US 2004O161818A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0161818A1 Horne et al. (43) Pub. Date: Aug. 19, 2004

(54) PHOSPHOTRIESTERASE FROM (30) Foreign Application Priority Data AGROBACTERIUM RADIOBACTER P230 May 15, 2001 (AU)...... PR 5023 (76) Inventors: Irene Horne, Yass (AU); Tara Sutherland, Watson (AU); Rebecca Publication Classification Harcourt, North Ryde (AU); Robyn Russell, Wanniassa (AU); John (51) Int. Cl." ...... C12N 9/16; CO7H 21/04; Oakeshott, Wanniassa (AU) C12N 1/20 (52) U.S. Cl...... 435/69.1; 435/196; 435/252.2; 435/320.1; 536/23.2 Correspondence Address: Greenlee Winner & Sullivan (57) ABSTRACT Suite 201 5370 Manhattan Circle The present invention provides enzymes capable of hydrol ysing organophosphate (OP) molecules. In particular, the Boulder, CO 80303 (US) invention provides a phosphotriesterase enzyme identified from an Agrobacterium radiobacter strain isolated from soil (21) Appl. No.: 10/477,469 that hydrolyses OP pesticides, and the gene encoding that enzyme. The invention also provides mutants of the identi (22) PCT Fed: May 15, 2002 fied phosphotriesterase enzyme which have altered Substrate Specificity. The use of these enzymes in bioremediation (86) PCT No.: PCT/AU02/005.94 Strategies is also provided. Patent Application Publication Aug. 19, 2004 Sheet 1 of 4 US 2004/0161818A1

S O | O O---Ot O OH S

OF --> as HO-P-OEt c-S C CO OEt CH CH

Figure 1 Patent Application Publication Aug. 19, 2004 Sheet 2 of 4 US 2004/0161818A1

1. AGCAAACGA GAAGAGATGC ACTTAAGTCT GCGGCCGCAA AACTCTGCT 51 CGGCGGCTTG GCTGGGGTG CAAGCATGGC CCGACCAATC GGTACAGGCG 101 ATCTGATTAA TACTGTTCGC GGCCCCATTC CAGTTTCGGA AGCGGGCTTC 151 ACACTGACCC ATGAGCATAT CTGCGGCAGT TCGGCGGGAT TCCTACGTGC 201 GTGGCCGGAG TTTTTCGGTA GCCGCAAAGC TCTAGCGGAA AAGGCTGTGA 251 GAGGATTACG CCATGCCAGA TCGGCTGGCG TGCAAACCAT CGTCGATGTG 301 TCGACTTTCG ATATCGGTCG TGACGTCCGT TTATTGGCCG AAGTTTCGCG 35 GGCCGCCGAC GTGCATATCG TGGCGGCGAC TGGCTTATGG TTCGACCCGC 4 O1 CACTTTCAAT GCGAATGCGC AGCGTCGAAG AACTGACCCA GTTCTTCCTG 451. CGTGAAATCC AACATGGCAT CGAAGACACC GGTATTAGGG CGGGCATTAT 5 O1. CAAGGTCGCG ACCACAGGGA AGGCGACCCC CTTTCAAGAG TTGGTGTTAA 551 AGGCAGCCGC GCGGGCCAGC TTGGCCACCG GTGTTCCGGT AACCACTCAC 6O1 ACGTCAGCAA GTCAGCGCGA TGGCGAGCAG CAGGCAGCCA TATTGAAC 651 CGAAGGTTTG AGCCCCT CAC GGGTTTGTAT CGGTCACAGC GATGATACTG 7 O1 ACGATTTGAG CTACCTAACC GGCCTCGCTG CGCGCGGATA CCTCGTCGGT 751. TTAGATCGCA TGCCGTACAG TGCGATTGGT CTAGAAGGCA ATGCGAGGC 8O1 ATTAGCGCTC TTTGGTACTC GGTCGTGGCA AACAAGGGCT CTCTTGATCA 851 AGGCGCTCAT CGACCGAGGC TACAAGGATC GAAICCCGT CTCCCATGAC 901 TGGCTGITCG GGTTTTCGAG CTATGTCACG AACATCATGG ACGTAATGGA 951 TCGCATAAAC CCAGATGGAA TGGCCTTCGT CCCTCTGAGA GTGATCCCAT 1001 TCCTACGAGA GAAGGGCGTC CCGCCGGAAA CGCTAGCAGG CGTAACCGTG 1051 GCCAATCCCG CGCGGTTCTT GT CACCGACC GTGCGGGCCG TCGTGACACG 1101 ATCTGAAACT TCCCGCCCTG CCGCGCCTAT TCCCCGTCAA GATACCGAAC 1151 GATGA

Figure 2 Patent Application Publication Aug. 19, 2004 Sheet 3 of 4 US 2004/0161818A1

MQTRRDALKS AAAITLLGGL AGCASMARP, GTGDLINTVR GPIPWSEAGF 5. TILTHEHCGS SAGFLRAWPE FEGSRKALAE KAVRGRHAR SAGVQTIVDV 1.01 STFDIGRDVR LLAEWSRAAD WHIVAATGLW FDPPLSMRMR SVEELTQFFL 15. REIQHGIEDT GIRAGIIKVA TTGEXATPFQE LVLKAAARAS LATGWPWTTH 2O TSASQRDGEQ QAAIFESEGL SPSRWCIGHS DDTDDLSYLT GLAARGYLVG 251 LDRMPYSAIG LEGNASALAL EGTRSWOTRA LLIKALIDRG YKDRILVSHD 301 WLFGFSSYWT NIMDWMDRIN PDGMAEWPIR WIPFLREKGW PPETLAGWTW 351 ANPARFISPT WRAWVTRSET SRPAAPIPRO DTER

Figure 3 Patent Application Publication Aug. 19, 2004 Sheet 4 of 4 US 2004/0161818A1

OPD 1 MQTRRVWLKSAAAAGTLLGGLAGCASVAGSIGTGDRINTVRGPITISEAG 50 . : . | | | | | | | | | | | | . : OpdA 1 MQTRRDALKSAAAI.TLLGGLAGCASMARPIGTGDLINTVRGPIPVSEAG 49 OPD 51. FTLTHEHICGSSAGFLRAWPEFFGSRKALAEKAVRGLRRARAAGVRTIVD 100 | | | | | | | | | | | | | | | | | | | | | | : . . . . . OpdA 50 FTL THEHICGSSAGFLRAWPEFFGSRKALAEKAVRGLRHARSAGVQTIVD 99 OPD lo1 VSTFDIGRDVSLLAEVSRAADVHIVAATGLWFDPPLSMRLRSVEELTOFF 150 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | : | | | | | | | | | | OpdA 100 VSTFDIGRDVRLIAEVSRAADVHIVAATGLWFDPPLSMRMRSVEELTQFF 149 OPD 151 LREIQYGIEDTGIRAGIIKVATTGKATPFOELVLKAAARASILATGVPVTT 200 . | | | | | | | | | | | | | | | | | | | | | | | | | | | | OpdA 150 LREIQHGIEDTGIRAGIIKVATTGKATPFQELVLKAAARASLATGVPVTT 199 OPD 201 HTAASQRDGEQQAAIFESEGLSPSRVCIGHSDDTDDLSYLTALAARGYLI 250 . | | | | | | | | | | | | | | | | | | | : : OpdA 200 HTSASQRDGEQQAAIFESEGLSPSRVCIGHSDDTDDLSYLTGLAARGYLV 249 OPD 251 GLDHIPHSAIGLEDNASASALLGIRSWQTRALLIKALIDQGYMKQII.VSN 300 : : . : | | | | | | : ...... : OpdA 250 GLDRMPYSAIGLEGNASALALFGTRSWQTRALLIKALIDRGYKDRILVSH 299 OPD 301 DWLFGFSSYVTNIMDVMEDRVNPDGMAFIPLRVEPFLREKGVPQETLAGIT 350 | | | | | | | | | | | | | | : | | | | | | : | | | | | | | | | | | | | . : opdA 300 DWLFGFSSYVTNIMDVMDRINPDGMAFVPLRVIPFLREKGVPPETLAGVT 349 OPD 35. VTN PARFLSPTLRAS 365 . : OpdA 350 VANPARFLSPTVRAVVTRSETSRPAAPIPRODTER 384

