WO2005059125A1

WO2005059125A1 - Variants of phosphotriesterases with enhanced and/or altered substrate specificity

Info

Publication number: WO2005059125A1
Application number: PCT/AU2003/001675
Authority: WO
Inventors: Irene Horne; Jeevan Lal Khurana; Tara Deane Sutherland; Robyn Joyce Russell; John Graham Oakeshott
Original assignee: Commonwealth Scientific and Industrial Research Organization CSIRO
Current assignee: Commonwealth Scientific and Industrial Research Organization CSIRO
Priority date: 2003-12-16
Filing date: 2003-12-16
Publication date: 2005-06-30
Anticipated expiration: 2006-06-16
Also published as: AU2003285217A1

Abstract

The present invention provides enzymes capable of hydrolysing organophosphate (OP) and fungicidal carbamate molecules. In particular, 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. The use of these enzymes in bioremediation strategies is also provided.

Description

VARIANTS OF PHOSPHOTRIESTERASES WITH ENHANCED AND/OR ALTERED SUBSTRATE SPECIFICITY

FIELD OF THE INVENTION: This invention relates to enzymes capable of hydrolysing organophosphate (OP), fungicidal carbamate and insecticidal carbamate molecules. In particular, 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.

BACKGROUND OF THE INVENTION: 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. The most thoroughly studied OP degrading enzyme is bacterial organophosphate hydrolase (OPH), which is encoded by identical genes on dissimilar plasmids in both Flavobacterium sp. ATCC 27551 and Brevundimonas diminuta MG (Harper et al., 1988; Mulbry and Karns, 1989). OPH is a homodimeric protein that is capable of hydrolysing a wide range of phosphate triesters (both oxon and thion OPs) (Dumas et al., 1989a, b). Its reaction mechanism directly or indirectly involves metal ions, preferably Zn⁺⁺. OPH has no detectable activity with phosphate monoesters or diesters (Dumas et al., 1989a, b; 1990). OPH homologues (phosphotriesterase homology proteins, or PHPs) have been identified in the genomes of Escherichia coli (ePHP), Mycobacterium tuberculosis (mtPHP) and Mycoplasma pneumoniae (mpPHP), although only ePHP has been tested for phosphotriesterase activity (Scanlan and Reid, 1995; Buchbinder et al., 1998). No activity was detected in ePHP crude lysates with any of the substrates tested, such as p-nitrophenyl acetate, bis(p-nitrophenyl) phosphate, paraoxon and p-nitrophenyl phosphate. 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). 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. One, a prolidase from Alteromonas sp., normally functions in hydrolysis of X-Pro dipeptides. Its activity for insecticidal OPs is reported as modest, although it has not been reported in terms of k_ca K_m specificity constants (Cheng et al., 1999). The other, an aryldialkylphosphatase (ADPase) 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). It has high activity for paraoxon, with a specificity constant of around 10⁶ M^"1sec^"1 (Doom et al., 1999; Hong and Raushel, 1999). There is also evidence for other, so-called diisopropyl fluorophosphatase (DFPase) enzymes in a wide range of vertebrates, invertebrates and microorganisms (Wang et al., 1998; Hoskin et al., 1999; Billecke et al., 1999). These enzymes are notably diverse in many of their biochemical properties but are all characterised by their hydrolytic activity against OP chemical warfare agents. Limited sequence data suggest that they are unrelated to all the other OP hydrolytic enzymes described above. OP resistant blowflies and houseflies have been the source of esterase enzymes with activity against oxon OPs like chlorfenvinfos (CVP) and carboxylester OPs like malathion (Newcomb et al., 1997; Campbell et al., 1998; Claudianos et al., 1999; WO 95/19440; WO 97/19176). A Gly to Asp substitution at residue 137 in blowfly esterase E3 (and its housefly ortholog, ALI) resulted in the acquisition of activity for CVP, while a Trp to Leu/Ser mutation at residue 251 in the same enzyme resulted in activity against malathion. However, 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.

