ENZYMES AND METHODS FOR PREVENTING AND TREATING PYRETHROID EXPOSURE IN ANIMALS

Abstract

Compositions and methods for treating an animal who has been exposed to pyrethroid are provided. An effective amount of a pyrethroid detoxifying enzyme to prevent or treat pyrethroid toxicity. The pyrethroid detoxifying enzyme is one or more pyrethroid-hydrolyzing carboxylesterases.

Claims

1. A method for treating an animal who has been exposed to pyrethroid comprising administering an effective amount of a pyrethroid detoxifying enzyme to prevent or treat pyrethroid toxicity.

2. The method of claim 1, wherein the pyrethroid detoxifying enzyme is one or more hydrolyzing carboxylesterases.

3. The method of claim 1 wherein the animal is selected from the group consisting of an aquatic species, a poultry species, a porcine species, a bovine species, an ovine species, an equine species, and a companion animal.

4. The method of claim 3 wherein the animal is a companion animal.

5. The method of claim 4 wherein the animal is a canine species or a feline species.

6. The method of claim 5, wherein the animal is a feline species.

7. The method of claim 1, wherein the pyrethroid is one or more of pyrethrin, tetramethrin, allethrin, phenothrin, barthrin, dimethrin, bioresmethrin, permethrin fenpropathrin, bifenthrin, cyfluthrin, beta-cylfuthrin, phenothrin, imiprothrin, flumethrin, momfluorothrin, bioallethirn, deltamethrin, cypermethrin, and tetramethrin.

8. A composition for the administering to an animal who has been exposed to pyrethroid, the composition comprising a pharmaceutical carrier suitable for administration in an animal and an effective amount of a hydrolyzing carboxylesterase.

9. The composition of claim 8, wherein the composition is administered to an animal is selected from the group consisting of an aquatic species, a poultry species, a porcine species, a bovine species, an ovine species, an equine species, and a companion animal.

10. The composition of claim 9, wherein the animal is a companion animal.

11. The composition of claim 10, wherein the animal is a feline species.

12. The composition of claim 8, wherein the animal has been exposed to is one or more of pyrethrin, tetramethrin, allethrin, phenothrin, barthrin, dimethrin, bioresmethrin, permethrin, fenpropathrin, bifenthrin, cyfluthrin, beta-cylfuthrin, phenothrin, imiprothrin, flumethrin, momfluorothrin, bioallethirn, deltamethrin, cypermethrin, and tetramethrin.

13. A method for treating an animal to prevent pyrethroid toxicity, the method comprising: administering an effective amount of a pyrethroid detoxifying enzyme to prevent pyrethroid toxicity.

14. The method of claim 13, further comprising: administering an effective amount of a one or more hydrolyzing carboxylesterases to an animal to prevent the toxic effect of synthetic pyrethroids.

15. The method of claim 14, wherein the administration of an effective amount of one or more hydrolyzing carboxylesterases is done before the animal is exposed to pyrethroids.

16. A method of claim 15, wherein administering an effective amount of one or more hydrolyzing carboxylesterases an animal to reduces the toxic effect of synthetic pyrethroids.

17. The method of claim 13, wherein the pyrethroid detoxifying enzyme is one or more hydrolyzing carboxylesterases.

18. The method of claim 13, comprising feeding an animal, the method comprising the step of administering to the animal a feed composition or drinking water comprising an effective amount of an additive one or more hydrolyzing carboxylesterases wherein the one or more hydrolyzing carboxylesterases causes an effect selected from the group consisting of preventing or treating pyrethroid toxicity.

19. The method of claim 13, comprising administering to the animal an effective amount of hydrolyzing carboxylesterase by injection.

20. The method of claim 13, comprising administering to the animal an effective amount of hydrolyzing carboxylesterase by topical administration.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Illustrative aspects of the present invention are described in detail below with reference to the attached drawing figures, and wherein:

[0012] FIG. 1 is graph of enzymes expressed with a tag (e.g., a polyhistidine (His)) to facilitate purification.

[0013] FIG. 2 is a graph of absorbance.

[0014] FIGS. 3 and 4 are graphs of a permethrin standard curve.

[0015] FIG. 5 is a graph of deltamethrin reaction with PYTH2.

[0016] FIG. 6 is a graph of estherase activity.

[0017] FIG. 7 is graph of hydrolysis of trans permethrin.

[0018] FIGS. 8 and 9 are graphs of scuds exposed to permethrin and esterases.

[0019] FIG. 10 is a graph of a nucleotide amplicon.

[0020] FIG. 11 is a graph of a plasmid.

DETAILED DESCRIPTION

[0021] The present invention is generally directed as a method for administering an effective amount of detoxifying enzymes and compositions to an animal to prevent or treat pyrethroid toxicity in animals. The present invention is also directed to treating animals with permethrin exposure.

