ZA200406576B - Degradation of hydrophobic ester pesticides and toxins - Google Patents

Description

DEGRADATION OF HYDROPHOBEC ESTER PESTICIDES AND TOXINS

Field of the Invention:

This invention relates to enzynmes and methods for degrading hydrophobic ester pesticides and toxins. In particular, the present invention relates to the use of insect esterases, s uch as a-carboxylesterases, and mutants thereof, in the bioremediation of pyrethroid residues contaminating the environment and horticultural commeodities.

Background of the Invention:

Pyrethroids constitute a major class of chemical pesticides. They are synthetic analogues of the natural pyrethrins, which are produced in the flowers of the pyrethrum plant (Tanaecetum cinerariifolium). Modification of their structure has yielded compoundls that retain the intrinsically modest vertebrate toxicity of the natural prod_ucts but are both more stable and more potent as pesticides. In the thirty yea_rs since their introduction they have risen to about 10-20% of insecticide s-ales worldwide and they are projected to retain substantial market share into tlhe forseeable future. They are now widely used across agricultural production and processing systems in many countries and have caused residue in. cidents in diverse commodities ranging from cotton and horticulture through to wool.

Residues of pyrethroid pesticidies are undesirable contaminants of the environment and a range of commodities. They are undesirable because of the broad target range of the pesticide across invertebrates and their significant toxicity to vertebrates, although they are generally considered to be amongst the safest pesticides to m ammals. Areas of particular sensitivity include contamination of soil, irrigation tailwater that is re-cycled, used by irrigators downstream or simply allowved to run off-farm, and residues above permissible levels in horticultural ex—ports. Animal industries also have problems with pesticide-contaminatesd commodities arising through either their own pesticide use or their reliarce on crop products and by-products as fodder. Processing wastes from food processing plants, carpet dye baths and ’ animal dips are also contaminated, sometimes quite heavily, with pesticide residues. Bioremediation strategies are therefore required for eliminating or reducing these pesticide residues.

One proposed bioremediation strategy involves the use of enzymes ’ capable of immobilising or degrading the pesticide residues. Such enzymes may be employed, fo r example, in bioreactors through which contaminated ’ water could be passe d, or in washing solutions after post-harvest disinfestation of fruit, vegetables or animal products to reduce residue levels and withholding times. Suitable enzymes for degrading pesticide residues include OP hydrolases from bacteria, vertebrates and organophosphate (OP) resistant insects. It is desirable that the hydrolytic enzymes degrade the pesticide residues at a rapid rate.

Organophosph ate resistance in the sheep blowfly, Lucilia cuprina, is conferred by two different mutations in the gene encoding carboxylesterase

E3. The two mutant enzymes differ in their substrate specificities but between them can detoxify two major subtypes of OPs. The E3 gene from L. cuprina was cloned bby Newcomb et al. (1997) and, using a combination of

DNA sequencing, baculovirus expression and in vitro mutagenesis, these workers identified the two resistance mutations. One is an Asp for Gly substitution at residue 137 in the oxyanion hole region of the active site (Newcomb et al., 199 7). The other is a Leu for Trp substitution at residue 251 in the substrate-binding region (Campbell et al., 1998), which results in an increase in malathiora carboxylesterase activity as well as the acquisition of

OP hydrolase activity.

There is a need for methods and enzymes which can be used for the bioremediation of, fox example, soils, foodstuff and water samples contaminated with hydrophobic ester pesticides and toxins.

Summary of the Inve ntion:

The present inwentors have now found that insect esterases, and mutants thereof, are able to hydrolyse hydrophobic ester pesticides and toxins such as pyreth roids. The activity of the insect esterases, and mutants thereof, show a degre e of chiral specificity, which differed between mutants. : It is therefore possible to provide a suite of insect esterases, or mutants thereof, that are able to degrade hydrophobic ester pesticides and toxins that : can act, alone or together, as effective bioremediation agents for hydrophobic ester pesticides and toxins such as pyrethroids.

Accordingly, im a first aspect, the present invention provides a method of eliminating or redixcing the concentration of a hydrophobic ester pesticide or toxin in a samples, the method comprising contacting the sample with an ) insect esterase, or a. mutant thereof.

In a preferred embodiment of the first aspect, the insect esterase ds a member of the carb oxyl/cholinesterase multi-gene family of enzymes. More preferably, the insect esterase is an a-carboxylesterase. Even more preferably, the insect esterase i.s a member of the a-carboxylesterase cluster which forms a sub-clade within #his multi-gene family (Oakeshott et al., 1999). Esterases which form this sulb-clade include at least a-carboxylesterases which caan be isolated from species of Diptera, Hemiptera and Hymenoptera. Specific enzymes which are found in this sub-clade include, but are not limited to, the

E3 or EST23 esterases. However, orthologous esterases of E3 and EST2 3 from other insect species can also be used in the methods of the present invention.

Preferably, thme a-carboxylesterases can be isolated from a species of

Diptera. Accordingly, examples of preferred a-carboxylesterases for us+e in the present invention are the E3 esterase (SEQ ID NO:1) which is deriveed from Lucilia cuprinea, or the EST23 esterase (SEQ ID NO:2) which is derived from Drosophila melanogaster.

In a further pereferred embodiment, the mutant insect esterase has a mutation(s) in the oxyanion hole, acyl binding pocket or anionic site re gions of the active site, or- any combination thereof.

In a further pereferred embodiment, the mutant a-carboxylesteras e is selected from the group consisting of: E3G137R, E3G137H, E3W251L,

E3W251S, E3W251-G, E3W251T, E3W251A, E3W251L/F309L,

E3W2511/G137D, F23W251L/P250S, E3F309L, E3Y148F, E3E217M, E3F 354W,

E3F354L, and ESTZ3W251L. Preferably, the mutant a-carboxylesterase is

E3W251L, E3F3091., E3W251L/F309L or EST23W251L.

In another preferred embodiment of the first aspect, the a- carboxylesterase, or~ mutant thereof, has a sequence selected from the group consisting of: i) a sequence as shown in SEQ ID NO:1, ) ii) a sequences as shown in SEQ ID NO:2, and iii) a sequence which is at least 40% identical to i) or ii) which is capable of hydrolyssing a hydrophobic ester pesticide or toxin. More preferably, the poly~peptide is at least 50% identical, more preferably at least 60% identical, mores preferably at least 70% identical, more preferably atleast 80% identical, and more preferably at least 90% identical, more preferably at

. least 95% ideratical, and even more preferably at least 97% identical t oi) or ii).

As the skilled addressee would be aware, the method of the first aspect can be perforrmed using more than one insect esterase, or mutants the=reof.

This is partictalarly the case where different insect esterases, or mutamts thereof, have clifferent hydrolytic activity for different stereo-isomers of the hydrophobic ester pesticide or toxin.

The hydrophobic ester pesticide or toxin can be any molecule “which is hydrophobic i n nature, contains an ester group and has some level of” toxicity towards livingg organisms. A particularly preferred hydrophobic ester: pesticide or tosxin is a pyrethroid. The pyrethroid can be a Type I or "Type II pyrethroid. Pareferably, the Type I pyrethroid is selected from the gro up consisting of: 1S/1R trans permethrin, 1S/1R cis permethrin, NRDC157 1S cis, and NRDC157 1R cis. Preferably, the Type II pyrethroid is deltameth rin.

Preferably, the sample is a soil sample, a water sample or a bio logical sample. Prefertred biological samples include matter derived from plants such as seeds, vegetables or fruits, as well as matter derived from animals such as meat.

Preferably, the method is performed in a liquid containing environment.

The sample can be exposed to the insect esterase, or mutant thereof, by any appropriate means. This includes providing the insect esterase, or mutant thereof, directly to the sample, with or without carriers or excipients etc. The insect esterase, or mutant thereof, can also be provided in tkne form of a host cell, typically a microorganism such as a bacterium or a fun gus, which expresses a polynucleotide encoding the insect esterase, or mutant thereof.

The insect esterase, or mutant thereof, can also be as provided a polymeric sponge or foam, the foam or sponge comprising the insect esterase, or mutant the reof, immobilized on a polymeric porous support. ’ Preferalbly, the porous support comprises polyurethane.

In a presferred embodiment, the sponge or foam further comprises ’ carbon embedded or integrated on or in the porous support.

It is enwisaged that the use of a surfactant in the method of thes present invention may liberate hydrophobic ester pesticides and/or toxins from any, for example, sediment in the sample. Thus increasing efficiency of the

] method of the present invention. Accordingly, in anotiner preferred embocliment, the method comprises the presence of a swarfactant when the hydro phobic ester pesticide or toxin is contacted with tThe insect esterase, or mutart thereof. More preferably, the surfactant is a bio surfactant. 5 Further, hydrophobic ester pesticide or toxin in a sample can also be degracled by exposing the sample to a transgenic plant which produces the insect esterase, or mutant thereof.

In a second aspect the present invention provides a substantially purified polypeptide which is a mutant of an insect estesrase, wherein one or more Tmutations are within a region of the esterase selec:ted from the group consisting of: oxyanion hole, acyl binding pocket and amnionic site, wherein the m-utant insect esterase is capable of hydrolysing a hydrophobic ester pestic ide or toxin, with the proviso that the mutant insesct esterase is not

E3W2 51L, E3W2518S, E3W251G or E3G137D.

Preferably, the insect esterase is an a-carboxylesteerase.

Preferably, the polypeptide is selected from the gmroup consisting of: i) a mutant of a sequence as shown in SEQ ID N(®:1, and ii) a mutant of sequence as shown in SEQ ID NO: 2, wherein the mutant is at least 40% identical to at least one of SEQ ID

NO's: 1 or 2. More preferably, the mutant is at least 8095 identical to at least one of SEQ ID NO's:1 or 2. Even more preferably, the mautant is at least 90% identi cal to at least one of SEQ ID NO's:1 or 2.

Preferably, the mutation is a point mutation.

Preferably, the polypeptide selected from the grovap consisting of:

E3G137R, E3G137H, E3W251T, E3W251A, E3W251L/F309L,

E3W2.51L/G137D, E3W251L/P250S, E3F309L, E3Y148F, E3E217M, E3F354W,

E3F35 4L, ESTZ3W251L.

In a third aspect, the present invention provides a fusion polypeptide comprising a polypeptide according to the second aspect fused to at least one other “polypeptide sequence.

In a fourth aspect the present invention provides an isolated polynwicleotide encoding a polypeptide according to thes second or third aspects.

In a fifth aspect the present invention provides a ~vector for replication and/or expression of a polynucleotide according to the fourth aspect.

In a sixth aspect the present invention provides a heost cell transformed or transfected with the vector of the fifth aspect.