SUMMARY OF THE INVENTION: The present inventors have identified numerous variants of phosphotriesterases which possess enhanced and/or altered substrate specificity. Accordingly, in a first aspect 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 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 does not comprise the sequence provided as SEQ ID NO:3, and wherein the polypeptide is capable of hydrolysing an organophosphate molecule. In a preferred embodiment, 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. In one embodiment, 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. Preferably, the aliphatic non-vinyl organophosphate is selected from, but not limited to, the group consisting of: dimethoate and methamidophos. In another embodiment, 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. Preferably, the aliphatic vinyl organophosphate is dichlorvos. In a further embodiment, 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. Preferably, the aromatic vinyl organophosphate is selected from, but not limited to, the group consisting of: chlorfenvinphos, tetrachlorvinphos and dimethylvinphos. In a second aspect, 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. In one embodiment, 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. In another embodiment, 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. Preferably, 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. More preferably, 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. Even more preferably, the polypeptide of the second aspect comprises a Phe residue at an amino acid position corresponding to position 308 of SEQ ID NO:6. Preferably, 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. In a particularly preferred embodiment, the polypeptide of the second aspect comprises a sequence as provided in SEQ ID NO:8. Preferably, the organophosphate is an aliphatic organophosphate. In a third aspect, 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. In one embodiment, 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 ;

W130, F131 , F305 or Y308. In another embodiment, 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. Preferably, 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. More preferably, 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. Even more preferably, 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. Preferably, with respect -to the third aspect, 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. In another aspect, a fusion polypeptide is provided which comprises a polypeptide according to the present invention fused to at least one other polypeptide sequence. Preferably, 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. Preferably, the at least one other polypeptide is the maltose-binding protein or glutathione S-transferase (GST). In another aspect, the present invention provides an isolated polynucleotide encoding a polypeptide according to the invention. Preferably, 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. In a further aspect, a vector is provided which comprises a polynucleotide according to the invention. Preferably, 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. In another aspect, a host cell is provided which comprises a vector according to the invention. In a further aspect, 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. In another aspect, 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. In another aspect, 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. Accordingly, in a further aspect 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. Preferably, the polypeptide is provided as a composition of the invention. In addition, it is preferred that the method further comprises exposing the organophosphate to a divalent cation. Preferably, 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. 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 of the invention. Usually, 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. Thus, in a further aspect a transgenic plant is provided which produces a polypeptide according to the invention. In a further aspect, 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. In a further aspect, 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. Preferably, the porous support comprises polyurethane. In a preferred embodiment, the sponge or foam further comprises carbon embedded or integrated on or in the porous support. In a further aspect, 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. In another aspect, 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. Thus, in a further aspect, 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. In a further aspect, the present invention provides a polypeptide produced according to the above method. Surprisingly, the present inventors have also discovered that OpdA, and polypeptides related thereto, are capable of hydrolysing fungicidal and insecticidal carbamates. Thus, in a further aspect, 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). In a preferred embodiment, 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). Preferably, 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. Preferably, the fungicidal carbamate is benomyl or carbendazim. Preferably, the insecticidal carbamate is methomyl or fenoxycarb. Preferably, the method further comprises exposing the carbamate to a divalent cation. Preferably, 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. Similarly, fungicidal or insecticidal carbamate contaminated soil or water can be treated with a polypeptide as described herein. Preferably, 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. Usually, the polypeptide will be provided as a composition of the invention. In yet another aspect, 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. In a further aspect, 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. In yet another aspect, 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. In yet another aspect, 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). It is preferred that the polypeptide is at least produced in the roots of the transgenic plant. In another aspect, 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). In yet another aspect, 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 described hterein can be mutated, and the resulting mutants screened for altered activity such as changes in substrate specificity against different fungicidal or insecticidal carbamates. Thus, in a further aspect, 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 comprising a sequence which is greater than 70% identical to any one of (i) to (iv), b) determining the ability of the mutant to hydrolyse a fungicidal or insecticidal carbamate molecule, and c) selecting a mutant with enhanced ability to hydrolyse the fungicidal or insecticidal carbamate molecule or altered substrate specificity for the fungicidal or insecticidal carbamate molecule, when compared to the first polypeptide. In another aspect, the present invention provides a polypeptide produced according to the above method. As will be apparent, preferred features and characteristics of one aspect of the invention are applicable to many other aspects of the invention. Throughout this specification the word "comprise", or variations such as

"comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. The invention will hereinafter be described by way of the following non- limiting Figures and Examples.

