the enzyme of the present invention can also be produced by a chemical synthesis technique. For example, all or part of the amino acid sequence shown in SEQ ID NO: 3, 5, 9, or 11 is synthesized using a peptide synthesizer, and the resulting polypeptide is reconstructed under appropriate conditions. Can be prepared.

  Whether the protein produced as described above has a desired function or activity is determined by measuring the aromatic ring dioxygenase activity, the aromatic ring dihydrodiol / dehydrogenase activity, and the aromatic ring cleavage dioxygenase activity, respectively. Can be evaluated. Any known method can be used as a method for measuring the activity, and it can be evaluated by detecting the presence or absence of a substrate conversion reaction by reacting with a substrate. For example, it can be performed by measuring the conversion activity of biphenyl. Specifically, taking the aromatic ring dioxygenase activity as an example, the conversion activity of biphenyl is measured by, for example, confirming the formation of yellow HOPDA, which is a degradation product of biphenyl, in the presence of BphB and BphC. be able to. Moreover, it can also carry out by detecting the amount of oxygen consumption accompanying the initial oxidation of an aromatic compound. However, it is not limited to this.

  As demonstrated in the Examples, the aromatic ring dioxygenase of the present invention catalyzes the initial oxidation in the biphenyl decomposition reaction shown in FIG. Furthermore, it is understood that the aromatic ring dioxygenase of the present invention also degrades PCBs that are degraded by co-metabolism with biphenyl. In addition, it is an enzyme with a wide substrate specificity that has a degradation activity for PAH. Furthermore, the aromatic ring-cleaving substance is oxidatively decomposed by aromatic ring dihydrodiol dehydrogenase and aromatic ring-cleaving dioxygenase.

Here, the enzyme of the present invention is cloned from the KBC101 strain capable of degrading a high-chlorine-substituted PCB having a large number of substituted chlorines (especially, 5 chlorine substitutions or more), which has been difficult to decompose by a conventionally known biodegradation processing technique. It has been done. The resolution of PCB was found for the first time in the genus Paenibacillus , and was found to be a novel enzyme that is also phylogenetically different from the conventionally known enzyme derived from PCB-degrading bacteria (FIG. 2). . Therefore, it is expected that the substrate specificity is different from that of a conventionally known enzyme having PCB degradation activity. In view of the high PCB-degrading activity of the KBC101 strain, the enzyme of the present invention can catalyze the degradation of highly chlorine-substituted PCB, which has been considered difficult by biodegradation treatment, and has a broader substrate specificity than known enzymes. It is understood that the enzyme has

  PCB to be decomposed is a generic name for hydrogen in a biphenyl skeleton in which two benzene rings are connected and substituted with 1 to 10 chlorines. Theoretically 209 isomers depending on the number and position of substituted chlorines And all isomers and mixtures thereof are included. The PAH to be decomposed here is a general term for compounds having two or more aromatic rings, including compounds that form a heterocyclic ring with a nitrogen atom, an oxygen atom, a sulfur atom, and specifically, , Dibenzofuran, xanthene, phenanthrene, fluorene and the like.

[4] Gene group involved in the aromatic compound oxidative degradation pathway of the present invention The gene group involved in the aromatic compound oxidative degradation pathway of the present invention is a gene encoding the aromatic ring dioxygenase of the present invention, an aromatic ring dihydro A gene encoding diol dehydrogenase and a gene encoding the aromatic ring-cleaving dioxygenase are included.

  And preferably, the above-mentioned four genes form a gene cluster expressed on one operon. Therefore, the present invention includes an aromatic compound oxidative degradation operon as an operon comprising at least one of these genes. Furthermore, it includes at least one gene of the present invention described above, and other genes include those functionally equivalent to those described above. In addition, those comprising a gene encoding a protein involved in the oxidative degradation pathway of an aromatic ring compound other than those identified in the present invention are also included. For example, a gene encoding ferredoxin or ferredoxin reductase may be included. Specific examples of the aromatic compound oxidative degradation operon containing the gene group of the present invention include those containing a DNA consisting of the base sequence shown in SEQ ID NO: 1 in the Sequence Listing. Therefore, in the present specification, the term “gene” is interpreted as a concept including an operon unless otherwise specified.

  Examples of each gene group constituting the operon include those containing DNA encoding a protein having the amino acid sequence described above. In particular, the gene encoding the α subunit of the aromatic ring dioxygenase of the present invention is exemplified by a gene containing DNA comprising the base sequence shown in SEQ ID NO: 2 in the sequence listing.

  Examples of the gene encoding the β subunit of the aromatic ring dioxygenase of the present invention include those containing DNA consisting of the base sequence shown in SEQ ID NO: 4 in the sequence listing.

  Examples of the gene encoding the aromatic ring dihydrodiol dehydrogenase of the present invention include those containing DNA consisting of the base sequence shown in SEQ ID NO: 8 in the sequence listing.

  Examples of the gene encoding the aromatic ring-cleaving dioxygenase of the present invention include those containing DNA consisting of the base sequence shown in SEQ ID NO: 10 in the sequence listing.

  In addition, the gene of the present invention encodes a protein that hybridizes to the DNA comprising the above base sequence under stringent conditions and has a function or activity as the above-described aromatic compound oxidative degradation enzyme. The modified gene containing DNA to be included is also included in the present invention. Such a modified gene can be prepared by using a known mutation introduction technique. For example, a known mutagenesis technique such as a site-directed mutagenesis method, a PCR abrupt induction method that introduces a point mutation using a PCR method, or a transposon insertion mutagenesis method can be used. Commercially available mutagenesis kits (for example, QuikChange (registered trademark) Site-directed Mutagenesis Kit (manufactured by Stratagene), etc.) may also be used, and DNA chemical synthesis methods such as the conventional phosphoramidite method may be used. Thus, it can also be constructed by synthesizing a DNA having a desired modification, or a DNA comprising the nucleotide sequence shown in SEQ ID NO: 1, particularly SEQ ID NO: 2, 4, 8, or 10. In addition, the modified gene can be obtained by acting exonuclease on the base sequence shown in SEQ ID NO: 1, particularly SEQ ID NO: 2, 4, 8, or 10. Alternatively, a gene containing a plurality of bases deleted, substituted, added or inserted is also included.

  Here, the stringent condition refers to a condition in which DNA having the identity of 60% or more, preferably 70%, more preferably 80% or more, particularly preferably 90% or more in the base sequence can hybridize. . The stringency can be suitably adjusted by those skilled in the art by appropriately changing the salt concentration and temperature during the hybridization reaction and washing. For example, the conditions for Southern hybridization described in Molecular Cloning: A Laboratory Manual 2nd edition (Sambrook et al., Cold Spring Harbor Laboratory Press, 1989) can be mentioned.

  More specifically, preferably after hybridization at preferably 60 to 68 ° C., more preferably 65 ° C. in the presence of 150 to 1000 mM, more preferably 700 to 900 mM sodium chloride. This means washing at 60 to 68 ° C., more preferably 65 ° C. in 1-2 × SSC, but is not limited thereto.

