WO2009125462A1 - Microorganisme apte à décomposer des composés aromatiques et procédé de décomposition de composés aromatiques l'utilisant - Google Patents

Microorganisme apte à décomposer des composés aromatiques et procédé de décomposition de composés aromatiques l'utilisant Download PDF

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WO2009125462A1
WO2009125462A1 PCT/JP2008/056861 JP2008056861W WO2009125462A1 WO 2009125462 A1 WO2009125462 A1 WO 2009125462A1 JP 2008056861 W JP2008056861 W JP 2008056861W WO 2009125462 A1 WO2009125462 A1 WO 2009125462A1
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aromatic compound
strain
microorganism
decomposing
aromatic
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PCT/JP2008/056861
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Japanese (ja)
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祐子 向井
八千代 川本
豊一 横田
学 佐見
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アサヒビール株式会社
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    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
    • A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
    • A62D2101/20—Organic substances
    • A62D2101/22—Organic substances containing halogen
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the present invention is characterized by cleaving a heterocycle when a substrate is an aromatic heterocycle by introducing two hydroxyl groups into the aromatic ring, and cleaving an ether bond when the substrate is phenol ether.
  • the present invention relates to a microorganism having a gene encoding a characteristic enzyme and a novel method for decomposing aromatic compounds including dioxins using the microorganism.
  • One method of purifying soil and riverbed mud contaminated with dioxins is bioaugmentation.
  • This is a method for purifying dioxins by spraying microorganisms that decompose dioxins on contaminated soil, and the microorganisms to be sprayed are white-rot fungi (Patent Document 1 “Method for Degrading Chlorinated Dioxins”)
  • Patent Document 2 The microorganism must have a dioxin-degrading ability, such as the dioxin-degrading bacterium Sphingomonas sp. KA1 strain (FERM P-19744).
  • Such a decomposition method is cheaper in processing cost than a generally known physicochemical purification method.
  • the period required for the purification is generally long, and in order to maintain the decomposition ability of microorganisms. , Soil aeration, moisturizing, and application of nutrients.
  • the degrading enzyme is almost always an inducing enzyme, it is necessary to realize a state in which the microbial enzyme is induced in the environment.
  • dioxin degrading enzyme is induced in the presence of carbazole, so that the bacterium is cultured with carbazole, and dioxin is effectively decomposed by adding carbazole to contaminated soil.
  • culturing and spraying with carbazole to increase the degradation effect not only increases the treatment cost, but may cause new environmental contamination by carbazole, which is a major problem in practical use.
  • CARDO carbazole 1,9a-dioxygenase
  • CARDOI and CARDOII are multi-component enzymes composed of ferredoxin and ferredoxin reductase in addition to the main body (terminal oxygenase), and the genes encoding them are all found on the plasmid pCAR3 held by the KA1 strain.
  • the terminal oxygenase genes are called carAaI and carAaII, respectively, and the homology between them is about 75% at the amino acid level. As a result of the examinations so far, it has been revealed that they show almost the same activity.
  • the operon formed by these gene groups is called a carbazole degrading operon.
  • the operon promoter site is normally suppressed by the repressor protein CarRI produced by the transcriptional control gene carRI.
  • the problem to be solved by the present invention is to provide a microorganism that produces a dioxin-degrading enzyme without requiring an inducer such as carbazole that may cause environmental pollution.
  • the present inventors succeeded in breeding a mutant strain of KA1 strain that is always produced by dioxin degrading enzyme. That is, the KA1 strain was subcultured in a culture system containing carbazole, which is considered to have been released from suppression of CARDO expression. As a result, mutant strains KA1G1 and KA1G2 carrying plasmids having a size different from that of pCAR3 were obtained at the 63rd passage. In these mutants, carAaI was deleted from the two cardo terminal oxygenase genes carAaI and carAaII encoded on pCAR3.
  • the present inventors have also succeeded in breeding mutant strains that are more active than the KA1G1 and KA1G2 strains while constantly producing dioxin degrading enzymes. That is, the KA1G2 strain was subcultured in a culture system containing carbazole, and as a result, a mutant strain KA1G2-13012 having a plasmid of a size different from KA1G2 was obtained at the number of passages 67 times. While this mutant strain maintained the property of constitutive expression of CARDO, the CARDO activity was about twice that of the KA1 strain at the time of induction.
