WO2005059125A1 - Variants de phosphotriesterases a specificite de substrat amelioree et/ou modifiee - Google Patents

Variants de phosphotriesterases a specificite de substrat amelioree et/ou modifiee Download PDF

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WO2005059125A1
WO2005059125A1 PCT/AU2003/001675 AU0301675W WO2005059125A1 WO 2005059125 A1 WO2005059125 A1 WO 2005059125A1 AU 0301675 W AU0301675 W AU 0301675W WO 2005059125 A1 WO2005059125 A1 WO 2005059125A1
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polypeptide
seq
sequence provided
sequence
organophosphate
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PCT/AU2003/001675
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English (en)
Inventor
Irene Horne
Jeevan Lal Khurana
Tara Deane Sutherland
Robyn Joyce Russell
John Graham Oakeshott
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Commonwealth Scientific And Industrial Research Organisation
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Priority to PCT/AU2003/001675 priority Critical patent/WO2005059125A1/fr
Priority to AU2003285217A priority patent/AU2003285217A1/en
Publication of WO2005059125A1 publication Critical patent/WO2005059125A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C1/00—Reclamation of contaminated soil
    • B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8259—Phytoremediation
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase

Definitions

  • This invention relates to enzymes capable of hydrolysing organophosphate (OP), fungicidal carbamate and insecticidal carbamate molecules.
  • the invention relates to variants of a phosphotriesterase enzyme which have enhanced and/or altered substrate specificity when compared to the wild-type molecule isolated from an Agrobacterium radiobacter strain.
  • Residues of organophosphate insecticides, as well as fungicidal and insecticidal carbamates, are undesirable contaminants of the environment and a range of commodities. Areas of particular sensitivity include contamination of soil, irrigation tailwater that is re-cycled, used by irrigators downstream or simply allowed to run off-farm, and residues above permissible levels in agricultural and horticultural exports. Poisoning with organophosphates presents a problem for agricultural workers that are exposed to these chemicals, as well as military personnel exposed to organophosphates used in chemical warfare. Furthermore, the stockpiling of organophosphorus nerve agents has resulted in the need to detoxify these stocks.
  • Bioremediation strategies are therefore required for eliminating or reducing these organophosphate residues and/or stockpiles.
  • One proposed strategy involves the use of enzymes capable of immobilising or degrading the organophosphate residues. Such enzymes may be employed, for example, in bioreactors through which contaminated water could be passed, or in washing solutions after post-harvest disinfestation of fruit, vegetables or animal products to reduce residue levels and withholding times.
  • Suitable enzymes for degrading organophosphate residues include OP hydrolases from bacteria (Mulbry, 1992; Mulbry and Kearney, 1991 ; Cheng et al., 1999; Home et al., 2002, 2003; US 5,484,728; US 5,589,386; PCT/AU02/00594), vertebrates (Wang et al., 1993; 1998; Gan et al., 1991 ; Broomfield et al., 1999) and OP resistant insects (WO 95/19440 and WO 97/19176). It is desirable that the OP hydrolases degrade the organophosphate residues at a rapid rate.
  • OPH bacterial organophosphate hydrolase
  • OPH has no detectable activity with phosphate monoesters or diesters (Dumas et al., 1989a, b; 1990).
  • OPH homologues phosphotriesterase homology proteins, or PHPs
  • ePHP Escherichia coli
  • mtPHP Mycobacterium tuberculosis
  • mpPHP Mycoplasma pneumoniae
  • OPH homologues have also been identified in vertebrates (Davies et al., 1997), although their function in these organisms is unknown. OPH, ePHP, mtPHP and mammalian PHPs are 27-30% identical at the amino acid level, while mpPHP is less similar. Amino acid residues involved in Zn ++ binding are conserved across the six members of the phosphotriesterase family identified to date (Buchbinder et al., 1998).
  • OP hydrolysing enzymes Three other distinct OP hydrolysing enzymes have been isolated from bacteria with a history of exposure to OPs (Mulbry and Karns, 1989; Mulbry, 1992; Cheng et al., 1999). The two for which sequence data are available are unrelated to each other and to OPH.
  • ADPase aryldialkylphosphatase from Nocardia sp.
  • strain B-1 has a turnover number for ethyl parathion that is 4500-fold lower than that reported for OPH (Mulbry and Karns, 1989; Mulbry, 1992).
  • Paraoxonase, or PON1 is a distinct OP hydrolysing enzyme found in mammals. Like OPH it is a metalloenzyme, preferring Ca ++ in this case, which is associated with low density lipoproteins in plasma and normally involved in metabolism of oxidised lipid compounds (Gan et al., 1991 ; Sorenson et al., 1995).
  • DFPase diisopropyl fluorophosphatase
  • the specificity constants of these enzymes for their OP substrates are orders of magnitude less than those of OPH for paraoxon.
  • the present inventors isolated a strain of Agrobacterium radiobacter (isolate P230) from contaminated soil that is capable of using coumaphos as the sole phosphorus source.
  • the enzyme (OpdA) responsible for this coumaphos hydrolytic activity was isolated, characterised and shown to be 90% identical in amino acid sequence to OPD (WO 02/092803). Whilst OpdA has activity towards many OPs, there is a need for further enzymes with altered and/or enhanced substrate specificity.
  • the present invention provides a substantially purified polypeptide comprising a sequence provided as SEQ ID NO:6 or SEQ
  • polypeptide comprises at least one of the following: i) a Gly residue at an amino acid position corresponding to position 237 of SEQ ID NO:6, ii) an Asp, Glu, Lys, Arg or His residue at an amino acid position corresponding to position 119 of SEQ ID NO:6, iii) a Phe residue at an amino acid position corresponding to position 130 of SEQ ID NO:6, iv) a Trp residue at an amino acid position corresponding to position 236 of SEQ ID NO:6, v) a Thr residue at amino acid position corresponding to position 307 of SEQ ID NO:6, and vi) a Phe, Trp, His, Arg, Glu, Gin, Leu, Ser, Gly, Ala, Lys, Val, lie or Thr residue at an amino acid position corresponding to position 308 of SEQ ID NO:6, wherein the polypeptide comprises at least one of the following: i) a Gly residue at an amino acid position corresponding to position 237 of SEQ ID NO:6,
  • the polypeptide is greater than 75%, more preferably greater than 80%, more preferably greater than 85%, more preferably greater than 90%, more preferably greater than 92%, more preferably greater than 95%, more preferably greater than 97%, and even more preferably greater than 99%, identical to the sequence provided as SEQ ID NO:6 or SEQ ID NO:7.