Moreover, since the base sequence has been clarified in the present invention, the gene of the present invention can be prepared based on such sequence information. For example, a bacterium belonging to the genus Paenibacillus having PCB resolution, particularly preferably KBC101 strain, by a hybridization method using a DNA probe prepared based on a part or all of the base sequence shown in SEQ ID NO: 2 or 4 The polynucleotide of the present invention can be prepared from the genomic DNA. It can also be similarly prepared by a PCR method using a primer prepared based on a partial sequence of the nucleotide sequence shown in SEQ ID NO: 1, particularly SEQ ID NO: 2, 4, 8, or 10. The design and preparation of the primer used at this time can be performed based on a conventional method as described above. For example, the primers used in the examples described below can be suitably used.

  Furthermore, the gene of the present invention can be chemically synthesized using a DNA synthesis method such as a conventional phosphoramidite method.

  Whether the gene produced as described above encodes a protein having a desired function or activity can be determined by measuring the activity of the protein produced based on the gene. At this time, it is preferably carried out by expressing together with other subunit genes and measuring the aromatic ring dioxygenase activity, aromatic ring dihydrodiol / dehydrogenase activity, and aromatic ring cleavage dioxygenase activity.

[5] Recombinant vector of the present invention The recombinant vector of the present invention can be obtained by incorporating at least one of the above-described genes of the present invention or an operon into an appropriate vector. It can also be obtained by incorporating any suitable region on the operon. The vector that can be used is not particularly limited as long as it can incorporate foreign DNA and can replicate autonomously in a host cell. Therefore, the vector contains a sequence of at least one restriction enzyme site into which the gene of the present invention can be inserted. For example, plasmid vectors (pEX system, pUC system, pBR system, etc.), phage vectors (Charomid, λgt10, λgt11, λZAP, etc.), cosmid vectors, virus vectors (vaccinia virus, baculovirus, etc.) and the like are included. . A shuttle vector such as pHY300PLK can be used.

  The recombinant vector of the present invention is incorporated so that the gene of the present invention can express its function. For example, the recombinant vector of the present invention is constructed so that the above four components of the aromatic ring dioxygenase, including the α subunit gene and the β subunit gene of the aromatic ring dioxygenase of the present invention, can be expressed in the host. Is done. The order may be arbitrary, and the direction is also arbitrary. Ferredoxin and ferredoxin reductase are not particularly limited as long as they are genes encoding ferredoxin and ferredoxin reductase. Moreover, the thing derived from a host can also be utilized and does not need to be contained on a recombinant vector. In addition, other known base sequences necessary for the expression of gene function may be included. For example, a promoter sequence, a leader sequence, a signal sequence, a ribosome binding sequence and the like can be mentioned. As the promoter sequence, for example, when the host is Escherichia coli, lac promoter, trp promoter and the like are preferably exemplified. However, the present invention is not limited to this, and a known promoter sequence can be used. Furthermore, the recombinant vector of the present invention can also include a marking sequence that can confer phenotypic selection in the host. Examples of such marking sequences include sequences encoding genes such as drug resistance and auxotrophy. Specifically, kanamycin resistance gene, chloramphenicol resistance gene, ampicillin resistance gene and the like are exemplified.

  For insertion of the gene of the present invention into a vector, for example, a method in which the gene of the present invention is cleaved with an appropriate restriction enzyme and inserted into a restriction enzyme site or a multicloning site of an appropriate vector is used. However, it is not limited to this. For ligation, a known method such as a method using DNA ligase can be used. Commercially available ligation kits such as DNA Ligation Kit (Takara-bio) can also be used.

[6] Transformant of the present invention The transformant of the present invention is a host cell transformed with a recombinant vector containing the gene of the present invention. Here, the host cell is not particularly limited as long as it can efficiently express the aromatic ring dioxygenase of the present invention. Prokaryotes can be preferably used, and in particular, E. coli can be used. In addition, Bacillus subtilis, Bacillus bacteria, Pseudomonas bacteria, and the like can also be used. As E. coli , for example, E. coli DH5α, E. coli BL21, E. coli JM109 and the like can be used. Furthermore, eukaryotic cells can be used without being limited to prokaryotes. For example, yeasts such as Saccharomyces cerevisiae, insect cells such as Sf9 cells, animal cells such as CHO cells, COS-7 cells, and the like can be used. As the transformation method, known methods such as an electroporation method, a calcium chloride method, a liposome transfection method, and a microinjection method can be used. In the case of using a phage vector, after ligation of the gene of the present invention and the phage vector DNA, the phage protein is folded in vitro by packaging in vitro and enclosed in the outer shell of the mature phage, and then the phage infection system is used. Utilizing the gene of the present invention, it can be introduced into a host cell.

[7] Process for producing the enzyme of the present invention using the transformant of the present invention The activity of the aromatic ring dioxygenase, aromatic ring dihydrodiol dehydrogenase and aromatic ring-cleaving dioxygenase of the present invention using the transformant of the present invention The production method is carried out by culturing the transformant of the present invention and collecting a protein having an aromatic ring dihydrodiol dehydrogenase activity and an aromatic ring cleavage dioxygenase activity from the obtained culture. That is, the method includes a culture process for culturing the transformant of the present invention and a recovery process for recovering the protein expressed in the culture process. Thus, by expressing in an appropriate host, the enzyme of the present invention can be produced at low cost and in large quantities.

  The culturing step is carried out by inoculating the transformant of the present invention in an appropriate medium and culturing according to a conventional method. For the culture of the transformant of the present invention, the culture conditions may be selected in consideration of the nutritional physiological properties of the host cells. The medium to be used is not particularly limited as long as it contains nutrients that can be assimilated by the host cell and can efficiently express the protein in the transformant. Therefore, a medium containing a carbon source, a nitrogen source and other essential nutrients necessary for the growth of the host cell is preferable, regardless of whether it is a natural medium or a synthetic medium. Examples of the carbon source include glucose, dextran, starch, and the like, and examples of the nitrogen source include ammonium salts, nitrates, amino acids, peptone, and casein. As other nutrients, inorganic salts, vitamins, antibiotics, and the like can be included as desired. When the host cell is Escherichia coli, LB medium, M9 medium and the like can be preferably used. Moreover, although there is no restriction | limiting in particular also about a culture form, A liquid medium can be utilized suitably from a viewpoint of mass culture.

  Selection of a host cell carrying the recombinant vector of the present invention can be performed by, for example, the presence or absence of expression of a marking sequence. For example, when a drug resistance gene is used as the marking sequence, it can be performed by culturing in a drug-containing medium corresponding to the drug resistance gene.

  In the purification step, the aromatic ring dioxygenase, aromatic dihydrodiol dehydrogenase and aromatic ring-cleaving dioxygenase of the present invention are recovered from the culture of the transformant obtained in the above-described culture step, that is, isolated and purified. Can be done. That is, according to the fraction in which the aromatic ring dioxygenase, aromatic ring dihydrodiol / dehydrogenase, or aromatic ring-cleaving dioxygenase of the present invention is present, a technique according to a general protein isolation and purification method may be applied. Specifically, when the enzyme of the present invention is produced outside the host cell, the culture solution is used as it is, or the host cell is removed by means such as centrifugation or filtration to obtain a culture supernatant. Subsequently, the enzyme of the present invention can be isolated and purified by appropriately selecting a known protein purification method for the culture supernatant. For example, known isolation and purification techniques such as ammonium sulfate precipitation, dialysis, SDS-PAGE electrophoresis, gel filtration, various types of chromatography such as hydrophobicity, anion, cation, and affinity chromatography can be applied alone or in appropriate combination. . In addition, when the enzyme of the present invention is produced in a host cell, the host cell is recovered by means of centrifugation, filtration or the like of the culture. Subsequently, the host cells are disrupted by an enzymatic disruption method such as lysozyme treatment or a physical disruption method such as ultrasonic treatment, freeze-thawing, and osmotic shock. After crushing, the solubilized fraction is collected by means such as centrifugation or filtration. The obtained solubilized fraction can be isolated and purified by treating in the same manner as in the case where it can be produced extracellularly.