BRIEF DESCRIPTION OF THE DRAWINGS: 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.

KEY TO THE SEQUENCE LISTING:

SEQ ID NO:1 - Polypeptide sequence of OpdA.

SEQ ID NO:2 - Polypeptide sequence of OpdA minus the signal sequence.

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:8 - Polypeptide sequence of OpdB.

SEQ ID NO's 9 to 48 - Oligonucleotide primers.

SEQ ID NO: 49 - Polynucleotide sequence encoding OpdA.

DETAILED DESCRIPTION OF THE INVENTION:

General Techniques Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques, chemistry and biochemistry). Unless otherwise indicated, the recombinant DNA techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991 ), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (Editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley- Interscience (1988, including all updates until present).

Organophosphates Organophosphates are synthetic organophosphorus esters and related compounds such as phosphoroamidates. They have the general formula (RR'X)P=0 or (RR'X)P=S, where R and R¹ are short-chain groups. For insecticidal organophosphates 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. Accordingly, it is envisaged that the polypeptides of the present invention will also be useful for hydrolysis of organophosphates which are not pesticides. In a particularly preferred embodiment, the polypeptides of the invention (especially OpdB) are used to hydrolyse O-ethyl S-(2-diisopropyamino)ethyl methylphosphonothiolate (VX).

Fungicidal and Insecticidal Carbamates 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) are herbicidal. 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. Finally, carbamates with a benzimidazole group coming from the amine and a small methyl group coming from the carboxylester linkage are fungicidal (Figure 8).

Polypeptides By "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. Preferably, 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. More preferably, 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. For example, 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. coli XL-1 red (Stratagene) and propagating the transformed bacteria for a suitable number of generations. In another example, the polynucleotides 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. In designing amino acid sequence mutants, 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. For instance, 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. However, since 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. As used herein, 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.

Furthermore, if desired, 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, α-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, β-alanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, Coo-methyl amino acids, Nα-methyl amino acids, and amino acid analogues in general. Also included within the- scope of the invention are 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. In one embodiment, 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.

Polynucleotides By "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. Preferably, 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. Furthermore, the term "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. As used herein, 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 % NaDodS0₄ 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 % SDS.

Vectors 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. Such 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. As used herein, 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. Preferably, 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. In particular, recombinant molecules of the present invention include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important 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, phosphate and nitrate transcription control sequences as well as other sequences capable of controlling gene expression in prokaryotic or eukaryotic cells. Additional 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. Examples of 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. In addition, 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.

Transgenic Plants As generally described in WO 99/53037, the levels of organophosphates in a sample can be reduced by exposing the sample to a transgenic plant expressing a suitable enzyme. Typically, the sample is soil. Accordingly, 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. Similarly, transgenic plants expressing a polypeptide described herein can also be used to reduce fungicidal or insecticidal carbamates in a sample. The term "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. Furthermore, depending on the use, 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

(nos) gene of Agrobacterium tumefaciens. Several techniques are available for the introduction of the expression construct containing a DNA sequence encoding a protein of interest into the target plants. Such techniques include but are not limited to transformation of protoplasts using the calcium/polyethylene glycol method, electroporation and microinjection or (coated) particle bombardment. In addition to these so-called direct DNA transformation methods, transformation systems involving vectors are widely available, such as viral and bacterial vectors (e.g. from the genus Agrobacterium). After selection and/or screening, the protoplasts, cells or plant parts that have been transformed can be regenerated into whole plants, using methods known in the art. The choice of the transformation and/or regeneration techniques is not critical for this invention.