In a seventh aspect the present invention provides a composition for h ydrolysing a hydrophobic ester pesticide or toxin, the co mposition comprising a polypeptide according to the second or third aspects, and one or more acceptable carriers.

In an eighth aspect the present invention provides & method for g-enerating and selecting an enzyme that hydrolyses a hyd rophobic ester p esticide or toxin, the method comprising (i) introducing one or more mutations into an insect esterase, or an insect esterase that has already been mutated, and (ii) determining the ability of the mutant insect este-rase to hydrolyse a hydrophobic ester pesticide or toxin.

Preferably, the one or more mutations enhances hydrolytic activity amd/or alters the stereospecificty of the esterase.

Such one or more mutations can be introduced by a variety of techniques known to the skilled addressee. These techniques include, but axe not limited to, site directed mutagenesis, random mutagenesis, or the use of DNA shuffling in in vitro evolution techniques, each of which are p erformed on a polynucleotide encoding the insect esterase or insect esterase that has already been mutated.

In a preferred embodiment of the eighth aspect, the insect esterase is an o -carboxylesterase. More preferably, the a-carboxylestera_se is an E3 or

E ST23 esterase. More preferably, the a-carboxylesterase nas a sequence selected from the group consisting of: i) a sequence as shown in SEQ ID NO:1, ii) a sequence as shown in SEQ ID NO:2, and iii) a sequence which is at least 40% identical to i) orii). More preferably, the polypeptide is at least 50% identical, more preferably at least 6©0% identical, more preferably at least 70% identical, more preferably at least 8W0% identical, and more preferably at least 90% identical, more preferably at least 95% identical, and even more preferably at least 9796 identical to i) or ’ ii).

Preferably, the one or more mutations are within a region of the esterase selected from the group consisting of: oxyanion h-ole, acyl binding pocket and anionic site.

In a further preferred embodiment, the insect: esterase that has already bee n mutated is selected from the group consisting of: E3G137R, E3G137H,

E3wWV251L, E3W2518S, E3W251G, E3W251T, E3W25 “1A, E3W251L/F309L,

E3WV251L/G137D, E3W251L/P250S, E3F309L, E3Y1-48F, E3E217M, E3F354W,

E3F354L, and EST23W251L.

In a further preferred embodiment of the eiglmth aspect, the mutation is a point mutation.

In a ninth aspect the present invention provicles an enzyme obtained by a method according to the eighth aspect.

Throughout this specification the word "comporise", or variations such as "ecomprises" or "comprising", will be understood t=o imply the inclusion of a stateed element, integer or step, or group of elementss, integers or steps, but not the exclusion of any other element, integer or step, eor group of elements, inte=gers or steps. : The invention is hereinafter described by waxy of the following non- limi ting example and with reference to the accompanying figures.

Brief Description of the Accompanying Drawings:

Figuare 1: Amino acid sequence alignment of the E3 (SEQ ID NO:1) and

Torpedo californica acetylcholinesterase (SEQ ID N(O:3) enzymes. The sequience around the active site serine and residues Gly137, Trp251 and

Phe -309 are shown in bold and underlined.

Figiare 2: Proposed configuration of active site of Lc-E3 carboxylesterase in an acyl ation reaction.

Figiare 3: Results of representative titration experiments performed on cell extr.acts containing baculovirus expressed esterases.

Figure 4: Molecular structures for 1R/S cis and trarms permethrin, 1R/S cis and trans NRDC157 and the four stereoisomers of ci.s deltamethrin. ’ Figuwre 5: Hydrolysis of cis and trans permethrin (0.5uM) by E3W251L.

Key to Sequence Listing:

SEQ» ID NO:1 - Amino acid sequence of Lucilia cupr&na E3 a-carboxylesterase.

SEQ ID NO:2 - Amino acid sequence of Drosophila melanogaster EST23 a- carboxylesterase.

SEQ ID NO:3 - Partial amino acid sequence of Torpedo californica acetylcholinesterase.

Detailed Description of the Inven tion:

General Techniques

Unless otherwise indicated _ the recombinant DNA techniques utilized in the present invention are stand ard procedures, well known to those skillecl in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular

Cloning, John Wiley and Sons (19 84), J. Sambrook et al., Molecular Cloning:

A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A.

Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glower and B.D. Hames (editors), DNA Cloning=

A Practical Approach, Volumes 1—4, IRL Press (1995 and 1996}, and F.M.

Ausubel et al. (Editors), Current Protocols in Molecular Biology, Greene Pub.

Associates and Wiley-Interscience= (1988, including all updates until present) and are incorporated herein by reference.

Pyrethroids

Pyrethroids are synthetic araalogs of pyrethrum pesticides. For example, pyrethroids include (in each case common name in accordance with

The Pesticide Manual, 12th Editio n): permethrin, fenvalerate, esfenvalerate, cypermethrin, alpha-cypermethrir, deltamethrin, fenpropathrin, fluvalinate, flucythrinate, cyfluthrin, acrinathain, tralomethrin, cycloprothrin, lambda- cyhalothrin, tefluthrin, bifenthrin,., transfluthrin, zeta-cypermethrin, and halfenprox. :

Type I pyrethroid compounads (e.g., permethrin) differ from type II pyrethroid compounds in that typee II compounds possess a cyano group on the a-carbon atom of the phenoxy¥benzyl moiety. Some examples of type II pyrethroids are cypermethrin, deltamethrin, and fenvalerate.

Examples of pyrethroid pesticides which can be hydrolysed using the methods of the present invention finclude, but are not restricted to these compounds; 3-phenoxybenzyl(1RS)-cis, trans-3-(2,2-dichlorovinyl)-2,2- dimethylcyclopropane carboxylates [permethrin], o-cyano-3-phenoxybenzyl-1.--

(4-ethoxyphenyl)—2,2-dichlorocyclopropane carboxylate [cyloprothr-in], (RS)- a-cyano-3-phenoscybenzyl(RS)-2-(4-chlorophenyl)-3-isovalerate [fermvalerate], (S)-a-cyano-3-phe=noxybenzyl(S)-2-(4-chlorophenyl)isovalerate [esfenvalerate], o.- cyano-3-phenoxybenzyl(S)-2-(4-difluoromethoxyphenyl) isovalerate [flucythrinate], a-cyano-3-phenoxybenzyl 2-(2-chloro-4- trifluoromethylan iline)isovalerate [fluvalinate], (RS)-a-cyano-3- phenoxybenzyl 2, 2,3,3-tetramethylcyclopropane carboxylate [fenpreopathrin], 3-phenoxybenzyl( 1R)-cis,trans-chrysanthemate [d-fenothrin], (RS)-cx-cyano-3- phenoxybenzyl(1R)-cis,trans-chrysanthemate [cyfenothrin], (RS)3-allyl-2- methyl-4-oxocyclopento-2-enyl(1RS)-cis, trans-chrysanthemate [alle thrin], o- cyano-3-phenoxylbenzyl(1R)-cis,trans-3-phenoxybenzyl(1R)-cis, tran s- 3-(2,2- dichlorovinyl)-2,2--dimethylcyclopropane carboxylate [cypermethrir], (S)-o- cyano-3-phenoxybenzyl(1R)-cis-3-(2,2-dibromovinyl)-2,2-dimethy lcyclopropane carboxylate [deltamethrin], (S)-a-cyano-3-phenoxybesnzyl(1R)- cis-2,2-dimethyl-3-(1,2,2,2-tetrabro moethyl)cyclopropane carboxylate [tralomethrin], 3,4,5,6-tetrahydro imidomethyl(1RS) -cis,trans- chrysanthemate [tetramethrin}, 5-benzyl-3-furylmethyl(1RS)-cis,trams- chrysanthemate [resmethrin], a-cyano-4-fluoro-3-phenoxybenzyl(1R,trans)- 2,2~-dimethyl-3-(2,.2-dichl orovinyl)cyclopropane carboxylate [cyflut-hrin].

Polypeptides

By "substan tially purified" we mean a polypeptide that has besen separated from most of the lipids, nucleic acids, other polypeptides _, and other contaminating molecules with which it is associated in its native state.

The % iden tity of a polypeptide is determined by GAP (Needleman and

Wunsch, 1970) amalysis (GCG program) with a gap creation penalty =5, and a gap extension permalty=0.3. The query sequence is at least 15 amin-o acids in length, and the GAAP analysis aligns the two sequences over a region of at least 15 amino acids. More preferably, the query sequence is at least 50 @mino acids in length, arad the GAP analysis aligns the two sequences over a region of at least 50 amiro acids. More preferably, the query sequence is ant least 100 amino acids in leragth and the GAP analysis aligns the two sequences over a region of at least M00 amino acids. More preferably, the query sequence is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a wegion of at least 250 amino acids. Even more pref erably, the query sequence is at least 500 amino acids in length and the CGAP analysis aligns the two se=quences over a region of at least 500 amino a.cids.

As used hesrein, the term "mutant thereof" refers to mut ants of a naturally occurring insect esterase which maintains at least seome hydrolytic activity towards a hydrophobic ester pesticide or toxin when compared to the naturally occurring insect esterase from which they are deriveed. Preferably, the mutant has e=nhanced activity and/or altered stereospecifiecity when compared to the naturally occurring insect esterase from which they are derived.

Amino acid sequence mutants of naturally occurring irasect esterases can be prepared by introducing appropriate nucleotide chang es into a nucleic acid of the present invention, or by in vitro synthesis of the desired polypeptide. Su«ch mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A «combination of deletion, insertion and substitution can be made to arrive at the final construct, provicled that the final protein product possesses tke desired characteristics.

In designimg amino acid sequence mutants, the locatior of the mutation site and the natu_re of the mutation will depend on characterisstic(s) to be modified. In a p articularly preferred embodiment, naturally occurring insect esterases are mutated to increase their ability to hydrolyse a Inydrophobic ester pesticide ox toxin, particularly a pyrethroid. The sites fOr mutation can be modified indi vidually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other rezsidues adjacent to the located site. Example s of such mutants include 5 E3G137R, E3G137H, E3W251L, E3W251S, EE3W251G,

E3W251T, E3SW251A, E3W251L/F309L, E3W251L/G137D, E3WNV251L/P2508S,

E3F309L, E3Y148F, E3E217M, E3F354W, E3F354L, and EST2: 3W251L.

Mutants u seful for the methods of the present inventiomn can also be obtained by the wise of the DNA shuffling technique (Patten et al., 1997).

DNA shuffling iss a process for recursive recombination and mutation, performed by raradom fragmentation of a pool of related genes, followed by reassembly of the fragments by primerless PCR. Generally, D NA shuffling provides a means for generating libraries of polynucleotides v-vhich can be selected or screened for, in this case, polynucleotides encodirag enzymes which cam hydrolyse a hydrophobic ester pesticide or toxin. "The stereospe=cificity of the selected enzymes can also be screened. .