  • the organophosphate is an aliphatic non-vinyl organophosphate and the polypeptide comprises at least one of the following: i) a Gly residue at an amino acid position corresponding to position 237 of SEQ ID NO:6, ii) an Asp, Glu, Lys, Arg or His residue at an amino acid position corresponding to position 119 of SEQ ID NO:6, iii) a Phe residue at an amino acid position corresponding to position 130 of SEQ ID NO:6, iv) a Trp residue at an amino acid position corresponding to position 236 of SEQ ID NO:6, v) a Thr residue at amino acid position corresponding to position 307 of SEQ ID NO:6, and vi) a Phe, Trp, His, Arg, Glu, or Gin residue at an amino acid position corresponding to position 308 of SEQ ID NO:6.
  • the aliphatic non-vinyl organophosphate is selected from, but not limited to, the group consisting of: dimethoate and methamidophos.
  • the organophosphate is an aliphatic vinyl organophosphate and the polypeptide comprises at least an Glu, Lys, Arg or His residue at an amino acid position corresponding to position 119 of SEQ ID NO:6 and a Phe, Trp, His, Arg, Asp, Glu or Gin residue at an amino acid position corresponding to position 308 of SEQ ID NO:6.
  • the aliphatic vinyl organophosphate is dichlorvos.
  • the organophosphate is an aromatic vinyl organophosphate and the polypeptide comprises a Leu, Ser, Gly, Ala, Lys, Val, lie or Thr residue at an amino acid position corresponding to position 308 of SEQ ID NO:6.
  • the aromatic vinyl organophosphate is selected from, but not limited to, the group consisting of: chlorfenvinphos, tetrachlorvinphos and dimethylvinphos.
  • the present invention provides a substantially purified polypeptide comprising a sequence provided as SEQ ID NO:6 or SEQ ID NO:7, or a sequence which is greater than 70% identical to a sequence provided as SEQ ID NO:6 or SEQ ID NO:7, wherein the polypeptide has a leaving group pocket which is smaller in size than a polypeptide comprising a sequence provided as SEQ ID NO:1 or SEQ ID NO:5, and wherein the polypeptide is capable of hydrolysing an organophosphate molecule.
  • the polypeptide of the second aspect of the invention comprises a mutation of at least one of the following residues of SEQ ID NO:1 ; W130, F131 , F305 or Y308.
  • the polypeptide of the second aspect of the invention comprises a mutation of at least one of the following residues of SEQ ID NO:5; W131 , F132, F306 or Y309.
  • the polypeptide of the second aspect comprises a Phe, Trp, His, Arg, Glu or Gin residue at an amino acid position corresponding to position 308 of SEQ ID NO: 6.
  • the polypeptide of the second aspect comprises a Phe or Trp residue at an amino acid position corresponding to position 308 of SEQ ID NO:6.
  • the polypeptide of the second aspect comprises a Phe residue at an amino acid position corresponding to position 308 of SEQ ID NO:6.
  • the polypeptide of the second aspect further comprises an Asp, Glu, Lys, Arg or His residue at an amino acid position corresponding to position 119 of SEQ ID N0:6.
  • the polypeptide of the second aspect comprises a sequence as provided in SEQ ID NO:8.
  • the organophosphate is an aliphatic organophosphate.
  • the present invention provides a substantially purified polypeptide comprising a sequence provided as SEQ ID NO:6 or SEQ ID NO:7, or a sequence which is greater than 70% identical to a sequence provided as SEQ ID NO:6 or SEQ ID NO:7, wherein the polypeptide has a leaving group pocket which is larger in size than a polypeptide comprising a sequence provided as SEQ ID NO:1 or SEQ ID NO:5, and wherein the polypeptide is capable of hydrolysing an organophosphate molecule.
  • the polypeptide of the third aspect of the invention comprises a mutation of at least one of the following residues of SEQ ID NO:1 ;
  • the polypeptide of the third aspect of the invention comprises a mutation of at least one of the following residues of SEQ ID NO:5; W131 , F132, F306 or Y309.
  • the polypeptide of the third aspect comprises a Leu, Ser, Gly, Ala, Lys, Val, lie or Thr residue at an amino acid position corresponding to position 308 of SEQ ID NO:6.
  • the polypeptide of the third aspect comprises a Leu, Ser, Gly, Ala, Lys, Val, or lie or residue at an amino acid position corresponding to position 308 of SEQ ID NO:6.
  • the polypeptide of the third aspect comprises a Gly or Ala residue at an amino acid position corresponding to position 308 of SEQ ID NO:6.
  • the organophosphate is an aromatic vinyl organophosphate. More preferably, the aromatic vinyl organophosphate is selected from, but not limited to, the group consisting of: chlorfenvinphos, tetrachlorvinphos and dimethylvinphos.
  • a fusion polypeptide is provided which comprises a polypeptide according to the present invention fused to at least one other polypeptide sequence.
  • the at least one other polypeptide is selected from the group consisting of: a polypeptide that enhances the stability of the polypeptide of the invention, a polypeptide which can be used as a marker for the fusion protein, and a polypeptide that assists in the purification of the fusion polypeptide.
  • the at least one other polypeptide is the maltose-binding protein or glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • the present invention provides an isolated polynucleotide encoding a polypeptide according to the invention.
  • the polynucleotide is a variant of SEQ ID NO:49, wherein the variant comprises nucleic acid changes such that it encodes the desired polypeptide of the invention.
  • Methods of producing such nucleic acid changes are known in the art and are also described herein.
  • a vector is provided which comprises a polynucleotide according to the invention.
  • the vector is suitable for the replication and/or expression of a polynucleotide.
  • the vectors may be, for example, a plasmid, virus or phage vector provided with an origin of replication, and preferably a promotor for the expression of the polynucleotide and optionally a regulator of the promotor.
  • the vector may contain one or more selectable markers, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian expression vector.
  • the vector may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.
  • a host cell which comprises a vector according to the invention.
  • the present invention provides a process for preparing a polypeptide of the invention, the process comprising cultivating a host cell of the invention under conditions which allow production of the polypeptide, and recovering the polypeptide. Such cells can be used for the production of commercially useful quantities of the encoded polypeptide.
  • the present invention provides a composition for hydrolysing an organophosphate molecule, the composition comprising a polypeptide according to the invention, and one or more acceptable carriers.
  • the present invention provides a composition for hydrolysing an organophosphate molecule, the composition comprising a host cell of the invention, and one or more acceptable carriers. It will be appreciated that the present invention can be used to hydrolyse organophosphates in a sample. For instance, after a crop has been sprayed with an organophosphate pesticide, the organophosphate residue can be hydrolysed from seeds, fruits and vegetables before human consumption. Similarly, organophosphate contaminated soil or water can be treated with a polypeptide of the invention.
  • the present invention provides a method for hydrolysing an organophosphate molecule, the method comprising exposing the organophosphate molecule to a polypeptide according to the invention.
  • the polypeptide is provided as a composition of the invention.
  • the method further comprises exposing the organophosphate to a divalent cation.
  • the divalent cation is zinc.