Figure 4 US 2004/0161818 A1 Aug. 19, 2004

PHOSPHOTRIESTERASE FROM Substrates tested, Such as p-nitrophenyl acetate, bis(p-nitro AGROBACTERIUM RADIOBACTER P230 phenyl) phosphate, paraoxon and p-nitrophenyl phosphate. 0006 OPD homologues have also been identified in FIELD OF THE INVENTION vertebrates (Davies et al., 1997), although their function in 0001. This invention relates to enzymes capable of these organisms is unknown. OPD, ePHP, mtPHP and mam hydrolysing organophosphate (OP) molecules. In particular, malian PHPs are 27-30% identical at the amino acid level, the invention relates to a phosphotriesterase enzyme iden while mpFHP is less similar. Amino acid residues involved tified from an Agrobacterium radiobacter strain isolated in Zn" binding are conserved across the six members of the from Soil that hydrolyses OP pesticides, and the gene encod phosphotriesterase family identified to date (Buchbinder et ing that enzyme. The invention also relates to mutants of the al., 1998). identified phosphotriesterase enzyme which have altered 0007 Three other distinct OP hydrolysing enzymes have Substrate Specificity. been isolated from bacteria with a history of exposure to OPS (Mulbry and Karns, 1989; Mulbry, 1992; Cheng et al., BACKGROUND OF THE INVENTION 1999). The two for which sequence data are available are unrelated to each other and to OPD. One, a prolidase from 0002 Residues of organophosphate insecticides are Alteromonas sp., normally functions in hydrolysis of X-Pro undesirable contaminants of the environment and a range of dipeptides. Its activity for insecticidal OPs is reported as commodities. Areas of particular Sensitivity include con tamination of Soil, irrigation tailwater that is re-cycled, used modest, although it has not been reported in terms of ka/K by irrigators downstream or simply allowed to run off-farm, specificity constants (Cheng et al., 1999). The other, an and residues above permissible levels in agricultural and aryldialkylphosphatase (ADPase) from Nocardia sp. strain horticultural exports. Poisoning with organophosphates pre B-1, has a turnover number for ethyl parathion that is Sents a problem for agricultural workers that are exposed to 4500-fold lower than that reported for OPD (Mulbry and these chemicals, as well as military perSonnel exposed to Karns, 1989; Mulbry, 1992). organophosphates used in chemical warfare. Furthermore, 0008 Paraoxonase, or PON1, is a distinct OP hydrolys the Stockpiling of organophosphorus nerve agents has ing enzyme found in mammals. Like OPD it is a metalloen resulted in the need to detoxify these stocks. Bioremediation Zyme, preferring Ca" in this case, which is associated with Strategies are therefore required for eliminating or reducing low density lipoproteins in plasma and normally involved in these organophosphate residues and/or stockpiles. metabolism of oxidised lipid compounds (Gan et al., 1991; Sorenson et al., 1995). It has high activity for paraoxon, with 0003. One proposed strategy involves the use of enzymes a specificity constant of around 10'M' sec" (Doom et al., capable of immobilising or degrading the organophosphate 1999; Hong and Raushel, 1999). residues. Such enzymes may be employed, for example, in bioreactors through which contaminated water could be 0009. There is also evidence for other, so-called diiso passed, or in Washing Solutions after post-harvest disinfes propyl fluorophosphatase (DFPase) enzymes in a wide range tation of fruit, vegetables or animal products to reduce of vertebrates, invertebrates and microorganisms (Wang et residue levels and withholding times. Suitable enzymes for al., 1998; Hoskin et al., 1999; Billecke et al., 1999). These degrading organophosphate residues include OP hydrolases enzymes are notably diverse in many of their biochemical from bacteria (Mulbry, 1992; Mulbry and Kearney, 1991; properties but are all characterised by their hydrolytic activ Cheng et al., 1999; U.S. Pat. No. 5,484,728; U.S. Pat. No. ity against OP chemical warfare agents. Limited Sequence 5,589.386), vertebrates (Wang et al., 1993; 1998; Gan et al, data Suggest that they are unrelated to all the other OP 1991; Broomfield et al., 1999) and OP resistant insects (WO hydrolytic enzymes described above. 95/19440 and WO 97/19176). It is desirable that the OP 0010 OP resistant blowflies and houseflies have been the hydrolases degrade the organophosphate residues at a rapid Source of esterase enzymes with activity against Oxon OPS rate. like chlorfenvinfos (CVP) and carboxylester OPs like 0004. The most thoroughly studied OP degrading enzyme malathion (Newcomb et al., 1997; Campbell et al. 1998; is bacterial organophosphate dihydrolase (OPD), which is Claudianos et al. 1999; WO95/19440; WO 97/19176). A encoded by identical genes on dissimilar plasmids in both Gly to Asp Substitution at residue 137 in blowfly esterase E3 Flavobacterium sp. ATCC 27551 and Brevundimonas (and its housefly ortholog, ALI) resulted in the acquisition of diminuta MG (Harper et al., 1988; Mulbry and Karns, 1989). activity for CVP, while a Trp to Leu/Ser mutation at residue OPD is a homodimeric protein that is capable of hydrolysing 251 in the same enzyme resulted in activity against a wide range of phosphate triesters (both oxon and thion malathion. However, the Specificity constants of these OPs) (Dumas et al., 1989a, b). Its reaction mechanism enzymes for their OPSubstrates are orders of magnitude less directly or indirectly involves metal ions, preferably Zn". than those of OPD for paraoxon. OPD has no detectable activity with phosphate monoesters 0011. There is a need for further OP degrading enzymes or diesters (Dumas et al., 1989a, b; 1990). which can be used in bioremediation Strategies. 0005 OPD homologues (phosphotriesterase homology proteins, or PHPs) have been identified in the genomes of SUMMARY OF THE INVENTION Escherichia coli (ePHP), Mycobacterium tuberculosis 0012. The present inventors have developed a rapid and (mtPHP) and Mycoplasma pneumoniae (mpPHP), although sensitive fluorimetric assay for coumaphos (a thion OP only ePHP has been tested for phosphotriesterase activity insecticide) hydrolysis and used it to isolate a bacterium (Scanlan and Reid, 1995; Buchbinder et al., 1998). No from contaminated Soil that is capable of using OPS as the activity was detected in ePHP crude lysates with any of the Sole phosphorus Source. 16S rRNA sequencing identified the US 2004/0161818 A1 Aug. 19, 2004 bacterium. (isolate P230) as a strain of Agrobacterium 0031 (i) a sequence of nucleotides shown in SEQ radiobacter. The present inventors have also isolated and ID NO:5; characterized the enzyme responsible for this coumaphos hydrolytic activity and provide methods for the use of this 0032 (ii) a sequence of nucleotides shown in SEQ enzyme in bioremediation Strategies. ID NO:6; 0013 In one aspect, the present invention provides a 0033 (iii) a sequence of nucleotides shown in SEQ Substantially purified polypeptide, the polypeptide being ID NO:7; Selected from: 0034 (iv) a sequence of nucleotides shown in SEQ 0014 (i) a polypeptide comprising a sequence pro ID NO:8; vided in SEQ ID NO:1; 0035 (v) a sequence encoding a polypeptide accord 0015 (ii) a polypeptide comprising a sequence pro ing to the present invention; or vided in SEQ ID NO:2; 0036 (vi) a sequence which is at least 90% identical 0016 (iii) a polypeptide comprising a sequence pro to any one of (i) to (V), vided in SEQ ID NO:3: 0037 wherein the polynucleotide encodes a polypeptide 0017 (iv) a polypeptide comprising a sequence pro capable of hydrolysing an organophosphate molecule. vided in SEQ ID NO:4; or 0038 Preferably, the polynucleotide is at least 95% iden 0018 (v) a polypeptide comprising a sequence tical, more preferably at least 97% identical, and even more which is greater than 90% identical to any one of (i) preferably at least 99% identical to any one of (i) to (v). to (iv), 0039. In a further aspect, a vector is provided which 0.019 wherein the polypeptide is capable of hydrolysing comprises a polynucleotide according to the invention. an organophosphate molecule. 0040 Preferably, the vector is suitable for the replication 0020 Preferred organophosphate molecules include, but and/or expression of a polynucleotide. The vectors may be, are not limited to, coumaphos, coroXon, paraoxon, par for example, a plasmid, Virus or phage vector provided with athion, parathion-methyl, phoSmet, fenthion, diazinon, chlo an origin of replication, and preferably a promotor for the rpyrifos, dMUP, DFP, dimethoate, malathion, and malaoxon. expression of the polynucleotide and optionally a regulator More preferably, the organophosphate is phoSmet or of the promotor. The vector may contain one or more fenthion. Selectable markers, for example an amplicillin resistance 0021. In a preferred embodiment, the polypeptide can be gene in the case of a bacterial plasmid or a neomycin purified from an Agrobacterium sp. resistance gene for a mammalian expression vector. The vector may be used in vitro, for example for the production 0022. In a further preferred embodiment, the polypeptide of RNA or used to transfect or transform a host cell. is at least 95% identical to any one of (i) to (iv), more preferably at least 97% identical, and even more preferably 0041. In another aspect, a host cell is provided which at least 99% identical to any one of (i) to (iv). comprises a vector according to the invention. 0023. In another aspect, the present invention provides a 0042. In a further aspect, the present invention provides Substantially purified polypeptide, the polypeptide being a process for preparing a polypeptide of the invention, the Selected from: process comprising cultivating a host cell of the invention under conditions which allow production of the polypeptide, 0024 (i) a polypeptide comprising the sequence and recovering the polypeptide. Such cells can be used for provided in SEQ ID NO:1; the production of commercially useful quantities of the 0025 (ii) a polypeptide comprising the sequence encoded polypeptide. provided in SEQ ID NO:2; or 0043. In another aspect, the present invention provides a composition for hydrolysing an organophosphate molecule, 0026 (iii) a polypeptide which is greater than 90% the composition comprising a polypeptide according to the identical to (i) or (ii). invention, and one or more acceptable carriers. 0027. In another aspect, a fusion polypeptide is provided 0044) In another aspect, the present invention provides a which comprises a polypeptide according to the present composition for hydrolysing an organophosphate molecule, invention fused to at least one other polypeptide Sequence. the composition comprising a host cell of the invention, and 0028 Preferably, the at least one other polypeptide is one or more acceptable carriers. Selected from the group consisting of a polypeptide that 0045. It will be appreciated that the present invention can enhances the Stability of the polypeptide of the invention, be used to hydrolyse organophosphates in a Sample. For and a polypeptide that assists in the purification of the fusion instance, after a crop has been sprayed with an organophos polypeptide. phate pesticide, the organophosphate residue can be 0029 Preferably, the at least one other polypeptide is the hydrolysed from Seeds, fruits and vegetables before human maltose-binding protein. consumption. Similarly, organophosphate contaminated Soil 0.030. In another aspect, the present invention provides an or water can be treated with a polypeptide of the invention. isolated polynucleotide, the polynucleotide comprising a 0046 Accordingly, in a further aspect the present inven Sequence Selected from: tion provides a method for hydrolysing an organophosphate US 2004/0161818 A1 Aug. 19, 2004 molecule in a Sample, the method comprising exposing the method as disclosed herein. Alternatively, probes and/or Sample to a polypeptide according to the invention. primers can be designed based on the polynucleotides of the present invention to identify bacteria which produce natu 0047 Preferably, the polypeptide is provided as a com rally occurring variants of the polypeptides of the present position of the invention. invention. 0048. Further, it is preferred that the method further 0061 Accordingly, in a further aspect the present inven comprises exposing the Sample to a divalent cation. Prefer tion provides an isolated bacterium which produces a ably, the divalent cation is zinc. polypeptide according to the invention. 0049 Preferably, the sample is selected from the group consisting of; Soil, water, biological material, or a combi 0062 Preferably, the bacterium is an Agrobacterium sp. nation thereof. Preferred biological Samples include matter More preferably, the bacterium is a strain of Agrobacterium derived from plants Such as Seeds, vegetables or fruits, as radiobacter: well as matter derived from animals. Such as meat. 0063. In a further aspect, the present invention provides the use of an isolated naturally occurring bacterium which 0050 Preferred organophosphate molecules include, but produces a polypeptide according to the invention for are not limited to, coumaphos, coroXon, paraoxon, par hydrolysing an organophosphate in a Sample. athion, parathion-methyl, phoSmet, fenthion, diazinon, chlo rpyrifos, dMUP, DFP, dimethoate, malathion, and malaoxon. 0064. In a further aspect, the present invention provides More preferably, the organophosphate is phoSmet or a polymeric Sponge or foam for hydrolysing an organophos fenthion. phate molecule, the foam or Sponge comprising a polypep tide according to the invention immobilized on a polymeric 0051. The sample can be exposed to the polypeptide via porous Support. any available avenue. This includes providing the polypep tide directly to the Sample, with or without carriers or 0065 Preferably, the porous support comprises polyure excipients etc. The polypeptide can also be provided in the thane. form of a host cell, typically a microorganism Such as a 0066. In a preferred embodiment, the sponge or foam bacterium or a fungus, which expresses a polynucleotide further comprises carbon embedded or integrated on or in encoding the polypeptide of the invention. Usually, the the porous Support. polypeptide will be provided as a composition of the inven tion. 0067. In a further aspect, the present invention provides a method for hydrolysing an organophosphate molecule in a 0.052 Organophosphate molecules in a sample can also Sample, the method comprising exposing the sample to a be hydrolysed by exposing the Sample to a transgenic plant Sponge or foam according to the invention. which produces a polypeptide of the present invention. 0068. In another aspect, the present invention provides a 0.053 Thus, in a further aspect a transgenic plant is biosensor for detecting the presence of an organophosphate, provided which produces a polypeptide according to the the biosensor comprising a polypeptide of the invention, and invention. a means for detecting hydrolysis of an organophosphate 0054. In a further aspect, the present invention provides molecule by the polypeptide. a method for hydrolysing an organophosphate molecule in a 0069. In yet another aspect, the present invention pro Sample, the method comprising exposing the Sample to a vides a method for Screening for agents which hydrolyse an transgenic plant according to the invention. organophosphate molecule, the method comprising 0055) Preferably, the sample is soil. 0070 (i) exposing the organophosphate to a candi 0056 Further, it is preferred that the polypeptide is at date agent, and least produced in the roots of the transgenic plant. 0071 (ii) measuring a fluorescent signal produced 0057. In yet another aspect, the present invention pro from Step (i), wherein the fluorescent signal is vides an isolated strain of Agrobacterium radiobacter indicative of hydrolysis of the organophosphate. deposited under NM01/21112 on 20 Apr. 2001 at Australian 0072 Preferably, the organophosphate is coumaphos or Government Analytical Laboratories. COOXO. 0.058. In another aspect, the present invention provides a composition for hydrolysing an organophosphate molecule, 0073. Further, it is preferred that the agent is a polypep the composition comprising the Agrobacterium radiobacter tide or a micro-organism. Strain of the invention, and one or more acceptable carriers. 0074 The polypeptide of the present invention can be 0059. In yet another aspect, the present invention pro mutated, and the resulting mutants Screened for altered vides a method for hydrolysing an organophosphate mol activity Such as changes in Substrate Specificity. ecule in a Sample, the method comprising exposing the 0075 Thus, in a further aspect, the present invention Sample to an Agrobacterium radiobacter Strain according to provides a method of producing a polypeptide with the invention. enhanced ability to hydrolyse an organophosphate or altered Substrate Specificity for an organophosphate, the method 0060. The disclosure of the present invention can readily comprising be used to isolate other bacterial Species/strains which hydrolyse organophosphates. For example, other bacterial 0076 i) mutating one or more amino acids of a first Species/strains may be isolated using a fluorimetric Screening polypeptide according to the present invention, US 2004/0161818 A1 Aug. 19, 2004