Amino acid sequence deletions generally range from ab-out 1 to 30 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguowis residues.

Substitution mutants have at least one amino acid residlue in the polypeptide molecule removed and a different residue inserte d in its place.

The sites of greatest interest for substitutional mutagenesis ineclude sites identified as the active or binding site(s). Other sites of interest are those in which particular residues obtained from various strains or spescies are identical. These positions may be important for biological activity. These sites, esp ecially those falling within a sequence of at least threse other identically conserved sites, can be substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1 under the heading of "exemplary substitutions".

Fu rthermore, if desired, unnatural amino acids or chemical amino acid analogue s can be introduced as a substitution or addition into the insect esterase, or mutants thereof. Such amino acids include, but aare not limited to, the D—isomers of the common amino acids, 2,4-diaminobu-tyric acid, a- amino iseobutyric acid, 4-aminobutyric acid, 2-aminobutyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic aczid, ornithine, norleucire, norvaline, hydroxyproline, sarcosine, citrulline, Faomocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cysclohexylalanine,

R-alanine, fluoro-amino acids, designer amino acids such as [3-methyl amino acids, Cox-methyl amino acids, Na-methyl amino acids, and a mino acid analogue=s in general.

Also included within the scope of the invention are insect esterases, or mutants thereof, which are differentially modified during or after synthesis, e.g., by beiotinylation, benzylation, glycosylation, acetylation, phosphozylation, derivatization by known protecting/blockin_g groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. The se modifications may serve to increase the stability and/or bioactivity of the polypeptide of the invention. :

TABTLE 1

Original Exemplary

Glu(® fap

Too) yr

Insect esterases, and mutants thereof, can be pr-oduced in a variety of ways, including production and recovery of natural pmroteins, production and recoveery of recombinant proteins, and chemical syntlmesis of the proteins. In one embodiment, an isolated polypeptide encoding the insect esterase, or : mutarat thereof, is produced by culturing a cell capabl_e of expressing the polypeptide under conditions effective to produce the: polypeptide, and recovesring the polypeptide. A preferred cell to cultures is a recombinant cell of the present invention. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature=, pH and oxygen conditions that permit protein production. An effective medium refers to any medium in which a cell is cultured to produce a polypeptide of the present invention. Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate source:s, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells producing the insect esterase, or mutant thereof, can be cultured din conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing; conditions are within the expertise of one of ordinary skill in the art.

Polynucleotides

By "isolated polynucleotide”, we mean a poly nucleotide separated from the polynucleotide sequences with which it is associated or linked in its native state. Furthermore, the term "polynucleotid €” is used interchangeably herein with the term "nucleic acid molecule".

The 9 identity of a polynucleotide is determmined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequeznce is at least 45 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 45 nucleotides. Preferably, the query sequence is at least 150 nucleotides in length, and the GAP analysis al gns the two sequences over a region of at least 150 nucleotides. More prefferably, the query sequence is at least 300 nucleotides in length and the GAP armalysis aligns the two sequences over a region of at least 300 nucleotides.

Recombinant Vectors

Recombinant vectors can be used to express an insect esterase, or mutant thereof, for use in the methods of the presemnt invention. In addition, in another embodiment of the present invention includes a recombinant vector, which includes at least one isolated polynu-cleotide molecule of the present invention, inserted into any vector capable of delivering the polynucleotide molecule into a host cell. Such vecstors contain heterologous polynucleotide sequences, that is polynucleotide seequences that are not naturally found adjacent to polynucleotide encodirag the insect esterase, or mutant thereof, and that preferably are lerived from a species other than the species from which the esterase is deriveed. The vector can be either RNA or

DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid.

One type of recombinant vector comprises a polynucleotide encoding an insect esterase, or mutant thereof, opeeratively linked to an expression vector. The phrase operatively linked rexfers to insertion of a polynucleotide molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into & host cell. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified polynucleotide molecule.

Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids. Expressio n vectors of the present invention include any vectors that function (i.e., d-irect gene expression) in recombinant cells of the present invention, including in bacterial, fungal, endoparasite, arthropod, other animal, and plant cells. Preferred expression vectors of the present invention can direct gene expression in bacterial, yeast, arthropod and mammalian cells and more preferab-ly in the cell types disclosed herein.

Expression vectors of the present dnvention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell amd that control the expression of polynucleotide molecules of the present invention. In particular, expression vectors which comprise a polynucleotide encoding an insect esterase, or mutant thereof, include transcription comtrol sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transecription initiation, such as promoter, enhancer, operator and repressor sequemces. Suitable transcription control sequences include any transcription con. trol sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are knowvn to those skilled in the art.

Preferred transcription control sequence s include those which function in bacterial, yeast, arthropod and mammali an cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda, bacteriophage

T7, T7lac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01,

metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virtis subgenomic promoters), antibiotic resistance gene, baculovirus, Helieothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other gpoxvirus, adenovirus, cytomegalovirus (such as intermediate early promoters), simian virus 40, retrovirus, actin, retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphate and nitrate transcription control sequences as well as other sequences capable of controlling gene expre:-ssion in prokaryotic or eukaryotic cells. Additional suitable transcription confrol sequences include tissue- specific promoters and enhancers.

Polynucleotide encoding an insect es€erase, or mutant thereof, may also (a) contain secretory signals (i.e., signal segrmnent nucleic acid sequences) to enable an expressed insect esterase, or mutant thereof, to be secreted from the cell that produces the polypeptide and/or (b ) contain fusion sequences.

Examples of suitable signal segments includle any signal segment capable of directing the secretion of an insect esterase, or mutant thereof. Preferred signal segments include, but are not limited to, tissue plasminogen activator (t-PA), interferon, interleukin, growth hormeone, histocompatibility and viral envelope glycoprotein signal segments, as wrell as natural signal sequences.

In addition, polynucleotides encoding an in sect esterase, or mutant thereof, can be joined to a fusion segment that directs the encoded protein to the proteosome, such as a ubiquitin fusion segnment.

Host Cells

Another embodiment of the present imvention includes a recombinant cell comprising a host cell transformed with one or more polynucleotides encoding an insect esterase, or mutant theresof. Transformation of a polynucleotide molecule into a cell can be accomplished by any method by which a polynucleotide molecule can be inserted into the cell.

Transformation techniques include, but are not limited to, transfection, ' electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain uni cellular or may grow into a tissue, organ or a multicellular organism. A transformed polynucleotide encoding an insect esterase, or mutant thereof, can remazin extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e.,

: recombinant) cell in such a manmer that their ability to be expressed is retained.

Suitable host cells to trans form include any cell that can be transformed with a polynucleoticle encoding an insect esterase, or mutant thereof. Host cells of the present: invention either can be endogenously (i.e., naturally) capable of producing an insect esterase or mutant thereof, or can be capable of producing such proteins after being transformed with at least one polynucleotide encoding an inse«ct esterase, or mutant thereof. Host cells of the present invention can be any cell capable of producing at least one insect esterase, or mutant thereof, and i nclude bacterial, fungal (including yeast), parasite, arthropod, animal and plant cells. Preferred host cells include bacterial, mycobacterial, yeast, amthropod and mammalian cells. More preferred host cells include Salnronella, Escherichia, Bacillus, Listeria,

Saccharomyces, Spodoptera, Myc-obacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells (normal dog kidney cell line for canine herpesvirus cultivation), CRFK cells (normal «cat kidney cell line for feline herpesvirus cultivation), CV-1 cells (African rnonkey kidney cell line used, for example, to culture raccoon poxvirus), COS (-e.g., COS-7) cells, and Vero cells.

Particularly preferred host cells a:re E. coli, including E. coli K-12 derivatives;

Salmonella typhi; Salmonella typ_himurium, including attenuated strains;

Spodoptera frugiperda; Trichoplussia ni; BHK cells; MDCK cells; CRFK cells;

CV-1 cells; COS cells; Vero cells; and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246). Additional appropriate mammalian cell hosts include other kidney cell lines, other fibroblast cell lines (e.g., human, murine or chicken embryo fibrob last cell lines), myeloma cell lines, Chinese hamster ovary cells, mouse NIH/3T3 cells, LMTK cells and/or HeLa cells.

Recombinant DNA technol ogies can be used to improve expression of a transformed polynucleotide mole-cule by manipulating, for example, the number of copies of the polynucl-eotide molecule within a host cell, the efficiency with which those polymucleotide molecules are transcribed, the efficiency with which the resultamt transcripts are translated, and the efficiency of post-translational meodifications. Recombinant techniques useful for increasing the expression of a polynucleotide encoding an insect esterase, or mutant thereof, include, but are not limited to, operatively linking polynucleotide molecules to high -copy number plasmids, integration of the polynucleotide molecule into ones or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of transl ational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of polynucleotide molecules of the present invention to correspond to the codon usage of the host cell, and the deletion of sequences that destabilize transcripts.

Compositions

Compositions useful for the methods of the present invention, or which comprise a polypeptide of the present fnvention, include excipients, also referred to herein as "acceptable carriems". An excipient can be any material that the animal, plant, plant or animal material, or environment (including soil and water samples) to be treated can tolerate. Examples of such : excipients include water, saline, Ringe r's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions.

Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other wseful formulations include suspensions containing viscosity enhamcing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as suibstances that enhance isotonicity and chemical stability. Examples of buffer s include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal or o-cresol, formalin and benzyl alcohol. Excipients can also be used to increase the half-life of a composition, for example, but are not limited to, polymeric controlled release vehicles, biodegradable implants, liposomes, bacteria, viruses, other cells, oils, esters, and glwcols.

Furthermore, the insect esterase , or mutant thereof, can be provided in a composition which enhances the rate and/or degree of degradation of hydrophobic ester pesticides or toxins , or increases the stability of the polypeptide. For example, the insect esterase, or mutant thereof, can be ) immobilized on a polyurethane matri>xc (Gordon et al., 1999), or encapsulated in appropriate liposomes (Petrikovics et al. 2000a and b). The insect esterase, ) or mutant thereof, can also be incorporated into a composition comprising a foam such as those used routinely in fire-fighting (LeJeune et al., 1998).

As would be appreciated by the skilled addressee, the insect es terase, or mutant thereof, could readily be used in a sponge or foam as disclosed in

WO 00/64539, the coratents of which are incorporated herein in their entirety.