  • the method can be performed upon, for example, a sample selected from the group consisting of; soil, water, biological material, or a combination thereof.
  • Preferred biological samples include matter derived from plants such as seeds, vegetables or fruits, as well as matter derived from animals such as meat.
  • the organophosphate can be exposed to the polypeptide via any available avenue.
  • the polypeptide can also be provided in the form of a host cell, typically a microorganism such as a bacterium or a fungus, which expresses a polynucleotide encoding the polypeptide of the invention.
  • a host cell typically a microorganism such as a bacterium or a fungus
  • the polypeptide will be provided as a composition of the invention.
  • Organophosphate molecules can also be hydrolysed by exposing the organophosphate to a transgenic plant which produces a polypeptide of the present invention.
  • a transgenic plant is provided which produces a polypeptide according to the invention.
  • the present invention provides a method for hydrolysing an organophosphate molecule, the method comprising exposing the organophosphate molecule to a transgenic plant of the invention. Further, it is preferred that the polypeptide is at least produced in the roots of the transgenic plant.
  • the present invention provides a polymeric sponge or foam for hydrolysing an organophosphate molecule, the foam or sponge comprising a polypeptide of the invention immobilized on a polymeric porous support.
  • the porous support comprises polyurethane.
  • the sponge or foam further comprises carbon embedded or integrated on or in the porous support.
  • the present invention provides a method for hydrolysing an organophosphate molecule, the method comprising exposing the organophosphate molecule to a sponge or foam of the invention.
  • the present invention provides a biosensor for detecting the presence of an organophosphate, the biosensor comprising a polypeptide according to the invention, and a means for detecting hydrolysis of an organophosphate molecule by the polypeptide.
  • a polypeptide of the present invention can be mutated, and the resulting mutants screened for altered activity such as changes in substrate specificity.
  • the present invention provides a method of producing a polypeptide with enhanced ability to hydrolyse an organophosphate or altered substrate specificity for an organophosphate, the method comprising a) mutating one or more amino acids of a first polypeptide of the invention, b) determining the ability of the mutant to hydrolyse an organophosphate, and c) selecting a mutant with enhanced ability to hydrolyse the organophosphate or altered substrate specificity for the organophosphate, when compared to the first polypeptide.
  • the present invention provides a polypeptide produced according to the above method.
  • the present invention provides a method of hydrolysing a fungicidal or insecticidal carbamate, the method comprising exposing the carbamate to a polypeptide selected from the group consisting of: i) a polypeptide comprising a sequence provided in SEQ ID NO:1 ; ii) a polypeptide comprising a sequence provided in SEQ ID NO:2; iii) a polypeptide comprising a sequence provided in SEQ ID NO:4; iv) a polypeptide comprising a sequence provided in SEQ ID NO:5; and v) a polypeptide comprising a sequence which is greater than 70% identical to any one of (i) to (iv).
  • the polypeptide is greater than 75%, more preferably greater than 8O%, more preferably greater than 85%, more preferably greater than 90%, more preferably greater than 92%, more preferably greater than 95%, more preferably greater than 97%, and even more preferably greater than 99%, identical to any one of (i) to (iv).
  • the polypeptide comprises at least one of the following: i) a Gly residue at an amino acid position corresponding to position 237 of
  • SEQ ID NO:6 ii) an Asp, Glu, Lys, Arg or His residue at an amino acid position corresponding to position 119 of SEQ ID NO:6, iii) a Phe residue at an arnino acid position corresponding to position 130 of SEQ ID NO:6, iv) a Trp residue at an arnino acid position corresponding to position 236 of SEQ ID NO:6, v) a Thr residue at amino acid position corresponding to position 307 of SEQ ID NO:6, vi) a Phe, Trp, His, Arg, Glu, Gin, Leu, Ser, Gly, Ala, Lys, Val, lie or Thr residue at an amino acid position corresponding to position 308 of SEQ ID NO:6, or vii) a polypeptide comprising a sequence provided in SEQ ID NO:3.
  • the fungicidal carbamate is benomyl or carbendazim.
  • the insecticidal carbamate is methomyl or fenoxycarb.
  • the method further comprises exposing the carbamate to a divalent cation.
  • the divalent cation is zinc. It will be appreciated that the present invention can be used to hydrolyse fungicidal or insecticidal carbamates in a sample. For instance, after a crop has been sprayed with a fungicidal or insecticidal carbamate, any carbamate residue can be hydrolysed from seeds, fruits and vegetables before human consumption.
  • fungicidal or insecticidal carbamate contaminated soil or water can be treated with a polypeptide as described herein.
  • the sample is selected from the group consisting of; soil, water, biological material, or a combination thereof.
  • Preferred biological samples include matter derived from plants such as seeds, vegetables or fruits, as well as matter derived from animals such as meat.
  • the carbamate can be exposed to the polypeptide via any available avenue. This includes providing the polypeptide directly to a sample, with or without carriers or excipients etc.
  • the polypeptide can also be provided in the form of a host cell, typically a microorganism such as a bacterium or a fungus, which expresses a polynucleotide encoding the polypeptide.
  • a host cell typically a microorganism such as a bacterium or a fungus, which expresses a polynucleotide encoding the polypeptide.
  • the polypeptide will be provided as a composition of the invention.
  • the present invention provides a composition for hydrolysing a fungicidal or insecticidal carbamate molecule, the composition comprising a polypeptide selected from the group consisting of: i) a polypeptide comprising a sequence provided in SEQ ID NO:1 ; ii) a polypeptide comprising a sequence provided in SEQ ID NO:2; iii) a polypeptide comprising a sequence provided in SEQ ID NO:4; iv) a polypeptide comprising a sequence provided in SEQ ID NO:5; v) a polypeptide comprising a sequence which is greater than 70% identical to any one of (i) to (iv), and dimethylsulfoxide.
  • a polypeptide selected from the group consisting of: i) a polypeptide comprising a sequence provided in SEQ ID NO:1 ; ii) a polypeptide comprising a sequence provided in SEQ ID NO:2; iii) a polypeptide comprising a
  • the present invention provides for the use of composition for hydrolysing a fungicidal or insecticidal carbamate molecule, the composition comprising a polypeptide selected from the group consisting of: i) a polypeptide comprising a sequence provided in SEQ ID NO:1 ; ii) a polypeptide comprising a sequence provided in SEQ ID NO:2; iii) a polypeptide comprising a sequence provided in SEQ ID NO:4; iv) a polypeptide comprising a sequence provided in SEQ ID NO:5; and v) a polypeptide comprising a sequence which is greater than 70% identical to any one of (i) to (iv), and one or more acceptable carriers.