0077 ii) determining the ability of the mutant to DETAILED DESCRIPTION OF THE hydrolyse an organophosphate, and INVENTION 0078 iii) selecting a mutant with enhanced ability to 0.098 General Techniques hydrolyse the organophosphate or altered Substrate Specificity for the organophosphate, when compared 0099. Unless otherwise indicated, the recombinant DNA to the first polypeptide. techniques utilized in the present invention are Standard procedures, well known to those skilled in the art. Such 0079. As outlined in the Example section, this method techniques are described and explained throughout the lit has been Successfully applied to produce the polypeptides erature in Sources such as, J. Perbal, A Practical Guide to provided as SEQ ID NO:2 and SEQ ID NO:3. Molecular Cloning, John Wiley and Sons (1984), J. Sam brook et al., Molecular Cloning: A Laboratory Manual, Cold 0080 Preferably, the first polypeptide is selected from Spring Harbour Laboratory Press (1989), T. A. Brown any one if SEQ ID NO's: 1 to 4. (editor), Essential Molecular Biology: A Practical Approach, 0081. In a further aspect, the present invention provides Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. a polypeptide produced according to the above method. Hames (editors), DNA Cloning: A Practical Approach, Vol 0082 Throughout this specification the word “comprise’, umes 1x, IRL Press (1995 and 1996), and F. M. Ausubel et or variations Such as “comprises' or “comprising”, will be al. (Editors), Current Protocols in Molecular Biology, understood to imply the inclusion of a Stated element, Greene Pub. Associates and Wiley-Interscience (1988, integer or Step, or group of elements, integers or Steps, but including all updates until present). not the exclusion of any other element, integer or Step, or 0100 Organophosphates group of elements, integers or Steps. 0101 Organophosphates are Synthetic organophosphorus 0083) The invention will hereinafter be described by way esters and related compounds Such as phosphoroamidates. of the following nonlimiting Figures and Examples. They have the general formula (RRX)P=O or (RRX)P=S, where R and R' are short-chain groups. For insecticidal BRIEF DESCRIPTION OF THE DRAWINGS organophosphates X is a good leaving group, which is a 0084) FIG. 1: Structure of coumaphos and its hydrolysis requirement for the irreversible inhibition of acetylcho products. linesterase. 0085 FIG. 2: The DNA sequence of opdA (SEQ ID 0102) The polypeptides of the present invention hydrol NO:5). The region encoding the signal peptide domain is yse the phosphoester bonds of organophosphates. These given in bold, with the remaining Sequence being referred to organophosphates can be, but are not limited to, OXon and herein as SEO ID NO:6. thion OPS. The organophosphate can have aromatic or aliphatic leaving groups (X). 0.086 FIG. 3: Amino acid sequence of OpdA (SEQ ID NO:1). The signal peptide is given in bold. 0.103 Although well known for their use as pesticides, organophosphates have also been used as nerve gases 0087 FIG. 4: Amino acid sequence alignment of OPD against mammals. Accordingly, it is envisaged that the (SEQ ID NO:17) and OpdA. The secretion signals are given polypeptides of the present invention will also be useful for in bold. hydrolysis of organophosphates which are not pesticides. KEY TO THE SEQUENCE LISTING 0104 Polypeptides 0088 SEQID NO: 1-Polypeptide sequence of OpdA. 0105. By “substantially purified polypeptide' we mean a 0089 SEQ ID NO: 2-Polypeptide sequence of OpdA polypeptide that has generally been Separated from the minus the Signal Sequence. lipids, nucleic acids, other polypeptides, and other contami nating molecules with which it is associated in its native 0090 SEQ ID NO: 3-Polypeptide sequence of State. Preferably, the Substantially purified polypeptide is at OpdA1. least 60% free, more preferably at least 75% free, and most 0091 SEQ ID NO: 4-Polypeptide sequence of preferably at least 90% free from other components with OpdA2. which they are naturally associated. 0106 The % identity of a polypeptide is determined by 0092) SEQ ID NO: 5-Polynucleotide sequence FASTA (Pearson and Lipman, 1988) analysis (GCG pro encoding OpdA. gram) using the default Settings and a query sequence of at 0093 SEQ ID NO: 6-Polynucleotide sequence least 50 amino acids in length, and whereby the FASTA encoding OpdA minus the Signal Sequence. analysis aligns the two Sequences over a region of at least 50 amino acids. More preferably, the FASTA analysis aligns the 0094 SEQ ID NO: 7-Polynucleotide sequence two Sequences over a region of at least 100 amino acids. encoding OpdA1. More preferably, the FASTA analysis aligns the two 0.095 SEQ ID NO: 8-Polynucleotide sequence Sequences over a region of at least 250 amino acids. Even encoding OpdA2. more preferably, the FASTA analysis aligns the two Sequences over a region of at least 350 amino acids. 0.096 SEQ ID NO’s: 9 to 16-PCR primers. 0107 Amino acid sequence mutants of the polypeptides 0097 SEQ ID NO: 17-Polypeptide sequence of OPD of the present invention can be prepared by introducing from Flavobactelium sp. appropriate nucleotide changes into a nucleic acid Sequence, US 2004/0161818 A1 Aug. 19, 2004 or by in vitro synthesis of the desired polypeptide. Such mutants include, for example, deletions, insertions or Sub TABLE 1-continued Stitutions of residues within the amino acid Sequence. A combination of deletion, insertion and Substitution can be Exemplary substitutions made to arrive at the final construct, provided that the final Original Exemplary protein product possesses the desired characteristics. Residue Substitutions Examples of mutants of the present invention are provided Pro (P) gly in Example 8. Ser (S) thr Thr (T) Se 0108. In designing amino acid sequence mutants, the Trp (W) tyr location of the mutation site and the nature of the mutation Tyr (Y) trp; phe will depend on characteristic(s) to be modified. The sites for Val (V) ille; leu; met; phe, ala 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 0112 Furthermore, if desired, unnatural amino acids or results achieved, (2) deleting the target residue, or (3) chemical amino acid analogues can be introduced as a inserting other residues adjacent to the located Site. Substitution or addition into the polypeptide of the present invention. Such amino acids include, but are not limited to, 0109) Amino acid sequence deletions generally range the D-isomers of the common amino acids, 2,4-diaminobu from about 1 to 30 residues, more preferably about 1 to 10 tyric acid, C.-amino isobutyric acid, 4-aminobutyric acid, residues and typically about 1 to 5 contiguous residues. 2-aminobutyric acid, 6-amino hexanoic acid, 2-amino isobu 0110 Substitution mutants have at least one amino acid tyric acid, 3-amino propionic acid, Ornithine, norleucine, residue in the polypeptide molecule removed and a different norvaline, hydroxyproline, Sarcosine, citrulline, homocitrul residue inserted in its place. The Sites of greatest interest for line, cysteic acid, t-butylglycine, t-butylalanine, phenylgly Substitutional mutagenesis include Sites identified as the cine, cyclohexylalanine, B-alanine, fluoroamino acids, active site(s). Other sites of interest are those in which designer amino acids Such as B-methyl amino acids, CC-me particular residues obtained from various Species are iden thyl amino acids, NC.-methyl amino acids, and amino acid tical. These positions may be important for biological activ analogues in general. ity. These sites, especially those falling within a Sequence of 0113 Also included within the scope of the invention are at least three other identically conserved sites, are preferably polypeptides of the present invention which are differen Substituted in a relatively conservative manner. Such con tially modified during or after Synthesis, e.g., by biotinyla servative Substitutions are shown in Table 1 under the tion, benzylation, glycosylation, acetylation, phosphoryla heading of “exemplary Substitutions'. tion, amidation, derivatization by known protecting/ blocking groups, proteolytic cleavage, linkage to an 0111 Since the sequence of SEQ ID NO:1 is 90% iden antibody molecule or other cellular ligand, etc. These modi tical to that of the Flavobactedium OPD enzyme it is fications may serve to increase the Stability and/or bioactiv possible that SEQ ID NO:1 could be used to design mutants ity of the polypeptide of the invention. of the Flavobacterium OPD enzyme which have the desired activity but are less than 90% identical. More specifically, 0114 Polypeptides of the present invention can be pro those amino acids important for hydrolysing an organophoS duced in a variety of ways, including production and recov phate molecule could be changed to match the polypeptides ery of natural proteins, production and recovery of recom of the present invention and other amino acids not affecting binant proteins, and chemical Synthesis of the proteins. In this activity could also be changed to ensure the identity one embodiment, an isolated polypeptide of the present levels do not exceed 90%. Examples of such OPD mutants invention is produced by culturing a cell capable of express include the amino acid changes L272F and/or H257Y. Such ing the polypeptide under conditions effective to produce the mutants are also included in the present invention. polypeptide, and recovering the polypeptide. Effective cul ture conditions include, but are not limited to, effective TABLE 1. media, bioreactor, temperature, pH and oxygen conditions that permit protein production. An effective medium refers Exemplary Substitutions to any medium in which a cell is cultured to produce a Original Exemplary polypeptide of the present invention. Such medium typically Residue Substitutions comprises an aqueous medium having assimilable carbon, nitrogen and phosphate Sources, and appropriate Salts, min Ala (A) val; leu; ille; gly Arg (R) lys erals, metals and other nutrients, Such as Vitamins. Cells of Asn (N) gln; his; the present invention can be cultured in conventional fer Asp (D) glu mentation bioreactors, Shake flaskS, test tubes, microtiter Cys (C) Se dishes, and petri plates. Culturing can be carried out at a Gln (Q) asn; his temperature, pH and oxygen content appropriate for a Glu (E) asp Gly (G) pro, ala recombinant cell. Such culturing conditions are within the His (H) asn; gln. expertise of one of ordinary skill in the art. Ile (I) leu; val; ala Leu (L) ille; val; met; ala; phe 0115 Polynucleotides Lys (K) arg Met (M) leu; phe; 0116. By “isolated polynucleotide' we mean a polynucle Phe (F) leu; val; ala otide which have generally been Separated from the poly nucleotide Sequences with which it is associated or linked in US 2004/0161818 A1 Aug. 19, 2004 its native state. Preferably, the isolated polynucleotide is at 0123. One type of recombinant vector comprises a least 60% free, more preferably at least 75% free, and most nucleic acid molecule of the present invention operatively preferably at least 90% free from other components with linked to an expression vector. The phrase operatively linked which they are naturally associated. Furthermore, the term refers to the insertion of a nucleic acid molecule into an “polynucleotide' is used interchangeably herein with the expression vector in a manner Such that the molecule is able term “nucleic acid molecule'. to be expressed when transformed into a host cell. AS used 0117 Polynucleotides of the present invention may pos herein, an expression vector is a DNA or RNA vector that is sess one or more mutations when compared to SEQ ID capable of transforming a host cell and effecting expression NO’s: 5 to 8. These mutations can be deletions, insertions, of a specified nucleic acid molecule. Preferably, the expres or Substitutions of nucleotide residues. Mutants can be either Sion vector is also capable of replicating within the host cell. naturally occurring (that is to say, isolated from a natural Expression vectors can be either prokaryotic or eukaryotic, Source) or Synthetic (for example, by performing site-di and are typically viruses or plasmids. Expression vectors of rected mutagenesis on the nucleic acid). It is thus apparent the present invention include any vectors that function (i.e., that polynucleotides of the invention can be either naturally direct gene expression) in recombinant cells of the present occurring or recombinant. invention, including in bacterial, fungal, endoparasite, 0118. The % identity of a polynucleotide is determined arthropod, other animal, and plant cells. Preferred expres by FASTA (Pearson and Lipman, 1988) analysis (GCG Sion vectors of the present invention can direct gene expres program) using the default Settings and a query sequence of Sion in bacterial, yeast, plant and mammalian cells. at least 150 nucleotides in length, and whereby the FASTA 0.124 Expression vectors of the present invention contain analysis aligns the two Sequences over a region of at least regulatory Sequences Such as transcription control 150 nucleotides. More preferably, the FASTA analysis aligns Sequences, translation control Sequences, origins of replica the two Sequences over a region of at least 300 nucleotides. tion, and other regulatory Sequences that are compatible Even more preferably, the FASTA analysis aligns the two with the recombinant cell and that control the expression of Sequences over a region of at least 1050 nucleotides. nucleic acid molecules of the present invention. In particu lar, recombinant molecules of the present invention include 0119 Oligonucleotides of the present invention can be transcription control Sequences. Transcription control RNA, DNA, or derivatives of either. The minimum size of Sequences are Sequences which control the initiation, elon Such oligonucleotides is the size required for the formation gation, and termination of transcription. Particularly impor of a stable hybrid between an oligonucleotide and a comple tant transcription control Sequences are those which control mentary Sequence on a nucleic acid molecule of the present transcription initiation, Such as promoter, enhancer, operator invention. The present invention includes oligonucleotides and repressor Sequences. Suitable transcription control that can be used as, for example, probes to identify nucleic Sequences include any transcription control Sequence that acid molecules or primers to produce nucleic acid mol can function in at least one of the host cells of the present ecules. invention. A variety of Such transcription control Sequences 0120 Oligonucleotides and/or polynucleotides of the are known to those skilled in the art. Preferred transcription present invention may selectively hybridise to the Sequences control Sequences include those which function in bacterial, set out in SEQ ID NO’s: 5 to 8 under high stringency. As yeast, plant and mammalian cells, Such as, but not limited to, used herein, Stringent conditions are those that (1) employ tac, lac, trp, trc, oxy-pro, Omp/lpp, rrnB, bacteriophage low ionic Strength and high temperature for Washing, for lambda, bacteriophage T7, T7lac, bacteriophage T3, bacte example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% riophage SP6, bacteriophage SPO1, metallothionein, alpha NaDodSO, at 50° C.; (2) employ during hybridisation a mating factor, Pichia alcohol oxidase, alphavirus Subge denaturing agent Such as formamide, for example, 50% nomic promoters (such as Sindbis virus Subgenomic (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% promoters), antibiotic resistance gene, baculovirus, Helio Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phos this zea insect virus, vaccinia virus, herpesvirus, raccoon phate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium poxvirus, other poxvirus, adenovirus, cytomegalovirus citrate at 42° C.; or (3) employ 50% formamide, 5xSSC (Such as intermediate early promoters), Simian virus 40, (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium retrovirus, actin, retroviral long terminal repeat, Rous Sar phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Den coma virus, heat Shock, phosphate and nitrate transcription hardt's solution, Sonicated salmon sperm DNA (50 g/ml), control Sequences as well as other Sequences capable of 0.1% SDS and 10% dextran Sulfate at 42°C. in 0.2xSSC and controlling gene expression in prokaryotic or eukaryotic O.1% SDS. cells. Additional Suitable transcription control Sequences 0121 Vectors include tissue-specific promoters and enhancers. 0.122 One embodiment of the present invention includes 0.125 Recombinant molecules of the present invention a recombinant vector, which includes at least one isolated may also (a) contain Secretory signals (i.e., signal Segment nucleic acid molecule of the present invention, inserted into nucleic acid sequences) to enable an expressed polypeptide any vector capable of delivering the nucleic acid molecule of the present invention to be secreted from the cell that into a host cell. Such a vector contains heterologous nucleic produces the polypeptide and/or (b) contain fusion acid Sequences, that is nucleic acid Sequences that are not Sequences which lead to the expression of nucleic acid naturally found adjacent to nucleic acid molecules of the molecules of the present invention as fusion proteins. present invention and that preferably are derived from a Examples of Suitable Signal Segments include any Signal Species other than the Species from which the nucleic acid Segment capable of directing the Secretion of a protein of the molecule(s) are derived. The vector can be either RNA or present invention. Preferred Signal Segments include, but are DNA, either prokaryotic or eukaryotic, and typically is a not limited to, tissue plasminogen activator (t-PA), inter Virus or a plasmid. feron, interleukin, growth hormone, histocompatibility and US 2004/0161818 A1 Aug. 19, 2004

Viral envelope glycoprotein Signal Segments, as well as efficiency with which those polynucleotide molecules are natural Signal Sequences. In addition, a nucleic acid mol transcribed, the efficiency with which the resultant tran ecule of the present invention can be joined to a fusion Scripts are translated, and the efficiency of post-translational Segment that directs the encoded protein to the proteoSome, modifications. Recombinant techniques useful for increas Such as a ubiquitin fusion Segment. Recombinant molecules ing the expression of polynucleotide molecules of the may also include intervening and/or untranslated Sequences present invention include, but are not limited to, operatively Surrounding and/or within the nucleic acid Sequences of linking polynucleotide molecules to high-copy number plas nucleic acid molecules of the present invention. mids, integration of the polynucleotide molecules into one or more host cell chromosomes, addition of vector Stability 0126) Host Cells Sequences to plasmids, Substitutions or modifications of 0127. Another embodiment of the present invention transcription control signals (e.g., promoters, operators, includes a recombinant cell comprising a host cell trans enhancers), Substitutions or modifications of translational formed with one or more recombinant molecules of the control signals (e.g., ribosome binding sites, Shine-Dalgarno present invention. Transformation of a nucleic acid molecule Sequences), modification of polynucleotide molecules of the into a cell can be accomplished by any method by which a present invention to correspond to the codon usage of the nucleic acid molecule can be inserted into the cell. Trans host cell, and the deletion of Sequences that destabilize formation techniques include, but are not limited to, trans transcripts. The activity of an expressed recombinant protein fection, electroporation, microinjection, lipofection, adsorp of the present invention may be improved by fragmenting, tion, and protoplast fusion. A recombinant cell may remain modifying, or derivatizing polynucleotide molecules encod unicellular or may grow into a tissue, organ or a multicel ing Such a protein. lular organism. Transformed nucleic acid molecules of the 0130 Transgenic Plants present invention can remain extrachromosomal or can integrate into one or more Sites within a chromosome of the 0131) As generally described in WO99/53037, the levels transformed (i.e., recombinant) cell in Such a manner that of organophosphates in a Sample can be reduced by exposing their ability to be expressed is retained. the Sample to a transgenic plant expressing a Suitable enzyme. Typically, the Sample is Soil. Accordingly, the 0128 Suitable host cells to transform include any cell polynucleotide of the present invention can be expressed in that can be transformed with a polynucleotide of the present a transgenic plant, particularly the roots of the plant, for invention. Host cells can be either untransformed cells or hydrolysing organophosphate molecules in the sample. cells that are already transformed with at least one nucleic acid molecule (e.g., nucleic acid molecules encoding one or 0132) The term “plant” refers to whole plants, plant more proteins of the present invention). Host cells of the organs (e.g. leaves, stems roots, etc), Seeds, plant cells and present invention either can be endogenously (i.e., naturally) the like. Plants contemplated for use in the practice of the capable of producing proteins of the present invention or can present invention include both monocotyledons and dicoty be capable of producing Such proteins after being trans ledons. Exemplary dicotyledons include cotton, corn, formed with at least one nucleic acid molecule of the present tomato, tobacco, potato, bean, Soybean, and the like. invention. Host cells of the present invention can be any cell 0.133 Transgenic plants, as defined in the context of the capable of producing at least one protein of the present present invention include plants (as well as parts and cells of invention, and include bacterial, fungal (including yeast), said plants) and their progeny which have been genetically parasite, arthropod, animal and plant cells. Preferred host modified using recombinant DNA techniques to cause or cells include bacterial, mycobacterial, yeast, plant and mam enhance production of at least one protein of the present malian cells. More preferred host cells include Agrobacte invention in the desired plant or plant organ. rium, Salmonella, Escherichia, Bacillus, Listeria, Saccharo myces, Spodoptera, Mycobacteda, Trichoplusia, BHK (baby 0134) The polypeptide of the present invention may be hamster kidney) cells, MDCK cells (normal dog kidney cell expressed constitutively in the transgenic plants during all line for canine herpesvirus cultivation), CRFK cells (normal Stages of development. Depending on the use of the plant or cat kidney cell line for feline herpesvirus cultivation), CV-1 plant organs, the proteins may be expressed in a stage cells (African monkey kidney cell line used, for example, to Specific manner. Furthermore, depending on the use, the culture raccoon poxvirus), COS (e.g., COS-7) cells, and proteins may be expressed tissue-specifically. Vero cells. Particularly preferred host cells are E. coli, including E. coli K-12 derivatives; Salmonella typhi; Sal 0.135 The choice of the plant species is determined by the monella typhimurium, including attenuated Strains, intended use of the plant or parts thereof and the amenability Spodoptera frugiperda, Trichoplusia ni; BHK cells; MDCK of the plant species to transformation. cells, CRFK cells; CV-1 cells; COS cells; Vero cells; and 0.136 Regulatory sequences which are known or are non-tumorigenic mouse myoblast G8 cells (e.g., ATCCCRL found to cause expression of a gene encoding a protein of 1246). Additional appropriate mammalian cell hosts include interest in plants may be used in the present invention. The other kidney cell lines, other fibroblast cell lines (e.g., choice of the regulatory Sequences used depends on the human, murine or chicken embryo fibroblast cell lines), target plant and/or target organ of interest. Such regulatory myeloma cell lines, Chinese hamster ovary cells, mouse Sequences may be obtained from plants or plant viruses, or NIH/3T3 cells, LMTK cells and/or HeLa cells. may be chemically Synthesized. Such regulatory Sequences are well known to those skilled in the art. 0129 Recombinant DNA technologies can be used to improve expression of transformed polynucleotide mol 0.137 Other regulatory sequences such as terminator ecules by manipulating, for example, the number of copies Sequences and polyadenylation signals include any Such of the polynucleotide molecules within a host cell, the Sequence functioning as Such in plants, the choice of which US 2004/0161818 A1 Aug. 19, 2004