  The performance of the isolated and purified protein can be confirmed by measuring the aromatic ring dioxygenase activity, the aromatic ring dihydrodiol / dehydrogenase activity, and the aromatic ring cleavage dioxygenase activity. Any known method can be used as a method for measuring the activity, and it can be evaluated by detecting the presence or absence of a substrate conversion reaction by reacting with a substrate. For example, it can be performed by measuring the conversion activity of biphenyl as described above.

[8] Aromatic compound decomposing agent of the present invention In addition, an aromatic compound decomposing agent containing at least one of the aromatic ring dioxygenase, aromatic ring dihydrodiol dehydrogenase, and aromatic ring-cleaving dioxygenase of the present invention as an active ingredient Can be offered as. Preferably, all three enzymes are included as active ingredients. Here, as an active ingredient, a crude enzyme can be used in addition to a purified enzyme. Moreover, as a crude enzyme, in addition to various intermediate purified products obtained in the course of the purification process, a culture of the transformant produced by a genetic engineering technique can be used as appropriate. In addition, a product obtained by subjecting the aromatic ring dioxygenase of the present invention to an appropriate treatment such as freeze-drying can also be used. The active ingredient concentration can be adjusted to an appropriate concentration and is not particularly limited.

  And in preparation of the decomposition agent of this invention, it can prepare in various forms, such as a tablet, a powder agent, a granule, a liquid agent, a wettable powder, using the aromatic ring dioxygenase of this invention as an active ingredient. It can be formulated in accordance with a known formulation technique, and appropriate excipients, stabilizers and the like can be appropriately contained. It can also be provided in a form immobilized on a suitable insolubilized carrier. Any immobilization carrier can be used as long as it can be immobilized while retaining the biological activity of the aromatic ring dioxygenase of the present invention. Methods known to those skilled in the art can be used as the immobilization method. For example, there are no limitations on the form such as covalent bonding, ionic bonding, and carrier bonding by physical adsorption, crosslinking, and inclusion. Specific examples include DEAE cellulose, silica, carrier binding by adsorption of titanium oxide, crosslinking by glutaraldehyde, inclusion by a photocatalytic resin prepolymer, and the like.

  Since the aromatic compound decomposing agent of the present invention contains the aromatic ring dioxygenase, aromatic dihydrodiol dehydrogenase, and aromatic ring-cleaving dioxygenase of the present invention, which are enzymes having a wide substrate specificity, as an active ingredient, PCB and It can be applied to the decomposition treatment of various aromatic compounds such as PAH, and can also be suitably used for the decomposition treatment of a high chlorine substituted PCB having a large number of substituted chlorines (particularly, 5 chlorine substitutions or more). Also, PCB and PAH can be decomposed simultaneously. Therefore, the decomposing agent of the present invention can be particularly suitably used for environmental pollution purification treatment, and can be effectively used for complex pollution systems.

[9] Method for Decomposing Aromatic Compound of the Present Invention The aromatic compound of the present invention is decomposed by at least one of the aromatic ring dioxygenase, aromatic ring dihydrodiol dehydrogenase and aromatic ring-cleaving dioxygenase of the present invention and the decomposition target. The aromatic compound is subjected to biodegradation treatment by contacting the aromatic compound. Here, the aromatic ring dioxygenase, the aromatic ring dihydrodiol / dehydrogenase, and the aromatic ring-cleaving dioxygenase are preferably used in the form of the above-described decomposing agent, and particularly preferably immobilized on a suitable insolubilizing carrier.

  In the decomposition method of the present invention, the aromatic ring dioxygenase of the present invention is preferably converted into an aromatic compound simultaneously with the aromatic ring dihydrodiol / dehydrogenase of the present invention or the aromatic ring-cleaving dioxygenase or after a certain period of time. It is done in contact.

  The aromatic compounds to be subjected to the aromatic compound decomposition method of the present invention are PCB, PAH, and various other aromatic compounds. And the high chlorine substitution body PCB with many substituted chlorine numbers (especially 5 chlorine substitution or more) can also be decomposed | disassembled suitably.

  Moreover, it can utilize suitably also when the aromatic compound used as the decomposition | disassembly object of this invention is contained in the contaminated soil or contaminated water by the said aromatic compound. Here, the contaminated soil or contaminated water is not particularly limited, and includes soil, bottom sediment, groundwater, river water, lake water, drainage from factories, etc. It can use suitably for these. In addition, since PCB and PAH can be decomposed simultaneously, it can be effectively used for a combined PCB and PAH contamination system.

  Contact of the enzyme of the present invention with the aromatic compound in the contaminated soil or contaminated water can be performed as long as the enzyme of the present invention is capable of exhibiting the resolution of aromatic compounds including PCB and PAH. It is possible to adopt. For example, in the case of degradation treatment of aromatic compounds in contaminated soil, in-situ bioremediation method in which contaminated soil etc. is repaired without excavation at the site, excavated contaminated soil etc., on-site or off-site The present invention can be applied to any case of a general bioremediation technique including an on-site / off-site bioremediation method and a slurry reactor method in which a remedial treatment is performed at a soil treatment plant in the world. As an input form of the aromatic ring dioxygenase of the present invention, for example, when purifying by adopting the above-mentioned in-situ bioremediation method, for example, two or more wells are excavated in a saturated layer in the vicinity of an underground contamination site, A method in which the aromatic ring dioxygenase of the present invention is injected into at least a part of groundwater pumped from a pumping well drilled downstream of the flow, and this is injected into an injection well drilled upstream and circulated. When adopting the site / off-site bioremediation method, for example, a method of spraying the aromatic ring dioxygenase of the present invention directly on soil, a method of charging through a pipe embedded in soil, etc. Either can be used suitably.

  In the case of the decomposition treatment of the aromatic compound in the contaminated water, for example, a reactor capable of passing water is constructed, and the contaminated water and the aromatic ring dioxygenase of the present invention are brought into contact in the reactor. There is no particular limitation on the form of the reactor, and either a batch system or a continuous system can be adopted.

    The amount of enzyme input is appropriately set according to the degree of contamination of contaminated soil and contaminated water, the type of contaminated medium, etc., and about 0.01 to 10% of the enzyme of the present invention is administered per contaminated soil or contaminated water. It is preferred to continue the process until it is reduced to any contamination level.

  Further, the treatment temperature is not particularly limited as long as the biological activity of the enzyme of the present invention can be maintained, but is preferably 20 to 37 ° C.