  • a polypeptide selected from the group consisting of: i) a polypeptide comprising a sequence provided in SEQ ID NO:1 ; ii) a polypeptide comprising a sequence provided in SEQ ID NO:2; iii) a polypeptide comprising
  • the present invention provides for the use of a composition for hydrolysing an a fungicidal or insecticidal carbamate molecule, the composition comprising a host cell encoding a polypeptide selected from the group consisting of: i) a polypeptide comprising a sequence provided in SEQ ID NO:1 ; ii) a polypeptide comprising a sequence provided in SEQ ID NO:2; iii) a polypeptide comprising a sequence provided in SEQ ID NO:4; iv) a polypeptide comprising a sequence provided in SEQ ID NO:5; and v) a polypeptide comprising a sequence which is greater than 70% identical to any one of (i) to (iv), and one or more acceptable carrier.
  • a polypeptide selected from the group consisting of: i) a polypeptide comprising a sequence provided in SEQ ID NO:1 ; ii) a polypeptide comprising a sequence provided in SEQ ID NO:2; i
  • the present invention provides a method for hydrolysing a fungicidal or insecticidal carbamate molecule, the method comprising exposing the carbamate to a transgenic plant which produces a polypeptide selected from the grou consisting of: i) a polypeptide comprising at sequence provided in SEQ ID NO:1 ; ii) a polypeptide comprising a sequence provided in SEQ ID NO:2; iii) a polypeptide comprising a sequence provided in SEQ ID NO:4; iv) a polypeptide comprising a sequence provided in SEQ ID NO:5; and v) a polypeptide comprising a seq uence which is greater than 70% identical to any one of (i) to (iv).
  • the p resent i nvention provides a method for hydrolysing a fungicidal or insecticidal carbamate molecule, the method comprising exposing the carbamate to a s-ponge or foam which comprises a polypeptide selected from the group consisting of: i) a polypeptide comprising a sequence provided in SEQ ID NO: 1 ; ii) a polypeptide comprising a sequence provided in SEQ ID NO:2; iii) a polypeptide comprising a sequence provided in SEQ ID NO:4; iv) a polypeptide comprising a sequence provided in SEQ ID NO:5; and v) a polypeptide comprisin g a sequence which is greater than 70% identical to any one of (i) to (iv).
  • the present invention provides a biosensor for detecting the presence of a fungicidal or insecticidal carbamate molecule, the biosensor comprising a polypeptide selected from the group consisting of: i) a polypeptide comprising a sequence provided in SEQ ID NO:1 ; ii) a polypeptide comprising a sequence provided in SEQ ID NO:2; iii) a polypeptide comprising a sequence provided in SEQ ID NO:4; iv) a polypeptide comprising a sequence provided in SEQ ID NO:5; and v) a polypeptide comprisin g a sequence which is greater than 70% identical to any one of (i) to (iv), and a means for detecting hydrolys-is of the carbamate by the polypeptide.
  • a polypeptide selected from the group consisting of: i) a polypeptide comprising a sequence provided in SEQ ID NO:1 ; ii) a polypeptide comprising a sequence provided in
  • t he present invention provides a method of producing a polypeptide with enhanced ability to hydrolyse a fungicidal or insecticidal carbamate molecule or altered substrate specificity for a fungicidal or insecticidal carbamate molecule, the method comprising a) mutating one or more amino acids of a first polypeptide selected from the group consisting of: i) a polypeptide comprising a sequence provided in SEQ ID NO:1 ; ii) a polypeptide comprising a sequence provided in SEQ ID NO:2; iii) a polypeptide comprising a sequence provided in SEQ ID NO:4; iv) a polypeptide comprising a sequence provided in SEQ ID NO:5; and v) a polypeptide
  • Figure 1 The chemical classes of organophosphate pesticides.
  • Figure 2 Plasmid maps of pCYopdA, pCYmutl and pCYmut2. The plasmids were used for combining various combinations of mutants. The opdA gene is shown as a shaded semi-circle with the remainder of the circle representing vector sequence representing pCY76. Relevant restriction sites are shown, as are the mutations (filled triangle).
  • Figure 3 Relative dimethoate activities of OpdA mutants.
  • Figure 4 Relative dimethoate activities of the various OpdA mutants at residue 119.
  • Figure 5 Relative dimethoate activities of the various active site mutants of OpdA.
  • Figure 6 Chemical structures of chlorfenvinphos, tetrachlorvinphos and dimethylvinphos.
  • Figure 7 The chlorfenvinphos hydrolytic activity, relative to that of a Y308L mutant, of various OpdA mutants.
  • Figure 8 The chemical classes of carbamates, their primary targets and examples of each.
  • Figure 9 The chemical structure of dimethyl formamide.
  • Figure 10 Amino acid sequence alignment of OPH (SEQ ID NO:5) and OpdA (SEQ ID NO:1). The secretion signal is provided in bold.
  • SEQ ID NO:3 Polypeptide sequence of OpdA1.
  • SEQ ID NO:4 Polypeptide sequence of OpdA2.
  • SEQ ID NO:5 Polypeptide sequence of OPH from Flavobacterium sp (also known in the art as OPD).
  • SEQ ID NO:6 Framework polypeptide sequence of various OpdA mutants of the present invention.
  • SEQ ID NO:7 Framework polypeptide sequence of various OPH mutants of the present invention.
  • SEQ ID NO: 49 Polynucleotide sequence encoding OpdA.
  • X is a good leaving group, which is a requirement for the irreversible inhibition of acetylcholinesterase.
  • the polypeptides of the present invention hydrolyse the phosphoester bonds of organophosphates.
  • the organophosphate can have aromatic or aliphatic leaving groups (X) and can also contain vinyl groups ( Figure 1 ). Although well known for their use as pesticides, organophosphates have also been used as nerve gases against mammals.
  • polypeptides of the present invention will also be useful for hydrolysis of organophosphates which are not pesticides.
  • the polypeptides of the invention (especially OpdB) are used to hydrolyse O-ethyl S-(2-diisopropyamino)ethyl methylphosphonothiolate (VX).
  • Fungicidal and Insecticidal Carbamates are pesticides that possess an amide linkage, with the carbonyl group also forming a carboxylester linkage ( Figure 8).
  • Different constituents from either the amine group or the carboxyl ester group determine the target organism of these compounds.
  • Carbamates with aromatic groups from both the amine and carboxylester eg phenmedipham
  • Carbamates with an aromatic group coming from the carboxylester group and a small group, such as a methyl group, coming from the amine (such as carbaryl) are insecticidal.
  • carbamates with a benzimidazole group coming from the amine and a small methyl group coming from the carboxylester linkage are fungicidal ( Figure 8).
  • substantially purified polypeptide we mean a polypeptide that has generally been separated from the lipids, nucleic acids, other polypeptides, and other contaminating molecules with which it is associated in its native state.
  • the substantially purified polypeptide is at least 50% free, more preferably at least 60% free, more preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.
  • the % identity of a polypeptide is determined by FASTA (Pearson and Lipman, 1988) analysis (GCG program) using the default settings and a query sequence of at least 50 amino acids in length, and whereby the FASTA analysis aligns the two sequences over a region of at least 50 amino acids.