would be obvious to the skilled addressee. An example of controlled release vehicles include, but are not limited to, Such Sequences is the 3' flanking region of the nopaline biocompatible polymers, other polymeric matrices, cap Synthase (noS) gene of Agrobacterium tumefaciens. Sules, microcapsules, microparticles, bolus preparations, oSmotic pumps, diffusion devices, liposomes, lipospheres, 0.138. Several techniques are available for the introduc and transdermal delivery systems. Preferred controlled tion of the expression construct containing a DNA sequence encoding a protein of interest into the target plants. Such release formulations are biodegradable (i.e., bioerodible). techniques include but are not limited to transformation of 0.143 A preferred controlled release formulation of the protoplasts using the calcium/polyethylene glycol method, present invention is capable of releasing a composition of electroporation and microinjection or (coated) particle bom the present invention into Soil or water which is in an area bardment. In addition to these so-called direct DNA trans Sprayed with an organophosphate pesticide. The formulation formation methods, transformation Systems involving vec is preferably released over a period of time ranging from tors are widely available, Such as viral and bacterial vectors about 1 to about 12 months. A preferred controlled release (e.g. from the genus Agrobacterium). After Selection and/or formulation of the present invention is capable of effecting Screening, the protoplasts, cells or plant parts that have been a treatment preferably for at least about 1 month, more transformed can be regenerated into whole plants, using preferably for at least about 3 months, even more preferably methods known in the art. The choice of the transformation for at least about 6 months, even more preferably for at least and/or regeneration techniques is not critical for this inven about 9 months, and even more preferably for at least about tion. 12 months. 013:9 Compositions 0144. The concentration of the polypeptide, vector, or host cell of the present invention that will be required to 0140 Compositions of the present invention include produce effective compositions for hydrolysing an organo excipients, also referred to herein as "acceptable carriers'. phosphate will depend on the nature of the Sample to be An excipient can be any material that the animal, plant, plant decontaminated, the concentration of the organophosphate or animal material, or environment (including Soil and water Samples) to be treated can tolerate. Examples of Such in the Sample, and the formulation of the composition. The excipients include water, Saline, Ringer's Solution, dextrose effective concentration of the polypeptide, vector, or host Solution, Hank's Solution, and other aqueous physiologically cell within the composition can readily be determined balanced Salt Solutions. Nonacqueous vehicles, Such as fixed experimentally, as will be understood by the skilled artisan. oils, Sesame oil, ethyl oleate, or triglycerides may also be 0145 Biosensors used. Other useful formulations include Suspensions con taining Viscosity enhancing agents, Such as Sodium car 0146 Biosensors are analytical devices typically consist boxymethylcellulose, Sorbitol, or dextran. Excipients can ing of a biologically active material Such as an enzyme and also contain minor amounts of additives, Such as Substances a transducer that converts a biochemical reaction into a that enhance isotonicity and chemical Stability. Examples of quantifiable electronic signal that can be processed, trans buffers include phosphate buffer, bicarbonate buffer and Tris mitted, and measured. A general review of biosensors which buffer, while examples of preservatives include thimerosal have been used for the detection of orangophosphorus or o-creSol, formalin and benzyl alcohol. Excipients can also compounds is provided by Rekha et al. (2000), the entire be used to increase the half-life of a composition, for contents of which are incorporated by reference. The example, but are not limited to, polymeric controlled release vehicles, biodegradable implants, liposomes, bacteria, polypeptide of the present invention can be adapted for use Viruses, other cells, oils, esters, and glycols. in Such biosensors. 0141 Furthermore, the polypeptide of the present inven EXAMPLES tion can be provided in a composition which enhances the rate and/or degree of organophosphate hydrolysis, or Example 1 increases the Stability of the polypeptide. For example, the polypeptide can be immobilized on a polyurethane matrix Enriching Soil Samples for Microorganisms with (Gordon et al., 1999), or encapsulated in appropriate lipo Coumaphos Hydrolytic Activity somes (Petrikovics et al. 2000a and b). The polypeptide can also be incorporated into a composition comprising a foam 0147 Fluorimetric Assay for Coumaphos Hydrolysis Such as those used routinely in fire-fighting (LeJeune et al., 1998). As would be appreciated by the skilled addressee, the 0.148 Phosphotriesterase enzymes catalyse the cleavage polypeptide of the present invention could readily be used in of a phosphoester bond in organophosphate (OP) molecules to yield a phosphodiester and an alcohol. In the case of a sponge or foam as disclosed in WO 00/64539, the contents coumaphos (3-chloro-4-methyl-7-coumarinyl diethyl phos of which are incorporated herein in their entirety. phorothioate), phosphotriesterase hydrolysis yields dieth 0142. One embodiment of the present invention is a ylthiophosphate and the fluorescent alcohol, chlorferon controlled release formulation that is capable of slowly (3-chloro-7-hydroxy-4-methyl coumarin; FIG. 1). Couma releasing a composition of the present invention into an phoS hydrolysis can therefore be measured fluorimetrically animal, plant, animal or plant material, or the environment by the production of chlorferon, as measured by excitation (including Soil and water Samples). AS used herein, a con at a wavelength of 355 nm and an emission intensity of 460 trolled release formulation comprises a composition of the nm. Chlorferon fluorescence was linear over the range 0.01 present invention in a controlled release vehicle. Suitable uM to 2.5uM. US 2004/0161818 A1 Aug. 19, 2004

0149 All fluorescence measurements were performed in Example 2 a POLARstar fluorimeter (BMG Technologies Pty Ltd, Australia) using 96-well white microtitre plates (Fluoro Identification of Isolate P230 Nunc plates with PolySorp Surface, Nalge Nunc Interna tional) and final reaction volumes of 100 ul. Stock solutions 0154 Isolate P230 was a Gram negative, catalase posi of coumaphos and chlorferon (0.4 mM) were prepared in tive and oxidase positive, rod-shaped bacterium. To deter 20% methanol. Crude assays of whole cells were performed mine the identity of isolate P230, sequence analysis of the in 100 uM coumaphos, 0.5% Triton X-100 and 50 mM 16S rRNA gene was performed. DNA was extracted from Tris-HCl, pH8.0. Coumaphos hydrolytic assays of cell isolate P230 according to the method of Rainey et al. (1992). lysates were performed without the Triton X-100. Cells of a P230 culture that had been grown in low salt LB medium (2 ml) overnight at 28° C. were pelleted by cen 0150. The fluorescence of bacterial colonies and stained polyacrylamide gels was examined using a hand-held long trifugation in a microfuge (12 000 rpm/2 minutes). The cell wavelength (approximately 340 nm) UV light (Gelman pellet was resuspended in 400 ul STE buffer (10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, pH8.0) and 5 ul of Sciences). a freshly-prepared lysozyme Solution (0.3 ug/ul) was added. 0151. Enrichment Cultures After incubation at 37° C. for 20 minutes, Proteinase K (15 0152 The phosphodiester hydrolysis products of phos ul of a 1% solution) and SDS (10 ul of a 25% solution) were photriesterases can be used as phosphorus Sources by a wide added and the reactions incubated at 60° C. for 30 minutes. range of bacteria (Cook et al., 1978; Rosenberg and Alex DNA preparations were then extracted Sequentially with an ander, 1979). An enrichment culture was therefore estab equal Volume of buffer-Saturated phenol, and then an equal lished in which 1 g of Soil obtained from a domestic yard, volume of chloroform. which had previously been exposed to diazinon (a diethyl thion OP), served as an inoculum for 50 ml enrichment 0155 The 16S rRNA gene was amplified from the medium (Table 2), in which coumaphos was the only added extracted DNA by PCR using bacterial universal primers 27f phosphorus Source. (5' AGAGTTTGATCMTGGCTCAG 3) (SEQ ID NO: 9) and 1492r (5'TACGGYTACCTTGTTACGACTT 3) (SEQ TABLE 2 ID NO: 10), the names of which are based on the numbering system of the E. coli 16S rRNA gene (Lane, 1991). Approxi Qomposition of coumaphos enrichment media. mately 1320 bp of the 16S rRNA gene from isolate P230 was Medium (per litre) Trace element solution (per litre) obtained and Sequence Similarities were performed using the Tris 6.05 g (NH4)Mo,O.4H2O 20 mg FASTA algorithm (Pearson and Lipman, 1988). The 16S NHCI 1 g HBO 50 mg rRNA gene of isolate P230 was very similar in sequence to FeCl 20 tug ZnCl2 30 mg KCI 0.5 g. CoCl2.6HO 3 mg that of other Agrobactedium strains (Table 3). Sodium acetate 0.68 g MnCl2.4H2O 10 mg MgSO 0.1 g Cupric acetate 10 mg TABLE 3 p-aminobenzoate 0.9 mg nicotinic acid 0.9 mg Trace element solution 10 ml Nucleic acid sequence comparisons of the 16S rRNA genes of Coumaphos 100 mM isolate P230 with those of various Agrobacterium strains. pH7.0 Sequence Agrobacterium strains Identity (%) 0153 Ten percent of the enrichment culture was subcul tured twice into 50 ml fresh enrichment medium containing Agrobacterium radiobacter LMG383 1OO coumaphos as the Sole phosphorus Source. Coumaphos was Agrobacterium sp. LMG 11936 99.7 then replaced with diazinon (at 100 mM) in the enrichment Agrobacterium sp. MSMC211 99.5 medium and the culture subcultured as before. After 3 days Agrobacterium sp. LMG 11915 99.3 incubation at 28 C., the enrichment culture was further subcultured into media in which 100 mM parathion (another diethyl thion OP) was the sole phosphorus source. It was noted that after two days the culture had turned yellow 0156 These results suggested that isolate P230 was an (presumably due to the production of p-nitrophenol). This Agrobacterium Strain. The utilization of carbon Sources by culture was then diluted in phosphorus-free medium and isolate P230 was examined using the Biolog system plated onto low Salt LB plates (10 g/l tryptone, 5 g/l yeast (Oxoid), according to procedures recommended by the extract and 2.5 g/l NaCl). After three days of growth at 28 C., approximately 100 colonies were picked randomly, re manufacturer. The carbon utilization profile was then com Streaked to ensure purity and then assayed for coumaphos pared with that of known species of Agrobacterium (Table 4; hydrolytic activity using the microtitre plate assay described Krieg and Holt, 1984). The isolate was capable of using above. Fluorescence was measured after 8 hours at room Sucrose, ornithine and glucose as carbon Sources. This, along temperature. One isolate (designated P230) demonstrated with a positive oxidase reaction determined using Kovac's Significant fluorescence and this isolate was examined fur method (Kovac, 1956), Suggested that the isolate was most ther. Colonies of this isolate also demonstrated fluorescence similar to either A. tumefaciens biovar1 or A. radiobacter on an agar plate containing coumaphos. biovar 1. US 2004/0161818 A1 Aug. 19, 2004 10

TABLE 4 Carbon utilization profiles and oxidase status of isolate P230 and known Agrobacterium species. Agrobacterium spp.

Carbon A. A. A. A. A. sourcef tumefaciens tumefaciens radiobacter radiobacter rhizogenes Isolate test biovar1 biowar2 biowar1 biowar2 biowar2 A. rubi P230

Tween 80 Sucrose ------Ornithine ------D-glucose ------Oxidase ------test (Kovac)

O157 A. tumefaciens biovar1 and A. radiobacter biovar1 pH8.0. Cells were disrupted by Sonication (five 15 second can be distinguished by the presence of a tumour-inducing bursts at 4 C.) and large cell debris or intact cells removed plasmid in the former. The tumour-inducing ability of Strain by centrifugation (8000 g for 15 minutes). An aliquot of the P230 was tested in a tomato seedling by transferring a heavy resultant Supernatant (containing 5 ug protein) was then Suspension of bacteria in water to the leaves and using a separated on a 10% (29:1 acrylamide:bis) SDS-PAGE gel. Sterile needle to pierce the Surface of the leaves through the Prior to loading, neither SDS nor B-mercaptoethanol was Suspension. No evidence of tumours was seen after a period added to the Sample and furthermore, the Sample was not of four weeks. A. tumefaciens C58 was used as a positive boiled as in conventional SDS-PAGE. After electrophoresis control and produced tumours in this period of time. A cured the gel was equilibrated for 5 min in 50 mM Tris-HCl pH8.0, Strain of A. tumefacienS C58 was used as a negative control and then incubated for a further 5 min in 50 mM Tris-HCl and produced the same effects in the test plant as isolate pH8.0, containing 8 uM coumaphos. The gel was then P230. Therefore isolate P230 was designated as a strain of examined under UV light as described above. A major Agrobacterium radiobacter biovar1. fluorescent band was detected, indicating that the P230 isolate contains a Single enzyme with coumaphoS hydrolytic Example 3 activity. This enzyme had an apparent molecular mass of 66 kDa. Constitutive Expression of Coumaphos Hydrolytic Activity TABLE 5 0158. In order to determine if the phosphotriesterase Parathion hydrolytic activities of P230 cultures grown in the activity of A. radiobacter P230 was constitutively presence or absence of parathion to an OD at S95 nm of 0.280. expressed, regardless of the presence of OPs, the parathion hydrolytic activities of cultures of A. radiobacter P230 were Parathion hydrolytic examined in the presence and absence of parathion (Table activity (umol/min/mg 5). Growth was monitored by measuring the optical density Culture protein) of the cultures at 595 nm in a BioPad Model 3550-UV +parathion 3.36 - 0.18 microplate Spectrophotometer. Parathion hydrolytic activity -parathion 3.13 - O.O7 was assayed according to the procedure of Serdar et al. (1989). This involved measuring the formation of p-nitro phenol from parathion at 405 nm in a BioPad Model Example 5 3550-UV microplate spectrophotometer. The reaction mix ture contained 880 uM parathion in 50 mM Tris-HCl pH8.0 Cloning the Gene Responsible for Coumaphos (this reaction also contained 5% methanol). Table 5 shows Hydrolytic Activity that parathion hydrolytic activity was constitutively expressed in isolate P230 and that the majority of this 0.160) Cloning Techniques and DNA Preparations activity was expressed in early- to mid-log phase. 01.61 General cloning techniques, unless otherwise indi Example 4 cated, were Standard and as described by Sambrook et al. (1989). Chromosomal DNA was extracted from A. radio bacter P230 according to the method of Gardiner et al. Native Polyacrylamide Gel Electrophoresis (PAGE) (1996). Briefly, an overnight culture (100 ml) of A. radio of P230 Extracts bacter P230 was pelleted by centrifugation at 5000 g for 20 0159. To demonstrate that a single enzyme was involved minutes, washed twice with 10 ml ice-cold STE buffer (see in coumaphos hydrolysis, native gels of A. radiobacter P230 above), and finally resuspended in 10 ml STE. Lysozyme cell extracts were stained for coumaphoS hydrolytic activity. (20mg) was added and the cells incubated for 2 hours at 37 A culture (50 ml) of A. radiobacter P230 in low salt LB C. An equal volume of STE was added along with SDS (to broth was pelleted by centrifugation at 8000 g for 15 minutes a final concentration of 2% w/v) and RNase (to a final and the cell pellet resuspended in 2 ml 50 mM Tris-HCl concentration of 20 ug/ml) and the cell lysate incubated at US 2004/0161818 A1 Aug. 19, 2004