[10] Method for detecting aromatic compound-degrading bacterium according to the present invention The method for detecting an aromatic compound-degrading bacterium according to the present invention uses the aromatic ring dioxygenase, aromatic ring dihydrodiol dehydrogenase and aromatic ring-cleaving dioxygenase of the present invention as indicators. , To detect bacteria having aromatic compound resolution.

Here, the bacterium having an aromatic compound resolution to be detected is preferably a bacterium belonging to the genus Paenibacillus having PCB resolution, and particularly preferably the above-mentioned KBC101 strain is specifically detected. That is, since the enzyme of the present invention is a novel enzyme isolated from the above-mentioned KBC101 strain, the KBC101 strain using the aromatic ring dioxygenase, aromatic ring dihydrodiol dehydrogenase, and aromatic ring-cleaving dioxygenase of the present invention as indicators. Can be specifically detected.

  Moreover, it can utilize for the detection of the bacterium which expresses not only KBC101 stock | strain but the enzyme of high homology with the enzyme of this invention. It is understood that a bacterium expressing such an aromatic ring dioxygenase possesses an enzyme having an activity similar to that of the aromatic ring dioxygenase, aromatic ring dihydrodiol dehydrogenase, or aromatic ring cleavage dioxygenase of the present invention. . Therefore, it is understood that such bacteria can degrade a wide variety of aromatic compounds such as PCB and PAH including the high chlorine substitution PCB as well as the KBC101 strain.

  The method for detecting an aromatic compound-degrading bacterium according to the present invention preferably uses the expression of the aromatic ring dioxygenase gene, aromatic ring dihydrodiol / dehydrogenase gene, aromatic ring cleaved dioxygenase gene of the present invention as an index to determine the resolution of aromatic compounds. It is to detect bacteria having. Specifically, the gene of the present invention is detected by utilizing an oligonucleotide having a part or all of the base sequence of the gene of the present invention or an oligonucleotide complementary to the oligonucleotide. The detection method of the present invention is performed by detecting the gene of the present invention derived from the KBC101 strain contained in the sample by contacting the test sample with the oligonucleotide. For example, it can be performed by a hybridization method using the oligonucleotide as a probe. Moreover, it can carry out by PCR method using the said oligonucleotide as a primer.

  Examples of the test sample of the detection method of the present invention include environmental samples such as water and soil, bacterial cells separated from the environmental samples, or nucleic acids prepared from the environmental samples or bacterial cells. Examples of nucleic acids prepared from environmental samples include genomic DNA, total RNA, mRNA, and cDNA. Nucleic acid preparation from an environmental sample can be performed based on a known method.

  Oligonucleotides suitable for use in the detection method of the present invention are those having a part or all of the nucleotide sequence shown in SEQ ID NO: 1, particularly SEQ ID NO: 2, 4, 8 or 10. Alternatively, an oligonucleotide complementary to the oligonucleotide can be preferably used.

  Here, the term “complementary” means that the gene of the present invention to be detected and the oligonucleotide can specifically bind according to the base pairing rule to form a stable double-stranded structure. Here, not only perfect complementarity, but only a few nucleobases are sufficient as long as it is sufficient for the gene of the present invention to be detected and the oligonucleotide to form a stable duplex structure with each other. Even partial complementarity that conforms to the rules of base pairing is acceptable. Therefore, it means that 70% or more, preferably 80% or more, more preferably 90% or more, particularly preferably 95% or more of the base sequences are matched according to the base pairing rule between the base sequences.

  The oligonucleotide is not limited to DNA, RNA, or PNA, but is preferably DNA. In addition, the oligonucleotide is not limited to a single strand or a double strand, but is preferably a single strand.

  Further, the oligonucleotide may be a naturally occurring one or a chemically synthesized one. Therefore, when the gene fragment of the present invention isolated by a known cloning method from nature or the gene of the present invention to be detected has already been obtained from nature, it is cleaved by physical means or restriction enzyme digestion. It may be a fragment. In the case of chemical synthesis, a phosphoramidite method or the like can be used, and a commercially available automatic synthesizer can be used. The chemical synthesis is preliminarily designed based on sequence information, but can be easily performed by those skilled in the art. If necessary, it is possible to use primer design support software.

  Further, the size of the oligonucleotide is appropriately selected according to the detection method. When the oligonucleotide is used as a probe used in a hybridization reaction, it is not particularly limited, but it is necessary to have a certain base length to avoid non-specific hybridization. Preferably it is 10-100 bp, More preferably, it is 20-50 bp. In addition, when the oligonucleotide is used as a primer used in a nucleic acid amplification reaction, it is not particularly limited as long as the characteristic sequence of the gene of the present invention can be amplified, but preferably 10 to 50 bp, more preferably 15-30 bp.

The oligonucleotide is preferably labeled to increase detection sensitivity.
The type of label is not particularly limited. For example, fluorescent labels such as FITC, rhodamine, and Texas Red, radioisotope labels such as ³² P and ³ H, hapten labels such as biotin and digigosigenin, alkali Examples of the enzyme label include phosphatase and peroxidase.

  When detecting by hybridization, for example, the sample and the oligonucleotide probe are hybridized under stringent conditions, and the aromatic compound-degrading bacteria in the target sample, particularly KBC101, depending on the presence / absence and intensity of the hybridization. The presence or absence of the strain is detected, or the abundance is quantitatively detected. Hybridization itself can be performed by a known method. Examples include Southern, Northern blot hybridization, colony hybridization, and plaque hybridization. Here, examples of the stringent conditions include the conditions as described above.

  When detecting by a nucleic acid amplification method, for example, a target sample and an oligonucleotide primer are contacted, a nucleic acid amplification reaction is performed, the presence or absence of a specific amplification product, and the strength, aromatic compound-degrading bacteria in the target sample, especially The presence or absence of the KBC101 strain is detected, or the abundance is quantitatively detected. The nucleic acid amplification method can be performed by a method known per se. For example, polymerase chain reaction (PCR), ligase chain reaction (LCR), etc. are mentioned. When an amplification reaction is performed by the PCR method, the temperature setting, time, and cycle number are appropriately selected depending on the amount of template DNA to be used and the primer pair. Further, the annealing temperature in the PCR reaction is preferably adapted to the above stringent conditions.

  The primer set preferably has a complementary base sequence at both ends of the target sequence to be amplified. In addition, detection accuracy can be further improved by using a plurality of types of primers capable of amplifying different regions of the gene of the present invention as a primer and performing a plurality of amplification reactions.

  When quantitatively detecting the detection method of the present invention, for example, an aromatic compound degrading bacterium to be detected, particularly a genomic DNA standard sample of KBC101 strain, can be used simultaneously as a control. Compare the hybridization intensity or specific nucleic acid amplification product amount obtained using the control DNA sample with the hybridization intensity or specific nucleic acid amplification product amount obtained using the detection target sample, It is possible to calculate the amount of decomposing bacteria in the subject.

  The above-described method for detecting an aromatic compound-degrading bacterium of the present invention can be applied to a contamination purification system by bioremediation. That is, bioremediation conditions suitable for the purification of PCB and PAH can be set by detecting an aromatic compound-degrading bacterium having PCB and PAH resolution, particularly the KBC101 strain, by the above detection method.