One embodimemt of the present invention is a controlled release formulation that is capable of slowly releasing a composition comprising an insect esterase, or mutant thereof, into an animal, plant, animal or plant material, or the envirosnment (including soil and water samples). As vised herein, a controlled re lease formulation comprises an insect esterase, or mutant thereof, in a controlled release vehicle. ‘Suitable controlled re lease vehicles include, but are not limited to, biocompatible polymers, othe=r polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes; lipospherees, and transdermal delivery s ystems. Preferred controlled release formulatiosns are biodegradable (i.e., bioerodible).

A preferred controlled release formulation of the present invention is capable of releasing ara insect esterase, or mutant thereof, into soil or wvater which is in an area sprayed with a hydrophobic ester pesticide or toxin. The formulation is preferably released over a period of time ranging from about 1 to about 12 months. As preferred controlled release formulation of the present invention is capable of effecting a treatment preferably for at least abo-ut 1 month, more preferably for at least about 3 months, even more preferably for at least about 6 months, even more preferably for at least about 9 mon ths, and even more preferably for at least about 12 months.

The concentration of the insect esterase, or mutant thereof, (or Inost cell expressing the insect e sterase, or mutant thereof) that will be required to produce effective compositions for degrading a hydrophobic ester pesticide or toxin will depend on the nature of the sample to be decontaminated, t he concentration of the hydrophobic ester pesticide or toxin in the sampl e, and the formulation of the composition. The effective concentration of thes insect esterase, or mutant the reof, (or host cell expressing the insect esterase. or mutant thereof) within a composition can readily be determined experimentally, as will be understood by the skilled artisan.

Surfactants

It is envisaged thuat the use of a surfactant in the method of the present invention may liberate hydrophobic ester pesticides and/or toxins, frorm any,

for example, sediment in the sample. Thus increasing efficiency of tle method of the present i nvention.

Surfactants are ammphipathic molecules with both hydrophilic &and hydrophobic (generally hydrocarbon) moieties that partition preferen_tially at the interface between fl uid phases and different degrees of polarity amd hydrogen bonding sucha as oil/water or air/water interfaces. These properties render surfactants capa ble of reducing surface and interfacial tensiom and forming microemulsiorm where hydrocarbons can solubilize in water or where water can solubilize in hydrocarbons. Surfactants have a number of —useful properties, including d&spersing traits.

Biosurfactants aree a structurally diverse group of surface-actives molecules synthesized "by microorganisms. These molecules reduce ssurface and interfacial tensions in both aqueous solutions and hydrocarbon rmixtures.

Biosurfactants have several advantages over chemical surfactants, such as lower toxicity, higher b iodegradability, better environmental compatability, higher foaming, high se=lectivity and specificity at extreme temperatures, pH and salinity, and the ability to be synthesized from a renewable source.

Biosurfactants useful in the bioremediation methods of the pre-sent invention include, but are not limited to; glycolipids such as rhamno lipids (from, for example, Pseudomonas aeruginosa), trehalolipids (from, for example, Rhodococcus -erythropolis), sophorolipids (from, for examples,

Torulopsis bombicola), and cellobiolipids (from, for example, Ustilage zeae); lipopeptides and lipoproteins such as serrawettin (from, for example» Serratia marcescens), surfactin from, for example, Bacillus subtilis); subtilisir (from, for example, Bacillus subtilis), gramicidins (from, for example, Bacillt 1s brevis), and polymyxinss (from, for example, Bacillus polymyxa); fatty acids, neutral lipids, and phosspholipids; polymeric surfactants such as emialsan (from, for example, Acimetobacter calcoaceticus), biodispersan (from, for example, Acinetobacter calcoaceticus), mannan-lipid-protein (from, for example, Candida tropfcalis), liposan (from, for example, Candida lypoolytica), protein PA (from, for example, Pseudomonas. aeruginosa); and particsulate biosurfactants such as wesicles and fimbriae from, for example, A. calcoaceticus.

Transgenic Plants

The term "plant’ refers to whole plants, plant orgams (e.g. leaves, stems roots, etc), seeds, plant cells and the like. Plants contemplated for use in the practice of the present invention include both monocotyledons and dicoty-ledons. Exemplary monocotyledons include wheat, barley, rye, triticale, oats, rice, and the like.

Transgenic plants, as defined in the context of the present invention includ_e plants (as well as parts and cells of said plants) and their progeny which. have been genetically modified using recombinant DNA techniques to either i) cause the production of an the insect esterase, ox mutant thereof, in the de sired plant or plant organ.

Several techniques exist for introducing foreign ge netic material into a plant cell. Such techniques include acceleration of genetic material coated onto microparticles directly into cells (see, for example, WS 4,945,050 and US 5,141,7131). Plants may be transformed using Agrobacterfum technology (see, for example, US 5,177,010, US 5,104,310, US 5,004,863, US 5,159,135).

Electroporation technology has also been used to transform plants (see, for example, WO 87/06614, US 5,472,869, 5,384,253, WO 92=/09696 and WO 93/21335). In addition to numerous technologies for trarasforming plants, the type of tissue which is contacted with the foreign genes rmay vary as well.

Such tissue would include but would not be limited to eambryogenic tissue, callus tissue type I and II, hypocotyl, meristem, and the Like. Almost all plant tissues may be transformed during development and/or differentiation using appropriate techniques described herein. _A number of vectors suitable for stable transfectior of plant cells or for the establishment of transgenic plants have been describ ed in, e.g., Pouwels et al., «Cloning Vectors: A Laboratory Manual, 1985, supp . 1987; Weissbach and Wreissbach, Methods for Plant Molecular Biology, Academic Press, 1989; and Gelvin et al., Plant Molecular Biology Manual, Kluweer Academic

Publis hers, 1990. Typically, plant expression vectors include, for example, one or- more cloned plant genes under the transcriptional control of 5' and 3' regula-tory sequences and a dominant selectable marker. Such plant expres sion vectors also can contain a promoter regulator-y region (e.g., a regula-tory region controlling inducible or constitutive, emvironmentally- or developmentally-regulated, or cell- or tissue-specific exp ression), a transcription initiation start site, a ribosome binding site , an

RNA processing signal, a transcription termination site, and/or a polyadenylation signal. “Examples of plant promoters include, but are not limited to ribulose- 1,6-bis phosphate carboxylase small subunit, beta-=onglycinin promoter, phaseolin promoter, ADH promoter, heat-shock promoters and tissue specific promoters. Promoters may also contain certain enhancer sequerce elements that may improve the transcripttion efficiency. Typical enhanecers include but are not limited to Adh-introm 1 and Adh-intron 6.

Constitutive promoters direct continuous gere expression in all cells types and at all times (e.g., actin, ubiquitin, CaMV 35S). Tissue specific promo-ters are responsible for gene expression in specific cell or tissue types, such a_s the leaves or seeds (e.g., zein, oleosin, napi n, ACP, globulin and the like) amd these promoters may also be used. Promoters may also be active duringg a certain stage of the plants’ development ass well as active in plant tissues and organs. Examples of such promoters in clude but are not limited to poll en-specific, embryo specific, corn silk specific, cotton fiber specific, root specific, seed endosperm specific promoters arad the like. “Under certain circumstances it may be desirable to use an inducible promo ter. An inducible promoter is responsible for expression of genes in respomase to a specific signal, such as: physical stirmulus (heat shock genes); light (RUBP carboxylase); hormone (Em); metabolites; and stress. Other desirabole transcription and translation elements theat function in plants may be used.

Hn addition to plant promoters, promoters from a variety of sources can be used efficiently in plant cells to express foreign genes. For example, promo-ters of bacterial origin, such as the octopine ssynthase promoter, the nopalime synthase promoter, the mannopine synthase promoter; promoters of viral origin, such as the cauliflower mosaic virus (3385S and 19S) and the like : may be used. ~The following examples are offered for illustration purposes, and are not intended to limit or define the invention in any~ manner.

EXAMPLES

Example 1: Construction of Mutants

An alignment of the amino acid sequencee of the E3 enzyme with that of a vertebrate acetylcholinesterase (TcAChE, for which the three dimensional structure is known; Sussman et al., 1991) is given in Figure 1. Mutants of E3 and EST23 were constructed using the QuickC hange™ Site-Directed

Mutagenesis Kit of Stratagene and are named a ccording to the number of the residue that has been changed, and the nature «of that change. For example, mutant E3W251L is an E3 mutant in which thes Trp residue at position 251 in the wild-type enzyme (i.e. E3WT) has been mu tated to Leu.

E3 and EST23 enzymes were expressed using the baculovirus expression system as described by Newcomb et al. (1997), but using the HyQ

SFX-insect serum-free medium (HyClone) for imcreased expression. Cell extracts were prepared by lysing the cells at a concentration of 10° cells ml™* in 0.1M phosphate buffer pH 7.0 containing 0.05% Triton X-100. Extracts were then titrated for the number of esterase mmolecules using a fluorometric assay based on the initial release of coumarin ( a fluorescent compound) upon phosphorylation of the enzyme by diethylcounnaryl phosphate (dECP).

Figure 2 illustrates the proposed configu ration of the active site of E3 (based on the three dimensional structure of vexrtebrate AChE) in an acylation reaction. We have examined mutations in seveen E3 residues in regions corresponding to three distinct subsites of the lknown AChE active site.

These are the oxyanion hole (E3 residue 137), &he anionic site (E3 residues 148, 217 and 354) and acyl binding pocket (E3 residues 250, 251 and 309).

The anionic site and acyl binding pocket corresspond to the p1 and p2 subsites in the nomenclature of Jarv (1984).

Mutations in the Oxyanion Hole

In TcAChE the oxyanion hole comprises Gly118, Gly119 and Ala201, which corresponds to Gly136, Gly137 and Ala=219 in E3. These residues are ’ highly conserved throughout the carboxyl/chol inesterase multigene family : (Oakeshott ef al., 1999) and there is empirical evidence for the conservation of the oxyanion hole structure from X-ray crystallographic studies of several cholinesterases and lipases (Cygler and Schrag , 1997), albeit the structure does change during interfacial activation in somne lipases (Derewenda et al. 1992). There is also empirical structural evide nce for their function in stabilising the oxyanion formed by the ecarbonyl oxygen of the carboxylester substrate as the first transition state during catalysis (Grochulski ef al., 1993;

Martinez et al., 1994). This stabilisatiom is achieved by a network of hydrogen bonds to the amide groups of the three key residues in the peptide chain (Ordentlich et al., 1998). Recently Koellner et al. (2000) have also shown that both Gly residues in the ACShE oxyanion hole make hydrogen bonds with buried “structural” water molecules, which are retained during catalysis and thought to act as lubricants to facilitate traffic of substrates and products within the active site.