[0022] The enzymes can be used in methods for detoxifying pyrethroids. The present invention relates to proteins having pyrethroid degradation abilities (esterase) including proteins containing a targeting sequence having protease properties.

[0023] An esterase is a hydrolase that splits esters into acids and alcohols. An esterase can act as a pyrethroid hydrolase, hydrolyzing permethrin into (3-phenoxyphenyl) methanol and (1S,3R)-3-(2,2-dichloroethenyl)-2,2 dimethylcyclopropanecarboxylate. Enzymes were purified from an E. coli strain optimized for recombinant protein expression.

[0024] The specific reason for the sensitivity of the cat is also unknown. Species susceptibility to permethrin is likely dependent on the nature of the tissue esterase, the level of activity detected, the substrate specificity, and the rate of hydrolysis encountered. Since hydrolytic enzymes degrade pyrethroid esters, it is suspected that the species susceptibility of permethrin could be due to the rate of hydrolysis being slower in cats than other species. Cats naturally lack the production of glucuronidyltransferase in the liver and this invention would provide an enzyme that would metabolize the pyrethroid to prevent toxicosis.

Detoxifying Enzymes

[0025] As described in greater detail below, the genomes of microbes in microbial libraries that contained microorganisms capable of degrading pyrethroid, particularly pyrethrin, were screened to identify enzymes capable of degrading pyrethroids. This screening resulted in identification of a number of pyrethroid-hydrolyzing carboxylesterases.

[0026] Pyrethroid-hydrolyzing carboxylesterases can be used to detoxify pyrethroids and degrade pyrethroids in animals. For example, pyrethroid-hydrolyzing carboxylesterases can be used to promote accelerated degradation of pyrethroid, as compared to the rate of degradation that would occur in the absence of the esterase. The mechanism for detoxifying pyrethroid by an esterase is shown 1) hydrolysis of ester bound between dibromo- or dichloro-vinyl cyclopropane carboxylate.

[0027] A reaction scheme for detoxification by esterase is shown below:

##STR00001##

Selected Esterase Activity for Permethrin Degradation

[0028] ##STR00002##

Initial Screen

[0029] Initial screen to identify esterase candidates was performed. The initial screen was performed by the ability to grow in the presence of permethrin 0.2% on a solid plate. Over 30 expected tolerant strains were tested. The next step was to look for liquid growth of the identified candidates

[0030] Permethrin was not soluble in water and once dissolved in a carrier such as methanol, DMSO, or xylene it was challenging to keep the chemical in solution when diluted into a media appropriate for bacterial growth. Thus, a six hour growth assay with BHI and 0.2% Permethrin dissolved in methanol was used to screen the same strains that were initially tolerant. This assay was performed three times to look for consistency in performance.

[0031] Candidates were moved to a minimalist media to force the bacteria to use the permethrin as a carbon source. Bacterial CFU at 0, 6, 24, 30, 48 and 72 hours were collected to assess growth. This assay was performed three times. At this point five (5) candidates emerged as possible degrading strains of permethrin. Those candidate strains were then screened using pyrethrin as the sole carbon source to look for general pyrethroid degrading ability

[0032] Correct controls were created for the newly created bacterial growth assay as assessment for degradation of chemicals. At this point, one of the five microbial candidate enzymes was identified as a specific carboxylesterase for degradation in plasma. This enzyme was cloned into an expression vector system so a Spectral Analysis of the degradation of permethrin by the enzyme over time in a cell free assay could be performed.

[0033] The five enzymes having pyrethroid degradation abilities are identified in Table 1.

TABLE-US-00001 TABLE 1 DNA Abbrev- Amino Acid SEQ iation Esterase Source SEQ ID No. ID No. E1 pytZ Ochrobactrum sp. SEQ. ID No. 1 E2 pnbA B. subtilis SEQ. ID No. 2 E3 PytH2 Sphingobium SEQ. ID No. 3 SEQ. (pytH) wenxiniae ID No. 6 E4 MsEl Methylobacterium sp. SEQ. ID No. 4 E5 EstP Klebsiella sp. SEQ. ID No. 5

[0034] With reference to nucleic acids of the invention, the term “isolated nucleic acid” is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5′ and 3′ directions) in the naturally occurring genome of the organism from which it originates. For example, the “isolated nucleic acid” may comprise a DNA or cDNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the DNA of a prokaryote or eukaryote.

[0035] With respect to protein, the term “isolated protein” or “isolated and purified protein” is sometimes used herein. This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in “substantially pure” form.

[0036] The term “vector” refers to a small carrier DNA molecule into which a DNA sequence can be inserted for introduction into a host cell where it will be replicated. An “expression vector” is a specialized vector that contains a gene or nucleic acid sequence with the necessary regulatory regions needed for expression in a host cell.

[0037] For ease of reference, amino acid sequences of the enzymes are provided in Table 2.