Compositions 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. Examples of such 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. Examples of 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. Furthermore, 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. For example, 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). As would be appreciated by the skilled addressee, the polypeptide of the present invention could readily be used in a sponge or foam as disclosed in WO 00/64539, the contents of which are incorporated herein in their entirety. One embodiment of the present invention is 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). As used herein, 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. The 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 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 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.

EXAMPLES:

Example 1 - Analysis of the various mutations in OpdAL The various mutations^' contained in OpdA1 (WO 02/092803) were examined individually and in various combinations for their effect on aliphatic OP hydrolysis.

(a) Construction of mutants of OpdAI (i) OpdA1a (OpdA P42S) The plasmids pCYmutl and pCYopdA were digested with ZΞcoRV and Xho\ (see Figure 2). After electrophoresis, the 800 bp fragment from pCYopdA and the 3 kb fragment from pCYmutl were excised from an agarose gel using the QIAgen QIAquick PCR purification kit according to the manufacturer's instructions. These two fragments were pooled and ligated with T4 DNA Ligase (NEB) at 4°C overnight.

(ϋ) OpdA1 be (OpdA P134S, A170S) 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).

(iii) OpdA1d (OpdA S237G) The plasmids pCYmutl and pCYopd/\ were digested with Sfi\ and Xho\

(see Figure 2). After electrophoresis, the 6O0 bp fragment from pCYmutl and the 3 kb fragment from pCYopdA were excised as described above and ligated together with T4 DNA ligase (NEB) at 4°C overnight.

(iv) QpdAlad (OpdA P42S, S237G) The plasmids pCYopdA and pCYmutl were digested with EcoRV and Sfi\

(see Figure 2). After electrophoresis, the 300 bp fragment from pCYopdA and the 3 kb fragment from pCYmutl were excised as described above and ligated at 4°C overnight with T4 DNA Ligase (NEB).

(v) QpdAlbcd (OpdA P134S. A170S. S237G) The plasmids pCYmutl and pCYopdA were digested with EcoRV and Xho\ (see Figure 2). After electrophoresis, the 800 bp fragment from. pCYmutl and the 3 kb fragment from pCYopdA were excised as described above and ligated overnight at 4°C with T4 DNA Ligase (NEB).

(vi) 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).

All of the ligations were transformed into E. coli DH10B cells and clones selected on LB agar plates containing 100 μg/ml ampicillin. Clones were confirmed by sequence analysis.

(b) Analysis of dimethoate hydrolytic activity of OpdA1 variants Clones were grown in 500 ml of LB containing 100 μg/ml ampicillin, purified as described above and examined for their activity towards the aliphatic OPs, dimethoate, malathion and malaoxon (Table 2). Only those mutants containing the "d" mutation (S237G) demonstrated significant aliphatic OP hydrolytic activity (shaded), although this activity was enhanced by one or more of the other mutations as well. Table 2. The specific activity (nmol/hr/mg) of purified OpdA1 variants against various aliphatic OPs.

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.

(b) Construction of GST translational fusions To enable quantitation of OpdA variant protein concentrations when expressing the mutants in E. coli and to allow for comparison between samples without purifcation, OpdA variants were expressed as GST (glutathione S- transferase) fusion proteins. OP hydrolytic activity was expressed as "per GST activity" and this ratio was compared between samples. The opdA variant genes were amplified from their respective templates using PCR with the upstream (Popdδ) and downstream (PopdEco) primers, 5'GATCGTGGATCCCCAATCGGTACAGGCGATCTG (SEQ ID NO:9) and 5'GATCGTGAATTCTCATCGTTCGGTATCTTGACGGGGAAT (SEQ ID NO:10), respectively. 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 μg/ml ampicillin. Transformants were confirmed by restriction analysis and DNA sequence analysis.