Three further mutations were ma_de to the Gly137 of E3 in addition to the G137D found naturally in OP resistant L. cuprina. First, Glu was substituted as the other acidic amino acid, in G137E. The mutant G137H was also constructed, because His is also nosn-protonated at neutral pH (pK, about 6.5 cf 4.4 for Asp and Glu) and it was f-ound to confer some OP hydrolysis on human butyrylcholinesterase when subwstituted for either Gly in its oxyanion hole (Broomfield et al., 1999). Finally, _Arg (pK, around 12) was substituted at position 137, to examine the effects of he most strongly basic substitution possible.

Mutations in the Acyl Binding Pocket

The acyl binding pockets of strucsturally characterised cholinesterases are formed principally from four non-polar residues, three of which are generally also aromatic. Together they create a strongly hydrophobic pocket to accommodate the acyl moiety of boumd substrate. The four residues in

TcAChE are Trp233, Phe288, Phe290 amd Val400 corresponding to Trp251,

Val307, Phe309 and Phe422 in E3. Simmilar arrays of hydrophobic residues appear to be conserved at the correspormding sites of most carboxyl/cholinesterases (Oakeshott et cal., 1993; Robin et al., 1996; Yao et al., 1997; Harel et al., 2000). In particular Hp is strongly conserved at residue 233/251 and 290/309 is Phe in cholinestterases and most carboxylesterases, albeit a Leu or Ile in several lipases and_ a few carboxylesterases. The residue corresponding to TcAChE Phe288 is typically a branched chain aliphatic amino acid in cholinesterases that show a preference for longer chain esters such as butyrylcholine. This includes rnammalian butyrylcholinesterase and some insect acetylcholinesterases, which have a butyrylcholinesterase-like substrate specificity. The branched chain aliphatic amino acid appears to

2-4 provide a greater space in the acyl-bind ing pocket to accommodate the larger acyl group.

Mutational studies of 288/307 and 290/309 in several cholinesterases confirm their key role in determining aspects of substrate specificities related to acyl group identity. In human AChE: replacement of the Phe at either position with a smaller residue like Ala improves the kinetics of the enzyme for substrates like propyl- or butyl- (thi-o)choline with larger acyl groups than the natural acetyl (thio)choline substrate (Ordentlich et al., 1993). In AChE from D. melanogaster and the housefly, Musca domestica, natural mutations of their 290/309 equivalent to the bulki-er, polar Tyr that contributes to target site OP resistance have lower reactivity~ to both acetylcholine and OPs (Fournier et al., 1992; Walsh ef al., 20071). For D. melanogaster AChE, substitution of this Phe residue with th e smaller Leu gave the predicted increase in OP sensitivity, although surprisingly replacement with other small residues like Gly, Ser or Val did mot (Villatte et al., 2000).

Trp 233/251 has received much Less attention in mutational studies of cholinesterases but our prior work on 3 shows its replacement with a smaller Leu residue again increases rea ctivity for carboxylester substrates with bulky acyl moieties as in malathion, or for OPs (Campbell et al., 1998a, b; Devonshire et al., 2002). A mutatiora to Gly has also been found in a homologue from the wasp, Anisopteromalus calandrae, that shows enhanced malathion carboxylesterase (MCE) kine=tics (Zhu et al., 1999) while a Ser has been found in a homologue from M. do_mestica that may be associated with malathion resistance (Claudianos et al. , 2002). In respect of OP hydrolase activity Devonshire et al. (2002) propossed that the particular benefit of such mutations is to accommodate the inversion about the phosphorus that must occur for the second hydrolysis stage of the reaction to proceed. Notably

Devonshire et al. (2002) found that the k,, for OP hydrolase activity of

E3W251L is an order of magnitude higher for dMUP, with its smaller dimethyl phosphate group than for dECCP, which has a diethyl phosphate ) group. This suggests that there remain_ tight steric constraints on the inversion even in a mutant with a larger acyl pocket. ’ We have mutated both the W251. and F309 residues of E3 as well as the

P250 immediately adjacent to W251. Im addition to the previously characterised natural W251L mutation we have now analysed substitutions with four other small amino acids in W/251S, W251G, W251T and W251A. A double mutant of W251L an«d P250S was also analysed, because a natural variant of the ortholog of E3 in M. domestica with high MCE activity has Ser and Leu at positions 250 ancl 251, respectively. Only one F309 substitution was examined, F309L, which the AChE results suggest should enhance MCE and OP hydrolyse activities. F309L was analysed alone and as a double mutant with W251L.

Mutations in the Anionic Site

The anionic site of ch olinesterases is sometimes called the quaternarsy binding site (for the quaternary ammonium in acetylcholine), or the p1 subsite in the original nome nclature of Jarv (1984). It principally involves

Trp 84, Glu 199 and Phe 330, with Phe 331 and Tyr 130 (TcAChE nomenclature) also involved. Except for Glu 199 it is thus a highly hydrophobic site. Glu 199 i s immediately adjacent to the catalytic Ser 200.

The key residues are highly conserved across cholinesterases and to a lesser extent, many carboxylestera_ses (Oakeshott et al., 1993; Ordentlich et al, 1995; Robin et al., 1996; Clasudianos et al., 2002). Except for Trp 84 (the sequence alignment in Figuwe 1 shows that E3 is missing residues corresponding to AChE resiclues 74-85), E3 has identical residues to TcACImE at the corresponding positiosns (217, 354 and 148, respectively). Interestingly the equivalent of Glu 199 is Gln and the equivalent of the Phe 330 is Leuin_ some lipases and certain carboxylesterases, whose substrates are known to have small leaving groups (Thomas et al., 1999; Campbell et al., 2001;

Claudianos et al., 2002).

Structural and mutati onal studies have provided a detailed picture off the role of the anionic site im cholinesterase catalysis. The key residues foram part of a hydrogen bonded raetwork at the bottom of the active site, with Tyr 130 and Glu 199 also sharimg contact with a structural water molecule (Ordentlich et al., 1995; Koesllner et al., 2000). The anionic site undergoes a conformational change whe n substrate binds a peripheral binding site at th_e lip of the active site gorge, tthe new conformation accommodating the cholime (leaving) group of the substmrate and facilitating the interaction of its carbonyl carbon with the catalytic Sesr 200 (Shafferman et al., 1992; Ordentlich et al. , 1995; 1996). Consequently the site functions mainly in the first, enzyme acylation, stage of the reaction and, in particular, in the formation of the nop- covalent transition state (Nair et al., 1994). Therefore mutations of the key residues mainly affect K, rather than k,,. The interactions with the choline leaving group are mainly me=diated through non-polar and m—electron interactions, principally involving Trp 84 and Phe 330 (Ordentlich et al., 1995).

Studies with OP inhibeitors suggest that the anionic site of cholinesterases also accommodates their leaving group but there is some evidence that part of the site (mainly Glu 199 and Tyr 130; also possibly Ser 226) may also then affect the reactivity of the phosphorylated enzyme (Qian and Kovach, 1993; and see a 1so Ordentlich et al., 1996; Thomas et al., 1999).

There has been little mutational analysis of carboxylesterase sites corresponding to the AChE anionic site but one interesting exception involves the EST6 carboxyle sterase of D. melanogaster, which has a His at th e equivalent of Glu 199. A muatant in which this His is replaced by Glu shows reduced activity against varieous carboxylester substrates but has acquired some acetylthiocholine hydr olytic activity (Myers et al., 1993). The E4 carboxylesterase of the aphicl, Myzus persicae, has a Met at this position and this enzyme is unusually reactive to OPs (Devonshire and Moores, 1982).

However, it is not known whether the Met contributes to the OP hydrolase activity. Similarly, a Y148F substitution is one of several recorded in the E3 ortholog in an OP resistant strain (ie also G137D) of M. domestica but it is no t known whether this change directly contributes to OP hydrolase activity (Claudianos et al., 1999).

The Y148, E217 and F-354 residues in E3 have now been mutated.

E217M and Y148F mutations were made to test whether the corresponding mutations in the M. persicae and M. domestica enzymes above contribute directly to their OP reactivity. Y148F is also tested in a G137D double mutarat since this is the combinatiorm found in the resistant M. domestica. F354 was mutated both to a smaller Le u residue and a larger Trp, Leu commonly being: found at this position in lipa ses (see above).

Example 2: Enzyme Titratioms

Four 100ul reactions vwere set up for each expressed esterase in microplate columns 1-4: plate well blank containing 0.025% Triton X-100, 0.1M phosphate buffer pH a5 7.0;

: substrate blank containing 100uM dECP in 0.025% Tritora X-100, 0.1M phosphate buaffer pH 7.0; cell blank comtaining 50ul cell extract mixed 1:1 with 0.12M phosphate buffer

PH 7.0; titration reaction containing 50pl cell extract mixed 1:1 with 0.1M phosphate buffer pH 7.08 containing 200uM dECP.

All cormponents except dECP (freshly prepared at a. concentration of 200puM in buiffer) were placed in the wells. Several enzyrmes were assayed simultaneously in a plate, and the reactions were started by adding dECP simultaneoussly to the 2nd and 4th wells down a column. The interval to the first reading @ typically 1 minute) was noted for the subsequent calculations.

The mean value for the plate well blank (A) was subtracted from all readings before further calculations. Preliminary experim ents with various cell extracts showed that they gave some fluorescence at <460nm and that their additior to solutions of the assay product, 7-hydrox-ycoumarin, quenched flu orescence by 39(+7)%. Fluorescence values in the titration reactions (D) were therefore corrected for this quenching effect after subtraction o:f the intrinsic fluorescence of the cell extracts (C). Finally, the substrate blamk (B), taken as the mean from all the simult-aneous assays ina plate, was sulbbtracted to give the corrected fluorescence caused by the esterase-relea sed coumarin. These corrections were most important for cell lines expressing esterase at very low level (<1pmol/ul extract).

The fully corrected data were plotted as a progress curve, and the equilibrium slope extrapolated back to zero time to deterrmine the amount of esterase, base=d on its stoichiometric interaction with the inhibitor (the 100 uM concentration of dECP gave full saturation of the estexrase catalytic sites of all these enzymes in 10-20 minutes). A calibration curve for 7- hydroxycourrarin was prepared alongside the reactions im all plates, and used to calculate nmmolar concentration of enzyme and product formation.

Figure 3 shows the results of representative titratiom experiments performed on: cell extracts containing baculovirus expressed esterases.

Example 3: P.ermethrin Hydrolysis Assays

Expresssed enzymes were tested for permethrin hyd rolytic activity using a radiometric partition assay for acid-labelled compounds, or a TLC based assay for those labelled in the alcohol moiety (Devonshire aand Moores, 1982).