42 C. for 1 hour. Proteinase K (50 ug/ml final concentra assayed for coumaphoS hydrolytic activity in a microtitre tion) was then added and the lysate incubated at 55° C. until plate. This involved resuspending the mating mix in an assay the Solution became translucent. An equal Volume of buffer buffer containing 0.5% Triton X-100, 100 uM coumaphos Saturated phenol/chloroform (1:1) was added, the sample and 50 mM Tris-HCl pH8.0. The microtitre plates were then thoroughly mixed and then centrifuged at 5000 g for 1 hour incubated for 8 hours at room temperature, and the amount at 4 C. The upper aqueous layer was transferred to a clean of fluorescence noted as described above. One mating mix tube using a broken pipette in order to prevent shearing of (containing clone p65) demonstrated significant fluores DNA. To precipitate the chromosomal DNA, 3M sodium CCCC. acetate, pH5.2, (0.1 volume) and ice-cold ethanol (2.5 Volumes) were added, the Solution mixed gently and placed 0168 Coumaphos Hydrolytic Activity of E. coli DH10? at -20° C. for 1 hour. The DNA was then removed using a p65 Cell-Free Extracts "hooked' pasteur pipette. This precipitate was washed with 0169 Cell-free extracts of E. coli DH10? p65, and con 70% ethanol and air-dried for 5 minutes. TE buffer (pH8.0; trol eXtracts containing the pBlueScript vector alone, were 1 ml) was added and the DNA left-to dissolve at 4 C. prepared from cells grown to mid-log phase on LB medium overnight. containing amplicillin (100 lug/ml). The 50 ml cultures were 0162 Library Construction in E. coli pelleted by centrifugation at 8000 g for 15 minutes and resuspended in 2 ml 50 mM Tris-HCl pH8.0. The cells were 0163 A partial Sau3AI digest of A. radiobacter P230 disrupted by Sonication (five 15 second bursts at 4 C.) and chromosomal DNA was prepared by digesting 37.5 ug of large cell debris or intact cells were removed by centrifu DNA with 4, 2, 1, 0.5 and 0.25 units of Sau3AI restriction gation (8000 g for 15 minutes). Aliquots (containing 15 ug endonuclease for 10 minutes at 37 C. DNA fragments in the protein) of the Supernatants were assayed for coumaphos Size range of 10-12 kb were excised from a 0.7% agarose gel hydrolytic activity. The increase in fluorescence over time and extracted using a QIAGENPCR purification/gel extrac was measured and the amount of activity determined. It can tion kit, according to the manufacturer's instructions. p3lue be seen from Table 6 that cell-free extracts of E. coli DH103 Script KS+ plasmid DNA (Stratagene), prepared using the containing clone p65 displayed Significant coumaphos GeneworkS Ultraclean Plasmid miniprep kit, was digested hydrolytic activity compared to that of the vector-only with BamHI for 1 hour at 37 C. and dephosphorylated using calf intestinal alkaline phosphatase (Boehringer Mannheim). controls. The phosphatase was then removed using the QIAquick 0170 Localisation of the Gene Encoding the OP Hydro PCR purification kit (QIAGEN). The size-fractionated P230 lytic Activity in Clone p65 DNA fragments were ligated to the BamHI digested, phos 0171 Clone p65 DNA was digested to completion with phatase treated pBlueScript vector, using T4DNA ligase and HindIII and the resultant four fragments 5.5 kb (containing the T4 ligase buffer provided by New England Biolabs. pBluescript vector), 4 kb, 3.5 kb and 1.4 kb separated and Ligations were performed for 20 hours at 4 C. Subsequently excised from a 1% agarose gel. The fragments 0164. The ligated DNA was then transformed into E. coli were extracted using the QIAquick PCR purification kit DH103 using the revised Hanahan transformation method (QIAGEN) and ligated to HindIII digested pBluescript DNA (Sambrook et al., 1989). The transformation mix was plated prepared as described above. The ligation mixes were trans onto LB agar plates containing amplicillin (100 ug/ml), formed into E. coli DH103 and individual clones assayed for X-Gal (5-bromo-4-chloro-3-indolyl-B-D-galactoside; 40 coumaphoS hydrolytic activity. Several of the clones con Aug/ml) and IPTG (isopropyl-f-D-thiogalactoside; 40 ug/ml), taining the 4 kb HindIII fragment demonstrated coumaphos and incubated at 37 C. overnight. Approximately 350 white hydrolytic activity, depending on the orientation of the colonies were revealed. fragment in p3lueScript relative to the lac promoter. 0.165 Triparental Matings for Expression in A. tumefa cienS TABLE 6 0166 Since it was possible that the gene encoding the OP Coumaphos hydrolytic activity of cell-free extracts of E. coli hydrolytic activity in A. radiobacter would not be expressed DH10? po5 and control extracts containing sufficiently in E. coli to allow identification on the basis of the plueScript vector alone. expression, the pBlueScript plasmids from the E. coli library Coumaphos hydrolytic above were transferred into a closely-related Strain, A. activity (nmol/min/mg tumefaciens C58. A. tumefaciens C58 is a non-OP hydrol Strain protein) ysing Strain of Agrobacterium (Zimmerer et al., 1966). Briefly, the white colonies identified above on LB plates E. coli DH10? (pBluescript) O.78 O.O4 containing amplicillin, X-Gal and IPTG were patched onto E. coli DH10? (p65) 3.30 O.O7 LB plates, without any additions. Also patched onto the Same plates at the same places were A. tumefacienS C58 and E. coli JM109 (Yanisch-Perron et al., 1985) containing the Example 6 conjugative, cointegrative plasmid, pR751::Tn813 (Bowen and Pemberton, 1985). It was intended that the conjugative Sequence of opdA cointegrative plasmid would move into E. coli DH100 0172 The nucleotide sequence of the 4 kb HindIII frag containing the pBlueScript-derivatives and form a cointe ment identified above was determined using primers grate. The cointegrates would then be transferred into A. complementary to the T3 and T7 promoters in the vector and tumefaciens C58. primer walking. DNA was Sequenced using the BigDye 0167 The tri-parental matings were incubated overnight Terminator system (Applied BioSystems) on the Applied at 28 C. The mating mixtures were then Scraped up and BioSystems ABIPRISM377 automated DNA sequencer. An US 2004/0161818 A1 Aug. 19, 2004 open reading frame (ORF) was identified in the same fragment was subsequently cloned into the Pst-HindIII orientation as the lacZ promoter in clones possessing activ cloning sites of pMAL-c, to generate the recombinant plas ity and in the opposite orientation in clones lacking activity. mid, pmal-opdA. The open reading frame contains 1152 nucleotides (FIG. 2) 0180. The opd gene (Mulbry and Karns, 1989) was and, when translated, would encode a protein of 384 amino cloned into the pMAL-2X vector (New England Biolabs) in acids (FIG. 3) and 41.4 kDa. a similar way. The opd gene, without the Signal peptide 0173 Sequence similarities were calculated using the domain, was amplified using PCR. The upstream and down FASTA algorithm (Pearson and Lipman, 1988). This indi stream oligonucleotide primers, 5'GATCGTGGATCCTC cated that the ORF had 88% nucleotide sequence identity to GATCGGCACAGGCGATCGG (SEQ ID NO: 13) and opd, a previously identified phosphotriesterase gene from 5'GATCGTAAGCTTTCATGACGCCCGCAAGGTCGG Flavobacterium sp. ATCC27551 (Mulbry and Karns, 1989). (SEQ ID NO: 14), respectively, were designed to contain a Furthermore, the inferred amino acid sequence of the ORF BamHI restriction site at the opd start codon and a HindIII was 90% identical to that of the Flavobacterium OPD restriction site at the stop codon (underlined bases). The enzyme (FIG. 4). For this reason we have named this open PCR fragment was subsequently cloned into the BamHI reading frame opdA. HindIII restriction sites of pMAL-c2X to generate the 0.174 Some notable differences were observed between recombinant plasmid, pFmal. the Flavobactenum Opd Sequence and that of opdA from A. 0181 Purification of OpdA and OPD Proteins radiobacter P230 (FIGS. 2 and 3). There appears to be one less amino acid in the putative Signal Sequence of the OpdA 0182 Both MBP fusion proteins were expressed in E. protein and the Signal cleavage site is also different. Fur coli DH10? cells. Optimal production of MBP fusion pro thermore, a frameshift near the 3' end of the opdA gene gives teins was obtained when mid-log cells (ODoo=0.6) were OpdA an additional 16 amino acids. This region has been induced with 0.1 mM isopropyl-B-D-thiogalactopyranoside Sequenced multiple times to ensure that the extra base in for 5 hours at 37 C. Harvested cells were disrupted by opdA is not a Sequencing error. Sonication and the Soluble fraction loaded onto an amylose resin (New England Biolabs), equilibrated with 50 mM 0175. The native OPD enzyme is a homodimer that Tris-HCl pH7.5. MBP fusion proteins were eluted with 10 contains two Zinc ions per monomeric Subunit (Benning et mM maltose in 50 mM Tris-HCl pH7.5. Fractions contain al., 1995). The two His residues at positions 254 and 257 in ing coumaphoS hydrolytic activity were pooled and cleaved the OPD protein sequence are located near the bimetallic with Xa protease (10 ug/ml; New England Biolabs) for 5 active site present in each monomer and are thought to hours. The cleaved fractions were then passaged through a interact with active Site residues and the Substrate in the DEAE Sepharose ion exchange resin. Cleaved OpdA and Substrate binding pocket. Replacement of each of these His OPD proteins did not bind to this resin and eluted with the residues with Arg and Leu, respectively, resulted in enzymes Void volume. Fractions from this Sample appeared to be pure possessing only two metal atoms per dimer (diSioudi et al., as judged by SDS-PAGE. The amount of protein in purified 1999). The Opda protein has Arg and Tyr at the positions Samples was calculated according to the method of Gill and corresponding to His 254 and His257 in OPD (FIG. 4). It von Hippel (1989). would therefore be expected that the OpdA native enzyme would contain only two metal ions per dimer rather than 0183 Kinetic Analyses of OPD and OpdA four, as in native OPD. 0184 (i) Substrates Example 7 0185. Kinetic parameters were determined for the hydrolysis by Opda and OPD of the following substrates: Activity of the Purified OpdA Protein coumaphos, parathion (O,O-diethyl p-nitrophenyl phospho rothioate; Riedel de Haan), parathion-methyl (O,O-dimethyl 0176) To confirm that the open reading frame in FIG. 3 p-nitrophenyl phosphorothioate; Riedel de Haan), paraoxon encoded the protein responsible for OP hydrolytic activity, (O,O-diethyl p-nitrophenyl phosphate, Sigma), coroxon the protein was expressed and purified as a fusion protein (3-chloro-4-methyl-7-coumarinyl diethyl phosphate; with maltose-binding protein. Alltech), fenthion (O,O-dimethyl O-3-methyl 4-(methylth 0177 Expression of OpdA and OPD as Fusion Proteins io)phenylphosphorothioate; Riedel de Haan), diazinon (labelled-O,O-diethyl-O-(2-isopropylmethyl-6-pyrimidi 0178. The OpdA and OPD proteins were expressed in nyl)-phosphorothioate; Alltech), dMUP (O,O-dimethyl Escherichia coli using the pMAL protein fusion and puri 4-methyl-umbelliferyl phosphate; a gift from Alan Devon fication system of New England Biolabs, which results in the shire), chlorpyrifos (O,O-diethyl O-3,5,6-trichloro-2-py expression of maltose-binding protein (MBP) fusion pro ridyl phosphorothioate; Alltech) and phosmet (S-(1,3-dihy teins. dro-1,3-dioxo-2H-isoindol-2-yl)methyl O,O-dimethyl 0179 To clone the opdA gene into the pMAL-c vector, phosphorodithioate; Alitech). the opdA gene (without the Signal peptide domain) was amplified by the polymerase chain reaction (PCR) using the 0186 (ii) Assays upstream and downstream primers, 5'GATCGTCTGCAGC 0187 All reactions contained organophosphates dis CAATCGGTACAGGCGATCTG (SEQ ID NO: 11) and Solved in methanol, except for phoSmet, which was dis 5'GATCGTAAGCTTTCATCGTTCGGTATCT Solved in acetone. The concentration of acetone or methanol TGACGGGGAAT (SEQ ID NO: 12), respectively. A PstI in the reactions was constant at 5%, where appropriate. All cloning Site was inserted at the Start codon and a HindIII reactions were performed in 50 mM Tris-HCl pH 8.0 at 25° cloning site at the stop-codon (underlined bases). The PCR C. US 2004/0161818 A1 Aug. 19, 2004 13