(c) Analysis of dimethoate hydrolytic activity of OpdA2 and OpdA2-1d All GST fusion proteins were expressed in E. coli DH10B cells. Optimal production of GST fusion proteins was obtained when mid-log cells (OD₆oo = 0.6) were induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside for 5 hours at 37°C. Harvested cells were disrupted by sonication and large cell debris and unbroken cells removed by centrifugation (15 min, 7000g). The supernatants were then assayed for GST activity and dimethoate activity (according to the method described earlier). GST activity was measured according to the method of Habig et al. (1974) which follows the conjugation of of 1-chloro-2,4- dinitrobenzene (CDNB) with glutathione. Reaction mixtures consisted of 0.1 M potassium phosphate buffer pH6.6, 1 mM CDNB and 1 mM reduced glutathione. To this mixture, aliquots of samples were added and the change in absorbance at 340 nm noted. The activity of glutathione S-transferase was determined using the extinction coefficient of 9.6 mM^"1cm^"1 for the CDNB-glutathione product (Habig et al., 1974). The activity of the mutants towards dimethoate was expressed as per GST activity. OpdA2 (A119D) had the highest activity (Figure 3). A combination of the S237G/A119D (OpdA2-1d) mutations resulted in a mutant with higher activity than the S237G (OpdAld) mutant alone but less activity than OpdA2 (A119D) (Figure 3).

Example 3 - Site saturation mutagenesis at OpdA residue A119.

(a) Construction of mutants Site-saturation mutagenesis was undertaken at position 119. This involved changing amino acid 119 to all other possible amino acids. During random mutagenesis, there are only a certain number of codons that can be generated by a single nucleotide change, and therefore only a certain number of amino acids can be placed at any one position. It was reasoned, therefore, that perhaps another mutation at position 119 other than A to D might generate a mutant of greater activity. All site-directed mutants were created using the QuikChange Site-directed Mutagenesis kit from Stratagene. The primers used are shown in Table 3. The reaction mixture consisted of 5 μl 10X Pfu reaction buffer, 10 ng pCYmut2 (template DNA), 125 ng of each primer, 200 μM each dNTP and 2.5 U Pfu Turbo DNA polymerase (Stratagene) in a 50 μl 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 μg/ml). Site-directed mutagenesis was then confirmed by sequence analysis.

Table 3. Primers used for site-saturation mutagenesis.

(b) Transfer of mutants to pGEX4T-1 All of the mutant opdA variants were amplified by PCR using the primers Popd5 and PopdEco and the conditions described previously. The PCR reactions were then digested with BamHI-EcoRI and ligated with appropriately digested pGEX4T-1. The ligations were then transformed into E. coli DH10B and transformants selected on LB agar plates containing ampicillin (1 O0 μg/ml). Transformants were confirmed by restriction analysis.

(c) Comparison of activities towards dimethoate All GST fusion proteins were expressed in E. coli DH10B cells . Optimal production of GST fusion proteins was obtained when mid-log cells (ODβoo = 0.6) were induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside for 5 hours at 37°C. Harvested cells were disrupted by sonication and large cell debris and unbroken cells removed by centrifugation (15 min, 7000g). The supernatants were then assayed for GST activity and dimethoate activity (accord ing to the methods described earlier). All dimethoate hydrolytic activities were normalised for GST activity and are expressed in Figure 4 relative to OpdA2 (A119D) activity. The only mutants demonstrating activity were A119D, A119EΞ, A119H, A119K and A119R, with A119H having the highest activity (approximately 18- fold higher than the original mutant).

Example 4 - Site-directed mutagenesis of active site residues.

(a) Construction of mutants The active site residues in the leaving group pocket of O pdA were targeted for mutagenesis. The leaving group pocket is lined wit i aromatic amino acids (W130, F131 , F305 and Y308). It was reasoned that mutating W130 and Y308 to phenylalanine may decrease the size of the leaving group pocket by increasing the hydrophobic interactions of these amino acid residues. Site-directed mutagenesis was carried out as described above with the primers listed in Table 4. The templates used for mutagenesis were pCYOpdA and pCYmut2H (OpdA with the A119H mutation). Table 4. Primers used for site-directed mutagenesis of active site residues.