TABLE-US-00002 TABLE 2  Esterase name and source Amino Acid Sequence EstP MEICTKGSRKHLTSRASEP SYNVPENQYVLYVVSSTLSIVICQLVKV (Klebsiella sp.) AESKKSRFSGAIEKLNERLDSLKDYRIINRDLVVKDLERLKKRFDT SEQ. ID No. 5 EVINAELSEQLAKINVNLSRSYSEKGYLRLEKATGSENEWWAK1K PSQNDDWQQFEPDGYTIFSSRDHYASLEKSYSDYEIEAKIPLPLRR GICAVVLYPEYISRICVLPESRSIQREQENFTKLRDKGIALSKKDWQ AKLTIDELAEQEKERATINKRLGYFATEHEKVGIVHKALEPKLICP FQQIEKEWRQCKVKSKSTFPNSMTFVQNPAYQAVHSGFKKLKEQI GLADEDILLSLEKIEAIGLVNMPLLYERWCLLQIIKVLTQAFRYQPE DNWKRKLIANIQGNEEQISIQFFNPSVSRAITLQYEPFLANGKRPDF VLDVEAITKSGNQISKRLVVDAKYYSAAYLKQRGGIGGVIHELYN GKDYSECQENSVFVLHPVLDAVEKVVSPQEWAKDSYLGELSMFD WEPAHHQRQATNYGAVCANPMKSQRYLDEIQRMLGMFLQYGIE DNTSFRGASDDTHAVNFCVSCGSEKVVDVTKSMSSNNQKRWYR CNECTHFTVYTHC GTCNTRLIKNGEYWTYLS LMPMS SINIKCPNC ESPV MsE1 MTQDTTGFIFIREEP GPDTTRPLMLLHGTGGDENDLLPLGRMVAPE (Methylobacterium sp.) AALLAPRGGVSENGMPRFFRRLAEGVFDEADLRRRTGDLAAFVA SEQ. ID No. 4 ASRARYGLGAPLALGFSNGANIAASLLMLRPETLTGAVLIRPMVPF AEPPAADLAGRPVLIL SGAMDPIVPVENARRLAQQLSASGARVEH RILPAGHGLSQADVSQALAWLRSLPGPEAA PnbA MTHQIVTTQYGKVKGTTENGVHKWKGIPYAKPPVGQWRFKAPEP (Bacillus subtilis) PEVWEDVLDATAYGSICPQPSDLLSLSYTELPRQSEDCLYVNVFAP SEQ. ID No. 2 DTP SKNLPVMVWIHGGAFYLGAGSEPLYDGSKLAAQGEVIVVTL NYRLGPFGELHLSSFNEAYSDNLGLLDQAAALKWVRENISAFGGD PDNVTVFGESAGGMSIAALLAMPAAKGLFQICAIMESGASRTMTIC EQAASTSAAFLQVLGINEGQLDKLHTVSAEDLLKAADQLRIAEKE N1FQLFFQPALDPKTLPEEPEKAIAEGAASGIPLLIGTTRDEGYLFFT PDSDVHSQETLDAALEYLLGKPLAEKVADLYPRSLESQIHMMTDL LEWRPAVAYASAQSHYAPVWMYREDWHPKKPPYNKAFHALELP FVFGNLDGLERMAKAEITDEVKQLSHTIQSAWITFAKTGNPSTEA VNWPAYHEETRETLILDSEITIENDPESEICRQKLEPSKGE pPytH2 MTVTDIILIHGALNRGACYDAVVPLLEARGYRVHAPDLTGHTPGD (Sphingobium wenxiniae) GGHLSVVDMEHYTRPVADILARAEGQSILLGHSLGGASISWLAQH SEQ. ID No. 3 HYDKVAGLIYLTAVLTAPGITPETFVLPGEPNRGTPHALDLIQPVDE GRGLQADFSRLERLREVFMGDYPGEGMPPAEQFIQTQSTVPFGTP NPMEGRALEIPRLY1EALDDVVIPIAVQRQMQKEFPGPVAVVSLPA SHAPYYSMPERLAEAIADFADAPAEYRQTATKAGPDRPAGADGG RADRADLP PytZ MTTQTYEHRLKAGAKGAPLFIVEHGTGGDENQFFGLAEQLLPDAT (Ochrobactrum sp.) IMSPRGDVSEYGAARFFRRTGEGVYDMEDLARATDKMAGFIAAL SEQ. ID No. 1 AAEYKTSEVIGLGYSNGANIMANLLIEKGRVEDKAALLHPLVPFRP KDNPALEGAKILMTAGRMDPICPPDLTEALAQYFERQKADVELV WHPGGHELRQTELAAVQSLLAY

Compositions

[0038] A method for treating an animal who has been exposed to pyrethroid comprises administering an effective amount of a pyrethroid detoxifying enzyme to prevent or treat pyrethroid toxicity. The pyrethroid detoxifying enzyme is one or more hydrolyzing carboxylesterases.