  • the FASTA analysis aligns the two sequences over a region of at least 100 amino acids. More preferably, the FASTA analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the FASTA analysis aligns the two sequences over a region of at least 350 amino acids.
  • Mutant (altered) polypeptides can be prepared using any technique known in the art.
  • the polynucleotide provided as SEQ ID NO:8 can be subjected to in vitro mutagenesis.
  • Such to in vitro mutagenesis techniques include sub-cloning the polynucleotide into a suitable vector, transforming the vector into a "mutator" strain such as the E.
  • polypeptides of the invention are subjected to DNA shuffling techniques as broadly described by Harayama (1998). Protein products derived from mutated/altered DNA can readily be screened using techniques described herein to determine if they have enhanced and/or altered substrate specificity.
  • Amino acid sequence mutants of the polypeptides of the present invention can also be prepared by introducing appropriate nucleotide changes into a nucleic acid sequence, or by in vitro synthesis of the desired polypeptide. Such mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence.
  • a combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final protein product possesses the desired characteristics.
  • the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified.
  • the sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.
  • Amino acid sequence deletions generally range from about 1 to 30 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues.
  • Substitution mutants have at least one amino acid residue in the polypeptide molecule removed and a different residue inserted in its place.
  • the sites of greatest interest for substitutional mutagenesis include sites identified as the active site(s). Other sites of interest are those in which particular residues obtained from various species are identical. These positions may be important for biological activity. These sites, especially those falling within a sequence of at least three other identically conserved sites, are preferably substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1 under the heading of "exemplary substitutions".
  • the term "corresponding to” is used in the context of the present invention to refer to amino acid residues of polypeptides related to OpdA (for example greater than 70% identical to SEQ ID NO:1), however, the relative residue numbering of the related polypeptide may be different to that of OpdA.
  • one embodiment of the invention relates to mutants of OpdA where the tyrosine at position 308 (Y308) is replaced with, for example, a phenylalanine (Y308F).
  • the present invention also encompasses mutants/variants of OPH (SEQ ID NO: 5) with the same substitution.
  • OPH has one more amino acid residue in the signal sequence (see Figure 10) the residue of OPH corresponding to Y308 of OpdA is Y309.
  • Other amino acids at positions corresponding to designated positions of OpdA can readily be determined by aligning OpdA or SEQ ID NO:6 with the related polypeptide such as shown in Figure 10.
  • the leaving pocket group of OpdA is W130, F131, F305 and Y308 (Yang et al., 2003), whereas the leaving pocket group pocket of OPH is W131 , F132, F306 and Y309. Table 1. Exemplary substitutions.
  • unnatural amino acids or chemical amino acid analogues can be introduced as a substitution or addition into the polypeptide of the present invention.
  • Such amino acids include, but are not limited to, the D- isomers of the common amino acids, 2,4-diaminobutyric acid, ⁇ -amino isobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid, omithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, ⁇ -alanine, fluoro-amino acids, designer amino acids such as ⁇ -methyl amino acids, Coo-methyl amino acids, N ⁇ -methyl amino acids, N
  • polypeptides of the present invention which are differentially modified during or after synthesis, e.g., by biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. These modifications may serve to increase the stability and/or bioactivity of the polypeptide of the invention.
  • Polypeptides of the present invention can be produced in a variety of ways, including production and recovery of natural proteins, production and recovery of recombinant proteins, and chemical synthesis of the proteins.
  • an isolated polypeptide of the present invention is produced by culturing a cell capable of expressing the polypeptide under conditions effective to produce the polypeptide, and recovering the polypeptide.
  • Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production.
  • An effective medium refers to any " medium in which a cell is cultured to produce a polypeptide of the present invention.
  • Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins.
  • Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.
  • isolated polynucleotide we mean a polynucleotide which has generally been separated from the polynucleotide sequences with which it is associated or linked in its native state.
  • the isolated polynucleotide is at least 50% free, more preferably at least 60% free, more preferably at least 75% free, and most preferably at least 90% free from other components with which they are naturally associated.
  • polynucleotide is used interchangeably herein with the term “nucleic acid molecule”.
  • the % identity of a polynucleotide is determined by FASTA (Pearson and Lipman, 1988) analysis (GCG program) using the default settings and a query sequence of at least 150 nucleotides in length, and whereby the FASTA analysis aligns the two sequences over a region of at least 15O nucleotides. More preferably, the FASTA analysis aligns the two sequences over a region of at least 300 nucleotides. Even more preferably, the FASTA analysis aligns the two sequences over a region of at least 1050 nucleotides.
  • Polynucleotides of the present invention may selectively hybridise to the sequences encoding polypeptides of the invention under high stringency.
  • stringent conditions are those that (1 ) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCI/0.0015 M sodium citrate/0.1 % NaDodS0 4 at 50°C; (2) employ during hybridisation a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1 % bovine serum albumin, 0.1% Ficoll, 0.1 % polyvinylpyrroli one, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCI, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCI, 0.075 IV1 sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and 10% dextran sulfate at 42°C in 0.2 x SSC and 0.1
  • formamide for
  • One embodiment of the present invention includes a recombinant vector, which includes at least one isolated nucleic acid molecule of the present invention, inserted into any vector capable of delivering the nucleic acid molecule into a host cell.
  • a vector contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to nucleic acid molecules of the present invention and that preferably are derived from a species other than the species from which the nucleic acid molecule(s) are derived.
  • the vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid.
  • One type of recombinant vector comprises a nucleic acid molecule of the present invention operatively linked to an expression vector.
  • the phrase operatively linked refers to the insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell.
  • an expression vector is a DNA or RNA vector that is capable of transforming a host cell and effecting expression of a specified nucleic acid molecule.
  • the expression vector is also capable of replicating within the host cell.
  • Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids.
  • Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, endoparasite, arthropod, other animal, and plant cells.
  • Preferred expression vectors of the present invention can direct gene expression in bacterial, yeast, plant and mammalian cells.
  • Expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present invention.
  • recombinant molecules of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription.
  • transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences.
  • Suitable transcription control sequences include any transcription control sequence that can function in at least one of the host cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art.
  • Preferred transcription control sequences include those which function in bacterial, yeast, plant and mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda, bacteriophage T7, T7lac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01 , metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters), antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as intermediate early promoters), simian virus 40, retrovirus, actin, retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphat
  • Suitable transcription control sequences include tissue-specific promoters and enhancers.
  • Recombinant molecules of the present invention may also (a) contain secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed polypeptide of the present invention to be secreted from the cell that produces the polypeptide and/or (b) contain fusion sequences which lead to the expression of nucleic acid molecules of the present invention as fusion proteins.
  • suitable signal segments include any signal segment capable of directing the secretion of a protein of the present invention.