  • KA1G1 strain, KA1G2 strain and KA1G2-13012 strain were deposited internationally at the National Institute of Advanced Industrial Science and Technology, Patent Microorganism Depositary, respectively, with accession numbers FERM BP-10861 (deposit date: July 19, 2006), FERM BP -10862 (Deposit date: July 19, 2006) and FERM BP-10954 (Deposit date: March 4, 2008).
  • the present invention has the following gist.
  • a method for decomposing an aromatic compound comprising using a microorganism having a carAaI or carAaII derived from a Sphingomonas sp. Strain KA1 and a promoter whose expression suppression mechanism is canceled by mutation or deletion of the transcriptional control gene carRI.
  • the degradation method according to item (1), wherein the microorganism is a Sphingomonas sp. KA1 strain or a strain derived therefrom.
  • Item 6 The method for decomposing an aromatic compound according to any one of Items (1) to (3), wherein the ether bond of phenol ether is cleaved.
  • (6) 6.
  • the decomposition method according to claim 1. (9) Sphingomonas sp. KA1G1 strain (FERM BP-10861), an aromatic compound-degrading microorganism. (10) An aromatic compound-degrading microorganism which is Sphingomonas sp. KA1G2 strain (FERM BP-10862).
  • the aromatic compound is at least one selected from benzene, toluene, xylene, naphthalene, biphenyl, anthracene, dibenzofuran, dibenzothiophene, fluorene, fluoranthene, xanthene, phenoxathiin, and diphenyl ether, any of (12) to (15) Decomposition method according to the above.
  • the microorganism that constitutively expresses the dioxin-degrading enzyme of the present invention has the following advantages compared to the microorganism that is induced and expressed by conventional carbazole. (1) Any medium may be used for the growth as long as the microorganism can grow, and the bacterial cells can be produced at low cost. (2) There is no worry of causing new environmental pollution by the inducer.
  • various pollutants in the soil for example, aromatic compounds such as dioxins, benzene, toluene, naphthalene, anthracene, and fluoranthene can be efficiently purified. .
  • the aromatic compound to be decomposed according to the present invention is selected from chlorinated dioxins or benzene, toluene, xylene, naphthalene, biphenyl, anthracene, dibenzofuran, dibenzothiophene, fluorene, fluoranthene, xanthene, phenoxathiin and diphenyl ether. At least one.
  • the microorganism used may be a promoter that does not require induction and that has carAaI or carAaII derived from the Sphingomonas sp. KA1 strain under its control.
  • a promoter that does not require induction is a promoter in which transcription of mRNA is not repressed even if the strain is cultured under any culture conditions.
  • the nptII promoter, and the like It includes a promoter that is in a state in which induction suppression is not caused by mutation or deletion.
  • the gene expressed under the control of such a promoter may be any gene as long as it contains the enzyme genes carAaI or carAaII that are the main components of dioxin degradation.
  • these enzymes are combined with ferredoxin that conducts electrons and ferredoxin reductase. It is better to have it.
  • ferredoxin examples include carAcI and carAcII possessed by the KA1 strain.
  • ferredoxin reductase include fdrI and fdrII possessed by the KA1 strain.
  • Sphingomonas sp. KA1G1, KA1G2 and KA1G2-13012 are mutant strains obtained by mutation from the dioxin-degrading bacterium Sphingomonas sp. KA1 in order to satisfy these conditions.
  • CARDO thus expressed promotes the spontaneous cleavage of the heterocycle by introducing two hydroxyl groups into the benzene ring of carbazole and produces 2′-aminobiphenyl-2,3-diol.
  • This enzyme has a relatively wide substrate specificity and can introduce two hydroxyl groups into an aromatic compound having a similar reaction site structure.
  • the heteroatom is an oxygen atom instead of a nitrogen atom as in dibenzofuran
  • the heterocycle is cleaved in the same manner as carbazole.
  • dibenzo-p-dioxin and its chlorinated compound the corresponding cleavage compound is produced.
  • the decomposed bacteria and the pollutant may be contacted by spraying the decomposing bacteria over the contaminated sites.
  • the spraying method may be any form such as spraying the culture solution as it is if the degrading bacteria are alive, or immobilizing on activated carbon, diatomaceous earth, or the like.
  • the treatment temperature may be in a range called normal temperature, but is preferably 10 ° C. to 35 ° C., more preferably 25 ° C. to 30 ° C.