  For example, in a contaminated site contaminated with PCB and PAH, the method for detecting an aromatic compound-decomposing bacterium of the present invention is used to detect an aromatic compound-degrading bacterium having PCB and PAH resolution, particularly the KBC101 strain. If the detection results confirm the presence of aromatic compound-degrading bacteria having PCB and PAH resolution, biostimulation is applied to determine the optimal growth conditions for aromatic compound-degrading bacteria having PCB and PAH resolution. To construct. Therefore, the method for detecting an aromatic compound-degrading bacterium of the present invention can be effectively used to determine whether biostimulation is possible. On the other hand, if the detection result does not detect an aromatic compound-degrading bacterium having PCB and PAH resolution, particularly KBC101 strain, or if the amount of living is small, bioaugmentation is applied and PCB and PAH resolution is provided. Add aromatic compound-degrading bacteria, especially KBC101 strain.

  Furthermore, the detection method of the present invention is useful for grasping the behavior and fate of degrading bacteria in both biostimulation and bioaugmentation applications. In addition, in order to ascertain whether or not the bacterium is expressing sufficient purification activity, that is, sufficient aromatic ring dioxygenase activity, aromatic ring dihydrodiol / dehydrogenase activity, and aromatic ring cleavage dioxygenase activity, Detection methods are useful. And based on a detection result, the environment of a purification | cleaning field can be adjusted so that a decomposing bacteria can demonstrate the maximum purification | cleaning capability. For example, it becomes possible to monitor the dynamics of degrading bacteria in the purification process and control the pollution purification system exemplified by the control of nutrient source dosage, bacterial dosage, temperature, pH, etc. based on the monitoring result. . That is, a highly efficient environmental pollution purification system can be constructed, and the purification processing time can be shortened.

  In addition, various types of information related to pollution purification including information obtained by using the above method can be stored in a database, and a purification system suitable for the situation of the pollution site can be constructed by using such a database. For example, based on information such as the type of contaminant at the contaminated site, the contamination status including the amount of contaminated material, and the physical properties of the contaminated site, the optimal input amount of degrading bacteria is set, and the set input amount Applies to contaminated sites. Thereby, a highly efficient environmental pollution purification system can be constructed, and the purification processing time can be shortened.

  The method for detecting an aromatic compound degrading bacterium of the present invention uses an antibody that specifically reacts with the aromatic ring dioxygenase, aromatic ring dioxygenase, aromatic ring dihydrodiol / dehydrogenase, or aromatic ring cleavage dioxygenase of the present invention. Can also be done. For example, an antibody specific for the enzyme of the present invention can be prepared based on a conventional method, and the sample can be detected based on a fluorescent antibody method or the like. Here, the antibody used is not particularly limited as long as it has an ability to specifically bind to the enzyme of the present invention, and may be a polyclonal antibody or a monoclonal antibody. In addition, antibodies modified by known techniques and antibody derivatives such as Fab fragments, single chain antibodies and the like can also be used.

[11] Biosensor for detecting aromatic compound-decomposing bacteria of the present invention The method for detecting aromatic compound-degrading bacteria of the present invention can be carried out by using a biosensor. As the biosensor of the present invention, a sensor having the above-described hybridization probe of the present invention immobilized on a support can be used. The biosensor of the present invention can be configured as a DNA microarray.

  The detection of the aromatic compound-degrading bacterium of the present invention is carried out, for example, by a hybridization obtained by contacting a labeled nucleic acid derived from a test sample with the biosensor for detecting an aromatic compound-degrading bacterium of the present invention. By analyzing the hybridization pattern, the abundance of aromatic compound-degrading bacteria having PCB and PAH resolution, particularly KBC101 strain, is calculated.

  The above-mentioned hybridization conditions can be used. As a control, for example, quantitative detection is possible by simultaneously fixing a genomic DNA standard sample of the KBC101 strain to a support.

  As said support body, a glass plate, a quartz plate, a silicon wafer etc. can be utilized, for example. As a method for immobilizing a nucleic acid molecule on a support, an appropriate method is selected depending on the type of nucleic acid molecule to be immobilized and the type of carrier. For example, when the nucleic acid molecule is DNA, a method of electrostatically binding to a solid support surface-treated with a polycation such as polylysine or polyethyleneimine using the charge of DNA, an amino group, an aldehyde group, etc. For example, a method of covalently bonding a nucleic acid molecule into which a modifying group such as an amine group, an aldehyde group, or an SH group is introduced can be used on the introduced support surface.

[12] Method for detecting aromatic compound of the present invention The method for detecting the aromatic compound of the present invention is a substrate for the aromatic ring dioxygenase, aromatic ring dioxygenase, aromatic ring dihydrodiol dehydrogenase, or aromatic ring-cleaving dioxygenase of the present invention. The presence of an aromatic compound is detected using the conversion reaction as an index.

  The aromatic ring dioxygenase, aromatic ring dioxygenase, aromatic ring dihydrodiol dehydrogenase, and aromatic ring-cleaving dioxygenase of the present invention are aromatics specifically exemplified by biphenyl as demonstrated in Examples 9 and 10 described below. It is an inducible enzyme expressed as an operon in the presence of a family compound. Therefore, examples of detection targets include aromatic compounds, particularly PCB and PAH.

  Substrate conversion ability for an aromatic compound is generated, for example, by contacting a sample with at least one of the above-mentioned aromatic ring dioxygenase, aromatic ring dihydrodiol dehydrogenase, and aromatic ring-cleaving dioxygenase and converting the substrate. This can be done by detecting the substance to be treated. For example, it can be carried out by detecting the formation of yellow HOPDA due to the decomposition of biphenyl. Thereby, the presence of biphenyl and PCB in the test sample can be detected. For example, it can be carried out by detecting oxygen consumption accompanying the initial oxidation of the aromatic ring dioxygenase of the present invention. However, it is not limited to these.

  Here, the test sample can be applied without particular limitation as long as it is a sample suspected of being mixed with an aromatic compound. Examples include environmental samples such as water and soil. Therefore, the method for detecting an aromatic compound of the present invention can be used for sensing pollutants in the field of environmental pollution, and those skilled in the art can use the detection results as a selection material for a pollution treatment method. Moreover, it can utilize for the monitoring of the purification | cleaning condition in the contamination purification process of an aromatic compound. This makes it possible to construct a highly efficient pollution purification system.

[13] A biosensor for detecting an aromatic compound of the present invention The method for detecting an aromatic compound of the present invention can be performed by using a biosensor. For example, the aromatic ring dioxygenase, aromatic ring dioxygenase, aromatic ring dihydrodiol dehydrogenase, and aromatic ring-cleaving dioxygenase of the present invention immobilized on an appropriate support can be used.

  Here, there is no restriction | limiting in particular as a support body, and there is no restriction | limiting in particular also about the fixing method, In addition to what was mentioned above, any well-known thing can be utilized. It can also be used as an enzyme column packed with an immobilized enzyme. For detection, an oxygen electrode, an optical fiber, a photodiode, or the like can be used.

  Examples of the present invention will be described below, but the scope of the present invention is not construed as being limited by these Examples.