Features of th e assays include keeping the concentration of ~ permethrin below its published .solubility in aqueous solution (0.5 uM), the concentration of detergent (usexd to extract the enzyme from the insect cells Zin which itis expressed) below the critical micelle concentration (0.02% for Triton X100), and performing the assays quickly (ie within 10-30 minutess) to minimise the substrate stick<ing to the walls of the assay tubes (glass tube s were used to minimise stickiness). At these permethrin concentrations tne enzyme is not saturated by the substrate, so K,, values could not be deternmined. However, specificity comstants (k,./K,,) could be calculated accurately= for each of the enzymes withe permethrin activity, which allows direct comparison of their efficiency at l-ow substrate concentrations. The power of thes analyses was increased by separating permethrin into its cis and trans isomers. (a) Separation of cis and trans Isomers of Permethrin

Commercial preparations of permethrin contain four stereoisomers: 1S cis, 1R cis, 1S trans, 1R trans (Figure 4). Preparative thin layer chromatograpehy (TLC) on silica was used to separate the is omers into two enantiomer pairs: 1S/1R cis and 1S/1R trans. The enantiormers could not be separated further. Enzyme preparations could then be assaiyed for the hydrolysis of each enantiomer pair. (b) Assay Protocol

Pyrethroids radiolabelled in the acid moiety

This as say (Devonshire and Moores, 1982) is used for permethrin isomers. It re:lies on incubating the expressed esterase witla radiolabelled substrate and then measuring the radioactive cyclopropanescarboxylate anion in the aqueous phase after extracting the unchanged substrate into organic solvent. Base=d on previous experience, the best extraction protocol utilises a 2:1 (by volunne) mixture of methanol and chloroform. Whesn mixed in the appropriate p-roportion with aliquots of the assay incubatiosn, the consequent mixture of buffer, methanol and chloroform is monophasic=, which serves the purpose of stopping the enzyme reaction and ensuring the complete solubilizatiom of the pyrethroid. Subsequent addition of ara excess of chloroform ard buffer exceeds the capacity of the methanoel to hold the

W/O 03/066874 PCT/AU02/00114 phases together, so that the organic phase car be removed and the product measured in the aqueous phase. In detail, the protocol is as follows.

Phosphate buffer (0.1M, pH 7.0) was aclded to radiolabelled permethrin (50uM in acetone) to give a 1uM solution andl the assay then started by adding an equal volume of expressed esterase appropriately diluted in the same buffer. Preliminary work had established that the concentration of detergent (Triton X-100 used to extract esterase from the harvested cells} in the incubation had to be below its CMC (critical micelle concentration of 0.02%) to avoid the very lipophilic pyrethroicd partitioning into the micelles and becoming unavailable to the enzyme. Twpically, the final volume of the assay was 500-1000pl, with substrate and acetone concentrations 0.5uM and 1%, respectively. At intervals ranging from 30 seconds to 10 minutes at a temperature of 30°, 100ul aliquots of the incubation were removed, added to tubes containing 300pul of the 2:1 methanol chloroform mixture and vortex- mixed. The tubes were then held at room ternperature until a batch could be further processed together, either at the end of the incubation or during an extended sampling interval. After adding 50gul buffer and 100pl chloroform, the mixture was vortex-mixed, centrifuged amd the lower organic phase removed with a 500ul Hamilton syringe and «discarded. The extraction was repeated after adding a further 100pl chloroform, and then 200ul of the upper aqueous phase was removed (using a pipettoz with a fine tip) for scintillation counting. It is critical to avoid taking any of the organic phase. Since the final volume of the aqueous phase was 260pL (including some methanol), the total counts produced in the initial 100u! alicjuot were corrected accordingly.

Pyrethroids radiolabelled in the alcohol moietty i) Type I pyrethroids - dibromo analogues (NFEDC157) of permethrin:

The 3-phenoxbenzyl alcohol formed om hydrolysis of these esters does not partition into the aqueous phase in the chloroform methanol extraction procedure. It was therefore necessary to separate this product from the substrate by TLC on silica (Devonshire and NMooers, 1982). In detail, the protocol is as follows. ) Incubations were set up as for the acid_-labelled substrates. The reactions were stopped at intervals in 100ul aliquots taken from the incubation by immediately mixing with 2001] acetone at -79° (solid CO,).

Then 100pl of the mixture was transferred, together with 3ul non-radioactive

3-phenoxbenzyl alcohol (2% in acetone), con to the loading zone of LinearQ channelled silica F254 plates (Whatman). After developing in a 10:3 mixture of toluene (saturated with formic acid) wifth diethy! ether, the substrate and product were located by radioautography for 6-7 days (confirming an identical mobility of the product to the co 1d standard 3-phenoxbenzyl alcohol revealed under UV light). These areas of the TLC plate were then impregnated with Neatan (Merck) and drieed, after which they were peeled from the glass support and transferred to wials for scintillation counting. The counts were corrected for the 3-fold diluti on of the initial 100ul by acetone 710 before spotting on the silica. ii) Type II pyrethroids - deltamethrin isomesrs:

Preliminary experiments, in which -incubations were analysed by TLC as above, showed primarily the formation of 3-phenoxbenzoic acid, in line 215 with literature reports that the initial cyamohydrin hydrolyis product is rapidly converted non-enzymically to the acid. Since the TLC assay is more protracted than the chloroform-methanol eextraction procedure, the latter (as described above for acid-labelled pyrethroids) was adopted to measure the 3- phenoxbenzoate anion produced from the se substrates.

For all assays the molar amount of product formed was calculated from the known specific activity of the radiolabeelled substrate. Early experiments on the expressed E3WT esterase showed t hat the rate of hydrolysis was directly proportional to the concentration of 1RS cis or 1RS trans permethrin in the assay up to 0.5uM, i.e. there was noe accumulation of Michaelis complex. Assays at concentrations greater than 0.5uM, which approximates the published aqueous solubility of permesthrin, gave erratic results so precluding the measurement of K,, and kK, Furthermore, with the racemic substrates, the rate of hydrolysis slowed d ramatically once approximately 50% of the substrate had been hydrolysed , indicating that only one of the two enantiomers (1R or 1S present in equal amounts in a racemic mixture) was readily hydrolysed, in line with previously published data for an esterase from aphids (Devonshire and Moores, 198 2). Assay conditions were therefore adjusted to measure the hydrolysis of the zmore-readily hydrolysed m5 enantiomer in each pair. Sequential inculoation of trans permethrin with

E3WT homogenates confirmed that both s howed preference for the 1S trans enantiomer. In all cases, the rate of hydrolysis at 0.5uM (or 0.25uM for the one enantiomer in racemic substrates), together with the molar amount of esterase determined by titration witla dECP, were used to calculate the specificity constant (k.,/ K,) since it was not possible to separate these kinetic parameters. The same consideratioras about substrate solubility and proportionality of response to its coracentration were assumed for all enzymes and substrates. (c) Calculation of Specificity Constants

Figure 5 presents the results o f an experiment in which the trans- and cis- isomers of permethrin were hydmolysed by the E3W251L enzyme.

Since the rate of hydrolysis of permethrin isomers was directly proportional to the concentration of substrate used up to 0.5uM (i.e. there was no significant formation of Michaelis complex), it was not possible to measure K and k,, as independent paramters. At concentrations well below the K,,, the Michaelis-Menten equati on simplifies to: k v=g [S] [E] m

The specificity constant (ie k,.,/K,) can therefore be calculated from the above equation using the initial hydarolysis rate (pmol/min, calculated from the known specific activity of the radliolabelled substrate) and the concentrations of substrate and enzyme in the assay. The diffusion-limited maximum value for a specificity comstant is 10°-10° M*sec” (Stryer, 1981).

Example 4: Malathion Hydrolysis Assays

MCE activity was assayed as described by Campbell et al. (1998), but without diluting the specific activity of the **C malathion (25mGCi mmol™) for : enzymes that appeared to have a lows K,,. This was an end-point assay in which malathion was extracted into an organic phase while radiolabelled : 30 malathion carboxylic acids, the hydrolysis products remained, in the aqueous phase. Activity was measured over the range 50nM to 1uM to determine the

K, and k,,, and analysed by non-linear regression using the Enzfitter 1.05 software (Elsevier-Biosoft), with graphical output to reveal any deviation from

Michaelis-Menten kinetics. Specificity constants were calculated directly - from the K, and k_, values. ) Example 5: Permethrin Hyd rolytic Activity of E3 and EST23 Variants

Table 2 summarises thee kinetic data obtained for eighteen E3 and three

EST23 variants using cis- and trans- permethrin as substrates. The malathion hydrolytic activity of the enz-ymes is also given for comparison. In each case the data represent the hydrol-ysis of the enantiomer that is hydrolysed the fastest out of each of the 1S/1 R cis and 1S/1R trans isomer pairs (see above).

The E3WT enzyme fou nd in OP susceptible blowflies, and its EST23 D. melanogaster orthologue, shoswed significant levels of permethrin hydrolytic activity, which was specific f«or the trans isomers. The wild-type enzymes showed at least an order of mmagnitude higher activity for malathion (although this high MCE activity does mot confer malathion resistance on the blowfly because the enzyme is readils inhibited by the malaoxon produced in vivo by the fly; Campbell et al., 1998). Mutations in either the acyl binding pocket or anionic site regions of the acttive site of the E3 enzyme resulted in significant increases in activity for both -the trans and cis isomers of permethrin. These increases in permethrin hydrolysis were not in the main correlated with increases in malathion hydrolytic activity. a) Oxyanion hole mutations

The E3G137D mutation is responsible for diazinon resistance in the sheep blowfly. In this mutan-t the very small, aliphatic, neutral Gly residue in the oxyanion hole region of the active site of the enzyme is replaced by an acidic Asp, allowing hydrolyssis of a bound oxon OP molecule. However, this mutant (and its D. melanogaster orthologue) had reduced activity for trans- permethrin in particular, corrmpared to that of the wild-type enzyme. This activity was not increased by substitution of Gly-137 with either His or Glu.

However, substitution of Gly- 137 with Arg did not affect the activity for either cis- or frans-permethrira appreciably. The linear nature of Arg might mean that it can fold easily arid not interfere with binding of permethrin to : the active site. The MCE activity of this group of mutants correlated broadly with their activity for trans permethrin in particular, indicating effects of

G137 substitutions on the accommodation and stabilisation of the substrate acyl group. Effects are germerally smaller for permethrin than malathion but ) this is consistent with the somewhat smaller acyl group for permethrin. ’ b) Acyl binding pocket mwmtations

The E3W251L mutation, which replaces the large aromatic Trp reside with the smaller aliphatic Ieu in the acyl pocket of the active site, resulted in a 7-fold increase in trans-p ermethrin hydrolysis and the acquisition of substantial cis-permethrin hydrolysis. This is the mutation responsible for the acquisition of malathion resistance in the sheep blowfly. The MCE activity of this mutant was 2-fold higher than that of the wild-type enzyme.