[0039] Animals may include humans, aquatic species, a poultry species, a porcine species, a bovine species, an ovine species, an equine species, and companion animals, such as canines and felines.

[0040] The pyrethroid detoxifying enzyme may be administered by enteric/enteral route, parenteral or topical route.

[0041] The pyrethroid detoxifying enzymes of the present invention may be incorporated into pharmaceutical compositions that may be delivered to a subject, so as to allow delivery of a biologically active enzyme. In a particular embodiment of the present invention, pharmaceutical compositions comprising sufficient genetic material to enable a recipient to produce a therapeutically effective amount of a pyrethroid detoxification. An effective amount of the enzyme may be directly injected or infused into a patient in need thereof. The compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents.

[0042] The pharmaceutical compositions may also contain a pharmaceutically acceptable excipient. Such excipients include any pharmaceutical agent which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, sugars and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

[0043] Pharmaceutical formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0044] The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc.

[0045] The pharmaceutical preparation may contain dosages of between IV push or injection 0.001 mg-1 g/kg/dose of pytH2 for IV push or injection. An oral dose pharmaceutical preparation may contain 1 mg-5 g/kg pytH2 per dose.

[0046] In one embodiment, the enzyme is administered to an animal orally or through feeding. The enzyme is administered to the animal a feed composition or drinking water comprising an effective amount of an additive one or more hydrolyzing carboxylesterases wherein the one or more hydrolyzing carboxylesterases causes an effect selected from the group consisting of preventing or treating pyrethroid toxicity.

[0047] In one embodiment, the enzyme administered to an animal comprises or consists of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4 or 5, an allelic variant thereof; or is a fragment thereof able to degrade one or more pyrethroids.

[0048] The isolated enzyme can comprise an amino acid sequence having at least 75% identity to SEQ. ID Nos. 1, 2, 3, 4, or 5.

[0049] The isolated enzyme can comprise an amino acid sequence having at least 80% identity to SEQ. ID Nos. 1, 2, 3, 4, or 5.

[0050] The isolated enzyme can comprise an amino acid sequence having at least 85% identity to SEQ. ID Nos. 1, 2, 3, 4, or 5.

[0051] The isolated enzyme can comprise an amino acid sequence having at least 90% identity to SEQ. ID Nos. 1, 2, 3, 4, or 5.

[0052] The isolated enzyme can comprise an amino acid sequence having at least 95% identity to SEQ. ID Nos. 1, 2, 3, 4, or 5.

[0053] The isolated enzyme can comprise an amino acid sequence having at least 98% identity to SEQ. ID Nos. 1, 2, 3, 4, or 5.

[0054] The isolated enzyme can comprise an amino acid sequence having at least 99% identity to SEQ. ID Nos. 1, 2, 3, 4, or 5.

[0055] Enzymes can be prepared by a number of standard biochemical and molecular biology methods which are generally known in the art. For example, a gene encoding an enzyme can be amplified from chromosomal DNA using the polymerase chain reaction (PCR), and cloned into a suitable vector (e.g., a plasmid vector). The vector suitably comprises a multiple cloning site into which the DNA molecule encoding the fusion protein can be easily inserted. The vector also suitably contains a selectable marker, such as an antibiotic resistance gene, such that microorganisms transformed, transfected, or mated with the vector can be readily identified and isolated. Where the vector is a plasmid, the plasmid suitably also comprises an origin of replication. Alternatively, DNA coding for the enzyme protein can be integrated into the chromosomal DNA of the microorganism host.

[0056] The host can then be cultured and enzyme harvested from the cultures. A crude cell extract can be used or the enzyme can be partially or substantially purified using standard biochemical techniques.

[0057] Suitable hosts for large-scale production of enzymes include but are not limited to Bacillus species (e.g., Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus megaterium, Bacillus thuringiensis, Bacillus fusiformis, Bacillus cereus, or Bacillus mycoides), Escherichia coli, Aspergillus niger, Aspergillus oryzae, Streptomyces species, Klebsiella species, Mucor species, Rhizopus species, Mortierella species, Kluyveromyces species, Candida species, Penicillium chrysogenum, Trichoderma species Saccharomyces cerevisiae, Pichiapastoris, Hansenula polymorpha, Kluyveromyces lactis, Yarrowia lipolytica, Schizosaccharomyces pombe, and Candida utilitis.