(Example 1)
Construction of genome library of Paenibacillus genus KBC101 strain
Paenibacillus genus KBC101 strain (Accession number: FERM BP-08636) was inoculated into 5 ml of LB liquid medium and cultured at 30 ° C. for 2 days. After culturing, the cells were collected and the genomic DNA of KBC101 strain was prepared. To 50 mg of the prepared genomic DNA of KBC101 strain, 250 units of restriction enzyme Sau3AI was added and reacted at 37 ° C. for 30 minutes to perform partial digestion, and the cleavage state was confirmed by agarose gel electrophoresis. Next, the obtained Sau3AI partial cleavage fragment was ligated to Charomid9-36 (manufactured by Nippon Gene) cleaved with the restriction enzyme BamHI . For the connection, TaKaRaligation kit Ver.2 (manufactured by Takara Bio Inc.) was used. After ligation, packaging was performed using in vitro packaging kit LAMBDA INN (manufactured by Nippon Gene Co., Ltd.), and the transformant obtained by infecting E. coli DH5 strain was used as the genomic library of KBC101 strain.

(Example 2)
Cloning of the α-subunit gene fragment of the aromatic oxidase first oxidase From the alignment of the amino acid sequence of the α-subunit of the first oxidase of a known aromatic compound-degrading bacterium, a highly conserved region between the proteins of this group Based on the amino acid sequence in the region, the following degenerate primers were designed and synthesized.

Primer DO-1a:
5'-TG (TC) AG (TC) T (AT) (TC) CA (TC) GG (GATC) TGG-3 '(sequence recognition number 12)
Primer DO-1s:
5'-TC (GATC) (GA) C (GATC) GC (AG) AA (TC) TTCCA (AG) TT-3 '(SEQ ID NO: 13)

  Primer DO-1a is an 18-mer primer designed on the basis of the amino acid sequence of a region containing a cysteine residue and a histidine residue that is considered to be involved in iron-sulfur cluster formation near the N-terminus of the α subunit. Primer DO-1s is a 20-mer primer designed based on the amino acid sequence of the homologous region at the center of the α subunit.

  Using the genomic DNA prepared in Example 1 above as a template, PCR reaction was performed using Ex Taq Premix (Takara Bio) according to the manufacturer's instructions. As a result of the PCR reaction, a DNA fragment corresponding to the expected about 300 bp was amplified. The amplified DNA fragment was cloned into pGEM-T Easy vector using pGEM-T Easy vector system I (Promega) according to the manufacturer's instructions.

  In order to determine the base sequence of the cloned amplified fragment, the following primers were designed and synthesized based on the base sequence outside the insert DNA in the vector.

Primer UP-1s:
5'-GAAGTCATCATGACCGTTCTGCA-3 '(SEQ ID NO: 14)
Primer UP-2sr:
5'-AGCAGGGTACGGATGTGCGAGCC-3 '(SEQ ID NO: 15)

  The base sequence of the insert DNA was determined using the above primers. The base sequence was determined using PRISM-3100, 3700 (Applied Biosystems) based on the dye terminator method. The amino acid sequence was estimated based on the determined base sequence, and homology search of the deduced amino acid sequence was performed. As a result of the homology search, it showed significant homology to a part of the amino acid sequence of the α subunit of the first oxidase derived from aromatic compound-degrading bacteria. Therefore, it was suggested that the amplified DNA fragment obtained above was a partial fragment of the aromatic primary oxidase α subunit gene.

(Example 3)
Identification of clone containing coding sequence of primary oxidase α subunit of aromatic compound One of the α subunit genes obtained in Example 2 was added to the colony of the genomic library of KBC101 strain obtained in Example 1 above. Hybridization was performed using the amplified fragment considered to be a part as a probe. As a result, one positive clone was obtained. A DIG DNA labeling and detection kit (Roche) was used for probe labeling, hybridization, and signal detection from the label.

Example 4
Determination of the base sequence of the gene group encoding the aromatic ring primary oxidase From the above results, in order to determine the base sequence of the cloned DNA fragment, based on the sequence of the multicloning site outside the insert DNA in the vector, The primers were designed and synthesized.

Primer EcoRI-R:
5'-AAAATAGGCGTATCACGAGG-3 '(SEQ ID NO: 16)
Primer XbaI-F:
5'-TGACAGCTTGTATGTTTCTG-3 '(SEQ ID NO: 17)

The nucleotide sequence of the DNA insert was determined according to the procedure described in Example 2 using the primer. As the analysis of the base sequence progressed, new primers were sequentially designed, followed by the synthesis of primers and the base sequence analysis, and the base sequence of about 4.5 kb region was determined. The nucleotide sequence is shown as sequence recognition number 1. As a result of the analysis, five ORFs were found. The amino acid sequence was estimated based on the base sequence of ORF, and homology search was performed based on the estimated amino acid sequence. As a result of the homology search, the five ORFs were found to be α-subunit, β-subunit, ORF of unknown function, dihydrodiol dehydrogenase, 2,3-dihydroxybiphenyl 1, It showed significant homology to 2-dioxygenase. Therefore, these ORFs are named bphA1 , bphA2 , orf3 , bphB and bphC , respectively, their respective base sequences are shown in sequence recognition numbers 2, 4, 6, 8 and 10 in the sequence listing, and these gene groups are represented by bphA It is called a gene group. And the information-ized part of a 4.5 kb area | region is illustrated in FIG. The arrow indicates the transcription direction. The proteins encoded by the ORF are referred to as BphA1, BphA2, ORF3, BphB, and BphC, respectively, and the deduced amino acid sequences are shown in SEQ ID NOs: 3, 5, 7, 9, and 11 in the sequence listing.

(Example 5)
Homology search Based on the deduced amino acid sequence of the ORF confirmed in Example 4 above, a homology search with known proteins was performed. Blast was used as a homology search tool.

  The results are shown in Table 1.

As a result of the homology search, BphA1, BphA2, BphB and BphC showed homology with known BphA1, BphA2, BphB and BphC at an amino acid level of about 70 to 80%. From this, it was suggested that the gene group acquired in the said Example codes the enzyme involved in the oxidative degradation with respect to an aromatic compound, especially biphenyl. In addition, it is an enzyme capable of decomposing PCB for the first time in the genus Paenibacillus , and at the same time it is a novel oxidative degradation enzyme that is phylogenetically different from the conventionally known oxidative degradation enzymes of PCB-degrading bacteria. found.

(Example 6)
Construction of Expression Plasmids Containing bphA1A2 , bphB and bphC Regions In order to construct a plasmid containing the gene region identified in Example 4 above, the following primers were designed and synthesized.

bphA1A2 region amplification primer primer bphA1F-SalI:
5'-ACGC GTCGAC GGTGTACACCATGACTCAGC-3 '(SEQ ID NO: 18)
Primer bphA2R-XbaI:
5'-CTAG TCTAGA AACCATGGCAAACTCGTTG-3 '(SEQ ID NO: 19)

Primer primer for amplification of bphB region bphBF-EcoRI:
5'-G GAATT CCTGGATCAGACTACCTTGG-3 '(SEQ ID NO: 20)
Primer bphBR-BamHI:
5'-CG GGATCC CTCCATGTTAGGCGTGACG-3 '(SEQ ID NO: 21)

Primer primer for amplification of bphC region bphCF-EcoRI:
5'-G GAATTC GCCTGGGCTTATGTGTACC-3 '(sequence recognition number 22)
Primer bphCR-BamHI:
5'-CG GGATCC ATCCTTCAAGCTCTCCAGG-3 '(SEQ ID NO: 23)

  The underlined portion in the primer sequence indicates a restriction enzyme site.