0188 The initial rates of reactions of purified OPD and All dichloromethane. The upper aqueous phase (150 ul) was OpdA with coumaphos and coroXon were determined using removed and quantitated by liquid Scintillation. the fluorimetric assay described above. 0195 Results 0189 The initial rates of reaction of both OPD and OpdA with dMUP were determined using a fluorimetric assay to 0.196 Results of the kinetic analyses are given in Table 7. quantitate the formation of the hydrolysis product, 4-methyl OpdA and OPD were able to hydrolyse the Substrates umbelliferone (Roth, 1969). The fluorescence was measured coumaphos, coroXon, paraoxon, parathion, parathion-me using an excitation wavelength of 355 nm and an emission thyl, diazinon, chlorpyrifos and dMUP. OpdA had a higher intensity of 460 nm. k for both parathion-methyl and dMUP, and OPD was 0190. The initial rates of reaction of both purified unable to hydrdolyse either phosmet or fenthion. A lack of enzymes with parathion, parathion-methyl and paraoxon hydrolysis of the latter two substrates was also observed were measured spectrophotometrically by quantitating the over a 24 hour period by thin layer chromatography (Mun formation of the hydrolysis product, pnitrophenol, at 405 nm necke and Hsieh, 1976). This involved the extraction of a 100 ul reaction containing 0.4 mM Substrate with an equal and using an extinction coefficient of 17 000 M' cm Volume of ethyl acetate. The upper organic phase was gently (Dumas et al., 1989b). dried with a nitrogen Stream, and the remaining residue was 0191 The initial rates of reaction of OpdA with fenthion dissolved in 10 ul acetone and then applied to a neutral Silica were quantitated Spectrophotometrically by monitoring a gel FTLC plate (Alltech, NSW, Australia). The plate was reduction in absorbance at 252 nm (Ibrahim and Cavagnol, then developed in hexane-chloroform-methanol (7:2:1) and 1966). A lack of hydrolysis offenthion and phosmet by OPD compounds Visualised by Short wavelength ultra-violet light. was confirmed by thin layer chromatography after 24 hour Hydrolysis of both phosmet and fenthion were consistently incubation of the Substrate with OPD. observed for OpdA and no hydrolysis was seen for OPD. 0192 The reaction rates of OpdA and OPD with chlo 0197). In summary, several differences in substrate speci rpyrifos were measured spectrophotometrically by monitor ficity between OpdA and OPD were observed. OpdA ing the increase in absorbance at 276 nm (Dumas et al., hydrolysed fenthion and phosmet whereas OPD did not. 1989b). Furthermore, there was a Significant difference between 0193 The hydrolysis of phosmet was measured by quan OpdA and OPD in the k values for dimethyl OPs, with titating the formation of free thiols during the course of the OpdA possessing a higher k for methyl-parathion and reaction using DTNB (Ellman's reagent; 5'5 dithio-bis-(2- dMUP than OPD. We would also expect OpdA, like OPD nitro benzoic acid)) as has been described previously for (Dumas et al., 1989a; Yang et al., 1995), to hydrolyse OP monitoring P-S hydrolysis in organophosphates (Lai et al., nerve agentS. 1995). This involved the addition of DTNB (80 ul of 1 0198 As discussed above, the two His residues at posi mg/ml in 50 mM sodium phosphate pH7.5 and methanol, tions 254 and 257 in the OPD protein sequence are located 1:1 (V/v)) to 20 ul aliquots of the reaction taken at various near the bimetallic active site present in each monomer and times. are thought to interact with active site residues and the 0194 The hydrolysis of diazinon was monitored using Substrate in the Substrate binding pocket (Benning et al., radiolabelled diazinon (ethyl-1-'C; 14.8 MBq/mmol) in the 1995). Replacement of each of these His residues with Arg radiometric partition assay previously used for radiolabelled and Leu, respectively, resulted in enzymes with only one OP substrates (Campbell et al., 1998). At various times metal ion per monomer, increased catalytic activity for during the reaction, an aliquot (50 ul) was removed and larger Substrates Such as demeton, and decreased activity for diluted with 150 ul water. This was then extracted with 500 Smaller

TABLE 7

Kinetic parameters of purified Opda and OPD enzymes for various OP substrates.

Substratef K (uM) ka (min')

Structure OpdA OPD OpdA OPD

coumaphos 8.3 - 18 21.4 - 6.0 12.4 - 0.6 14.1 + 2.6 i O O o--on OEt 1S CH US 2004/0161818 A1 Aug. 19, 2004 14

TABLE 7-continued Kinetic parameters of purified Opda and OPD enzymes for various OP substrates.

Substratef K (uM) ka (min')

Structure OpdA OPD OpdA OPD

COOXO. 15.9 - 1.9 25.3 + 1.3 22.7 - 0.1 39.5 - 5.3

O O O- -OEt

OEt C N

CH paraoxon 242 61 225 - 14 33.5 - O.S 46.0 O.4

O

ON O- -OEt

OEt parathion 92.6 6.4 50.6 - 12.2 21.9 2.0 23.5 + 0.2

S

ON O- -OEt

OEt parathion-methyl 61.2 - 23 32.9 - 1.7 94.2 - 0.8 5.46 - 0.05

S

ON O- -OMe

OMe phosmet 208.3 - 13.2 O.1OO O.OO2

S N-CH-S-P-OMe C. OMe fenthion 148.6 17.2 1.63 O.O1

3C HCS O | O Me OMe diazinon 51.9 - 4.5 54.2 + 5.4 65.2 + 6.7 56.5 2.9

o S N \ / O- -OEt N OEt US 2004/0161818 A1 Aug. 19, 2004 15

TABLE 7-continued Kinetic parameters of purified Opda and OPD enzymes for various OP substrates. Substratef Km (LM min' Structure OpdA OPD OpdA OPD chlorpyrifos 32.6 8.1 47.2 3.0 O.525 O.OO5 O.90 O.O1

S C N o--or, n OEt C 21 C dMUP 66.O. 9.1 46.7 2.8 81.7 - 9.1 20.5 + 2.3

O O O o--o. bu. N

CH

0199 substates like paraoxon (diSioudi et al., 1999). It hydrolytic activity as described above. Two colonies (des was postulated that changes in the number of bound metal ignated pmal-opdA1 and pmal-opdA2) were Selected and ions may enhance Structural flexibility and improve acceSS examined further. of larger Substrates to the active Site, while Simultaneously 0203 The sequences of the two mutants were examined decreasing activity for Smaller Substrates. The OpdA protein and compared with that of wild-type OpdA. OpdA1 con has Arg and Tyr at the positions corresponding to His 254 tained 4 mutations (P42S, P134S, A170S and S237G) (SEQ and His257 in OPD (FIG. 4). It is therefore surprising that ID NO: 3) and OpdA2 contained one mutation (A119D) OpdA possessed a higher k for methyl-parathion than (SEQ ID NO: 4). The numbering system is based on Opda OPD, yet its k for the larger ethyl-parathion Substrate was numbering of amino acid residues, taking into account the similar to that of OPD. Similar results were obtained for the Signal Sequence. To correlate the numbering with OPD, add coumaphos/dMUP substrate pair. Clearly, differences one to each number. between the OpdA and OPD amino acid sequences other than those at residues 253/254 and 256/257 affect catalytic 0204 OpdA and the two mutants OpdA1 and Opda2 activity. were purified after expression in the plasmid pCY76. The genes were amplified by PCR using the primers pFTopdA5 Example 8 (5'GATCGTGAATTCCATATGCCAATCGGTACA, with EcoRI site underlined and NdeI double underlined) (SEQID Identification of OpdA Mutants with Altered NO: 15) and pETopdA3 (5"GATCGTGGATCCTCATCGT. Specificity TCGGTATCTTG, with BamHI site underlined) (SEQ ID NO:16). PCR fragments were digested with EcoRI and 0200. The plasmid pmal-opdA was transformed into the BamHI and ligated with similarly-digested pBluescript. E. Colimutator Strain XL1-red. The plasmid was propagated Sequence of the fragments were confirmed in this vector. in this Strain for 120 generations, with plasmid extractions The pBS-derivatives were then digested with Nde-BamHI occurring after every 24 generations. These plasmids were and ligated with Nde-Bg/II-digested pCY76. Positive then transformed into E. coli DH103 and the transformation clones were grown in 500 ml of LB. After the cultures had mix diluted to 50 ml in LB containing ampicillin. grown for 24 hours, they were pelleted by centrifugation at 0201 When the culture reached an ODsos of 0.3, fusion 7000 g, 15 minutes at 4 C. The pellets were resuspended in protein expression was induced with 0.1 mM IPTG, and 4 ml 50 mM Tis-HCl pH7.5 and broken by sonication induction allowed to occur for 5 hours. The culture was then (Harcourt et al., 2002). Cell-free extracts were then charged pelleted by centrifugation, resuspended in 2 ml of sterile 50 onto a DEAE Sepharose column that was pre-equilibrated mM Tris-HCl pH7.5 with the addition of malathion to a final with 50 mM Tris-HCl pH7.5. OpdA and variants did not concentration of 440 uM. This assay mixture was left for 1 bind to this column and the eluant was collected and placed hour and the hydrolysis of malathion detected using Ell on a heparin Sepharose column pre-equilibrated with 50 mM man's reagent (DTNB) (Lai et al., 1995). Tris-HCl pH7.5 (Pharmacia). OpdA and variants were 0202 Pools containing activity were then diluted and bound by this column and eluted with 50 mM Tris-HCl plated onto LB plates with amplicillin. Individual colonies pH7.5/0.1 M NaCl. After this column step, OpdA was were then Selected and tested for malathion and dimethoate judged to be pure by SDS-PAGE. The kinetics of the US 2004/0161818 A1 Aug. 19, 2004 proteins were examined against the aliphatic OPs, present specification is Solely for the purpose of providing a dimethoate, malathion, malaoxon and DFP (diisopropyl context for the present invention. It is not to be taken as an fluorophosphate) (Table 8). Both mutants were active admission that any or all of these matters form part of the against dimethoate, malathion and malaoxon, whereas the prior art base or were common general knowledge in the wild-type Opda was not. Furthermore, the mutants had field relevant to the present invention as it existed in increased activity for DFP compared to that of wild-type Australia before the priority date of each claim of this OpdA. application.

TABLE 8 The kinetic parameters of purified Opda and the mutants, OpdA1 and OpdA2, for various OP substrates. Substrate Km (LM min' OpdA OpdA1 OpdA2 OpdA OpdA1 OpdA2 DFP 2.3 O.4 18.1 - 0.6 9.6 - 5.6 1.36 0.04 45.9 0.9 27.5 O.7 r--or, OPir dimethoate ind 78.9 17.7 14.3 + 4.2 ind 1.22 O.O7 1.4 + 0.1

O

HC-NH ls C-S-P-OMei H OMe malathion ind 33.3 14.2 40.9 7.2 ind 1.21 O.O7 1.43 O.O7

O S H EtO C-C-S-P-OMe H2 OMe EtO O malaoxon ind 159.2 - 28.2 45.7 8.9 ind 1.98 - 0.05 1.84 OO6

O O H EtO C-C-S-P-OMe H2 OMe EtO O 'nd = not detected

0205. It will be appreciated by persons skilled in the art REFERENCES that numerous variations and/or modifications may be made to the invention as shown in the Specific embodiments 0208 Benning, M. M., Kuo, J. M., Raushel, F. M. and without departing from the Spirit or Scope of the invention as Holden, H. M. (1995). Biochemistry 34: 7973-7978. broadly described. The present embodiments are, therefore, 0209 Billecke, S. S., Primo-Parmo, S. L., Dunlop, C. to be considered in all respects as illustrative and not S., Doorn, J. A., La Du, B. N. and Broomfield, C. A. restrictive. (1999). Chemico-Biological Interactions 120: 251-256. 0206 All publications discussed above are incorporated herein in their entirety. 0210 Bowen, A. R. St.G. and Pemberton, J. M. (1985). Mercury resistance transposon Tn813 mediates chro 0207 Any discussion of documents, acts, materials, moSome transfer in Rhodopseudomonas Sphaeroides devices, articles or the like which has been included in the and intergeneric transfer in pBR322. In Helsinki, D.R., US 2004/0161818 A1 Aug. 19, 2004 17

S. N. Cohen, D. B. Clewell, D. A. Jackson and A. 0229 Hong, S. B. and Raushel, F. M. (1999). Hollaender (ed.), Plasmids in Bacteria. Plenum Press, Chemico-Biological Interactions 120: 225-234. New York, p105-115. 0230 Hoskin, F. C. G., Walker, J. E. and Mello, C. M. 0211 Broomfield, C.A., Lockridge, O. and Millard, C. (1999). Chemico-Biological Interactions 120:399-404. B. (1999). Chemico-Biological Interactions 119-120: 413-418. 0231 Ibrahim, F. B. and Cavagnol, J. C. (1966). Jour 0212 Buchbinder, J. L., Stephenson, R. C., Dresser, nal of Agricultural and Food Chemistry 14: 369-371. M. J., Pitera, J. W., Scanlan, T. S. and Fletterick, R. J. (1998). Biochemistry 37: 5096-5160. 0232 Kovac, N. (1956). Nature 178: 703. 0213 Campbell, P. M., Newcomb, R. D., Russell, R. J. 0233 Krieg, N. R. and Holt, J. G. (ed.) 1984. Bergey’s and Oakeshott, J. G. (1998). Insect Biochemistry and Manual of Determinative Bacteriology. The Williams Molecular Biology 28: 139-150. & Wilkins Co., Baltimore. 0214 Cheng, T., DeFrank, J. J. and Rastogi, V. K. 0234) Lai, K., Stolowich, N.J. and Wild, J. R. (1995). 1999).... Chemico-BiologicalChemicO-BOOgical IInteractOnS 119-120:: 455 Archives of Biochemistry and Biophysics 318: 59-64. 462. 0235 Lane, D. J. (1991). 16S/23S rRNA sequencing, 0215 Claudianos, C., Russell, R. J. and Oakeshott, J. p115-175. In E. Stackebrandt and M. Goodfellow (ed.) G. (1999). Insect Biochemistry and Molecular Biology Nucleic acid techniques in bacterial Systematics. John 29: 675-686. Wiley & Sons, New York. 0216 Cook, A.M., Daughton,9. C. G. and Alexander, M. 0236 Le Juene, K. E., Wild, J. R. and Russell, A. J. (1978). Applied and Environmental Microbiology 36: (1998). Nature 395: 27-28. 668-672. 0237 Mulbry, W. W. (1992) Gene 121:149-153. 0217. Davies, J. A., Buchman, V. L., Krylova, O. and 0238) Mulbry, W. W. and Karns, J. S. (1989). Journal Ninkina, N. N. (1997). FEBS Letters 410: 378-382. of Bacteriology 171: 6740-6746. 0218 disioudi, B., Grimsley, J. K., Lai, K. and Wild, J. R. (1999). Biochemistry 38: 2866-2872. 0239). Mulbry, W. W. and Kearney, P. C. (1991). Crop Protection 10: 334-345. 0219) Doorn, J. A., Sorenson, R. C., Billecke, S. S., Hsu, C. and La Du, B. N. (1999). Chemico-Biological 0240 Munnecke, D. M. and Hsieh, D. P. (1976). Interactions 120: 235-241. Applied and Environmental Microbiology 31: 63-69. 0220 Dumas, D. P., Wild, J. R. and Raushel, F. M. 0241 Newcomb, R. D., Campbell, P. M., Ollis, D. L., (1989a). Biotechnology and Applied Biochemistry 11: Cheah, E., Russell, R. J. and Oakeshott, J. G. (1997). 235-243. Proceedings of the National Academy of Sciences USA 94: 7464-7468. 0221) Dumas, D. P., S. R. Caldwell, J. R. Wild and F. M. Raushel. (1989b). Journal of Biological Chemistry 0242) Pearson, W. R. and D. J. Lipman. (1988). Pro 264: 19659-19665. ceedings of the National Academy of Sciences USA85: 2444-2448. 0222 Dumas, D. P., Wild, J. R. and Raushel, F. M. (1990). Experientia 46: 729-731. 0243 Petrikovics, I., Cheng, T. C., Papahadjopoulos, D., Hong, K., Yin, R., DeFrank, J. J., Jaing, J., Zong, Z. 0223 Gan, K. N., Smolen, A., Eckerson, H. W. and H., McGuinn, W. D., Sylvester, D., Pei, L., Madec, J., Bert, N. L. (1991). Drug Metabolism and Disposition Tamulinas, C., Jaszberenyi, J. C., Barcza, T. and Way, 19: 100-106. J. L. (2000a). Toxicology Science 57: 16-21. 0224 Gardiner, A.T., MacKenzie, R. C., Barrett, S.J., Kaiser, K. and Cogdell, R. J. (1996). Photosynthesis 0244 Petrikovics, I., McGuinn, W. D., Sylvester, D., Research 49: 223-235. YuZapavik, P., Jaing, J., Way, J. L., Papahadiopoulos, D., Hong, K., Yin, R., Cheng, T. C., and DeFrank, J. J. 0225 Gill, S. C. and von Hippel, P. H. (1989). Ana (2000b). Drug Delivery 7: 83-89. lytical Biochemistry 182: 319-326. 0245 Rainey, F. A., M. Dorsch, H. W. Morgan and E. 0226 Gordon, R. K., Feaster, S. R., Russell, A. J., Stackebrandt. (1992). Systematic and Applied Micro LeJeune, K. E., Maxwell, M.D., Lenz, D. E., Ross, M. biology 15: 197-202. and Doctor, B. P. (1999). Chemical-Biological Interac tions 14: 463-470. 0246 Rekha, M., Thakur, M. S., and Karanth, N. G. (2000). Critical Reviews in Biotechnology 20: 213 0227 Harcourt, R. L., Horne, I., Sutherland, T. D., 235. Hammock, B. D., Russell, R. J. and Oakeshott, J. G. (2002) Lett. Appl. Microbiol. 34: 263-268. 0247 Rosenberg, A. and Alexander, M. (1979). Applied and Environmental Microbiology 37: 886-891. 0228) Harper, L. L., McDaniel, S., Miller, C. E. and Wild, J. R. (1988). Applied and Environmental Micro 0248 Roth, M. (1969). Methods of Biochemical biology. 54: 2586-2589. Analysis 17: 189-285. US 2004/0161818 A1 Aug. 19, 2004 18