[0058] Enzymes can be expressed with a tag (e.g., a polyhistidine (His)) to facilitate purification as can be seen in FIG. 1.

pNBA Method

[0059] Each of the identified enzymes were evaluated for esterase activity assay using pNBA method described below. Determination of carboxylesterase activity of cargo enzyme in Bacillus thuringiensis spore productions or of free enzyme productions. Three active carboxylesterases cloned into pBCm (Bt EO) and pET28a (E. coli BL21) systems: pnbA (B. subtilis), PytH2 (Sphingobium wenxiniae strain), and MsE1 (Methylobacterium sp). p-Nitrophenyl acetate was the substrate in assays for esterase activity. The hydrolysis of p-nitrophenyl acetate releases p-nitrophenol and acetate, and while p-nitrophenyl acetate is colorless, p-nitrophenol is yellow. This allows to follow the reaction progress by measuring the generation of yellow color, the absorbance may be monitored between 400 and 410 nm where it shows the max absorbance in basic conditions as shown in FIG. 2.

[0060] 6 mg p-Nitrophenyl acetate (pNPA, CAS #830-03-5, MW: 181.1 g/mol) was weighed and dissolved in 1 mL methanol (CAS #67-56-1, MW: 32.04 g/mol). Stored at −20° C. and kept on ice when preparing reactions.

Phosphate Buffer Solution (PBS)

[0061] 8.0 g sodium chloride (NaCl, CAS #7647-14-5, MW: 58.44 g/mol), 1.44 g sodium phosphate dibasic was dissolved (Na2HPO4, CAS #7558-79-4, MW: 141.96 g/mol), and 0.24 g potassium phosphate monobasic (KH2PO4, CAS #7778-77-0, 136.09 g/mol) in 800 mL DI H20, then adjust pH to 7.4 using hydrochloric acid (HCl, CAS #7647-01-0, MW: 36.46 g/mol). Make 100 mL aliquots and autoclave.

1 mg/mL Esterase from Rabbit Liver in PBS

[0062] Esterase from rabbit liver (CAS #9016-18-6, Sigma E0887-powder). Store at −20° C. To prepare a stock dissolve 1 mg in 1 mL PBS, kept on ice when using.

[0063] Up to 1 mL spore production was pelleted up for 5 min at max speed. Supernatant was discarded without disturbing spore pellet and suspend pellet in ice-cold PBS. Spores kept suspended on ice until use.

[0064] The assay was run at 30° C. for 30 minutes. The assay was shaken continuously. Activity was read on (A) 400 BioTek Synergy/HTX microplate reader was used. The results of pNPA hydrolysis are shown in Table 3 depicting carboxylesterase enzyme activity for each of the five strains using pNPA hydrolysis.

TABLE-US-00003 TABLE 3 Abbrev- pNPA hydrolysis [nmol iation Enzyme Source pNPA/min/1E+8 spores E1 pytZ Ochrobactrum sp. BDL E2 pnbA B. subtilis 19.7 ± 1.4 E3 PytH2 Sphingobium 26.3 ± 0.9 wenxiniae E4 MsE1 Methylobacterium 30.6 ± 0.0 sp. E5 EstP Klebsiella sp. BDL BDL—below the detectable limit of the assay

Permethrin Hydrolysis

[0065] The three enzymes, pnbA, pytH2 and msE1 and control were identified, expressed and tested in the quantifiable esterase assay.

[0066] Permethrin hydrolysis was then performed for control as shown in FIGS. 3 and 4, and three esterases pnbA, pytH2 and MsE1. 3 μL of 83,333 ppm permethrin was added to 497 μL MeOH to make 500 μL of 500 ppm permethrin. 10 μL of 500 ppm permethrin was added to 940 μL PBS. 50 μL 2.63 mg/mL pPytH2PytH2 was added to the PBS/permethrin mixture to make a 5 ppm permethrin, 132 μg/mL pytH2 enzymatic reaction mixture. The enzymatic mixture was then incubated at 37 C for 1 hour. Samples of the enzymatic reaction were taken before and after the reaction (T0 before enzymatic reaction and T1) 1 hour after reaction.

[0067] 400 μL of the enzymatic reaction was added to 600 uL MeOH to make a 2 ppm permethrin solution in an HPLC sample vial. A volume of each sample was injected into Waters 2695 Separations Module with a 2996 Photodiode Array Detector. Samples were run isocratically (85% acetonitrile and 15% water). Analysis was carried out at 254 nm and compared to a 5-point 2-fold standard curve for permethrin (Trans-permethrin and Cis-permethrin). The treatments from the permethrin hydrolysis are fully described in Table 4 below. They are reported as absolute remaining from the initial starting concentration and the percent permethrin concentration relative to the T0 control treatment.

TABLE-US-00004 TABLE 4 Permethrin hydrolysis by esterase proteins expressed in E0 Enzymatic reaction 1 mL reactions (PBS:MeOH) 20 ppm (20 ug/mL) 2.44 product Incubation 1 h Esterase % Permethrin (2.44 1E+8 spores) hydrolysis BcIA.sub.20-35-E2 7.50 BcIA.sub.20-35-E3 9.18 BcIA.sub.20-35-E4 4.75

Cypermethrin Degradation

[0068] 50 ppm of cypermethrin was placed in 1 mL PBS with 10 μL pPytH2, PytH2 and reacted for 15 minutes at 37 C with shaking at 300 rpm. HPLC was run on the sample after being diluted 1:1 MeOH using instrument method described above for permethrin for 10 minutes. Cypermethrin was degraded at a rate of 12.69 nmol/min/μg pPytH2,PytH2.