  • Preferred signal segments include, but are not limited to, the native signal sequence, tissue plasminogen activator (t-PA), interferon, interleukin, growth hormone, histocompatibility and viral envelope glycoprotein signal segments, as well as natural signal sequences. In other circumstances, it may be desirable that the polypeptide be encoded without any secretion signal sequence.
  • a nucleic acid molecule of the present invention can be joined to a fusion segment that directs the encoded protein to the proteosome, such as a ubiquitin fusion segment. Recombinant molecules may also include intervening and/or untranslated sequences surrounding and/or within the nucleic acid sequences of nucleic acid molecules of the present invention.
  • Host Cells Another embodiment of the present invention includes a recombinant cell comprising a host cell transformed with one or more recombinant molecules of the present invention. Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism.
  • Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate i nto one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.
  • Suitable host cells to transform include any cell that can be transformed with a polynucleotide of the present invention.
  • Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule (e.g., nucleic acid molecules encoding one or more proteins of the present invention).
  • Host cells of the present invention either can be endogenously (i.e., naturally) capable of producing proteins of the present invention or can be capable of producing such proteins after being transformed with at least one nucleic acid molecule of the present invention.
  • Host cells of the present invention can be any cell capable of producing at least one protein of the present invention, and include bacterial, fungal (including yeast), parasite, arthropod, animal and plant cells.
  • Preferred host cells include bacterial, mycobacterial, yeast, plant and mammalian cells.
  • More preferred host cells include Agrobacterium, Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells (normal dog kidney cell line for canine herpesvirus cultivation), CRFK cells (normal cat kidney cell line for feline herpesvirus cultivation), CV-1 cells (African monkey kidney cell line used, for example, to culture raccoon poxvirus), COS (e.g., COS-7) cells, and Vero cells.
  • Particularly preferred host cells are E. coli, including E.
  • coli K-12 derivatives Salmonella typhi; Salmonella typhimurium, including attenuated strains; Spodoptera frugiperda; Trichoplusia ni; BHK cells; MDCK cells; CRFK cells; CV-1 cells; COS cells; Vero cells; and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246).
  • Additional appropriate mammalian cell hosts include other kidney cell lines, other fibroblast cell lines (e.g., human, murine or chicken embryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, mouse NIH/3T3 cells, LMTK cells and/or HeLa cells.
  • Recombinant DNA technologies can be used to improve expression of transformed polynucleotide molecules by manipulating, for example, the number of copies of the polynucleotide molecules within a host cell, the efficiency with which those polynucleotide molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications.
  • Recombinant techniques useful for increasing the expression of polynucleotide molecules of the present invention include, but are not limited to, operatively linking polynucleotide molecules to high-copy number plasmids, integration of the polynucleotide molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of polynucleotide molecules of the present invention to correspond to the codon usage of the host cell, and the deletion of sequences that destabilize transcripts.
  • the activity of an expressed recombinant protein of the present invention may be improved by fragmenting, modifying, or derivatizing polynucleotide molecules encoding such a protein.
  • the levels of organophosphates in a sample can be reduced by exposing the sample to a transgenic plant expressing a suitable enzyme.
  • the sample is soil.
  • the polynucleotide of the present invention can be expressed in a transgenic plant, particularly the roots of the plant, for hydrolysing organophosphate molecules in the sample.
  • transgenic plants expressing a polypeptide described herein can also be used to reduce fungicidal or insecticidal carbamates in a sample.
  • plant refers to whole plants, plant organs (e.g. leaves, stems roots, etc), seeds, plant cells and the like.
  • Plants contemplated for use in the practice of the present invention include both monocotyledons and dicotyledons.
  • Exemplary dicotyledons include cotton, corn, tomato, tobacco, potato, bean, soybean, and the like.
  • Transgenic plants, as defined in the context of the present invention include plants (as well as parts and cells of said plants) and their progeny which have been genetically modified using recombinant DNA techniques to cause or enhance production of at least one protein of the present invention in the desired plant or plant organ.
  • the polypeptide of the present invention may be expressed constitutively in the transgenic plants during all stages of development. Depending on the use of the plant or plant organs, the proteins may be expressed in a stage-specific manner.
  • the proteins may be expressed tissue-specifically.
  • the choice of the plant species is determined by the intended use of the plant or parts thereof and the amenability of the plant species to transformation.
  • Regulatory sequences which are known or are found to cause expression of a gene encoding a protein of interest in plants may be used in the present invention.
  • the choice of the regulatory sequences used depends on the target plant and/or target organ of interest.
  • Such regulatory sequences may be obtained from plants or plant viruses, or may be chemically synthesized.
  • Such regulatory sequences are well known to those skilled in the art.
  • Other regulatory sequences such as terminator sequences and polyadenylation signals include any such sequence functioning as such in plants, the choice of which would be obvious to the skilled addressee.
  • An example of such sequences is the 3' flanking region of the nopaline synthase
  • compositions of the present invention include excipients, also referred to herein as "acceptable carriers".
  • An excipient can be any material that the animal, plant, plant or animal material, or environment (including soil and water samples) to be treated can tolerate.
  • excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions.
  • Nonaqueous vehicles such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used.
  • Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran.
  • Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability.
  • buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal or o-cresol, formalin and benzyl alcohol.
  • Excipients can also be used to increase the half-life of a composition, for example, but are not limited to, polymeric controlled release vehicles, biodegradable implants, liposomes, bacteria, viruses, other cells, oils, esters, and glycols.
  • the polypeptide of the present invention can be provided in a composition which enhances the rate and/or degree of organophosphate, or fungicidal and insecticidal carbamate, hydrolysis, or increases the stability of the polypeptide.
  • the polypeptide can be immobilized on a polyurethane matrix (Gordon et al., 1999), or encapsulated in appropriate liposomes (Petrikovics et al., 2000a and b).
  • the polypeptide can also be incorporated into a composition comprising a foam such as those used routinely in fire-fighting (LeJeune et a/.,- 1998).
  • a controlled release formulation that is capable of slowly releasing a composition of the present invention into an animal, plant, animal or plant material, or the environment (including soil and water samples).
  • a controlled release formulation comprises a composition of the present invention in a controlled release vehicle.
  • Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems.
  • Preferred controlled release formulations are biodegradable (i.e., bioerodible).
  • a preferred controlled release formulation of the present invention is capable of releasing a composition of the present invention into soil or water which is in an area sprayed with an organophosphate, or fungicidal or insecticidal carbamate. The formulation is preferably released over a period of time ranging from about 1 to about 12 months.
  • a preferred controlled release formulation of the present invention is capable of effecting a treatment preferably for at least about 1 month, more preferably for at least about 3 months, even more preferably for at least about 6 months, even more preferably for at least about 9 months, and even more preferably for at least about 12 months.