Using the genomic DNA of KBC101 strain prepared in Example 1 above as a template, PCR reaction was performed using the above primers to obtain amplified fragments containing the entire regions. The PCR reaction was carried out in the same manner as in Example 2 above. The obtained amplified fragment was cleaved with a restriction enzyme. Specifically, the amplified fragments of the bphA region were cleaved with SalI and XbaI , and the amplified fragments of the bphB and bphC regions were cleaved with EcoRI and BamHI , respectively, to obtain restriction enzyme cleaved fragments. The obtained cleaved fragment was ligated to pHY300PLK (manufactured by Takara Bio Inc.) cleaved with each corresponding restriction enzyme to construct an expression plasmid. PHY300PLK is an E. coli - Bacillus shuttle vector. Here, bphA1A2, bphB, a plasmid containing the bphC area, referred to as respectively PAA1, pAB1, pAC1.

(Example 7)
Expression was prepared by introducing the above Example 6 to Paenibacillus validus JCM9077 strain plasmid bphA1A2, bphB, the PAA1, pAB1, pAC1 an expression plasmid containing the bphC gene, by electroporation Paenibacillus validus JCM9077 strain having no biphenyl resolution This was introduced to obtain a transformant. Here, electroporation was performed at 200Ω and 25 μF.

(Example 8)
Enzyme activity of bphA gene product
activity bphA gene product was carried out BphB, and by coupling the assay with bphC gene product by confirming visually HOPDA yellow resulting from biphenyl. Specifically, the transformant containing each gene prepared in Example 7 was inoculated into 5 mL of LB liquid medium containing 5 mg / L tetracycline and cultured at 30 ° C. overnight. After culturing, the cells were collected and suspended in 500 μL of 50 mM phosphate buffer to prepare resting cells. The prepared resting cells and an appropriate amount of biphenyl were mixed to confirm the formation of HOPDA. As a control, formation of HOPDA was also confirmed in the sample not containing the bphA gene product.

The results are shown in Table 2.

From the results of Table 2, accumulation of yellow substances derived from HOPDA was confirmed only when each gene product of bphA , bphB and bphC was included. That is, the enzyme activity derived from bphA was confirmed.

From the above results, it was confirmed that bphA is a gene encoding a protein having dioxygenase activity with respect to biphenyl.

Example 9
Or bph genes obtained by RT-PCR analysis above embodiment of bph genes of KBC101 strain that are involved in biphenyl degradation, or if you are involved, transcription of these genes is as a series of operon RT-PCR analysis was performed to see if it was done.

The RT-PCR reaction was performed using TaKaRa RNA PCR kit (AMV) ver3.0 (manufactured by Takara Bio Inc.) according to the attached manual. Specifically, the reaction was performed in two stages. First, as the first step, total RNA was prepared from KBC101 strain cultured in biphenyl-added medium and LB medium, and then cDNA was synthesized by reverse transcription reaction using the prepared RNA as a template. Total RNA was prepared using Sepasol-RNA I kit (Nacalai Tesque, Kyoto, Japan) according to the attached manual. As a second step, PCR was carried out to the cDNA synthesized in the first stage (including orf3) bphB from bphA1 from bphA2 and bphA2 as template and amplifying a bphC region from BphB. After completion of the amplification reaction, a part of the PCR product was subjected to agarose gel electrophoresis to confirm the amplified fragment. Here, the PCR reaction was carried out by repeating 30 cycles of a heat denaturation at 94 ° C. for 30 seconds, an annealing reaction at 55 ° C. for 30 seconds, and an extension reaction at 72 ° C. for 60 seconds as one cycle. It was. Table 3 shows the primers used for PCR amplification.

  As a control, in order to confirm whether genomic DNA was contaminated in the RNA prepared as described above, RT-PCR reaction was similarly performed on a system from which reverse transcriptase was excluded.

The results are shown in FIG.
Lanes 1 to 3 show the results of amplification of the bphA1 to bphA2 regions, lane 1 shows the control, lane 2 shows the culture in the LB medium, and lane 3 shows the culture in the biphenyl-added medium.
Lanes 4 to 6 show the results of amplification of the bphA2 to bphB region (including orf3 ), lane 4 is the control, lane 5 is the culture in the LB medium, and lane 6 is the culture in the biphenyl-added medium. Show.
Lanes 7 to 9 show the results of amplification of the bphB to bphC region, lane 7 shows the results of control, lane 8 shows the culture in the LB medium, and lane 9 shows the culture in the biphenyl-added medium.

From the results shown in FIG. 3, amplified fragments of the expected size between the genes were observed only when cultured in a biphenyl-added medium (comparison between lanes 3, 6, 9 and other lanes). From the above, it was confirmed that the gene region from bphA1 to bphC was inducibly expressed as one operon in the KBC101 strain. Furthermore, it was also found that expression was regulated by biphenyl. That is, the expression of bph genes of the present invention is suppressed in the absence of biphenyl, be derepressed was suggested by the addition of biphenyl.

(Example 10)
Primer extension reaction analysis of bph gene group of KBC101 strain In Example 9 above, primer extension reaction analysis was performed to confirm the 5 ′ end structure of RNA whose operon structure was confirmed. The primer extension reaction was carried out using the RNA prepared in Example 9 as a template and the method described in Kulakov et al. (Web-Type Evolution of Rhodococcus Gene Clusters Associated with Utilization of Naphthalene, Applied and Environmental Microbiology, April 2005, No. 71 Vol. 4, No. 1, pp. 1754-1764). Here, the reagents used are shown below. All were made by Invitrogen.

Superscript III RNaseH Reverse transcriptase
5 x First strand buffer
dNTP mix
Ribonuclease inhibitor (RNase out): Recombinant type

Primer labeled on the 5 'side with Well Red Dye
5′-CTGCCCGCGATAATTTGCTTTCGTCCG-3 ′ (SEQ ID NO: 30) was used. The label was outsourced to Proligo.

The results are shown in FIG.
FIG. 4 (A) shows the results when cultured in a biphenyl-added medium, and FIG. 4 (B) shows the results when cultured in an LB medium.

  As a result of FIG. 4, when the KBC101 strain was cultured in a biphenyl-added medium, the transcription start point of the bph gene group was confirmed 190 bp upstream (FIG. 5) from the start codon of bphA1 (FIG. 4 (A)). On the other hand, the transcription start point of the bph gene group could not be confirmed during the culture in the LB medium (FIG. 4B). This result is consistent with the result of Example 9, and a result strongly suggesting that the series of bph gene groups obtained in the present invention is regulated by biphenyl.

  In addition, since the transcription start point has been determined, it becomes important information for the analysis of regions involved in transcriptional regulation, and elucidation of the transcriptional control mode of genes encoding enzymes involved in aromatic compound oxidative degradation pathways. It is possible to proceed.