(b) Transfer of mutants into pGEX4T-1 All of the mutant opdA variants were amplified by PCR using the primers Popdδ and PopdEco and the conditions described previously. The PCR reactions were then digested with SamHI-EcoRI and ligated with appropriately digested pGEX4T-1. The ligations were then transformed into E. coli DH10B 0 and transformants selected on LB agar plates containing ampicillin (100 μg/ml). Transformants were confirmed by restriction analysis.

(c) Comparison of activities towards dimethoate All GST fusion proteins were expressed in E. coli DH10B cells. Optimal 5 production of GST fusion proteins was obtained when mid-log cells (OD₆oo = 0.6) were induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside for 5 hours at 37°C. Harvested cells were disrupted by sonication and large cell debris and unbroken cells removed by centrifugation (15 min, 7000g). The supernatants were then assayed for GST activity and dimethoate activity (according to the 0 methods described earlier). All dimethoate hydrolytic activities were normalised for GST activity and are expressed in Figure 5 relative to OpdA2 (A119D) activity. A Y308F mutation conferred a small amount of dimethoate hydrolytic activity on the wild-type OpdA enzyme but only about 2% of the activity displayed by OpdA2 (A119D), whereas a W130F OpdA mutant had no activity at 5 all (data not shown). However, the Y308F mutation had a substantial enhancing effect on dimethoate hydrolytic activity in an A119H background, while the W130F mutation abolished most of the dimethoate hydrolytic activity in the same background. The OpdA mutant with the A119H/Y308F mutations is now referred to as OpdB (SEQ ID NO:8), and the plasmid, pGopdB. 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 μg/ml) until an OD₆oo of 0.6. The cultures were then induced for 5 hours with 0.1 mM IPTG. Harvested cells were disrupted by sonication in 50 mM Tris-HCI pH7.5 and large cell debris and unbroken cells removed by centrifugation (15 min, 7000g). The reaction mixtures consisted of 0.5 mM OP (either dichlorvos, methamidophos or propetamphos) with 10 μl cell extract and made up to 100 μl with H₂0. After a 3 hour incubation bromothymol blue was added to a final concentration of 0.015 g/l. The colour of the reaction was then noted. Bromothymol blue is a pH indicator which is blue under alkaline conditions and yellow under acidic conditions. This transition occurs between pH6 and 7.6. The hydrolysis of OPs releases the relatively acidic diethyl phosphosphate (or diethyl phosphorothioate in the case of thion OPs), which results in a decrease of pH. Therefore hydrolysis of OPs has occurred if the bromothymol blue turns yellow. OpdA had no detectable hydrolysis against dichlorvos, methamidophos or propetamphos as seen by a blue colour of the bromothymol blue (data not shown). OpdB, however, demonstrated hydrolytic. activity towards dichlorvos and methamidophos.

Example 6 - Other mutants displaying aliphatic OP hydrolytic activity.

(a) Mutants of OpdA generated by error-prone PCR Error-prone PCR was used to generate a mutant library of OpdA. The reactions were carried out essentially as described by Yang et al. (2003). In a 50 μl reaction, dNTPs (200 μM), 5 μl Taq DNA polymerase buffer (Gibco BRL), 5 mM MgCI₂, 250 μM MnCI₂, 100 pmol Popdδ, 100 pmol PopdEco with 500 ng pCYopdA as the template and 5 U Taq DNA polymerase (Gibco BRL) were combined to begin mutagenesis. 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 μg/ml). Approximately 2500 colonies were picked and grown for 16 hours in 96-well growth blocks with 1 ml LB containing ampicillin (100 μg/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 μl of the culture with the CDNB/glutathione mixture described above. Dimethoate hydrolytic activity was measured using 200 μl of the culture with the required amount of dimethoate (to a final concentration of 440 μM). 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.

