DNA fragment carrying toluene monooxygenase, gene, recombinant plasmid, transformed microorganism, method for degrading chlorinated aliphatic hydrocarbon compounds and aromatic compounds, and method for environmental remediation
AssigneeCanon Kabushiki Kaisha
InventorsTetsuya Yano, Tsuyoshi Nomoto, Takeshi Imamura
Life of this patent
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Abstract
A recombinant DNA is constructed by using a toluene monooxygenase gene isolated from Ralstonia eutropha strain TB64 and employed to provide the transformant which can express toluene monooxygenase useful for cleaning of aqueous media such as drain and waste water containing halogenated aliphatic hydrocarbon compounds or aromatic compounds, for remediation of soil polluted with such compounds, and cleaning of air (gas phase) polluted with volatile organic chlorine compounds.
Claims50
A DNA fragment of about 5.3 Kb containing a toluene monooxygenase gene, having 3 BamHI, 1 ClaI, 1 EcoRI, 3 KpnI, 2 NcoI, 2 NspV, 2 ScaI, 2 SmaI, 2 SphI, 1 StuI, 0 DraI, 0 EcoRV, 0 HindIII, OHpaI, 0 NdeI, 0 PvuII, 0 ScaI, 0 Sse83871, 0 XbaI, 0 XhoI restriction sites, and having a restriction map of:
- 3
A DNA fragment having a nucleotide sequence of SEQ ID NO:
- 1 in the Sequence Listing.
A DNA fragment having a nucleotide sequence of SEQ ID NO:
- 1 with deletion, substitution, and/or addition of one or more nucleotides encoding a protein having a toluene monooxygenase activity.
A DNA fragment containing a portion encoding a toluene monooxygenase, the portion comprising a region encoding a polypeptide TomL having an amino acid sequence of SEQ ID NO:
- 3, a region encoding a polypeptide TomM having an amino acid sequence of SEQ ID NO:
- 4, a region encoding a polypeptide TomN having an amino acid sequence of SEQ ID NO:
- 5, a region encoding a polypeptide TomO having an amino acid sequence of SEQ ID NO:
- 6, and a region encoding a polypeptide TomP having an amino acid sequence of SEQ ID NO:
- 7 of the Sequence Listing, and the regions are aligned so that expressed TomL-TomP polypeptides can form an active monooxygenase protein.
A DNA fragment comprising a region encoding a polypeptide TomK which has an amino acid sequence of SEQ ID NO:
- 2 and a property to enhance the toluene monooxygenase activity of a protein comprised of at least TomL to TomP;
- or a region encoding a variant TomK in which the amino acid sequence of SEQ ID NO:
- 2 is altered with the proviso that the property to enhance the toluene monooxygenase activity is not impaired.
A recombinant DNA comprising a vector, a promoter, and a DNA fragment,
- wherein the vector and the promoter are functionally ligated to the DNA fragment to enable expression of the toluene monooxygenase encoded by the DNA fragment,
- the DNA fragment comprises a region encoding a polypeptide TomL having an amino acid sequence of SEQ ID NO:
- 3, a region encoding a polypeptide TomM having an amino acid sequence of SEQ ID NO:
- 4, a region encoding a polypeptide TomN having an amino acid sequence of SEQ ID NO:
- 5, a region encoding a polypeptide TomO having an amino acid sequence of SEQ ID NO:
- 6, and a region encoding a polypeptide TomP having an amino acid sequence of SEQ ID NO:
- 7 of the Sequence Listing.
A recombinant DNA comprising a vector, a promoter, a first DNA fragment, and a second DNA fragment;
- wherein the first DNA encodes a toluene monooxygenase and comprises a region encoding a polypeptide TomL having an amino acid sequence of SEQ ID NO:
- 3, a region encoding a polypeptide TomM having an amino acid sequence of SEQ ID NO:
- 4, a region encoding a polypeptide TomN having an amino acid sequence of SEQ ID NO:
- 5, a region encoding a polypeptide TomO having an amino acid sequence of SEQ ID NO:
- 6, and a region encoding a polypeptide TomP having an amino acid sequence of SEQ ID NO:
- 7 of the Sequence Listing,
- the second DNA fragment encodes a polypeptide TomK having an amino acid sequence of SEQ ID NO:
- 2 and a property to enhance the toluene monooxygenase activity of a protein comprised of at least TomL to TomP;
- or encodes a variant TomK polypeptide in which the amino acid sequence of SEQ ID NO:
- 2 is altered with the proviso that the property to enhance the toluene monooxygenase activity is not impaired;
- the first DNA fragment is functionally linked to the promoter to express the toluene monooxygenase activity, and the second DNA fragment is functionally linked to the promoter to express the property to enhance the activity of the toluene monooxygenase encoded by the first DNA fragment.