Diagram showing the biphenyl oxidative degradation pathway Evolutionary phylogenetic tree of aromatic ring dioxygenase Information structure diagram of aromatic ring oxidative degradation enzyme gene on 4.5 kb DNA fragment and electrophoretic diagram showing the result of Example 9 in which RT-PCR analysis of bph gene group of KBC101 strain was performed (A) KBC101 strain diagram showing the results (biphenyl-supplemented medium) of Example 10 was subjected to primer extension reaction analysis of bph genes of implementation was performed primer extension reaction analysis of bph genes of KBC101 strain (B) Example The figure which shows the result (LB culture medium) of 10 Sequence diagram showing the transcription start point identified by Example 10 in which primer extension reaction analysis of bph gene group of KBC101 strain was performed

Claims (22)

An aromatic compound oxidative degradation operon containing the following DNA (I) or (II):
(I) DNA comprising the base sequence shown in sequence recognition number 1 in the sequence listing
(II) comprising DNA that hybridizes with DNA consisting of the base sequence shown in sequence recognition number 1 in the sequence listing under stringent conditions and encodes a protein having oxidative degradation activity against aromatic compounds DNA forming an aromatic compound oxidative degradation operon   The aromatic compound oxidative degradation operon according to claim 1, comprising DNA encoding a protein having an oxidative degradation activity for at least one of polychlorinated biphenyls and polycyclic aromatic hydrocarbons. A gene encoding the protein shown in (A) or (B) below.
(A) a protein having the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing (B) substitution, deletion, insertion of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing, or A protein comprising an amino acid sequence including an addition and having a function as an α subunit of an aromatic ring dioxygenase A gene comprising the following DNA (a) or (b):
(A) DNA comprising the base sequence shown in sequence recognition number 2 in the sequence listing
(B) a DNA that hybridizes with a DNA comprising the nucleotide sequence shown in SEQ ID NO: 2 in the sequence listing under stringent conditions and encodes a protein having a function as an α subunit of an aromatic ring dioxygenase   The gene according to claim 3 or 4, which encodes a protein having a function as an α subunit of an aromatic ring dioxygenase having a degrading activity for at least one of polychlorinated biphenyls and polycyclic aromatic hydrocarbons. A gene encoding the protein shown in (C) or (D) below.
(C) a protein having the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing (D) substitution, deletion, insertion of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing, or A protein comprising an amino acid sequence including an addition and having a function as a β subunit of an aromatic ring dioxygenase A gene comprising the following DNA (c) or (d):
(C) DNA consisting of the base sequence represented by sequence recognition number 4 in the sequence listing
(D) a DNA that hybridizes with a DNA comprising the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing under stringent conditions and encodes a protein having a function as a β subunit of an aromatic ring dioxygenase   The gene according to claim 6 or 7, which encodes a protein having a function as a β subunit of an aromatic ring dioxygenase having a degrading activity on at least one of polychlorinated biphenyls and polycyclic aromatic hydrocarbons. A gene encoding the protein shown in (E) or (F) below.
(E) a protein having the amino acid sequence shown in SEQ ID NO: 9 in the sequence listing (F) substitution, deletion, insertion of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 9 in the sequence listing, or A protein comprising an amino acid sequence including an addition and having a function as an aromatic ring dihydrodiol dehydrogenase A gene comprising DNA of (e) or (f) below.
(E) DNA comprising the base sequence shown in SEQ ID NO: 8 in the sequence listing
(F) a DNA that hybridizes with a DNA comprising the base sequence shown in SEQ ID NO: 8 in the sequence listing under stringent conditions and encodes a protein having a function as an aromatic ring dihydrodiol dehydrogenase   The gene according to claim 9 or 10, which encodes a protein having a function as an aromatic ring dihydrodiol dehydrogenase having a decomposition activity on at least one of polychlorinated biphenyls and polycyclic aromatic hydrocarbons. A gene encoding a protein shown in (G) or (H) below.
(G) a protein having the amino acid sequence shown in SEQ ID NO: 11 in the sequence listing (H) substitution, deletion, insertion of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 11 in the sequence listing, or A protein comprising an amino acid sequence including an addition and having a function as an aromatic ring-cleaving dioxygenase A gene comprising DNA of (g) or (h) below.
(G) DNA comprising the base sequence shown in SEQ ID NO: 10 in the sequence listing
(H) a DNA that hybridizes with a DNA comprising the nucleotide sequence shown in SEQ ID NO: 10 in the sequence listing under a stringent condition and encodes a protein having a function as an aromatic ring-cleaving dioxygenase   The gene according to claim 12 or 13, which encodes a protein having a function as an aromatic ring-cleaving dioxygenase having a decomposition activity on at least one of polychlorinated biphenyls and polycyclic aromatic hydrocarbons. An aromatic ring dioxygenase comprising a protein complex comprising the following (A) or (B) and (C) or (D) protein.
(A) a protein having the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing (B) substitution, deletion, insertion of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing, or Protein (D) sequence recognition number of protein (D) sequence list which has amino acid sequence shown in sequence recognition number 5 of protein (C) sequence list which consists of amino acid sequence including addition and has a function as α-subunit of aromatic ring dioxygenase 5. A protein comprising an amino acid sequence including substitution, deletion, insertion, or addition of one or several amino acid residues in the amino acid sequence shown in 5 and having a function as a β subunit of aromatic ring dioxygenase   The aromatic ring dioxygenase according to claim 15, which has a resolution for at least one of polychlorinated biphenyls and polycyclic aromatic hydrocarbons. An aromatic ring dihydrodiol dehydrogenase comprising the following protein (E) or (F):
(E) a protein having the amino acid sequence shown in SEQ ID NO: 9 in the sequence listing (F) substitution, deletion, insertion of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 9 in the sequence listing, or A protein comprising an amino acid sequence including an addition and having a function as an aromatic ring dihydrodiol dehydrogenase   The aromatic ring dihydrodiol dehydrogenase according to claim 17, which has a resolution for at least one of polychlorinated biphenyls and polycyclic aromatic hydrocarbons. An aromatic ring-cleaving dioxygenase comprising the following protein (G) or (H):
(G) a protein having the amino acid sequence shown in SEQ ID NO: 11 in the sequence listing (H) substitution, deletion, insertion of one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 11 in the sequence listing, or A protein comprising an amino acid sequence including an addition and having a function as an aromatic ring-cleaving dioxygenase   The aromatic ring-cleaving dioxygenase according to claim 19, which has a resolution for at least one of polychlorinated biphenyls and polycyclic aromatic hydrocarbons.   The aromatic ring dioxygenase according to claim 15 or 16, at least one selected from the aromatic ring dihydrodiol dehydrogenase according to claim 17 or 18, and the aromatic ring cleavage dioxygenase according to claim 19 or 20. And a method for decomposing an aromatic compound by contacting the aromatic compound. At least one of the base sequence shown in sequence recognition number 2 in the sequence listing, the base sequence shown in sequence recognition number 4 in the sequence listing, the base sequence shown in sequence recognition number 8 and the base sequence shown in sequence recognition number 10 in the sequence listing Bacterial detection for detecting Paenibacillus genus KBC101 strain deposited under accession number FERM BP-08636 using an oligonucleotide having a part or all of the base sequence in the base sequence, or an oligonucleotide complementary to the oligonucleotide Method.