0249 Sambrook, Fritsch, J. E. F. and Maniatis, T. 0253 Wang,9. O., Sun, M., Zhang,9. H. and Huang,9. C. (1989). Molecular cloning-A laboratory Manual. 2" (1998). Journal of Biochemistry and Molecular Toxi Ed. Cold Spring Harbour Laboratory Press, USA. cology 12: 213-217. 0250 Scanlen, C. S. and Reid, R. C. (1995). Chemistry 0254 Wang, F., Xiao, M. and Shaofeng, M. (1993). and Biology 2: 71-75. Journal of Biochemistry and Toxicology 8: 161-166. 0251 Serdar, C.M., Murdock, D.C. and Rohde, M. F. 0255 Yang, F., Wild, J. R. and Russell, A. J. (1995). (1989). Bio/Technology 7: 1151-1155. Biotechnology Progress 11: 471-474. 0252) Sorenson, R. C., Primo-Parmo, S. L., Kuo, C-L, 0256 Yanisch-Perron, Vieira, C. J. and Messing, J. Adkins, S., Lockridge, O and La Du, B. N. (1995). (1985). Gene 33: 103-119. Proceedings of the National Academy of Science USA 0257 Zimmerer, R. P., Hamilton, R. H. and Pootjes, C. 92: 7187-71.91. (1966). Journal of Bacteriology 92: 746-750.

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS : 17 <21 Oc SEQ ID NO 1 <211 LENGTH 384 <212> TYPE PRT ORGANISM: Agrobacterium radiobacter <400 SEQUENCE: 1

Met Glin Thr Arg Arg Asp Ala Teu Lys Ser Ala Ala Ala Ile Thr Teu 1 5 10 15

Leu Gly Gly Telu Ala Gly Ala Ser Met Ala Arg Pro Ile Gly Thr 20 25 30

Gly Asp Teu Ile Asn Thr Wall Arg Gly Pro Ile Pro Wall Ser Glu Ala 35 40 45

Gly Phe Thir Leu Thr His Glu. His Ile Cys Gly Ser Ser Ala Gly Phe 5 O 55 60

Teu Arg Ala Trp Pro Glu Phe Phe Gly Ser Arg Ala Lieu Ala Glu 65 70 75

Ala Val Arg Gly Lieu Arg His Ala Arg Ser Ala Gly Wall Glin Thr 85 90 95

Ile Wall Asp Wall Ser Thr Phe Asp Ile Gly Asp Val Arg Teu Teu 100 105 110

Ala Glu Wall Ser Arg Ala Ala Asp Wall His Ile Wall Ala Ala Thr Gly 115 120 125

Leu Trp Phe Asp Pro Pro Leu Ser Met Arg Met Arg Ser Wall Glu Glu 130 135 14 O

Teu Thr Glin Phe Phe Leu Arg Glu Ile Glin His Gly Ile Glu Asp Thr 145 15 O 155 160

Gly Ile Arg Ala Gly Ile Ile Wall Ala Th Thr Gly Ala Thr 1.65 17 O 175

Pro Phe Glin Glu Telu Wall Teu Ala Ala Ala Arg Ala Ser Leu Ala 18O 185 190

Thr Gly Wall Pro Wall. Thir Thr His Thr Ser Ala Ser Glin Arg Asp Gly 195 200

Glu Glin Glin Ala Ala Ile Phe Glu Ser Glu Gly Leu Ser Pro Ser Arg 210 215 220

Wall Ile Gly His Ser Asp Asp Thr Asp Teu Ser Teu Thr 225 230 235 240

Gly Lieu Ala Ala Arg Gly Leu Wall Gly Telu Asp Arg Met Pro 245 25 O 255 US 2004/0161818 A1 Aug. 19, 2004 19

-continued

Ser Ala Ile Gly Lieu Glu Gly Asn Ala Ser Ala Leu Ala Lieu Phe Gly 260 265 27 O Thr Arg Ser Trp Glin Thr Arg Ala Lieu Lieu. Ile Lys Ala Lieu. Ile Asp 275 280 285 Arg Gly Tyr Lys Asp Arg Ile Leu Val Ser His Asp Trp Lieu Phe Gly 29 O 295 3OO Phe Ser Ser Tyr Val Thr Asn Ile Met Asp Val Met Asp Arg Ile Asn 305 310 315 320 Pro Asp Gly Met Ala Phe Val Pro Leu Arg Val Ile Pro Phe Leu Arg 325 330 335 Glu Lys Gly Val Pro Pro Glu Thr Leu Ala Gly Val Thr Val Ala Asn 340 345 35 O Pro Ala Arg Phe Leu Ser Pro Thr Val Arg Ala Val Val Thr Arg Ser 355 360 365 Glu Thir Ser Arg Pro Ala Ala Pro Ile Pro Arg Glin Asp Thr Glu Arg 370 375 38O

<210> SEQ ID NO 2 &2 11s LENGTH 356 &212> TYPE PRT <213> ORGANISM: Agrobacterium radiobacter <400 SEQUENCE: 2 Pro Ile Gly Thr Gly Asp Leu Ile Asn Thr Val Arg Gly Pro Ile Pro 1 5 10 15 Val Ser Glu Ala Gly Phe Thr Leu Thr His Glu His Ile Cys Gly Ser 2O 25 30 Ser Ala Gly Phe Leu Arg Ala Trp Pro Glu Phe Phe Gly Ser Arg Lys 35 40 45 Ala Lieu Ala Glu Lys Ala Val Arg Gly Lieu Arg His Ala Arg Ser Ala 50 55 60 Gly Val Glin Thr Ile Val Asp Val Ser Thr Phe Asp Ile Gly Arg Asp 65 70 75 8O Val Arg Lieu Lieu Ala Glu Val Ser Arg Ala Ala Asp Wal His Ile Val 85 90 95 Ala Ala Thr Gly Leu Trp Phe Asp Pro Pro Leu Ser Met Arg Met Arg 100 105 110 Ser Val Glu Glu Leu Thr Glin Phe Phe Leu Arg Glu Ile Glin His Gly 115 120 125 Ile Glu Asp Thr Gly Ile Arg Ala Gly Ile Ile Lys Val Ala Thr Thr 130 135 1 4 0 Gly Lys Ala Thr Pro Phe Glin Glu Lieu Val Lieu Lys Ala Ala Ala Arg 145 15 O 155 160 Ala Ser Leu Ala Thr Gly Val Pro Val Thr Thr His Thr Ser Ala Ser 1.65 170 175 Glin Arg Asp Gly Glu Glin Glin Ala Ala Ile Phe Glu Ser Glu Gly Lieu 18O 185 19 O Ser Pro Ser Arg Val Cys Ile Gly His Ser Asp Asp Thr Asp Asp Lieu 195 200 2O5 Ser Tyr Lieu. Thr Gly Lieu Ala Ala Arg Gly Tyr Lieu Val Gly Lieu. Asp 210 215 220 Arg Met Pro Tyr Ser Ala Ile Gly Lieu Glu Gly Asn Ala Ser Ala Lieu US 2004/0161818 A1 Aug. 19, 2004 2O

-continued

225 230 235 240 Ala Lieu Phe Gly Thr Arg Ser Trp Glin Thr Arg Ala Leu Lieu. Ile Lys 245 250 255 Ala Lieu. Ile Asp Arg Gly Tyr Lys Asp Arg Ile Leu Val Ser His Asp 260 265 27 O Trp Leu Phe Gly Phe Ser Ser Tyr Val Thr Asn Ile Met Asp Val Met 275 280 285 Asp Arg Ile Asn Pro Asp Gly Met Ala Phe Val Pro Leu Arg Val Ile 29 O 295 3OO Pro Phe Leu Arg Glu Lys Gly Val Pro Pro Glu Thir Leu Ala Gly Val 305 310 315 320 Thr Val Ala Asn Pro Ala Arg Phe Leu Ser Pro Thr Val Arg Ala Val 325 330 335 Val Thr Arg Ser Glu Thir Ser Arg Pro Ala Ala Pro Ile Pro Arg Glin 340 345 35 O Asp Thr Glu Arg 355

<210> SEQ ID NO 3 &2 11s LENGTH 384 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Mutant of OpdA <400 SEQUENCE: 3 Met Glin Thr Arg Arg Asp Ala Lieu Lys Ser Ala Ala Ala Ile Thr Lieu 1 5 10 15 Leu Gly Gly Lieu Ala Gly Cys Ala Ser Met Ala Arg Pro Ile Gly Thr 2O 25 30 Gly Asp Lieu. Ile Asn Thr Val Arg Gly Ser Ile Pro Val Ser Glu Ala 35 40 45 Gly Phe Thr Leu Thr His Glu His Ile Cys Gly Ser Ser Ala Gly Phe 50 55 60 Leu Arg Ala Trp Pro Glu Phe Phe Gly Ser Arg Lys Ala Lieu Ala Glu 65 70 75 8O Lys Ala Val Arg Gly Lieu Arg His Ala Arg Ser Ala Gly Val Glin Thr 85 90 95 Ile Val Asp Val Ser Thr Phe Asp Ile Gly Arg Asp Val Arg Lieu Lieu 100 105 110 Ala Glu Val Ser Arg Ala Ala Asp Wal His Ile Val Ala Ala Thr Gly 115 120 125 Leu Trp Phe Asp Pro Ser Leu Ser Met Arg Met Arg Ser Val Glu Glu 130 135 1 4 0 Leu Thr Glin Phe Phe Leu Arg Glu Ile Glin His Gly Ile Glu Asp Thr 145 15 O 155 160 Gly Ile Arg Ala Gly Ile Ile Llys Val Ser Thr Thr Gly Lys Ala Thr 1.65 170 175 Pro Phe Glin Glu Lieu Val Lieu Lys Ala Ala Ala Arg Ala Ser Lieu Ala 18O 185 19 O Thr Gly Val Pro Val Thr Thr His Thr Ser Ala Ser Glin Arg Asp Gly 195 200 2O5 Glu Glin Glin Ala Ala Ile Phe Glu Ser Glu Gly Lieu Ser Pro Ser Arg US 2004/0161818 A1 Aug. 19, 2004 21

-continued

210 215 220 Val Cys Ile Gly His Ser Asp Asp Thr Asp Asp Leu Gly Tyr Lieu. Thr 225 230 235 240 Gly Lieu Ala Ala Arg Gly Tyr Lieu Val Gly Lieu. Asp Arg Met Pro Tyr 245 250 255 Ser Ala Ile Gly Lieu Glu Gly Asn Ala Ser Ala Leu Ala Lieu Phe Gly 260 265 27 O Thr Arg Ser Trp Glin Thr Arg Ala Lieu Lieu. Ile Lys Ala Lieu. Ile Asp 275 280 285 Arg Gly Tyr Lys Asp Arg Ile Leu Val Ser His Asp Trp Lieu Phe Gly 29 O 295 3OO Phe Ser Ser Tyr Val Thr Asn Ile Met Asp Val Met Asp Arg Ile Asn 305 310 315 320 Pro Asp Gly Met Ala Phe Val Pro Leu Arg Val Ile Pro Phe Leu Arg 325 330 335 Glu Lys Gly Val Pro Pro Glu Thr Leu Ala Gly Val Thr Val Ala Asn 340 345 35 O Pro Ala Arg Phe Leu Ser Pro Thr Val Arg Ala Val Val Thr Arg Ser 355 360 365 Glu Thir Ser Arg Pro Ala Ala Pro Ile Pro Arg Glin Asp Thr Glu Arg 370 375 38O

<210> SEQ ID NO 4 &2 11s LENGTH 384 &212> TYPE PRT <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: Mutant of OpdA <400 SEQUENCE: 4 Met Glin Thr Arg Arg Asp Ala Lieu Lys Ser Ala Ala Ala Ile Thr Lieu 1 5 10 15 Leu Gly Gly Lieu Ala Gly Cys Ala Ser Met Ala Arg Pro Ile Gly Thr 2O 25 30 Gly Asp Lieu. Ile Asn Thr Val Arg Gly Pro Ile Pro Val Ser Glu Ala 35 40 45 Gly Phe Thr Leu Thr His Glu His Ile Cys Gly Ser Ser Ala Gly Phe 50 55 60 Leu Arg Ala Trp Pro Glu Phe Phe Gly Ser Arg Lys Ala Lieu Ala Glu 65 70 75 8O Lys Ala Val Arg Gly Lieu Arg His Ala Arg Ser Ala Gly Val Glin Thr 85 90 95 Ile Val Asp Val Ser Thr Phe Asp Ile Gly Arg Asp Val Arg Lieu Lieu 100 105 110 Ala Glu Val Ser Arg Ala Asp Asp Wal His Ile Val Ala Ala Thr Gly 115 120 125 Leu Trp Phe Asp Pro Pro Leu Ser Met Arg Met Arg Ser Val Glu Glu 130 135 1 4 0 Leu Thr Glin Phe Phe Leu Arg Glu Ile Glin His Gly Ile Glu Asp Thr 145 15 O 155 160 Gly Ile Arg Ala Gly Ile Ile Llys Val Ala Thr Thr Gly Lys Ala Thr 1.65 170 175 Pro Phe Glin Glu Lieu Val Lieu Lys Ala Ala Ala Arg Ala Ser Lieu Ala US 2004/0161818 A1 Aug. 19, 2004 22