A recombinant DNA comprising a vector, a first promoter and a first DNA fragment, and a second promoter and a second DNA fragment;
- wherein the first DNA comprises a region encoding a polypeptide TomL having an amino acid sequence of SEQ ID NO:
- 3, a region encoding a polypeptide TomM having an amino acid sequence of SEQ ID NO:
- 4, a region encoding a polypeptide TomN having an amino acid sequence of SEQ ID NO:
- 5, a region encoding a polypeptide TomO having an amino acid sequence of SEQ ID NO:
- 6, and a region encoding a polypeptide TomP having an amino acid sequence of SEQ ID NO:
- 7 of the Sequence Listing,
- the second DNA fragment encodes a polypeptide TomK having an amino acid sequence of SEQ ID NO:
- 2 and a property to enhance the toluene monooxygenase activity of a protein comprised of at least TomL to TomP;
- or encodes a variant TomK polypeptide in which the amino acid sequence of SEQ ID NO:
- 2 is altered with the proviso that the property to enhance the toluene monooxygenase activity is not impaired;
- the first DNA fragment is functionally linked to the first promoter to express the toluene monooxygenase activity, and the second DNA fragment is functionally linked to the second promoter to express the property to enhance the activity of the toluene monooxygenase encoded by the first DNA fragment.
A transformant obtainable by introducing into a host microorganism a recombinant DNA which comprises a vector, a promoter, and a DNA fragment, and the vector and the promoter are functionally ligated to the DNA fragment to enable expression of the toluene monooxygenase encoded by the DNA fragment in the host, where the DNA fragment comprises a region encoding a polypeptide TomL having an amino acid sequence of SEQ ID NO:
- 3, a region encoding a polypeptide TomM having an amino acid sequence of SEQ ID NO:
- 4, a region encoding a polypeptide TomN having an amino acid sequence of SEQ ID NO:
- 5, a region encoding a polypeptide TomO having an amino acid sequence of SEQ ID NO:
- 6, and a region encoding a polypeptide TomP having an amino acid sequence of SEQ ID NO:
- 7 of the Sequence Listing.
A method for producing a toluene monooxygenase, comprising a step of making a transformant produce a toluene monooxygenase being a genetic product of a recombinant DNA introduced into the transformant;
- wherein the transformant is obtainable by introducing a recombinant DNA comprising a vector, a promoter, and a DNA fragment where the vector and the promoter are functionally ligated to the DNA fragment to enable expression of the toluene monooxygenase encoded by the DNA fragment in the host,
- wherein the DNA fragment comprises a region encoding a polypeptide TomL having an amino acid sequence of SEQ ID NO:
- 3, a region encoding a polypeptide TomM having an amino acid sequence of SEQ ID NO:
- 4, a region encoding a polypeptide TomN having an amino acid sequence of SEQ ID NO:
- 5, a region encoding a polypeptide TomO having an amino acid sequence of SEQ ID NO:
- 6, and a region encoding a polypeptide TomP having an amino acid sequence of SEQ ID NO:
- 7 of the Sequence Listing.
A method for degrading at least one of a halogenated aliphatic hydrocarbon compound and an aromatic compound in a medium comprising a degradation step by a transformant obtainable by introducing into a host microorganism a recombinant DNA;
- wherein the recombinant DNA comprises a vector, a promoter, and a DNA fragment where the vector and the promoter are functionally ligated to the DNA fragment to enable expression of the toluene monooxygenase encoded by the DNA fragment in the host,
- the DNA fragment comprises a region encoding a polypeptide TomL having an amino acid sequence of SEQ ID NO:
- 3, a region encoding a polypeptide TomM having an amino acid sequence of SEQ ID NO:
- 4, a region encoding a polypeptide TomN having an amino acid sequence of SEQ ID NO:
- 5, a region encoding a polypeptide TomO having an amino acid sequence of SEQ ID NO:
- 6, and a region encoding a polypeptide TomP having an amino acid sequence of SEQ ID NO:
- 7 of the Sequence Listing.
A method for remedying an environment polluted with a pollutant being at least either of a halogenated aliphatic hydrocarbon compound or an aromatic compound, comprising a step of degrading the pollutant by bringing a transformant into contact with the pollutant,
- wherein the transformant is obtainable by introducing into a host microorganism a recombinant DNA which comprises a vector, a promoter, and a DNA fragment where the vector and the promoter are functionally ligated to the DNA fragment to enable expression of the toluene monooxygenase encoded by the DNA fragment in the host,
- the DNA fragment comprises a region encoding a polypeptide TomL having an amino acid sequence of SEQ ID NO:
- 3, a region encoding a polypeptide TomM having an amino acid sequence of SEQ ID NO:
- 4, a region encoding a polypeptide TomN having an amino acid sequence of SEQ ID NO:
- 5, a region encoding a polypeptide TomO having an amino acid sequence of SEQ ID NO:
- 6, and a region encoding a polypeptide TomP having an amino acid sequence of SEQ ID NO:
- 7 of the Sequence Listing.
A component polypeptide having any one of amino acid sequences of SEQ ID NOs:
- 2 to 8 in the sequence listing, capable of being a component of a toluene monooxygenase.
A toluene monooxygenase comprising at least component polypeptides TomL to TomP having amino acid sequences of SEQ ID NOs:
- 3 to 7 in the Sequence Listing.
Dependencies are inferred from the claim text.