Deltamethrin Degradation

[0069] Using the method and instruments described above for permethrin, 40% of deltamethrin was degraded by pPytH2, PytH2 esterase as shown in FIG. 5.

Fenpropathrin Degradation

[0070] 1000 ppm fenpropathrin was diluted to 5 point curve which was able to detect on HPLC the gradient of 20% water and 80% using the instrument method described above for permethrin. 20 ppm of fenpropathrin was placed in 1 uL PBS with 10 μL fenpropathrin and reacted for 15 minutes at 37 C with shaking. HPLC was run on the sample after being diluted 1:1 MeOH using instrument method described above for permethrin. A 90% degradation of fenpropathrin was detected with PytH2.

Various Matrices and Permethrin

[0071] An assay was developed to determine the effect of matrix components on esterase activity with identified enzymes, pnbA, pytH2 and msE1 and control were identified, expressed and tested with various matrices. The results are shown in FIG. 6.

Biological Permethrin Assay

[0072] A biological permethrin assay was performed for control, and three esterases pnbA, pytH2 and MsE1. The sensitivity of scuds (Hyalella Azteca) to permethrin was tested with three esterases pnbA, pytH2 and MsE1.

[0073] The LD50 of the crickets by permethrin was determined and a delivery mechanism was created. Each of the three esterases pnbA, pytH2 and MsE1 were tested to determine their ability to prevent lethality to the insects as seen in FIG. 7.

Analytical Demonstration of Permethrin Degradation

[0074]

TABLE-US-00005 Bioassay methods Analytical methods Cricket killing Reverse Phase Chromotography Drosophila killing Scud killing (preferred) Spectroscopic analysis

[0075] Insecticides have broad-spectrum toxicities and are lethal to multiple non target aquatic invertebrates in contaminated areas. Standard methods have been developed for assessing the toxicity of contaminants associated with sediments using amphipods and other invertebrates. The amphipod Hyalella azteca (scuds) is broadly used to assess such toxicities because scuds are relatively sensitive to chemicals, have contact with sediment, are easy of culture in laboratory, and are tolerant to varying culture conditions.

[0076] Two fish tanks with clean aquarium gravel were prepared and half of scuds were transferred to each tank. Two volumes of scud water were added to each tank. The culture was aerated and a bubbler was introduced and turned on in each tank. Scuds were fed every other week by adding ¼ full spoon of fish food.

[0077] Scuds that are close to 3 mm in length were selected and transferred to each container. Ten scuds were placed back in large glass pan with 3 mm.sup.2 grid underneath. Each scud was individually selected and placed in a 150 mL beaker that is filled with 50 mL scud water. A separate beaker was used for one scud for one treatment.

[0078] For control and each of the three esterases pnbA, pytH2 and MsE1, ten scuds were exposed to permethrin and one of pnbA, pytH2 or MsE1.

Biological Permethrin Assay for Esterase Activity

[0079]

TABLE-US-00006 Esterases: Activity units Control  8.1 pnbA 31.4 pytH2 31.6 MsE1 39.5

Reaction mix:

[0080] 100 μL 50% MeOH in PBS [0081] 0.2 μg/μL

Incubation:

[0082] 37 degree C. [0083] 18 hr [0084] Shanking 1000 rpm
Transferred 20 μL rxn mix [4 μg] to 100 mL [containing 10 scuds]
At time, 24 h and 48 h the ten scuds were scored:
A=Alive and Moving actively
BA=Barely Alive—Difficulty swimming away; only legs are moving
D=Dead—Not moving at all
The results of which are shown in FIGS. 8 and 9.

Producing pPytH2PytH2 Carboxylesterase

[0085] The esterase gene (PytH2) of Sphingobium wenxiniae was synthesized as a DNA fragment containing an open reading frame (ORF) of 873 bp. The ORF of PytH2 was isolated by PCR and incorporated into the pET28a plasmid by homologous recombination. This plasmid was transformed into E. coli BL21 (DE3) and verified by sequencing. The esterase was expressed in Escherichia coli, under the control of T7 system. The expression plasmid, pET28a, used to introduce the PytH2 gene in the recipient strain is based on the replication origin of E. coli, on pBR322 and is selected for with the bacterial resistance gene KanR. The plasmid contains the expression cassette consisting of a T7Iac promoter, a fragment of the Sphingobium wenxiniae PytH2 (Pyrethroid hydrolase) gene encoding the esterase activity, N-terminal His tag, and the transcriptional terminator of T7.