The effect of W251L in EST23 was essentially the same as for E3.

Replacement of Trp-251 with even smaller residues in E3 (Thr, Ser, Ala and

Gly in decreasing order of size) also resulted in an increase in permethrin hydrolytic activity, although the activity of these mutants was not as high as that of E3W251L. Clearly, steric factors are not the only consideration in the activity of the mutants. For example, Thr and Ser both contain hydroxyl groups and are hydrophilic. Furthermore, Ala is both aliphatic and hydrophobic (like Leu) and even smaller than Leu, yet this mutant was as active for permethrin as the W251L mutant. Opening up the oxyanion hole of the W251L mutant (ie E3P250S/W251L) also decreased its activity for both cis- and trans-permethrin, although the activity was still higher than that of the wild type. It is interesting to note that increases in specificity constants for permethrin for all W252 mutants in E3 as well as W251L in EST23 compared to those of the wild types were uniformly more pronounced for the cis isomers. Whereas the wild type enzymes yielded trans:cis ratios of at least 20:1, these ratios were only 2-6:1 for the W251 mutants. The extra space in the acyl pocket provided bys these mutants was apparently of greatest benefit for the hydrolysis of the otlaerwise more problematic cis isomers.

The MCE activity of the E3-251 mutants was not correlated with permethrin hydrolytic activity, Of this group of mutants, E3W251G had approximately 10-fold higher MCE activity than the remainder of the group, and yet its permethrin hydrolytic activity was among the lowest. : Combination of both the W251L and G137D mutations on to the same

E3 molecule increased the activity of the enzyme for cis permethrin over wild-type levels, but decreased the activity for trans-permethrin and also malathion. However, the activity of the double mutant was not as great as that of the muitant containing the E3W251L mutation alone (i.e. &he mutations did not act ad_ditively).

Some lipases are known to have a Leu residue at the position : corresponding to Phe 309 in L. cuprina E3. The E3F309L mutant was therefore constructed with the aim of conferring activity for lipophilic substrates likes pyrethroids. As can be seen from Table 2, the E3F'309L mutant was much better than E3WT for both isomers. It was even more active for trans-permetharin than E3W251L, though not as active for the cis isomers.

However, the MCE activity of this mutant was less than half that of the wild- type enzyme. Combination of both the F309L and W251L mutatZons on the same E3 molescule increased the activity for cis-permethrin and d ecreased the activity for tra ns-permethrin to E3W251L levels. In other words, the F309L mutation had very little effect on the activity of the W251L mutamt for permethrin, bwit decreased its activity for malathion. ¢) Anionic site mutations

Some ligpases are known to have a Leu residue at the position corresponding to Phe 354 in L. cuprina E3. However, substitutiorh of Phe 354 for Leu in E3 did not increase its activity for permethrin appreciably, but greatly reduced its activity for malathion. Substitution of Phe 35<1 for the bulkier aromatic residue, Trp, on the other hand, increased activi-ty for both cis- and trans-permethrin 3-4-fold, but decreased MCE activity sli ghtly. Itis perhaps surpri sing that F354W, not F354L, should show increases in activity against the very lipohilic permethrin, given that it is a Leu that replaces Phe in some naturally occurring lipases.

Although Y148F is of little consequence for MCE activity it has large effects on permethrin kinetics and the effects are opposite in direction depending on genetic background. As a single mutant compared to wild type it shows 5-6 folld enhancement of activity for both cis and trans permethrin.

Asa double mutant with G137D (which as a single mutant gives values much : lower than wild type), it shows a further two fold reduction for trans permethrin and and almost obliterates activity for cis permethrin. These latter results clearly imply a strong interaction of Y148 with the oxyanion hole in respect of permethrin hydrolysis.

Glu-217, the residue immediately adjacent to the catalytic serine, is thought to be irmportant in stabilising the transition state intermed_iate in hydrolysis reactions. However, mutating this residue to Met (E3E2217M), as found naturallw in the esterase E4 of the aphid M. persicae, had li ttle effect on permethrin activity but greatly reduced its MCE activity.

Example 6: Hydrolysis of Bromo-Permethrin Analogue :

Table 2 also summarises the kinetic data obtained for the E-3 and EST23 variants using &he two cis -dibromovinyl analogues of permethrin (NRDC157).

The 18 cis isomer of this dibromo analogue of permethrin was hy drolysed with similar efficiency to the 1R/1S cis permethrin by all enzymes except

E3F309L and F 309L/W251L. This indicates that the larger bromire atoms did not substantial ly obstruct access of this substrate to the catalytic centre.

Although the activities with the E3WT and EST23WT enzymes were too low for significant comparison between isomers, all other enzymes ex cept

E3F309L and F 309L/W251L showed 10 to 100-fold faster hydrolysis of the 1S isomer. This is the same preference for this configuration at C1 o f the cyclopropane ring as found previously for 1S trans permethrin in M. persicae (Devonshire an d Moores, 1982).

F309L slmowed a dramatic effect on NRDC157 kinetics. The single mutant showed little difference from wild type for 1S cis and the double with

W251L showed less activity than W251L alone for this isomer. However, the 1S/1R preferenece was reversed, with values of 0.7:1 in the single mutant and 0.4:1 in the double. The result is the two highest values for 1R ciss activities in all the data set. The value for the double mutant is in fact abouat 10 fold higher than those for either mutant alone.

Example 7: Hydrolysis of Type II Pyrethroids by Expressed Enz-ymes

Table 3 summarises the kinetic data obtained for a sub-set of the E3 and EST23 vari ants using the four deltamethrin cis isomers. With the exception of EZW251L and E3F309L, the 1R cis isomers of deltammethrin (whether aS or aR) were hydrolysed with similar efficiency to the 1R cis

NRDGC157 (which can be considered intermediate in character between permethrin andl deltamethrin in that it has dibromovinyl substitusent but lacks the a cyano group). Activity against 1R cis isomers was always grreater with the oR than thes aS conformation. E3W251L and E3F309L were markedly less efficient with tlhe 1R cis isomers of deltamethrin than with the comresponding isomers of NRIDC157.

VWVO 03/066874 PCT/AU02/00114

TABLE 2: Specificity constants of natural amd synthetic variants of L. cuprina esterase E3 and D. melanogaster EST 23 for the cis- and trans-isomers of permethrin, malathion and the two cis -dib romovinyl analogues of permethrin (NRDC157). Ratios of the specificity constants for trans and cis permethrin, and for 1S cis and 1R cis NRDC157 are also indicated.

Enzyme Specificity Constant ( k./K,, M?sec”) 1S/1R 1S/1R cis- m_alathion | NRDC15 | NRDC157 trans- permethrin 7 1R cis permethri (trans:cis 1S cis (1S:1R n ratio) ratio) 90000 | 3400 2 600 000 4700 | 630 (8:1) 27:1

Oxyanion hole mutants:

E3G137D 9 600 1 800 5100 ND? ND (5:1)

E3G137R 85 000 3900 (22:1) 1 200 000 ND ND

E3G137H 26 000 1 600 8 800 ND ND (16:1)

E3G137E 2 400 280 19 000 ND ND (9:1)

Acyl binding pocket mutants:

E3w251L 900 000 460 000(2:1) 4 800 000 370 000 | 5 400 (68:1)

E3W251S 140 000 36 000 (4:1) 6 500 000 35 000 | 2 900 {12:1)

E3W251G 35 G00 24000 (4:1) | 57 000000 27 000 | 1 700 (16:1)

E3W251T 150 000 | 24 000 (6:1) 4 500 000 24 000 | 900 (26:1)

E3W251A 300 000 72000 (4:1) 5 400 000 67 000 | 1 200 (56:1)

E3F309L 1 200 000 | 48 000 (25:1) 1 000000 5700 | 8000 (0.7:1)

E3W251L 810 000 430 000(2:1) 1 400 000 26 000 {69 100 /F300L (0.4:1)

E3W251L 24 000 11 000 60 000 12 000 | 1 100 /G137D 2:1) (11:1)

E3P250S 340 000 | 110 000 1 400 000 /W251L (3:11

Anionic site mutants:

E3Y148F 580 000 17 000 (34:1) 3 100 000 ND ND

E3Y148F 4100 47 12 000 ND ND /G137D (87:1) . E3E217M 93 000 4 400 77 000 ND ND (21:1)

E3F354W 350 000 8 800 (40:1) 1 600 000 ND ND

E3F354L 104400 | 2700 (38:1 7106 000 ND ND

Cb recontnued ones,

VVO 03/066874 PCT/AU02/00114

EST23WT 21 000 890 2 7600 000 990 | 330 (24:1) (3:1) ‘ EST28W251 260 000 | 160 000 (2:1) 2 3800 000 72 000 | 1200 (60:1)

EST23G137D 2500 - - ND ND ! Not determined 2 Not substantially different from values obtained vasing control cell extracts

Significantly, the 251 mutant with the hi ghest deltamethrin activities was W2518, while W251L (highest for the othe 1 two pyrethroids), and

W251G (highest for malathion) gave the lowest deltamethrin activities of the five 251 mutants. This suggests that accommodation of the acyano moiety of the leaving group may be the major impedimemt to efficient deltamethrin hydrolysis, sufficient to prevent any significant hydrolysis by wild type E3.

Accommodation of substrate requires significarmtly different utilisation of ~ space across the active site compared to other ssubstrates, such that substitution of W251 in the acyl pocket with a ssmaller residue allows useful accommodation, particularly for oR isomers. Immportantly, however, the details of the spatial requirements, and thereforre the most efficacious mutants, differ from those for the other pyrethr oids.

The activity of all enzymes with the 1S c_is isomers of deltamethrin was dramatically less than with the corresponding i somer of NRCC157 lacking the o cyano group. This dramatic influence of the o cyano group appears to be expressed with this 1S conformation at C1 of tle cyclopropane group. With the exception of some of the least active mutants, activity against 1S cis isomers was again always greater with the aR tThan the aS conformation.

Example 8 - General Discussion

Together, the permethrin and NRDC157 results for the 251 series mutants generate some quite strong and simple: inferences about acyl binding constraints in E3/EST23. Overall, as with mala _thion, 251 replacements that : should generate a more spacious acyl pocket do facilitate the accommodation/stabilisation of the bulky acyl groups of these substrates.