  • concentration of the polypeptide, vector, or host cell of the present invention that will be required to produce effective compositions for hydrolysing an organophosphate, or fungicidal or insecticidal carbamate, will depend on the nature of the sample to be decontaminated, the concentration of the organophosphate, or fungicidal or insecticidal carbamate, in the sample, and the formulation of the composition.
  • the effective concentration of the polypeptide, vector, or host cell within the composition can readily be determined experimentally, as will be understood by the skilled artisan.
  • Biosensors are analytical devices typically consisting of a biologically active material such as an enzyme and a transducer that converts a biochemical reaction into a quantifiable electronic signal that can be processed, transmitted, and measured.
  • a biologically active material such as an enzyme
  • a transducer that converts a biochemical reaction into a quantifiable electronic signal that can be processed, transmitted, and measured.
  • a general review of biosensors which have been used for the detection of orangophosphorus compounds is provided by Rekha et al. (2000), the entire contents of which are incorporated by reference.
  • the polypeptide of the present invention can be adapted for use in such biosensors.
  • the plasmids pCYmutl and pCYopdA were digested with EcoRV and Sfil (see Figure 2). After electrophoresis of the digest, the 300 bp fragment from pCYmutl and the 3 kb fragment from pCVopdA were excised as described above and ligated together overnight at 4°C with T4 DNA ligase (NEB).
  • QpdAlabc (OpdA P42S. P134S, A170S)
  • the plasmids pCYmutl and pCYopdA were digested with Sfi ⁇ and Xho ⁇ (see Figure 2). After electrophoresis, the 600 bp fragment from pCYopdA and the 3 kb fragment from pCYmutl were excised as described above and ligated overnight at 4°C with T4 DNA Ligase (NEB).
  • Example 2 Combination of OpdA1 and OpdA2 mutations.
  • (a) Construction of plasmid pCYmut2-1d encoding OpdA2-1d The plasmids pCYmut2 (containing OpdA2; see Figure 2) and pCYmutl d (containing OpdAld) were digested with Sfi ⁇ and Xho ⁇ . After electrophoresis the 600 bp fragment from pCYmutl d was excised from the agarose gel using the QIAgen QIAquick PCR purification kit. Digested pCYmut2 was dephosphorylated using calf intestinal alkaline phosphatase (Boehringer Mannheim).
  • the phosphatase was then removed using the QIAquick PCR purification kit (QIAGEN) and the plasmid ligated with the 600 bp fragment from pCYmutl d overnight at 4°C with T4 DNA Ligase (NEB), to create pCYmut2-1d. After transformation, positive clones were identified by sequence analysis.
  • a BamVW cloning site was inserted at the start codon and an EcoRI cloning site at the stop codon (underlined bases).
  • the PCR fragments were subsequently cloned into the Saml-EcoRI cloning sites of pGEX-4T1 (Amersham-Pharmacia).
  • the ligations were transformed into E. coli DH10B cells and transformants selected on LB agar plates with 100 ⁇ g/ml ampicillin. Transformants were confirmed by restriction analysis and DNA sequence analysis.
  • the reaction mixture consisted of 5 ⁇ l 10X Pfu reaction buffer, 10 ng pCYmut2 (template DNA), 125 ng of each primer, 200 ⁇ M each dNTP and 2.5 U Pfu Turbo DNA polymerase (Stratagene) in a 50 ⁇ l reaction.
  • the cycling conditions were as follows: 1 cycle 95°C 3 minutes and 16 cycles of 95°C 30 seconds, 55°C 1 minute and 68°C 12 minutes. Any template DNA was destroyed by digestion with Dpnl at 37°C. The digestions were then transformed into E. coli DH10B with transformants selected on LB agar plates containing ampicillin (100 ⁇ g/ml). Site-directed mutagenesis was then confirmed by sequence analysis.
  • OpdB SEQ ID NO:8
  • Other mutations which would decrease the size of the leaving pocket should be useful mutations for hydrolysing aliphatic organophosphates, these mutants include Y308W, Y308H, Y308R, Y308N and Y308E.
  • Example 5 Hydrolysis of additional aliphatic OPs by OpdB.
  • the activity of OpdB was examined towards the aliphatic, non-vinyl OP, methamidophos, and the aliphatic vinyl OPs, dichlorvos and propetamphos.
  • the method of analysis was a pH indicator assay.
  • E. coli DH10B (pGopdB) and E. coli DH10B (pGAfull) were grown at 37°C in LB with ampicillin (100 ⁇ g/ml) until an OD 6 oo of 0.6. The cultures were then induced for 5 hours with 0.1 mM IPTG.
  • Example 6 Other mutants displaying aliphatic OP hydrolytic activity.
  • the reaction conditions were as follows: initial denaturation of 94°C for 3 minutes, followed by addition of Taq DNA polymerase and then 30 cycles of 94°C/1 minute, 48°C/1 minute 35 seconds and 72°C/2 minutes plus a final extension of 72°C/5 minutes.
  • the PCR reaction was digested with SamHI-EcoRI and ligated with appropriately digested pGEX4T-1.
  • the ligations were transformed into E. coli DH10B and transformants selected on LB agar plates with ampicillin (100 ⁇ g/ml). Approximately 2500 colonies were picked and grown for 16 hours in 96-well growth blocks with 1 ml LB containing ampicillin (100 ⁇ g/ml) at 37°C.
  • the clones were then induced with 0.1 mM IPTG for 3 hours, after which the cultures were assayed for GST activity and dimethoate hydrolytic activity.
  • GST activity was measured using 100 ⁇ l of the culture with the CDNB/glutathione mixture described above.
  • Dimethoate hydrolytic activity was measured using 200 ⁇ l of the culture with the required amount of dimethoate (to a final concentration of 440 ⁇ M). Dimethoate hydrolytic activity was normalised against GST activity. The majority of the mutants demonstrated no dimethoate activity.
  • One mutant was identified that had acquired dimethoate hydrolytic activity. This mutant was shown by sequence analysis to have a single amino acid substitution L236W.
  • coli DH10B with transformants selected on LB agar plates containing ampicillin. One transformant was confirmed to contain the S307T mutation and was examined for malathion hydrolytic activity.
  • GST activity was assayed as described above. Malathion hydrolysis was assayed with radioactive malathion ( 14 C-malathion) in the radiometric partition assay previously used for radiolabelled OP substrates (Campbell et al., 1998). At various times during the reaction, an aliquot (50 ⁇ l) was removed and diluted with 150 ⁇ l water. This was then extracted with 500 ⁇ l dichloromethane. The upper aqueous phase (50 ⁇ l) was removed and quantitated by liquid scintillation. An E. coli DH10B culture was used as a negative control. The activity of the S307T mutant of OpdA was 74 ⁇ mol/hr/ ⁇ mol protein.
  • Example 7 Mutagenesis of active site residues for aromatic vinyl OP hydrolytic activity.