-continued

18O 185 19 O Thr Gly Val Pro Val Thr Thr His Thr Ser Ala Ser Glin Arg Asp Gly 195 200 2O5 Glu Glin Glin Ala Ala Ile Phe Glu Ser Glu Gly Lieu Ser Pro Ser Arg 210 215 220 Val Cys Ile Gly His Ser Asp Asp Thr Asp Asp Leu Ser Tyr Lieu. Thr 225 230 235 240 Gly Lieu Ala Ala Arg Gly Tyr Lieu Val Gly Lieu. Asp Arg Met Pro Tyr 245 250 255 Ser Ala Ile Gly Lieu Glu Gly Asn Ala Ser Ala Leu Ala Lieu Phe Gly 260 265 27 O Thr Arg Ser Trp Glin Thr Arg Ala Lieu Lieu. Ile Lys Ala Lieu. Ile Asp 275 280 285 Arg Gly Tyr Lys Asp Arg Ile Leu Val Ser His Asp Trp Lieu Phe Gly 29 O 295 3OO Phe Ser Ser Tyr Val Thr Asn Ile Met Asp Val Met Asp Arg Ile Asn 305 310 315 320 Pro Asp Gly Met Ala Phe Val Pro Leu Arg Val Ile Pro Phe Leu Arg 325 330 335 Glu Lys Gly Val Pro Pro Glu Thr Leu Ala Gly Val Thr Val Ala Asn 340 345 35 O Pro Ala Arg Phe Leu Ser Pro Thr Val Arg Ala Val Val Thr Arg Ser 355 360 365 Glu Thir Ser Arg Pro Ala Ala Pro Ile Pro Arg Glin Asp Thr Glu Arg 370 375 38O

<210 SEQ ID NO 5 &2 11s LENGTH 1155 &212> TYPE DNA <213> ORGANISM: Agrobacterium radiobacter <400 SEQUENCE: 5 atgcaaacga gaagagatgc acttaagttct gcggcc.gcaa taactctgct c gg.cggcttg 60 gctgggtgtg caag catggc cc gaccaatc ggtacaggcg atctgattaa tactgttc.gc 120 ggcc cc attc cagtttcgga agcgggctitc acact gacco atgag catat citgcggcagt 18O toggcgggat to citacgtgc gtggc.cggag tttitt.cggta gcc.gcaaagc tictagoggaa 240 aaggctgttga gaggattacg ccatgccaga toggctdgcg togcaaac cat cqtcgatgtg 3OO togactitt.cg atatoggtog togacgtocgt ttattggccg aagtttc.gc g g g c cqc.cgac 360 gtgcatatog toggcgg.cgac togct tatgg titc gaccc.gc. cactittcaat gc gaatgcgc 420 agcgtogaag aactgaccca gttctitcc to cqtgaaatcc aac atgg cat cqaag acacc 480 gg tattaggg cqggcattat caaggtogcg accacaggga aggcg accoc ctittcaagag 540 ttggtottaa agg cago.cgc gcgggccago ttggccaccg gtgttcc.ggit aaccacticac 600 acgtcago aa gtcagogcga tiggcgagcag caggcago.ca tatttgaatc cqaaggtttg 660 agcc cctoac gggtttgtat cqgtolacago gatgatact g acgatttgag citaccta acc 720 ggccitc.gctg. c.gc.gcggata cotcgtoggt ttagatcgca toccgtacag td.cgattggit 78O citagaaggca atgcgagtgc attagcgcto tttgg tacto ggtogtggca aacaagggct 840 citcttgatca aggcgctcat cq accgaggc tacaaggatc gaatcct cqt citcc.catgac 9 OO

US 2004/0161818 A1 Aug. 19, 2004 25

-continued gataccgaac gatga 1155

<210 SEQ ID NO 9 &2 11s LENGTH 2.0 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: PCR primer for 16S rRNA gene <400 SEQUENCE: 9 agagtttgat cmtggctcag 20

<210> SEQ ID NO 10 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: PCR primer for 16S rRNA gene <400 SEQUENCE: 10 tacggy tacc ttgttacgac tt 22

<210> SEQ ID NO 11 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: PCR primer for OpdA gene <400 SEQUENCE: 11 gatcgtctgc agccaatcgg tacaggcgat citg 33

<210> SEQ ID NO 12 &2 11s LENGTH 39 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: PCR primer for OpdA gene <400 SEQUENCE: 12 gatcgtaagc titt catcgtt cqgitatcttg acggggaat 39

<210> SEQ ID NO 13 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: PCR primer for OpdA gene <400 SEQUENCE: 13 gatcgtggat cotcgatcgg cacaggcgat cqg 33

<210> SEQ ID NO 14 &2 11s LENGTH 33 &212> TYPE DNA <213> ORGANISM: Artificial Sequence &220s FEATURE <223> OTHER INFORMATION: PCR primer for OpdA gene <400 SEQUENCE: 14 gatcgtaagc titt catgacg ccc.gcaaggit cqg 33

<210 SEQ ID NO 15 US 2004/0161818 A1 Aug. 19, 2004 26

-continued

LENGTH 30 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: PCR primer <400 SEQUENCE: 15 gatcgtgaat to catatgcc aatcggtaca 30

SEQ ID NO 16 LENGTH 30 TYPE DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: PCR primer <400 SEQUENCE: 16 gatcgtggat cotcatcgtt cqgitatcttg 30

SEQ ID NO 17 LENGTH 365 TYPE PRT ORGANISM: Flavobacterium sp. <400 SEQUENCE: 17

Met Glin Thr Arg Arg Val Val Leu Lys Ser Ala Ala Ala Ala Gly Thr 1 5 10 15

Leu Lieu Gly Gly Lieu Ala Gly Cys Ala Ser Wall Ala Gly Ser Ile Gly 2O 25 30

Thr Gly Asp Arg Ile Asn Thr Val Arg Gly Pro Ile Thr Ile Ser Glu 35 40 45

Ala Gly Phe Thr Leu Thr His Glu His Ile Gly Ser Ser Ala Gly 50 55 60

Phe Leu Arg Ala Trp Pro Glu Phe Phe Gly Ser Arg Ala Telu Ala 65 70 75

Glu Lys Ala Val Arg Gly Lieu Arg Arg Ala Arg Ala Ala Gly Wall Arg 85 90 95

Thir Ile Val Asp Val Ser Thr Phe Asp Ile Gly Arg Asp Wall Ser Telu 100 105 110

Leu Ala Glu Val Ser Arg Ala Ala Asp Wall His Ile Wall Ala Ala Thr 115 120 125

Gly Leu Trp Phe Asp Pro Pro Leu Ser Met Arg Teu Arg Ser Wall Glu 130 135 1 4 0 Glu Leu Thr Glin Phe Phe Leu Arg Glu Ile Glin Gly Ile Glu Asp 145 15 O 155 160

Thr Gly Ile Arg Ala Gly Ile Ile Lys Wall Ala Thr Thr Gly Lys Ala 1.65 170 175

Thr Pro Phe Glin Glu Leu Val Leu Lys Ala Ala Ala Arg Ala Ser Telu 18O 185 19 O

Ala Thr Gly Val Pro Val Thir Thr His Thr Ala Ala Ser Glin Arg Asp 195 200

Gly Glu Glin Glin Ala Ala Ile Phe Glu Ser Glu Gly Teu Ser Pro Ser 210 215 220

Arg Val Cys Ile Gly. His Ser Asp Asp Thr Asp Asp Teu Ser Telu 225 230 235 240

Thr Ala Lieu Ala Ala Arg Gly Tyr Lieu Ile Gly Teu Asp His Ile Pro US 2004/0161818 A1 Aug. 19, 2004 27

-continued

245 250 255

His Ser Ala Ile Gly Lieu Glu Asp Asn Ala Ser Ala Ser Ala Telu Telu 260 265 27 O

Gly Ile Arg Ser Trp Glin Thr Arg Ala Leu Lieu. Ile Lys Ala Lieu. Ile 275 280 285

Asp Glin Gly Tyr Met Lys Glin Ile Telu Wall Ser Asn Asp Trp Leu Phe 29 O 295

Gly Phe Ser Ser Wall Thr Asn Ile Met Asp Wall Met Asp Wall 305 310 315 320

Asn Pro Asp Gly Met Ala Phe Ile Pro Telu Arg Wall Ile Pro Phe Telu 325 330 335

Arg Glu Lys Gly Val Pro Glin Glu Thr Leu Ala Gly Ile Thr Wall Thr 340 345 35 O

Asn. Pro Ala Arg Phe Teu Ser Pro Thr Telu Arg Ala Ser 355 360 365

1. A Substantially purified polypeptide, the polypeptide (vi) a sequence which is at least 90% identical to any one being Selected from: of (i) to (v), wherein the polynucleotide encodes a polypeptide capable of hydrolysing an organophos (i) a polypeptide comprising a sequence provided in SEQ phate molecule. ID NO:1; 7. A vector comprising a polynucleotide according to (ii) a polypeptide comprising a sequence provided in SEQ claim 6. ID NO:2; 8. The vector of claim 7 which is a viral vector. 9. The vector of claim 7 which is a plasmid vector. (iii) a polypeptide comprising a sequence provided in 10. A host cell comprising a vector according to any one SEQ ID NO:3: of claims 7 to 9. (iv) a polypeptide comprising a sequence provided in 11. A process for preparing a polypeptide according to any SEQ ID NO:4; or one of claims 1 to 5, the process comprising cultivating a host cell according to claim 10 under conditions which allow (v) a polypeptide comprising a sequence which is greater production of the polypeptide, and recovering the polypep than 90% identical to any one of (i) to (iv), tide. 12. A composition for hydrolysing an organophosphate wherein the polypeptide is capable of hydrolysing an molecule, the composition comprising a polypeptide accord organophosphate molecule. ing to any one of claims 1 to 5, and one or more acceptable 2. The polypeptide of claim 1, wherein the organophoS carriers. phate molecule is Selected from the group consisting of: 13. A composition for hydrolysing an organophosphate coumaphos, coroXon, paraoxon, parathion, parathion-me molecule, the composition comprising a host cell according thyl, phosmet, fenthion, diazinon, chlorpyrifos, dMUP, DFP, to claim 10, and one or more acceptable carriers. dimethoate, malathion, and malaoxon. 14. A method for hydrolysing an organophosphate mol 3. The polypeptide of claim 1 or claim 2, wherein the ecule in a Sample, the method comprising exposing the polypeptide can be purified from an Agrobacterium sp. Sample to a polypeptide according to any one of claims 1 to 4. A fusion polypeptide comprising a polypeptide accord 5. ing to any one of claims 1 to 3 fused to at least one other 15. The method of claim 14, wherein the polypeptide is polypeptide Sequence. provided as a composition according to claim 12 or claim 5. The fusion polypeptide of claim 4, wherein the at least 13. one other polypeptide is a maltose-binding protein. 16. The method of claim 14 or claim 15, further com 6. An isolated polynucleotide, the polynucleotide com prising exposing the Sample to a divalent cation. prising a Sequence Selected from: 17. The method of claim 16, wherein the divalent cation is zinc. (i) a sequence of nucleotides shown in SEQ ID NO:5; 18. The method according to any one of claims 14 to 17, (ii) a sequence of nucleotides shown in SEQ ID NO:6; wherein Sample is Selected from the group consisting of; Soil, water, biological material, or a combination thereof. (iii) a sequence of nucleotides shown in SEQ ID NO:7; 19. A transgenic plant which produces a polypeptide according to any one of claims 1 to 5. (iv) a sequence of nucleotides shown in SEQ ID NO:8; 20. A method for hydrolysing an organophosphate mol (v) a Sequence encoding a polypeptide according to any ecule in a Sample, the method comprising exposing the one of claims 1 to 5; or Sample to a transgenic plant according to claim 19. US 2004/0161818 A1 Aug. 19, 2004 28

21. The method of claim 20, wherein the polypeptide is at 32. A biosensor for detecting the presence of an organo least produced in the roots of the transgenic plant. phosphate, the biosensor comprising a polypeptide accord 22. An isolated strain of Agrobacterium radiobacter ing to any one of claims 1 to 5, and a means for detecting deposited under NM01/21112 on 20 Apr. 2001 at Australian hydrolysis of an organophosphate molecule by the polypep Government Analytical Laboratories. tide. 23. A composition for hydrolysing an organophosphate 33. A method for Screening for agents which hydrolyse an molecule, the composition comprising the Agrobacterium organophosphate molecule, the method comprising radiobacter Strain of claim 22, and one or more acceptable carriers. (i) exposing the organophosphate to a candidate agent, 24. A method for hydrolysing an organophosphate mol and ecule in a Sample, the method comprising exposing the (ii) measuring a fluorescent signal produced from Step (i), Sample to an Agrobacterium radiobacter Strain according to claim 22. wherein the fluorescent Signal is indicative of hydrolysis 25. An isolated bacterium which produces a polypeptide of the organophosphate. according to any one of claims 1 to 3. 34. The method of claim 33, wherein the organophosphate 26. The isolated bacterium of claim 25, wherein the is coumaphoS or coroXon. bacterium is a strain of Agrobacterium radiobacter. 35. The method of claim 33 or claim 34, wherein the agent 27. Use of an isolated naturally occurring bacterium is a polypeptide or a micro-organism. which produces a polypeptide according to any one of 36. A method of producing a polypeptide with enhanced claims 1 to 3 for hydrolysing an organophosphate in a ability to hydrolyse an organophosphate or altered Substrate Sample. Specificity for an organophosphate, the method comprising 28. A polymeric Sponge or foam for hydrolysing an (i) mutating one or more amino acids of a first polypeptide organophosphate molecule, the foam or Sponge comprising according to any one of claims 1 to 5, a polypeptide according to any one of claims 1 to 5 immo bilized on a polymeric porous Support. (ii) determining the ability of the mutant to hydrolyse an 29. The polymeric sponge or foam of claim 28, wherein organophosphate, and the porous Support comprises polyurethane. (iii) Selecting a mutant with enhanced ability to hydrolyse 30. The polymeric sponge or foam of claim 28 or claim the organophosphate or altered Substrate Specificity for 29, wherein the Sponge or foam further comprises carbon the organophosphate, when compared to the first embedded or integrated on or in the porous Support. polypeptide. 31. A method for hydrolysing an organophosphate mol 37. A polypeptide produced according to the method of ecule in a Sample, the method comprising exposing the claim 36. Sample to a Sponge or foam according to any one of claims 28 to 30.