  • the degrading bacteria do not always survive in the contaminated area for a long time and do not always continue to decompose the pollutants. It is possible to further purify by repeatedly spraying the decomposed bacteria cultured on the surface.
  • Two mutant strains KA1G1 and KA1G2 were isolated from the main culture. When plasmids were extracted from each of them and subjected to DNA sequencing, it was found that both mutants had a large portion of plasmid pCAR3 possessed by KA1 strain deleted at least about 60 kb.
  • the deletion site included an operon containing carAaI and a catechol-degrading operon responsible for metabolism downstream of carbazole.
  • KA1G2 strain a portion not related to the carbazole degradation gene was further deleted from the KA1G1 strain by about 300b.
  • the mutant strain was able to grow using carbazole as the sole carbon source because the operon containing another carbazole-degrading gene carAaII on the plasmid and on the chromosome downstream of catechol. This is because a gene group exists. Furthermore, as a result of intensive studies, it was found that the deletion site contained carRI that regulates the transcription of operons including carAaI and carAaII. It was inferred that this gene was lost because carAaII was constitutively expressed.
  • Dioxin-degrading gene expression level (activity) When CARDO acts on naphthalene, it produces cis-1,2-dihydroxy-1,2-dihydronaphthalene.
  • CARDO acts on naphthalene
  • naphthalene it produces cis-1,2-dihydroxy-1,2-dihydronaphthalene.
  • the KA1 strain, KA1G1 strain, KA1G2 strain, and KA1G2-13012 strain were compared in CARDO activity.
  • the results are shown in Table 1.
  • the unit unit used here represents the ng number of cis-1,2-dihydroxy-1,2-dihydronaphthalene produced in one hour in this reaction system.
  • the KA1G1 and KA1G2 strains showed a production rate of about 40% of the KA1 strain induced with carbazole without adding carbazole.
  • the KA1G2-13012 strain showed about twice the production rate of the KA1 strain. From these results, it was confirmed that all these mutants expressed constitutive expression of CARDO.
  • the KA1G1 and KA1G2 strains are less active than the KA1 strain in which CARDO was induced because one of the two enzyme genes was deleted in the mutant strain. it is conceivable that.
  • the high activity of the KA1G2-13012 strain is considered to be due to duplication of carAaII in the genome. It is not clear whether the effect is only due to the large number of genes or whether the transcriptional activity is also increased.
  • Table 2 shows the ratio (%) of the recovered remaining substrate. All strains degraded the test substance well. In particular, in the KA1G2-113012 strain, many substances were degraded at a high degradation rate. On the other hand, the detected decomposition products were dibenzofuran, dibenzothiophene and diphenyl ether, except for compounds to dihydrodiol, dibenzofuran to trihydroxybiphenyl and dibenzofurandihydrodiol, dibenzothiophene to dibenzothiophene dihydrodiol and dibenzothiophene-5- To oxide, diphenyl ether was converted to catechol and phenol. As a result, it was confirmed that these mutant strains degraded the aromatic compound by the same mechanism as the parent strain KA1.
  • tetrachlorinated dioxin 5 ml of 50 mM phosphate buffer (pH 7.2) was placed in a 30 ml Erlenmeyer flask, and cultured in the same manner as in Example 3.
  • 10 ng of 2,3,7,8-TeCDD was added and stirred at 30 ° C. for 24 hours. Residual substrates were extracted and quantified from these samples, and the decomposition rates as shown in the table below were obtained.
  • As a control cells obtained by autoclaving the KA1G1 strain at 121 ° C. for 5 minutes were used, and the degradation rate was calculated based on the ratio to the recovered amount from this control.
  • any of the obtained mutant strains decomposes 2,3,7,8-TeCDD even when a culture medium without carbazole is used for the culture.
  • the KA1G2-113012 strain decomposed the added 2,3,7,8-TeCDD by 60% or more in 24 hours and was shown to have a very strong 2,3,7,8-TeCDD degradation ability.
  • the amount of dioxin added is 10 ng for tetrachlorinated dioxins, pentachlorinated dioxins, tetrachlorinated dibenzofurans, pentachlorinated dibenzofurans, and coplanar PCBs, 50 ng for octachlorinated dioxins and octachlorinated dibenzofurans, and other dioxins for dioxins 20 ng.
  • the residual substrate was extracted and quantified, and the residual ratio as shown in FIG. 1 was obtained.