[0086] Protein is a carboxylesterase from Sphingobium wenxiniae produced by PytH2 recombinant, expressed in E. coli BL21 (DE3) IUBMB: 3.1.1.88 CAS: 9016-18-6. The Chemical Composition of the carboxylesterase has Chemical purity: >90% buffer of 10 mM phosphate (pH 7.4), 2.7 mM KCI, 137 mM NaCI. Post translational procedures produced in a protease deficient E. coli strain and optimized for synthesis. The protein Appearance: clear, colorless solution:

Physical State: Liquid

[0087] pH: 7.4
Water Solubility: Not applicable

Storage: −20° C.

[0088] Unit definition: One unit is defined as the amount of enzyme required to generate 1 μmol/min of 4-p-nitrophenol from 4-nitrophenyl-L-acetate at 30° C. and pH 7.4.
Specific activity: 15±3 μ/mg

[0089] This enzyme is quite stable, and does not require a cofactor for its activity. It becomes inactive after incubation at 70° C. The enzyme is strongly inhibited by metal ions, but can recover activity. The enzyme is also deactivated by surfactant compounds such as SDS.

Activity of the enzyme: In order of hydrolysis efficiency: transpermethrin>cis-permethrin>fenpropathrin>trans-cypermethrin>cis-cypermethrin>cyhalothrin>fenvalerate>deltamethrin>bifenthrin.

[0090] An esterase gene (PytH2) of Sphingobium wenxiniae containing an open reading frame (ORF) of 873 bp. The ORF of PytH2 was expressed in Escherichia coli, under the control of the promoter T7. The amino acid sequence of PytH2 indicated that the esterase is a novel member of the A/B hydrolase family of enzymes and that the enzyme contains a catalytic triad, consisting of Ser78, Asp202, and His230 and a structural motif, GHSLG tight turn. E. coli BL21(DE3)/pET28 containing PytH2 expressed a novel 31.4-kDa protein corresponding to PytH2 in an N-terminal fusion with the His-tag peptide. The recombinant Sphingobium wenxiniae PytH2 protein was purified to electrophoretic homogeneity in a one-step affinity chromatography procedure on Ni-NTA Resin. The optimum pH and temperature of the purified enzyme were 7.0 and 37 degrees C., respectively. Among the pNP (p-nitrophenyl) esters tested, pNP-acetate (C(2)), was the best substrate. It was also active on pNP-butyrate(C(4) and pNPcaproate(C(6)).