These replacements are beneficial to the hydrolysis of all the isomers

TABLE 3: Specificity constants £or the four deltamethrin cis isomers

Enzyme Specificity Constant (k,./K, M7sec?) 1S cis aR 18 cis aS 1R cis aR 1R cis aS deltamethrin |deltamethrin |deltamethrin |deltamethrin

E3G137D - 890 560

E3G137R - 670 350

E3G137H ND ND ND

E3G137E ND ND ND

E3W251L 990 880 380 -

E3W2518 4 600 2 460 ND? ND

E3W251G 700 170 690 350

E3wW251T 2 900 520 2 100 1 300

E3W251A 2000 660 1300 730

E3F309L 2 400 810 1600 840

E3W251L 3 600 410 2700 1100 /G137D

Est23WT 450 750 -

Est23W251L 980 550 1000 430 ! Not substantially different from walues obtained using control cell extracts ? Not determined generated by the two stereocentress across the cyclopropane ring. While the : trans isomers are strongly preferred by wild type enzyme, the mutants can also hydrolyse at least part of the cis isomer mix relatively well. However, : within the cis isomers the improv-ements in the mutants is much more marked for the 1S cis isomers. The 1R cis isomers, which are the most problematic of all configurations for wild type enzyme, remain the most problematic for the mutants. Within the mutant series, the improved kinetics are not simply explained by the re duction in side chain size; the smallest substitution does not give the highest activities, as it does for malathion.

Indeed the best kinetics are obtain ed with W251L, although Leu has the greatest side chain size of all the replacements tested.

In contrast to the relatively simple and consistent patterns seen for permethrin and NRDC157, the del-tamethrin results for the 251 series mutants are quite complex and difficult to &nterpret. As might be expected from their enhanced kinetics for the other subostrates, they do show overall better activities than wild type for the four cis deltamethrin isomers, albeit as with wild type they are much lower in absolute terms than for the other substrates.

However, the preference for 1S over 1R isomers, which is so strong in respect of NRDC157, is weak at best in the deltamethrin data. On the other hand there is a clear trend across all the mutants for a preference for the aR over aS isomers. It is generally only of the order of 2:1, but notably it is opposite to the trend shown by wild type EST23. It is at first sight unexpected that : these presumptive acyl binding poecket replacements should affect aR/aS stereopreferences because the latter apply to the a-cynano moiety in the (alcohol) leaving group of the subs&rate.

Overall the F309L data clearBy show a major effect of this residue on the kinetics of pyrethroid hydrolysis. At one level there are parallels with the results for the W251 series mutantss, both data sets showing enhanced kinetics consistent with expectatioms based on the provision of greater space in the acyl binding pocket. Howeveer, there are also important differences, with the W251 series disproportiomately active for the cis vs trans isomers of permethrin and F309L disproportiosnately active with 1R vs 18 isomers of cis

NRDC157. The replacements at the two sites also show strong interactions, consistent with them contributing to a shared structure and function in the acyl binding pocket. For example, Toth the disproportionate enhancement of the W251 mutants for cis permethrin and the disproportionate enhancement of F309L for 1R cis NRDC157 behave as dominant characters in the double mutant. The 251 and 309 mutants thave quantitatively similar enhancing effects on activities and the same st-ereospecificities in respect of deltamethrin ’ hydrolysis and the stereospecific differences seen with the smaller pyrethroids are not seen. However, we argue that the additional bulk of the ocyano moiety in its leaving group mequires such a radical reallocation of space across the active site that the stereospecificities evident with the smaller pyrethroids are overridden.

It will be appreciated by persoras skilled in the art that numerous variations and/or modifications may bbe made to the invention as shown in the specific embodiments without depart-ing from the spirit or scope of the invention as broadly described. The gresent embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

All publications discussed abowe are incorporated herein in their entirety.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the peresent specification is solely for the purpose of providing a context for thes present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge i-n the field relevant to the present invention as it existed before the prioxity date of each claim of this application.

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Claims (40)

ow CLAIMS:

1. A method of eliminating or reducing -the concentration of a hydrophobic ester pesticide or toxin in a sample, the met "hod comprising contacting the sample with a Dipteran esterase, or a mutart thereof.

2. The method of claim 1, wherein the Deipteran esterase is an a- carboxylesterase.

3. The method of claim 1, wherein the mautant Dipteran esterase is an o- carboxylesterase, and has a mutation(s) in a_n oxyanion hole, acyl binding pocket or anionic site regions of an active site of the esterase, or any combination thereof.

4. The method of claim 3, wherein the nautant Dipteran esterase is selected from the group consisting of: E3G137R, E3G137H, E3Wa51L, E3W2518, E3W251G, E3W251T, E3W251A, E3W251L/F309L, E3W251L/G137D, E3W251L/P2508S, E3F30 9L, E3Y148F, E3E217M, E3F354W, E3F354L, and EST23W251L.

5. The method of claim 2 or claim 3, wherein the a-carboxylesterase, or mutant thereof, comprises a sequence select-ed from the group consisting of: i) a sequence as shown in SEQ ID NO=1, ii) a sequence as shown in SEQ ID NO»:2, and iii) a sequence which is at least 40% identical to i) or ii) which is capable of hydrolysing a hydrophobic ester pesticide or toxin.

6. The method of claim 6, wherein the se=quence is at least 80% identical to 1) or ii).

7. The method of claim 6, wherein the se-quence is at least 90% identical to i) or ii).

8. The method according to any one of cl aims 1 to 7, wherein the method is performed using two or more Dipteran esterases, or mutants thereof,

9. The method according to any one of claims 1 to 8, wherein the hydrophobic ester pesticide or toxin is a pyre=throid. AMENDED SHEET 2005 -11- 9 7

10. The method of claim 9, wherein the pyrethroid is a Type I or Type II pyrethroid.

11. The method of claim 10, whe=rein the Type I pyrethroid is selected from the group consisting of: 1S/1R trans permethrin, 1S/1R cis permethrin, NRDC157 1S cis, and NRDC157 1R cis.

12. The method of claim 10, whe rein the Type II pyrethroid is deltamethrin.

13. The method according to any” one of claims 1 to 12, wherein the method is performed in a liquid containing «environment.

14. The method according to any one of claims 1 to 13, wherein the Dipteran esterase, or mutant thereof, is provided directly to the sample.

15. The method according to any one of claims 1 to 13, wherein the Dipteran esterase, or mutant thereosf, is provided to the sample by expression of a polynucleotide encoding the Dippteran esterase, or mutant thereof, from a host cell comprising the polynucleotide.

16. The method according to any one of claims 1 to 13, wherein the Dipteran esterase, or mutant thereo, is provided as a polymeric sponge or foam, the foam or sponge comprisimg the Dipteran esterase, or mutant thereof, immobilized on a polymeric porous support.

17. The method according to any one of claims 1 to 16, wherein the method further comprises the presence of a surfactant when the hydrophobic ester pesticide or toxin is contacted with t he Dipteran esterase, or mutant thereof.

18. The method of claim 17, wherein the surfactant is a biosurfactant.

19. A substantially purified polypeptide which is a mutant of a Dipteran esterase, wherein one or more mutations are within a region of the esterase selected from the group consisting of: oxyanion hole, acyl binding pocket and anionic site, wherein the mutant Dipsteran esterase is capable of hydrolysing a hydrophobic ester pesticide or toxin, with the proviso that the mutant Dipteran esterase is not E3W251L, E.3W251S, E3W251G or E3G137D. AMENDED SHEET 2005 -1- 7

20. The polypeptide according to claim 19, wherein the Dipteran esterase is an o~-carboxylesterases.

21. The polypeptidle according to claim 20 selected from the group consisting of: i) a mutant of a sequence as shown in SEQ ID NO:1, and ii) a mutant of ssequence as shown in SEQ ID NO:2, wherein the muatant is at least 40% identical to at least one of SEEQ ID NO's:10r 2.

22. The polypeptide of claim 21, wherein the mutant is at least 80% identical to at least ome of SEQ ID NO's:1 or 2.

23. The polypeptidee of claim 21, wherein the mutant is at least 90% identical to at least orme of SEQ ID NO's:1 or 2.

24. The polypeptides according to any one of claims 19 to 23, whereim the mutation is a point mutation.

25. The polypeptide according to claim 21 comprising a sequence se Jected from the group consisting of: E3G137R, E3G137H, E3W251T, E3W251_A, E3W251L/F309L, E3WW251L/G137D, E3W251L/P250S, E3F309L, E3Y 148F, E3E217M, E3F354W, E3F354L, and EST23W251L.

26. A fusion polypeptide comprising a polypeptide according to any one of claims 19 to 25 fused t-o at least one other polypeptide sequence.

27. Anisolated polynucleotide encoding a polypeptide according to =any one of claims 19 to 26.

28. A vector for replication and/or expression of a polynucleotide acecording to claim 27.

29. A host cell transformed or transfected with the vector of claim 283. AMENDED SHEET 2005 -1- 07

3.0. A composition for hydrolysing a hydrophobic es’ ter pesticide or toxin, the composition comprising a polypeptide according tc any one of claims 19 to 2 6, and one or more acceptable carriers.

3 1. A method for generating and selecting an enzynme that hydrolyses a h_ydrophobic ester pesticide or toxin, the method comyorising (i) introducing one or more mutations into a Dijoteran esterase, or an Dipteran esterase that has already been mutated, and (ii) determining the ability of the mutant Dipteraan esterase to hydrolyse a hydrophobic ester pesticide or toxin.

3=. The method of claim 31, wherein the one or more mutations enhances hydrolytic activity and/or alters the stereospecificty of - the esterase.

3=3. The method of claim 31 or claim 32, wherein the= Dipteran esterase is an a—carboxylesterase.

3=. The method of claim 33, wherein the a-carboxyleesterase has a sequence selected from the group consisting of: i) a sequence as shown in SEQ ID NO:1, ii) a sequence as shown in SEQ ID NO:2, and iii) a sequence which is at least 40% identical to 3) or ii).

35. The method of claim 34, wherein the sequence iss at least 80% identical tos i) orii).

36. The method of claim 24, wherein the sequence iss at least 90% identical toe i) or ii).

37. The method of any one of claims 31 to 36, where®n the one or more m utations are within a region of the esterase selected from the group coensisting of: oxyanion hole, acyl binding pocket and amionic site.

38. The method of any one of claims 31 to 37, wherei_n the mutation is a point mutation.

39. The method of claim 31, wherein the Dipteran esterase that has already be=en mutated is selected from the group consisting of: BE3G137R, E3G137H, AMENDED SHEET 9005 41- 7

E3W251L, E3W251S, E3W251G, E3W251T, E3W 251A, E3W251L/F309L, E3W251L/G137D, E3W251L/P250S, E3F309L, F-3Y148F, E3E217M, E3F354W, E3F354L, and EST23W251L.

40. An enzyme obtained by a method according to any one of claims 31 to

39. AME NDED SHEET 2005 -11- 07