  • As a control microbial cells obtained by autoclaving the KA1G1 strain at 121 ° C. for 5 minutes were used, and the residual rate was calculated as a ratio to the recovered amount from this control.
  • the KA1, KA1G1 and KA1G2-13012 strains can decompose various toxic chlorinated dioxins other than 2,3,7,8-TeCDD. Particularly high in 2,3,7,8-TeCDF, 3,4,4 ′, 5-TeCB, 3,3 ′, 4,4′-TeCB, 3,3 ′, 4,4 ′, 5-PeCB, etc. The decomposition rate could be obtained.
  • the KA1G2 strain not used in this test has the same degradation activity as the KA1G1 strain.
  • TeCDF Tetrachlorodibenzofuran PeCDF: Pentachlorodibenzofuran HxCDF: Hexachlorodibenzofuran HpCDF: Heptachlorodibenzofuran OCDF: Octachlorodibenzofuran TeCDD: Tetrachlorodibenzo-p-dioxin PeCDD: Pentachlorodibenzo-p-dioxin HxCDD: Hexachlorodibenzo-p-dioxin HpCDD: Heptachlorodibenzo-p-dioxin OCDD: Octachlorodibenzo-p-dioxin TeCB: Tetrachlorobiphenyl PeCB: Pentachlorobiphenyl HxCB: Hexachlorobiphenyl HpCB: Heptachlorobiphenyl
  • the present invention can decompose aromatic compounds without causing environmental pollution, the present invention is extremely useful as an environmental purification technique.

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Abstract

L'invention vise à fournir un microorganisme apte à induire une enzyme par laquelle des composés aromatiques incluant de la dioxine peuvent être décomposés sans utiliser de carbazole susceptible de poser un risque de pollution environnementale. A cet effet, l'invention porte sur un procédé de décomposition de composés aromatiques caractérisé en ce qu'il consiste à utiliser un microorganisme qui possède un promoteur ne nécessitant pas d'induction et carAaI ou carAaII provenant de la souche KAI de Sphingomonas sp., et qui est de cette manière sous contrôle.
PCT/JP2008/056861 2008-04-07 2008-04-07 Microorganisme apte à décomposer des composés aromatiques et procédé de décomposition de composés aromatiques l'utilisant WO2009125462A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012111875A1 (fr) * 2011-02-15 2012-08-23 이화여자대학교 산학협력단 Microorganisme sphingomonas sp. inédit et procédé de décomposition du méthane ou de composés dégageant une odeur l'utilisant
KR101509861B1 (ko) 2012-08-24 2015-04-07 현대자동차주식회사 에어컨에서 나는 쉰 냄새의 검출 방법 및 쉰 냄새 재현 방법과 이에 제조된 쉰 냄새 조성물
CN107058155A (zh) * 2016-12-22 2017-08-18 江苏省农业科学院 一株二苯醚降解菌及其应用

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012111875A1 (fr) * 2011-02-15 2012-08-23 이화여자대학교 산학협력단 Microorganisme sphingomonas sp. inédit et procédé de décomposition du méthane ou de composés dégageant une odeur l'utilisant
KR101241546B1 (ko) 2011-02-15 2013-03-11 이화여자대학교 산학협력단 신규한 스핑고모나스 속 미생물 및 이를 이용한 메탄 또는 악취유발 화합물의 분해방법
JP2013532967A (ja) * 2011-02-15 2013-08-22 イファ ユニバーシティ−インダストリー コラボレーション ファウンデーション 新規なスフィンゴモナス属微生物及びそれを用いたメタン又は悪臭誘発化合物の分解方法
US8748154B2 (en) 2011-02-15 2014-06-10 Ewha University-Industry Collaboration Foundation Sphingomonas sp. microorganism and method for decomposing methane or odor-producing compounds using the same
KR101509861B1 (ko) 2012-08-24 2015-04-07 현대자동차주식회사 에어컨에서 나는 쉰 냄새의 검출 방법 및 쉰 냄새 재현 방법과 이에 제조된 쉰 냄새 조성물
CN107058155A (zh) * 2016-12-22 2017-08-18 江苏省农业科学院 一株二苯醚降解菌及其应用
CN107058155B (zh) * 2016-12-22 2019-06-07 江苏省农业科学院 一株二苯醚降解菌及其应用

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