(b) Mutations in the small subsite of the OpdA active site S307 was mutated to T using the QuikChange site-directed mutagenesis kit described above. The primers used for mutagenesis were S307Tfor (5'TTCGGGTTTTCGACCTATGTCACGAAC) (SEQ ID NO:35) and S307Trev (5'GTTCGTGACATAGGTCGAAAACCCGAA) (SEQ ID NO:36). The cycling protocol described above was. used for mutagenesis, with the template being pGAfull which contains opdA in pGEX4T-1. The template was removed from the PCR reaction by digestion with Dpπl and the resulting digests were transformed into E. 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. The GST fusion protein was expressed in E. coli DH10B cells. Optimal production of GST fusion proteins was obtained when mid-log cells (OD₆oo = 0.6) were induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside for 5 hours at 37°C. Harvested cells were disrupted by sonication and large cell debris and unbroken cells removed by centrifugation (15 min, 7000g). The supernatant was then assayed for GST activity and malathion hydrolytic activity. GST activity was assayed as described above. Malathion hydrolysis was assayed with radioactive malathion (¹⁴C-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 μl) was removed and diluted with 150 μl water. This was then extracted with 500 μl dichloromethane. The upper aqueous phase (50 μl) 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 μmol/hr/μmol protein.

Example 7 - Mutagenesis of active site residues for aromatic vinyl OP hydrolytic activity.

(a) Construction of mutants There are at least 3 aromatic vinyl OPs, chlorfenvinphos (CVP), tetrachlorvinphos and dimethylvinphos (Figure 6). All of these compounds are characterised by an aromatic group on the same side as the phosphoester linkage, resulting in a larger leaving group. The vinyl group also adds to the stringency of the molecule. Site-directed mutations targeting the active site of OpdA were constructed in order to increase the size of the leaving group pocket. Since the leaving group is lined with aromatic amino acids (W130, F131 , F305 and Y308), these were substituted with smaller residues (Table 5). Site-directed mutagenesis was performed using the QuikChange site- directed mutagenesis kit from Stratagene. 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 μg/ml). Mutagenesis was confirmed by sequence analysis.

(b) Analysis of CVP hydrolytic activity All GST fusion proteins were expressed in E. coli DH10B as described above. Harvested cells were disrupted by sonication and large cell debris and unbroken cells removed by centrifugation (15 min, 7000g). GST activity was measured as described above. CVP activity was measured using the radiometric partition assay of Campbell et al. (1998) with ¹⁴C-CVP. The activity is shown in Figure 7. All changes at Y308 conferred some vinyl aromatic OP hydrolytic activity on OpdA. Given that 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.

Table 5. Primers used for site-directed mutagenesis of active site residues.

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 μg of crude cell protein was incubated with 1 mM of each carbamate. The reactions were left for 3 hours and extracted with 100 μl of ethyl acetate. - After 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 μl acetone and spotted onto a silica gel TLC plate (Alltech F₂54)- 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. The OpdA mutants OpdA2 (A119D) and OpdA2H (A119H) also demonstrated benomyl hydrolytic activity. In addition, based on the structure of benomyl, 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.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. All publications discussed above are incorporated herein in their entirety. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application. REFERENCES:

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Claims

CLAIMS:

1. 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 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, Ile or Thr residue at an amino acid position corresponding to position 308 of SEQ ID NO:6, wherein the polypeptide does not comprise the sequence provided as SEQ ID NO:3, and wherein the polypeptide is capable of hydrolysing an organophosphate molecule.

2. The polypeptide of claim 1 , wherein 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.

3. The polypeptide of claim 1 or claim 2 which comprises a sequence which is greater than 90% identical to a sequence provided as SEQ ID NO:6 or SEQ

ID NO:7.

4. 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.

5. The polypeptide of claim 4 which comprises a Phe, Trp, His, Arg, Glu or Gin residue at an amino acid position corresponding to position 308 of SEQ ID NO:6.

6. The polypeptide of claim 4 or claim 5 which further comprises an Asp, Glu, Lys, Arg or His residue at an amino acid position corresponding to position

119 of SEQ ID NO:6.