TABLE-US-00007 PytH2  SEQ ID No. 6                                                 840 base pairs 5′-ATG ACT GTA ACC GAT ATC ATA CTC ATT CAC GGA GCG CTC AAC CGT GGT GCC TGT TAC GAT GCG GTC GTC CCG CTG TTA GAG GCC CGC GGA TAT CGT GTC CAT GCT CCT GAC CTG ACA GGG CAC ACA CCA GGC GAC GGG GGA CAT CTT AGT GTA GTA GAC ATG GAA CAC TAT ACT CGC CCA GTA GCA GAC ATA CTC GCT CGG GCT GAA GGC CAA TCC ATC CTG TTA GGA CAC AGC CTG GGC GGC GCC TCT ATC TCT TGG TTG GCC CAA CAC CAC CCA GAC AAA GTT GCT GGT CTC ATT TAT CTG ACC GCC GTT TTG ACA GCG CCA GGC ATA ACA CCG GAG ACA TTC GTT TTA CCA GGC GAG CCA AAT CGT GGA ACG CCT CAT GCA CTC GAC TTG ATC CAA CCA GTT GAT GAG GGA CGG GGA TTG CAG GCT GAT TTC TCC CGG TTA GAG AGA CTT CGT GAG GTC TTT ATG GGC GAC TAC CCT GGC GAA GGG ATG CCG CCT GCA GAG CAG TTT ATT CAA ACA CAG TCA ACA GTG CCG TTT GGT ACG CCT AAT CCA ATG GAG GGG CGT GCT CTT GAA ATT CCT CGC CTT TAT ATA GAA GCG CTT GAC GAT GTG GTC ATA CCG ATA GCC GTC CAA CGC CAA ATG CAA AAG GAG TTT CCA GGC CCG GTG GCT GTC GTA TCA CTC CCA GCT AGC CAC GCA CCT TAC TAT AGT ATG CCA GAG AGA CTT GCG GAG GCA ATT GCC GAT TTT GCA GAT GCC CCG GCC GAA TAT CGT CAG ACT GCC ACA AAG GCG GGC CCT GAT AGA CCG GCA GGG GCG GAC GGT GGC CGG GCT GAC CGT GCG GAT CTG CCA-3′ PytH2  SEQ. ID No. 5                                                                   280 as MTVTDIILIHGALNRGACYDAVVPLLEARGYRWAPDLTGHTPGDGGHL5VVDMEWYTRPVADILARAEGQSILLGESL GGASISWLAQHHPDKVAGLIYLTAVLTAPGITPETFVLFGEPNRGTPHALDLIQPVDEGRGLQADFSRLERLREVFMGDY PGEGMPPAEQFIQTQSTVPFGTPNPMEGRALEIPRLYIEALDDVVIPIAVQRQMQKEETGPVAVVSL2ASHAPYYSMPER LAEAIADFADAPAEYRQTATKAGPDRPAGADGGRADRADLP Primers below used for verification of PytH2 in plasmid via PCR: (S954) CATCATCACAGCAGCGGAATGACTGTAACCGATATCATACTCATTCACGGAGC (S955) TITCGGGCTTIGTTATGGCAGATCCGCACGGTCA Expected amplicon size: (pET28a-PytH2) 0.985 kb as seen in FIG. 10. Nucleotide sequence plasmid SEQ. ID No. 7 as seen in FIG. 11 Name-pET28a-PytH2 TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTAC ACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCA AGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAG GGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAA TAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGC CGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTT TACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATA TGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGAT TATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGC AAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATA AGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCC AGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGA TTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCG CAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCC CGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAA ATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAAC AACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATT TATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCT CATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTT TCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTG CTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCG AAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCA AGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTC GTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTG CACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCAC GCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGC TTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGA TGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGC CTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATA CCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTAT TTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCA TAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGC TGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATG TGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGC GATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGAT AAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGG GGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGG AACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGC GCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGT GCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTC GCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAAC CCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAA TGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGA ATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTG CCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCC ACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAAC TTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGC CAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAG CTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAA TCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATC CGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCAT CGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCC CGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAG AACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACC GTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGT GCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCG AGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCA GTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGC CAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGC TTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACA CCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTA TCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGC ATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATG GCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGG CGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCG GCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGT GAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCAGCAGCCATC ATCATCATCATCACAGCAGCggaATGACTGTAACCGATATCATACTCATTCACGGAGCGCTCAACCGTGGTGCCTGT TACGATGCGGTCGTCCCGCTGTTAGAGGCCCGCGGATATCGTGTCCATGCTCCTGACCTGACAGGGCACACACCAG GCGACGGGGGACATCTTAGTGTAGTAGACATGGAACACTATACTCGCCCAGTAGCAGACATACTCGCTCGGGCTG AAGGCCAATCCATCCTGTTAGGACACAGCCTGGGCGGCGCCTCTATCTCTTGGTTGGCCCAACACCACCCAGACAA AGTTGCTGGTCTCATTTATCTGACCGCCGTTTTGACAGCGCCAGGCATAACACCGGAGACATTCGTTTTACCAGGC GAGCCAAATCGTGGAACGCCTCATGCACTCGACTTGATCCAACCAGTTGATGAGGGACGGGGATTGCAGGCTGAT TTCTCCCGGTTAGAGAGACTTCGTGAGGTCTTTATGGGCGACTACCCTGGCGAAGGGATGCCGCCTGCAGAGCAGT TTATTCAAACACAGTCAACAGTGCCGTTTGGTACGCCTAATCCAATGGAGGGGCGTGCTCTTGAAATTCCTCGCCT TTATATAGAAGCGCTTGACGATGTGGTCATACCGATAGCCGTCCAACGCCAAATGCAAAAGGAGTTTCCAGGCCC GGTGGCTGTCGTATCACTCCCAGCTAGCCACGCACCTTACTATAGTATGCCAGAGAGACTTGCGGAGGCAATTGCC GATTTTGCAGATGCCCCGGCCGAATATCGTCAGACTGCCACAAAGGCGGGCCCTGATAGACCGGCAGGGGCGGAC GGTGGCCGGGCTGACCGTGCGGATCTGCCATAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGA GCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCC GGAT

Serum Analysis

[0091] Cat serum was acquired, permethrin was added to the serum and was assayed as a control by HPLC. After the cat serum control was analyzed three different concentrations of the enzyme were introduced to the cat serum control and then analyzed by HPLC. The results of the analysis show that 10 μl enzyme concentration added to the poisoned cat serum degraded all of the permethrin and the HPLC analyzed 0.0% permethrin in the control cat serum after the enzyme was applied. The results are shown in Table 5.

TABLE-US-00008 TABLE 5 Serum plus permethrin in HPLC Cat serum +permethrin +/−E3 15 min ↓ 37 degree C. Extraction w/MeOH +filtering ↓ HPLC Total Total % Trans- Cis- Total permethrin permethrin permethrin Sample permethrin permethrin Std (area) (expected 2 ppm) control No enzyme 16163.75 18519 13% 34682.75 1.402 30 μL E3 0 0 0 0 0.000 0.0% 10 μL E3 0 0 0 0 0.000 0.0% 2.5 μL E3 0 1624.5 24% 1624.5 0.098 7.0%
In the serum there appears to be either an inability to recover all of the permethrin or a native esterase that has a preference for the trans isomer