  • the primers used for mutagenesis are shown in Table 5. The reactions were performed as described above using pGAfull as a template. After mutagenesis, DNA templates were digested with Dpnl, the digested reactions transformed into E. coli DH10B and transformants selected on LB agar plates with ampicillin (100 ⁇ g/ml). Mutagenesis was confirmed by sequence analysis.
  • OpdA can have up to 20-fold greater activity for dimethyl OPs over diethyl OPs (Home et al., 2002), it would be expected that the activity of the Y308 mutants for dimethylvinphos could be up to 20-fold higher than that for CVP.
  • These two OPs differ only in whether they have dimethyl (in the case of dimethylvinphos) or diethyl groups (CVP) (see Figure 6).
  • Other mutations which would increase the size of the leaving pocket should be useful mutations for hydrolysing aromatic vinyl organophosphates, these mutants include Y308K, Y308V, Y308I, and Y308T.
  • Example 8 Activity of OpdA towards the fungicidal and insecticidal carbamates.
  • OpdA was expressed as a GST fusion in E. coli DH10B from the plasmid pGAfull. Induction with IPTG was performed as described above. Harvested cells were broken by sonication with large cell debris and unbroken cells removed by centrifugation (15 min, 7000g). Carbamate hydrolytic activity was determined using thin layer chromatography (TLC). E. coli DH10B was used as a control.
  • the carbamates tested were carbaryl, propoxur, carbofuran and pirimicab (all insecticidal) (dissolved in methanol), phenmedipham (herbicidal) (dissolved in methanol) and benomyl (fungicidal) (dissolved in dimethylsulfoxide).
  • Approximately 800 ⁇ g of crude cell protein was incubated with 1 mM of each carbamate.
  • the reactions were left for 3 hours and extracted with 100 ⁇ l of ethyl acetate.
  • centrifugation 5 min, 3000g
  • the upper aqueous organic layer was removed and transferred to a fresh tube. This organic layer was dried under a steady stream of nitrogen.
  • the dried fraction was resuspended in 10 ⁇ l acetone and spotted onto a silica gel TLC plate (Alltech F 2 54)- Separation on the TLC plate was performed in 3:1 chloroform- ethyl acetate. UV-absorbing spots were visualised using an ultraviolet light at 254 nm.
  • the benomyl hydrolysis product was also observed after being subjected to an iodine vapour chamber. OpdA was found to hydrolyse benomyl but not pirimicarb or phenmedipham. When benomyl was dissolved in dimethyl formamide (DMF, Figure 9), no hydrolysis was observed by OpdA. DMF may act as a competitive inhibitor for benomyl due to structural similarities.
  • OpdA mutants OpdA2 (A119D) and OpdA2H (A119H) also demonstrated benomyl hydrolytic activity.
  • OpdA, and variants thereof such as OpdA2 and OpdA2H, will also hydrolyse carabamate molecules closely related to benomyl such as carbendazim (which is also a fungicidal carabamate) as well as the insecticidal carbamates fenoxycarb and methomyl.
  • Petrikovics I., Cheng, T.C., Papahadjopoulos, D., Hong, K., Yin, R., DeFrank, J.J., Jaing, J., Zong, Z.H., McGuinn, W.D., Sylvester, D., Pei, L., Madec, J., Tamulinas, C, Jaszberenyi, J.C, Barcza, T. and Way, J.L. (2000a). Toxicology Science 57: 16-21.

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Abstract

L'invention concerne des enzymes capables d'hydrolyser des molécules d'organophosphate (OP), de carbamate fongicide et de carbamate insecticide. Plus précisément, l'invention concerne des variants d'une enzyme de la phosphotriestérase possédant une spécificité de substrat améliorée et/ou modifiée, comparativement à la molécule de type sauvage isolée d'une souche d'Agrobacterium radiobacter. L'invention concerne également l'utilisation de ces enzymes dans des stratégies de biorestauration.
PCT/AU2003/001675 2003-12-16 2003-12-16 Variants de phosphotriesterases a specificite de substrat amelioree et/ou modifiee WO2005059125A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/AU2003/001675 WO2005059125A1 (fr) 2003-12-16 2003-12-16 Variants de phosphotriesterases a specificite de substrat amelioree et/ou modifiee
AU2003285217A AU2003285217A1 (en) 2003-12-16 2003-12-16 Variants of phosphotriesterases with enhanced and/or altered substrate specificity

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US8822428B2 (en) 2005-09-21 2014-09-02 L'oreal Double-stranded RNA oligonucleotides which inhibit tyrosinase expression
US8410260B2 (en) 2005-09-21 2013-04-02 L'oreal Double-stranded RNA oligonucleotides which inhibit tyrosinase expression
EP1774959A1 (fr) * 2005-09-21 2007-04-18 L'Oréal Oligonucléotide d'ARN double brin inhibant l'expression de la tyrosinase
WO2008145865A2 (fr) * 2007-04-27 2008-12-04 Universite Henri Poincare Nancy 1 Phosphotriesterases hyperthermophiles mutees et leurs utilisations
WO2008145865A3 (fr) * 2007-04-27 2009-02-05 Univ Nancy 1 Henri Poincare Phosphotriesterases hyperthermophiles mutees et leurs utilisations
US8372618B2 (en) 2007-04-27 2013-02-12 Universite Henri Poincare Nancy 1 Mutated hyperthermophilic phosphotriesterases and their uses
FR2915489A1 (fr) * 2007-04-27 2008-10-31 Univ Henri Poincare Nancy I Et Phosphotriesterases hyperthermophiles mutees et leurs utilisations
US9796990B2 (en) 2011-07-20 2017-10-24 Commonwealth Scientific And Industrial Research Organization Enzymes for degrading organophosphates
WO2013010225A1 (fr) 2011-07-20 2013-01-24 Commonwealth Scientific And Industrial Research Organisation Enzymes pour dégrader des organosphosphates
CN103814127A (zh) * 2011-07-20 2014-05-21 联邦科学工业研究组织 用于降解有机磷酸酯的酶
WO2014096402A1 (fr) 2012-12-20 2014-06-26 Boumendil Olivier-Georges Enzyme présentant une activité d'antagoniste de récepteur nmda et/ou une activité anticholinergique
WO2020035865A1 (fr) * 2018-08-14 2020-02-20 Yeda Research And Development Co. Ltd. Hydrolases d'organophosphates préparées, efficaces et à large spécificité
US20210178207A1 (en) * 2018-08-14 2021-06-17 Yeda Research And Development Co. Ltd. Designed, efficient and broad-specificity organophosphate hydrolases
CN111837796A (zh) * 2019-12-17 2020-10-30 河南科技大学 一种提高一年生油用牡丹抗旱性的方法
CN111837796B (zh) * 2019-12-17 2022-01-04 河南科技大学 一种提高一年生油用牡丹抗旱性的方法

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