7. The polypeptide according to any one of claims 4 to 6 which comprises a sequence as provided in SEQ ID NO:8.

8. The polypeptide according to any one of claims 4 to 7, wherein the organophosphate is an aliphatic organophosphate.

9. 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.

10. The polypeptide of claim 9 which 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.

11. The polypeptide of claim 9 or claim 10, wherein the organophosphate is an aromatic vinyl organophosphate.

12. A fusion polypeptide comprising a polypeptide according to any one of claims 1 to 11 fused to at least one other polypeptide sequence.

13. An isolated polynucleotide encoding a polypeptide according to any one of claims 1 to 12.

14. A vector comprising a polynucleotide according to claim 13.

15. A host cell comprising a vector according to claim 14.

16. A process for preparing a polypeptide according to any one of claims 1 to 12, the process comprising cultivating a host cell according to claim 15 under conditions which allow production of the polypeptide, and recovering the polypeptide.

17. A composition for hydrolysing an organophosphate molecule, the composition comprising a polypeptide according to any one of claims 1 to 12, and one or more acceptable carriers.

18. A composition for hydrolysing an organophosphate molecule, the composition comprising a host cell according to claim 15, and one or more acceptable carriers.

19. A method for hydrolysing an organophosphate molecule, the method comprising exposing the organophosphate molecule to a polypeptide according to any one of claims 1 to 12.

20. The method of claim 19, wherein the polypeptide is provided as a composition according to claim 17 or claim 18.

21. The method of claim 19 or claim 20, further comprising exposing the organophosphate molecule to a divalent cation.

22. The method of claim 21 , wherein the divalent cation is zinc.

23. A transgenic plant which produces a polypeptide according to any one of claims 1 to 12.

24. A method for hydrolysing an organophosphate molecule, the method comprising exposing the organophosphate molecule to a transgenic plant according to claim 23.

25. The method of claim 24, wherein the polypeptide is at least produced in the roots of the transgenic plant.

26. A polymeric sponge or foam for hydrolysing an organophosphate molecule, the foam or sponge comprising a polypeptide according to any one of claims 1 to 12 immobilized on a polymeric porous support.

27. The polymeric sponge or foam of claim 26, wherein the porous support comprises polyurethane.

28. The polymeric sponge or foam of claim 26 or claim 27, wherein the sponge or foam further comprises carbon embedded or integrated on or in the porous support.

29. A method for hydrolysing an organophosphate molecule, the method comprising exposing the organophosphate molecule to a sponge or foam according to any one of claims 26 to 28.

30. A biosensor for detecting the presence of an organophosphate, the biosensor comprising a polypeptide according to any one of claims 1 to 12, and a means for detecting hydrolysis of an organophosphate molecule by the polypeptide.

31. 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 according to any one of claims 1 to 12, 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.

32. A polypeptide produced according to the method of claim 31.

33. 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).

34. The method of claim 33, wherein the fungicidal carbamate is benomyl or carbendazim.

35. The method of claim 33,^" wherein the insecticidal carbamate is methomyl or fenoxycarb.

36. The method according to any one of claims 33 to 35, further comprising exposing the carbamate to a divalent cation.

37. The method of claim 36, wherein the divalent cation is zinc.

38. 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.

39. 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.

40. 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.

41. 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 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).

42. A method for hydrolysing a fungicidal or insecticidal carbamate molecule, the method comprising exposing the carbamate to a sponge 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 comprising a sequence which is greater than 70% identical to any one of (i) to (iv).

43. 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 comprising a sequence which is greater than 70% i entical to any one of (i) to (iv), and a means for detecting hydrolysis of the carbamate by the polypeptide.

44. 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 comprising a sequence which is greater than 70% identical to any one of (i) to (iv), b) determining the ability of the mutant to hydrolyse a fungicidal or insecticidal carbamate molecule, and c) selecting a mutant with enhanced ability to hydrolyse the fungicidal or insecticidal carbamate molecule or altered substrate specificity for the fungicidal or insecticidal carbamate molecule, when compared to the first polypeptide.

45. A polypeptide produced according to the method of claim 44.